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Anthracnose, caused by Colletotrichum spp., is a threat to strawberry production globally. Unlike their chemical counterparts, microbial biofungicides offer a method of postharvest fungal disease control that is safe, sustainable and less affected by pathogen resistance. The present study evaluated the antifungal effects of three bacteria, Bacillus velezensis strains SH1 and SH2 and Pseudomonas azotoformans strain SH3, obtained from sheep manure compost tea. The bacteria or their cell‐free filtrates were tested against Colletotrichum acutatum and Colletotrichum gloeosporioides in bioassays and against strawberry anthracnose. In addition, precipitated or extracted extracellular fractions were tested to determine the effects on membrane permeability of Colletotrichum spp. spores. Confrontation assay results showed all bacteria inhibited mycelial growth, with B. velezensis SH1 and P. azotoformans SH3 being the most effective. All cell‐free filtrates inhibited mycelial growth with B. velezensis SH1 and SH2 resulting in the highest inhibition. The bacteria suppressed anthracnose lesions on strawberry fruit although effective treatments varied by causal mould. B. velezensis SH1 and SH2 significantly permeabilized spore membranes, indicating antibiosis as a possible mode of action. Investigation into antimicrobial compound production found various homologues of the lipopeptides fengycin, iturin and surfactin were produced by B. velezensis SH1 and SH2. Results suggest that lipopeptides produced by B. velezensis strains permeabilize Colletotrichum cell membranes, and that fengycins were the most inhibitory of the lipopeptides against Colletotrichum spp. [ABSTRACT FROM AUTHOR]

Bacillus spp. produce numerous antimicrobial metabolites. Among these metabolites, cyclic lipopeptides (CLP) including fengycins, iturins, and surfactins are known to have varying antifungal activity against phytopathogenic fungi. The differential activities of CLP have been attributed to diverse mechanisms of action on fungal membranes. However, the precise biochemical determinants driving their antifungal modes of action have not been conclusively identified. In this study, three plant pathogenic fungi of varying lipopeptide sensitivities, Alternaria solani , Cladosporium cucumerinum , and Fusarium sambucinum , were studied to determine how their cell membrane lipid compositions may confer sensitivity and/or tolerance to fengycin, iturin, and surfactin. Results indicated that sensitivity to all three lipopeptides correlated with lower ergosterol content and elevated phospholipid fatty acid unsaturation. Fungal sensitivity to surfactin was also notably different than fengycin and iturin, as surfactin was influenced more by lower phosphatidylethanolamine amounts, higher levels of phosphatidylinositol, and less by phospholipid fatty acyl chain length. Results from this study provide insight into the fungal membrane composition of A. solani , F. sambucinum , and C. cucumerinum and the specific membrane characteristics influencing the antifungal effectiveness of fengycin, iturin, and surfactin. Understanding of these determinants should enable more accurate prediction of sensitivity-tolerance outcomes for other fungal species exposed to these important CLP. • Fungi were differentially sensitive to Bacillus lipopeptides. • Lipopeptide sensitivity correlated with ergosterol content. • Phospholipid fatty acid unsaturation and chain length were sensitivity determinants. • Surfactin sensitivity was different and influenced by phospholipid polar headgroups. [ABSTRACT FROM AUTHOR]

This study aimed to evaluate how the combined presence of the synthetic fungicide azoxystrobin (AZ) and the biosurfactant-producing Bacillus sp. Kol B3 influences the growth of the phytopathogenic fungus Fusarium sambucinum IM 6525. The results showed a noticeable increase in antifungal effectiveness when biotic and abiotic agents were combined. This effect manifested across diverse parameters, including fungal growth inhibition, changes in hyphae morphology, fungal membrane permeability and levels of intracellular reactive oxygen species (ROS). In response to the presence of Fusarium and AZ in the culture, the bacteria changed the proportions of biosurfactants (surfactin and iturin) produced. The presence of both AZ and/or Fusarium resulted in an increase in iturin biosynthesis. Only in 72 h old bacterial–fungal co-culture a 20% removal of AZ was noted. In the fungal cultures (with and without the addition of the bacteria), the presence of an AZ metabolite named azoxystrobin free acid was detected in the 48th and 72nd hours of the process. The possible involvement of increased iturin and ROS content in antifungal activity of Bacillus sp. and AZ when used together are also discussed. Biosurfactants were analyzed by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Microscopy techniques and biochemical assays were also used. [ABSTRACT FROM AUTHOR]

Fusarium wilt, caused by Fusarium oxysporum f. sp. cucumerinum (FOC), poses a serious threat to cucumber productivity. Compared to traditional chemical pesticides, biological control strategies have attracted more attention recently owing to their effectiveness against pathogens and their environmental safety. This study investigated the effect of white rot fungi Pleurotus ostreatus P5 on the production of cyclic lipopeptides (CLPs) of Bacillus amyloliquefaciens B2 and the potential co-culture filtrate of strains B2 and P5 to control cucumber Fusarium wilt. A PCR amplification of CLP genes revealed that B. amyloliquefaciens B2 had two antibiotic biosynthesis genes, namely, ituA and srf, which are involved in iturin A and surfactin synthesis. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) revealed that CLPs derived from strain B2 contained two families, iturin A (C14, C15) and surfactin (C12–C17). The co-culture exhibited an enhanced accumulation of iturin A and surfactin compared to the monoculture of strain B2. Furthermore, the gene expressions of ituA and srf were both significantly upregulated when co-cultured with the fungus compared to monocultures. In an in vitro experiment, the co-culture filtrate and monoculture filtrate of B. amyloliquefaciens B2 inhibited mycelial growth by 48.2% and 33.2%, respectively. In a greenhouse experiment, the co-culture filtrate was superior to the monoculture filtrate in controlling cucumber Fusarium wilt disease and in the promotion of plant growth. Co-culture filtrate treatment significantly enhanced the microbial metabolic activity and decreased the abundance of FOC in the rhizosphere soil. These results show that the co-culture of P. ostreatus P5 and B. amyloliquefaciens B2 has great potential in cucumber Fusarium wilt disease prevention by enhancing the production of bacterial CLPs. [ABSTRACT FROM AUTHOR]

This comprehensive review examines iturin, a cyclic lipopeptide originating from Bacillus subtilis and related bacteria. These compounds are structurally diverse and possess potent inhibitory effects against plant disease-causing bacteria and fungi. Notably, Iturin A exhibits strong antifungal properties and low toxicity, making it valuable for bio-pesticides and mycosis treatment. Emerging research reveals additional capabilities, including anticancer and hemolytic features. Iturin finds applications across industries. In food, iturin as a biosurfactant serves beyond surface tension reduction, enhancing emulsions and texture. Biosurfactants are significant in soil remediation, agriculture, wound healing, and sustainability. They also show promise in Microbial Enhanced Oil Recovery (MEOR) in the petroleum industry. The pharmaceutical and cosmetic industries recognize iturin's diverse properties, such as antibacterial, antifungal, antiviral, anticancer, and anti-obesity effects. Cosmetic applications span emulsification, anti-wrinkle, and antibacterial use. Understanding iturin's structure, synthesis, and applications gains importance as biosurfactant and lipopeptide research advances. This review focuses on emphasizing iturin's structural characteristics, production methods, biological effects, and applications across industries. It probes iturin's antibacterial, antifungal potential, antiviral efficacy, and cancer treatment capabilities. It explores diverse applications in food, petroleum, pharmaceuticals, and cosmetics, considering recent developments, challenges, and prospects. [ABSTRACT FROM AUTHOR]

