naphthoquinones and aurofusarin

It is thought that fungi protect themselves from predation by the production of compounds that are toxic to soil-dwelling animals. Here, we show that a nontoxic pigment, the bis-naphthopyrone aurofusarin, protects Fusarium fungi from a wide range of animal predators. We find that springtails (primitive hexapods), woodlice (crustaceans), and mealworms (insects) prefer feeding on fungi with disrupted aurofusarin synthesis, and mealworms and springtails are repelled by wheat flour amended with the fungal bis-naphthopyrones aurofusarin, viomellein, or xanthomegnin. Predation stimulates aurofusarin synthesis in several Fusarium species and viomellein synthesis in Aspergillus ochraceus. Aurofusarin displays low toxicity in mealworms, springtails, isopods, Drosophila, and insect cells, contradicting the common view that fungal defence metabolites are toxic. Our results indicate that bis-naphthopyrones are defence compounds that protect filamentous ascomycetes from predators through a mechanism that does not involve toxicity.

Topics: Adaptation, Physiological; Animals; Arthropods; Aspergillus ochraceus; Food Preferences; Fusarium; Naphthoquinones; Pigments, Biological; Predatory Behavior

We staged the transfer of the aurofusarin and bikaverin biosynthetic gene clusters (BGCs) to Aspergillus nidulans with the aim of gaining functional insights into dynamics immediately following a horizontal gene transfer (HGT) event. While the introduction of both BGCs resulted in the production of detectable pathway metabolites in A. nidulans, the transferred aurofusarin BGC formed dimeric shunt products instead of aurofusarin. This was linked to low transcription of the cluster activator and insufficient activity of tailoring enzymes, demonstrating how a shift of the pathway bottleneck after HGT can result in metabolic innovation. The transferred bikaverin BGC readily produced bikaverin, providing a model system for studying the conservation of regulatory responses to environmental cues. Conserved PacC-mediated pH regulation of the bikaverin BGC was observed between original host Fusarium fujikuroi and A. nidulans. Contrary to strong nitrogen responses described in other hosts, the BGC appeared unresponsive to environmental nitrogen in A. nidulans. While F. fujikuroi and A. nidulans both form chlamydospore-like structures when exposed to ralsolamycin, specific induction of the bikaverin BGC was not observed in A. nidulans. We propose that the presence of compatible cis-regulatory elements in BGCs facilitates regulatory conservation after transfer, without which the chromosomal context would dictate expression.

Topics: Aspergillus nidulans; Fungal Proteins; Fusarium; Gene Expression Regulation, Fungal; Gene Transfer Techniques; Multigene Family; Naphthoquinones; Xanthones

Aurofusarin (AURO), a dimeric naphthoquinone, is produced by Fusarium fungi. Although frequently found in food and feed, toxicological studies are limited. Hence, the in vitro toxicity of AURO was investigated in the colon adenocarcinoma cell line HT29 and the non-tumorigenic colon cells HCEC-1CT. Cytotoxic effects were found at concentrations ≥1 μM by evaluating mitochondrial activity (WST-1) and cellular proliferation (sulforhodamine B assay). 10 μM of AURO induced a decrease of cells in the S-phase, measured by flow cytometry. Confocal microscopy revealed AURO-mediated increase of intracellular p53 protein. In accordance, DNA-damage was seen in the comet assay (≥1 μM) together with enhanced levels of formamidopyrimidine-DNA-glycosylase (fpg)-sensitive sites, indicative for oxidative stress. An increase of intracellular reactive oxygen species was observed in the dichlorofluorescein (DCF) assay (≥5 μM). The GSSG/GSH ratio was elevated, but no impact on redox-sensitive Nrf2-dependent genes (Nrf2, γ-GCL, NQO1) was found at the gene expression level. However, induction of cytochrome P450 monooxygenase (CYP) 1A1 was measured at the gene expression and protein level. In conclusion, these in vitro data suggest that, when co-occurring, AURO might be considered as a potential contributor to the overall toxicity of respective Fusarium mycotoxin mixtures.

