Plant-Microbe Interaction: Mining the Impact of Native Bacillus amyloliquefaciens WS-10 on Tobacco Bacterial Wilt Disease and Rhizosphere Microbial Communities

Abstract

Ralstonia solanacearum, the causative agent of bacterial wilt disease, has been a major threat to tobacco production globally. Several control methods have failed. Thus, it is imperative to find effective management for this disease. The biocontrol agent Bacillus amyloliquefaciens WS-10 displayed a significant control effect due to biofilm formation, and secretion of hydrolytic enzymes and exopolysaccharides. In addition, strain WS-10 can produce antimicrobial compounds, which was confirmed by the presence of genes encoding antimicrobial lipopeptides (fengycin, iturin, surfactin, and bacillomycinD) and polyketides (difficidin, bacilysin, bacillibactin, and bacillaene). Strain WS-10 successfully colonized tobacco plant roots and rhizosphere soil and suppressed the incidence of bacterial wilt disease up to 72.02% by reducing the R. solanacearum population dynamic in rhizosphere soil. Plant-microbe interaction was considered a key driver of disease outcome. To further explore the impact of strain WS-10 on rhizosphere microbial communities, V3-V4 and ITS1 variable regions of 16S and ITS rRNA were amplified, respectively. Results revealed that strain WS-10 influences the rhizosphere microbial communities and dramatically changed the diversity and composition of rhizosphere microbial communities. Interestingly, the relative abundance of genus Ralstonia significantly decreased when treated with strain WS-10. A complex microbial co-occurrence network was present in a diseased state, and the introduction of strain WS-10 significantly changed the structure of rhizosphere microbiota. This study suggests that strain WS-10 can be used as a novel biocontrol agent to attain sustainability in disease management due to its intense antibacterial activity, efficient colonization in the host plant, and ability to transform the microbial community structure toward a healthy state. IMPORTANCE The plant rhizosphere acts as the first line of defense against the invasion of pathogens. The perturbation in the rhizosphere microbiome is directly related to plant health and disease development. The introduction of beneficial microorganisms in the soil shifted the rhizosphere microbiome, induced resistance in plants, and suppressed the incidence of soilborne disease. Bacillus sp. is widely used as a biocontrol agent against soilborne diseases due to its ability to produce broad-spectrum antimicrobial compounds and colonization with the host plant. In our study, we found that the application of native Bacillus amyloliquefaciens WS-10 significantly suppressed the incidence of tobacco bacterial wilt disease by shifting the rhizosphere microbiome and reducing the interaction between rhizosphere microorganisms and bacterial wilt pathogen.

Keywords: Ralstonia solanacearum; biological control; disease incidence; microbiome; plant pathogens; plant-microbe interaction.

FIG 1

Analyses of disease index and population dynamics of Ralstonia solanacearum WS-001 in rhizosphere soil. Disease index (A) and R. solanacearum WS-001 population log/g of fresh soil (B). Disease index (DI), disease incidence (Di), control effect (CE), application of water (CK), application of R. solanacearum WS-001 (T1), and combined application of R. solanacearum WS-001 and Bacillus amyloliquefaciens WS-10 (T2). Different lowercase letters on error bars show significant differences among treatments according to Duncan's multiple range test at P < 0.05.

FIG 2

Fluorescence micrographs of flue-cured tobacco roots colonized by gfp-labeled Bacillus amyloliquefaciens WS-10. Fluorescence micrographs of control after 7 days under dark (A) and bright (B) fields. Fluorescence micrographs of treatment after 7 days under dark (C) and bright (D) fields. Fluorescence micrographs of treatment after 21 days under dark (E) and bright (F) fields. Here: Control; B. amyloliquefaciens WS-10 without gfp and treatment; gfp-labeled B. amyloliquefaciens WS-10. Colonization of gfp-labeled B. amyloliquefaciens WS-10 cells is shown by red arrows.

