Sequence divergence of the enniatin synthase gene in relation to production of beauvericin and enniatins in Fusarium species.
ABSTRACT: 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-1? 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.
Project description:Members of the fungal genus Fusarium can produce numerous secondary metabolites, including the nonribosomal mycotoxins beauvericin (BEA) and enniatins (ENNs). Both mycotoxins are synthesized by the multifunctional enzyme enniatin synthetase (ESYN1) that contains both peptide synthetase and S-adenosyl-l-methionine-dependent N-methyltransferase activities. Several Fusarium species can produce ENNs, BEA or both, but the mechanism(s) enabling these differential metabolic profiles is unknown. In this study, we analyzed the primary structure of ESYN1 by sequencing esyn1 transcripts from different Fusarium species. We measured ENNs and BEA production by ultra-performance liquid chromatography coupled with photodiode array and Acquity QDa mass detector (UPLC-PDA-QDa) analyses. We predicted protein structures, compared the predictions by multivariate analysis methods and found a striking correlation between BEA/ENN-producing profiles and ESYN1 three-dimensional structures. Structural differences in the ? strand's Asn789-Ala793 and His797-Asp802 portions of the amino acid adenylation domain can be used to distinguish BEA/ENN-producing Fusarium isolates from those that produce only ENN.
Project description:Totals of 158 corn and corn-based samples and 291 wheat and wheat-based samples from Shandong province, China in 2017 were analyzed for five mycotoxins including beauvericin (BEA), enniatin A (ENA), enniatin A? (ENA?), enniatin B (ENB), and enniatin B? (ENB?) by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). BEA was the predominant toxin detected, followed by ENB, ENA?, ENA, and ENB?. Corn and corn-based samples were more easily contaminated by BEA with an average concentration of 65.26 µg/kg, compared with that in wheat and wheat-based samples (average = 0.41 µg/kg). Concentrations of BEA, ENA, and ENB? in corn kernels, flours, and flakes were significantly different (Kruskal?Wallis Test, <i>p</i> < 0.05), as well as for BEA, ENA, ENB, and ENB? in wheat kernels, flours, and noodles (Kruskal?Wallis test, <i>p</i> < 0.05). Furthermore, 59.5% (94/158) and 59.8% (174/291) corn- and wheat-based samples were co-contaminated by at least two mycotoxins, respectively. Positive correlations in concentrations were observed in corn between levels of ENA and ENB?, ENA and ENB, ENA? and ENB?, as well as in wheat between BEA and ENA, BEA and ENA?, BEA and ENB, BEA and ENB?, ENA and ENA?, ENA and ENB, ENA and ENB?, ENA? and ENB, ENA? and ENB?, and ENB and ENB?. These results demonstrate that co-contamination of BEA and enniatins (ENNs) in corn- and wheat-based samples from Shandong, China is very common. More data on the contamination of five mycotoxins in cereal and cereal-based samples nationwide are needed.
Project description:Fungi from the Hypocreales order synthesize a range of toxic non-ribosomal cyclic peptides with antimicrobial, insecticidal and cytotoxic activities. Entomopathogenic Beauveria, Isaria and Cordyceps as well as phytopathogenic Fusarium spp. are known producers of beauvericins (BEAs), beauvenniatins (BEAEs) or enniatins (ENNs). The compounds are synthesized by beauvericin/enniatin synthase (BEAS/ESYN1), which shows significant sequence divergence among Hypocreales members. We investigated ENN, BEA and BEAE production among entomopathogenic (Beauveria, Cordyceps, Isaria) and phytopathogenic (Fusarium) fungi; BEA and ENNs were quantified using an LC-MS/MS method. Phylogenetic analysis of partial sequences of putative BEAS/ESYN1 amplicons was also made. Nineteen fungal strains were identified based on sequence analysis of amplified ITS and tef-1? regions. BEA was produced by all investigated fungi, with F. proliferatum and F. concentricum being the most efficient producers. ENNs were synthesized mostly by F. acuminatum, F. avenaceum and C. confragosa. The phylogeny reconstruction suggests that ancestral BEA biosynthesis independently diverged into biosynthesis of other compounds. The divergent positioning of three Fusarium isolates raises the possibility of parallel acquisition of cyclic depsipeptide synthases in ancient complexes within Fusarium genus. Different fungi have independently evolved NRPS genes involved in depsipeptide biosynthesis, with functional adaptation towards biosynthesis of overlapping yet diversified metabolite profiles.