Topics: Cell Culture Techniques; Cell Proliferation; Cell Survival; Colon; Comet Assay; DNA Damage; Flow Cytometry; Fusarium; HT29 Cells; Humans; Mutagens; Naphthoquinones; Oxidative Stress; S Phase

An efficient annulation involving pyrone addition to a quinone and Dieckmann condensation was developed for rapid assembly of a γ-naphthopyrone monomeric precursor to the bis-naphthoquinone natural product aurofusarin. Dimerization was achieved through Pd

Topics: Benzoquinones; Catalysis; Crystallography, X-Ray; Dimerization; Isomerism; Molecular Conformation; Naphthoquinones; Palladium

Topics: Fungal Proteins; Fusarium; Metabolic Engineering; Naphthoquinones; Pigments, Biological; Transcription Factors

Bio-guided screening is an important method to identify bioactive compounds from fungi. In this study we applied a fast digital time-lapse microscopic method for assessment of the antibacterial properties of secondary metabolites from the fungal genus

Topics: Anti-Bacterial Agents; Bacteria; Depsipeptides; Fusaric Acid; Fusarium; Mycotoxins; Naphthoquinones

Temperate species of Hypomyces and Cladobotryum that produce the red pigment aurofusarin are common on agaricoid and polyporoid basidiomata of species from five orders of Agaricomycetes. Several cause cobweb disease of commercially cultivated mushrooms resulting in serious losses. We sequenced rpb1, rpb2, tef1, and FG1093 regions in 90 wild strains and 30 strains from mushroom farms, isolated from Europe, North America, Africa, Asia, Australia, and New Zealand. Multigene analyses support the distinctness of five species but reveal Hypomyces rosellus to be paraphyletic, comprising several cryptic lineages. Hypomyces rosellus s. str. is characterised by wide dispersal and gene flow across Eurasia but does not occur in North America. Instead, the lineages from the West and the East Coast appear distinct, having given rise to species inhabiting the Southern Hemisphere. Our results reveal wide misuse of the name H. rosellus, especially for cobweb isolates. The majority of these belong to Hypomyces odoratus, including a weakly supported group of fungicide-resistant strains from Europe and North America sharing identical sequence data. New collections are presented for Cladobotryum rubrobrunnescens and Cladobotryum tenue as well as Cladobotryum multiseptatum and Hypomyces dactylarioides, all previously known only from their type material. The former species pair occurs in Europe and the latter in Australia and New Zealand. Separate lineages appear to be maintained by geographic isolation in North America and temperate Australasia but by host specialisation in the species occurring sympatrically in Europe and Asia. Both specialist and generalist host use strategies have evolved in the group. Although teleomorphs are known in most of the species and unnamed lineages, analyses of the five-gene regions suggest the prevalence of clonal reproduction in H. odoratus. This can be the reason for its success in mushroom farms, also facilitating the spread of fungicide resistance. While tef1 and rpb2 can be recommended for species delimitation, low variation, not exceeding 1 % in the whole ingroup, impeaches the use of ITS as a barcoding gene region in this group of fungi.

Topics: Agaricales; Biodiversity; Evolution, Molecular; Fungal Proteins; Genetic Variation; Host-Pathogen Interactions; Hypocreales; Molecular Sequence Data; Naphthoquinones; Phylogeny; Plant Diseases; Vegetables

Fusarium avenaceum is a widespread pathogen of important crops in the temperate climate zones that can produce many bioactive secondary metabolites, including moniliformin, fusarin C, antibiotic Y, 2-amino-14,16-dimethyloctadecan-3-ol (2-AOD-3-ol), chlamydosporol, aurofusarin and enniatins. Here, we examine the production of these secondary metabolites in response to cultivation on different carbon sources in order to gain insight into the regulation and production of secondary metabolites in F. avenaceum. Seven monosaccharides (arabinose, xylose, fructose, sorbose, galactose, mannose, glucose), five disaccharides (cellobiose, lactose, maltose, sucrose and trehalose) and three polysaccharides (dextrin, inulin and xylan) were used as substrates. Three F. avenaceum strains were used in the experiments. These were all able to grow and produce aurofusarin on the tested carbon sources. Moniliformin and enniatins were produced on all carbon types, except on lactose, which suggest a common conserved regulation mechanism. Differences in the strains was observed for production of fusarin C, 2-AOD-3-ol, chlamydosporol and antibiotic Y, which suggests that carbon source plays a role in the regulation of their biosynthesis.

