烟草角斑病菌拮抗细菌筛选及抗菌活性物质研究
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摘要
研究从黑龙江省健康烟草植株及其根际土壤和海南三亚采集的20种杂草中分别分离到287和98个细菌分离物,筛选出对烟草角斑病菌有拮抗活性的菌株22个,其中来自三亚稗草叶片中的一株细菌PA-2的抑菌效果最好,抑菌效果稳定。经形态学、生理生化及分子生物学鉴定,PA-2为铜绿假单胞杆菌(Pseudomonas aeruginosa)。筛选出最佳实验室发酵配方:氮源1%(牛肉膏??蛋白胨=5??3),葡萄糖0.3%,NaCl 0.5%,KNO30.3%;最佳培养条件:发酵温度28℃,培养基pH 7.0~7.5,接种量4%,装液量为250 mL三角瓶中装液50 mL。PA-2优化发酵液的田间防效达69.77%,比农用链霉素防效提高24.47%。采用硫酸铵分级沉淀法分离抑菌活性物质,结果表明,在(NH4)2SO4浓度为30%~90%各浓度梯度下均有抑菌活性物质沉淀,其中60%~70%(不含60%)是抑菌活性物质的最适盐析饱和度。抑菌活性物质具有较强的热稳定性、较好的蛋白酶耐受性、较好的耐碱性、较差的耐酸性,能溶于部分有机溶剂,经乙醇、乙醚、氯仿、乙酸乙酯、正丁醇和丙酮萃取后,该抑菌活性物质抑菌活性下降范围为1.4%~4%。确定该抑菌活性物质为小分子碱性、水溶性脂肽类物质。
In the present study,287 and 98 bacterial strains were isolated from healthy tobacco plants and their rhizospheric soil samples as well as 20 kinds of weeds from Hainan Province, in which 22 strains had antibacterial activity against Pseudomonas syingae pv.tabaci. The strain PA- 2 that was identified as Pseudomonas aeruginosa by morphological, physiological and biochemical as well as molecular biology tests was isolated from the leaf of Echinochloa crusgalli in Sanya and had the best inhibiting and stable由Pseudomonas syingae pv.tabaci(Wolf et Foster) effect. The optimal fermentation medium components for PA-2 constituted of 1% of nitrogen(beef extract:peptone=5:3), 0.1% of glucose, 0.5% of sodium chloride, and 0.3% of potassium nitrate. The optimal fermentation conditions: cultural temperature 28 ℃, optimum initial pH 7.0-7.5, inoculation seed liquid 4%and 50 mL of loading liquid in a 250 flask. The field effect of PA- 2 fermentation liquid against tobacco angular leaf spot was 69.77%, 24.47% increase over the streptomycin. We used the ammonium sulfate precipitation classification method to separate the antibacterial active substances, the results showed that each 30%-90% saturation of(NH4)2SO4had precipitated antibacterial active substances, in which 60%-70%(exclusive 60%) was the most appropriate saturation. The antibacterial active substances had stronger heat stability, better tolerance to proteinase K, better resistance to alkali, but poor tolerance to acid, dissolved in parts of the organic solvents. The decreasing range of the antibacterial activity of extractants of Ethyl alcohol, Ethyl ether, chloroform, ethyl acetate, n-butanol and acetone was 1.4%-4.0%. So they might be a kind of alkaline and water-solubility antibacterial substance.
In the present study,287 and 98 bacterial strains were isolated from healthy tobacco plants and their rhizospheric soil samples as well as 20 kinds of weeds from Hainan Province, in which 22 strains had antibacterial activity against Pseudomonas syingae pv.tabaci. The strain PA- 2 that was identified as Pseudomonas aeruginosa by morphological, physiological and biochemical as well as molecular biology tests was isolated from the leaf of Echinochloa crusgalli in Sanya and had the best inhibiting and stable由Pseudomonas syingae pv.tabaci(Wolf et Foster) effect. The optimal fermentation medium components for PA-2 constituted of 1% of nitrogen(beef extract:peptone=5:3), 0.1% of glucose, 0.5% of sodium chloride, and 0.3% of potassium nitrate. The optimal fermentation conditions: cultural temperature 28 ℃, optimum initial pH 7.0-7.5, inoculation seed liquid 4%and 50 mL of loading liquid in a 250 flask. The field effect of PA- 2 fermentation liquid against tobacco angular leaf spot was 69.77%, 24.47% increase over the streptomycin. We used the ammonium sulfate precipitation classification method to separate the antibacterial active substances, the results showed that each 30%-90% saturation of(NH4)2SO4had precipitated antibacterial active substances, in which 60%-70%(exclusive 60%) was the most appropriate saturation. The antibacterial active substances had stronger heat stability, better tolerance to proteinase K, better resistance to alkali, but poor tolerance to acid, dissolved in parts of the organic solvents. The decreasing range of the antibacterial activity of extractants of Ethyl alcohol, Ethyl ether, chloroform, ethyl acetate, n-butanol and acetone was 1.4%-4.0%. So they might be a kind of alkaline and water-solubility antibacterial substance.
