Microbial Cross-Talk: Dissecting the Core Microbiota Associated With Flue-Cured Tobacco (Nicotiana tabacum) Plants Under Healthy and Diseased State

Abstract

Bacterial wilt caused by Ralstonia solanacearum is a devastating disease of flue-cured tobacco production which poses significant yield losses all around the world. In this study, we evaluated the rhizosphere microbiome of healthy and bacterial wilt-infected (diseased) flue-cured tobacco plants through amplification of V3-V4 and ITS1-5f variable regions of 16S and internal transcribed spacer (ITS) rRNA. The study was based on the location (Qujing, Shilin, and Wenshan), plant components (rhizosphere soil and roots), and sample types (healthy and diseased) to assess the diversity of bacterial and fungal communities. Bacterial and fungal communities present in roots primarily emanated from rhizosphere soil. Healthy flue-cured tobacco plants exhibit high microbial diversity compared to diseased plants. Among three variables, plant components significantly influence the diversity of microbial communities, whereas rhizosphere soil harbors higher microbial diversity than roots. Bacterial phyla Cyanobacteria and Proteobacteria were found in high relative abundance in roots and rhizosphere soil samples, respectively. As far as fungi is concerned, a high relative abundance of Ascomycota and Basidiomycota was found in both rhizosphere soil and root. Bacterial genera such as Bacillus, Bradyrhizobium, Ensifer, Neorhizobium, and Lysobacter related to plant growth promotion and disease suppressing abilities were dominant than fungal genera. Analysis of relative abundance at specie-level revealed that most fungal species are pathogenic to flue-cured tobacco and could provide a conducive environment for wilt infection. In conclusion, R. solanacearum significantly influences the microbial diversity of flue-cured tobacco plants and negatively affects the bacterial community composition. Altogether, our study demonstrates the complexity of bacterial and fungal communities that possibly interact with each other (microbe-microbe) and host (host-microbe). This cross-talk could be helpful for healthy flue-cured tobacco plant growth and to induce resistance against bacterial wilt disease.

Keywords: Ralstonia solanacearum; disease resistance; flue-cured tobacco; locations; microbial diversity; plant components.

FIGURE 1

Rhizosphere soil and root samples were collected from three different locations in Yunnan Province: Kunming (Shilin), Qujing (Huize), and Wenshan (Qiubei), from healthy and bacterial wilt infected flue-cured tobacco plants. Healthy flue-cured tobacco plant with green leaves and white-colored xylem vessels (A,B). Disease flue-cured tobacco plant with typical bacterial wilt symptoms (yellowing, wilting of leaves, necrotic lesions on stem, and discoloration of xylem vessels) (C,D).

FIGURE 2

Principal coordinate analysis (PCoA) was based on the Bray–Curtis dissimilarity metrics showing the beta diversity analysis for all 12 composite samples (three replicates per sample) of flue-cured tobacco plants under three variables.

FIGURE 3

Boxplot of bacterial (top) and fungal (bottom) showing alpha diversity indexes of flue-cured tobacco plants under three variables. HS, healthy rhizosphere soil; DS, diseased rhizosphere soil; HR, healthy roots; DR, diseased roots.

FIGURE 4

Distribution of bacterial (top) and fungal (bottom) operational taxonomic units in three variables, i.e., locations (Qujing, Shilin, and Wenshan), plant components (rhizosphere soil and roots), and sample types (healthy and diseased).

FIGURE 5

Relative abundance bar plots at phylum level based on the species annotation results in 12 composite samples (average of three replicates per sample) of flue-cured tobacco plants under three variables. (A) Relative abundance at the phylum level in bacterial communities and (B) relative abundance at the phylum level in fungal communities. QJ, Qujing; SL, Shilin; WS, Wenshan; HS, healthy rhizosphere soil; DS, diseased rhizosphere soil; HR, healthy roots; DR, diseased roots.

FIGURE 6

Relative abundance heatmaps at the genus level for top 35 bacterial (A) and fungal (B) genera in group-wise comparison under three variables. HS, healthy rhizosphere soil; DS, diseased rhizosphere soil; HR, healthy roots; DS, diseased roots.

FIGURE 7

Relative abundance bar plots for top 10 bacterial (A) and fungal (B) species in group-wise comparison under three variables. HS, healthy rhizosphere soil; DS, diseased rhizosphere soil; HR, healthy roots; DS, diseased roots.

FIGURE 8

Co-occurrence network analysis of bacterial and fungal communities associated with healthy and diseased samples collected from different plant components (rhizosphere soil and roots). HS, healthy soil; DS, diseased soil; HR, healthy roots; DR, diseased roots.