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. 2024 Mar 26:15:1385067.
doi: 10.3389/fmicb.2024.1385067. eCollection 2024.

Genomic and biological control of Sclerotinia sclerotiorum using an extracellular extract from Bacillus velezensis 20507

Affiliations

Genomic and biological control of Sclerotinia sclerotiorum using an extracellular extract from Bacillus velezensis 20507

Yunqing Cheng et al. Front Microbiol. .

Abstract

Introduction: Sclerotinia sclerotiorum is a known pathogen that harms crops and vegetables. Unfortunately, there is a lack of effective biological control measures for this pathogen. Bacillus velezensis 20507 has a strong antagonistic effect on S. Sclerotiorum; however, the biological basis of its antifungal effect is not fully understood.

Methods: In this study, the broad-spectrum antagonistic microorganisms of B. velezensis 20507 were investigated, and the active antifungal ingredients in this strain were isolated, purified, identified and thermal stability experiments were carried out to explore its antifungal mechanism.

Results: The B. velezensis 20507 genome comprised one circular chromosome with a length of 4,043,341 bp, including 3,879 genes, 185 tandem repeats, 87 tRNAs, and 27 rRNAs. Comparative genomic analysis revealed that our sequenced strain had the closest genetic relationship with Bacillus velezensis (GenBank ID: NC 009725.2); however, there were significant differences in the positions of genes within the two genomes. It is predicted that B. velezensis 20507 encode 12 secondary metabolites, including difficidin, macrolactin H, fengycin, surfactin, bacillibactin, bacillothiazole A-N, butirosin a/b, and bacillaene. Results showed that B. velezensis 20507 produced various antagonistic effects on six plant pathogen strains: Exserohilum turcicum, Pyricularia oryzae, Fusarium graminearum, Sclerotinia sclerotiorum, Fusarium oxysporum, and Fusarium verticillioides. Acid precipitation followed by 80% methanol leaching is an effective method for isolating the antifungal component ME80 in B. velezensis 20507, which can damage the membranes of S. sclerotiorum hyphae and has good heat resistance. Using high-performance liquid chromatography, and Mass Spectrometry analysis, it is believed that fengycin C72H110N12O20 is the main active antifungal substance.

Discussion: This study provides new resources for the biological control of S. Sclerotiorum in soybeans and a theoretical basis for further clarification of the mechanism of action of B. velezensis 20507.