引文
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[14]沈萍,范秀荣,李广武.微生物学实验[M].3版.北京:高等教育出版社,2003:28-30,116-120.
[15]布坎南R E,吉本斯N E.伯杰细菌鉴定手册[M].8版.北京:科学出版社,1984:54-68.
[16]车秀珠,蔡妙英.常见细菌鉴定手册[M].北京:科学出版社,2001:364-398.
[17]Schroth N,Milton J G.Hancock.Importance of chitin synthesis for fungal growth and as a target for antifungal agents[J].Journal ofGeneralMicrobiology,1981,53(10):281-285.
[18]刘永峰,陈志谊,张杰,等.枯草芽孢杆菌B2916胞外抗菌蛋白质的性质[J].江苏农业学报,2005,21(4):288-293.
[19]张成省,孔凡玉,关小红,等.烟草叶围细菌Tpb55菌株的鉴定及其抑菌活性[J].中国生物防治,2008,24(1):63-68.
[20]Keer A.Interactions of soil microflora with cucurbit wilt special emphasison non-pathogenic Fusarium[J].1982(20):167-192.
[21]Klepper J W,Beauchamp C J.A review of issues related to measuring colonization of plant roots by bacteria[J].Can J Microbiol,1992,38:1219-1232.
[22]赵善欢,张兴.植物质杀虫剂对水稻三化螟的拒食及内吸毒力试验[J].中国农业科学,1982(2):55-62.
[23]张兴.试论无公害农药[J].西北农业大学学报,1995,23(6):90-95.
[2]刘雅婷,张世珖,李永忠,等.防治烟草野火病的药剂筛选及应用研究[J].湖南农业大学学报,2002(2):109-111.
[3]王家银,李明菊,刘玉斌,等.抗生素类药剂防治烟草野火病应用研究[J].云南农业科技,2000(1):11-12.
[4]张晓雯,高芬,魏颖颖,等.拮抗链霉菌H210发酵液抗菌谱及理化性质[J].湖北农业科学,2008,47(6):2283-2284.
[5]乔婵,陈星,张艳菊,等.PF7-5对烟草角斑病的室内及大田防治效果[J].中国林副特产,2010(5):18-19.
[6]Hiraoka H,Asaka O,Ano T,et al.Molecular cloning of a gene responsible for the biosynthesis of lipopeptide antibiotics iturin and surfactin[J].Journal of Fermentation and Bioengineering,1992,74:323-3261.
[7]Buchenauer H.Biological control of soil-borne disease by rhizobacteris[J].PlantDiseaseandProtection,1995,44:40-50.
[8]陈晓斌,张炳欣.植物根围促生细菌(PGPR)作用机制的研究进展[J].微生物学杂志,2000,20(1):38-41.
[9]Ran L X,Xing M L,Zhou B,et al.Siderophes are the main determinants of Pseudomonas fluorescens strain in suppression of grey mould in Eucalyptus urophylla[J].Plant Disease and Protection,2005,35(1):6-12.
[10]方中达.植病研究方法[M].3版.北京:中国农业出版社,1998:171-208,212-218.
[11]Edwards K,John stone C,Thompson C.A simple and rapid method for the preparation of plant genomic DNA for PCR analysis[M].NucleicAcidsResearch,1991,19:1349.
[12]文景芝,杨晓敏,李樱梅,等.烟草野火病菌拮抗细菌的筛选及其应用研究[J].东北农业大学学报,2012,43(10):156-160
[13]钱存柔,黄仪秀.微生物学实验教程[M].北京:北京大学出版社,2008:23-33,113-129.
[14]沈萍,范秀荣,李广武.微生物学实验[M].3版.北京:高等教育出版社,2003:28-30,116-120.
[15]布坎南R E,吉本斯N E.伯杰细菌鉴定手册[M].8版.北京:科学出版社,1984:54-68.
[16]车秀珠,蔡妙英.常见细菌鉴定手册[M].北京:科学出版社,2001:364-398.
[17]Schroth N,Milton J G.Hancock.Importance of chitin synthesis for fungal growth and as a target for antifungal agents[J].Journal ofGeneralMicrobiology,1981,53(10):281-285.
[18]刘永峰,陈志谊,张杰,等.枯草芽孢杆菌B2916胞外抗菌蛋白质的性质[J].江苏农业学报,2005,21(4):288-293.
[19]张成省,孔凡玉,关小红,等.烟草叶围细菌Tpb55菌株的鉴定及其抑菌活性[J].中国生物防治,2008,24(1):63-68.
[20]Keer A.Interactions of soil microflora with cucurbit wilt special emphasison non-pathogenic Fusarium[J].1982(20):167-192.
[21]Klepper J W,Beauchamp C J.A review of issues related to measuring colonization of plant roots by bacteria[J].Can J Microbiol,1992,38:1219-1232.
[22]赵善欢,张兴.植物质杀虫剂对水稻三化螟的拒食及内吸毒力试验[J].中国农业科学,1982(2):55-62.
[23]张兴.试论无公害农药[J].西北农业大学学报,1995,23(6):90-95.