Bacillus subtilis species complex is known as lipopeptide-producer with biotechnological potential for pharmaceutical developments. This study aimed to identify lipopeptides from a bacterial isolate and evaluate their antifungal effects. Here, we isolated and identified a lipopeptide-producing bacterium as a species of Bacillus subtilis complex (strain UL-1). Twenty lipopeptides (six iturins, six fengycins, and eight surfactins) were identified in the crude extract (CE) and fractions (F1, F2, F3, and F4), and the highest content of total lipopeptides was observed in CE and F2. The chemical quantification data corroborate with the hemolytic and antifungal activities that CE and F2 were the most hemolytic and inhibited the fungal growth at lower concentrations against Fusarium spp. In addition, they caused morphological changes such as shortening and/or atypical branching of hyphae and induction of chlamydospore-like structure formation, especially in Fusarium solani. CE was the most effective in inhibiting the biofilm formation and in disrupting the mature biofilm of F. solani reducing the total biomass and the metabolic activity at concentrations ≥ 2 µg/mL. Moreover, CE significantly inhibited the adherence of F. solani conidia on contact lenses and nails as well as disrupted the pre-formed biofilms on nails. CE at 100 mg/kg was nontoxic on Galleria mellonella larvae, and it reduced the fungal burden in larvae previously infected by F. solani. Taken together, the lipopeptides obtained from strain UL-1 demonstrated a potent anti-Fusarium effect inducing morphological alterations and antibiofilm activities. Our data open further studies for the biotechnological application of these lipopeptides as potential antifungal agents. Key points: • Lipopeptides inhibit Fusarium growth and induce chlamydospore-like structures. • Lipopeptides hamper the adherence of conidia and biofilms of Fusarium solani. • Iturins, fengycins, and surfactins were associated with antifungal effects. [ABSTRACT FROM AUTHOR]

This study aimed to evaluate how the combined presence of the synthetic fungicide azoxystrobin (AZ) and the biosurfactant-producing Bacillus sp. Kol B3 influences the growth of the phytopathogenic fungus Fusarium sambucinum IM 6525. The results showed a noticeable increase in antifungal effectiveness when biotic and abiotic agents were combined. This effect manifested across diverse parameters, including fungal growth inhibition, changes in hyphae morphology, fungal membrane permeability and levels of intracellular reactive oxygen species (ROS). In response to the presence of Fusarium and AZ in the culture, the bacteria changed the proportions of biosurfactants (surfactin and iturin) produced. The presence of both AZ and/or Fusarium resulted in an increase in iturin biosynthesis. Only in 72 h old bacterial–fungal co-culture a 20% removal of AZ was noted. In the fungal cultures (with and without the addition of the bacteria), the presence of an AZ metabolite named azoxystrobin free acid was detected in the 48th and 72nd hours of the process. The possible involvement of increased iturin and ROS content in antifungal activity of Bacillus sp. and AZ when used together are also discussed. Biosurfactants were analyzed by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Microscopy techniques and biochemical assays were also used.

The use of biocontrol agents based on endophytic bacteria against phloem-feeding insects is limited by a lack of knowledge and understanding of the mechanism of action of the endophyte community that makes up the plant microbiome. In this work, the mechanisms of the additive action of endophytic strains B. subtilis 26D and B. subtilis 11VM on the resistance of bread spring wheat against greenbug aphid Schizaphis graminum, was studied. It was shown that B. subtilis 26D secreted lipopeptide surfactin and phytohormones cytokinins, and B. subtilis 11VM produced iturin and auxins into the cultivation medium. Both strains and their lipopeptide-rich fractions showed direct aphicidal activity against greenbug aphid. For the first time, it was shown that B. subtilis 26D and B. subtilis 11VM in the same manner, as well as their lipopeptide-rich fractions, activated the expression of salicylate- and ethylene-dependent PR genes, and influenced plant redox metabolism, which led to an increase in plant endurance against aphids. The composition of endophytic strains B. subtilis 26D + B. subtilis 11VM had an additive effect on plant resistance to aphids due to an increase in the number of endophytic bacterial cells, and, as well as due to the synergistic effect of their mixture of lipopeptides − surfactin + iturin, both on the aphid mortality and on the expression of PR1 and PR3 genes. All these factors can be the reason for the observed increase in the growth of plants affected by aphids under the influence of B. subtilis 26D and B. subtilis 11VM, individually and in composition. The study demonstrates the possibility of creating in the future an artificial composition to enhance plant microbiome with endophytic bacteria, which combines growth-promoting and plant immunity stimulating properties against phloem-feeding insects. This direction is one of the most promising approaches to green pesticide discovery in the future. [ABSTRACT FROM AUTHOR]

Fungal infections pose severe and potentially lethal threats to plant, animal, and human health. Ergosterol has served as the primary target for developing antifungal medications. However, many antifungal drugs remain highly toxic to humans due to similarity in cell membrane composition between fungal and animal cells. Iturin A, lipopeptide produced by Bacillus subtilis , efficiently inhibit various fungi, but demonstrated safety in oral administration, indicating the existence of targets different from ergosterol. To pinpoint the exact antifungal target of iturin A, we used homologous recombination to knock out and overexpress erg3 , a key gene in ergosterol synthesis. Saccharomyces cerevisiae and Aspergillus carbonarius were transformed using the LiAc/SS-DNNPEG and Agrobacterium -mediated transformation (AMT), respectively. Surprisingly, increasing ergosterol content did not augment antifungal activity. Furthermore, iturin A's antifungal activity against S. cerevisiae was reduced while it pre-incubation with voltage-gated potassium (Kv) channel inhibitor, indicating that Kv activation was responsible for cell death. Iturin A was found to activate the Kv protein, stimulating K+ efflux from cell. In vitro tests confirmed interaction between iturin A and Kv protein. This study highlights Kv as one of the precise targets of iturin A in its antifungal activity, offering a novel target for the development of antifungal medications. [ABSTRACT FROM AUTHOR]

Fusarium wilt, caused by Fusarium oxysporum f. sp. cucumerinum (FOC), poses a serious threat to cucumber productivity. Compared to traditional chemical pesticides, biological control strategies have attracted more attention recently owing to their effectiveness against pathogens and their environmental safety. This study investigated the effect of white rot fungi Pleurotus ostreatus P5 on the production of cyclic lipopeptides (CLPs) of Bacillus amyloliquefaciens B2 and the potential co-culture filtrate of strains B2 and P5 to control cucumber Fusarium wilt. A PCR amplification of CLP genes revealed that B. amyloliquefaciens B2 had two antibiotic biosynthesis genes, namely, ituA and srf, which are involved in iturin A and surfactin synthesis. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) revealed that CLPs derived from strain B2 contained two families, iturin A (C14, C15) and surfactin (C12–C17). The co-culture exhibited an enhanced accumulation of iturin A and surfactin compared to the monoculture of strain B2. Furthermore, the gene expressions of ituA and srf were both significantly upregulated when co-cultured with the fungus compared to monocultures. In an in vitro experiment, the co-culture filtrate and monoculture filtrate of B. amyloliquefaciens B2 inhibited mycelial growth by 48.2% and 33.2%, respectively. In a greenhouse experiment, the co-culture filtrate was superior to the monoculture filtrate in controlling cucumber Fusarium wilt disease and in the promotion of plant growth. Co-culture filtrate treatment significantly enhanced the microbial metabolic activity and decreased the abundance of FOC in the rhizosphere soil. These results show that the co-culture of P. ostreatus P5 and B. amyloliquefaciens B2 has great potential in cucumber Fusarium wilt disease prevention by enhancing the production of bacterial CLPs.

This comprehensive review examines iturin, a cyclic lipopeptide originating from Bacillus subtilis and related bacteria. These compounds are structurally diverse and possess potent inhibitory effects against plant disease-causing bacteria and fungi. Notably, Iturin A exhibits strong antifungal properties and low toxicity, making it valuable for bio-pesticides and mycosis treatment. Emerging research reveals additional capabilities, including anticancer and hemolytic features. Iturin finds applications across industries. In food, iturin as a biosurfactant serves beyond surface tension reduction, enhancing emulsions and texture. Biosurfactants are significant in soil remediation, agriculture, wound healing, and sustainability. They also show promise in Microbial Enhanced Oil Recovery (MEOR) in the petroleum industry. The pharmaceutical and cosmetic industries recognize iturin’s diverse properties, such as antibacterial, antifungal, antiviral, anticancer, and anti-obesity effects. Cosmetic applications span emulsification, anti-wrinkle, and antibacterial use. Understanding iturin’s structure, synthesis, and applications gains importance as biosurfactant and lipopeptide research advances. This review focuses on emphasizing iturin’s structural characteristics, production methods, biological effects, and applications across industries. It probes iturin’s antibacterial, antifungal potential, antiviral efficacy, and cancer treatment capabilities. It explores diverse applications in food, petroleum, pharmaceuticals, and cosmetics, considering recent developments, challenges, and prospects.

