RESUMO
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 ß-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.
RESUMO
Exploration of endophytic bacteria with multiple plant growth promoting (PGP) attributes is considered as an eco-friendly and cost-effective alternative to agricultural chemicals for increasing crop productivity. In the present endeavor, healthy chickpea plants (Cicer arietinum L.) collected from district Birbhum, West Bengal, India were subjected for the isolation of endophytic bacteria having multifarious PGP properties. One potent endophytic Gram positive bacterial strain CNE6 was isolated from the nodule of chickpea and was identified as Bacillus siamensis based on 16S rDNA sequence homologies. The isolate showed a number of PGP properties like phosphate solubilization, IAA production, nitrogen fixation, hydroxamate type of siderophore production and ACC deaminase activities. The isolate CNE6 produced 33.27 ± 2.16 µg/mL of IAA in the presence of tryptophan. Production of IAA was also confirmed by HPLC analysis and it was found effective for inducing lateral root branching in chickpea. In addition, the isolate displayed significant antagonistic activity against a number of plant pathogenic fungi when tested by dual culture overlay and agar well diffusion assay. 50 % cell free supernatant of CNE6 was found effective for 60-80 % inhibition of radial growth of pathogenic fungi tested. Scanning electron microscopic observation revealed massive degradation of pathogenic fungal mycelia by the antifungal metabolites of CNE6. LC-MS analysis of bacterial lipopeptides suggested the production of antifungal antibiotics like surfactin, fengycin and iturin by the isolate. The presence of genes encoding antifungal lipopeptides was also confirmed by PCR amplification using specific primers. Green fluorescent protein (GFP) tagging of CNE6 using broad host range plasmid vector (pDSK-GFPuv) followed by colonization study indicated very good host colonization potential of the isolate and its probable movement through xylem vessels. Enhanced shoot and root length and chlorophyll content upon treatment with CNE6 as observed in in vivo pot experiments also supported the positive role of the endophytic isolate on overall development and growth of the chickpea plants. This is the first report of Bacillus siamensis as an endophyte of Cicer arietinum L. which can be successfully applied for improving the productivity of this crop plant.
