Zearalenone Production Research Articles

FgVelB globally regulates sexual reproduction, mycotoxin production and pathogenicity in the cereal pathogen Fusarium graminearum

The velvet genes are conserved in ascomycetous fungi and function as global regulators of differentiation and secondary metabolism. Here, we characterized one of the velvet genes, designated FgVelB, in the plant-pathogenic fungus Fusarium graminearum, which causes fusarium head blight in cereals and produces mycotoxins within plants. FgVelB-deleted (ΔFgVelB) strains produced fewer aerial mycelia with less pigmentation than those of the wild-type (WT) during vegetative growth. Under sexual development conditions, the ΔFgVelB strains produced no fruiting bodies but retained male fertility, and conidiation was threefold higher compared with the WT strain. Production of trichothecene and zearalenone was dramatically reduced compared with the WT strain. In addition, the ΔFgVelB strains were incapable of colonizing host plant tissues. Transcript analyses revealed that FgVelB was highly expressed during the sexual development stage, and may be regulated by a mitogen-activated protein kinase cascade. Microarray analysis showed that FgVelB affects regulatory pathways mediated by the mating-type loci and a G-protein alpha subunit, as well as primary and secondary metabolism. These results suggest that FgVelB has diverse biological functions, probably by acting as a member of a possible velvet protein complex, although identification of the FgVelB-FgVeA complex and the determination of its roles require further investigation.

The in vitro effects of zearalenone and T-2 toxins on Vero cells

The aim of the present study was to investigate in vitro, whether cytolethality and oxidative damage is enhanced by combination of both mycotoxins as compared to their individual effect. In our paper, we applied a tiered in vitro experimental approach in order to predict the possible health risk effects of two interactive fusarial toxins. Considering the concomitant production of zearalenone (ZEN) and T-2 toxin, it is very likely that humans and animals are always exposed to the mixture rather than to individual compounds. Our results clearly showed that cultured renal cells respond to individual (ZEN) or T-2 toxin exposure by a moderate inhibition of cell proliferation, respectively. However, when combined, they exert a more significant decrease in cell viability. Similar results were found for the investigated oxidative status endpoints. When combined, ZEN and T-2 toxin increased ROS production and heat shock protein (Hsp) 70 expression as compared to the effect of each mycotoxin taken alone. We can conclude that the mixture of ZEN and T-2 toxin increased their toxic effects. The health risk is heightened by the interactions between co-occurring mycotoxins.

Natural occurrence of zearalenone in Tunisian wheat grains

Fusarium mycotoxins are worldwide occurring in cereals and they are frequently reported in fresh or stored grains. Cereals represent a staple food for the Tunisian population; it therefore has a high social, economic and nutritional relevance. Zearalenone (ZEN) is a non-steroidal estrogenic mycotoxin produced by a variety of Fusarium fungi in temperate and warm countries. Fungi-producing ZEN contaminates corn, barley, wheat, sorghum and rice. A total of 205 samples of wheat were collected during the harvest year of 2010 from the major cropping areas in Tunisia and they were analyzed for zearalenone contamination. The aim of this study was to investigate for the first time the presence of ZEN in widely-consumed cereals in Tunisia, especially durum and tender wheat, to compare the levels of contamination by ZEN with the European norms and to suggest some factors that can promote the production of ZEN in Tunisia. To perform this study, we developed and validated in our laboratory conditions an HPLC method for quantitative analysis of ZEN in solid cereal samples. Our results showed that the incidence of ZEN contamination was 75%. The levels of contamination determined in the positive samples ranged between 3 and 560μg/kg with a mean value of 60μg/kg. These important amounts of ZEN in wheat can be attributed to the Tunisian climate, warm temperature and prolonged wetness witch are favor to Fusarium growth and mycotoxin production during the cultivation and the final ripening period of wheat grains.

