Production Of Beauvericin Research Articles

Mechanistic insight into human androgen receptor-mediated endocrine disrupting potential of cyclic depsipeptide mycotoxin, beauvericin, and influencing environmental factors for its biosynthesis in Fusarium oxysporum KFCC 11363P on rice cereal

In current study, Fusarium mycotoxin, beauvericin (BEA), has endocrine disrupting potential through suppressing the exogenous androgen receptor (AR)-mediated transcriptional activation. BEA was classified as an AR antagonist, with IC30 and IC50 values indicating that it suppressed AR dimerization in the cytosol. BEA suppress the translocation of cytosolic activated ARs to the nucleus via exogenous androgens. Furthermore, we investigated the impact of environmental conditions for BEA production on rice cereal using response surface methodology. The environmental factors affecting the production of BEA, namely temperature, initial moisture content, and growth time were optimized at 20.28 °C, 42.79 % (w/w), and 17.31 days, respectively. To the best of our knowledge, this is the first report showing that BEA has endocrine disrupting potential through suppressing translocation of cytosolic ARs to nucleus, and temperature, initial moisture content, and growth time are important influencing environmental factors for its biosynthesis in Fusarium strains on cereal.

Nematicidal effect of Beauveria species and the mycotoxin beauvericin against pinewood nematode Bursaphelenchus xylophilus

Introduction and main objectiveBursaphelenchus xylophilus, commonly known as pine wood nematode (PWN), is considered one of the greatest threats to European and Asian pines. Regarding its management, most efforts have been directed toward control measures for the major vector (Monochamus spp.) and screening for genetic resistance in its hosts. However, an integrated pest management strategy which also implements pinewood nematode control is currently lacking. The aim of this study was to evaluate the nematicidal effect of two Beauveria species, a genus well known for its entomopathogenic activity.Summary methodologyFor this purpose, in vitro antagonism tests of fungi (Beauveria bassiana and B. pseudobassiana) and the mycotoxin beauvericin (C45H57N3O9) on B. xylophilus populations were conducted. Finally, the production of beauvericin in B. bassiana and B. pseudobassiana strains was quantified by high-performance liquid chromatography - mass spectrometry (HPLC-MS).Results and discussionBoth the B. bassiana and B. pseudobassiana fungal species and the mycotoxin beauvericin showed a clear nematicidal effect on B. xylophilus populations, substantially reducing their survival rate and even attaining 100% mortality in one case. HPLC-MS analysis confirmed and quantified the production of beauvericin by B. bassiana and demonstrated for the first-time beauvericin production in B. pseudobassiana.Final conclusionThese findings highlight the potential of Beauveria species and the mycotoxin beauvericin to be implemented in an integrated pest management strategy to control both nematode and vector.

An explanation of the mystifying bakanae disease narrative for tomorrow's rice.

Rice production is severely hampered by the bakanae disease (Fusarium fujikuroi), formerly recognized as Fusarium moniliforme. F. moniliforme was called the F. fujikuroi species complex (FFSC) because it was later discovered that it had some separate species. The FFSC's constituents are also well recognized for producing phytohormones, which include auxins, cytokinin, and gibberellins (GAs). The normal symptoms of bakanae disease in rice are exacerbated by GAs. The members of the FFSC are responsible for the production of fumonisin (FUM), fusarins, fusaric acid, moniliformin, and beauvericin. These are harmful to both human and animal health. This disease is common around the world and causes significant yield losses. Numerous secondary metabolites, including the plant hormone gibberellin, which causes classic bakanae symptoms, are produced by F. fujikuroi. The strategies for managing bakanae, including the utilization of host resistance, chemical compounds, biocontrol agents, natural goods, and physical approaches, have been reviewed in this study. Bakanae disease is still not entirely preventable, despite the adoption of many different tactics that have been used to manage it. The benefits and drawbacks of these diverse approaches are discussed by the authors. The mechanisms of action of the main fungicides as well as the strategies for resistance to them are outlined. The information compiled in this study will contribute to a better understanding of the bakanae disease and the development of a more effective management plan for it.

Improvement of beauvericin production by Fusarium oxysporum AB2 under solid-state fermentation using an optimised liquid medium and co-cultures.