Microbial infection is one of the biggest causes of food spoilage. Thus, preventing food from microbial-related spoilage has been one of the main concerns for the food community and the public. Exploring safe and efficient bio-preservatives has been on the way to the improvement of food quality. Lipopeptides (LPs), mainly secreted by Bacillus spp., are promising potential bio-preservatives, as they not only cover a broad range of antimicrobial functions against foodborne pathogens but inhibit food-deteriorating microorganisms as well. This review presents a fine understanding of Bacillus -forming LPs covering their characteristics, biosynthesis and regulation, mechanisms of action in antimicrobial activity, and utilizations and fabrications as a bio-preservative agent in the food industry. LPs show good potential in preserving food from deterioration, mainly in keeping the quality of vegetables, fruits, drinks, and aquatic products. The notable effects of LPs against microbial growth and food spoilage depend greatly on their types, structures and dosage. Moreover, the yield of LPs has been enhanced by gene regulation and the optimization of fermentation parameters. The nano-encapsulation of LPs as the core material is promising in guaranteeing their sustained release upon various triggers and promoting the preservation of food better. Therefore, there is a great need to explore the prospects of LPs formed by Bacillus spp. in the improvement of food preservation. • We review the biosynthesis, structure, action mode and application trends of LPs. • Main action of LPs involves destroying membrane integrity and permeability. • Cutting-edge technology is valuable for improving the efficacy and stability of LPs. • LPs holds promise for being green and potent fresh-keeping agent. [ABSTRACT FROM AUTHOR]

The biosynthesis of antifungal lipopeptides iturin and fengycin has attracted broad interest; however, there is a bottleneck in its low yield in wild strains. Because the key metabolic mechanisms in the lipopeptides synthesis pathway remain unclear, genetic engineering approaches are all ending up with a single or a few gene modifications. The aim of this study is to develop a systematic engineering approach to improve the antifungal activity and biosynthesis of iturin and fengycin in Bacillus amyloliquefaciens. First, blocking the carbon overflow metabolic pathway to increase precursor supply of the branched-chain amino acids by knockout of bdh, disrupting sporulation to extend the stage for producing antifungal lipopeptides by deletion of kinA, blocking of siderophore synthesis to enhance the availability of amino acids and fatty acids by deletion of dhbF, and increasing Spo0A∼P by deletion of rapA, could improve the antifungal activity by 24%, 10%, 13% and 18%, respectively. Second, the double knockout strain ΔbdhΔkinA, triple knockout strain ΔbdhΔkinAΔdhbF and quadruple knockout strain ΔkinAΔbdhΔdhbFΔrapA could improve the antifungal activity by 38%, 44% and 53%, respectively. Finally, overexpression of sfp in ΔkinAΔbdhΔdhbFΔrapA further increased the antifungal activity by 65%. After purifying iturin and fengycin as standards for quantitative analysis of lipopeptides, we found the iturin titer was 17.0 mg/L in the final engineered strain, which was 3.2-fold of the original strain. After fermentation optimization, the titer of iturin and fengycin reached 31.1 mg/L and 175.3 mg/L in flask, and 123.5 mg/L and 1200.8 mg/L in bioreactor. Compared to the original strain, the iturin and fengycin titer in bioreactor increased by 22.8-fold and 15.9-fold in the final engineered strain, respectively. This study may pave the way for the commercial production of green antifungal lipopeptides, and is also favorable for understanding the regulatory and biosynthetic mechanism of iturin and fengycin.

We evaluated disease suppression by surfactin and iturin A against Pseudomonas syringae pv. maculicola MAFF 302783 in Arabidopsis thaliana and its mutants to gain insight into resistance pathways induced. Root treatments revealed that salicylic acid signaling via the isochorismate pathway was the major signaling pathway elicited by both cyclic lipopeptides. Disease suppression was negated after treatment with higher levels of surfactin or iturin A, and this negation of disease suppression was not associated with an antagonistic effect by induction of jasmonate signaling pathway in host plants. [ABSTRACT FROM AUTHOR]

The use of biocontrol agents based on endophytic bacteria against phloem-feeding insects is limited by a lack of knowledge and understanding of the mechanism of action of the endophyte community that makes up the plant microbiome. In this work, the mechanisms of the additive action of endophytic strains B. subtilis 26D and B. subtilis 11VM on the resistance of bread spring wheat against greenbug aphid Schizaphis graminum, was studied. It was shown that B. subtilis 26D secreted lipopeptide surfactin and phytohormones cytokinins, and B. subtilis 11VM produced iturin and auxins into the cultivation medium. Both strains and their lipopeptide-rich fractions showed direct aphicidal activity against greenbug aphid. For the first time, it was shown that B. subtilis 26D and B. subtilis 11VM in the same manner, as well as their lipopeptide-rich fractions, activated the expression of salicylate- and ethylene-dependent PR genes, and influenced plant redox metabolism, which led to an increase in plant endurance against aphids. The composition of endophytic strains B. subtilis 26D + B. subtilis 11VM had an additive effect on plant resistance to aphids due to an increase in the number of endophytic bacterial cells, and, as well as due to the synergistic effect of their mixture of lipopeptides − surfactin + iturin, both on the aphid mortality and on the expression of PR1 and PR3 genes. All these factors can be the reason for the observed increase in the growth of plants affected by aphids under the influence of B. subtilis 26D and B. subtilis 11VM, individually and in composition. The study demonstrates the possibility of creating in the future an artificial composition to enhance plant microbiome with endophytic bacteria, which combines growth-promoting and plant immunity stimulating properties against phloem-feeding insects. This direction is one of the most promising approaches to green pesticide discovery in the future.

Accession numbers for whole-genome sequence of Brevibacillus sp. strain GI9 and SKDU10 are CAGD01000001 to CAGD01000061 and LSSO00000000, respectively. Members of the genus Brevibacillus have been demonstrated to produce a variety of bioactive compounds including polyketides, lipopeptides and bacteriocins. Lipopeptides are non-ribosomally synthesized surface-active compounds with antimicrobial, antitumor, and immune-stimulatory activities. They usually exhibit strong antifungal and antibacterial activities and are considered as promising compounds in controlling fungal diseases. In this study, we have characterized two lipopeptides from Brevibacillus sp. strains GI9 and SKDU10. The corresponding lipopeptides were purified by reverse-phase high-performance liquid chromatography. Mass analysis and characterization by MALDI-TOF-MS (Matrix-assisted laser desorption ionization time-of-flight mass spectrometry) analysis revealed production of an iturin-like lipopeptide by strain GI9 and bogorol-like lipopeptide by strain SKDU10. Both lipopeptides exhibited broad spectrum antibacterial activity and inhibited the growth of various fungi. They showed minimum inhibitory concentration (MIC) values between 90 and 300 μg/ml against indicator strains of bacteria and drug-resistant Candida indicator strains. The lipopeptides did not show phytotoxic effect in seed germination experiments but caused hemolysis. Further, both lipopeptides inhibited the growth of fungi on fruits and vegetables in in vitro experiments, thereby exhibited potential use in biotechnological industry as effective biocontrol agents. [ABSTRACT FROM AUTHOR]

A new biosurfactant was obtained from a moderately halophilic bacterium identified as Bacillus tequilensis ZSB10 that was isolated from a saline water pond located in Tehuacan-Cuicatlan valley, Mexico. A kinetic analysis of the bacterial growth of the ZSB10 strain showed a maximum growth at 24 h regardless of the initial pH (5, 7.4, and 9). The best results were found at pH = 7.4 in terms of bacterial growth, besides which the produced biosurfactant showed emulsifying and surfactant properties with an emulsification index (E24) and surface tension change (ΔST) of 54 ± 0% and 26 mN m-1, respectively. Extracted ZSB10 crude biosurfactant had a yield of 106 ± 6 mg L-1, an E24 = 58.4 ±0.2%, and a ΔST = 26 mN m-1 with a critical micelle concentration (CMC) of 44.82 mg L-1. Also, its structure was characterized by MALDI-TOF mass spectrometry as a surfactin, iturin A, and fengycin mixture whose main isoform was leu/ile-7 C15 surfactin [M + Na]+. Finally, the ZSB10 crude biosurfactant showed antifungal activity against Helminthosporium sp., with a 79.3% growth inhibition and an IC50 of 1.37 mg per disc. Therefore, this biosurfactant could be used as biopesticide. [ABSTRACT FROM AUTHOR]