A putative ABC transporter gene, ZRA1, is required for zearalenone production in Gibberella zeae

Zearalenone (ZEA) is a secondary metabolite produced by various Fusarium species and causes estrogenic disorders in humans and animals. Recent studies have identified the ZEA biosynthesis gene cluster in F. graminearum, but other genes such as transporters responsible for ZEA export have not been identified in the cluster. In this study, we performed microarray analyses from the wild-type strain with and without ZEA supplementation and ZEA-nonproducing strain zeb2 to discover other genes responsible for ZEA biosynthesis. Three putative ABC transporters were significantly down-regulated in the zeb2 and were under positive regulation of the ZEB2 gene, which functions as a transcriptional activator for ZEA production in this fungus. However, only one gene (ZRA1) was found to be up-regulated by 20-fold in the wild-type strain supplemented with ZEA, and deletion of ZRA1 resulted in reduced ZEA production. Deletions of the other two genes showed similar ZEA productions as the wild-type strain. ZRA1 localized to the plasma membrane and vacuoles indicating possible roles of ZRA1 as a transporter. This study indicated that ZRA1 is involved in ZEA production and shares a common regulatory mode with ZEA cluster genes by ZEB2.

The neem [Azadirachta indica a: juss (meliaceae)] oil reduction in the in vitro production of zearalenone by Fusarium graminearum

Zearalenone, a mycotoxin produced by fungi of the genus Fusarium, including F. graminearum, triggers reproduction disorders in certain animals and hyperestrogen syndromes in humans. Current research investigates three concentrations of neem oil extract (0.1, 0.25 and 0.5%) in reducing the production of zearalenone. Neem oil extract decreased zearalenone amount in the three concentrations but highest inhibition (59.05%) occurred at 0.1%.

Effects of infection time by Fusarium graminearum on ear blight, deoxynivalenol and zearalenone production in wheat

Although it is generally agreed that most Fusarium head blight infection takes place during the wheatflowering period, very rainy conditions at the end of the plant growth, such as occurred in France in 2007,raise the question of the possibility of late infections on the development of the disease and the production oftoxins. Such infections are not taken into account by predictive models. To explore the effect of infection timeon disease severity, Fusarium-damaged kernels (FDK), desoxynivalenol (DON) and zearalenone (ZON), anexperiment was set up in 2009 on two varieties. The experiment had a split-plot design. Plots were inoculatedby spraying Fusarium graminearum conidia at different stage, from heading to ripening. Inoculation time hada significant effect on disease severity, FDK, DON and ZON levels (P<0.0001), as well as interaction betweenvariety and inoculation time. The results of this trial have shown no significant difference between infection atheading, anthesis and post-anthesis until 15 days for disease severity, FDK and mycotoxins levels. Nevertheless, this trial has suggested different behaviour between the two varieties according to infection time andtoxin accumulation. The period of high susceptibility to Fusarium graminearum has appeared unstable between varieties, ranging from closed to flowering to a large period from heading to several days after flowering.

Evaluation of sorghum straw hemicellulosic hydrolysate for biotechnological production of xylitol by Candida guilliermondii.

Zearalenone, a mycotoxin produced by fungi of the genus Fusarium, including F. graminearum, triggers reproduction disorders in certain animals and hyperestrogen syndromes in humans. Current research investigates three concentrations of neem oil extract (0.1, 0.25 and 0.5%) in reducing the production of zearalenone. Neem oil extract decreased zearalenone amount in the three concentrations but highest inhibition (59.05%) occurred at 0.1%.