The production of beauvericin (BEA) by Fusarium oxysporum AB2 in liquid medium (SmF) was compared to that on solid medium (SSF) on inert support (polyurethane foam or PUF), using a previously optimised medium. The analysis included two different concentrations of the medium (1 × and 3 ×). Under SSF, the production of BEA (22.8mg·L-1) was higher relative to SmF (0.8mg·L-1). The production increased proportionally in the concentrated medium (3 ×) (65.3mg·L-1); using the concentrated medium in SmF, the production of BEA was completely inhibited, although more biomass was produced. The peak of BEA production was reached on day 7 and remained stable until day 11; sustained production after several days has not been achieved in similar reports. The presence of BEA was corroborated by high-performance liquid chromatography (HPLC) and mass spectrometry. The BEA production profile is shown performing mixed cultures of Fusarium oxysporum AB2 and Epicoccum nigrum TORT using the same system, increasing the production of BEA up to 84.6mg·L-1. We propose SSF using polyurethane foam (PUF) as a solid support as a new culture system for obtaining secondary metabolites such as BEA.

The regulation of BbLaeA on the production of beauvericin and bassiatin in Beauveria bassiana.

Beauvericin and bassiatin are two valuable compounds with various bioactivities biosynthesized by the supposedly same nonribosomal peptide synthetase BbBEAS in entomopathogenic fungus Beauveria bassiana. To evaluate the regulatory effect of global regulator LaeA on their production, we constructed BbLaeA gene deletion and overexpression mutants, respectively. Deletion of BbLaeA resulted in a decrease of the beauvericin titer, while overexpression of BbLaeA increased its production by 1-2.26 times. No bassiatin could be detected in ΔBbLaeA and wild type strain of B. bassiana, but 4.26-5.10µg/mL bassiatin was produced in OE::BbLaeA. Furthermore, additional metabolites with increased production in OE::BbLaeA were isolated and identified as primary metabolites. Among them, 4-hydroxyphenylacetic acid showed antibacterial bioactivity against Ralstonia solanacearum. These results indicated that BbLaeA positively regulates the production of beauvericin, bassiatin and various bioactive primary metabolites.

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.

Identification and Production of Beauvericin by Fusarium subglutinansand F. sacchari from Sugarcane

HIGHLIGHTS Isolation two species of pathogens Fusarium from sugarcane. Identification the difference in morphological characterization between two species of Fusarium. Determination the ability of pathogens Fusarium to produce mycotoxin Beauvericin. Determination of the toxicity Beauvericin on the brine shrimp.

Divergence of Beauvericin Synthase Gene among Fusarium and Trichoderma Species.

Beauvericin (BEA) is a cyclodepsipeptide mycotoxin, showing insecticidal, antibiotic and antimicrobial activities, as well as inducing apoptosis of cancer cell lines. BEA can be produced by multiple fungal species, including saprotrophs, plant, insect and human pathogens, particularly belonging to Fusarium, Beauveria and Isaria genera. The ability of Trichoderma species to produce BEA was until now uncertain. Biosynthesis of BEA is governed by a non-ribosomal peptide synthase (NRPS), known as beauvericin synthase (BEAS), which appears to present considerable divergence among different fungal species. In the present study we compared the production of beauvericin among Fusarium and Trichoderma strains using UPLC methods. BEAS fragments were sequenced and analyzed to examine the level of the gene’s divergence between these two genera and confirm the presence of active BEAS copy in Trichoderma. Seventeen strains of twelve species were studied and phylogenetic analysis showed distinctive grouping of Fusarium and Trichoderma strains. The highest producers of beauvericin were F. proliferatum and F. nygamai. Trichoderma strains of three species (T. atroviride, T. viride, T. koningiopsis) were minor BEA producers. The study showed beauvericin production by Fusarium and Trichoderma species and high variance of the non-ribosomal peptide synthase gene among fungal species from the Hypocreales order.

Application of untargeted tandem mass spectrometry with molecular networking for detection of enniatins and beauvericins from complex samples

An untargeted LC-MS/MS-based molecular networking method was established for the automatic determination of variants of enniatin and beauvericin from both fungal cultures and naturally contaminated samples. Using this method, a large number of samples can be efficiently analyzed for the presence of enniatin- and beauvericin-related compounds. As proof of concept, 26 cultures, derived from 13 fungal strains in the genera of Fusarium, Beauveria, and Diaporthe, as well as 46 food samples were analyzed. Four enniatin- and three beauvericin-producing fungi were newly discovered. Among them, the production of beauvericin by Fusarium sp. 190-20-2 was further confirmed by the presence of a beauvericin biosynthesis gene cluster in its genomic sequence. Additionally, 17 enniatin congeners, including one new isomer of enniatin A, and three previously unreported bassianolide analogues were detected from an enniatin-producing fungus, Fusarium sp. 17-048, and a beauvericin-producing fungus, Beauveria sp. 186-069, respectively. The structures of the detected compounds were tentatively determined by a series of product ions of their sodium adducts. The new isomer of enniatin A was further confirmed by NMR spectra. A preliminary survey of food samples showed that enniatins were prevalent in the tested wheat flour and noodle samples, whereas beauvericin was only discovered in cornflour powder samples.

