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. 2017 Sep 15;83(19):e01075-17.
doi: 10.1128/AEM.01075-17. Print 2017 Oct 1.

Bacillomycin D Produced by Bacillus amyloliquefaciens Is Involved in the Antagonistic Interaction with the Plant-Pathogenic Fungus Fusarium graminearum

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Bacillomycin D Produced by Bacillus amyloliquefaciens Is Involved in the Antagonistic Interaction with the Plant-Pathogenic Fungus Fusarium graminearum

Qin Gu et al. Appl Environ Microbiol. .

Abstract

Fusarium graminearum (teleomorph: Ascomycota, Hypocreales, Gibberella, Gibberella zeae) is a destructive fungal pathogen that threatens the production and quality of wheat and barley worldwide. Controlling this toxin-producing pathogen is a significant challenge. In the present study, the commercially available strain Bacillus amyloliquefaciens (Bacteria, Firmicutes, Bacillales, Bacillus) FZB42 showed strong activity against F. graminearum The lipopeptide bacillomycin D, produced by FZB42, was shown to contribute to the antifungal activity. Purified bacillomycin D showed strong activity against F. graminearum, and its 50% effective concentration was determined to be approximately 30 μg/ml. Analyses using scanning and transmission electron microscopy revealed that bacillomycin D caused morphological changes in the plasma membranes and cell walls of F. graminearum hyphae and conidia. Fluorescence microscopy combined with different dyes showed that bacillomycin D induced the accumulation of reactive oxygen species and caused cell death in F. graminearum hyphae and conidia. F. graminearum secondary metabolism also responded to bacillomycin D challenge, by increasing the production of deoxynivalenol. Biological control experiments demonstrated that bacillomycin D exerted good control of F. graminearum on corn silks, wheat seedlings, and wheat heads. In response to bacillomycin D, F. graminearum genes involved in scavenging reactive oxygen species were downregulated, whereas genes involved in the synthesis of deoxynivalenol were upregulated. Phosphorylation of MGV1 and HOG1, the mitogen-activated protein kinases of F. graminearum, was increased in response to bacillomycin D. Taken together, these findings reveal the mechanism of the antifungal action of bacillomycin D.IMPORTANCE Biological control of plant disease caused by Fusarium graminearum is desirable. Bacillus amyloliquefaciens FZB42 is a representative of the biocontrol bacterial strains. In this work, the lipopeptide bacillomycin D, produced by FZB42, showed strong fungicidal activity against F. graminearum Bacillomycin D caused morphological changes in the plasma membrane and cell wall of F. graminearum, induced accumulation of reactive oxygen species, and ultimately caused cell death in F. graminearum Interestingly, when F. graminearum was challenged with bacillomycin D, the deoxynivalenol production, gene expression, mitogen-activated protein kinase phosphorylation, and pathogenicity of F. graminearum were significantly altered. These findings clarified the mechanisms of the activity of bacillomycin D against F. graminearum and highlighted the potential of B. amyloliquefaciens FZB42 as a biocontrol agent against F. graminearum.

Keywords: Bacillus amyloliquefaciens; Fusarium graminearum; bacillomycin D; cell death; fungus-bacterium interactions; mitogen-activated protein kinases; reactive oxygen species.