Plant growth-promoting rhizobacteria (PGPR) represent a set of microorganisms that play significant role in improving plant growth and controlling the phytopathogens. Unpredictable performance after the application of PGPR has been observed when these were shifted from in-vitro to in-vivo conditions due to the prevalence of various abiotic stress conditions. During growing period, the potato crop is subjected to a combination of biotic and abiotic stresses. Rhizoctonia solani, a soil-borne plant pathogen, causes reduced vigor and yield of potato crop worldwide. In the current study, multi-stress-tolerant rhizobacterial strain, Bacillus subtilis PM32, was isolated from field-grown potato with various plant growth promoting (PGP) traits including zinc and potassium solubilization, biological nitrogen fixation, ammonia and siderophore, as well as extracellular enzyme productions (cellulase, catalase, amylase, protease, pectinase, and chitinase). The strain PM32 exhibited a distinct potential to support plant growth by demonstrating production of indole-3-acetic acid (102.6 μM/mL), ACC-deaminase activity (1.63 μM of α-ketobutyrate/h/mg protein), and exopolysaccharides (2.27 mg/mL). By retarding mycelial growth of R. solani the strain PM32 drastically reduced pathogenicity of R. solani. The strain PM32 also suppressed the pathogenic activity significantly by impeding mycelial expansion of R. solani with inhibition co-efficient of 49.87. The B. subtilis PM32 also depicted significant tolerance towards salt, heavy metal (Pb), heat and drought stress. PCR based amplification of ituC and acds genes coding for iturin and ACC-deaminase activity respectively indicated potential of strain PM32 for lipopeptides production and ACC deaminase enzyme activity. Results of both in-vitro and pot experiments under greenhouse conditions depicted the efficiency of B. subtilis PM32 as a promising bio-control agent for R. solani infection together with enhanced growth of potato plants as deciphered from biomass accumulation, chlorophyll a, b, and carotenoid contents. Therefore, it was envisioned that application of indigenous multi-stress tolerant PGPR may serve to induce biotic and abiotic stress tolerance in crops/plants for pathogen control and sustainable global food supply. [ABSTRACT FROM AUTHOR]

Accession numbers for whole-genome sequence of Brevibacillus sp. strain GI9 and SKDU10 are CAGD01000001 to CAGD01000061 and LSSO00000000, respectively. Members of the genus Brevibacillus have been demonstrated to produce a variety of bioactive compounds including polyketides, lipopeptides and bacteriocins. Lipopeptides are non-ribosomally synthesized surface-active compounds with antimicrobial, antitumor, and immune-stimulatory activities. They usually exhibit strong antifungal and antibacterial activities and are considered as promising compounds in controlling fungal diseases. In this study, we have characterized two lipopeptides from Brevibacillus sp. strains GI9 and SKDU10. The corresponding lipopeptides were purified by reverse-phase high-performance liquid chromatography. Mass analysis and characterization by MALDI-TOF-MS (Matrix-assisted laser desorption ionization time-of-flight mass spectrometry) analysis revealed production of an iturin-like lipopeptide by strain GI9 and bogorol-like lipopeptide by strain SKDU10. Both lipopeptides exhibited broad spectrum antibacterial activity and inhibited the growth of various fungi. They showed minimum inhibitory concentration (MIC) values between 90 and 300 μg/ml against indicator strains of bacteria and drug-resistant Candida indicator strains. The lipopeptides did not show phytotoxic effect in seed germination experiments but caused hemolysis. Further, both lipopeptides inhibited the growth of fungi on fruits and vegetables in in vitro experiments, thereby exhibited potential use in biotechnological industry as effective biocontrol agents.

Candida albicans (C. albicans) is a fungal pathogen that is difficult to cure clinically due to lack of effective antifungal agents with low toxicity. In this study, iturin, a cyclic peptide having wide antifungal spectrum, was used to synthesize nanosilver particles (AgNPs), and a complex of iturin-AgNPs was formed. The antifungal activity of iturin-AgNPs against C. albicans and its mechanisms were tested in vitro. Iturin-AgNPs were also loaded in chitosan (CS) composite dressing and applied to skin wound healing in mice. As results, iturin-AgNPs showed excellent antifungal activity with the minimum inhibitory concentrations (MIC) of 1.25, 2.5, and 5 μg/mL at C. albicans concentrations of 1×105, 1×106, and 1×107 CFU/mL, respectively. The MIC value still kept at 2.5 μg/mL against C. albicans (105 CFU/mL) after 15 regeneration, showing less induction of drug resistance to the pathogenic fungus. The antifungal mechanisms of iturin-AgNPs against C. albicans were identified as the increase of membrane permeability, damage of cell membrane integrity, and leakage of cellular protein and nucleic acids. No toxicity was found for iturin-AgNPs to HaCaT cells at concentrations of lower than 10 μg/mL. In wound healing application, iturin-AgNP CS composite dressing significantly accelerated the healing of C. albicans infected skin wounds at the early 10 days. In conclusion, iturin-AgNPs were developed as an efficient antifungal agent against C. albicans in vitro and in vivo and showed potential application in wound healing promotion. [ABSTRACT FROM AUTHOR]

Abstract Lipopeptides possess excellent broad spectrum antimicrobial activity. Different lipopeptides have their own unique chemical structures, properties and biological activities. Quantitative analysis of the lipopeptides iturin and surfactin and their homologues produced by Bacillus natto NT–6 subjected to different culture media, shaking speed of rotary shaker, and liquid and solid fermentation methods was conducted using LC–MS. For iturins, liquid-state fermentation in Landy medium at a shaking speed of 160 r min−1 was the most suitable for maximal homologue production. Addition of 0.4% attapulgite powder increased production by 1.92-fold; activated carbon significantly reduced production. For surfactin homologues, solid-state fermentation in potato dextrose broth medium at shaking speed > 160 r min−1 was the best. Addition of 0.4% attapulgite powder increased production by 1.96-fold; activated carbon had no effect. Thus it is clear that fermentation conditions can be manipulated to maximize iturin and surfactin production.

ABSTRACT Microbes produce a plethora of secondary (or specialized) metabolites that, although not essential for primary metabolism, benefit them to survive in the environment, communicate, and influence cell differentiation. Biosynthetic gene clusters (BGCs), responsible for the production of these secondary metabolites, are readily identifiable on bacterial genome sequences. Understanding the phylogeny and distribution of BGCs helps us to predict the natural product synthesis ability of new isolates. Here, we examined 310 genomes from the Bacillus subtilis group, determined the inter- and intraspecies patterns of absence/presence for all BGCs, and assigned them to defined gene cluster families (GCFs). This allowed us to establish patterns in the distribution of both known and unknown products. Further, we analyzed variations in the BGC structures of particular families encoding natural products, such as plipastatin, fengycin, iturin, mycosubtilin, and bacillomycin. Our detailed analysis revealed multiple GCFs that are species or clade specific and a few others that are scattered within or between species, which will guide exploration of the chemodiversity within the B. subtilis group. Surprisingly, we discovered that partial deletion of BGCs and frameshift mutations in selected biosynthetic genes are conserved within phylogenetically related isolates, although isolated from around the globe. Our results highlight the importance of detailed genomic analysis of BGCs and the remarkable phylogenetically conserved erosion of secondary metabolite biosynthetic potential in the B. subtilis group. IMPORTANCE Members of the B. subtilis species complex are commonly recognized producers of secondary metabolites, among those, the production of antifungals, which makes them promising biocontrol strains. While there are studies examining the distribution of well-known secondary metabolites in Bacilli, intraspecies clade-specific distribution has not been systematically reported for the B. subtilis group. Here, we report the complete biosynthetic potential within the B. subtilis group to explore the distribution of the biosynthetic gene clusters and to reveal an exhaustive phylogenetic conservation of secondary metabolite production within Bacillus that supports the chemodiversity within this species complex. We identify that certain gene clusters acquired deletions of genes and particular frameshift mutations, rendering them inactive for secondary metabolite biosynthesis, a conserved genetic trait within phylogenetically conserved clades of certain species. The overview guides the assignment of the secondary metabolite production potential of newly isolated Bacillus strains based on genome sequence and phylogenetic relatedness.

