Control of Sexual Reproduction in Gibberella zeae (Fusarium roseum “Graminearum”)

Sexual reproduction in Gibberella zeae (Fusarium roseum) is regulated by the fungal sex hormone zearalenone, which is known to be synthesized only by species of Fusarium. The presence of cyclic adenosine 3′,5′-monophosphate (cAMP) in mycelium of this fungus has been confirmed by analyses with thin-layer and gas-liquid chromatography, fluorescent properties, ultraviolet absorption, competitive protein-binding tests, and degradation by cyclic phosphodies-terase. cAMP but not cyclic guanosine monophosphate increased both the number of perithecia formed and the incorporation of [1-¹⁴C]acetate into zearalenone. It is proposed that cAMP stimulates the synthesis of zearalenone which then exerts its effect directly or indirectly on formation of perithecia.     

Media for identification of Gibberella zeae and production of F-2-(Zearalenone).

Media are described for the isolaton of Fusarium graminearum in the perithecial state, Gibberella zeae, and for the production of F-2 (zearalenone) by Fusarium species. On soil extract-corn meal agar isolated medium, G. Zeae produced perithecia in 9 to 14 days under a 12-h photoperiod. Species of Fusarium were screened for F-2 production on a liquid medium. From strains that produced F-2, the yields, from stationary cultures of G. zeae and F. culmorum after 12 days of incubation, ranged from 22 to 86 mg/liter. Three strains produced no F-2. Glumatic acid, starch, yeast extract,and the proper ratio of medium volume-to-flask volume were necessary for F-2 synthesis.     

Media for identification of Gibberella zeae and production of F-2-(Zearalenone).

Media are described for the isolaton of Fusarium graminearum in the perithecial state, Gibberella zeae, and for the production of F-2 (zearalenone) by Fusarium species. On soil extract-corn meal agar isolated medium, G. Zeae produced perithecia in 9 to 14 days under a 12-h photoperiod. Species of Fusarium were screened for F-2 production on a liquid medium. From strains that produced F-2, the yields, from stationary cultures of G. zeae and F. culmorum after 12 days of incubation, ranged from 22 to 86 mg/liter. Three strains produced no F-2. Glumatic acid, starch, yeast extract,and the proper ratio of medium volume-to-flask volume were necessary for F-2 synthesis.     

Heterotrimeric G protein β subunit GPB1 and MAP kinase MPK1 regulate hyphal growth and female fertility in Fusarium sacchari

Heterotrimeric GTP-binding proteins (G proteins) and mitogen-activated protein kinase (MAPK) cascades involve vegetative hyphal growth, development of infection-related structure, colonization in host plant and female fertility in phytopathogenic ascomycete fungi. In this study, a heterotrimeric G protein β subunit (Gβ), GPB1, and MAPK, MPK1, were characterized from Fusarium sacchari (= Gibberella sacchari; mating population B of the G. fujikuroi-species complex). GPB1 and MPK1 showed high homology to known Gβ and Fus3/Kss1 MAP kinases of other filamentous ascomycetes, respectively. Disruption (Δ) of gpb1 suppressed hyphal branching and accelerated aerial hyphae formation in F. sacchari. Oppositely, disruption of mpk1 caused delayed aerial hyphae formation. These indicated that GPB1 regulates vegetative hyphal growth negatively, and MPK1 does positively in F. sacchari. Both Δgpb1 and Δmpk1 showed female sterility. Level of intracellular cAMP in Δgpb1 was lower than wild type. Exogenous cyclic AMP (cAMP) partially restored enhanced aerial hyphae formation. These suggested that abnormal hyphal growth was caused by depletion of intracellular cAMP in Δgpb1. cAMP has been reported to suppress development of perithecia in crossing between wild type strains. Thus, precise regulation of intracellular cAMP level via Gβ/MAPK is essential for normal hyphal growth and fertility.     

