烈性青枯雷尔氏菌噬菌体的分离与生物学特性分析
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摘要
为采用噬菌体疗法防治烟草青枯病,以烟草青枯菌TB15-15为宿主菌,从土壤中筛选分离得到噬菌体,并采用双层平板法检测了噬菌体的效价、最佳感染复数(MOI)、一步生长曲线以及噬菌体对温度和酸碱度的敏感性。结果表明:从长期植烟田块土壤中分离得到1株青枯雷尔氏菌烈性噬菌体,命名为RS-PⅡ-1。经检测该噬菌体最佳感染复数为1;一步生长曲线显示噬菌体潜伏期为10 min,爆发期持续100 min,裂解量为1 411;该噬菌体40℃活性最高,裂解量也最大,当温度超过40℃时活性逐渐下降,直至在60℃时活性丧失;噬菌体对酸碱均具有一定的耐受能力,pH在4~8范围内噬菌体均有很好裂解活性,当p H小于4或大于8,裂解活性均减小,特别是在p H大于11时,噬菌体裂解活性完全丧失,因此噬菌体RS-PⅡ-1不适合与碱性溶液混合应用。
To utilize phage therapy in tobacco bacterial wilt controlling, R. solanacearum strain TB15-15 was used as a host bacterium to screen a phage from tobacco soil. Phage titer, multiplicity of infection(MOI), onestep growth curve and phage sensitivity to temperature and p H were measured by a two-layer plating method.The results showed that a virulent phage infecting TB15-15 strain was isolated from soil of a long-term tobacco planting field and named as RS-PⅡ-1. The MOI, phage latent period, burst phase and burst size of RS-PⅡ-1 were 1, 10 min, 100 min, 1 411, respectively. The lytic activity and burst size of RS-PⅡ-1 was the highest at40 ℃. When temperature was higher than 40 ℃, the activity of RS-PⅡ-1 decreased gradually until it was lost at60 ℃. Phage had a certain tolerance to p H change, RS-PⅡ-1 showed a good lytic activity in the p H range of 4-8. The lytic activity was reduced when p H <4 or >8, and the phage completely lost lytic activity at p H>11.These results suggested that using RS-PⅡ-1 in an alkaline solution was not suitable.
To utilize phage therapy in tobacco bacterial wilt controlling, R. solanacearum strain TB15-15 was used as a host bacterium to screen a phage from tobacco soil. Phage titer, multiplicity of infection(MOI), onestep growth curve and phage sensitivity to temperature and p H were measured by a two-layer plating method.The results showed that a virulent phage infecting TB15-15 strain was isolated from soil of a long-term tobacco planting field and named as RS-PⅡ-1. The MOI, phage latent period, burst phase and burst size of RS-PⅡ-1 were 1, 10 min, 100 min, 1 411, respectively. The lytic activity and burst size of RS-PⅡ-1 was the highest at40 ℃. When temperature was higher than 40 ℃, the activity of RS-PⅡ-1 decreased gradually until it was lost at60 ℃. Phage had a certain tolerance to p H change, RS-PⅡ-1 showed a good lytic activity in the p H range of 4-8. The lytic activity was reduced when p H <4 or >8, and the phage completely lost lytic activity at p H>11.These results suggested that using RS-PⅡ-1 in an alkaline solution was not suitable.
引文
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[11]蔡刘体,汪汉成,袁赛飞,等.青枯菌特异性噬菌体的研究进展与应用[J].生物技术通讯,2012,23(6):887-890.CAI Liuti,WANG Hancheng,YUAN Saifei,et al.Research and application of special bacteriophage of Ralstonia solanacearum[J].Letters in Biotechnology,2012,23(6):887-890.
[12]王盛,童贻刚.噬菌体治疗研究进展[J].微生物学通报,2009,36(7):1019-1024.WANG Sheng,TONG Yigang.Recent advance in bacteriophage therapy[J].Microbiology,2009,36(7):1019-1024.
[13]Tanaka H,Negishi H,Maeda H.Control of tobacco bacterial wilt by an avirulent strain of Pseudomonas solanacearum M4S and its bacteriophage[J].Japanese Journal of Phytopathology,1990,56(2):243-246.
[14]Lee J,Park T H.Isolation and characterization of bacteriophages infecting Ralstonia solanacearum from potato fields[J].,2016,22(4):236-242.
[15]高苗,杨金广,刘旭,等.一株裂解性青枯雷尔氏菌噬菌体的分离及生物学特性分析[J].中国农业科学,2015,48(7):1330-1338.GAO Miao,YANG Jinguang,LIU Xu,et al.Isolation and biological properties of a lytic phage infecting Ralstonia solanacearum[J].Scientia Agricultura Sinica,2015,48(7):1330-1338.
