引用本文:赵远超,叶 茂,夏 冰,焦文涛,冯彦房,都瑞军,杜道林,张忠云,黄 丹,孙明明,武 俊,胡 锋,蒋 新,杜良成.多价噬菌体分离筛选及靶向灭活 土壤病原菌的应用研究[J].土壤学报,2019,56(2):420-431.
ZHAO Yuanchao,YE Mao,XIA Bing,JIAO Wentao,FENG Yanfang,都瑞军,杜道林,ZHANG Zhongyun,HUANG Dan,SUN Mingming,WU Jun,HU Feng,JIANG Xin,DU Liangcheng.Targeted Inactivation of Pathogenic Bacteria in the Farmland Soil by Polyvalent Phage Therapy[J].Acta Pedologica Sinica,2019,56(2):420-431
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多价噬菌体分离筛选及靶向灭活 土壤病原菌的应用研究
赵远超,叶茂
南京农业大学\中国科学院南京土壤研究所,中国科学院南京土壤研究所
摘要:
农田土壤中残留和滋生多种病原菌会对人体健康和生态环境带来显著的安全隐患,开展针对性的生物修复研究十分迫切。噬菌体疗法靶向灭活土壤中病原菌的技术为修复此类污染土壤提供了全新途径。本研究以南京城郊某奶牛场牛粪堆积池周边,粪肠杆菌和假单胞菌复合污染农田土壤为例,首先筛选和纯化获得两株专一型噬菌体(YSZ1和YSZ5),再人为加速其宿主谱的表达过程,获得对应的多价噬菌体(YSZ1R和YSZ5K),并进行生物学特性(形态、核酸、最佳感染复数、一步生长曲线等)鉴定,发现:在水相和污染土壤中不同噬菌体对于同步灭活病原菌的能力依次是YSZ5K>YSZ1R>YSZ5>YSZ1,并且施用多价噬菌体疗法有助于维护和改善修复后土壤微生物生态功能多样性与稳定性。本研究结果可为噬菌体疗法靶向灭活土壤中多种病原菌提供切实可行的修复技术。
关键词:  病原菌  土壤  噬菌体疗法  靶向灭活
DOI:10.11766/trxb201801020006
分类号:
基金项目:2017年度江苏省环保科研课重点项目(2017005)、2017年江苏省农业科技自主创新资金项目(CX(17)3047)、中国科学院青年创新促进会(2018350)、国家自然科学基金面上项目(41771350)资助
Targeted Inactivation of Pathogenic Bacteria in the Farmland Soil by Polyvalent Phage Therapy
zhaoyuanchao and yemao
Nanjing Agricultural University, Nanjing 210095, China/Institute of Soil Science,Chinese Academy of Sciences,Institute of Soil Science,Chinese Academy of Sciences
Abstract:
【Objective】 Given the drastic development of livestock industry in China, animal manure amount increased significantly in the last few decades. Without proper environmental management, the manure as well as relevant wastes have made the agricultural soil near the livestock site a hotspot of pathogens, which has posed potential threat against the public health and environmental safety. Therefore, it is urgent to carry out bioremediation technologies to effectively inactivate the targeted pathogens in the soil. Among various techniques, phage therapy provides a novel way for eliminating pathogenic bacteria in the soil. In this work, polyvalent phages were firstly isolated, and further screened for their capacity of inactivating various host pathogenic bacteria, in an attempt to apply the polyvalent phage therapy for controlling the pathogens contamination in the soil. 【Method】In this work, double-layer agar was employed to isolate phages from the soil applied with cow manure in a dairy farm in Nanjing Eastern China,which was contaminated by Enterobacteriaceae and Pseudomonas pathogenic bacteria. Two host-specific phages (YSZ1 and YSZ5) were isolated from the polluted soil. Then with the accelerated human-directed evolution in the laboratory, two polyvalent (broad host range) phages (YSZ1R and YSZ5k) were obtained each corresponding to YSZ1 and YSZ5. Meanwhile, the biological characteristics including morphology and molecular technology were carried out for the identification of the four phages obtained. Moreover, by studying the optimal multiplicity of infection and growth characteristics, the capacity of inactivating pathogenic bacteria among four phages were also determined. Meanwhile, Biolog ECO plates were used to investigate the impact of phage therapy on the diversity of the soil microbial community. 【Result】The morphological and molecular identification indicated that the four phages described above all belonged to Stylovinidaeacid. Meanwhile, in both aqueous system and contaminated soil, the four phages exhibited significacnt but varying capacity of inactivating Fecal Coliform and Pseudomonas, following the order of YSZ5K>YSZ1R>YSZ5>YSZ1. In addition, compared with the control, host-specific phage inoculation (YSZ5 and YSZ1) slightly decreased the AWCD value, while polyvalent phage inoculation (YSZ5K and YSZ1R) significantly increased the AWCD value after 120 h of incubation. Shannon index showed the same trend of YSZ5K/YSZ1R> CK> YSZ5/YSZ1. Therefore, polyvalent phages therapy applied in this work was able to maintain and increase the diversity and stability of the indigenous microbial community in the soil.【Conclusion】The polyvalent phages isolated from the pathogen contaminated soil were determined as Stylovinidaeacid. Both polyvalent phages (YSZ5K and YSZ1R) could significantly stimulate the dissipation of the pathogenic bacteria in both aqueous and soil systems. Meanwhile, the diversity of the indigenous bacteria community in the soil could be clearly enhanced after polyvalent phage therapy, suggesting that the polyvalent phage therapy developed in this work was an applicable technique to inactivate broad range pathogenic bacteria in the soil.
Key words:  Pathogenic bacteria  Phage therapy  Soil  Targeted inactivation