Microbial community succession patterns and metabolite profiles in cigar tobacco during different mildew stages

Mildew in cigar tobacco leaves (CTLs) degrades both quality and market value. This research systematically examines dynamic changes in key metabolic compounds and microbial community succession throughout the mildew process, categorized into three stages: un-mildew (d0), early-mildew (d4 and d8), and late-mildew (d12 and d16). As mildew progresses, carbohydrates decrease, nitrogen metabolism is hindered, the carbon-nitrogen ratio (C/N) declines, and pH rises, making the tobacco weakly alkaline. The total amount of volatile flavor compounds (VFCs) increases, with the proportion of nitrogen-containing compounds such as nicotine rising, while neophytadiene, ketones, and alcohols decrease, leading to a disruption in the coordination of various aroma substances. Microbial diversity declines, with shifts in populations of Staphylococcus, Pseudomonas, Aspergillus, and Sampaiozyma. Six fungal and five bacterial genera are the characteristic dominate microorganisms at different stages. Co-occurrence network analysis shows that complexity decreases and stability declines, while microbial diversity peaks at the early-mold stage and is severely compromised at the late-mold stage in terms of stability and functional diversity. Two-way Orthogonal Partial Least Squares (O2PLS) identified 12 fungal and 3 bacterial genera as key drivers of metabolic changes. Partial Least Squares Structural Equation Modeling (PLS-SEM) emphasized the role of fungi in CTL degradation and the impact of C/N ratio on fungal metabolism. This study, for the first time, elucidates the complex relationship between microbial succession and metabolite compounds during mildew process, providing a reference for dynamic monitoring of fermented tobacco quality.