[16]Nakayama N,Okumura M,Inoue K,et al.Abundance of bacteriophages of common heterotrophic bacteria in the floodwater of a Japanese paddy field[J].Soil Science and Plant Nutrition,2007,53(5):595-605.
[17]路荣,顾敬敏,刘晓贺,等.NDM-1肺炎克雷伯菌裂解性噬菌体的分离鉴定及其对小鼠菌血症的治疗研究[J].中国兽医学报,2012,32(12):1747-1751.LU Rong,GU Jingmin,LIU Xiaohe,et al.Using lytic bacteriophage to control murine bacteremia caused by NDM-l-producing Klebsiella pneumoniae strain BAA-2146[J].Chinese Journal of Veterinary Science,2012,32(12):1747-1751.
[18]余静丹.鲍曼不动杆菌噬菌体的分离鉴定及生物学特性的研究[D].长春:吉林大学,2013.YU Jingdan.Isolation,characterization and biological features of lytic bacteriophages infecting Acinetobacter baumannii clinical isolates[D].Changchun:Jilin University,2013.
[19]王丹.阴沟肠杆菌噬菌体的分离鉴定及生物学特性的初步研究[D].长春:吉林大学,2013.WANG Dan.Preliminary research on isolation and biological characteristics of a lytic bacteriophage against Enterobacter cloacae[D].Changchun:Jilin University,2013.
[20]Gu J M,Xu W,Lei L C,et al.Lys GH15,a novel bacteriophage lysin,protects a murine bacteremia model efficiently against lethal methicillin-resistant Staphylococcus aureus infection[J].Journal of Clinical Microbiology,2011,49(1):111-117.
[21]Lu Z,Breidt F Jr,Fleming H P,et al.Isolation and characterization of a Lactobacillus plantarum bacteriophage,ΦJL-1,from a cucumber fermentation[J].International Journal of Food Microbiology,2003,84(2):225-235.
[22]Weiss B D,Capage M A,Kessel M,et al.Isolation and characterization of a generalized transducing phage for Xanthomonas campestris pv.campestris[J].Journal of Bacteriology,1994,176(11):3354-3359.
[23]Van Truong Thi B,Khanh N H P,Namikawa R,et al.Genomic characterization of Ralstonia solanacearum phage?RS138 of the family Siphoviridae[J].Archives of Virology,2016,161(2):483-486.
[24]Van T T B,Yoshida S,Miki K,et al.Complete genome sequence of a filamentous bacteriophage,RS611,that infects the phytopathogen Ralstonia solanacearum[J].Archives of Virology,2015,160(3):865-867.
[2]刘雅婷,张世光.烟草青枯病的研究进展[J].云南农业大学学报,2001,16(1):72-76.LIU Yating,ZHANG Shiguang.New progress of tobacco bacterial wilt[J].Journal of Yunnan Agricultural University,2001,16(1):72-76.
[3]余静丹,史红艳,王丹,等.鲍曼不动杆菌噬菌体生物学特性的研究[J].微生物学杂志,2013,33(2):17-23.YU Jingdan,SHI Hongyan,WANG Dan,et al.Biologic characteristics of bacteriophages specific to Acinetobacter baumannii[J].Journal of Microbiology,2013,33(2):17-23.
[4]郭坚华,孙平华,吴云波,等.植物细菌性青枯病的生物防治机制和途径[J].中国生物防治学报,1997,13(1):42-46.GUO Jianhua,SUN Pinghua,WU Yunbo,et al.Mechanisms and methods biocontrol of plant bacterial wilt diseases[J].Chinese Journal of Biological Control,1997,13(1):42-46.
[5]张竹青,罗宽,高必达,等.七株抗青枯病菌生防菌的初步鉴定[J].湖南农业大学学报(自然科学版),2002,28(6):512-513.ZHANG Zhuqing,LUO Kuan,GAO Bida,et al.Studies on the identification of some bacteria for biological control of tobacco bacterial wilt[J].Journal of Hunan Agricultural University(Natural Sciences),2002,28(6):512-513.
[6]卢洪兴,曾军,邱志丹,等.烟草青枯病发生与药剂防治研究[J].福建省农科院学报,1996,11(3):41-45.LU Hongxing,ZENG Jun,QIU Zhidan,et al.Studies on the occurrence and chemical control of tobacco bacterial wilt(P.solanacearum)[J].Journal of Fujian Academy of Agricultural Sciences,1996,11(3):41-45.
[7]纪成灿,方树民,顾钢,等.烟草品种抗青枯病鉴定中的相关因素分析[J].中国烟草科学,2000,21(2):1-4.JI Chengcan,FANG Shumin,GU Gang,et al.Analysis of correlated factors in the identification of tobacco bacterial wilt resistance[J].Chinese Journal of Tobacco Science,2000,21(2):1-4.