Fusarium subglutinans f. sp. Pini represents a distinct mating population in the gibberella fujikuroi species complex

Fusarium strains in the Gibberella fujikuroi species complex cause diseases on a variety of economically important plants. One of these diseases, pitch canker of Pinus spp., is caused by strains identified as Fusarium subglutinans f. sp. pini. Fertile crosses were detected between F. subglutinans f. sp. pini strains from South Africa, California, and Florida. F. subglutinans f. sp. pini strains were not cross-fertile with the standard tester strains of six of the seven other mating populations of G. fujikuroi. Sporadic perithecia with ascospores were obtained in two crosses with the mating population B tester strains. These perithecia were homothallic, and the ascospores derived from these perithecia were vegetatively compatible with the mating population B tester strain parent. We concluded that fertile F. subglutinans f. sp. pini isolates represent a new mating population (mating population H) of G. fujikuroi and that they belong to a unique biological species in a distinct taxon.     

Isolation and characterization of zearalenone sulfate produced by Fusarium spp.

A water-soluble compound related to zearalenone was isolated from a culture of Fusarium graminearum 30 grown in rice. The structure of the novel metabolite was determined to be zearalenone-4-sulfate on the basis of fast-atom-bombardment mass spectrometry, proton nuclear magnetic resonance, UV spectroscopy, and by chemical and enzymatic reactions. Strains representing Fusarium equiseti, Fusarium sambucinum, and Fusarium roseum produced the sulfate conjugate as well. In the rat uterus enlargement bioassay, the metabolite or its hydrolysis product was found to retain the estrogenic activity characteristic of zearalenone. Natural occurrence of this novel metabolite might be significant because analytical methods devised for zearalenone in grain cannot detect the conjugate but the conjugate retains the biological properties of the mycotoxin when ingested by animals.     

Isolation and characterization of zearalenone sulfate produced by Fusarium spp.

A water-soluble compound related to zearalenone was isolated from a culture of Fusarium graminearum 30 grown in rice. The structure of the novel metabolite was determined to be zearalenone-4-sulfate on the basis of fast-atom-bombardment mass spectrometry, proton nuclear magnetic resonance, UV spectroscopy, and by chemical and enzymatic reactions. Strains representing Fusarium equiseti, Fusarium sambucinum, and Fusarium roseum produced the sulfate conjugate as well. In the rat uterus enlargement bioassay, the metabolite or its hydrolysis product was found to retain the estrogenic activity characteristic of zearalenone. Natural occurrence of this novel metabolite might be significant because analytical methods devised for zearalenone in grain cannot detect the conjugate but the conjugate retains the biological properties of the mycotoxin when ingested by animals.     

Zearalenon in Lebensmitteln

The research project “Methods of Analysis and occurrence of important Fusarium toxins (deoxynivalenol and zearalenone) as well as the intake of these toxins by the German consumer” supported by the Federal Ministry of Consumer Protection, Food and Agriculture (BMVEL) is processed by the institutions mentioned above. This work represents a comprehensive summary of the contamination of food by zearalenone (ZEA).     

Sexual reproduction in the soybean sudden death syndrome pathogen Fusarium tucumaniae

We investigated the sexual reproductive mode of the two most important etiological agents of soybean sudden death syndrome, Fusarium tucumaniae and Fusarium virguliforme. F. tucumaniae sexual crosses often were highly fertile, making it possible to assign mating type and assess female fertility in 24 South American isolates. These crosses produced red perithecia and oblong-elliptical ascospores, as is typical for sexual members of the F. solani species complex. Genotyping of progeny from three F. tucumaniae crosses confirmed that sexual recombination had occurred. In contrast, pairings among 17 U.S. F. virguliforme isolates never produced perithecia. Inter-species crosses between F. tucumaniae and F. virguliforme, in which infertile perithecia were induced only in one of the two F. tucumaniae mating types, suggest that all U.S. F. virguliforme isolates are of a single mating type. We conclude that the F. tucumaniae life cycle in S. America includes a sexual reproductive mode, and thus this species has greater potential for rapid evolution than the F. virguliforme population in the U.S., which may be exclusively asexual.     

Incidence of zearalenol (Fusarium mycotoxin) in animal feed.

