繁殖青枯菌噬菌体无毒菌株的筛选及应用
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摘要
利用噬菌体防治植物细菌性病害,关键技术是噬菌体的扩大繁殖,利用宿主菌繁殖,应用时存在一定的风险。本研究通过继代培养筛选致病性丧失的青枯菌菌株作为青枯菌噬菌体扩繁的宿主,结果表明,通过对野生青枯菌株RSsw326连续超过20代的培养,获得了一株菌落圆形、游动性弱的变异菌株;再通过刺叶、注射和伤根等方法接种烟苗,30 d后无萎蔫症状出现,将该菌株命名为RSsw326-2;测试表明,该菌株对青枯病菌没有拮抗作用,可被从福建不同烟区分离纯化的8株噬菌体裂解。利用菌株RSsw326-2作为宿主,进行青枯菌噬菌体的扩大繁殖,培养36h,效价可达1010 PFU/mL。将无毒菌株RSsw326-2+噬菌体的共培养液刺叶、伤根和不伤根接种烟苗,35 d后均无症状出现,而将毒性菌株RSsw326+噬菌体的共培养液刺叶接种后14 d、伤根接种后28 d发病率都为100%,不伤根接种烟苗35 d,发病率为66.7%。无毒菌株RSsw326-2+噬菌体的共培养液对盆栽烟苗防病试验结果表明,发病时间推迟8d,并且在接种后21 d时对烟草青枯病的防效达74.1%,显著高于对照组。本研究采用继代培养筛选获得的无毒菌株作为噬菌体扩大繁殖的宿主,为今后研制噬菌体制剂的生产提供了参考。
Phage could be used to control plant bacterial diseases, but how to multiply propagate is a key technology. At present, the host bacteria of the bacteriophage are mostly used to reproduce the phage and it could bring risk when the phage is used. In this study, the wild Ralstonia solanacearum strain RSsw326 had been continuously cultivated for 20 generations and a mutant strain with circular colony and a weak motility was obtained. The tobacco was inoculated with the mutant strain by stabbing, injecting and wounding root, and there were no withered symptoms produced on the tobacco. The result proved the strain, named as RSsw326-2, was an avirulent strain. The strain also had no antagonistic effect on other R. solanacearum strains and could be infected by eight phage strains which were isolated from different tobacco-growing areas in Fujian province. Using strain RSsw326-2 as a host to breed the phage, the phage titer could be 1010 PFU/mL after culturing for 36 h. When tobacco was treated with the culture solution of avirulent strain RSsw326-2+bacteriophage by stabbing, wounding roots and without wounding roots, there were no wilting symptoms on the tobacco in 35 d. But the disease incidence was 100%, 100% and 66.7% in 14 d after inoculation by stabbing, in 28 d by wounding and in 35 d without wounding roots with the culture solution of RSsw326+bacteriophage, respectively. The results also showed that the culture solution of avirulent strain RSsw326-2+bacteriophage performed good control potential against tobacco bacterial wilt disease with 74.1% control efficiency in pot experiment in 21 d after treatment. Compared with the treatment by the culture solution of the virulent strain RSsw326+bacteriophage, the symptoms delayed for 8 d. Taking an avirulent strain as host of phage multiple propagation might provide a significant basis for development and preparation of highly effective bacterial phage agent.
Phage could be used to control plant bacterial diseases, but how to multiply propagate is a key technology. At present, the host bacteria of the bacteriophage are mostly used to reproduce the phage and it could bring risk when the phage is used. In this study, the wild Ralstonia solanacearum strain RSsw326 had been continuously cultivated for 20 generations and a mutant strain with circular colony and a weak motility was obtained. The tobacco was inoculated with the mutant strain by stabbing, injecting and wounding root, and there were no withered symptoms produced on the tobacco. The result proved the strain, named as RSsw326-2, was an avirulent strain. The strain also had no antagonistic effect on other R. solanacearum strains and could be infected by eight phage strains which were isolated from different tobacco-growing areas in Fujian province. Using strain RSsw326-2 as a host to breed the phage, the phage titer could be 1010 PFU/mL after culturing for 36 h. When tobacco was treated with the culture solution of avirulent strain RSsw326-2+bacteriophage by stabbing, wounding roots and without wounding roots, there were no wilting symptoms on the tobacco in 35 d. But the disease incidence was 100%, 100% and 66.7% in 14 d after inoculation by stabbing, in 28 d by wounding and in 35 d without wounding roots with the culture solution of RSsw326+bacteriophage, respectively. The results also showed that the culture solution of avirulent strain RSsw326-2+bacteriophage performed good control potential against tobacco bacterial wilt disease with 74.1% control efficiency in pot experiment in 21 d after treatment. Compared with the treatment by the culture solution of the virulent strain RSsw326+bacteriophage, the symptoms delayed for 8 d. Taking an avirulent strain as host of phage multiple propagation might provide a significant basis for development and preparation of highly effective bacterial phage agent.
