Production Of Enniatins Research Articles

Encapsulation of Ammoides pusila Essential Oil into Mesoporous Silica Particles for the Enhancement of Their Activity against Fusarium avenaceum and Its Enniatins Production

Essential oils (EOs) that have antifungal activity and mycotoxin reduction ability are candidates to develop bioactive alternatives and environmentally friendly treatment against Fusarium species in cereals. However, their practical use is facing limitations such as high volatility, UV sensitivity, and fast oxidation. Encapsulation techniques are supposed to provide protection to the EOs and control their release into the environment. Ammoides pusilla essential oil (AP-EO) proved to be an efficient inhibitor of Fusarium avenaceum growth and its enniatins (ENNs) production. In the present work, AP-EO was encapsulated, using the impregnation method, into mesoporous silica particles (MSPs) with narrow slit pores (average diameter = 3.1 nm) and coated with chitosan. In contact assays using an agar medium, the antifungal activity of AP-EO at 0.1 µL mL-1 improved by three times when encapsulated into MSPs without chitosan and the ENNs production was significantly inhibited both in coated and non-coated MSPs. Controls of MSPs also inhibited the ENNs production without affecting the mycelial growth. In fumigation experiments assessing the activity of the EO volatile compounds, encapsulation into MSPs improved significantly both the antifungal activity and ENNs inhibition. Moreover, coating with chitosan stopped the release of EO. Thus, encapsulation of an EO into MSPs improving its antifungal and antimycotoxin properties is a promising tool for the formulation of a natural fungicide that could be used in the agriculture or food industry to protect plant or food products from the contamination by toxigenic fungi such as Fusarium sp. and their potential mycotoxins.

Inhibition of the Growth of Fusarium tricinctum and Reduction of Its Enniatin Production by Erwinia gerundensis Isolated from Barley Kernels

Fusarium tricinctum is among the pathogenic fungi that contaminate cereal crops, including barley. It produces toxic metabolites such as enniatins (ENNs) that affect the quality and safety of barley-based products such as malt and beer, thus representing a significant concern for the cereal industries. Therefore, it is critical to reduce Fusarium spp. development and enniatin contamination in cereal-based products. Fungicides are commonly used during agricultural practices, but they are facing growing fungal resistance and environmental and health concerns. Hence, there is need to define environmentally friendly control strategies. This study highlights the ability of a bacterial strain isolated from barley kernels, namely Erwinia gerundensis 6E4, to inhibit the growth of Fusarium tricinctum and to reduce its toxigenic potential. First, E. gerundensis 6E4 exhibited a significant fungistatic activity against pathogenic fungal strains during co-cultures, with fungal growth reduced by up to 100% on Petri dishes and by about 69% in liquid medium. The bacterial strain also led to a drastic reduction (74 to 100%) of ENNs production by F. tricinctum strains over 72 h co-cultures. Based on these results, our study supports the use of E. gerundensis 6E4 as a biocontrol agent in strategies aiming at reducing the presence of Fusarium tricinctum and enniatins in cereal-based products, or as a food and feed supplement for the bio-detoxification of enniatins.

Ammoides pusilla Essential Oil: A Potent Inhibitor of the Growth of Fusarium avenaceum and Its Enniatin Production.

Enniatins are mycotoxins produced by Fusarium species contaminating cereals and various agricultural commodities. The co-occurrence of these mycotoxins in large quantities with other mycotoxins such as trichothecenes and the possible synergies in toxicity could lead to serious food safety problems. Using the agar dilution method, Ammoides pusilla was selected among eight Tunisian plants for the antifungal potential of its essential oil (EO) on Fusarium avenaceum mycelial growth and its production of enniatins. Two EO batches were produced and analyzed by GC/MS-MS. Their activities were measured using both contact assays and fumigant tests (estimated IC50 were 0.1 µL·mL−1 and 7.6 µL·L−1, respectively). The A. pusilla EOs and their volatiles inhibited the germination of spores and the mycelial growth, showing a fungistatic but not fungicidal activity. The accumulation of enniatins was also significantly reduced (estimated IC50 were 0.05 µL·mL−1 for the contact assays and 4.2 µL·L−1 for the fumigation assays). The most active batch of EO was richer in thymol, the main volatile compound found. Thymol used as fumigant showed a potent fungistatic activity but not a significant antimycotoxigenic activity. Overall, our data demonstrated the bioactivity of A. pusilla EO and its high potential to control F. avenaceum and its enniatins production in agricultural commodities.

