Effects of earthworms on soil virus-associated ARGs and resistance phenotypes in long-term field cropping systems

Long-term effects of earthworms on soil resistomes under realistic field conditions remain poorly understood. Here, we conducted a two-year corn-wheat rotation study within a long-term field experiment established in 2018 to systematically investigate how a one-time earthworm addition durably modulates resistomes. Integrated metagenomics and viromics analyses revealed that diverse ARGs and virulence factor genes (VFGs) were consistently higher in rhizosphere than bulk soils, identifying the rhizosphere as a hotspot for resistance dissemination. Despite limited metagenomic shifts, long-term earthworm activity suppressed virus-associated ARGs and VFGs, as well as high-risk ARGs, especially in bulk soils. Notably, early phenotypic resistance in the rhizosphere increased markedly in Year 1 (ciprofloxacin + 38.9 %, meropenem + 31.3 %) without concurrent genotypic changes, whereas significant genotypic shifts emerged only in Year 2, indicating that phenotypic resistance preceded genotypic changes. Long-term earthworm addition reshaped microbial life-history strategies from R- to K-strategist traits, enhanced lysogeny proportions that dominated 72.73-85.05 % of the viromes throughout the 2-year crop rotation, and stabilized virus-host networks. Caudovirales infecting Streptomyces and Pseudomonas acted as hubs linking ARGs to nutrient-cycling taxa, with earthworms reinforcing cooperative cross-kingdom interactions. These findings highlight earthworms' long-term regulatory role in microbial adaptation and resistome dynamics, informing resistance risk management under the One Health framework.