Metabolic profiling of Fusarium oxysporum f. sp. conglutinans race 2 in dual cultures with biocontrol agents Bacillus amyloliquefaciens, Pseudomonas aeruginosa, and Trichoderma harzianum

There are increasing efforts to identify biocontrol-active microbial metabolites in order to improve strategies for biocontrol of phytopathogens. In this work, Fusarium oxysporum f. sp. conglutinans was confronted with three different biocontrol agents: Trichoderma harzianum, Bacillus amyloliquefaciens, and Pseudomonas aeruginosa in dual culture bioassays. Metabolites produced during the microbial interactions were screened by a matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). T. harzianum exhibited the strongest inhibition of growth of F. oxysporum resulting in overlay of the pathogen colony with its mycelium. Recorded metabolite profiles suggested a direct attack of F. oxysporum mycelium by T. harzianum and B. amyloliquefaciens by means of membrane-attacking peptaibols and a set of antimicrobial lipopeptides and siderophores, respectively. The direct mode of the biocontrol activity of T. harzianum and B. amyloliquefaciens corresponded to their ability to suppress F. oxysporum production of mycotoxin beauvericin suggesting that this ability is not specific only for Trichoderma species. In the case of P. aeruginosa, siderophores pyoverdine E/D and two rhamnolipids were produced as major bacterial metabolites; the rhamnolipid production was blocked by F. oxysporum. The results showed that this type of biocontrol activity was the least effective against F. oxysporum. The effective application of MALDI-MS profiling to the screening of nonvolatile microbial metabolites produced during the interaction of the phytopathogen and the biocontrol microorganisms was demonstrated.

Pharmacological and Therapeutic Potential of Beauvericin: A Short Review

An entomopathogenic fungus, Beauveria spp. has been known to have numerous pharmacological and therapeutic implications, especially, in terms of human health making it a suitable for ethnopharmacological use. Beauvericin is a cyclic hexadepsipeptide mycotoxin, a novel bio-metabolite derived from this fungus, exhibiting a very potent anticancer, cytotoxic activities, antiplatelet aggregation and antimicrobial activities. The current review discusses the therapeutic potential of beauvericin including pharmacological and biological activities which will certainly draw the attention of scientific community to improve the production of beauvericin for its use in medical fields.

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.

Paraconiothyrium variabile, an ascomycete endophyte, suppresses mycotoxin production in the plant pathogen Fusarium oxysporum

Fungal plant pathogens produce more than 70% of all major crop diseases and destroy 15% of global agricultural production through yield losses and mycotoxin contamination, making them by far the most harmful class of plant pathogens. Fusarium is a cosmopolitan genus of filamentous ascomycete fungi that includes many toxin-producing plant pathogens of agricultural importance [1]. Mycotoxin contamination of agricultural products makes them unsuitable for food or feed. As phytopathogens, fusaria employ a broad range of infection strategies and host specificity varies among Fusarium species. The ubiquitous soil-borne F. oxysporum causes vascular wilt disease in over 100 different plant species and it is also an emerging pathogen of humans that can cause invasive infections in immunocompromised patients [2]. Research in our laboratory focuses on secondary metabolites produced by endophytic fungi, which live in plants without provoking disease symptoms. We showed that the endophyte Paraconiothyrium variabile, when co-cultured with F. oxysporum, has an antagonistic growth effect on the phytopathogen and actively suppresses the production of beauvericin, a mycotoxin of F. oxysporum involved in virulence [3]. Our study also showed that the oxylipin 13-oxo-9,11-octadecadienoic acid (13-oxo-ODE) is overproduced during this antagonistic interaction and is responsible for over 50% of beauvericin suppression. The genome sequencing of the endophyte P. variabile is in progress and we have already established a genetic transformation system in this fungus. Using molecular approaches, we are currently trying to elucidate the biosynthesis of the 13-oxo-ODE oxylipin in P. variabile and its mode of action on beauvericin regulation in F. oxysporum.

Mitogen-activated protein kinases are associated with the regulation of physiological traits and virulence in Fusarium oxysporum f. sp. cubense.

Fusarium oxysporum f. sp. cubense (FOC) is an important soil-borne fungal pathogen causing devastating vascular wilt disease of banana plants and has become a great concern threatening banana production worldwide. However, little information is known about the molecular mechanisms that govern the expression of virulence determinants of this important fungal pathogen. In this study, we showed that null mutation of three mitogen-activated protein (MAP) kinase genes, designated as FoSlt2, FoMkk2 and FoBck1, respectively, led to substantial attenuation in fungal virulence on banana plants. Transcriptional analysis revealed that the MAP kinase signaling pathway plays a key role in regulation of the genes encoding production of chitin, peroxidase, beauvericin and fusaric acid. Biochemical analysis further confirmed the essential role of MAP kinases in modulating the production of fusaric acid, which was a crucial phytotoxin in accelerating development of Fusarium wilt symptoms in banana plants. Additionally, we found that the MAP kinase FoSlt2 was required for siderophore biosynthesis under iron-depletion conditions. Moreover, disruption of the MAP kinase genes resulted in abnormal hypha and increased sensitivity to Congo Red, Calcofluor White and H2O2. Taken together, these results depict the critical roles of MAP kinases in regulation of FOC physiology and virulence.

