Characterization and discrimination of microbial community and co-occurrence patterns in fresh and strong flavor style flue-cured tobacco leaves.

Fermentation, also known as aging, is vital for enhancing the quality of flue-cured tobacco leaves (FTLs). Aged FTLs demonstrate high-quality sensory characteristics, while unaged FTLs do not. Microbes play important roles in the FTL fermentation process. However, the eukaryotic microbial community diversity is poorly understood, as are microbial associations within FTLs. We aimed to characterize and compare the microbiota associated with two important categories, fresh and strong flavor style FTLs, and to reveal correlations between the microbial taxa within them. Based on 16S and 18S rRNA Illumina MiSeq sequencing, the community richness and diversity of prokaryotes were almost as high as that of eukaryotes. The dominant microbes of FTLs belonged to seven genera, including Pseudomonas, Bacillus, Methylobacterium, Acinetobacter, Sphingomonas, Neophaeosphaeria, and Cladosporium, of the Proteobacteria, Firmicutes, and Ascomycota phyla. According to partial least square discriminant analysis (PLS-DA), Xanthomonas, Franconibacter, Massilia, Quadrisphaera, Staphylococcus, Cladosporium, Lodderomyces, Symmetrospora, Golovinomyces, and Dioszegia were significantly positively correlated with fresh flavor style FTLs, while Xenophilus, Fusarium, unclassified Ustilaginaceae, Tilletiopsis, Cryphonectria, Colletotrichum, and Cyanodermella were significantly positively correlated with strong flavor style FTLs. Network analysis identified seven hubs, Aureimonas, Kocuria, Massilia, Brachybacterium, Clostridium, Dietzia, and Vishniacozyma, that may play important roles in FTL ecosystem stability, which may be destroyed by Myrmecridium. FTL microbiota was found to be correlated with flavor style. Species present in lower numbers than the dominant microbes might be used as microbial markers to discriminate different flavor style samples and to stabilize FTL microbial communities. This research advances our understanding of FTL microbiota and describes a means of discriminating between fresh and strong flavor FTLs based on their respective stable microbiota.