Changes in the community composition and function of the rhizosphere microbiome in tobacco plants with Fusarium root rot

Abstract

Introduction: Tobacco root rot caused by Fusarium spp. is a soil-borne vascular disease that severely affects tobacco production worldwide. To date, the community composition and functional shifts of the rhizosphere microbiome in tobacco plants infected with Fusarium root rot remain poorly understood.

Methods: In this study, we analyzed the differences in the compositions and functions of the bacterial and fungal communities in the rhizosphere and root endosphere of healthy tobacco plants and tobacco with Fusarium root rot using amplicon sequencing and metagenomic sequencing.

Results and discussion: Our results showed that Fusarium root rot disrupted the stability of bacteria-fungi interkingdom networks and reduced the network complexity. Compared to healthy tobacco plants, the Chao1 index of bacterial communities in the rhizosphere soil of diseased plants increased by 4.09% (P < 0.05), while the Shannon and Chao1 indices of fungal communities decreased by 13.87 and 8.17%, respectively (P < 0.05). In the root tissues of diseased plants, the Shannon index of bacterial and fungal communities decreased by 17.71-27.05% (P < 0.05). Additionally, we observed that the rhizosphere microbial community of diseased tobacco plants shifted toward a pathological combination, with a significant increase in the relative abundance of harmful microbes such as Alternaria, Fusarium, and Filobasidium (89.46-921.29%) and a notable decrease in the relative abundance of beneficial microbes such as Lysobacter, Streptomyces, Mortierella, and Penicillium (48.48-81.56%). Metagenomic analysis further revealed that the tobacco rhizosphere microbial communities of diseased plants played a significant role in basic biological metabolism, energy production and conversion, signal transduction, and N metabolism, but their functions involved in C metabolism were significantly weakened. Our findings provide new insights into the changes in and interactions within the rhizosphere and root endosphere microbiomes of tobacco plants under the stress of Fusarium soil-borne fungal pathogens, while laying the foundation for the exploration, development, and utilization of beneficial microbial resources in healthy tobacco plants in the future.

Keywords: Fusarium spp.; microbial diversity; microbial interactions; rhizosphere; soil-borne fungal disease.

Figure 1

Field symptoms of tobacco Fusarium root rot and sample collection. (A) After being infected with Fusarium, the leaves of diseased tobacco plants turn yellow, the stems turn black, and in severe cases, the whole plant wilts and dies. (B) Healthy flue-cured tobacco plants with green leaves and white-colored roots. (C) Disease flue-cured tobacco plants with root rot symptoms (such as yellowing, and wilting of leaves, and necrotic lesions on the stem and root). Root tissues from healthy and diseased tobacco plants were cut off respectively to be used as root tissue samples. (D, E) After removing the bulk soil, a brush was used to carefully collect the tiny soil particles attached to the roots of healthy and diseased tobacco plants as rhizosphere soil. The red arrow points to our sampling location.

Figure 2

The microbial community structures (Beta - diversity) in the rhizosphere soil (A) and root endosphere (B) of tobacco plants under diseased and healthy conditions. Principal coordinate analysis (PCoA) plots based on the Bray-Curtis dissimilarity matrices with permutational analysis of variance (PERMANOVA), showing the changes in the structure of the bacterial (left) and fungal (right) community composition.

Figure 3

Microbial community composition in tobacco plants rhizosphere soil under diseased and healthy conditions. (A) The number of unique, shared, and common bacterial and fungal operational taxonomic units at different groups. (B) The bar plots of relative abundance illustrate the composition of bacterial (up) and fungal (down) communities in the rhizosphere soil at the phylum level under diseased and healthy conditions. Low abundance genera with less than 1% of the total sequences across all samples are grouped into “Other”. (C, D) The non-parametric Wilcoxon rank-sum test shows the differences in the average relative abundances of the same bacterial (C) and fungal (D) species (at the genus level) between the diseased group and the healthy group. * Stands for 0.01 ≤ p < 0.05, and ** stands for 0.001 ≤ p < 0.01.

Figure 4

Microbial community composition in tobacco plants root endosphere under diseased and healthy conditions. (A) The number of unique, shared, and common bacterial and fungal operational taxonomic units at different groups. (B) The bar plots of relative abundance illustrate the composition of bacterial (up) and fungal (down) communities in the root endosphere of tobacco plants at the phylum level under diseased and healthy conditions. Low abundance genera with less than 1% of the total sequences across all samples are grouped into “Other”. (C, D) The non-parametric Wilcoxon rank-sum test shows the differences in the average relative abundances of the same bacterial (C) and fungal (D) species (at the genus level) between the diseased group and the healthy group. * Stands for 0.01 ≤ p < 0.05, and ** stands for 0.001 ≤ p < 0.01.

Figure 5

Co-occurrence networks of microbial communities in the rhizosphere soil and root endosphere of tobacco plants under diseased and healthy conditions. (A) The inter-domain networks between bacteria and fungi communities in the rhizosphere soil (up) and root endosphere (down) of tobacco plants under diseased and healthy conditions. Both the diseased networks in the rhizosphere soil and root endosphere showed a lower number of nodes and edges than the healthy networks. Edge color represents positive (red) and negative (green) correlations. (B) The intra-domain networks of bacterial-bacterial and fungal-fungal in the rhizosphere soil and root endosphere of tobacco plants under diseased and healthy conditions. (C) The proportion of bacterial–fungal, bacterial–bacterial, and fungal–fungal correlations in the healthy and diseased networks. Red and green colors of the column indicate positive and negative correlations, respectively. (D) Average degree of the healthy and diseased networks. Green, orange and blue represent the bacterial–fungal, bacterial–bacterial, and fungal–fungal networks, respectively.

Figure 6

Functional profiles of microbiomes in the rhizosphere soil of tobacco plants under diseased and healthy conditions. (A) NMDS ordinations of functional genes based on Bray-Curtis distance matrices of KO functional genes show the distinct functions of microbial communities in the rhizosphere soil of tobacco plants under diseased and healthy conditions. (B) The boxplot shows the functional diversity (including KO, COG and CAZy) of the rhizosphere microbiomes of tobacco plants under diseased and healthy conditions. (C, D) Differential abundance analysis of COG (C) and CAZy (D) functional genes of the rhizosphere microbiomes of of tobacco plants under diseased and healthy conditions. * Stands for 0.01 ≤ p < 0.05, ** stands for 0.001 ≤ p < 0.01, and *** stands for p < 0.001 according to Student's t-test.

Figure 7

Heatmap exhibiting the relative abundance of functional genes (based on KO) involved in carbon (A) and nitrogen (B) metabolism in the rhizosphere microbial communities of tobacco plants under diseased and healthy conditions. Each row of the heatmap corresponds to a specific gene, while each column represents a different sample. The colors in the heatmap, red representing higher relative abundance levels of the gene in that sample, and green representing lower relative abundance levels, indicate the variation in gene relative abundance.