Zearalenol, the reduction product of zearalenone produced by Fusarium roseum growing in cereals, was found for the first time naturally occurring in oats and corn. This metabolite is three to four times more active estrogenically than zearalenone.     

Incidence of zearalenol (Fusarium mycotoxin) in animal feed.

Zearalenol, the reduction product of zearalenone produced by Fusarium roseum growing in cereals, was found for the first time naturally occurring in oats and corn. This metabolite is three to four times more active estrogenically than zearalenone.     

Natural occurrence of Fusarium toxins in feedstuff.

The mycotoxins diacetoxyscirpenol, deoxynivalenol, and zearalenone, produced by Fusarium roseum, were found naturally occuring in mixed feed samples. In all cases analyzed, deoxynivalenol occurred together with zearalenone. The natural occurrence of zearalenone in sesame seed is reported for the first time. Strains of F. roseum isolated in various parts of the world form feed implicated in animal mycotoxicosis produced monoacetoxyscirpenol, diacetoxyscirpenol, deoxynivalenol, and zearalenone.     

Preferential binding of radiolabeled zearalenone to a protein fraction of Fusarium roseum graminearum.

Zearalenone [6-(10-hydroxy-6-oxo-trans-1-undecenyl)beta-resorcyclic acid lactone] is a hormone produced by Fusarium spp. which regulates the sexual stage in F. roseum. 3H- and 14C-labeled zearalenone were found to bind preferentially to one of two peaks containing uncharacterized proteins obtained from the cytosol of young mycelium and resolved by gel column chromatography. The proteins were partially purified by successive resolution on Sephadex G-100, Sephadex G-200, and BioGel P-300. Free zearalenone (37%) was reisolated from the purified proteins after resolution by thin-layer chromatography or partitioning with ethyl acetate.     

Characterization of two polyketide synthase genes involved in zearalenone biosynthesis in Gibberella zeae

Zearalenone, a mycotoxin produced by several Fusarium spp., is most commonly found as a contaminant in stored grain and has chronic estrogenic effects on mammals. Zearalenone is a polyketide derived from the sequential condensation of multiple acetate units by a polyketide synthase (PKS), but the genetics of its biosynthesis are not understood. We cloned two genes, designated ZEA1 and ZEA2, which encode polyketide synthases that participate in the biosynthesis of zearalenone by Gibberella zeae (anamorph Fusarium graminearum). Disruption of either gene resulted in the loss of zearalenone production under inducing conditions. ZEA1 and ZEA2 are transcribed divergently from a common promoter region. Quantitative PCR analysis of both PKS genes and six flanking genes supports the view that the two polyketide synthases make up the core biosynthetic unit for zearalenone biosynthesis. An appreciation of the genetics of zearalenone biosynthesis is needed to understand how zearalenone is synthesized under field conditions that result in the contamination of grain.     

Effects of Temperature and Moisture on Development of Fusarium graminearum Perithecia in Maize Stalk Residues

Fusarium graminearum is the predominant component of the Fusarium head blight complex of wheat. F. graminearum ascospores, which initiate head infection, mature in perithecia on crop residues and become airborne. The effects of temperature (T) and moisture on perithecium production and maturation and on ascospore production on maize stalk residues were determined. In the laboratory, perithecia were produced at temperatures between 5 and 30°C (the optimum was 21.7°C) but matured only at 20 and 25°C. Perithecia were produced when relative humidity (RH) was ≥75% but matured only when RH was ≥85%; perithecium production and maturation increased with RH. Equations describing perithecium production and maturation over time as a function of T and RH (R² > 0.96) were developed. Maize stalks were also placed outdoors on three substrates: a grass lawn exposed to rain; a constantly wet, spongelike foam exposed to rain; and a grass lawn protected from rain. No perithecia were produced on stalks protected from rain. Perithecium production and maturation were significantly higher on the constantly wet foam than on the intermittently wet lawn (both exposed to rain). Ascospore numbers but not their dispersal patterns were also affected by the substrate.     

