Enhanced control efficacy of Bacillus subtilis NM4 via integration of chlorothalonil on potato early blight caused by Alternaria solani.

Early blight caused by Alternaria solani is a common foliar disease of potato around the world, and serious infections result in reduced yields and marketability due to infected tubers. The major aim of this study is to figure out the synergistic effect between microorganism and fungicides and to evaluate the effectiveness of Bacillus subtilis NM4 in the control of early blight in potato. Based on its colonial morphology and a 16S rRNA analysis, a bacterial antagonist isolated from kimchi was identified as B. subtilis NM4 and it has strong antifungal and anti-oomycete activity against several phytopathogenic fungi and oomycetes. The culture filtrate of strain NM4 with the fungicide effectively suppressed the mycelial growth of A. solani, with the highest growth inhibition rate of 83.48%. Although exposure to culture filtrate prompted hyphal alterations in A. solani, including bulging, combining it with the fungicide caused more severe hyphal damage with continuous bulging. Surfactins and fengycins, two lipopeptide groups, were isolated and identified as the main compounds in two fractions using LC-ESI-MS. Although the surfactin-containing fraction failed to inhibit growth, the fengycin-containing fraction, alone and in combination with chlorothalonil, restricted mycelial development, producing severe hyphal deformations with formation of chlamydospores. A pot experiment combining strain NM4, applied as a broth culture, with fungicide, at half the recommended concentration, resulted in a significant reduction in potato early blight severity. Our results indicate the feasibility of an integrated approach for the management of early blight in potato that can reduce fungicide application rates, promoting a healthy ecosystem in agriculture.

Microbial biosurfactants for oil spill remediation: pitfalls and potentials.

Spillage of fossil-based oils during their conveyance through water conduits are sporadic, but significant environmental disasters. As the viscous hydrocarbons of the crude oils spread on water surface and choke aquatic life to death, their effective degradation is crucial for ecological balance. Though chemical and mechanical means are conventional ways to tackle the issues, they are riddled with limitations. In this scenario, coercing the biosurfactant-producing bacteria and fungi are promising avenues. Biosurfactants, the amphiphilic compounds, are capable of reducing interfacial tension, dispersing the oil particles, and degrading them into non-toxic debris. Among the vast array of biosurfactants, the trio of rhamnolipid, sophorolipid, and surfactin have been characterized well. Among the microbes, only Pseudomonas, Bacillus, and Candida have been evaluated, while there can be other exploitable candidates. In this regard, this review discusses the scopes and hurdles in utilization of the microbial surface-active compounds for oil spill management.

Antibacterial, anti-adherent and cytotoxic activities of surfactin(s) from a lipolytic strain Bacillus safensis F4.

The bacterial strain F4, isolated from olive oil-contaminated soil, has been found to produce biosurfactants as confirmed by oil displacement test and the emulsification index results. The identification of the strain F4, by 16S ribosomal RNA gene, showed a close similarity to Bacillus safensis, therefore the strain has been termed Bacillus safensis F4. The Thin Layer Chromatography (TLC) and the High Pressure Liquid Chromatography-Mass Spectrometry (HPLC-MS/MS) demonstrated that the biosurfactant had a lipopeptide structure and was classified as surfactin. The present study showed also that the produced biosurfactant has an important antibacterial activity against several pathogen strains as monitored with minimum inhibitory concentration (MIC) micro-assays. In particular, it presented an interesting anti-planktonic activity with a MIC of 6.25 mg mL-1 and anti-adhesive activity which exceeded 80% against the biofilm-forming Staphylococcus epidermidis S61 strain. Moreover, the produced lipopeptide showed an antitumor activity against T47D breast cancer cells and B16F10 mouse melanoma cells with IC50 of 0.66 mg mL-1 and 1.17 mg mL-1, respectively. Thus, our results demonstrated that Bacillus safensis F4 biosurfactant exhibited a polyvalent activity via a considerable antibiofilm and antitumoral potencies.

Screening of Bacillus mojavensis biofilms and biosurfactants using laser ablation electrospray ionization mass spectroscopy.

AIMS: Biofilms are composed of micro-organisms within a matrix of chemically complex polymer compounds and from these structures many unknown competitive factors are suggested that many considered are important consequences for biological control. This research was undertaken to study further the endophyte, Bacillus mojavensis and its relationships to biofilm and two classes of lipopeptides considered relevant for biocontrol of plant pathogens. METHODS AND RESULTS: Laser ablation electrospray ionization mass spectrometry and conventional MS/MS were used to study in situ biofilm production and the production of lipopeptides fengycin and surfactin in different strains of B. mojavensis in plate and test tube culture on two media. All strains were capable of producing biofilm in vitro along with the accumulation of surfactin and fengycin although no concentration-dependent relationship between lipopeptide accumulation and biofilm was observed. CONCLUSION: All strains studied produce biofilms in culture with the accumulated surfactin and fengycin, demonstrating that endophytic bacteria also produced biofilms. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates that this endophytic species produced biofilms along with two biocontrol compounds of which one, surfactin, considered by others as a quorum sensor, highlighting its ecological role as a signalling mechanism in planta.