Topics: Carbohydrates; Cyclobutanes; Depsipeptides; Ergosterol; Fusarium; Mycotoxins; Naphthoquinones; Polyenes; Pyrones; Secondary Metabolism; Sphingolipids

Trichothecenes are a group of toxic secondary metabolites produced mainly by Fusarium graminearum (teleomorph: Gibberella zeae) during the infection of crop plants, including wheat, maize, barley, oats, rye and rice. Some fungal genes involved in trichothecene biosynthesis have been shown to encode regulatory proteins. However, the global regulation of toxin biosynthesis is still enigmatic. In addition to the production of secondary metabolites belonging to the trichothecene family, F. graminearum produces the red pigment aurofusarin. The gene regulation underlying the production of aurofusarin is not well understood. The velvet gene (veA) is conserved in various genera of filamentous fungi. Recently, the veA gene from Aspergillus nidulans has been shown to be the key component of the velvet complex regulating development and secondary metabolism. Using blast analyses, we identified the velvet gene from F. graminearum, FgVe1. Disruption of FgVe1 causes several phenotypic effects. However, the complementation of this mutant with the FgVe1 gene restores the wild-type phenotypes. The in vitro phenotypes include hyperbranching of the mycelium, suppression of aerial hyphae formation, reduced hydrophobicity of the mycelium and highly reduced sporulation. Our data also show that FgVe1 modulates the production of the aurofusarin pigment and is essential for the expression of Tri genes and the production of trichothecenes. Pathogenicity studies performed on flowering wheat plants indicate that FgVe1 is a positive regulator of virulence in F. graminearum.

Topics: Amino Acid Sequence; Cell Membrane; Chromatography, High Pressure Liquid; Fungal Proteins; Fusarium; Gene Deletion; Gene Expression Regulation, Fungal; Genes, Fungal; Genetic Complementation Test; Hydrophobic and Hydrophilic Interactions; Molecular Sequence Data; Naphthoquinones; Phenotype; Sequence Alignment; Spores, Fungal; Trichothecenes

Chromatin modifications and heterochromatic marks have been shown to be involved in the regulation of secondary metabolism gene clusters in the fungal model system Aspergillus nidulans. We examine here the role of HEP1, the heterochromatin protein homolog of Fusarium graminearum, for the production of secondary metabolites. Deletion of Hep1 in a PH-1 background strongly influences expression of genes required for the production of aurofusarin and the main tricothecene metabolite DON. In the Hep1 deletion strains AUR genes are highly up-regulated and aurofusarin production is greatly enhanced suggesting a repressive role for heterochromatin on gene expression of this cluster. Unexpectedly, gene expression and metabolites are lower for the trichothecene cluster suggesting a positive function of Hep1 for DON biosynthesis. However, analysis of histone modifications in chromatin of AUR and DON gene promoters reveals that in both gene clusters the H3K9me3 heterochromatic mark is strongly reduced in the Hep1 deletion strain. This, and the finding that a DON-cluster flanking gene is up-regulated, suggests that the DON biosynthetic cluster is repressed by HEP1 directly and indirectly. Results from this study point to a conserved mode of secondary metabolite (SM) biosynthesis regulation in fungi by chromatin modifications and the formation of facultative heterochromatin.

