Synergistic bioremediation of metolachlor-contaminated soil by Penicillium oxalicum MetF1 and earthworms: Enhanced degradation, microbial community restructuring, and ring-opening metabolic activation.

The chloroacetamide herbicide metolachlor (MET) is a widespread environmental contaminant posing risks to aquatic and soil ecosystems. Although the fungal strain Penicillium oxalicum MetF1 efficiently degrades MET in liquid culture, its performance in complex soil environments remains unexplored. Recognizing the multifunctional roles of earthworms as soil ecosystem engineers-including their capacity to modulate microbial activity and pollutant bioavailability-we incorporated Eisenia fetida into a synergistic bioremediation system with P. oxalicum MetF1 in MET-contaminated soil. The synergistic treatment achieved 93.88% MET removal within 14 days, significantly outperforming treatments with the fungus (81.75%) or earthworms (30.10%) alone. Metabolite dynamics revealed that earthworms accelerated the transformation of MET intermediates (M2H, MESA, MDES), with a unique enrichment of genes encoding key ring-opening enzymes (3-hydroxyphenylacetate 6-hydroxylase, tyrosine aminotransferase). Earthworms also decreased soil pH toward neutrality, mobilization of available phosphorus, and stabilization of organic carbon, and may partially offset microbial alkali-hydrolyzable nitrogen consumption. High-throughput sequencing demonstrated that the synergistic system alleviated pesticide-induced stress, increased microbial alpha diversity, enriched beneficial degraders (Sphingomonas, Flavobacterium, Fusarium), suppressed pathogenic Blumeria, and fostered tighter co-occurrence networks with more positive interactions. Functional prediction revealed upregulation of carbohydrate and amino acid metabolism pathways as well as the xenobiotics biodegradation pathway, with phenylacetate and tyrosine degradation pathways providing an efficient metabolic route for MET ring-opening. Earthworms exhibited low MET bioaccumulation (BAF < 1) and net weight gain, indicating limited bioaccumulation potential and the absence of obvious lethal effects. However, sublethal effects and the toxicity of MET metabolites (M2H, MESA, and MDES) require further investigation. Collectively, this study demonstrates a powerful earthworm-fungus synergistic system for MET remediation, unravels its microbial and biochemical regulatory networks governing enhanced performance, and offers a mechanistic framework for deploying multi-organism strategies in the remediation of persistently contaminated soils.