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In the present study, a deep-sea bacterial strain designated Bacillus sp. wsm-1 was screened to exhibit strong antifungal activity against many plant pathogenic fungi, and corresponding antifungal agents were thereby purified and determined by tandem mass spectrometry to be two cyclic lipopeptide homologs. These two lipopeptide homologs were identified to possess identical amino acid sequences of β-amino fatty acid-Asn-Ser-Asn-Pro-Tyr-Asn-Gln, which were different from any previously reported lipopeptides, and deduced as two novel lipopeptides designated as C14 iturin W and C15 iturin W, respectively. Electron microscopy observation indicated that both iturin W homologs caused obvious morphological changes and serious disruption of plasma membrane toward fungal cells, while C15 iturin W exhibited more serious cell damages than C14 iturin W did, which was consistent well with the results of the antifungal activity assays. To improve the yield and antifungal activity of iturin W, the effects of different carbon and nitrogen sources and amino acids on production of C14 iturin W and C15 iturin W were investigated, respectively. The results indicated that supplements of most of the detected carbon and nitrogen sources could increase the yield of C14 iturin W, but inhibit the yield of C15 iturin W, while supplements of tryptone and most of the detected amino acids could increase the yield of both C14 iturin W and C15 iturin W. [ABSTRACT FROM AUTHOR]

Abstract A strong antibiotic producer, Bacillus subtilis strain Bbv 57 (KF718836), has been utilized for the management of wilt-nematode complex (Fusarium oxysporum f. sp. gerberae, Meloidogyne incognita) in gerbera under greenhouse conditions in the Department of Floriculture, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India. The strain strongly inhibited F. oxysporum f. sp. gerberae (KM523669) mycelial growth to an extent of 44.33 and 63.33%, at 10 and 100 μl, of culture filtrate, respectively. Further, the culture filtrate at 100% concentration exerted lethal effect on nematode eggs (7.00 hatched) and juveniles (87% mortality) compared to control. The analysis of TLC revealed that Bbv 57 showed the cyclic antimicrobial peptides surfactin and iturin that were confirmed by PCR. Strain Bbv 57 was able to produce antifungal and anti-nematicidal activity with reduced wilt incidence (15.33%) and thus holds a great potential for use in the biocontrol of Fusarium wilt-root-knot nematode disease complex in gerbera under greenhouse conditions.

Surfactin, a lipopeptide produced by Bacillus species, has been used for the oral delivery of insulin. In this study, another lipopeptide of iturin was tested for its ability to orally delivery insulin alone or plus surfactin. Iturin could form co-precipitate with insulin at acidic pH values. After treatment by ultrasonification, the structure of coprecipitate was destroyed that led to a significant decrease in hypoglycemic effect after oral administration. Iturin weakly binds to (Kd = 257 μM) and induce insulin structure more compact that is favorable for insulin uptake by the intestine. After being coated with Acryl-Eze by lyophilization, the coprecipitate formed the spherical enteric-coated insulin microparticles delivered by iturin with a relative oral bioavailability of 6.84% in diabetic mice. For further improving oral hypoglycemic effect, surfactin was added to form the spherical enteric-coated insulin microparticles in a formulation containing insulin, Acryl-Eze, iturin and surfactin at a ratio of 1:1:0.5: 0.5 (w/w), with an insulin encapsulation efficiency of 66.22%. The enteric-coated insulin microparticles delivered by iturin plus surfactin showed a classical profile for controlled release in the intestine with a relative bioavailability of 7.67% after oral administration, which could effectively control the postprandial blood glucose at a level about 50% of the initial one just like the subcutaneous injection. Collectively, iturin plus surfactin is more efficient for oral delivering insulin than the sole one, and the resultant enteric-coated insulin microparticles are potential for the development of oral insulin to control postprandial blood glucose in diabetic patients.

Lipopeptides are a group of second metabolites of Bacillus and have multiple activities such as inhibiting fungi, bacteria, viruses, and tumors, showing a great potential application in agricultural and biomedical fields. However, low production severely restrained their application in practice. Deeply understanding the key elements of lipopeptide synthesis and the regulatory strategies is essential to target the improvement of lipopeptide production. The synthetic pathways of different lipopeptides are different, but closely and mutually interfered. The loading of fatty acid chains and the extension of peptide chains are two key steps for the synthesis of different lipopeptides by Bacillus. The selection of fatty acid chains, the loading order of amino acids, and the recognition of the final cyclization site are the critical steps to determine the end products of different lipopeptides. In order to find the key elements for precisely directing the formation of different lipopeptides by Bacillus, the key structural elements and possible regulatory strategies that have been reported in the production of different lipopeptides, mainly surfactin, iturin, and fengycin by Bacillus, were summarized and compared. The possible ways to improve the production of different targeted lipopeptides were proposed. Key points: • The selectivity of fatty acids is determined by specific domains. • The COM domain and the PKS docking domain determine the order of amino acids. • The regulation patterns of different domains for lipopeptide synthesis are different. • The regulation of different lipopeptide products overlaps each other. [ABSTRACT FROM AUTHOR]

We report the inclusion of cyclolipopeptides (CL) from Bacillus subtilis in the production of an easily removed alginate coating for the preservation of vulnerable berries. The sodium alginate-CL (SA-CL) coating films exhibited potent antifungal and freshness-maintaining properties during the storage of blueberries, the 3% CL-added film decreased total fungal count to 2.5 × 103 cfu/g at the end of the storage. The SA-CL films had poor mechanical properties (i.e., tensile strength and elongation at break), low water vapor permeability and high water solubility. The addition of CL (3%) decreased the tensile strength and elongation at break of the SA-CL film to 0.37 mPa and 2.57%, respectively, and increased the water solubility to 28.92%. But the water vapor permeability of the 3% film was lowered to 398.10 g/m2/day. These results implied the film was suitable for the coating of vulnerable berries. Further research revealed that ionic and hydrogen bonding interactions between CL and alginate stabilized the network of the coating film while the fatty acid moiety weakened the strength and crystallinity and restricted the water transfer of the coating film. Our findings provide an effective route to an easily washed alginate coating film through inclusion of amphiphilic antifungal substances. • Cyclolipopeptides is added to alginate to make easily-cleaned active coating film. • The coating film suppresses fungal growth on blueberry and maintains its quality. • The coating film has weak mechanical strength and high water solubility. • Addition of cyclolipopeptides improves the water barrier capacity of film. [ABSTRACT FROM AUTHOR]

Rice monoculture in acid sulfate soils (ASSs) is affected by a wide range of abiotic and biotic constraints, including rice blast caused by Pyricularia oryzae. To progress towards a more sustainable agriculture, our research aimed to screen the biocontrol potential of indigenous Bacillus spp. against blast disease by triggering induced systemic resistance (ISR) via root application and direct antagonism. Strains belonging to the B. altitudinis and B. velezensis group could protect rice against blast disease by ISR. UPLC–MS and marker gene replacement methods were used to detect cyclic lipopeptide (CLiP) production and construct CLiPs deficient mutants of B. velezensis, respectively. Here we show that the CLiPs fengycin and iturin are both needed to elicit ISR against rice blast in potting soil and ASS conditions. The CLiPs surfactin, iturin and fengycin completely suppressed P. oryzae spore germination resulting in disease severity reduction when co-applied on rice leaves. In vitro microscopic assays revealed that iturin and fengycin inhibited the mycelial growth of the fungus P. oryzae, while surfactin had no effect. The capacity of indigenous Bacillus spp. to reduce rice blast by direct and indirect antagonism in ASS conditions provides an opportunity to explore their usage for rice blast control in the field.