Topics: Fungal Proteins; Fusarium; Gene Deletion; Gene Expression Regulation, Fungal; Heterochromatin; Histones; Naphthoquinones; Trichothecenes

Metabolites of toxigenic fungi and bacteria occur as natural contaminants (e.g. mycotoxins) in feedstuffs making them unsafe to animals. The multi-toxin profiles in 58 commercial poultry feed samples collected from 19 districts in 17 states of Nigeria were determined by LC/ESI-MS/MS with a single extraction step and no clean-up. Sixty-three (56 fungal and seven bacterial) metabolites were detected with concentrations ranging up to 10,200 µg kg⁻¹ in the case of aurofusarin. Fusarium toxins were the most prevalent group of fungal metabolites, whereas valinomycin occurred in more than 50% of the samples. Twelve non-regulatory fungal and seven bacterial metabolites detected and quantified in this study have never been reported previously in naturally contaminated stored grains or finished feed. Among the regulatory toxins in poultry feed, aflatoxin concentrations in 62% of samples were above 20 µg kg⁻¹, demonstrating high prevalence of unsafe levels of aflatoxins in Nigeria. Deoxynivalenol concentrations exceeded 1000 µg kg⁻¹ in 10.3% of samples. Actions are required to reduce the consequences from regulatory mycotoxins and understand the risks of the single or co-occurrence of non-regulatory metabolites for the benefit of the poultry industry.

Topics: Aflatoxins; Animal Feed; Animals; Anti-Bacterial Agents; Bacterial Toxins; Chickens; Chromatography, High Pressure Liquid; Food Contamination; Food Inspection; Fusarium; Limit of Detection; Mycotoxins; Naphthoquinones; Nigeria; Poultry; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry; Trichothecenes; Valinomycin

Previous studies have reported the functional characterization of 9 out of 11 genes found in the gene cluster responsible for biosynthesis of the polyketide pigment aurofusarin in Fusarium graminearum. Here we reanalyze the function of a putative aurofusarin pump (AurT) and the two remaining orphan genes, aurZ and aurS. Targeted gene replacement of aurZ resulted in the discovery that the compound YWA1, rather than nor-rubrofusarin, is the primary product of F. graminearum polyketide synthase 12 (FgPKS12). AurZ is the first representative of a novel class of dehydratases that act on hydroxylated γ-pyrones. Replacement of the aurS gene resulted in accumulation of rubrofusarin, an intermediate that also accumulates when the GIP1, aurF, or aurO genes in the aurofusarin cluster are deleted. Based on the shared phenotype and predicted subcellular localization, we propose that AurS is a member of an extracellular enzyme complex (GIP1-AurF-AurO-AurS) responsible for converting rubrofusarin into aurofusarin. This implies that rubrofusarin, rather than aurofusarin, is pumped across the plasma membrane. Replacement of the putative aurofusarin pump aurT increased the rubrofusarin-to- aurofusarin ratio, supporting that rubrofusarin is normally pumped across the plasma membrane. These results provide functional information on two novel classes of proteins and their contribution to polyketide pigment biosynthesis.

Topics: Fungal Proteins; Fusarium; Genes, Fungal; Multienzyme Complexes; Naphthoquinones; Polyketide Synthases

Wet apple core rot (wACR) is a well-known disease of susceptible apple cultivars such as Gloster, Jona Gold, and Fuji. Investigations in apple orchards in Slovenia identified Fusarium avenaceum, a known producer of several mycotoxins, as the predominant causal agent of this disease. A LC-MS/MS method was developed for the simultaneous detection of thirteen F. avenaceum metabolites including moniliformin, acuminatopyrone, chrysogine, chlamydosporol, antibiotic Y, 2-amino-14,16-dimethyloctadecan-3-ol (2-AOD-3-ol), aurofusarin, and enniatins A, A1, B, B1, B2, and B3 from artificially and naturally infected apples. Levels of moniliformin, antibiotic Y, aurofusarin, and enniatins A, A1, B, and B1 were quantitatively examined in artificially inoculated and naturally infected apples, whereas the remaining metabolites were qualitatively detected. Metabolite production was examined in artificially inoculated apples after 3, 7, 14, and 21 days of incubation. Most metabolites were detected after 3 or 7 days and reached significantly high levels within 14 or 21 days. The highest levels of moniliformin, antibiotic Y, aurofusarin, and the combined sum of enniatins A, A1, B, and B1 were 7.3, 5.7, 152, and 12.7 microg g(-1), respectively. Seventeen of twenty naturally infected apples with wACR symptoms contained one or more of the metabolites. Fourteen of these apples contained moniliformin, antibiotic Y, aurofusarin, and enniatins in levels up to 2.9, 51, 167, and 3.9 microg g(-1), respectively. Acuminatopyrone, chrysogine, chlamydosporol, and 2-AOD-3-ol were detected in 4, 11, 4, and 10 apples, respectively. During wet apple core rot, F. avenaceum produced high amounts of mycotoxins, which may pose a risk for consumers of apple or processed apple products.