Keywords: Bacillus velezensis; Sclerotinia sclerotiorum; antagonism; biological control; whole genome sequencing.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Genomic cycle diagram of Bacillus velezensis 20507. The outermost circle of the circle chart is an indicator of genome size, with each scale being 0.5 Mb. The second and third circles represent CDS on positive and negative chains, with different colors indicating the functional classification of different COGs in CDS. The fourth circle contains rRNA and tRNA. The fifth circle shows the GC content. The innermost circle is the GC skew value.
FIGURE 2
FIGURE 2
Gene Ontology annotation of Bacillus velezensis 20507 genome.
FIGURE 3
FIGURE 3
Cluster of orthologous groups of proteins [COG, (A)] and Kyoto encyclopedia of genes and genomes (B) annotation of Bacillus velezensis 20507 genome.
FIGURE 4
FIGURE 4
Comparative genomics analysis of Bacillus velezensis 20507. (A) Phylogenetic analysis of Bacillus velezensis 20507. (B) Venn diagram based on gene family analysis of Bacillus velezensis 20507 and four nearby species. (C) Collinearity analysis of at gene level. (C) The lines in the figure represent the positional connections of homologous genes between two species, and the colors do not represent specific meanings. Colored areas have a span greater than 100 in the contiguous area. JDF, Bacillus velezensis 20507; NC_009725.2, Bacillus velezensis; NZ_CP082278.1, Bacillus amyloliquefaciens; NZ_CP019663.1, Bacillus subtilis subsp. subtilis str. 168; NC_016047.1, Bacillus spizizenii.
FIGURE 5
FIGURE 5
Inhibitory effect of antagonistic bacteria Bacillus velezensis 20507 on growth of six plant pathogens. For (A2,B2,C2,D2,E2,F2), the plant pathogenic fungi were inoculated in the center of the Petri dish, and the biocontrol bacteria were inoculated on the four corners of the pathogenic fungi. (A1) Exserohilum turcicum; (A2) Exserohilum turcicum and Bacillus velezensis 20507; (B1) Pyricularia oryzae; (B2) Pyricularia oryzae and B. velezensis 20507; (C1) Fusarium graminearum; (C2) Fusarium graminearum and Bacillus velezensis 20507; (D1) Sclerotinia sclerotiorum; (D2) Sclerotinia sclerotiorum and B. velezensis 20507; (E1) Fusarium oxysporum; (E2) Fusarium oxysporum and B. velezensis 20507; (F1) Fusarium verticillioides; (F2) Fusarium verticillioides and B. velezensis 20507. (G) Inhibition rate of B. velezensis 20507 against six pathogenic fungi. In the bar chart, different lowercase letters above the columns indicate significant differences at P = 0.05 level.
FIGURE 6
FIGURE 6
Inhibitory effect of antagonistic bacteria B. velezensis 20507 on growth of six plant pathogens. (A–F) The inhibitory effects of crude extract from different ammonium sulfate saturation conditions on the growth of S. sclerotiorum. (A–F) represents ammonium sulfate saturation of 20, 30, 40, 50, 60, and 70%, respectively. (G–L) The inhibitory effects of crude extract from different concentration methanol leaching conditions on the growth of S. sclerotiorum. (G–L) Represents methanol concentration of 50, 60, 70, 80, 90, and 100%. (M) Inhibition rate of crude extract from B. velezensis 20507 against Sclerotinia sclerotiorum using different ammonium sulfate saturation conditions. AS20, AS30, AS40, AS50, AS60, and AS70 represents ammonium sulfate saturation of 20, 30, 40, 50, 60, and 70%, respectively. (N) Inhibition rate of crude extract from B. velezensis 20507 against Sclerotinia sclerotiorum using different concentration methanol leaching conditions. ME50, ME60, ME70, ME80, ME90, and ME100 represents 50, 60, 70, 80, 90, and 100% methanol leaching conditions, respectively. In the bar chart, different lowercase letters above the columns indicate significant differences at P = 0.05 level.
FIGURE 7
FIGURE 7
Effect of 80% methanol leaching treatment on the integrity of S. sclerotiorum cell membrane. (A1) Control hypha of S. sclerotiorum, observed with fluorescence microscope under visible light conditions. (A2) Control hypha of S. sclerotiorum, observed with fluorescence microscope under fluorescence conditions. (B1) Treatment hypha of S. sclerotiorum, observed with fluorescence microscope under visible light conditions. (B2) Treatment hypha of S. sclerotiorum, observation with fluorescence microscope under fluorescence conditions. (C1) Control hypha of S. sclerotiorum, observed with scanning electron microscope. (C2) Treatment hypha of S. sclerotiorum, observed with scanning electron microscope. White Arrows indicate damaged hyphae. Control, hypha of S. sclerotiorum was treated with distilled water for 20 min. Treatment, hypha of S. sclerotiorum was treated with 100 μg/mL ME80 aqueous solution for 20 min. An excitation wavelength was 535 nm.
FIGURE 8
FIGURE 8
High performance liquid chromatography (HPLC) analysis of antibacterial components in 80% methanol leaching of Bacillus velezensis 20507. (A) HPLC spectrum of ME80 of Bacillus velezensis 20507. (B) The spectrum of component 1 collected, and component 2 has been removed. (C) Plate antagonism experiment using obtained component 1 and component 2; 1 CK, component 1 at ordinary temperature; 1HT, component 1 was heated at 121°C for 20 min; 2CK, component 2 at ordinary temperature; 2HT, component 2 was heated at 121°C for 20 min. HPLC, high-performance liquid chromatography; ME80, Acid precipitation followed by 80% methanol leaching.
FIGURE 9
FIGURE 9
Triple TOF-MS/MS analysis of main antibacterial in Bacillus velezensis 20507. Component 1 in ME80 was used as a sample. Using liquid chromatography columns, the substance collection solution from 12 to 15 min was mixed to obtain component 1.

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