[8]刘焕利,何礼远,毛国璋,等.植物青枯病原细菌胞外蛋白相关基因的克隆[J].植物病理学报,1999,29(2):110-114,119.LIU Huanli,HE Liyuan,MAO Guozhang,et al.Cloning of the gene related to extracellular proteins secreted by Ralstonia solanacearum[J].Acta Phytopathologica Sinica,1999,29(2):110-114,119.
[9]Jones J B,Jackson L E,Balogh B,et al.Bacteriophages for plant disease control[J].Annual Review of Phytopathology,2007,45:245-262.
[10]Breitbart M,Rohwer F.Here a virus,there a virus,everywhere the same virus?[J].Trends in Microbiology,2005,13(6):278-284.
[11]蔡刘体,汪汉成,袁赛飞,等.青枯菌特异性噬菌体的研究进展与应用[J].生物技术通讯,2012,23(6):887-890.CAI Liuti,WANG Hancheng,YUAN Saifei,et al.Research and application of special bacteriophage of Ralstonia solanacearum[J].Letters in Biotechnology,2012,23(6):887-890.
[12]王盛,童贻刚.噬菌体治疗研究进展[J].微生物学通报,2009,36(7):1019-1024.WANG Sheng,TONG Yigang.Recent advance in bacteriophage therapy[J].Microbiology,2009,36(7):1019-1024.
[13]Tanaka H,Negishi H,Maeda H.Control of tobacco bacterial wilt by an avirulent strain of Pseudomonas solanacearum M4S and its bacteriophage[J].Japanese Journal of Phytopathology,1990,56(2):243-246.
[14]Lee J,Park T H.Isolation and characterization of bacteriophages infecting Ralstonia solanacearum from potato fields[J].,2016,22(4):236-242.
[15]高苗,杨金广,刘旭,等.一株裂解性青枯雷尔氏菌噬菌体的分离及生物学特性分析[J].中国农业科学,2015,48(7):1330-1338.GAO Miao,YANG Jinguang,LIU Xu,et al.Isolation and biological properties of a lytic phage infecting Ralstonia solanacearum[J].Scientia Agricultura Sinica,2015,48(7):1330-1338.
[16]Nakayama N,Okumura M,Inoue K,et al.Abundance of bacteriophages of common heterotrophic bacteria in the floodwater of a Japanese paddy field[J].Soil Science and Plant Nutrition,2007,53(5):595-605.
[17]路荣,顾敬敏,刘晓贺,等.NDM-1肺炎克雷伯菌裂解性噬菌体的分离鉴定及其对小鼠菌血症的治疗研究[J].中国兽医学报,2012,32(12):1747-1751.LU Rong,GU Jingmin,LIU Xiaohe,et al.Using lytic bacteriophage to control murine bacteremia caused by NDM-l-producing Klebsiella pneumoniae strain BAA-2146[J].Chinese Journal of Veterinary Science,2012,32(12):1747-1751.
[18]余静丹.鲍曼不动杆菌噬菌体的分离鉴定及生物学特性的研究[D].长春:吉林大学,2013.YU Jingdan.Isolation,characterization and biological features of lytic bacteriophages infecting Acinetobacter baumannii clinical isolates[D].Changchun:Jilin University,2013.
[19]王丹.阴沟肠杆菌噬菌体的分离鉴定及生物学特性的初步研究[D].长春:吉林大学,2013.WANG Dan.Preliminary research on isolation and biological characteristics of a lytic bacteriophage against Enterobacter cloacae[D].Changchun:Jilin University,2013.
[20]Gu J M,Xu W,Lei L C,et al.Lys GH15,a novel bacteriophage lysin,protects a murine bacteremia model efficiently against lethal methicillin-resistant Staphylococcus aureus infection[J].Journal of Clinical Microbiology,2011,49(1):111-117.
[21]Lu Z,Breidt F Jr,Fleming H P,et al.Isolation and characterization of a Lactobacillus plantarum bacteriophage,ΦJL-1,from a cucumber fermentation[J].International Journal of Food Microbiology,2003,84(2):225-235.
[22]Weiss B D,Capage M A,Kessel M,et al.Isolation and characterization of a generalized transducing phage for Xanthomonas campestris pv.campestris[J].Journal of Bacteriology,1994,176(11):3354-3359.
[23]Van Truong Thi B,Khanh N H P,Namikawa R,et al.Genomic characterization of Ralstonia solanacearum phage?RS138 of the family Siphoviridae[J].Archives of Virology,2016,161(2):483-486.
[24]Van T T B,Yoshida S,Miki K,et al.Complete genome sequence of a filamentous bacteriophage,RS611,that infects the phytopathogen Ralstonia solanacearum[J].Archives of Virology,2015,160(3):865-867.