一株青枯菌专性噬菌体的分离及应用效果研究

doi:10.13560/j.cnki.biotech.bull.1985.2020-0816

生物技术通报 ›› 2020, Vol. 36 ›› Issue (9): 194-201.doi: 10.13560/j.cnki.biotech.bull.1985.2020-0816

• 根际微生物专题(专题主编：张瑞福研究员) • 上一篇下一篇

一株青枯菌专性噬菌体的分离及应用效果研究

王孝芳¹, 侯玉刚^1,2, 杨可铭¹, 王佳宁¹, 韦中¹, 徐阳春¹, 沈其荣¹

1.南京农业大学资源与环境科学学院作物免疫重点实验室江苏省固体有机废弃物资源化高新技术研究重点实验室江苏省有机固体废弃物资源化协同创新中心资源节约型肥料教育部工程研究中心国家有机类肥料工程技术研究中心,南京 210095;
2.中化作物保护品有限公司,上海 200125

收稿日期:2020-07-03 出版日期:2020-09-26 发布日期:2020-09-30
作者简介:王孝芳,女,博士研究生,研究方向：土壤微生物与土传病害防控;E-mail：wangxf@njau.edu.cn
基金资助:
国家自然科学基金项目（41671248）,中央高校基本科研业务费专项资金（KYXK202003）,江苏省自然科学基金项目（BK20170085,SBK2020042856）

Isolation of Specific Phage of Ralstonia solanacearum and Its Effects on Control of Soil-borne Bacterial Wilt Disease

WANG Xiao-fang¹, HOU Yu-gang^1,2, YANG Ke-ming¹, WANG Jia-ning¹, WEI Zhong¹, XU Yang-chun¹, SHEN Qi-rong¹

1. College of Resources and Environmental Science Nanjing Agricultural University,Key Laboratory of Plant Immunity,Jiangsu Provincial Key Lab of Organic Solid Waste Utilization,Jiangsu Collaborative Innovation Center for Solid Waste Resource Utilization,Educational Ministry Engineering Center of Resource-saving Fertilizers,National Engineering Research Center for Organic-based Fertilizers,Nanjing 210095;
2. Sinochem International Crop Care Company Limited,Shanghai 200125

Received:2020-07-03 Published:2020-09-26 Online:2020-09-30

摘要/Abstract

摘要： 土传青枯病是最严重的细菌性病害之一,专性侵染细菌的病毒——噬菌体在青枯病的防控中发挥重要作用。前期研究发现不同地区的青枯菌在遗传型和生理型上呈现高度多态性,但噬菌体宿主专一性强,对来自同一地方的青枯菌的侵染能力较强。拟针对特定地区的病原青枯菌,从原位筛选高效稳定的噬菌体,并探究其抑菌的稳定性。以分离自南京麒麟大棚的一株病原青枯菌为宿主,从同一地区病土中筛选其专性噬菌体;并从中筛选出一株抑菌能力最强的噬菌体NJ-P3,室内检测了其基础生物学特性、稳定性和最佳保存方式,盆栽试验探究其最佳应用方式。噬菌体NJ-P3属于裂解性短尾噬菌体,基因组中含有特殊的蛋白可能与其强裂解性相关;抗逆性强,能够耐受50℃高温、4-10的pH、紫外线80 min的照射;在SM缓冲液中低温保存效果稳定,常温条件可短期保存;盆栽试验结果表明与传统灌根法相比,茎部注射方式接种噬菌体NJ-P3应用效果更好,对土传青枯病的防控效果提高了37.5%。从原位筛选到一株高效稳定的青枯菌专性噬菌体,系统地研究了其最佳保存条件和应用方式,为建立青枯菌噬菌体资源库防控土传病害的广泛应用奠定理论基础。

关键词: 土传青枯病, 专性噬菌体, 高效稳定, 应用方式

Abstract: Soil-borne Ralstonia solanacearum is one of the most serious bacterial diseases,and phage,a virus specifically infecting bacteria,plays an important role in the control of R. solanacearum. Previous studies revealed that R. solanacearum in different regions have high polymorphism,while the phage is highly specific to host and its infection to R. solanacearum in the same region is strong. This study intends to in situ screen efficient and stable phage against R. solanacearum in a specific region,and to explore its antibacterial stability. A strain of R. solanacearum isolated from the Kirin Greenhouse in Nanjing was used as the host to screen its obligate phage from the diseased soil in the same area. A phage NJ-P3 with the strongest bacteriostatic ability was screened from it,its basic biological characteristics,stability and best preservation methods were tested indoors,and pot experiment to explore its optimal application methods. The phage NJ-P3 was a lytic podoviridae and there was a special protein in the genome that may be related to its strong lysis. Phage NJ-P3 had strong resistance,and tolerated to high temperature of 50℃,4-10 of pH,and 80 min of ultraviolet irradiation. NJ-P3 stored in SM buffer showed the highest stability in low temperature but can be stored in short time under normal temperature condition. Pot experiment demonstrated that the control effect on the soil-borne R. solanacearum was better compared to traditional soil drench（SD）method and increased by 37.5% on the average by the stem injection（SI）method for inoculating phage NJ-P3. In sum,we obtained a highly efficient and stable R. solanacearum-specific phage,and systematically explored the optimal storage conditions and application methods,which lays a theoretical foundation for the establishment of phage resource library of R. solanacearum and widespread application of phage in the prevention and control of soil-borne diseases in the future.

Key words: soil-borne Ralstonia solanacearum, specific phages, efficiency and stability, application methods

王孝芳, 侯玉刚, 杨可铭, 王佳宁, 韦中, 徐阳春, 沈其荣. 一株青枯菌专性噬菌体的分离及应用效果研究[J]. 生物技术通报, 2020, 36(9): 194-201.

WANG Xiao-fang, HOU Yu-gang, YANG Ke-ming, WANG Jia-ning, WEI Zhong, XU Yang-chun, SHEN Qi-rong. Isolation of Specific Phage of Ralstonia solanacearum and Its Effects on Control of Soil-borne Bacterial Wilt Disease[J]. Biotechnology Bulletin, 2020, 36(9): 194-201.

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一株青枯菌专性噬菌体的分离及应用效果研究

Isolation of Specific Phage of Ralstonia solanacearum and Its Effects on Control of Soil-borne Bacterial Wilt Disease

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