Diazotrophic community restructuring and soil metabolome reprogramming drive enhanced nitrogen fixation in pecan...

Biological nitrogen fixation (BNF) is a key ecological process underpinning sustainable agroforestry by reducing dependence on synthetic nitrogen fertilizers. We used integrated high-throughput sequencing and untargeted metabolomics to examine how stand age affects BNF activity, diazotrophic community structure, and soil metabolomic profiles in pecan (Carya illinoinensis) orchards at two developmental stages (7year and 12-year stands). Pecan age significantly increased diazotrophic α-diversity (by 23.52%, p < 0.01) and nitrogenase activity (p < 0.001). Pseudomonadota dominated across all samples at the phylum level (>90% relative abundance). At the class level, aging was associated with a decline in Alphaproteobacteria and concurrent increases in Betaproteobacteria, Deltaproteobacteria, and Gammaproteobacteria (p < 0.01). Co-occurrence network analysis revealed reduced interactions and a shift from predominantly negative correlations (~90%) toward balanced positive-negative associations (~50%) with increasing stand age. Metabolomic profiling showed enrichment of galactose metabolism, isoflavonoid, linoleic acid, and phenylpropanoid biosynthesis, as well as aromatic compound degradation pathways. Nitrogenase activity correlated positively with diazotrophic diversity (p < 0.01), and community assembly was primarily shaped by soil pH, total potassium, and available phosphorus. Abundances of Bacilli and Actinomycetota correlated strongly with nitrogenase activity (p < 0.001), suggesting their potential as biofertilizer candidates. Overall, pecan stand maturation drives coordinated restructuring of diazotrophic communities and soil metabolic networks, jointly enhancing BNF capacity. Identifying key taxa, metabolic pathways, and environmental drivers provides actionable targets for BNF optimization, advancing mechanistic understanding of nitrogen cycling in agroforestry systems and offering practical insights for sustainable pecan orchard management.