Zearalenone risk in European wheat

Zearalenone is an oestrogenic mycotoxin produced by several Fusarium species which can infect cereals, in particular wheat and maize. The predominant species responsible for zearalenone production is Fusarium graminearum, which also produces the trichothecene mycotoxin, deoxynivalenol. The infection and subsequent mycotoxin production by Fusarium spp. is largely dependent on rainfall at flowering and before harvest. High concentrations of zearalenone in particular, appear to be produced during delayed wet harvests. There has been a recent and ongoing increase in F. graminearum incidence resulting in increased occurrence of deoxynivalenol and zearalenone across northern Europe, where delayed wet harvests are more common. Zearalenone contamination of grain is largely restricted to the outer layers of wheat grain and therefore is partitioned into the bran fraction during milling. This results in higher concentrations of zearalenone in high fibre cereal products. After the delayed wet harvest in the UK in 2008, 29% of wheat at harvest exceeded the European limit for unprocessed cereals of 100 µg/kg. This resulted in difficulties in sourcing bran which would allow production of high fibre breakfast cereals within zearalenone limits and the European Commission provided a temporary derogation for high fibre breakfast cereals of 100 µg/kg zearalenone until 31 October 2009. Rainfall data and zearalenone concentrations in UK wheat from the last ten years were used to predict the occurrence of high zearalenone in wheat (10% or more of unprocessed wheat greater than 100 µg/ kg zearalenone). High zearalenone was predicted to occur one year in five for northern Europe which matches the observed incidence for the UK within the last ten years. As a consequence, current legislative limits for zearalenone in cereals and cereal products could impact on the availability of high fibre cereal products one year in five.

Solid substrate bioassay to evaluate the effect of Trichoderma and Clonostachys on the production of zearalenone by Fusarium species

Fusarium head blight and maize ear rot are destructive diseases in various cereal production regions, leading to significant yield losses and to the contamination of cereal grain with Fusarium mycotoxins. The mycotoxin zearalenone belongs to the three most important mycotoxins produced by Fusarium species worldwide; moreover, its hormonal oestrogenic activity is higher than its toxicity. The compound also exhibits fungitoxic activity. Toxigenic Fusarium species sporulate on cereal crop residues and produce inoculum to infect the next crop. Reduction of mycotoxin production and pathogen sporulation may be influenced by saprophytic fungal antagonists. Selected Trichoderma and Clonostachys isolates in dual culture bioassays on rice reduced zearalenone, α-zearalenol and zearalenone sulphate production by two isolates of Fusarium graminearum Schwabe and two isolates of F. culmorum (W.G. Smith) Saccardo belonging to three different chemotypes, by over 97%. Two Trichoderma isolates reduced the amount of zearalenone produced by the same four Fusarium isolates by 5-83% in solid substrate bioassays, whereas several Clonostachys isolates reduced the amount of zearalenone by over 96%. Examination of the dynamics of zearalenone reduction showed that the reduction occurred at the fastest rate during the first four days of incubation. Selected non-toxigenic Trichoderma and Clonostachys isolates are candidates for biocontrol agents against toxigenic Fusarium pathogens of cereals and may be useful for decomposition of zearalenone in contaminated cereal grain and cereal products.

Dynamics of deoxynivalenol and zearalenone production by Fusarium graminearum under laboratory conditions

Toxicological investigations encompassed two cultures of Fusarium graminearum: (i) D2 isolate, originating from air was obtained on Sabouraud medium during a routine control of laboratory sterility conditions at the Department of Microbiology of the Center for Bio-Ecology in 2006, and (ii) GZ-LES control isolate, a well known producer of zearalenone (ZON) and deoxynivalenol (DON), was isolated from maize kernel collected at Leskovac in 1975. Preliminary analysis of fungal potential for the production of DON and ZON were performed by the modified rapid screening method of Filtenborg et al. (1983). Dynamics of DON and ZON biosynthesis was tested under different conditions of isolate cultivation: (i) in a basic liquid semi-synthetic medium with 2% yeast extract and 15% sucrose, pH 6.5 (YES), (ii) in broth with same concentrations of yeast extract and sucrose supplemented with 0.23 mg/l ZnSO4 x 5 H2O, pH 6.5 (YESZn) and (iii) on natural solid substrates such as wet sterilized maize and rice kernels. The quantitative determination of DON and ZON was performed in both liquid and natural solid substrates with thin-layer chromatographic methods (TLCs). The maximum yield of DON was recorded after three weeks of cultivation on maize kernels at 27?1?C. Contrary to the D2 isolate, which did not show the potential for the DON biosynthesis, the control isolate GZ-LES produced 645.6 ppb of the same type B trichothecene under previously mentioned conditions. The ZON biosynthesis by the isolate D2 (1.2 ppb) was observed after 2 weeks of the stationary cultivation in YES and YESZn at room temperature (17-19?C). The same isolate produced 0.74 ppb and 17.35 ppb ZON on maize and rice kernels after only 7 and 28 days of cultivation at the room temperature ranging from 17 to 19?C and from 15 to 23?C, respectively.

