枯草芽孢杆菌Bs01发酵条件的优化及对胶孢炭疽菌的抑制
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摘要
Bs01是前期研究分离的一株对果实胶孢炭疽菌具有较强抑制作用的枯草芽孢杆菌菌株。本研究以其发酵后的发酵液上清对胶孢炭疽菌的离体抑菌活性为评价指标,优化了Bs01发酵培养基的氮源、碳源和无机盐成分及配比,同时采用正交试验优化了发酵时间、起始pH、接种量、装液量及温度等发酵条件,并对比测定了最佳发酵条件获得的发酵液上清对胶孢炭疽菌的抑制效果。结果表明,可获得具最强离体抑菌活性发酵液上清的发酵培养基最优氮源、碳源及无机盐分别为细菌蛋白胨、葡萄糖和NaCl,最佳的发酵培养基配方为:2%葡萄糖、3%细菌蛋白胨和0.5%NaCl。最佳的发酵条件组合是:起始培养基pH为7.0,接种量15%,装液量1/10,32℃、200 r/min振荡培养48 h。采用优化后的最佳发酵培养基及发酵条件组合获得的发酵液上清可显著抑制活体接种的胶孢炭疽菌病斑扩展,降低病情指数,显著优于优化前获得的发酵液上清的抑制效果。
Bacillus subtilis strain Bs01,which isolated from our previous research,showed inhibitive effects on postharvest pathogen Colletotrichum gloeosporioides.In this work,the culture medium formula(nitrogen sources,carbon sources and inorganic salts)and fermentation conditions(fermentation time,initial pH,inoculation volume,loading volume and fermentation temperature) of Bs01 strain were optimized by single factor and orthogonal tests with antifungal activity of fermentation supernatant on C.gloeosporioides in vitro as the assessment criteria.And in vivo inhibiting effects of the fermentation supernatant obtained under optimized fermentation condition on disease development in tomato and avocado caused by C.gloeosporioides was compared.The results showed that,the optimal medium formula were:glucose 2%,bacterium peptone 3% and sodium chloride 0.5%,and the optimal fermentation conditions were:pH 7.0,inoculum volume 15%,loading volume 1/10,fermentation temperature 32 ℃,fermentation time 48 h,and shaking speed 200 r/min.The fermentation supernatant obtained under the above fermentation conditions could significantly inhibit lesion expansion of anthracnose inoculated with C.gloeosporioides in vivo and reduce disease index,which was obviously superior to previous supernatant.
Bacillus subtilis strain Bs01,which isolated from our previous research,showed inhibitive effects on postharvest pathogen Colletotrichum gloeosporioides.In this work,the culture medium formula(nitrogen sources,carbon sources and inorganic salts)and fermentation conditions(fermentation time,initial pH,inoculation volume,loading volume and fermentation temperature) of Bs01 strain were optimized by single factor and orthogonal tests with antifungal activity of fermentation supernatant on C.gloeosporioides in vitro as the assessment criteria.And in vivo inhibiting effects of the fermentation supernatant obtained under optimized fermentation condition on disease development in tomato and avocado caused by C.gloeosporioides was compared.The results showed that,the optimal medium formula were:glucose 2%,bacterium peptone 3% and sodium chloride 0.5%,and the optimal fermentation conditions were:pH 7.0,inoculum volume 15%,loading volume 1/10,fermentation temperature 32 ℃,fermentation time 48 h,and shaking speed 200 r/min.The fermentation supernatant obtained under the above fermentation conditions could significantly inhibit lesion expansion of anthracnose inoculated with C.gloeosporioides in vivo and reduce disease index,which was obviously superior to previous supernatant.
引文
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[18]WU S,JIA S,SUN D,et al.Purification and characterization of two novel antimicrobial peptides subpeptin JM4-A and subpeptin JM4-B produced by Bacillus subtilis JM4[J].Current Microbiology,2005,51(5):292-296.DOI:10.1007/s00284-005-0004-3.
[19]马瑜,王长晔,柯杨,等.生防细菌枯草芽孢杆菌A97对小鼠的急性毒理作用研究[J].保鲜与加工,2015,15(2):12-17.DOI:10.3969/J.ISSN.1009-6221.2015.02.003.
[20]GRISOLIA C K,OLIVEIRA-FILNO E C,RAMOS F R,et al.Acute toxicity and cytotoxicity of Bacillus thuringiensis and Bacillus sphaericus strains on fish and mouse bone marrow[J].Ecotoxicology,2009,18(1):22-26.DOI:10.1007/s10646-008-0252-7.
[21]AKPA E,JACQUES P,WATHELET B,et al.Influence of culture conditions on lipopeptide production by Bacillus subtilis[J].Applied Biochemistry and Biotechnology,2001,91(1-9):551-561.DOI:10.1385/ABAB:91-93:1-9:551.
[22]鹿秀云,李社增,栗秋生,等.玉米叶斑病拮抗细菌的筛选及其发酵培养基优化[J].中国生物防治,2006,22(S1):47-53.
