Leaf-year-driven microbial network rewiring promotes nutrient conversion during tobacco leaf aging: The critical role of mid-stage-aging community succession

Long-term aging enhances tobacco leaf flavor through microbial-driven biochemical reactions, yet the regulatory mechanisms of microbial community succession remain unclear. To address this, this study integrated stoichiometric and molecular ecological network analyses to track microbial dynamics in tobacco leaves aged for one year (Y1), two years (Y2), and three years (Y3). Key findings are as follows: the mid-stage-aging (Y2) represents a critical turning point in tobacco quality formation, characterized by the lowest levels of carbon source nutrients (Total Sugar, Reducing Sugar, and Starch) and the peak levels of nitrogen source nutrients (total nitrogen and protein), providing an optimal environment for aroma precursor synthesis. Correspondingly, microbial community structure and function reach their optimal state during this period. The richness indices (Observed and Chao1) and the complexity of interaction molecular ecological networks are significantly higher than in the early (Y1) and late (Y3) stages. Concurrently, microbial community composition undergoes succession, transitioning from a Rhizopus -dominated fungal community in the early stage to a bacterial-fungal synergistic system centered on Sampaiozyma and Sphingomonas in the mid stage, and ultimately evolving into a Methylobacterium -dominated bacterial community in the late stage. Bacteria are pivotal engineers in quality enhancement during aging, with their correlation to key nutrients (Total Sugar, Nicotine, Sugar alkali ratio) continuously strengthening throughout the process, and exhibiting significantly higher sensitivity to environmental factors compared to fungi. This study systematically elucidates the microbial-driven mechanism of tobacco quality enhancement, centered on bacteria during the mid-aging period, providing a theoretical foundation for targeted regulation of aging processes. • The carbon source nutrient content reaches its lowest level and the nitrogen source nutrient content peaks during the mid-stage of tobacco aging(Y2), providing an optimal environment for aroma precursor synthesis. • During the middle stage of tobacco leaf aging(Y2), the diversity of the microbial community reaches its peak, and the molecular ecological network of the interaction between bacteria and fungi is the most complex. • Microbial community succession from fungal dominance to eventual bacterial dominance, enhancing bacterial-fungal interactions. • Bacteria has strong association with key nutrients and environmental sensitivity, have been identified as the core engineering microbiota regulating aging quality.