Decade-long fertilization and Bradyrhizobium inoculation reconfigure soybean rhizosphere microecology through fungal community assembly and metabolic niche partitioning

Soil microbial-metabolite interactions influence crop productivity, yet their responses to long-term nutrient management in legume systems warrant further investigation. This study examined how fertilization and Bradyrhizobium inoculation reshape soybean rhizosphere fungal-metabolite networks to improve soil health. Through a decade-long field trial utilizing internal transcribed spacer (ITS) sequencing and liquid chromatography-mass spectrometry (LC-MS) metabolomics, four treatments were evaluated: no fertilizer application (CK); phosphorus and potassium fertilization (PK); PK chemical fertilizers combined with urea (PK+N); PK fertilization with Bradyrhizobium japonicum 5821 inoculation (PK+R). Results indicated that nitrogen fertilization increased fungal diversity at maturity and enhanced co-occurrence network complexity (displaying the highest node and edge counts), while Bradyrhizobium inoculation promoted stochastic assembly. Soil fungi exhibited notable correlations with 3-hydroxymethylantipyrine, chrysophanol, 3,7-dihydroxyflavone and triethylamine. Metabolite profiling revealed nitrogen suppression of stress-resistant flavonoids (3-hydroxymethylantipyrine, chrysophanol, 3,7-dihydroxyflavone), whereas Bradyrhizobium enhanced these key metabolites. KEGG enrichment identified tryptophan and caffeine metabolism as central during flowering–podding stage, coordinating nitrogen assimilation and defense responses. Additionally, the key metabolites correlated significantly with soil total nitrogen, organic matter, and available nitrogen. These findings reveal that Bradyrhizobium acts synergistically with fertilization to activate fungal-driven metabolic pathways, offering a microbiome-based approach to enhance nitrogen efficiency and reduce agrochemical dependency in soybean systems.