Management of Black Root Disease-Causing Fungus Fusarium solani CRP1 by Endophytic Bacillus siamensis CNE6 through Its Metabolites and Activation of Plant Defense Genes

Abstract

Black root rot disease of Cicer arietinum L. is accountable for substantial loss in chickpea production worldwide. Endophytic Bacillus siamensis CNE6 has previously shown multifaceted plant growth-promoting, broad-spectrum antifungal, and chickpea plant-colonizing potential. In the present study, the strain Bacillus siamensis CNE6 was used for controlling black root rot disease caused by Fusarium solani CRP1 in chickpea. CNE6 showed strong antagonistic potential against CRP1 both in vivo and in vitro. Scanning electron microscopic studies indicated cellular deformation of CRP1 due to production of β-glucanase, protease, and other secondary metabolites. A total of five compounds were detected from the cell-free supernatant (CFS) of the ethyl acetate (EA) fraction of CNE6 through gas chromatography-mass spectrometry analysis. A confocal microscopic study demonstrated strong inhibition of biofilm formation of the pathogen CRP1 by the EA fraction of CFS of CNE6. Molecular docking analysis revealed that one compound, (2E)-6-methoxy-2-[(4-methoxyphenyl)methylidene]-2,3-dihydro-1-benzofuran-3-one, may inhibit the activity of lanosterol 14-alpha demethylase, which is involved in ergosterol biosynthesis and beta-tubulin assembling. In vivo experiments also showed the efficacy of CNE6 for increasing chickpea growth as well as upregulation of four defense genes (CHI1, PAMP, PR2B, and TF1082) upon pathogenic challenge. Thus, our results strongly suggest a positive role for CNE6 as a prospective biocontrol agent for combating Fusarium solani in chickpea. IMPORTANCE The present work was undertaken to explore an effective biocontrol agent against the destructive black root rot disease of chickpea. We have used an efficient bacterial endophyte, CNE6, which can colonize in the chickpea root system, produce secondary metabolites and enzymes to degrade pathogenic cellular integrity, inhibit pathogenic establishment by rupturing biofilm formation, and induce host immunity upon treatment. Interaction of the bacterial metabolite was also observed with lanosterol 14-alpha demethylase, which is an important component in fungal membrane functioning. Being an endophyte, Bacillus siamensis CNE6 fulfills a suitable criterion as a biocontrol agent to control black root rot disease in chickpea and has huge prospects for use commercially.

Keywords: Cicer arietinum L.; Fusarium solani CRP1; biocontrol; biofilm inhibition; black root rot disease; host defense genes; in vivo experiment; molecular docking; secondary metabolites.

FIG 1

Characterization and identification of isolated pathogen. A neighbor joining phylogenetic tree of isolated pathogen CRP1 was constructed using nucleotide sequence of the ITS region of rRNA.

FIG 2

Pathogenecity test of isolated pathogen CRP1 in chickpea plants. (a) In vitro laboratory assessment; (b) in vivo pot experiment. C, untreated control; T, treated with CRP1 (n = 3).

FIG 3

Zones of inhibition produced by B. siamensis CNE6 against isolated pathogen F. solani CRP1. (a) Dual culture overlay assay; (b) agar well diffusion assay (upper two wells, 24-h-grown CFS; lower two wells, 48-h-grown CFS; middle well, untreated control). Values are expressed as the mean ± SD (n = 3).

FIG 4

Corrected growth inhibition of F. solani CRP1 at different concentrations (%, vol/vol) of CFS of B. siamensis CNE6. Values are expressed as the mean ± SD (n = 3). Significance level, P < 0.001. LSD, Fisher’s least significant differences.

FIG 5

SEM observation of morphological deformations of fungal phytopathogen caused by B. siamensis CNE6. (a) Untreated control; (b) treated with CNE6. (Micrographs with the highest clarity were selected from several randomly taken micrographs.)

FIG 6

Characterization of antifungal metabolites produced by CNE6. (a) Determination of the nature of antifungal metabolites produced by CNE6 against the pathogen CRP1. Well numbers signify the following: 1, control; 2, normal CFS; 3, boiled CFS; 4, PK only; 5, CFS treated with PK. (b to d) Determination of β-glucanase production on a 1% laminarin-containing plate. (b) Negative control (laboratory isolate, unidentified strain CNE3); (c) halo zone produced by CNE6 indicating β-glucanase production; (d) positive control (Bacillus velezensis SEB1). (e to g) Determination of protease production on peptone gelatin agar plate. (e) Negative control (strain CNE3); (f) halo zone produced by CNE6 indicating protease production; (g) positive control (Bacillus velezensis SEB1); (h) zone produced by crude antifungal metabolites extracted by ethyl acetate and chloroform against CRP1. DMSO was used as a control. Values are expressed as the mean ± SD (n = 3).

FIG 7

Inhibition of biofilm formation of F. solani CRP1 at different concentrations of EA extract of CNE6. (a) Biofilm observed in 24-well polystyrene cell culture plate after safranin staining; (b) quantification of biofilm formation at different concentrations of EA extract; (c) biofilm morphologies of F. solani CRP1 observed by CLSM after treatment. Values are expressed as the mean ± SD (n = 3), Significance level, P < 0.001.

FIG 8

Molecular docking analysis showing interaction of the compounds. (a and d) 3D and 2D representations of docked complex of CE and lanosterol 14- alpha demethylase; (b and e) 3D and 2D representations of docked complex of CE and beta-tubulin; (c and f) 3D and 2D representations of docked complex of CC and beta-tubulin.

FIG 9

Effect of endophytic bacterium CNE6 in controlling black root disease and growth parameters (shoot and root length). (a) In vivo challenge experiment; (b) shoot and root lengths of different treated plants compared to those of untreated control set. Values are expressed as the mean ± SD (n = 6). Significance level, P < 0.001.

FIG 10

Ability of endophytic bacterium CNE6 to induce chickpea defense genes (expression of defense genes was monitored by qPCR analysis). (a) CHI1; (b) PAMP; (c) PR2B; (d) TF1063; (e) TF1082; (f) PAL. Values are expressed as the mean ± SD (n = 3). Means with different letters are significantly different. Significance level, P < 0.05. Con, control.