Fungal Responses to Neonicotinoid Seed Treatments in Soil: Temporal Shifts in Community Diversity and Composition

Abstract Neonicotinoid seed treatments are widely used in agriculture, yet their effects on soil fungal communities remain underexplored. This study investigated how three commonly applied neonicotinoids- imidacloprid, thiamethoxam, and clothianidin alter fungal community diversity, composition, and temporal dynamics in soil microcosms. Amplicon sequencing of fungal ITS regions was performed on soil samples collected across multiple time points following neonicotinoid exposure. While overall diversity indices remained largely stable across treatments, post-hoc comparisons identified reduction in fungal diversity under imidacloprid and thiamethoxam exposure on day 10. Beta diversity analyses revealed significant effects of sampling day and a treatment-by-time interaction, indicating temporally variable responses. While dominant phyla such as Ascomycota and Basidiomycota remained relatively stable, several less abundant phyla, including Mortierellomycota, Rozellomycota and Olpidiomycota, declined over time. Functional guild analysis further revealed a clothianidin-driven increase in saprotroph abundance and a decrease in symbiotroph abundance. Differential abundance analysis identified compound-specific taxonomic responses. Imidacloprid was associated exclusively with suppressed fungal biomarkers, whereas thiamethoxam induced both enriched and depleted taxa. Clothianidin was associated with the greatest number of discriminatory fungal biomarkers and was accompanied by increased saprotroph abundance and reduced symbiotroph abundance. These findings underscore the value of including fungal community responses when evaluating the broader ecological effects of neonicotinoids and highlight the need for long-term, functionally oriented studies in agroecosystem contexts.