Cloning and characterization of the gene cluster required for beauvericin biosynthesis in Fusarium proliferatum

Beauvericin, a cyclohexadepsipeptide-possessing natural product with synergistic antifungal, insecticidal, and cytotoxic activities. We isolated and characterized the fpBeas gene cluster, devoted to beauvericin biosynthesis, from the filamentous fungus Fusarium proliferatum LF061. Targeted inactivation of the F. proliferatum genomic copy of fpBeas abolished the production of beauvericin. Comparative sequence analysis of the FpBEAS showed 74% similarity with the BbBEAS that synthesizes the cyclic trimeric ester beauvericin in Beauveria bassiana, which assembles N-methyl-dipeptidol monomer intermediates by the programmed iterative use of the nonribosomal peptide synthetase modules. Differences between the organization of the beauvericin loci in F. proliferaturm and B. bassiana revealed the mechanism for high production of beauvericin in F. proliferatum. Our work provides new insights into beauvericin biosynthesis, and may lead to beauvericin overproduction and creation of new analogs via synthetic biology approaches.

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.

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.

Optimization of a liquid medium for beauvericin production in Fusarium redolens Dzf2 mycelial culture

Beauvericin (BEA) is a proven and potent antibiotic compound useful for bio-control and a potential antifungal and anticancer agent for human. This study was to evaluate and optimize the nutrient medium for BEA production in mycelial liquid culture of a high BEA-producing fungus Fusarium redolens Dzf2 isolated from a medicinal plant. Among various organic and inorganic carbon and nitrogen sources, glucose and peptone were found the most favorable for the F. redolens Dzf2 mycelial growth and BEA production. Through a Plackett-Burman screening test on a basal medium, glucose, peptone, and medium pH were identified as the significant factors for mycelial growth and BEA production. These factors were optimized through central composite design of experiments and response surface methodology, as 49.0 g/L glucose, 13.0 g/L peptone and pH 6.6, yielding 198 mg/L BEA (versus 156 mg/L in the basal medium). The BEA yield was further increased to 234 mg/L by feeding 10 g/L glucose to the culture during exponential phase. The results show that F. redolens Dzf2 mycelial fermentation is a feasible and promising process for production of BEA.

Identification, mycotoxin risk and pathogenicity of Fusarium species associated with fig endosepsis in Apulia, Italy

In a survey carried out on 87 rotted fig fruits samples collected in the Apulia region of Italy, the authors isolated 126 Fusarium strains identified as F. ramigenum (69 strains), F. solani (49), F. proliferatum (five) and three not identified. Investigation on the fertility of the strains belonging to F. proliferatum and F. ramigenum revealed that only strains of F. proliferatum were fertile. The identity of F. ramigenum strains was confirmed by sequencing a portion of the translation elongation factor-1α gene. When Fusarium species were analysed for their toxigenicity, 37/69 strains of F. ramigenum produced fusaric acid (FA) up to 525 mg kg−1; 30 strains produced beauvericin (BEA) up to 190 mg kg−1; 60 strains produced fumonisin B1 (FB1) and fumonisin B2 (FB2) up to 1575 mg kg−1 of total FBs; and two strains produced fusaproliferin (FUP) up to 345 mg kg−1; all five strains of F. proliferatum produced FA at low levels; two strains produced BEA up to 205 mg kg−1; one strain produced FB1 and FB2, 1100 and 470 mg kg−1, respectively; and one strain produced FUP, 820 mg kg−1; F. solani (30 strains) produced FA, 13 strains up to 215 mg kg−1. Few fungal extracts showed high toxicity toward brine shrimp larvae and in some cases in relation to BEA and FA content. A pathogenic assay on fig fruits showed that all three species were pathogenic, with higher virulence of F. ramigenum. These data report for the first time the production of BEA and FB1/FB2 by F. ramigenum and show that it is a main agent of fig endosepsis in Apulia and can contribute to fumonisin contamination of fresh and dried figs.

Production of beauvericin by a high-yield strain Fusarium sp. F-1 in static cultivations

Production of beauvericin by a high-yield strain Fusarium sp. F-1 in static cultivations