Bacillus subtilis RB14 was used as an antagonist against fungal pathogen Rhizoctonia solani K1 to control damping-off diseases in tomato plants. Tomato seeds were treated with B. subtilis RB14 culture. The concentrati...Bacillus subtilis RB14 was used as an antagonist against fungal pathogen Rhizoctonia solani K1 to control damping-off diseases in tomato plants. Tomato seeds were treated with B. subtilis RB14 culture. The concentration of bacterial cells for the treatment was about 10<sup>8</sup> cfu/ml. Treated tomato seeds showed 99% germination index similar to the untreated seeds. Scanning Electron Microscopic observations showed a clear evidence of the presence of B. subtilis RB14 on tomato seed surface. Clear inhibition zone was observed using treated seed in dual plate assay against R. solani K1. B. subtilis RB14 treated seed showed 80% reduction in disease incidence during in vivo plant experiments. B. subtilis RB14 produces lipopeptide antifungal antibiotic iturin A which could suppress R. solani K1. The phenomenon was supported by our observation where we found significant amount of iturin A from the root zone soil of the seed treated plants.展开更多
The present study was conducted with an aim to scale up the production of iturin A using soybean curd residue (okara). Iturin A was produced by indigenous bacterial strain Bacillus subtilis RB14-CS through glass colum...The present study was conducted with an aim to scale up the production of iturin A using soybean curd residue (okara). Iturin A was produced by indigenous bacterial strain Bacillus subtilis RB14-CS through glass column reactor (GCR) under solid state fermentation (SSF) was characterized. The enhanced iturin A production was observed with respect to enhanced substrate bed height when SSF was conducted in Erlenmeyer flask. To check the effect of substrate bed height on iturin A production under SSF of okara, GCR was introduced. Substrate bed height of 15 cm was suitable for iturin A production which was about 2700 mg/kg wet substrate. The observed iturin A production by the aerobic bacteria Bacillus subtilis in nearly anaerobic condition in such high substrate bed for SSF is a wonderful finding for development of SSF system in future.展开更多
Biocontrol microorganisms and their derived metabolites with antagonistic activity represent promising alternatives to chemical fungicides in managing plant pathogens.The lipopeptides(LPs)iturin and fengycin derived f...Biocontrol microorganisms and their derived metabolites with antagonistic activity represent promising alternatives to chemical fungicides in managing plant pathogens.The lipopeptides(LPs)iturin and fengycin derived from Bacillus amyloliquefaciens S76-3 exhibit highly inhibitory effects against pathogenic fungi,especially Fusarium graminearum(Fg),the primary pathogen causing Fusarium head blight(FHB)in cereals.However,the specific target of iturin and fengycin in Fg and the underlying mechanism of antagonistic activity remain unclear.Here,global transcriptome sequencing,combined with both genetic and chemical approaches,demonstrates that the LPs exhibit antagonism toward Fg by binding to multiple components in the cell membrane of Fg cells,including ergosterol,phospholipids,glycosylphosphatidylinositol,and ankyrin.Lipopeptides result in cell swelling by inducing cell wall remodeling and osmotic substance glycerol synthesis mediated by cell wall integrity and high-osmolarity glycerol signaling pathways.Furthermore,we found that LPs can activate the induced systemic resistance in wheat against FHB and deoxynivalenol accumulation.Additionally,LPs were able to promote wheat growth by regulating auxin,cytokinin,and gibberellin signaling pathways while also delaying seed germination through the stimulation of abscisic acid and ethylene signaling pathways.These findings advance knowledge on the underlying mechanism of iturin and fengycin antagonistic activity and provide a new avenue for developing agricultural and clinical broad-spectrum antifungal agents and identifying plant growth regulators in the future.展开更多
An antifungal lipopeptide iturin A with strong activity against Fusarium oxysporum was produced by honey isolated strain Bacillus amyloliquefaciens BH072. For large-scale biocontrol application, the antifungal effect ...An antifungal lipopeptide iturin A with strong activity against Fusarium oxysporum was produced by honey isolated strain Bacillus amyloliquefaciens BH072. For large-scale biocontrol application, the antifungal effect was deeply demonstrated by structure and mode of action. Cyclic structure and second structure were determined based on situ acid hydrolysis and Fourier Transformed-Infra Red (FT-IR) Spectra analysis. Structure of α-helix was predicted which might be associated with activity. Afterwards, antifungal mechanism of iturin A on F. oxysporum were investigated from fungal cell wall to the plasma membrane and finally to intracellular proteins by morphological, activity of alkaline phosphatase (AKP), conductivity, Malondialdehyde (MDA) and SDS-PAGE detection. Antifungal damage appears on not only the spore germination and mycelium growth of F. oxysporum, but also the leakage of cellular proteins. Moreover, growth of F. oxysporum could be inhibited in the presence of iturin A at a MIC of 2.5 mg/mL. Considered with its high production in previous work, B. amyloliquefaciens strain BH072 and iturin A might be a promising candidate for biocontrol.展开更多
