Bacterial pathogens are significant contributors to waterborne diseases globally. Viruses, as natural competitors and predators, interact with these pathogens through various infection cycles (lysogenic, lytic, chronic). Nutrient limitation, particularly phosphorus (P) deficiency, can heavily influence these interactions. P and nitrogen (N) are crucial growth-limiting factors in aquatic ecosystems, impacting both phototrophic and heterotrophic microbes. Liebig's law of the minimum suggests growth depends on the most limiting nutrient. Various criteria exist to define P-oligotrophic conditions based on total P (TP) and total N (TN) concentrations, with a high N:P ratio (e.g., exceeding 22.6) often indicating P limitation. P plays a vital role in ATP synthesis, nucleic acids, phospholipids, and other biomolecules; its deficiency represses cellular processes like DNA replication and protein synthesis. Viral infection cycles are tightly linked to nutrient supply and host availability. "Kill-the-winner" theory posits that lytic viruses target dominant hosts, while "piggyback-the-winner" suggests lysogeny prevails during host blooms. Metagenomic sequencing is increasingly used for rapid, large-scale pathogen detection in water quality surveillance, providing metagenome-assembled genomes (MAGs) for ecological insights. The Middle Route of the South-to-North Water Diversion Canal (MR-SNWDC) in China presents a unique case study due to its long-lasting P deficiency since its operation in 2015. This research investigates the impact of this P limitation on virus-pathogen dynamics and water quality along the 1432 km canal.

The literature review extensively discusses the roles of phosphorus and nitrogen in microbial growth, different criteria for defining phosphorus limitation in aquatic systems, and the contrasting theories explaining viral infection cycles (lytic vs. lysogenic) under various nutrient conditions. Existing studies on viral ecology suggest a correlation between viral lifestyles and environmental factors, with lysis being more prevalent in oligotrophic environments. The limitations of traditional pathogen detection methods are highlighted, paving the way for the application of metagenomic sequencing in water quality surveillance. The review also provides background on the MR-SNWDC, emphasizing its significance and the public concern surrounding water quality safety in this large-scale water transfer project.

The study utilized historical TP concentration data (2015-2021) from the MR-SNWDC, complemented by a special sampling campaign in August 2020 (autumn) and March 2021 (spring) at 32 monitoring stations along the canal. Water samples were filtered, and total genomic DNA was extracted and sequenced using the NovaSeq 6000 platform. Multiple environmental factors (pH, EC, turbidity, temperature, various nutrient concentrations, etc.) were measured. Bioinformatics analyses involved quality control of sequencing reads using TrimGalore, de novo assembly with MEGAHIT, and binning of contigs using metaWRAP. Prokaryotic MAGs were generated, and viral contigs were identified using several tools (viralVerify, VIBRANT, DeepVirFinder, PPR-Meta, Virsorter2), with quality assessed by CheckV. Non-redundant viral operational taxonomic units (vOTUs) were generated using CD-HIT. Taxonomic assignments of MAGs and vOTUs were conducted using GTDB-Tk and geNomad, respectively. Abundance profiles were calculated using Bowtie2 and CoverM. Virus-host linkages were predicted using three in silico methods (nucleotide sequence homology, tRNA match, CRISPR spacer match), validated against the Virus-Host Database. Antibiotic resistance genes (ARGs) and virulence factor genes (VFGs) were identified using BLASTP against SARG v2.2 and VFDB databases. Gene annotation was performed using eggNOG-mapper. Statistical analyses included NMDS, PERMANOVA, partial Mantel test, random forest, and co-occurrence network analysis. A regional growth factor (RGF) was calculated to assess bacterial population dynamics.

The study revealed long-lasting P limitation (TP < 0.02 mg/L and high N:P ratios) in the MR-SNWDC over seven years. Viral communities showed distinct spatial patterns, with most vOTUs originating from the Danjiangkou Reservoir. Bacterial richness decreased downstream, correlating with TP concentration. Viruses in the P-limited environment had significantly smaller genomes compared to those in other environments, suggesting adaptation to minimize resource costs. Bacterial pathogens showed repressed growth potential and reduced P-associated gene expression (e.g., phosphate transport system, ribonucleotide reductase). Viral predation was enhanced, with increased virus-to-host ratios and higher abundances of virus-encoded RNR genes downstream. The abundance of antibiotic-resistant "super pathogens" significantly decreased downstream. Viruses encoded auxiliary metabolic genes (AMGs) involved in P acquisition and nucleotide metabolism, indicating their ability to influence host metabolism.

The findings demonstrate a natural paradigm of water self-purification driven by virus-pathogen interactions under sustained P limitation. The reduced growth potential of pathogens due to P deficiency, coupled with enhanced viral predation, effectively decreased pathogen abundance and associated health risks. The smaller viral genomes and increased viral RNR gene expression highlight adaptive strategies for survival in P-limited environments. The virus-mediated P cycling, through viral shunt, likely played a role in maintaining the balance of the viral and bacterial communities. The results suggest the potential of harnessing natural viral predation for water quality management, similar to bacteriophage therapy used in other contexts.

This study reveals a natural water purification process in the MR-SNWDC driven by virus-pathogen dynamics under long-term P limitation. Viruses adapted to P scarcity with smaller genomes and enhanced lytic infection, reducing pathogen abundance and health risks. This research highlights the potential of leveraging natural viral predation for sustainable water resource management and drinking water safety. Future research could focus on quantifying the contribution of viral shunts to P cycling and developing more refined biophysical models for understanding these complex interactions.

While the study provides compelling evidence of virus-pathogen interactions and their impact on water quality, further research is needed to definitively quantify the contribution of viral shunts to the P cycle. The study primarily focused on the main canal and might not fully capture the variations occurring in branching canals. Long-term monitoring across multiple seasons is also recommended to confirm the seasonal trends and the long-term impact of P limitation on the microbial community.