Project description:A total of 470 edible vegetable oil samples including peanut, soybean, rapeseed, sesame seed, corn, blend, and others collected from eight provinces of China were analyzed for the concentrations of beauvericin (BEA), enniatin A (ENA), A1 (ENA1), B (ENB), and B1 (ENB1) by ultraperformance liquid chromatography/electrospray ionization tandem mass spectrometry (UPLC/ESI-MS/MS). Concentrations of BEA, ENB, and ENB1 (average = 5.59 μg/kg, 5.16 μg/kg, and 4.61 μg/kg) in all positive samples were higher than those for ENA and ENA1 (average = 0.85 μg/kg and 1.88 μg/kg). Frequencies of BEA and ENNs in all analyzed samples were all higher than 50% with the exception of ENA1 (36.6%, 172/470). Levels of BEA and ENNs in all analyzed samples varied based on their sample types and geographical distributions (Kruskal–Wallis test, p < 0.05). The soybean and peanut oil samples were found to be more easily contaminated by BEA and ENNs than other oil samples. Concentrations of BEA and ENNs in samples obtained from Heilongjiang, Shandong and Guizhou were higher than those found in samples from other provinces. Besides, frequencies of mycotoxin co-contaminations were high and their co-contamination types also varied by oil types. BEA-ENA-ENA1-ENB-ENB1 was the most commonly found toxin combination type, almost in one third of the analyzed samples (30%, 141/470). Overall, these results indicate that co-occurrence of BEA and ENNs in analyzed Chinese edible vegetable oil samples is highly common, and it is vital to monitor them, both simultaneously and on a widespread level.
Project description:Mycotoxins are secondary metabolites produced by a variety of fungi that contaminate food and feed resources, and are capable of inducing a wide range of toxicity. Here, we studied the developmental and behavioral toxicity in zebrafish (Danio rerio) embryos and larvae exposed to three mycotoxins: beauvericin (BEA), Enniatin A (ENN A), and Ennitain B (ENN B). Zebrafish embryos were collected after fertilization, treated individually from 1 to 6 dpf with BEA at 8, 16, 32 and, 64 μM and for both enniatins at 3.12, 6.25, 12.5 and, 25 μM. Mixture of mycotoxins were assayed as follows: i) for BEA + ENN A and BEA + ENN B at [32 + 12.5] μM and [16 + 6.25] μM; ii) for ENN A + ENN B at [12.5 + 12.5] μM and [6.25 + 6.25] μM and, iii) for BEA + ENN A + ENN B at [32 + 12.5 + 12.5] μM and [16 + 6.25 + 6.25] μM. Response was collected after a white light-flash intermittent coming on for 5 s during 2 h with a imaging platform. Outcomes measured were: time to death, response to light, and circadian rhythm. This last outcome was measured in a plate where embryos had evolved in natural intervals of light and dark until day 7 or in a plate maintained in darkness. Images of all stages and evolution were collected. Results indicated that mycotoxins induced toxicity at the concentrations tested. All exposed zebrafish induced developmental defects, specifically hatching time and motion activity. After exposure, fish showed enhanced baseline activity but they lost their responsiveness to light.
Project description: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.