Phylogeny and mycotoxin profile of Fusarium species isolated from sugarcane in Southern Iran

Sugarcane is an important crop in Southern Iran for agri-food, energy, and pharmaceutical industries. Among the pathogens that colonize sugarcane, mycotoxigenic Fusarium species are reason of serious concern for both their pathogenicity on plants and ability to produce harmful mycotoxins to humans and animals. We studied 104 Fusarium strains, selected within a wider Fusarium set isolated from sugarcane in Southern Iran, for molecular identification, phylogeny and mycotoxin analyses. Most of Fusarium strains belonged to Fusarium fujikuroi Species Complex (FFSC) and identified mainly as F. proliferatum, at minor extent as F. sacchari, and rarely as F. thapsinum, and F. verticillioides. Moreover, 14 strains identified as FFSC could not be assigned to any known species, although they were phylogenetically closely related to F. andiyazi, likely representing a new phylogenetic species. A subset of FFSC strains were analyzed for in vitro production of fumonisins (FBs), beauvericin (BEA), and enniatins (ENNs). Fusarium proliferatum strains produced FBs at high amount, and, at a lesser extent, BEA, and ENNs; F.sacchari produced only BEA and B ENNs at very low level; Fusarium sp. strains produced only B ENNs. The paper provides new insights on the genetic diversity of Fusarium species and their mycotoxin profile occurring on sugarcane in Iran.

Investigating the Efficiency of Hydroxycinnamic Acids to Inhibit the Production of Enniatins by Fusarium avenaceum and Modulate the Expression of Enniatins Biosynthetic Genes.

Enniatins (ENNs) that belong to the group of emerging mycotoxins are widespread contaminants of agricultural commodities. There is currently insufficient evidence to rule out health concerns associated with long-term exposure to ENNs and efforts must be strengthened to define a control strategy. While the potential of plant compounds to counteract the contamination with legislated mycotoxins has been reported, little remains known regarding ENNs. The present study evidenced for the first time the efficiency of hydroxycinnamic acids to inhibit the fungal growth and ENNs yield by Fusarium avenaceum. Notably, 0.5 mM of exogenous ferulic, caffeic, and p-coumaric acids led to a drastic reduction of ENNs synthesis in pH4 broths, with ferulic acid being the most potent. The ENNs production inhibitory activity of ferulic acid was shown to be associated with a significant down-regulation of the expression of ENNs biosynthetic genes. To further investigate the bioactivity of ferulic acid, its metabolic fate was characterized in fungal broths and the capacity of F. avenaceum to metabolize it through a C2-cleavage type degradation was demonstrated. Overall, our data support the promising use of ferulic acid in ENNs control strategies, either as part of an environmentally friendly plant-care product or as a biomarker of plant resistance.

Five‐year survey uncovers extensive diversity and temporal fluctuations among fusarium head blight pathogens of wheat and barley in Brazil

AbstractWe conducted a five‐year survey (2011–2015) of barley and wheat fields in Paraná state, Brazil, obtaining 754 Fusarium isolates from spikes with fusarium head blight (FHB)‐symptoms. Multilocus genotyping and TEF‐1α gene sequence analyses confirmed the dominance of the F. graminearum species complex (FGSC, 75.7%), but F. poae (11.5%), as well as F. avenaceum and related members of the F. tricinctum species complex (FTSC, 8.1%) appeared as substantial contributors to FHB. Within the FGSC, F. graminearum of the 15‐ADON genotype was dominant (63%), followed by F. meridionale of the NIV genotype (23.1%), F. cortaderiae of the NIV (7%) or 3‐ADON (2.6%) genotypes, and F. austroamericanum (3.8%) of the 3‐ADON genotype. Substantial variation in pathogen composition was observed across years, with F. poae and F. meridionale frequencies significantly elevated in some years. Most F. poae strains produced DAS, diANIV, and butenolide, but not neosolaniol, T‐2, or HT‐2. All FTSC species produced moniliformin. Enniatin production was widespread among FTSC species, with the single F. acuminatum strain found to be the strongest producer of enniatins. Our findings confirm FGSC as a major contributor to FHB and expand considerably our knowledge of the presence, frequency, and conditions under which other pathogens may emerge, altering the spectrum of toxins that may accumulate in grain.