In situ downstream strategies for cost-effective bio/surfactant recovery.

Since 60-80% of total costs of production are usually associated with downstream collection, separation, and purification processes, it has become advantageous to investigate how to replace traditional methods with efficient and cost-effective alternative techniques for recovery and purification of biosurfactants. In the traditional techniques, large volumes of organic solvents are usually used for increasing production cost and the overall environmental burden. In addition, traditional production and separation methods typically carried out in batch cultures reduce biosurfactant yields due to product inhibition and lower biosurfactants activity as a result of interaction with the organic solvents used. However, some in situ recovery methods that allow continuous separation of bioproducts from culture broth leading to an improvement in yield production and fermentation efficiency. For biosurfactants commercialization, enhancement of product capacity of the separation methods and the rate of product removal is critical. Recently, interest in the integration of separation methods with a production step as rapid and efficient techniques has been increasing. This review focuses on the technology gains and potentials for the most common methods used in in situ product removal: foam fractionation and ultrafiltration, especially used to recover and purify two well-known biosurfactants: glycolipids (rhamnolipids) and lipopeptides (surfactins).

Pumilacidins from the Octocoral-Associated Bacillus sp. DT001 Display Anti-Proliferative Effects in Plasmodium falciparum.

Chemical examination of the octocoral-associated Bacillus species (sp.) DT001 led to the isolation of pumilacidins A (1) and C (2). We investigated the effect of these compounds on the viability of Plasmodium falciparum and the mechanism of pumilacidin-induced death. The use of inhibitors of protein kinase C (PKC) and phosphoinositide 3-kinase (PI3K) was able to prevent the effects of pumilacidins A and C. The results indicated also that pumilacidins inhibit parasite growth via mitochondrial dysfunction and decreased cytosolic Ca2+.

Bacillus subtilis YZ-1 surfactins are involved in effective toxicity against agricultural pests.

BACKGROUND: Insect pests negatively affect crop quality and yield. The excessive use of chemical pesticides has serious impacts on the environment and food safety. Therefore, development of effective management strategies in the form of bio-agents have important agricultural applications. Tenebrio molitor, a storage pest, causes losses of grains, medicinal materials, and various agricultural and related products in the warehouse. Bacillus subtilis YZ-1 isolated from naturally deceased Pieris rapae has been found to exhibit significant toxicity against T. molitor. RESULTS: Treatment with B. subtilis YZ-1 fermentation broth resulted in a 90-95% mortality rate of T. molitor within 36 h post-treatment, indicating some active substances may have insecticidal activity in the bacterial supernatant. A bioactivity-guided fractionation method was used to isolate the insecticidal compounds from YZ-1, which led to the identification of surfactins. Additionally, a surfactin deletion mutant YZ-1△srfAA was constructed and the surfactin production by the mutant YZ-1△srfAA was verified through liquid chromatography-mass spectrometry (LC-MS). Further, YZ-1△srfAA exhibited loss of insecticidal activity against T. molitor, Plutella xylostella and Achelura yunnanensis. The insecticidal activity and surfactins contents of several strains of Bacillus sp. were also tested and correlation was found between varying surfactins yield and insecticidal activity exhibited by different strains. CONCLUSION: Conclusively, our results suggest that B. subtilis YZ-1 may provide a novel approach for plant protection against agricultural pests. © 2023 Society of Chemical Industry.

Molecular characterization and in silico evaluation of surfactins produced by endophytic bacteria from Phanera splendens.

The Phanera splendens (Kunth) Vaz. is a medicinal plant that is used in traditional medicine for the treatment of various diseases, such as malaria. This plant presents highly efficient endophytic bacterial isolates with biocontrol properties. Bacillus sp. is responsible for the production of a variety of non-ribosomal synthesized cyclic lipopeptides which highlight the surfactins. Surfactins have a wide range of antimicrobial activity, including antiplasmodial activity. There is scientific evidence that surfactin structure 2d-01 can be a potent inhibitor against a Plasmodium falciparum sirtuin (Sir2) by acting on the Sir2A protein as the target. The Pf genome encodes two known sirtuins, PfSir2A and PfSir2B, where PfSir2A is a regulator of asexual growth and var gene expression. Herein, we have identified six surfactins produced by endophytic bacteria and performed in silico analysis to elucidate the binding mode of surfactins at the active site of the PfSir2A enzyme. Among the characterized surfactins, 1d-02 showed the highest affinity for the PfSir2A enzyme, with binding energy values equal to -45.08 ± 6.0 and -11.95 ± 0.8 kcal/mol, using MM/GBSA and SIE methods, respectively. We hope that the information about the surfactin structures obtained in this work, as well as the potential binding affinity with an important enzyme from P. falciparum, could contribute to the design of new compounds with antimalarial activity.

