Azoxystrobin is a widely used strobilurin fungicide, yet how soil microorganisms respond to overexposure in long-term monocultures, and whether organic amendments can mitigate that response, remains poorly understood. We ran a 30-day greenhouse experiment with soil from a maize monoculture maintained since 1975 under conventional tillage (LC) and no-tillage (SD). Azoxystrobin was applied at the recommended (DR1X, 5 mg kg⁻¹) and tenfold (DR10X) doses, with or without vermicompost (VC, 50 g kg⁻¹). Analyses included 16S rRNA gene sequencing, fatty acid methyl ester (FAME) profiling and partial least squares structural equation modeling (PLS-SEM). DR10X significantly reduced the relative abundance of Gram-positive biomarkers in both systems and increased the fungi/bacteria ratio by 50% under LC, without reducing total microbial biomass. VC increased pH, electrical conductivity and, without fungicide, richness, and reversed the DR10X increase in the fungi/bacteria ratio under LC, but Chloroflexi and Planctomycetota reached their lowest relative abundances in VC-amended fungicide treatments under LC. Shannon diversity was unaffected, whereas Pielou’s evenness increased only under LC with DR10X, with or without VC. Soil chemistry significantly predicted FAME biomarkers only under SD; most other PLS-SEM paths were not significant. Microbial responses to azoxystrobin depended on soil management, and VC effects were taxon-specific rather than protective.

Context: Soil microbial communities are key indicators of bio-geochemical processes; however, their response to fungicides across various agro-management systems requires further investigation. Aims: Under controlled microcosm conditions, we evaluated the effects of the fungicides Carbendazim and Iprodione at recommended and higher rates of application on bacterial community composition, enzymatic activities, and predictive functional profiles in Typic Haplustoll soils from conventional monoculture and conservation crop rotation systems in Argentina. Methods: High-throughput 16S rRNA gene sequencing, enzymatic assays (β-glucuronidase, β-glucosidase, xylanase), and functional inference using Tax4Fun2 were used to assess microbial responses. Key results: Iprodione induced greater shifts in bacterial diversity and community structure, resulting in significant reductions in the phyla Proteobacteria and Acidobacteria, along with increases in the phylum Firmicutes. Enzymatic activity was more severely inhibited by Carbendazim, particularly within monoculture settings. Functional predictions indicated that Iprodione suppressed pathways related to amino acid and carbohydrate metabolism, energy production, and nitrogen cycling genes (nirK, nosZ), while Carbendazim exhibited milder effects. Although crop rotation alleviated some of these negative effects, both fungicides altered predicted microbial functional profiles. Conclusions: Iprodione had a stronger effect on the bacterial community structure and the predicted functional profiles, while Carbendazim showed stronger inhibition on enzymatic activity, especially in monoculture soils. Implications: These results provide mechanistic insight, under controlled microcosm conditions, into the potential susceptibility of soil microbial communities and enzyme activities of soil to fungicide exposure, highlighting the need of refined management strategies for preserving soil microbiological integrity.