Topics: Cyclobutanes; Depsipeptides; Fruit; Fusarium; Malus; Mycotoxins; Naphthoquinones; Plant Diseases

High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) can be used for simultaneous quantification of various mycotoxins in contaminated food samples. Therefore, multi-mycotoxin methods have been developed in the last couple of years. To enlarge these methods for further analytes, we have developed a LC-MS/MS method for the quantification of the mycotoxin aurofusarin. Additionally, further LC- MS(n) experiments were performed to demonstrate the fragmentation pattern of aurofusarin. Applicable multiple reaction monitoring (MRM) transitions of aurofusarin were found and optimized by parameter variation of the tandem mass spectrometer. The applicability of the developed method was tested by analysis of naturally contaminated wheat.

Topics: Chromatography, High Pressure Liquid; Mycotoxins; Naphthoquinones; Spectrometry, Mass, Electrospray Ionization; Triticum

Fusarium graminearum produces trichothecenes in aerial hyphae, a process which is markedly suppressed by NaCl without a significant effect on fungal growth. Here we report on the involvement of kinases of the two-component osmotic signal transduction pathway in the regulation of secondary metabolism in F. graminearum. While a deletion null mutant of FgOs1 (encoding the osmosensor histidine kinase) (deltaFgOs1) produced a reduced amount of the red pigment aurofusarin and was unaltered in its ability to produce trichothecenes, deletion null mutants of FgOs4 (encoding mitogen-activated protein kinase kinase kinase; MAPKKK), FgOs5 (MAPKK), and FgOs2 (MAPK) showed markedly enhanced pigmentation and failed to produce trichothecenes in aerial hyphae. Also, the transcript levels of PKS12 and GIP2 (aurofusarin biosynthetic pathway and regulatory genes, respectively) were significantly enhanced in the deltaFgOs4, deltaFgOs5, and deltaFgOs2 mutants and were reduced in the deltaFgOs1 mutant. In addition, expression of Tri4 and Tri6 (trichothecene biosynthetic pathway and regulatory genes) and production of trichothecenes in rice medium were markedly reduced in the former three protein kinase mutants. This is the first report demonstrating the involvement of a MAPK in the regulation of secondary metabolism.

Topics: Antifungal Agents; Blotting, Northern; Drug Resistance, Fungal; Fungal Proteins; Fusarium; Gene Expression Regulation, Fungal; Genotype; Histidine Kinase; MAP Kinase Kinase Kinases; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Models, Biological; Mutation; Mycotoxins; Naphthoquinones; Osmosis; Protein Kinases; Time Factors; Trichothecenes

Gibberella zeae (anamorph: Fusarium graminearum) is an important pathogen of maize, wheat, and rice. Colonies of G. zeae produce yellow-to-tan mycelia with the white-to-carmine red margins. In this study, we focused on nine putative open reading frames (ORFs) closely linked to PKS12 and GIP1, which are required for aurofusarin biosynthesis in G. zeae. Among them is an ORF designated GIP2 (for Gibberella zeae pigment gene 2), which encodes a putative protein of 398 amino acids that carries a Zn(II)2Cys6 binuclear cluster DNA-binding domain commonly found in transcription factors of yeasts and filamentous fungi. Targeted gene deletion and complementation analyses confirmed that GIP2 is required for aurofusarin biosynthesis. Expression of GIP2 in carrot medium correlated with aurofusarin production by G. zeae and was restricted to vegetative mycelia. Inactivation of the 10 contiguous genes in the DeltaGIP2 strain delineates an aurofusarin biosynthetic gene cluster. Overexpression of GIP2 in both the DeltaGIP2 and the wild-type strains increases aurofusarin production and reduces mycelial growth. Thus, GIP2 is a putative positive regulator of the aurofusarin biosynthetic gene cluster, and aurofusarin production is negatively correlated with vegetative growth by G. zeae.