In biological controls using <em>Bacillus</em> spp., cyclic lipopeptides play a role as elicitors to induce disease resistance on various host plants. However, it is still unclear the specificity between c...In biological controls using <em>Bacillus</em> spp., cyclic lipopeptides play a role as elicitors to induce disease resistance on various host plants. However, it is still unclear the specificity between cyclic lipopeptides and host plants to induce disease resistance. In this study, we aimed to clarify the specificity to induce disease resistance among cyclic lipopeptides on various host plants. Our data clearly showed both cyclic lipopeptides conferred disease suppression on most of host plants, but at different range of cyclic lipopeptide concentration. Our findings contribute to understanding the complex on the specificity of cyclic lipopeptide derived induced disease resistance.展开更多
Bacillus amyloliquefaciens generally secretes a mixture of multiple cyclic lipopeptides,including iturin,surfactin,and plipastatin,and it is challenging to discriminate each cyclic lipopeptide’s antimicrobial activit...Bacillus amyloliquefaciens generally secretes a mixture of multiple cyclic lipopeptides,including iturin,surfactin,and plipastatin,and it is challenging to discriminate each cyclic lipopeptide’s antimicrobial activity directly.In this study,a chassis strain Bacillus subtilis 1A751 WR without cyclic lipopeptides operons was constructed by CRISPR/Cas9.The iturin gene cluster of B.amyloliquefaciens HYM-12 was captured and assembled with sfp and degQ genes using transformation-associated recombination(TAR).It was then integrated into the chassis strain to obtain an iturin mono-producing strain that could synthesize six iturin isoforms.The frameshift sfp gene of strain 1A751Δpps was repaired by a scarless gene knock-in method to generate a surfactin mono-producing strain.Antimicrobial activity experiments indicated that the extracts of iturin,surfactin,and our previously constructed plipastatin mono-producing strains exhibited powerful inhibitory effects toward pathogenic bacteria.Interest-ingly,iturin showed significant antifungal activity,but surfactin and plipastatin exhibited weak antifungal ac-tivity.Compared with amphotericin B,iturin has a solid and durable antifungal activity.This study demonstrates that TAR cloning is efficient for cloning large gene clusters,proves the differences in the antimicrobial activity of three cyclic lipopeptides,and lays a theoretical basis for further elucidating their antimicrobial mechanism.展开更多
文摘Bacillus subtilis RB14 was used as an antagonist against fungal pathogen Rhizoctonia solani K1 to control damping-off diseases in tomato plants. Tomato seeds were treated with B. subtilis RB14 culture. The concentration of bacterial cells for the treatment was about 10<sup>8</sup> cfu/ml. Treated tomato seeds showed 99% germination index similar to the untreated seeds. Scanning Electron Microscopic observations showed a clear evidence of the presence of B. subtilis RB14 on tomato seed surface. Clear inhibition zone was observed using treated seed in dual plate assay against R. solani K1. B. subtilis RB14 treated seed showed 80% reduction in disease incidence during in vivo plant experiments. B. subtilis RB14 produces lipopeptide antifungal antibiotic iturin A which could suppress R. solani K1. The phenomenon was supported by our observation where we found significant amount of iturin A from the root zone soil of the seed treated plants.
文摘The present study was conducted with an aim to scale up the production of iturin A using soybean curd residue (okara). Iturin A was produced by indigenous bacterial strain Bacillus subtilis RB14-CS through glass column reactor (GCR) under solid state fermentation (SSF) was characterized. The enhanced iturin A production was observed with respect to enhanced substrate bed height when SSF was conducted in Erlenmeyer flask. To check the effect of substrate bed height on iturin A production under SSF of okara, GCR was introduced. Substrate bed height of 15 cm was suitable for iturin A production which was about 2700 mg/kg wet substrate. The observed iturin A production by the aerobic bacteria Bacillus subtilis in nearly anaerobic condition in such high substrate bed for SSF is a wonderful finding for development of SSF system in future.
基金supported by grants from the National Key R&D Program of China(2022YFD1400102and 2018YFD02005)the National Natural Science Foundation of China(32272170 and 31271717)+1 种基金China Postdoctoral Science Foundation(2021M701348)Hubei Hongshan Laboratory(2022hspy010).