GC/EI/MS and 1H NMR Metabolomics Reveal the Effect of an Olive Tree Endophytic Bacillus sp. Lipopeptide Extract on the Metabolism of Colletotrichum acutatum.

The transition to the Green Deal era requires the discovery of alternative sources of bioactivity and an in-depth understanding of their toxicity to target and non-target organisms. Endophytes have recently emerged as a source of bioactivity of high potential for applications in plant protection, used either per se as biological control agents or their metabolites as bioactive compounds. The olive tree endophytic isolate Bacillus sp. PTA13 produces an array of bioactive lipopeptides (LPs), which additionally exhibit reduced phytotoxicity, features that make them candidates for further research focusing on olive tree plant protection. Here, GC/EI/MS and 1H NMR metabolomics were employed to study the toxicity of a Bacillus sp. PTA13 LP extract on the olive tree pathogen Colletotrichum acutatum, which causes the devastating disease olive anthracnose. The discovery of resistant isolates of the pathogen to the applied fungicides makes the research on the development of improved sources of bioactivity of paramount importance. Analyses revealed that the applied extract affects the metabolism of the fungus by interfering with the biosynthesis of various metabolites and its energy production. LPs had a great impact on the aromatic amino acid metabolism, the energy equilibrium of the fungus and its fatty acid content. Additionally, the applied LPs affected the levels of pathogenesis-related metabolites, a finding that supports their potential for further research as plant protection agents.

Anti-proliferative activity of surfactins on human cancer cells and their potential use in therapeutics.

Surfactins are cyclic lipopeptides that are isolated from various Bacillus strains. They are made up of heptapeptides and ß-hydroxy fatty acids of variable chain lengths of carbon atoms. Therapeutically they are known to inhibit invasion, migration, and colony formation of human breast carcinoma cells. The role of surfactins is also known as anti-proliferative agents against human cancer cells through induction of apoptosis, arrest of the cell cycle, or suppression of survival signaling. The cytotoxic activity of surfactins is also perceived against human chronic myelogenous leukemia cells, human colon cancer cells, and hepatic carcinoma cells. Considering the wide spectrum of targets, the molecular effects of surfactins are diverse in different cancer cells and they can serve as promising chemotherapeutic agents for the treatment of cancer. Surfactins are being delivered to the targeted cancer cells through nano-carriers or nano-formulations. The present review article provides insight on different types and variations of surfactins, their molecular effect on different cancer cells, and their therapeutic use in the treatment of human cancer.

Effects of surfactins, Bacillus lipopeptides, on the behavior of an aphid and host selection by its parasitoid.

BACKGROUND: Lipopeptides extracted from the Bacillus genus are emerging biopesticides, especially in protecting crops against phytopathogens. Among the three main families of lipopeptides, surfactins have been identified as having insecticidal properties against several insect orders. However, the sublethal effects of these promising biopesticides on insect pests and their natural enemies remain largely unknown. The aim of this study was to evaluate the effects of surfactins topically applied on black bean aphid Aphis fabae mortality. First, the effects of surfactins on aphid mortality were determined by delivering increasing concentrations to adults and nymphs. Second, the sublethal effects of surfactins on locomotor activity and feeding behavior of surviving aphids were evaluated using the electropenetrography method. Finally, the effect of host exposure to surfactins on host selection behavior by Aphidius matricariae parasitoid females was analyzed. RESULTS: Four surfactins concentrations were studied (0.5, 1, 2.5 and 5 g L-1 ). There was concentration-dependent mortality in response to surfactins at 24 h after treatment. Surfactins impacted aphid behavior when delivered at 1 g L-1 by inducing a greater locomotor activity and a reduction in feeding activity. By contrast, at the third trophic level, exposure of aphid hosts to surfactins did not affect behaviors leading to host recognition and acceptance by parasitoid females. CONCLUSION: This study highlighted the consequences of aphid exposure to surfactins in the context of bottom-up regulation. Although surfactins could directly impact aphid behavior, they had no apparent consequences on the host selection behavior exhibited by parasitoid wasps.

A Metabolomic Approach to Assess the Toxicity of the Olive Tree Endophyte Bacillus sp. PTA13 Lipopeptides to the Aquatic Macrophyte Lemna minor L.