[2]NUNES C A.Biological control of postharvest diseases of fruit[J].European Journal of Plant Pathology,2012,133(1):181-196.DOI:10.1007/s10658-011-9919-7.
[3]SUN X,RUAN R,LIN L,et al.Genomewide investigation into DNA elements and ABC transporters involved in imazali resistance in Penicillium digitatum[J].FEMS Microbiology Letters,2013,348(1):11-18.DOI:10.1111/1574-6968.12235.
[4]JACOBSEN B J,ZIDACK N Z,LARSON B J.The role o Bacillus-based biological control agents in integrated pes management systems:plant diseases[J].Phytopathology,200494(11):1272-1275.DOI:10.1094/PHYTO.2004.94.11.1272.
[5]United States Environmental Protection Agency.Office of Pesticide Program:Biopesticide[DB/OL].[2017-12-23]http://www.epa.gov/pesticides/biopesticides.
[6]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].生物技术通讯,2010(1):24-29.
[7]黄海禅,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):213-215,219.DOI:10.3969/j.issn0528-9017.2005.03.022.
[8]林开春,徐雪莲,代鹏,等.一种含有枯草芽孢杆菌的防腐保鲜剂:200510011649.4[P].2007-11-28.
[9]杨佐忠,叶建仁.枯草芽孢杆菌PRS5菌剂控制水果贮藏期病害的研究[J].南京林业大学学报(自然科学版),200630(1):89-92.DOI:10.3969/j.jssn.1000-2006.2006.01.022.
[10]张荣意,谭志琼.一种防治香蕉冠腐病的芽孢杆菌及其应用:201210333094.5[P].2013-01-02.
[11]ZHENG G,SLAVIK M F.Isolation,partial purification and characterization of a bacteriocin produced by a newly isolated Bacillus subtilis strain[J].Letters in Applied Microbiology1999,28(5):363-367.DOI:10.1046/j.1365-2672.1999.00545.x.
[12]林福呈,李德葆.枯草芽孢杆菌(Bacillus subtilis)S9对植物病原真菌的溶菌作用[J].植物病理学报,2003,33(2):174-177.DOI:10.3321/j.issn:0412-0914.2003.02.016.
[13]谢栋,彭憬,王津红,等.枯草芽孢杆菌抗菌蛋白X98Ⅲ的纯化与性质[J].微生物学报,1998,31(1):13-19.
[14]林东,徐庆,刘忆舟,等.枯草芽孢杆菌S0113分泌蛋白的抑菌作用及抗菌蛋白的分离纯化[J].农业生物技术学报,2001,9(1):77-80.DOI:10.3969/j.issn.1674-7968.2001.01.018.
[15]刘永峰,陈志谊,张杰.枯草芽孢杆菌B-916胞外抗菌蛋白质的性质[J].江苏农业学报,2005,21(4):288-293.DOI:10.3969/j.issn.1000-4440.2005.04.009.
[16]REN J J,SHI G L,WANG X Q,et al.Identification and characterization of a novel Bacillus subtilis strain with potent antifungal activity of a flagellin-like protein[J].World Journal of Microbiology&Biotechnology,2013,29(12):2343-2352.DOI:10.1007/s11274-013-1401-6.
[17]VANITTANAKOM N,LOEFFLER W,KOCH U,et a1.Fengycin-a novel antifungal lipopeptide antibiotic produced by Bacillus subtilis F-29-3[J].Journal of Antibiotics,1986,39(7):888-901.DOI:10.7164/antibiotics.39.888.
[18]WU S,JIA S,SUN D,et al.Purification and characterization of two novel antimicrobial peptides subpeptin JM4-A and subpeptin JM4-B produced by Bacillus subtilis JM4[J].Current Microbiology,2005,51(5):292-296.DOI:10.1007/s00284-005-0004-3.
[19]马瑜,王长晔,柯杨,等.生防细菌枯草芽孢杆菌A97对小鼠的急性毒理作用研究[J].保鲜与加工,2015,15(2):12-17.DOI:10.3969/J.ISSN.1009-6221.2015.02.003.
[20]GRISOLIA C K,OLIVEIRA-FILNO E C,RAMOS F R,et al.Acute toxicity and cytotoxicity of Bacillus thuringiensis and Bacillus sphaericus strains on fish and mouse bone marrow[J].Ecotoxicology,2009,18(1):22-26.DOI:10.1007/s10646-008-0252-7.
[21]AKPA E,JACQUES P,WATHELET B,et al.Influence of culture conditions on lipopeptide production by Bacillus subtilis[J].Applied Biochemistry and Biotechnology,2001,91(1-9):551-561.DOI:10.1385/ABAB:91-93:1-9:551.
[22]鹿秀云,李社增,栗秋生,等.玉米叶斑病拮抗细菌的筛选及其发酵培养基优化[J].中国生物防治,2006,22(S1):47-53.