Enniatin Production Influences Fusarium avenaceum Virulence on Potato Tubers, but not on Durum Wheat or Peas.

Fusarium avenaceum is a generalist pathogen responsible for diseases in numerous crop species. The fungus produces a series of mycotoxins including the cyclohexadepsipeptide enniatins. Mycotoxins can be pathogenicity and virulence factors in various plant–pathogen interactions, and enniatins have been shown to influence aggressiveness on potato tubers. To determine the role of these mycotoxins in other F. avenaceum–host interactions, ENNIATIN SYNTHASE 1 (ESYN1) disruption and overexpression mutants were generated and their ability to infect wheat and peas investigated. As a preliminary study, the transformants were screened for their ability to cause potato tuber necrosis and, consistent with a previous report, enniatin production increased necrotic lesion size on the tubers. By contrast, when the same mutants were assessed in their ability to cause disease in pea roots or durum wheat spikes, no changes in disease symptoms or virulence were observed. While it is known that, at least in the case of wheat, exogenously applied enniatins can cause tissue necrosis, this group of mycotoxins does not appear to be a key factor on its own in disease development on peas or durum wheat.

Gaseous allyl isothiocyanate to inhibit the production of aflatoxins, beauvericin and enniatins by Aspergillus parasiticus and Fusarium poae in wheat flour

There is a growing concern about the presence of mycotoxins in foods, since up to 25% of cereals and cereal foods are contaminated with these compounds. Moreover, the general public is against the use of synthetic preservatives in foods and the use of natural antimicrobials in foods is a current trend. Allyl isothiocyanate (AITC) is a volatile antimicrobial derived from oriental and black mustard. The objective of this work was to evaluate the capacity of gaseous AITC in inhibiting the production of mycotoxins by Aspergillus parasiticus (aflatoxin producer) and Fusarium poae (beauvercin and enniatin producer) in wheat flour. Petri dish lids filled with 2 g of wheat flour were inoculated with 104 conidia/g of A. parasiticus or F. poae and placed in a 1 L mason jar. AITC was added at 0.1, 1 or 10 µL/L in the gaseous phase. Jars were hermetically closed and kept at 23 °C for 30 d. Mycotoxins were identified and quantified by LC-MS/MS. Even 0.1 µL/L of AITC was able to produce 6.9–23% reduction of mycotoxin production. In general, synthesis of aflatoxins and beauvericin was more affected than enniatins. The use of AITC at 10 µL/L completely inhibited the production of all mycotoxins for 30 d. AITC at low doses could be added to flour packages in order to inhibit the production of potentially dangerous mycotoxins.

OPTIMIZATION OF ENNIATINS PRODUCTION BY AN ENDOPHYTIC STRAIN FUSARIUM DIMERUM

The goal of this study was to find suitable composition of cultivation media for enniatin production by isolated endophytic strain Fusarium dimerum. In order to find optimal cultivation media, mono- and di- saccharides, complex nitrogen sources and L-amino acids directed biosynthesis of enniatins were tested. Submerged cultivation experiments were carried out in cultivation flasks. Most promising medium for enniatin accumulation contained fructose, malt extract and peptone for bacteriology. Finally, quite expensive carbon source fructose was replaced by more available syrups. Optimization resulted in 4-times elevated enniatin biosynthesis by metabolites production microorganism. Moreover, this is the strain obtained from Magnolia soulangeana, which has similar metabolites spectrum as the isolated Fusarium dimerum. Comparison of these results with published ones revealed that this endophyte is a potential strain for enniatins biosynthesis in submerged cultivation in which the maximum accumulation 1.27 g.L-1 of enniatin in culture medium was reached in a short period (96 h). The results proved that the endophytic strain F. dimerum may potentially be applied for efficient production of bioactive enniatins.

