Bacillus velezensis RC116 Inhibits the Pathogens of Bacterial Wilt and Fusarium Wilt in Tomato with Multiple Biocontrol Traits

Abstract

Soil-borne plant diseases seriously threaten the tomato industry worldwide. Currently, eco-friendly biocontrol strategies have been increasingly considered as effective approaches to control the incidence of disease. In this study, we identified bacteria that could be used as biocontrol agents to mitigate the growth and spread of the pathogens causing economically significant diseases of tomato plants, such as tomato bacterial wilt and tomato Fusarium wilt. Specifically, we isolated a strain of Bacillus velezensis (RC116) from tomato rhizosphere soil in Guangdong Province, China, with high biocontrol potential and confirmed its identity using both morphological and molecular approaches. RC116 not only produced protease, amylase, lipase, and siderophores but also secreted indoleacetic acid, and dissolved organophosphorus in vivo. Moreover, 12 Bacillus biocontrol maker genes associated with antibiotics biosynthesis could be amplified in the RC116 genome. Extracellular secreted proteins of RC116 also exhibited strong lytic activity against Ralstonia solanacearum and Fusarium oxysporum f. sp. Lycopersici. Pot experiments showed that the biocontrol efficacy of RC116 against tomato bacteria wilt was 81%, and consequently, RC116 significantly promoted the growth of tomato plantlets. Based on these multiple biocontrol traits, RC116 is expected to be developed into a broad-spectrum biocontrol agent. Although several previous studies have examined the utility of B. velezensis for the control of fungal diseases, few studies to date have evaluated the utility of B. velezensis for the control of bacterial diseases. Our study fills this research gap. Collectively, our findings provide new insights that will aid the control of soil-borne diseases, as well as future studies of B. velezensis strains.

Keywords: Rhizosphere microorganisms; antagonism activity; biocontrol genes; extracellular lyase; soil-borne disease.

Figure 1

Detection of antagonistic activity of RC116 against phytopathology: (A) the schematic of Oxford cup dual culture for phytopathogenic bacteria with RC116, NB liquid medium as a negative control, Gentamicin (30 μg·mL⁻¹) as a positive control; (B–D) antagonistic activity of RC116 (OD₆₀₀ = 2) against three plant pathogenic bacteria; (E) the schematic of confrontation dual culture for phytopathogenic fungi with RC116; (F–H) antagonistic activity of RC116 (OD₆₀₀ = 2) against three plant pathogenic fungi in dual culture test; (I) the schematic of confrontation dual culture for phytopathogenic fungi with NB medium; and (J–L) three plant pathogenic fungi in control dual culture test. The photos were taken at 24 h after inoculation.

Figure 2

Growth, morphology, and phylogenetic analysis of RC116: (A) colony morphology of strain RC116 on R2A medium, scar bar = 2 mm; (B) crystal violet straining result, scar bar = 5 μm; (C) transmission electron microscopy (TEM) images of RC116 cells, scar bar = 500 nm; (D) scanning electron microscopy (SEM) images of RC116 cells, scar bar = 5 μm; (E) phylogenetic trees analysis of RC116 and other closely related strains based on 16S rRNA gene sequences; (F) ANI heatmap of RC116 and other closely related strains; and (G) whole-genome-based phylogenetic trees analysis based on 92 single-copy orthologous genes. Bootstrap values are indicated at each node based on a total of 1000 bootstrap replicates.

Figure 3

Comparison of antagonistic activity of RC116 with other three B. velezensis strains against three phytopathogenic bacteria. Antagonistic activity assays were performed as described in the Materials and Methods section, the photos were taken after incubation at 30 °C for 24 h, and the diameter of inhibition zones were measured. (A,D) R. solanacearum GMI 1000; (B,E) P. syringae DC3000, (C,F) X. campestris badrii JCM 20466; and (D–F) statistical analysis of the inhibition zones diameters in (A–C), respectively. All assays were repeated thrice and the results represent the means of three independent experiments. Error bars represent standard deviation. * p < 0.05; ** p < 0.01; *** p < 0.001; ns represents no significant difference.

Figure 4

Biocontrol maker gene, CAZymes, and peptidase analysis in RC116 and the other three B. velezensis strains genomes. (A)The overview of Bacillus biocontrol maker gene amplified in four B. velezensis strains by the conventional PCR method. In heatmap, 1 indicates that the biocontrol maker gene can be detected in the genome of RC116, 0 in heatmap indicates that the biocontrol maker gene cannot be detected in the genome of RC116. (B) Total CAZy families in RC116 and FZB42 genomes. (C) Distribution of glycoside hydrolase (GH) family in RC116 and FZB42 genomes based on the CAZy database. (D) Distribution of proteases in the genomes of RC116 and FZB42 in the MEROPS database according to the catalytic sites. (E) The MEROPS categories of the metallopeptidases in RC116 and FZB42 genomes. (F) The MEROPS categories of the serine peptidases in RC116 and FZB42 genomes.

Figure 5

Determination the enzyme producing ability and plant-growth-promoting index of RC116. (A–F) Qualitative detection the ability of RC116 producing protease (A), α-amylase (B), lipase (C), chitinase ((D,E), (D): chitin as substrate, (E): colloidal chitin as substrate), and cellulose (F). (G–I) Changes of protease (G), α-amylase (H), and lipase (I) activities in the culture supernatant of RC116. (J) Qualitative detection the ability of RC116 dissolve inorganic phosphorus. (K) Siderophore qualitative detection of RC116 with the CAS method. (L,M) Changes in IAA concentration (L) and siderophores (M) with RC116. Error bars represent the standard deviation of three replications.

Figure 6

Lysis effect of RC116 extracellular proteins against plant pathogen: (A) lysis effect of RC116 extracellular proteins against Ralstonia solanacearum GMI 1000 (Rs); and (B) lysis effect of RC116 extracellular proteins against Fusarium oxysporum f. sp. Lycopersici (Fol). NA and TM were two agar plate; 0–40%, 40–60%, 60–80%, and 80–100% represent RC116 extracellular protein component precipitated by ammonium sulfate with different saturation; 40–60% saturation ammonium-sulfate-precipitated protein components were heat-treated at 100 °C for 10 min, then centrifugation at 12,000× g for 2 min as a heat-treated control.

Figure 7

Biocontrol effects of RC116 on tomato bacteria wilt (TBW) and tomato-growth-promoting effect of RC116 in greenhouse. (A) Phenotype of tomato plantlets in different inoculation treatment groups. Mock indicates inoculation with NB medium as a control; RC116 indicates inoculation only with the cultures of RC116; RC116 + GMI 1000 indicates simultaneous inoculation with the cultures of RC116 and the bacterial solution of Rs; GMI 1000 indicates inoculation only with and the bacterial solution of Rs. (B) The statistics of GMI 1000 colony in different treatment groups. (C) The statistics of disease investigation results. (D) Morphology of tomato roots in different groups at 28 days after inoculation. (E–H) Plant-growth-promoting effect of RC116 on tomato aboveground and belowground. For pot experiments, each treatment contains 10 tomato plantlets, and 5 experiments were carried out. Data represented the means ± standard error. The different lowercase letters within the same column indicate a significant difference according to Duncan’s test (p < 0.05); asterisks indicate a significant difference at the level of p < 0.0001 (****), p < 0.001 (***), p < 0.01 (**), and p < 0.05 (*); and ns represents no significant difference.