Fermentation process of tobacco leaves drives the specific changes of microbial community

Abstract

Background: The changes of microbial community on tobacco leaves are affected by several factors during fermentation. However, the relative contribution of different factors in determining microbial community is not clear. This study investigated the effects of fermentation time (fermentation for 0, 3, 6, 9 and 12 months), leaf position (middle and top tobacco leaves) and fermentation site (Longyan and Xiamen warehouses) on bacterial community of tobacco leaves using 16 S rDNA sequencing.

Results: The results demonstrated that fermentation time had a much stronger impact on bacterial diversity, composition, co-occurrence network and functional profiles than leaf position and fermentation site. With the fermentation progressed, the difference of bacterial community between middle and top tobacco leaves was gradually reduced or even disappeared. The bacterial community diversity and network complexity at three, six and nine months of fermentation were significantly lower than those at fermentation initiation. Specific bacterial genera with desired functions were recruited at different fermentation stages, such as Terribacillus, Pantoea and Franconibacter at three or six months of fermentation and Pseudomonas at nine months of fermentation. The recruited microorganisms would form biofilms on tobacco leaves and compete for polysaccharide or protein substances to accelerate the degradation of tobacco macromolecular substances.

Conclusions: In conclusion, fermentation time was an important factor in determining the composition and function of microbial community on tobacco leaves during the fermentation process.

Keywords: Co-occurrence network; Functional profiles; Microbial community; Plant fermentation; Tobacco leaves.

Fig. 1

The changes of bacterial community diversity on tobacco leaves during the fermentation process. A Principal coordinate analysis (PCoA) showing the effects of fermentation time, leaf position and fermentation site on the structures of bacterial community on tobacco leaves. B PCoA showing the effects of leaf position and fermentation site on the structures of bacterial community at different fermentation stages. Asterisks denote significant differences between samples from top and middle leaves (one-way ANOVA followed by a post hoc Tukey test, ***P < 0.001). C Shannon index and Chao1 showing the effects of fermentation time and leaf position on the α-diversity of bacterial community on tobacco leaves

Fig. 2

Deterministic and stochastic processes in bacterial community assembly during the fermentation process. A Relative contribution of determinism and stochasticity on bacterial community assembly on tobacco leaves during the fermentation process based on the β-Nearest Taxon Index (βNTI) values. B The relative importance of five ecological processes on bacterial community at different fermentation stages

Fig. 3

The changes of bacterial composition on tobacco leaves during the fermentation process. A The changes of bacterial composition at the phylum level. B The changes of bacterial composition at the genus level

Fig. 4

The enrichment and depletion patterns of bacterial composition during the fermentation process. A The LEfSe analysis with an LDA score threshold of 4 showing the specific bacterial genera of middle and top tobacco leaves at different fermentation stages. B The volcano plots showing the enriched or depleted bacterial ASVs of middle and top tobacco leaves at different fermentation stages (M3, M6, M9 and M12) compared with the original bacterial community (M0)

Fig. 5

The co-occurrence networks of bacterial community on tobacco leaves during the fermentation process. A Bacterial co-occurrence networks on tobacco leaves during the fermentation process. Nodes represent ASVs. The size of each node represents connections with other nodes and the colors of nodes represent different phyla. The edges between the nodes indicate strong and significant (P < 0.01) correlations. A blue line indicates a positive interaction, while a red line indicates a negative interaction. B Number of positive and negative interactions in bacterial networks at different fermentation stages. C The scatter plot of within-module connectivity (Zi) and among-module connectivity (Pi) showing the topological role of each ASV. D The number and types (positive or negative) of edges showing the interactions of module hubs and connectors on middle leaves with other ASVs. The nodes marked in red were bacteria that are significantly enriched at the corresponding fermentation stage compared with the original bacterial community (M0). E The number and types (positive or negative) of edges showing the interactions of module hubs and connectors on top leaves with other ASVs

Fig. 6

PICRUSt predicted metagenome functions of bacterial community on tobacco leaves at KO level. A Functional diversity of KO genes in bacterial community on top and middle leaves at different fermentation stages. Asterisks denote significant differences between samples from top and middle leaves (one-way ANOVA followed by a post hoc Tukey test, *P < 0.05; **P < 0.01). B PCoA analysis showing the effects of fermentation time, leaf position and fermentation site on the distribution of KO functional genes in bacterial community on tobacco leaves. C Heatmap showing the relative abundance of functional genes involved in carbohydrate metabolism, amino acid metabolism, cell motility, nucleotide metabolism and cell community-prokaryotes