The widespread application of chiral fungicides as seed-coating agents in agriculture has led to serious residue accumulation in soil, increasingly drawing attention to soil pollution remediation strategies for chiral pesticides. This study explored the role of earthworms and soil microorganisms in selectively accelerating the degradation of penflufen in soil. The results showed that soil microorganisms significantly accelerated penflufen enantiomer degradation, particularly the R-enantiomer. Nocardioides, Variovorax, Arthrobacter, and Pseudomonas were identified as key degrading microorganisms associated with the preferential degradation of the R-enantiomer. The addition of earthworms further significantly enhanced the preferential degradation of the R-enantiomer. Importantly, earthworms markedly promoted the growth and reproduction of the four aforementioned degrading microorganisms in soil treated with enantiomers. Notably, the relative abundance of these degrading microorganisms was significantly higher in R-enantiomer-treated soil with earthworms than in soil treated with the S-enantiomer. Additionally, earthworms significantly increased the relative abundance of degradation genes p450, bphA1, and benA in the soil, especially in the R-enantiomer treated soil. Nocardioides, Variovorax, Arthrobacter, and Pseudomonas were identified as potential hosts for the degradation gene benA. More importantly, twelve strains of penflufen-degrading bacteria were isolated from the treated soil, of which eight belonged to the aforementioned four microorganisms and exhibited a remarkable ability to preferentially degrade the R-enantiomer. This finding highlights the potential of adding earthworms to soil, which, in conjunction with key degrading microorganisms, preferentially accelerate penflufen R-enantiomer degradation.
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