Topics: Base Sequence; DNA, Fungal; Fungal Proteins; Gene Deletion; Gene Expression Regulation, Fungal; Genes, Fungal; Genetic Complementation Test; Gibberella; Multigene Family; Mycotoxins; Naphthoquinones; Transcription Factors

Fungal polyketide biosynthesis typically involves multiple enzymatic steps and the encoding genes are often found in gene clusters. A gene cluster containing PKS12, the polyketide synthase gene responsible for the synthesis of the pigment aurofusarin, was analysed by gene replacement using Agrobacterium tumefaciens-mediated transformation to determine the biosynthesis pathway of aurofusarin. Replacement of aurR1 with hygB shows that it encodes a positively acting transcription factor that is required for the full expression of PKS12, aurJ, aurF, gip1 and FG02329.1, which belong to the gene cluster. AurR1 and PKS12 deletion mutants are unable to produce aurofusarin and rubrofusarin. Bio- and chemoinformatics combined with chemical analysis of replacement mutants (DeltaaurJ, DeltaaurF, Deltagip1, DeltaaurO and DeltaPKS12) indicate a five-step enzyme catalysed pathway for the biosynthesis of aurofusarin, with rubrofusarin as an intermediate. This links the biosynthesis of naphthopyrones and naphthoquinones together. Replacement of the putative transcription factor aurR2 results in an increased level of rubrofusarin relative to aurofusarin. Gip1, a putative laccase, is proposed to be responsible for the dimerization of two oxidized rubrofusarin molecules resulting in the formation of aurofusarin.

Topics: Agrobacterium tumefaciens; Fungal Proteins; Fusarium; Genes, Fungal; Mutation; Naphthalenes; Naphthoquinones; Transcription Factors; Transformation, Bacterial

The red pigmentation of Fusarium graminearum and related species that cause stem and head blight of cereals is due to the deposition of aurofusarin in the cell walls. To determine the importance of this polyketide for fungal physiology and pathogenicity, aurofusarin deficient mutants were produced by random and targeted mutagenesis of F. pseudograminearum and F. graminearum. We show that a gene cluster, including the F. graminearum PKS12 gene, is responsible for the biosynthesis of aurofusarin. Three F. pseudograminearum aurofusarin deficient mutants were disrupted in a region upstream from a gene with sequence homology to the aflatoxin regulatory gene aflR. Comparative PCR analyses of the aurofusarin gene cluster in F. graminearum, F. culmorum, and F. pseudograminearum show conserved organization and expression analyses detected no PKS12 transcripts in any of the mutants. To confirm that PKS12 encodes the precursor for aurofusarin, targeted mutagenesis was carried out in F. graminearum. All disruptants showed an albino phenotype. The DeltaPKS12 mutants have higher growth rate and a 10-fold increase in conidia production compared to the wild type. Aurofusarin does not appear to aid in radiation protection and all the mutants are fully pathogenic on wheat and barley. HPLC analyses of aurofusarin deficient mutants confirm the absence of aurofusarin and show an increase in the level of the mycotoxin zearalenone.