Introduction: Different Bacillus subtilis products of secondary metabolits include iturin lipopeptids, that may control plant disease effectively. The aim of this study was to investigate the antifungal activity of Indigenous strains Bacillus subtilis against plant pathogenic fungi Pythium ultimum and Fusarium solani. Materials and methods: Seven soil samples were collected and B. subtilis were isolated from each soil sample. The isolates were screened by antifungal activity. Best strains were identified by 16srDNA sequence. The culture conditions were optimized for the best production of antifungal metabolites. The bacterial metabolites were then obtained from 4 days grown isolates, purified and were confirmed iturin existence by chromatography method. The iturin A (Sigma) was used as standards. Results: Totaly, 91 strains were isolated from soil samples, 23 spp were confirmed as B. subtilis by morphological and biochemical features. In subsequent experiments, two strains 48 and 83 showed the greatest activity against the Pythium ultimum and Fusarium solani respectively. 16srDNA sequence analyses for selected isolates confirmed 100% similarity to B. subtilis. Then nutrient broth with carbon and nitrogen sources glucose, yeast extract, neutral pH and 30⁰c incubation temperature were optimized for best production. The HPLC results showed the best productivity of iturin A for two isolates B. subtilis by comparing peaks and retention times between iturin A (Sigma) and native strains. Discussion and conclusion: Iranian native strains also have the ability to produce antifungal metabolites. Therefore, this strain can be a good candidate for biological control of plant pathogenic fungi and an alternative for chemical fungicides.

Genome shuffling is an effective method for the rapid improvement of the production of secondary metabolites. This study used the principle of gene shuffling to enhance the yield of iturin A produced by Bacillus amyloliquefaciens LZ-5. Improvements in lipopeptide yield were evident among four strains subjected to recursive protoplast fusion. The four strains were obtained through mutagenesis processes: nitrosoguanidine, ultraviolet irradiation, and atmospheric and room temperature plasma. A high yield strain with 179.22 mg/l of iturin A was obtained after two rounds of genome shuffling, which was a 2.03-fold increase compared with the wild strain. To evaluate the efficacy of iturin A for control of spoilage yeast in food, the anti-yeast efficacy of iturin A was evaluated in orange juice incubated with Saccharomyces cerevisiae. The juice treated with 0.76 mg/ml iturin A showed a significant (p < 0.05) control on yeast population during the storage, similar to that of the 0.30 mg/ml natamycin. In addition, iturin A showed a tiny effect on chemical-physical characteristics of orange juice. Our results provide a basis for the application of antimicrobial lipopeptide in juice products.

Bacillus subtilis widely exists in environment and shows a capability to deal with heavy metals and dyes in polluted waters by adsorption or biological oxidation and reduction. Little is known about the roles of lipopeptides in this capability of B. subtilis. In this study, we found that the lipopeptides produced by B. subtilis could reduce silver ions to silver nanoparticles (AgNPs) and iturin was identified as the major effective fraction. Furthermore, the synthesized AgNPs was successfully used to catalyze the reduction of organic dyes and reduce Pb2+ contamination in water. The formation of AgNPs was confirmed by the features analyzed by UV-vis spectroscopy, dynamic light scattering, high-resolution transmission electron microscopy (HR-TEM), and selected area electron diffraction (SAED). The formed AgNPs showed crystalline, with small size (~ 20 nm) and spherical shape. The biosynthesis of AgNPs was significantly accelerated by UV irradiation. A pH of 10 resulted in the highest formation rate, while pH 9.2 provided the most stability of AgNPs. In mechanisms, tyrosine and the polypeptide were identified as the major groups in iturin-A to form AgNPs via Ar–OH groups. The study revealed that iturin played important roles for the capability of B. subtilis to treat polluted water via a possible way by synthesizing AgNPs and then catalyzing the reduction of organic dyes and reducing the contamination of Pb2+. [ABSTRACT FROM AUTHOR]

Highlights • High cell density culture (HC) improves antimicrobial activity of B. subtilis. • HC's supernatant suppresses fungal growth on grapes and maintains grape quality. • HC of B. subtilis enhances the secretion of antifungal cyclic lipopeptides. • HC enriches iturins and surfactins rather than fengycins. Abstract This study investigated the effects of Bacillus subtilis grown in high-cell-density culture (HC) on mold growth suppression and postharvest quality improvement in Kyoho grapes. The HC supernatant (HS) of B. subtilis was more potent than the conventional B. subtilis culture supernatant (CS) in suppressing fungal growth on grapes. Specifically, HS was more effective than CS in maintaining the cosmetic qualities (colour, firmness and abscission rate) and physiological indices (respiration, weight loss, pectinases, antioxidant enzymes and titratable acidity). Chemical analyses showed that HC enhanced the secretion of lipopeptides in B. subtilis culture, and high-pressure liquid chromatography further demonstrated that iturin A and surfactin were increased by 1.78- and 1.23-fold in HS compared with CS, respectively. However, LC-MS/MS analysis revealed that HC only upregulated iturins and surfactins, but not fengycins. Therefore, HC may provide an effective method to enrich antifungal lipopeptides from B. subtilis for preservation of fruits and vegetables. [ABSTRACT FROM AUTHOR]

• Genetic marker development and LC-MS analysis rapidly detected antimicrobial compounds in 47 Bacillus strains. • Only 19 Bacillus strains possessed antimicrobial compounds among the screened strains. • Bacillus strains and their methanolic extracts showed suppression of Sclerotinia sclerotiorum during in vitro assays. Bacillus strains are well studied for antagonistic effect against fungal pathogens, but the selection of potential antifungal strains is laborious and time-consuming process. Newly developed genetic markers and LC-MS based detection was undertaken simultaneously to detect eight antimicrobial compounds viz., surfactin, bacillomycin, iturin, fengycin/plipastatin, bacilysin, bacillaene, bacillibactin and plantazolicin in forty-seven Bacillus strains. Out of these strains, 19 were positive for the presence of marker genes encoding antimicrobial compounds. Bacillus strains FZB42, EZ1509, EZ1507, VM10, GBAC46, VM49 and LLCG43 possessed genes for maximum number of antimicrobial compounds. LC-MS analysis of antimicrobial compounds showed corresponding results except OKB105 and 168. Contrary to marker-based detection of genes, LC-MS analysis revealed that OKB105 can produce surfactin but unable to synthesize fengycin, while 168 was deficit in both compounds. To assay antifungal potential, 19 Bacillus strains and their methanolic extracts were tested in vitro to inhibit mycelial growth of S. sclerotiorum. Results revealed that EZ1509, VM10, GBAC46, VM49 and FZB42 showed highest inhibitory activity. A bioassay on detached rapeseed leaves demonstrated that strains VM10, EZ1509, FZB42 and GBAC46 were excellent in reducing lesion diameter, while, OKB105 and 168 were completely ineffective to control S. sclerotiorum. Interestingly, antifungal activity of Bacillus strains was positively co-related to the number of antimicrobial genes, indicating their role in antifungal activity of Bacillus strains. Our findings suggest that combining genetic markers and LC-MS analysis can rapidly screen Potential Bacillus strains with antifungal attributes and this screening method can serve as foundation for the development of new biopesticides. [ABSTRACT FROM AUTHOR]

This study revealed that iturin A‐like lipopeptides produced by Bacillus subtillis induced both paraptosis and apoptosis in heterogeneous human epithelial colorectal adenocarcinoma (Caco‐2) cells. Autophagy was simultaneously induced in Caco‐2 cells treated with iturin A‐like lipopeptides at the early stage and inhibited at the later stage. A western blot analysis showed that the lipopeptides induced apoptosis in Caco‐2 cells via a mitochondrial‐dependent pathway, as indicated by upregulated expression of the apoptotic genes bax and bad and downregulated expression of the antiapoptotic gene bcl‐2. The induction of paraptosis in Caco‐2 cells was indicated by the occurrence of many cytoplasmic vacuoles accompanied by endoplasmic reticulum (ER) dilatation and mitochondrial swelling and dysfunction. ER stress also occurred with significant increases in reactive oxygen species and Ca2+ levels in cells. Autophagy was detected by a transmission electron microscopy analysis and by upregulated expression of LC3‐II and downregulated expression of LC3‐I. The inhibition of autophagy at the later stage was shown by upregulated expression of p62. This study revealed the capability of iturin A‐like B. subtilis lipopeptides to simultaneously execute antitumor potential via multiple pathways. Endoplasmic reticulum stress caused by Iturin A‐like lipopeptides human epithelial colorectal adenocarcinoma (Caco‐2) cells the appeared reaction, resulting in the increase of intracellular calcium and swelling and dysfunction of mitochondria. A large number of vacuoles appeared in the cell, and the parabed apoptosis occurred. Reactive oxygen species (ROS) was released in mitochondria, DNA was damaged and mitochondria permeability increased. Then the endogenous apoptosis pathway is triggered. A large number of damaged organelles and proteins in the cell lead to the activation of autophagy pathway. [ABSTRACT FROM AUTHOR]