文摘Biocontrol microorganisms and their derived metabolites with antagonistic activity represent promising alternatives to chemical fungicides in managing plant pathogens.The lipopeptides(LPs)iturin and fengycin derived from Bacillus amyloliquefaciens S76-3 exhibit highly inhibitory effects against pathogenic fungi,especially Fusarium graminearum(Fg),the primary pathogen causing Fusarium head blight(FHB)in cereals.However,the specific target of iturin and fengycin in Fg and the underlying mechanism of antagonistic activity remain unclear.Here,global transcriptome sequencing,combined with both genetic and chemical approaches,demonstrates that the LPs exhibit antagonism toward Fg by binding to multiple components in the cell membrane of Fg cells,including ergosterol,phospholipids,glycosylphosphatidylinositol,and ankyrin.Lipopeptides result in cell swelling by inducing cell wall remodeling and osmotic substance glycerol synthesis mediated by cell wall integrity and high-osmolarity glycerol signaling pathways.Furthermore,we found that LPs can activate the induced systemic resistance in wheat against FHB and deoxynivalenol accumulation.Additionally,LPs were able to promote wheat growth by regulating auxin,cytokinin,and gibberellin signaling pathways while also delaying seed germination through the stimulation of abscisic acid and ethylene signaling pathways.These findings advance knowledge on the underlying mechanism of iturin and fengycin antagonistic activity and provide a new avenue for developing agricultural and clinical broad-spectrum antifungal agents and identifying plant growth regulators in the future.
文摘An antifungal lipopeptide iturin A with strong activity against Fusarium oxysporum was produced by honey isolated strain Bacillus amyloliquefaciens BH072. For large-scale biocontrol application, the antifungal effect was deeply demonstrated by structure and mode of action. Cyclic structure and second structure were determined based on situ acid hydrolysis and Fourier Transformed-Infra Red (FT-IR) Spectra analysis. Structure of α-helix was predicted which might be associated with activity. Afterwards, antifungal mechanism of iturin A on F. oxysporum were investigated from fungal cell wall to the plasma membrane and finally to intracellular proteins by morphological, activity of alkaline phosphatase (AKP), conductivity, Malondialdehyde (MDA) and SDS-PAGE detection. Antifungal damage appears on not only the spore germination and mycelium growth of F. oxysporum, but also the leakage of cellular proteins. Moreover, growth of F. oxysporum could be inhibited in the presence of iturin A at a MIC of 2.5 mg/mL. Considered with its high production in previous work, B. amyloliquefaciens strain BH072 and iturin A might be a promising candidate for biocontrol.
文摘In biological controls using <em>Bacillus</em> spp., cyclic lipopeptides play a role as elicitors to induce disease resistance on various host plants. However, it is still unclear the specificity between cyclic lipopeptides and host plants to induce disease resistance. In this study, we aimed to clarify the specificity to induce disease resistance among cyclic lipopeptides on various host plants. Our data clearly showed both cyclic lipopeptides conferred disease suppression on most of host plants, but at different range of cyclic lipopeptide concentration. Our findings contribute to understanding the complex on the specificity of cyclic lipopeptide derived induced disease resistance.
基金supported by the special funds for Agro-product Quality Safety Risk Assessment of the Ministry of Agriculture of the People’s Republic of China(GJFP20191101)a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.
文摘Bacillus amyloliquefaciens generally secretes a mixture of multiple cyclic lipopeptides,including iturin,surfactin,and plipastatin,and it is challenging to discriminate each cyclic lipopeptide’s antimicrobial activity directly.In this study,a chassis strain Bacillus subtilis 1A751 WR without cyclic lipopeptides operons was constructed by CRISPR/Cas9.The iturin gene cluster of B.amyloliquefaciens HYM-12 was captured and assembled with sfp and degQ genes using transformation-associated recombination(TAR).It was then integrated into the chassis strain to obtain an iturin mono-producing strain that could synthesize six iturin isoforms.The frameshift sfp gene of strain 1A751Δpps was repaired by a scarless gene knock-in method to generate a surfactin mono-producing strain.Antimicrobial activity experiments indicated that the extracts of iturin,surfactin,and our previously constructed plipastatin mono-producing strains exhibited powerful inhibitory effects toward pathogenic bacteria.Interest-ingly,iturin showed significant antifungal activity,but surfactin and plipastatin exhibited weak antifungal ac-tivity.Compared with amphotericin B,iturin has a solid and durable antifungal activity.This study demonstrates that TAR cloning is efficient for cloning large gene clusters,proves the differences in the antimicrobial activity of three cyclic lipopeptides,and lays a theoretical basis for further elucidating their antimicrobial mechanism.