引文
[1]Hayward A C.Biology and epidemiology of bacterial wilt caused by Pseudomonas solanacearum[J].Annual Review of Phytopathology,1991,29(1):65-87.
[2]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.
[3]Bhunchoth A,Phironrit N,Leksomboon C,et al.Isolation of Ralstonia solanacearum-infecting bacteriophages from tomato fields in Chiang Mai,Thailand,and their experimental use as biocontrol agents[J].Journal of Applied Mcrobiology,2015,118(4):1023-1033.
[4]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.
[5]张乐,杨扬.梨火疫病菌噬菌体的初步研究[J].植物检疫,2001,15(1):14-17.
[6]王丽.两种水稻主要病原细菌噬菌体的分离、鉴定和特征化研究[D].杭州:浙江大学,2016,18.
[7]Fegan M,Prior P.How Complex Is the"Ralstonia solanacearum Species Complex"[M]//Allen C,Prior P,Hayward A C.Bacterial Wilt Disease and the Ralstonia solanacearum Cpecies Complex.St.Paul:APS,2005,449-462.
[8]Opina N,Tavner F,Hollway G,et al.A novel method for development of species and strain-specific DNA probes and PCR primers for identifying Burkholderia solanacearum(formerly Pseudomonas solanacearum)[J].Asia-Pacific Journal of Molecular Biology and Biotechnology,1997,5:19-30.
[9]方中达.植病研究方法[M].北京:中国农业出版社,1998,192.
[10]Huang Q,Yan X,Wang J F.Improved biovar test for Ralstonia solanacearum[J].Journal of Microbiological Methods,2012,88(2):271-274.
[11]郑继法,张建华,李连臣,等.烟草不同品种对青枯菌的抗性分析[J].山东农业大学学报,1995,26(1):23-29.
[12]Hayward A C.Characteristics of Pseudomonas solanacearum[J].Journal of Applied Microbiology,1964,27(2):265-277.
[13]王羽,肖崇刚.番茄青枯病病菌无致病力菌株的分离和控病研究[J].西南农业大学学报(自然科学版),2004,26(4):426-428.
[14]陈庆河,翁启勇,胡方平.无致病力青枯菌株对番茄青枯病的防治效果[J].中国生物防治,2004(1):42-44.
[15]杨宇红,刘俊平,杨翠荣,等.无致病力hrp-突变体防治茄科蔬菜青枯病[J].植物保护学报,2008,35(5):433-437.
[16]郑雪芳,刘波,朱育菁.青枯病植物疫苗对番茄根系土壤微生物群落结构的影响[J].中国生物防治学报,2017,33(3):385-393.
[2]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.
[3]Bhunchoth A,Phironrit N,Leksomboon C,et al.Isolation of Ralstonia solanacearum-infecting bacteriophages from tomato fields in Chiang Mai,Thailand,and their experimental use as biocontrol agents[J].Journal of Applied Mcrobiology,2015,118(4):1023-1033.
[4]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.
[5]张乐,杨扬.梨火疫病菌噬菌体的初步研究[J].植物检疫,2001,15(1):14-17.
[6]王丽.两种水稻主要病原细菌噬菌体的分离、鉴定和特征化研究[D].杭州:浙江大学,2016,18.
[7]Fegan M,Prior P.How Complex Is the"Ralstonia solanacearum Species Complex"[M]//Allen C,Prior P,Hayward A C.Bacterial Wilt Disease and the Ralstonia solanacearum Cpecies Complex.St.Paul:APS,2005,449-462.
[8]Opina N,Tavner F,Hollway G,et al.A novel method for development of species and strain-specific DNA probes and PCR primers for identifying Burkholderia solanacearum(formerly Pseudomonas solanacearum)[J].Asia-Pacific Journal of Molecular Biology and Biotechnology,1997,5:19-30.
[9]方中达.植病研究方法[M].北京:中国农业出版社,1998,192.
[10]Huang Q,Yan X,Wang J F.Improved biovar test for Ralstonia solanacearum[J].Journal of Microbiological Methods,2012,88(2):271-274.
[11]郑继法,张建华,李连臣,等.烟草不同品种对青枯菌的抗性分析[J].山东农业大学学报,1995,26(1):23-29.
[12]Hayward A C.Characteristics of Pseudomonas solanacearum[J].Journal of Applied Microbiology,1964,27(2):265-277.
[13]王羽,肖崇刚.番茄青枯病病菌无致病力菌株的分离和控病研究[J].西南农业大学学报(自然科学版),2004,26(4):426-428.
[14]陈庆河,翁启勇,胡方平.无致病力青枯菌株对番茄青枯病的防治效果[J].中国生物防治,2004(1):42-44.
[15]杨宇红,刘俊平,杨翠荣,等.无致病力hrp-突变体防治茄科蔬菜青枯病[J].植物保护学报,2008,35(5):433-437.
[16]郑雪芳,刘波,朱育菁.青枯病植物疫苗对番茄根系土壤微生物群落结构的影响[J].中国生物防治学报,2017,33(3):385-393.