Topics: DNA Mutational Analysis; DNA, Fungal; Fusarium; Genes, Fungal; Hordeum; Molecular Sequence Data; Multigene Family; Mutagenesis, Insertional; Mutagenesis, Site-Directed; Mutation; Naphthoquinones; Pigments, Biological; Plant Diseases; Plant Roots; RNA, Fungal; RNA, Messenger; Sequence Analysis, DNA; Virulence; Zearalenone

Mycelia of Gibberella zeae (anamorph, Fusarium graminearum), an important pathogen of cereal crops, are yellow to tan with white to carmine red margins. We isolated genes encoding the following two proteins that are required for aurofusarin biosynthesis from G. zeae: a type I polyketide synthase (PKS) and a putative laccase. Screening of insertional mutants of G. zeae, which were generated by using a restriction enzyme-mediated integration procedure, resulted in the isolation of mutant S4B3076, which is a pigment mutant. In a sexual cross of the mutant with a strain with normal pigmentation, the pigment mutation was linked to the inserted vector. The vector insertion site in S4B3076 was a HindIII site 38 bp upstream from an open reading frame (ORF) on contig 1.116 in the F. graminearum genome database. The ORF, designated Gip1 (for Gibberella zeae pigment mutation 1), encodes a putative laccase. A 30-kb region surrounding the insertion site and Gip1 contains 10 additional ORFs, including a putative ORF identified as PKS12 whose product exhibits about 40% amino acid identity to the products of type I fungal PKS genes, which are involved in pigment biosynthesis. Targeted gene deletion and complementation analyses confirmed that both Gip1 and PKS12 are required for aurofusarin production in G. zeae. This information is the first information concerning the biosynthesis of these pigments by G. zeae and could help in studies of their toxicity in domesticated animals.

Topics: Amino Acid Sequence; Culture Media; Gibberella; Hordeum; Laccase; Molecular Sequence Data; Mutation; Mycelium; Naphthoquinones; Pigments, Biological; Plant Diseases; Polyketide Synthases; Virulence

Polyketides are a class of secondary metabolites that exhibit a vast diversity of form and function. In fungi, these compounds are produced by large, multidomain enzymes classified as type I polyketide synthases (PKSs). In this study we identified and functionally disrupted 15 PKS genes from the genome of the filamentous fungus Gibberella zeae. Five of these genes are responsible for producing the mycotoxins zearalenone, aurofusarin, and fusarin C and the black perithecial pigment. A comprehensive expression analysis of the 15 genes revealed diverse expression patterns during grain colonization, plant colonization, sexual development, and mycelial growth. Expression of one of the PKS genes was not detected under any of 18 conditions tested. This is the first study to genetically characterize a complete set of PKS genes from a single organism.

Topics: Amino Acid Sequence; Fungal Proteins; Gene Expression Profiling; Gene Expression Regulation, Fungal; Gibberella; Molecular Sequence Data; Naphthoquinones; Pigments, Biological; Polyenes; Polyketide Synthases; Sequence Analysis, DNA; Zearalenone

The aim of this study was to evaluate effects of modified glucomannans (Mycosorb) on egg yolk and liver of the day-old quail after aurofusarin inclusion in the maternal diet. Fifty-four 45-day-old Japanese quail were divided into three groups and were fed ad libitum a corn-soya diet balanced in all nutrients. The diet of the experimental quail was supplemented with aurofusarin at the level of 26.4 mg/kg feed in the form of Fusarium graminearum culture enriched with aurofusarin or with aurofusarin plus Mycosorb at 1 g/kg feed. Eggs obtained after 8 weeks of feeding were analysed and incubated in standard conditions of 37.5 degrees C/55% RH. Samples of quail liver were collected from day-old hatchlings. Main polyunsaturated fatty acids (PUFAs) of the egg yolk were analysed by gas chromatography, and tocopherols and tocotrienols were analysed by HPLC-based methods. Inclusion of aurofusarin in the maternal diet was associated with decreased proportions of docosahexaenoic acid and increased proportions of linoleic acid in major lipid fractions of the egg yolk as well as with decreased concentrations of alpha- and gamma-tocopherols, alpha- and gamma-tocotrienols in egg yolk and liver of a day-old quail. Inclusion of modified glucomannans (Mycosorb) into the quail diet simultaneously with aurofusarin showed a significant protective effect against changes in PUFA and antioxidant composition in the egg yolk and liver of quail. It is suggested that a combination of mycotoxin adsorbents and natural antioxidants could be the next step in counteracting mycotoxins in animal feed.

