Effects of Agricultural Inputs on Soil Virome-associated Antibiotic Resistance and Virulence: A Focus on Manure, Microplastic and Pesticide

Soil viruses are increasingly recognized as crucial mediators of horizontal gene transfer, yet their role in disseminating antibiotic resistance genes (ARGs) and virulence factor genes (VFGs) under agricultural disturbances remains poorly understood. Here, we characterized the viromes and associated ARGs and VFGs in agricultural soils treated with low- and high-dose manures, microplastics, and pesticides. Using metagenomic sequencing coupled with advanced viral identification tools, we found that manure fertilization markedly altered viral community composition and increased viral diversity. Manure also enhanced the abundance of ARGs and VFGs in viromes by 2.0-9.8-fold and 2.0-8.1-fold, respectively, while microplastics and pesticides had limited impacts. Additionally, gene pathways related to human diseases and environmental adaptation were enriched in soil viromes treated with manures and high-dose pesticides. Virus-host prediction revealed that Actinomycetia dominated bacterial hosts of both ARG- and VFG-carrying viruses, with some VFG-carrying viruses linked to potential human pathogens, e.g., Escherichia albertii and Klebsiella pneumoniae. Co-occurrence network analysis indicated that these disturbances strengthened connections between bacteria, viruses, and ARGs (or VFGs). Our study provides a comprehensive profile of viromes and associated risks in agricultural soil under three disturbances, highlighting the role of viruses in spread of antibiotic resistance and pathogenic risks in agricultural soil.