Developing molecular tools to simultaneously monitor deoxynivalenol and zearalenone production byFusariumfungi

Deoxynivalenol (DON) and zearalenone (ZON) are mycotoxins frequently detected in Fusarium -infected cereal grain. Both toxins are produced by Fusarium culmorum and F. graminearum during the development of Fusarium Head Blight (FHB) disease. We are developing tools to allow us to simultaneously detect DON and ZON production by Fusarium species. We generated a transgenic F. culmorum strain that expresses fluorescent proteins under the control of promoters of genes that are essential for the biosynthesis of DON (trichodiene synthase; Tri5) and ZON (a promoter that drives two polyketide synthetases, Zea1 and Zea2). We developed a duplex real time PCR assays for the concurrent analysis of Zea1 and Tri4 (another gene essential for DON production). We are currently generating a multiplex real time PCR version of this assay to detect Zea1, Tri4 and a plant actin (Act1) gene. This assay can be used to detect DON and ZON producers in grain; Act1 serves as a positive control plant gene in the reaction. We are also developing a multiplex real time RT-PCR assay to detect Zea1, Tri4 and a F. culmorum/F. graminearum beta-tubulin (Btub) gene. Incorporation of the Btub enables normalization of Tri4 and Zea1 transcript expression, relative to fungal abundance.

Identification of up-regulated genes during zearalenone biosynthesis in Fusarium

The Fusarium genus includes devastating plant pathogenic fungi that cause diseases in cereals around the world. They produce several mycotoxins, including the estrogenic compound zearalenone. To better understand the molecular mechanisms determining zearalenone production, we performed differential display RT-PCR under conditions where Fusarium graminearum and F. culmorum produced high amounts of zearalenone. We found 133 expressed sequence tags (ESTs) and 54 of these were considered to be up-regulated during high zearalenone production. Several of the ESTs were confirmed to be up-regulated by real-time qPCR, but none showed any significant down-regulation in the zearalenone negative mutant ΔPKS4-T9, or were similar to typical gene expression patterns of previously described zearalenone-related genes. Some of the up-regulated ESTs were similar to genes involved in secondary metabolite production, lipid metabolism, transcriptional activation, provision of precursors, signal transduction, transport or detoxification. Several of the ESTs were also located adjacent to one another in the genome and therefore might represent genes involved in the same biosynthetic pathway. Members of six such putative pathways could be found. All sequences were compared to the MIPS F. graminearum Genome Database to verify autocalled gene predictions experimentally and to introduce new exons and gene structures.

Functional analyses of heterotrimeric G protein Gα and Gβ subunits in Gibberella zeae

The homothallic ascomycete fungus Gibberella zeae (anamorph: Fusarium graminearum) is a major toxigenic plant pathogen that causes head blight disease on small-grain cereals. The fungus produces the mycotoxins deoxynivalenol (DON) and zearalenone (ZEA) in infected hosts, posing a threat to human and animal health. Despite its agricultural and toxicological importance, the molecular mechanisms underlying its growth, development and virulence remain largely unknown. To better understand such mechanisms, we studied the heterotrimeric G proteins of G. zeae, which are known to control crucial signalling pathways that regulate various cellular and developmental responses in fungi. Three putative Gα subunits, GzGPA1, GzGPA2 and GzGPA3, and one Gβ subunit, GzGPB1, were identified in the F. graminearum genome. Deletion of GzGPA1, a homologue of the Aspergillus nidulans Gα gene fadA, resulted in female sterility and enhanced DON and ZEA production, suggesting that GzGPA1 is required for normal sexual reproduction and repression of toxin biosynthesis. The production of DON and ZEA was also enhanced in the GzGPB1 mutant, suggesting that both Gα GzGPA1 and Gβ GzGPB1 negatively control mycotoxin production. Deletion of GzGPA2, which encodes a Gα protein similar to A. nidulans GanB, caused reduced pathogenicity and increased chitin accumulation in the cell wall, implying that GzGPA2 has multiple functions. Our study shows that G. zeae heterotrimeric G protein subunits can regulate vegetative growth, sexual development, toxin production and pathogenicity.

