繁殖青枯菌噬菌体无毒菌株的筛选及应用

doi:10.16409/j.cnki.2095-039x.2018.06.014

中国生物防治学报 ›› 2018, Vol. 34 ›› Issue (6): 906-913.DOI: 10.16409/j.cnki.2095-039x.2018.06.014

繁殖青枯菌噬菌体无毒菌株的筛选及应用

林志坚¹, 夏志辉¹, 顾钢², 周挺², 胡方平¹, 蔡学清¹

1. 福建农林大学植物保护学院/闽台作物有害生物生态防控国家重点实验室, 福州 350002;
2. 福建省烟草公司烟草科学研究所, 福州 350003

收稿日期:2018-05-17 出版日期:2018-12-08 发布日期:2018-12-08
通讯作者: 蔡学清,博士,教授,E-mail:caixq90@163.com
作者简介:林志坚,硕士研究生,E-mail:705250271@qq.com
基金资助:
国家重点研发计划（2017YFD0201106-02）；中国烟草总公司福建省公司科技计划（闽烟司科-2017-2号）；福建农林大学科技创新项目（KFA17541A）

Screening of Avirulent Ralstonia solanacearum Strain to Culture Bacteriophage and Its Application

LIN Zhijian¹, XIA Zhihui¹, Gu Gang², Zhou Ting², HU Fangping¹, CAI Xueqing¹

1. State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops/College of Plant Protection, Fujian Agriculture and Forestry University, Fuzhou 350002, China;
2. Institute of Tobacco Science, Fujian Provincial Tobacco Company, Fuzhou 350003, China

Received:2018-05-17 Online:2018-12-08 Published:2018-12-08

摘要/Abstract

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36 h，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8 d，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。

关键词: 无毒青枯菌, 噬菌体, 致病性, 生物防治

Abstract: 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 21d 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.

Key words: avirulent Ralstonia solanacearum, phage, pathogenicity, biocontrol

中图分类号:

S476

林志坚, 夏志辉, 顾钢, 周挺, 胡方平, 蔡学清. 繁殖青枯菌噬菌体无毒菌株的筛选及应用[J]. 中国生物防治学报, 2018, 34(6): 906-913.

LIN Zhijian, XIA Zhihui, Gu Gang, Zhou Ting, HU Fangping, CAI Xueqing. Screening of Avirulent Ralstonia solanacearum Strain to Culture Bacteriophage and Its Application[J]. journal1, 2018, 34(6): 906-913.

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繁殖青枯菌噬菌体无毒菌株的筛选及应用

Screening of Avirulent Ralstonia solanacearum Strain to Culture Bacteriophage and Its Application

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