FIG 3

Box plot showing the alpha diversity indices of bacterial (top) and fungal (bottom) communities under different treatments. (A to D) Alpha diversity indices of bacterial communities Simpson, Shannon, Shannon evenness, and Chao 1, respectively. (E to H) Alpha diversity indices of fungal communities Simpson, Shannon, Shannon evenness, and Chao 1, respectively. Application of water (CK), application of Ralstonia solanacearum WS-001 (T1), and combined application of R. solanacearum WS-001 and Bacillus amyloliquefaciens WS-10 (T2). Different lowercase letters on each box plot represent the significant differences among treatments according to the Wilcoxon test at P < 0.05.

FIG 4

Principal coordinate analysis (PCoA) and permutational multivariate analysis of variance (PERMANOVA) based on the Bray-Curtis distance matrix demonstrating the separation between soil bacterial and fungal communities under different treatments. PCoA for bacterial (A) and fungal (B) communities. Overall differences among bacterial (C) and fungal (D) communities are shown by a pairwise PERMANOVA (P < 0.01). Application of water (CK), application of Ralstonia solanacearum WS-001 (T1), and combined application of R. solanacearum WS-001 and Bacillus amyloliquefaciens WS-10 (T2). Overall differences in microbial community composition between the treatments (X) and within the treatments (Y).

FIG 5

The dynamics of most dominant bacterial and fungal communities at the phyla level. Relative abundance bar plots for the top 10 most abundant bacterial (A) and fungal (B) phyla. The box plot shows the significant difference and relative abundance of the differentially abundant bacterial (C) and fungal (D) phyla under different treatments. The lowercase letters on each box plot display significant differences among treatments (Wilcoxon test, P < 0.05). Application of water (CK), application of Ralstonia solanacearum WS-001 (T1), and combined application of R. solanacearum WS-001 and Bacillus amyloliquefaciens WS-10 (T2).

FIG 6

Changes in the relative abundance of bacterial and fungal communities at the genera level. Chord diagram showing the interrelationships between relative abundance of top 10 bacterial (A) and fungal (B) genera under different treatments. The change in the width of the color bands indicates the change in the relative abundance of bacterial and fungal communities. Bar plots show the significant differences in the relative abundance of most abundant bacterial (C) and fungal (D) genera under different treatments. Significant differences among treatments are shown by different lowercase small letters on the error bars (Wilcoxon test, P < 0.05). Application of water (CK), application of Ralstonia solanacearum WS-001 (T1), and combined application of R. solanacearum WS-001 and Bacillus amyloliquefaciens WS-10 (T2).

FIG 7

Co-occurrence networks analysis of bacterial and fungal communities at genus level under different treatments. Nodes represent microbial genera, and edges represent the interaction between microbes within a specific treatment. Application of water (CK), application of Ralstonia solanacearum WS-001 (T1), and combined application of R. solanacearum WS-001 and Bacillus amyloliquefaciens WS-10 (T2).

FIG 8

Correlation analysis between bacterial-fungal genera and disease incidence according to Pearson correlation coefficient (PCC, P < 0.05). PCC between bacterial genera and disease incidence (A), and PCC between fungal genera and disease incidence (B). Asterisks indicates significant differences (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

FIG 9

Plant-microbe interaction and biocontrol mechanism of biocontrol agents. (A) Above ground plant parts represent a healthy and diseased state of plants in the presence of biocontrol agents and pathogens. Healthy plants, plants under no pathogen invasion or treated with a biocontrol agent. Diseased plants; plants under pathogen invasion. (B) The rhizosphere of plants shows the complexity of the microbial co-occurrence network under a healthy and diseased state in the presence of biocontrol agents and pathogens. Biocontrol agents suppress disease incidence through mechanisms of antibiosis, colonization, and produce antimicrobial compounds, biofilm formation, and engineering of the rhizosphere microbiome. Positive interactions between plant-microbes and microbe-microbe induce resistance in plants and help plants in combating with pathogen. Negative interactions between plant-microbes and microbe-microbe result in the imbalance of the rhizosphere microbiome breaking the first line of defense against pathogen invasion, and enhancing pathogen population, and disease development.