Transcriptional regulation of enniatins production by Fusarium avenaceum

The aim of this study was to analyse the transcriptional regulation of enniatins (ENs) production in Fusarium avenaceum. We develop a new method to quantify ENs in FDM agar medium. We performed an LC/MS/MS analysis to evaluate enniatin A, A1, B, B1 and B4 production by seven F. avenaceum strains and, in a time-course experiment, by ITEM 3404 to analyse the transcriptional regulation of the esyn1 gene. The expression profile, achieved by Real time reverse transcriptase assay, showed an activation of gene transcription at the seventh day of incubation, corresponding to the higher increase of total ENs production. Enniatin B was the most abundant ENs analogues, representing the 90% of total ENs. The relative percentage of ENs remained unaltered during the experiment. We reported a transcriptional regulation of esyn1 responsible for the modulation of ENs biosynthesis. Enniatins are cyclic depsipeptides metabolites with a wide range of biological activities. They are also widespread contaminants in grains and cereals due to infection by enniatin-producing Fusarium species. This is the first article describing the transcriptional regulation of esyn1 gene that modulates ENs production in Fusarium avenaceum and provides new knowledge about the molecular mechanism underlying the biosynthesis of these important fungal metabolites in this toxigenic fungal species.

Optimization of Enniatin Production by Solid-Phase Fermentation of Fusarium tricinctum

Enniatins are cyclic depsipeptides produced by fungi of the genus Fusarium that are of interest due to their pronounced biological activities; especially enniatins A, A1, B, and B1 possess anticarcinogenic and anti-HIV properties. In the present study, F. tricinctum was grown on seven solid media and in one liquid growth medium with or without addition of peptone or of amino acid precursors in order to identify favourable media with simple cultivation conditions for maximum enniatin production. Additionally, the optimal duration of growth was investigated for the highest yields of enniatins. From the different media analysed, white beans (Phaseolus vulgaris, solid medium) induced the highest accumulation of enniatins A, A1, B, and B1, that reached a maximum of 1,365 mg total enniatins in 1 L growth medium after 18 days of fermentation. Fermentation of F. tricinctum on white beans gave the highest yield of enniatins compared to all other media analysed in this study.

Sequence divergence of the enniatin synthase gene in relation to production of beauvericin and enniatins in Fusarium species.

Beauvericin (BEA) and enniatins (ENNs) are cyclic peptide mycotoxins produced by a wide range of fungal species, including pathogenic Fusaria. Amounts of BEA and ENNs were quantified in individual rice cultures of 58 Fusarium strains belonging to 20 species, originating from different host plant species and different geographical localities. The species identification of all strains was done on the basis of the tef-1α gene sequence. The main aim of this study was to analyze the variability of the esyn1 gene encoding the enniatin synthase, the essential enzyme of this metabolic pathway, among the BEA- and ENNs-producing genotypes. The phylogenetic analysis based on the partial sequence of the esyn1 gene clearly discriminates species producing exclusively BEA from those synthesizing mainly enniatin analogues.

Optimization of Enniatin Production by Solid-Phase Fermentation of Fusarium tricinctum

Enniatins are cyclic depsipeptides produced by fungi of the genus Fusarium that are of interest due to their pronounced biological activities; especially enniatins A, A1, B, and B1 possess anticarcinogenic and anti-HIV properties. In the present study, F. tricinctum was grown on seven solid media and in one liquid growth medium with or without addition of peptone or of amino acid precursors in order to identify favourable media with simple cultivation conditions for maximum enniatin production. Additionally, the optimal duration of growth was investigated for the highest yields of enniatins. From the different media analysed, white beans (Phaseolus vulgaris, solid medium) induced the highest accumulation of enniatins A, A1, B, and B1, that reached a maximum of 1,365 mg total enniatins in 1 L growth medium after 18 days of fermentation. Fermentation of F. tricinctum on white beans gave the highest yield of enniatins compared to all other media analysed in this study.