Effects of Root Isoquinoline Alkaloids from Hydrastis canadensis on Fusarium oxysporum Isolated from Hydrastis Root Tissue

Goldenseal (Hydrastis canadensis L.) is a popular medicinal plant distributed widely in North America. The rhizome, rootlets, and root hairs produce medicinally active alkaloids. Berberine, one of the Hydrastis alkaloids, has shown antifungal activity. The influence of a combination of the major Hydrastis alkaloids on the plant rhizosphere fungal ecology has not been investigated. A bioassay was developed to study the effect of goldenseal isoquinoline alkaloids on three Fusarium isolates, including the two species isolated from Hydrastis rhizosphere. The findings suggest that the Hydrastis root extract influences macroconidia germination, but that only the combined alkaloids--berberine, canadine, and hydrastine--appear to synergistically stimulate production of the mycotoxin zearalenone in the Fusarium oxysporum isolate. The Hydrastis root rhizosphere effect provided a selective advantage to the Fusarium isolates closely associated with the root tissue in comparison with the Fusarium isolate that had never been exposed to Hydrastis.

Zearalenone production and growth in drinking water inoculated with Fusarium graminearum

Zearalenone production and growth in drinking water inoculated with Fusarium graminearum

Zearalenone production and growth in drinking water inoculated with Fusarium graminearum

Production of the mycotoxin zearalenone (ZEN) was examined in drinking water inoculated with Fusarium graminearum. The strain employed was isolated from a US water distribution system. ZEN was purified with an immunoaffinity column and quantified by high-performance liquid chromatography (HPLC) with fluorescence detection. The extracellular yield of ZEN was 15.0 ng l−1. Visual growth was observed. Ergosterol was also indicative of growth and an average of 6.2 μg l−1 was obtained. Other compounds were also detected although remain unidentified. There is no equivalent information available. More work is required on metabolite expression in water as mycotoxins have consequences for human and animal health. The levels detected in this study were low. Water needs to be accepted as a potential source as it attracts high quality demands in terms of purity.

Zearalenone production during micro-malting of barley

The zearalenone (ZEA) content was determined during a micro-malting process (after steeping, germination, kilning and degermination, as well as in barley samples before micro-malting process) of two winter two-rowed barley samples, grown at Kragujevac location. In all phases of micro-malting isolation and determination of Fusarium spp. were performed. It was established that barley samples, before malting, were contaminated with zearalenone (barley sample 1-9.7 ?g/kg, barley sample 2-9.2 ?g/kg). The following Fusarium spp. were isolated: F. avenaceoum, F. culmorum, F. poae F. sporotrichioides, F. tricinctum and F. verticillioides. In both barley samples zearalenone content increased during steeping (86.5 ?g/kg and 37.4 ?g/kg), decreased during germination (12.5 ?g/kg and 26.8 ?g/kg), and increased after kilning (62.9 ?g/kg and 71.2 ?g/kg). In the finished malt the zearalenone content in sample 1 was 35.7 ?g/kg dry matter, and in sample 2 was 17.8 ?g/kg dry matter.