Verticillium dahliae is one of the most important soil pathogens, causing verticillium wilt. It is well known that the use of chemical products against this pathogen is not without side effects on the environment. In this regard, the present study was aimed to search for antagonistic rhizobacteria as an alternative of biological control against this causal agent. A total of 162 isolates were screened for their antagonistic activity according to the "double layer" and the "well diffusion" methods. Three of them (RS11, SF82 and ZO4), were subsequently selected as biological control agent (BCAs) according to their efficiency and were identified by 16S rRNA sequencing and Biolog microplate GEN III as Bacillus spp. Using 10 different lipopeptide gene primers, PCR reactions only revealed the involvement of genes responsible for iturins (ituA, ituD, ituC), bacillomycin (bmyA) and Bacilysin (bacA / B-F, bacA / B-R) biosynthesis. The Plant Growth Promoting Rhizobacteria traits [enzymatic activities, phytohormones production] of the three BCAs were also studied in vitro then on pepper plant (Capsicum annuum L.), indicating that Bacillus subtilis ZO4 was the most effective, enhancing leaf, stem and root growth comparing to the control. [ABSTRACT FROM AUTHOR]

Genome shuffling is an effective method for the rapid improvement of the production of secondary metabolites. This study used the principle of gene shuffling to enhance the yield of iturin A produced by Bacillus amyloliquefaciens LZ-5. Improvements in lipopeptide yield were evident among four strains subjected to recursive protoplast fusion. The four strains were obtained through mutagenesis processes: nitrosoguanidine, ultraviolet irradiation, and atmospheric and room temperature plasma. A high yield strain with 179.22 mg/l of iturin A was obtained after two rounds of genome shuffling, which was a 2.03-fold increase compared with the wild strain. To evaluate the efficacy of iturin A for control of spoilage yeast in food, the anti-yeast efficacy of iturin A was evaluated in orange juice incubated with Saccharomyces cerevisiae. The juice treated with 0.76 mg/ml iturin A showed a significant (p < 0.05) control on yeast population during the storage, similar to that of the 0.30 mg/ml natamycin. In addition, iturin A showed a tiny effect on chemical-physical characteristics of orange juice. Our results provide a basis for the application of antimicrobial lipopeptide in juice products. [ABSTRACT FROM AUTHOR]

Abstract A strain of bacteria in the Bacillus subtilis species complex was isolated from a building's air vent in the Washington DC area, USA, and produced strong antifungal activity with in vitro assays. This strain, designated (HU Biol-II), showed pronounced inhibitory effects on mycelial growth of a wide spectrum of fungi. The objectives of this study were to use genome sequencing to confirm the taxonomy of HU Biol-II, evaluate its antifungal activity and implement genome mining and HPLC-MS/MS to characterize the bioactive secondary metabolites. The strain, as determined by multilocus sequence alignment analysis, was identified as a member of Bacillus subtilis subsp. inaquosorum clade. Core genome phylogeny showed that the isolate is most closely related to B. subtilis subsp. inaquosorum strain DE111, a commercially produced human probiotic. The investigation identified eight bioactive metabolite clusters in the genome. HPLC MS/MS was able to confirm the production of seven of the metabolites. This study is the first to report the production of two antifungal cyclic lipopeptides (bacillomycin F and fengycin) from a member of B. subtilis subsp. inaquosorum. The strain also produced the antibacterial aurantinin B, which confirms the biosynthetic cluster responsible for its production. Comparative genomics and metabolomics demonstrated the commercial probiotic strain DE111 produced the same metabolites, with the exception of aurantinin B. These findings are the first description of the secondary metabolites produced by a strain of B. subtilis subsp. inaquosorum. [ABSTRACT FROM AUTHOR]

Surfactin, a lipopeptide produced by Bacillus species, has been used for the oral delivery of insulin. In this study, another lipopeptide of iturin was tested for its ability to orally delivery insulin alone or plus surfactin. Iturin could form co-precipitate with insulin at acidic pH values. After treatment by ultrasonification, the structure of coprecipitate was destroyed that led to a significant decrease in hypoglycemic effect after oral administration. Iturin weakly binds to (Kd = 257 μM) and induce insulin structure more compact that is favorable for insulin uptake by the intestine. After being coated with Acryl-Eze by lyophilization, the coprecipitate formed the spherical enteric-coated insulin microparticles delivered by iturin with a relative oral bioavailability of 6.84% in diabetic mice. For further improving oral hypoglycemic effect, surfactin was added to form the spherical enteric-coated insulin microparticles in a formulation containing insulin, Acryl-Eze, iturin and surfactin at a ratio of 1:1:0.5: 0.5 (w/w), with an insulin encapsulation efficiency of 66.22%. The enteric-coated insulin microparticles delivered by iturin plus surfactin showed a classical profile for controlled release in the intestine with a relative bioavailability of 7.67% after oral administration, which could effectively control the postprandial blood glucose at a level about 50% of the initial one just like the subcutaneous injection. Collectively, iturin plus surfactin is more efficient for oral delivering insulin than the sole one, and the resultant enteric-coated insulin microparticles are potential for the development of oral insulin to control postprandial blood glucose in diabetic patients. [ABSTRACT FROM AUTHOR]

The rapid and accurate quantification of lipopeptide families in biological samples are challenging. We present the development and validation of a method for simultaneous quantification of three families of lipopeptides (iturins, fengycins, and surfactins) and their isoforms, as well as the homologous series. The method was optimized in UPLC-MS for a column temperature at 65 °C, injection volume of 5 μL, and sample temperature of 10 °C. The SIM mode was used for detection and quantification of lipopeptides exhibiting ions [M + H]+ and [M + 2H]2+. Since the maximum mass detection threshold of the equipment is 1250 Da and the fengycins have ions between 1435 and 1505 Da, the ions [M + 2H]2+ were chosen for fengycin identification. The monitored ions were as follows: m/z 1043.5, 1057.5, 1071.5, 718.3, 725.4, 739.4, 732.4, 746.4, 753.4, 1008.6, 1022.6, and 1036.6. The compounds were separated by reverse-phase chromatography using a C18 analytical column in a total time of 19 min. Standard curves were linear with rw 0.99 for all analytes. Intra- and inter-day precision for samples (50, 250, and 750 μg L−1) were within recommended limits. The proposed analytical method was capable of simultaneously quantifying 12 isoforms and homologous series of lipopeptide families in biological samples, thus making it an important industrial tool in the evaluation of lipopeptide production processes.ᅟ [ABSTRACT FROM AUTHOR]

Abstract Biosurfactants are deemed as green replacements of synthetic surfactants. Lipopeptides, a class of biosurfactants, have excellent surface active properties along with anti-microbial and anti-tumor activities. Identifying microorganisms producing high titre of lipopeptide is of great importance in reducing production cost as currently lipopeptide production is not commercially competitive. A quantitative colorimetric assay method was developed for detecting lipopeptides and high-throughput screening of lipopeptide-producing strains by mixing bromothymol blue solution (pH 7.2) with lipopeptide-containing samples resulting in an instantaneous and stable color change. This color change is quantified spectrophotometrically at 410 nm and 616 nm showing linearly quantitative response to increased concentrations of 3 major lipopeptides (surfactin, iturin and fengycin) at 0–1 g/l. The assay was used on both cell-free broth and crude extracts of lipopeptide-producing strains and the titre obtained from the assay was supported by HPLC data. This assay can be used for screening lipopeptide-producing strains and quantitatively detecting lipopeptides either in purified form or in cell-free broth. Graphical abstract fx1 Highlights • A very simple method for rapid detection of biosurfactants. • No pre-incubation of samples is required. • Suitable for quantitative assay of lipopeptides and other surfactants. • Suitable for high throughput screening of biosurfactant-producing strains. [ABSTRACT FROM AUTHOR]