Production of enniatins A, A1, B, B1, B4, J1 by Fusarium tricinctum in solid corn culture: Structural analysis and effects on mitochondrial respiration

Enniatins (ENs) are secondary fungal metabolites with hexadepsipeptidic chemical structure and they possess a number of potent biological activities that can contaminate several kind of food and foodstuffs increasing the exposure risk for consumers. ENs are produced by several Fusariun strains including Fusarium subglutinans, Fusarium proliferatum and Fusarium tricinctum. Production of a mixture of ENs was performed by culturing F. tricinctum ITEM 9496 on white corn as substrate. The solid culture components were dried and subsequently extracted with water/methanol (50/50 v/v, 0.5% NaCl), homogenised, filtered, extracted by ethyl acetate and analysed by liquid chromatography with diode array detection (LC-DAD). The crude extract was first separated by low pressure liquid chromatography (LPLC) and then further purified by liquid chromatography (LC), resulting in six compounds with a purity higher than 95% as calculated by 1H NMR, and with a yield of 30–300mg per compound. The chemical structures of the ENs were determined by liquid chromatography coupled to mass spectrometry (LC–MS) and nuclear magnetic resonance (NMR). The biological activity of the resulting ENs was determined using a mitochondrial respiration test. We discovered that all the ENs studied induced an increase in the mitochondrial respiration resulting in uncoupling of the oxidative phosphorylation. This effect was most likely due to flux of K+ ions into the mitochondrial matrix. The order of potency of the ENs derivatives was: A1>B1>B>A>B4>J1. These results suggest a correlation between the chemical structures and bioactivity and confirm the severe risks for human associated with consumption of enniatins.

Effects of 14C-labelled precursor feeding on production of beauvericin, enniatins H, I, and MK1688 by Fusarium oxysporum KFCC11363P

The effects of ¹⁴C-labelled precursor feeding on the production of cyclic hexadepsipeptides were investigated by the mycelium of F. oxysporum KFCC11363P producing beauvericin along with enniatins H, I, and MK1688. Most ¹⁴C-phenylalanine and ¹⁴C-valine were incorporated easily into the biosynthetic pathway of ¹⁴C-labelled beauvericin in vivo by the mycelium. However, only a small amount of ¹⁴C-labelled enniatins could be detected by feeding of ¹⁴C-valine. When L-valine was fed as a precursor to the mycelium at large scale, the level of beauvericin increased to maximum followed by enniatins H and I. Feeding of L-valine did not affect the production of enniatin MK1688. These results suggest that L-valine feeding led to the production of D-hydroxyisovaleic acid in the mycelium and specifically enhanced the production of cyclic hexadepsipeptides containing D-hydroxyisovaleic acid, such as beauvericin and enniatins H and I.

Statistical optimization of culture conditions for the production of enniatins H, I, and MK1688 by Fusarium oxysporum KFCC 11363P

The aim of this study was to optimize the culture conditions for the production of biological cyclic hexadepsipeptides (enniatins H, I and MK1688) from Fusarium oxysporum KFCC 11363P. Tests of 10 complete or chemically defined liquid culture media revealed that Fusarium defined medium was the best for the production of enniatins (produced amounts: enniatin H, 185.4 mg/L; enniatin I, 349.1mg/L; enniatin MK1688, 541.1mg/L; and total enniatins, 1075.6 mg/L). On the eighth day after inoculation, the maximal production of enniatins was observed at 25°C in Fusarium defined medium. The optimal carbon and nitrogen sources for producing biological cyclic hexadepsipeptides (enniatins H, I, and MK1688) were sucrose and NaNO(3), respectively, and their optimal concentrations were determined by the principle of response surface methodology. It was confirmed that using the optimized growth medium compositions increased the amounts of enniatins H, I, and MK1688, and total enniatins produced to 695.2, 882.4, 824.8, and 2398.5mg/L, respectively. These findings will assist in formulating microbiological media useful for enniatin research.

Structural analysis of enniatin H, I, and MK1688 and beauvericin by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and their production by Fusarium oxysporum KFCC 11363P

The molecular structures of enniatins H, I, and MK1688 and beauvericin were investigated by liquid chromatography-tandem mass spectrometry (LC-MS/MS). MS fragmentation occurred by loss of -CO after opening of the cyclic molecule to carbonyl carbon, and cleavage of the peptide and ester bonds in the molecular structure. Fusarium oxysporum KFCC 11363P was tested for its ability to produce beauvericin and enniatins H, I, and MK1688 on five cereal substrates: rice, barley, maize, wheat, and Indian millet kernels. Furthermore, optimal conditions for the production of the four mycotoxins by the Fusarium isolate were examined on maize at four temperatures (15, 20, 25, and 30°C) and at three moisture contents (10, 20, and 40%). Large amounts of beauvericin and enniatin H were present in maize cultures at 25°C (232.4 and 196.4 µg g−1, respectively). Enniatins I and MK1688 were maximally formed at 20°C (221.5 and 180.2 µg g−1, respectively). The optimal moisture contents for the production of enniatins H (196.4 µg g−1) and MK1688 (165.6 µg g−1), were 40%.

