土壤噬菌体微生态研究方法进展与挑战

Methodological breakthroughs and challenges in research of soil phage microecology

摘要:

作为专性侵染细菌和古菌的一类病毒，噬菌体因其极高的丰度与遗传多样性成为地球生态系统的关键调控者，在物质循环、能量流动及生态服务功能中发挥了重要作用。在土壤微生态系统中，噬菌体更被视为微生物组中的“暗物质”，通过精准调控微生物群落结构与功能，维持着土壤-植物系统的生态平衡与健康稳态。近年来，噬菌体相关研究在土壤领域日益增长，但比例依然较低，其主要原因是研究人员对噬菌体微生态研究方法不熟悉、相关技术体系尚未完善以及土壤环境的特殊性。为此，本文从4个方面系统整合了土壤噬菌体研究的关键技术体系：(1) 基于切向流技术的土壤噬菌体富集策略；(2) 整合双层琼脂平板法、荧光显微计数和流式细胞仪的精准定量检测方法；(3) 基于宏基因组、宏病毒组的多组学联合分析技术；(4) 融合机器学习算法与生态进化理论的噬菌体-宿主互作解析框架。在此基础上，提出未来应关注噬菌体资源库构建、RNA噬菌体的挖掘、未知基因功能的解析以及多组学数据的深度整合与互作验证等方面的研究，为土壤噬菌体群落结构解析、功能挖掘及宿主互作机制研究提供系统性的方法与技术支撑。

Abstract:

Phages, as obligate bacterial and archaeal parasites, constitute a virus group of paramount ecological significance due to their exceptional abundance and genetic diversity. These biological entities serve as critical regulators in Earth’s ecosystems, driving biogeochemical cycles, energy fluxes, and ecosystem services across terrestrial and marine environments. Within soil microbiomes, phages function as microbial “dark matter,” maintaining the soil-plant system balance through precise modulation of the microbial community structure and functional dynamics. Despite the growing research interests in soil phages in recent years, the proportion of such studies in environmental virology remains disproportionately low, which is primarily attributed to researchers’ limited familiarity with the research methodologies for phage microecology, incomplete technical frameworks, and inherent challenges posed by soil environmental complexity. To address these challenges, this review synthesizes cutting-edge methodologies for soil phage investigation from four aspects: (1) tangential flow filtration (TFF)-based phage enrichment strategies; (2) integrated quantification approaches combining double-layer agar plating, epifluorescence microscopy, and flow cytometry; (3) multi-omics analytical pipelines leveraging metagenomics and viromics datasets; and (4) computational frameworks merging machine learning algorithms with eco-evolutionary theory for deciphering phage-host interaction networks. Through comparative analysis of methodological principles, technical merits, and application scopes, we establish a comprehensive workflow for soil phage research. Future research in this field should prioritize: (1) construction of soil phage resource libraries, (2) exploration of RNA phages based on transcriptomes, (3) functional characterization of unknown genes, and (4) deep integration and interaction validation of multi-omics data. This systematic methodological synthesis provides critical technical references for addressing fundamental challenges in characterizing soil phages regarding the community structure, functional potential, and interaction mechanisms with hosts.