Pesticides represent a major human input into the ecosystem, posing a serious risk to non-target organisms. Therefore, there is pressure toward the reduction in their use and the discovery of alternative sources of bioactivity. Endophytic microorganisms represent a source of bioactivity, whose potential for plant protection has been recently established. In this context, an olive tree endophytic Bacillus sp. was isolated, exhibiting superior antifungal activity, mainly attributed to its major surfactin, iturin, and fengycin and the minor gageotetrin and bacilotetrin groups of lipopeptides (LP). Based on the potential of LP and the lack of information on their toxicity to aquatic organisms, we have investigated the toxicity of an LP extract to the model macrophyte Lemna minor L. The extract exhibited low phytotoxicity (EC50 = 419 µg·mL-1), and for the investigation of its effect on the plant, GC/EI/MS metabolomics was applied following exposure to sub-lethal doses (EC25 and EC50). Results revealed a general disturbance of plants' biosynthetic capacity in response to LP treatments, with substantial effect on the amino acid pool and the defense mechanism regulated by jasmonate. There are no previous reports on the phytotoxicity of LP to L. minor, with evidence supporting their improved toxicological profile and potential in plant protection.

A cross-comparison of biosurfactants as marine oil spill dispersants: Governing factors, synergetic effects and fates.

Biosurfactant-based dispersants (BBDs) may be more effective, cost-efficient and environmentally friendly than dispersants currently used for oil spill response. An improved understanding of BBD performance is needed to advance their development and commercial use. In this study, the ability of four BBDs, i.e. sufactins, trehalose lipids, rhamnolipids and exmulsins, alone and as various combinations to disperse Arabian light crude oil and weathered Alaska North Slope crude oil was compared to a widely used commercial oil dispersant (Corexit 9500A). Surfactin and trehalose lipids, which have balanced surface activity/emulsification ability, showed dispersion efficacy comparable to Corexit 9500A. Rhamnolipids (primarily a surface-active agent) and exmulsins (primarily an emulsifier) when used alone had significantly lower efficacy. However, blends of these surfactants had excellent dispersion performance because of synergistic effects. Balanced surface activity and emulsification ability may be key to formulate effective BBDs. Of the BBDs evaluated, surfactins with an effective dispersant-to-oil ratio as low as 1:62.3 and trehalose lipids with high oil affinity, biodegradation rate, and low toxicity characteristics show the most promise for commercial development.

Discovering the Next-Generation Plant Protection Products: A Proof-of-Concept via the Isolation and Bioactivity Assessment of the Olive Tree Endophyte Bacillus sp. PTA13 Lipopeptides.

Endophytic microorganisms (EMs) have recently attracted interest for applications in plant protection, mainly due to their bioactive compound-producing capacity. Therefore, we underwent the task of isolating olive tree EMs and investigating their bioactivity against the devastating pathogen Colletotrichum acutatum. Several EMs were isolated; however, the Bacillus sp. PTA13 isolate exhibited the highest toxicity to the phytopathogen. Bacteria of the genus Bacillus exhibit superior bioactive metabolite-producing capacity, with the lipopeptides (LPs) of surfactin, iturin, and fengycin groups being the most studied. A total LP extract and several fractions were obtained, and their bioactivity was assessed against C. acutatum strains. LPs of the major surfactin, iturin, and fengycin groups and the minor gageotetrin and bacilotetrin groups were annotated. The results confirmed the bioactivity of the major LPs, with fengycins being the most fungitoxic. Interestingly, the minor LP fraction exhibited selective toxicity to the fungicide-resistant C. acutatum isolate, an observation that highlights the significance of our approach to comprehensively mine the total LP extract. This work represents a proof of concept of the exploitation of EMs in customized olive tree plant protection and aligns well with strategies that focus on the sustainability and safety of food production via the development of next-generation plant protection products.

Production and characterization of surfactin-type lipopeptides as bioemulsifiers produced by a Pinctada martensii-derived Bacillus mojavensis B0621A.

Bacillus mojavensis B0621A was isolated from the mantle of a pearl oyster Pinctada martensii collected from South China Sea. Semi-purified surfactins (225 mg L(-1)) were obtained by acid precipitation and vacuum flash chromatography. The component of the semi-purified surfactins was preliminarily analyzed by liquid chromatography mass spectrometer system, and the results showed that all these surfactins could be a group of homologues. Eight surfactin homologues were isolated and afforded by reversed phase high-performance liquid chromatography. Furthermore, their structure was characterized by mass spectrometry analysis combined with nuclear magnetic resonance spectroscopy techniques. These surfactins shared seven amino acids as peptide backbone and a saturated ß-hydroxy fatty acid chain residue (from C13 to C15), differed each other from peptide sequence in the position of Leu7 or Val7. All these surfactins had significant activity and stability of emulsification under various pH (from 7.0 to 12.0), temperature range (from 20 to 115 °C) and sodium chloride concentration (from 2.5 to 20.0 %, w/v). Taken all together, these results indicated that B. mojavensis B0621A have potential to be an alternative source as a biological-derived emulsifying agent.