Further data on the production of beauvericin, enniatins and fusaproliferin and toxicity to Artemia salina by Fusarium species of Gibberella fujikuroi species complex

The knowledge of toxigenic profiles of fungal plant pathogens is of extreme importance for evaluating the potential toxicity of infected plant products. Ninety-six fungal isolates belonging to 28 species in the Gibberella fujikuroi complex were studied for the production of beauvericin, enniatins and fusaproliferin in rice cultures. Toxin production ranged from 5 to 3000 μg/g for beauvericin, 2 to 131 μg/g for enniatins, and 4 to 440 μg/g for fusaproliferin. Beauvericin was the most common metabolite produced by 16 species followed by fusaproliferin with 11 species and enniatins with 4 species. The production of beauvericin by F. bulbicola, F. denticulatum, F. lactis, F. phyllophilum, F. pseudocircinatum, and F. succisae and fusaproliferin by F. antophilum, F. begoniae, F. bulbicola, F. circinatum, F. concentricum, F. succisae, and F. udum is reported here for the first time. Brine shrimp larvae were most sensitive to culture extracts of F. acutatum (up to 94 ± 3%) , F. concentricum (up to 99 ± 1%), F. denticuatum (up to 100%) and F. sacchari (up to 100%) . Toxicity towards brine shrimp was significantly correlated with the beauvericin content of the fungal extracts with few exceptions. These data indicate that beauvericin and fusaproliferin are common metabolites of species of the G. fujikuroi complex and pose a risk for a possible toxin accumulation in their respective host plant products. However, data from the brine shrimp bioassay showed that further toxic metabolites within this complex need to be characterized.

Enniatin Production by the Entomopathogenic Fungus Verticillium hemipterigenum BCC 1449.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Diversity in metabolite production by Fusarium langsethiae, Fusarium poae, and Fusarium sporotrichioides

The production of mycotoxins and other metabolites by 109 strains of Fusarium langsethiae, Fusarium poae, Fusarium sporotrichioides, and F. kyushuense was investigated independently in four laboratories by liquid or gas chromatography analyses of cultural extracts with UV diode array, electron capture, or mass spectrometric detection systems. From the compiled results, it was found that F. langsethiae consistently produced the trichothecenes diacetoxyscirpenol (DAS), T-2 toxin (T-2), HT-2 toxin (HT-2), and neosolaniol (NEO) and, to a lesser extent, some additional trichothecene derivatives. F. langsethiae also produced culmorins, chrysogine (CHRYS), aurofusarin (AUF), and enniatin (EN). F. sporotrichioides showed a metabolite profile similar to that of F. langsethiae, while F. poae had a different profile as 41 of 49 strains produced nivalenol (NIV) and other 8-keto trichothecenes, in addition to DAS and derivatives of this metabolite. Only a trace amount of NIV was detected from one strain of F. kyushuense. In summary, all the three core taxa of this joint study were found to produce trichothecenes. Fusarin C (F-C) was not detected from F. langsethiae, but it was produced by F. poae and F. sporotrichioides. Aurofusarin was only detected from a few strains of F. langsethiae, while nearly all strains of F. poae and F. sporotrichioides produced this compound. In contrast, chrysogine was not detected from F. poae, but was produced by the other two taxa. Production of enniatins was scattered among the three main taxa of this study, whereas beauvericin (BEA) was produced by many strains of F. poae and F. sporotrichioides. Only one odd strain of F. langsethiae (IBT 9959) produced beauvericin. However, the status of this strain is uncertain. By a polyphasic approach using species-specific metabolite profiles, the fruity odour of F. poae, and morphological observations, it was concluded that F. langsethiae, F. poae, and F. sporotrichioides should be regarded as three significant taxa at a species level.