A strong antibiotic producer, Bacillus subtilis strain Bbv 57 (KF718836), has been utilized for the management of wilt-nematode complex (Fusarium oxysporum f. sp. gerberae, Meloidogyne incognita) in gerbera under greenhouse conditions in the Department of Floriculture, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India. The strain strongly inhibited F. oxysporum f. sp. gerberae (KM523669) mycelial growth to an extent of 44.33 and 63.33%, at 10 and 100 µl, of culture filtrate, respectively. Further, the culture filtrate at 100% concentration exerted lethal effect on nematode eggs (7.00 hatched) and juveniles (87% mortality) compared to control. The analysis of TLC revealed that Bbv 57 showed the cyclic antimicrobial peptides surfactin and iturin that were confirmed by PCR. Strain Bbv 57 was able to produce antifungal and anti-nematicidal activity with reduced wilt incidence (15.33%) and thus holds a great potential for use in the biocontrol of Fusarium wilt-root-knot nematode disease complex in gerbera under greenhouse conditions. [ABSTRACT FROM AUTHOR]

The aim of the study was to identify and characterize lipopeptide (LP) biosurfactants produced by two Bacillus subtilis strains (KP7 and I′-1a) grown on various media prepared from renewable natural resources: two different brewery wastewaters (BW#4 and BW#6), 2% beet molasses (M), apple peels extract (APE) supplemented with 0.25% of yeast extract (YE) or 0.25% peptone (P), and similarly supplemented carrot peels extract (CPE). In all used media both strains retained their individual LP production signature characterized by surfactin and iturin overproduction exhibited by KP7 and I′-1a strain, respectively. The production level and the structural diversity of synthesized LPs were dependent on the medium composition. In the CPE+YE medium it was higher than the yield obtained in Luria-Bertani (140.6 and 100.3 mg L −1 , respectively). Surfactins were produced by both strains as a mixture of four homologues (C13-C16) with the domination of variant C14. All other broths prepared from renewable resources strongly stimulated the iturin production by I′-1a strain with the exception of BW media. The highest iturin concentration (428.7 mg L −1 ) obtained in the CPE+P culture of I′-1a strain was about seven-fold higher than in LB. In all cultures only iturin A was identified. Among four iturin homologues (C13-16) produced by I′-1a strain, the highest relative contents of C16 variant (70–80%) were calculated for samples obtained from APE+P and CPE+P media. The obtained data indicate that the waste composition has an influence on both the types and amounts of biosurfactants produced by studied B. subtilis strains. [ABSTRACT FROM AUTHOR]

Six Bacillus subtilis strains collected from broad-leaved trees rhizosphere and exhibiting an ability of surfactin production were investigated as potent plant biofertilizers with biocontrol activity. Lipopeptide (LP) biosurfactants were extracted by a modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) procedure proposed as a quick and effective method of isolating LP biosurfactants. Matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF-TOF) was applied to detect the lipopeptides. Predominant peaks at m / z 1016, 1030, 1044, 1058 and 1072 were putatively assigned as surfactin Na adducts possess a Glu-Leu-Leu-Val-Asp-Leu–Leu peptide and the β-OH fatty acid with a different length (C13-C16) whereas m / z 1016 was C13 surfactin Na adduct with Val in position 7 on the peptide moiety. Four other ions m / z 1051, 1065, 1079 and 1093 were recognized as iturin A Na adducts. Five fengycin homologues with Na adducts ( m / z 1471, 1485, 1499, 1513 and 1527) were also determined. The quantitative analyses of the compounds were performed with the use of liquid chromatography-mass spectrometry (LC–MS/MS). Strains IM 7, IM 16 and IM 17 cultivated in Luria-Bertani medium for 48 h produced surfactin alone at the concentrations below 10 mg l −1 . In contrast, strains KP7, IM 13 and IM 14 produced surfactin much more efficiently. Surfactin concentrations in 72-h-old cultures reached 100.3, 55.3 and 124.4 mg l −1 , respectively and were accompanied by the production of iturin in concentrations 5.7, 142.5 and 2.3 mg l −1 , respectively. Additionally, in Cooper’s medium, strain KP7 increased twofold the production of surfactin and iturin. Only surfactin overproducers (KP7, IM 13 and IM 14) exhibited swarming motility and formed biofilms, limited the growth of Botrytis cinerea and Aspergillus flavus strains, produced cellulases and inulinases and enhanced cucumber seed germination in vitro. All examined surfactin overproducers produced siderophores and, additionally, KP7 and IM 14 solubilized phosphates. Among all examined surfactin overproducers only IM 13 during the growth in the presence of F. oxysporum mycelium (in dual liquid culture) increased about twofold surfactin and iturin secretion and almost completely suppressed fungal biomass growth. The obtained results revealed that the studied surfactin overproducers were equipped with various plant growth-promoting (PGP) traits allowing them to promote plant development in a diverse manner. [ABSTRACT FROM AUTHOR]

Post-harvest diseases are responsible for significant losses worldwide, especially the plant pathogenic fungi Botrytis cinerea and Colletotrichum sp. are particularly severe and devastating. In this study, nine fungal pathogens were screened for growth inhibition by Bacillus subtilis EA-CB0015 strain and its metabolites. In vitro inhibitory assays showed that B. subtilis EA-CB0015 cells and the cell free supernatant (CFS) inhibited the growth of the tested fungal pathogens with different susceptibilities. Therefore, the antifungal activities of lipopeptides iturin A and fengycin C contained in the CFS, were tested against C. acutatum and B. cinerea . C. acutatum was more susceptible with minimal inhibitory concentrations (MIC) of 32 ppm (iturin A) and 128 ppm (fengycin C). Fruit and flower trials confirmed that B. subtilis EA-CB0015 cells and its lipopeptides reduced postharvest disease development but to differing degrees. Anthracnose symptoms caused by C. acutatum in tamarillo fruits were completely abolished by CFS, iturin A and fengycin C and reduced by 76% when treated with B. subtilis cells. In contrast, grey mold disease symptoms caused by B. cinerea in chrysanthemum flowers were inhibited by 72% when treated with lipopeptides and by 39% when applied with B. subtilis EA-CB0015 cells. Our results indicate that lipopeptides and cells of B. subtilis EA-CB0015 have a broad antifungal spectrum and control postharvest diseases caused by susceptible fungal pathogens. Our findings open the possibility of incorporating this biological control agent into different disease management programs. [ABSTRACT FROM AUTHOR]

[Display omitted] • A combined chemical-bacterial process was developed. • Hydrolysate from pepper straw was directly used by Cas02 without detoxification. • Extract with strong antifungal activity achieved by macroporous resin purification. • 1.58 g iturin-rich extract containing 259.22 mg/g iturin was obtained from 100 g straw. • Pepper straw is a promising feedstock for iturin production by B. amyloliquefaciens. A combined chemical-bacterial process was developed to convert vegetable straw waste to high value antifungal iturins. Straws from three widely cultivated vegetable (cucumber, tomato and pepper) were evaluated as feedstocks for iturin production. Microwave assisted hydrolysis with very dilute acid (0.2% w/w H 2 SO 4) achieved efficient reducing sugar recovery. The high glucose concentration in non-detoxified hydrolysate from pepper straw facilitated the optimal growth of Bacillus amyloliquefaciens strain Cas02 and stimulated the production of iturin. The fermentation parameters were optimised to enhance the iturin production efficiency. The obtained fermentation extract was further purified using macroporous adsorption resin, resulting in an iturin-rich extract that exhibited strong antifungal activity against Alternaria alternata with an IC50 of 176.44 μg/mL. Each iturin homologue was identified using NMR. Overall, 1.58 g iturin-rich extract containing 164.06 mg/g iturins was obtained from 100 g pepper straw, illustrating the great potential of valorising pepper straw via this process. [ABSTRACT FROM AUTHOR]