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Figures

FIG 1
FIG 1
Antagonistic activities against F. graminearum PH-1 of FZB42 and its mutants (A) and of the secondary metabolite extract (B). FZB42 and its mutants are described in Materials and Methods. CK, control (LB medium or methanol).
FIG 2
FIG 2
Purification and characterization of bacillomycin D from B. amyloliquefaciens CH02. (A) Bacillomycin D was purified from B. amyloliquefaciens CH02 using reverse-phase HPLC. (B) Five fractions (I to V) were separated and collected by reverse-phase HPLC and were used for the analysis of antagonistic activity. 45% acetonitrile (vol/vol) served as the control (CK). (C) The purity of bacillomycin D collected from peak I was analyzed by HPLC.
FIG 3
FIG 3
Effect of bacillomycin D on the morphology of F. graminearum PH-1 hyphae and conidia. (A) Effect of 9 μg/ml bacillomycin D on the morphology of F. graminearum PH-1 hyphae and conidia observed with a light microscope. (B and C) Ultrastructural effects of 30 μg/ml bacillomycin D on F. graminearum PH-1 after 12 h, as determined by scanning electron microscopy (B) and transmission electron microscopy (C). CW, cell wall; cy, cytoplasm; pm, plasma membrane; S, septum. In all of the experiments, 6.67% (vol/vol) methanol served as the control (CK).
FIG 4
FIG 4
Effect of 30 μg/ml bacillomycin D on ROS production by F. graminearum. (A) Detection of ROS was based on DCFH-DA staining after treatment with bacillomycin D for 5 h. (B) Quantitative real-time PCR analysis of the expression of five genes (fgsg_02881, fgsg_02974, fgsg_06554, fgsg_06733, and fgsg_12369) in F. graminearum PH-1 in response to bacillomycin D treatment. Values were normalized to the levels of the actin gene as an internal reference. The y axis represents the mean expression values ± standard deviations (SDs), relative to the control. The experiments were repeated independently three times. In all of the experiments, 6.67% (vol/vol) methanol served as the control (CK).
FIG 5
FIG 5
Detection of F. graminearum viability based on fluorescein diacetate and propidium iodide staining after treatment with bacillomycin D for 12 h. Live fungal cells with intact membranes show green fluorescence, and fungal cells with damaged membranes show red fluorescence; 6.67% (vol/vol) methanol served as the control (CK).
FIG 6
FIG 6
Analyses of F. graminearum conidiation (A) and conidial spore germination (B) for cultures treated with different concentrations of bacillomycin D. Data are expressed as means ± SDs. *, significant difference, compared with the control (P < 0.01). The experiment was repeated independently three times, and 6.67% (vol/vol) methanol served as the control (CK).
FIG 7
FIG 7
Bacillomycin D effects on F. graminearum PH-1 infection of corn silks, wheat seedlings, and wheat heads. (A) Corn silks were inoculated with a 0.6-cm-diameter plug containing F. graminearum PH-1 mycelia and then were treated with 30 to 90 μg/ml bacillomycin D; 6.67% (vol/vol) methanol served as the control. White arrows show the reddish-brown discoloration in the corn silks. (B) Wheat seedlings were inoculated with conidial suspensions of F. graminearum PH-1 and then were treated with 30 to 90 μg/ml bacillomycin D. A conidial suspension (106 conidia/ml) with 2% (wt/vol) gelatin and 6.67% (vol/vol) methanol served as the control. Black arrows show the black discoloration in the wheat seedlings. (C) Wheat heads were point inoculated with conidial suspensions of F. graminearum PH-1 and then were treated with 90 μg/ml bacillomycin D. A conidial suspension with 6.67% (vol/vol) methanol served as the control.
FIG 8
FIG 8
Effects of bacillomycin D on deoxynivalenol (DON) biosynthesis (A) and the expression of DON-related genes (B) of F. graminearum. Data are expressed as means ± SDs. *, significant difference, compared with controls (P < 0.01). In the quantitative real-time PCR analysis, values were normalized to the levels of the actin gene, as an internal reference; gene expression values are reported as means ± SDs, relative to control values. The experiments were repeated independently three times.
FIG 9
FIG 9
Phosphorylation of FgHOG1 and FgMGV1 in F. graminearum after exposure to bacillomycin D. (A) Western blotting assay. (B) Bar graphs representing quantification of the levels of phosphorylated FgHOG1 or FgMGV1, relative to the GAPDH reference. Prepared conidia of F. graminearum PH-1 were incubated in PDB with 30 μg/ml bacillomycin D, and 6.67% (vol/vol) methanol was used as the control (CK). Anti-phospho-p44/42 MAPK, anti-phospho-p38 MAPK, and anti-GAPDH were used as antibodies.

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