Project description:Emerging mycotoxins such as moniliformin (MON), enniatins (ENs), beauvericin (BEA), and fusaproliferin (FUS) may contaminate maize and negatively influence the yield and quality of grain. The aim of this study was to determine the content of emerging Fusarium mycotoxins in Serbian maize from the 2016, 2017, and 2018 harvests. A total of 190 samples from commercial maize production operations in Serbia were analyzed for the presence of MON, ENs, BEA, and FUS using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The obtained results were interpreted together with weather data from each year. MON, BEA, and FUS were major contaminants, while other emerging mycotoxins were not detected or were found in fewer samples (<20%). Overall contamination was highest in 2016 when MON and BEA were found in 50-80% of samples. In 2017 and 2018, high levels of MON, FUS, and BEA were detected in regions with high precipitation and warm weather during the silking phase of maize (July and the beginning of August), when the plants are most susceptible to Fusarium infections. Since environmental conditions in Serbia are favorable for the occurrence of mycotoxigenic fungi, monitoring Fusarium toxins is essential for the production of safe food and feed.
Project description:Enniatins are cyclohexadepsipeptides isolated largely from Fusarium species of fungi, although they have been isolated from other genera, such as Verticillium and Halosarpheia. They were first described over 60 years ago, and their range of biological activities, including antiinsectan, antifungal, antibiotic and cytotoxic, drives contemporary interest. To date, 29 enniatins have been isolated and characterized, either as a single compound or mixtures of inseparable homologs. Structurally, these depsipeptides are biosynthesized by a multifunctional enzyme, termed enniatin synthetase, and are composed of six residues that alternate between N-methyl amino acids and hydroxy acids. Their structure elucidation can be challenging, particularly for enniatins isolated as inseparable homologs; however, several strategies and tools have been utilized to solve these problems. Currently, there is one drug that has been developed from a mixture of enniatins, fusafungine, which is used as a topical treatment of upper respiratory tract infections by oral and/or nasal inhalation. Given the range of biological activities observed for this class of compounds, research on enniatins will likely continue. This review strives to digest the past studies, as well as, describe tools and techniques that can be utilized to overcome the challenges associated with the structure elucidation of mixtures of enniatin homologs.
Project description: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 <i>Fusarium avenaceum</i>. Notably, 0.5 mM of exogenous ferulic, caffeic, and <i>p</i>-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 <i>F. avenaceum</i> 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.
Project description:In 2017-2018, extensive symptoms of sudden decline and fruit rot were observed on date palms in southern Tunisia. Samples of diseased plants were randomly collected in six localities. Based on morphological identification, <i>Fusarium</i> was the most frequent fungal genus detected. A sequencing of translation elongation factor, calmodulin, and second largest subunit of RNA polymerase II genes was used to identify 63 representative <i>Fusarium</i> strains at species level and investigate their phylogenetic relationships. The main species detected was <i>Fusarium</i> <i>proliferatum</i>, and at a much lesser extent, <i>Fusarium</i> <i>brachygibbosum</i>, <i>Fusarium</i> <i>caatingaense</i>, <i>Fusarium</i> <i>clavum</i>, <i>Fusarium</i> <i>incarnatum</i><i>,</i> and <i>Fusarium</i> <i>solani</i>. Pathogenicity on the <i>Deglet</i> <i>Nour</i> variety plantlets and the capability to produce mycotoxins were also assessed. All <i>Fusarium</i> species were pathogenic complying Koch's postulates. <i>Fusarium</i> <i>proliferatum</i> strains produced mainly fumonisins (FBs), beauvericin (BEA), and, to a lesser extent, enniatins (ENNs) and moniliformin (MON). All <i>F.</i> <i>brachygibbosum</i> strains produced low levels of BEA, diacetoxyscirpenol, and neosolaniol; two strains produced also T-2 toxin, and a single strain produced HT-2 toxin. <i>Fusarium</i> <i>caatingaense</i>, <i>F.</i> <i>clavum</i>, <i>F.</i> <i>incarnatum</i> produced only BEA. <i>Fusarium</i> <i>solani</i> strains produced MON, BEA, and ENNs. This work reports for the first time a comprehensive multidisciplinary study of <i>Fusarium</i> species on date palms, concerning both phytopathological and food safety issues.