Enhanced mycotoxin production of a lipase-deficient Fusarium graminearum mutant correlates to toxin-related gene expression

Fusarium graminearum causes important diseases of small-grain cereals and maize and produces several mycotoxins. Among them, deoxynivalenol (DON) and zearalenone (ZEA) can accumulate in feedstuffs and foods to health-threatening levels. Although DON is important for fungal virulence in wheat, disease severity in the field does not correlate with mycotoxin concentrations. We compared gene expression and mycotoxin production of lipase-deficient mutants (Dfgl1), strongly reduced in virulence, and the respective wild-type isolate. Dfgl1 mutants exhibited up-regulated DON production during wheat head infection. On isolated wheat kernels, DON was only produced in low quantities, but higher in wild-type than in Dfgl1 mutants. In contrast, neither wild-type nor Dfgl1 mutants produced ZEA during wheat head infection. However, ZEA was clearly detectable on wheat kernels. Here, Dfgl1 mutants revealed a dramatically enhanced ZEA production. We could correlate the altered amounts of DON and ZEA directly with the expression of the toxin-related genes Tri5 for DON and PKS4 and PKS13 for ZEA. Based on Tri5 expression and the infection pattern of the wild-type and Dfgl1 mutants, we suggest that the transition zone of rachilla and rachis is important in the induction of DON synthesis. Gene expression studies indicate an involvement of the lipase FGL1 in regulation of 8 PKS genes and ZEA production. Abbreviations: FHB – Fusarium head blight; DON – deoxynivalenol; ZEA – zearalenone; PKS – polyketide synthase; Dpi – days post-inoculation

A Large Genomic Deletion in Gibberella zeae Causes a Defect in the Production of Two Polyketides but not in Sexual Development or Virulence

Gibberella zeae (anamorph: Fusarium graminearum) is an important pathogen of cereal crops. This fungus produces a broad range of secondary metabolites, including polyketides such as aurofusarin (a red pigment) and zearalenone (an estrogenic mycotoxin), which are important mycological characteristics of this species. A screen of G. zeae insertional mutants, generated using a restriction enzyme-mediated integration (REMI) procedure, led to the isolation of a mutant (Z43R606) that produced neither aurofusarin nor zearalenone yet showed normal female fertility and virulence on host plants. Outcrossing analysis confirmed that both the albino and zearalenone-deficient mutations are linked to the insertional vector in Z43R606. Molecular characterization of Z43R606 revealed a deletion of at least 220 kb of the genome at the vector insertion site, including the gene clusters required for the biosynthesis of aurofusarin and zearalenone, respectively. A re-creation of the insertional event of Z43R606 in the wild-type strain demonstrated that the 220-kb deletion is responsible for the phenotypic changes in Z43R606 and that a large region of genomic DNA can be efficiently deleted in G. zeae by double homologous recombination. The results showed that 52 putative genes located in the deleted genomic region are not essential for phenotypes other than the production of both aurofusarin and zearalenone. This is the first report of the molecular characterization of a large genomic deletion in G. zeae mediated by the REMI procedure.

The PKS4 gene of Fusarium graminearum is essential for zearalenone production.

Zearalenones are produced by several Fusarium species and can cause reproductive problems in animals. Some aurofusarin mutants of Fusarium pseudograminearum produce elevated levels of zearalenone (ZON), one of the estrogenic mycotoxins comprising the zearalenones. An analysis of transcripts from polyketide synthase genes identified in the Fusarium graminearum database was carried out for these mutants. PKS4 was the only gene with an enoyl reductase domain that had a higher level of transcription in the aurofusarin mutants than in the wild type. An Agrobacterium tumefaciens-mediated transformation protocol was used to replace the central part of the PKS4 gene with a hygB resistance gene through double homologous recombination in an F. graminearum strain producing a high level of ZON. PCR and Southern analysis of transformants were used to identify isolates with single insertional replacements of PKS4. High-performance liquid chromatography analysis showed that the PKS4 replacement mutant did not produce ZON. Thus, PKS4 encodes an enzyme required for the production of ZON in F. graminearum. Barley root infection studies revealed no alteration in the pathogenicity of the PKS4 mutant compared to the pathogenicity of the wild type. The expression of PKS13, which is located in the same cluster as PKS4, decreased dramatically in the mutant, while transcription of PKS4 was unchanged. This differential expression may indicate that ZON or its derivatives do not regulate expression of PKS4 and that the PKS4-encoded protein or its product stimulates expression of PKS13. Furthermore, both the lack of aurofusarin and ZON influenced the expression of other polyketide synthases, demonstrating that one polyketide can influence the expression of others.