Topics: Animals; Coturnix; Egg Yolk; Fatty Acids, Nonesterified; Female; Liver; Male; Mannans; Naphthoquinones; Ovum; Vitamin E

The effects of aurofusarin in the quail diet on the antioxidant systems of the developing embryo are investigated. Thirty eight 45-day-old Japanese quails (Coturnix japonica) were divided into two groups and were fed on a corn-soya diet or the same diet supplemented with aurofusarin at the level of 26.4 mg/kg feed in the form of Fusarium graminearum culture enriched with aurofusarin. Eggs obtained after 7 weeks of feeding were incubated. Samples of quail tissues were collected at day 17 of embryonic development and from day old hatchlings. Antioxidants and malondialdehyde were analysed by HPLC-based methods. Inclusion of aurofusarin in the maternal diet was associated with decreased concentrations of alpha- and gamma-tocopherols, alpha- and gamma-tocotrienols, retinol, lutein and zeaxanthin in egg yolk. The vitamin E (tocopherols and tocotrienols) concentration in the liver and yolk sac membrane (YSM) of the day 17 embryos and the hatchlings from aurofusarin-fed group was significantly decreased. Alpha-tocopherol concentration was also reduced in kidney, lung, heart, muscle and brain of day-old quails. In the liver of day-old quails, concentrations of lutein, zeaxanthin, retinol, retinyl linoleate, retinyl oleate, retinyl palmitate and retinyl stearate were also reduced. As a result of these diminished antioxidant concentrations, tissue susceptibility to lipid peroxidation was significantly increased. It is suggested that a compromised antioxidant system of the egg yolk and embryonic tissues could predispose quails to increased mortality at late stages of their embryonic development.

Topics: Animals; Animals, Newborn; Antioxidants; Carotenoids; Coturnix; Egg Yolk; Embryo, Nonmammalian; Female; Lipid Peroxidation; Liver; Malondialdehyde; Mycotoxins; Naphthoquinones; Quail; Tissue Distribution; Vitamin E; Yolk Sac

1. The effect of the mycotoxin aurofusarin on the antioxidant composition and fatty acid profile of quail eggs was investigated. 2. Thirty eight 45-d-old Japanese quails were divided into two groups (experimental and control, 15 females +4 males in each group) and were fed on a maize-soya diet balanced in all nutrients. The diet of the experimental quails was supplemented with aurofusarin at the level of 26.4 mg/kg feed in the form of Fusarium graminearum culture enriched with aurofusarin. At the beginning and after 2, 4 and 8 week supplementation periods, eggs were collected and analysed. After 8 weeks of supplementation, experimental quails were fed on unsupplemented diet during the next 4 weeks and eggs were collected after 2 and 4 weeks on such a diet and analysed. 3. Aurofusarin caused a significant (P<0.05) decrease in vitamins E, A, total carotenoid, lutein and zeaxanthin concentrations and significantly (P<0.05) increased egg yolk susceptibility to lipid peroxidation. During two weeks on the diet without aurofusarin the levels of carotenoids in the egg yolk returned to the initial level, vitamins A and E returned to the initial level during 4 weeks on the same unsupplemented diet. 4. Dietary supplementation with aurofusarin was associated with a significant (P<0.01) decrease in the docosahexaenoic acid proportion in the phospholipid, cholesteryl ester and free fatty acid fractions of the egg yolk. At the same time the proportion of linoleic acid in the phospholipid, free fatty acid and triacylglycerol fractions significantly (P<0.05) increased. 5. It is concluded that mycotoxin aurofusarin is detrimental to the nutritional quality of eggs.

Topics: Animals; Antioxidants; beta Carotene; Carotenoids; Coturnix; Egg Yolk; Eggs; Fatty Acids; Female; Lutein; Male; Mycotoxins; Naphthoquinones; Vitamin A; Vitamin E; Xanthophylls; Zeaxanthins