Role of zearalenone lactonase in protection of Gliocladium roseum from fungitoxic effects of the mycotoxin zearalenone

Zearalenone is a mycotoxin with estrogenic effects on mammals that is produced by several species of Fusarium. We found that zearalenone and its derivatives inhibit the growth of filamentous fungi on solid media at concentrations of < or =10 microg/ml. The fungitoxic effect declined in the order zearalenone > alpha-zearalenol > beta-zearalenol. The mycoparasitic fungus Gliocladium roseum produces a zearalenone-specific lactonase which catalyzes the hydrolysis of zearalenone, followed by a spontaneous decarboxylation. The growth of G. roseum was not inhibited by zearalenone, and the lactonase may protect G. roseum from the toxic effects of this mycotoxin. We inactivated zes2, the gene encoding zearalenone lactonase in G. roseum, by inserting a hygromycin resistance cassette into the coding sequence of the gene by means of Agrobacterium tumefaciens-mediated genetic transformation. The zes2 disruption mutants could not hydrolyze the lactone bond of zearalenone and were more sensitive to zearalenone. These data are consistent with a hypothesis that resorcylic acid lactones exemplified by zearalenone act to reduce growth competition by preventing competing fungi from colonizing substrates occupied by zearalenone producers and suggest that they may play a role in fungal defense against mycoparasites.     

Zearalenone induces apoptosis and necrosis in porcine granulosa cells via a caspase-3- and caspase-9-dependent mitochondrial signaling pathway

Zearalenone is a mycotoxin produced mainly by Fusarium. There are numerous incidences of mycotoxicosis in laboratory and domestic animals, especially in pigs. However, little is known about molecular mechanisms of zearalenone toxicity. Granulosa cells are the maximal cell population in follicles, and they play an essential role in the development and maturation of follicles. The objective of this study was to explore the effect of zearalenone at high concentrations on proliferation and apoptosis of porcine granulosa cells and uncover signaling pathway underlying the cytotoxicity of zearalenone. We found that zearalenone reduced the proliferation of porcine granulosa cells in a dose-dependent manner as shown by the MTT assay and zearalenone resulted in an obvious apoptosis and necrosis in porcine granulosa cells as determined by the TUNEL analysis and flow cytometry. In addition, TUNEL assay with caspase inhibitors showed that zearalenone triggered a caspase-3- and caspase-9-dependent apoptotic process in porcine granulosa cells. Fluorescence spectrophotometer displayed that zearalenone led to a loss of mitochondrial transmembrane potential of porcine granulosa cells but enhanced reactive oxygen species (ROS) levels of the cells. Notably, Western blots revealed that caspase-3 and caspase-9 were activated by zearalenone in porcine granulosa cells. Collectively, our results suggest that zearalenone induces apoptosis and necrosis of porcine granulosa cells in a dose-dependent manner via a caspase-3- and caspase-9-dependent mitochondrial pathway. This study thus offers a novel insight into molecular mechanisms by which zearalenone has adverse cytotoxicity on reproduction.     

alpha-Zearalenol, a major hepatic metabolite in rats of zearalenone, an estrogenic mycotoxin of Fusarium species

The chemical and biological properties of the hepatic metabolite of zearalenone, an estrogenic and non-steroidal fungal toxin produced by Fusarium species, were investigated by employing TLC, GC/MS, high pressure liquid chromatography and fluorospectral analyses, as well as uterine weight bioassay in immature mice. All the chemical and physical data supported the view that the major metabolite, obtained by incubating zearalenone with S-9 and microsomes of rat liver in the presence of NADPH, is C-6'-alpha-hydroxylated zearalenone (alpha-zearalenol). The estrogenic activity of this metabolite was several times higher than that of the parent zearalenone, and the results of biological and toxicological evaluations of alpha-zearalenol are discussed.     

Natural occurrence of deoxynivalenol, 15-acetyl-deoxynivalenol, and zearalenone in refusal factor corn stored since 1972.

Two samples of "refusal factor" corn, one stored frozen in Minnesota and one stored dry in Indiana since 1972 or 1973, were analyzed for the presence of Fusarium spp. and Fusarium toxins. Both samples were from corn refused by swine in Indiana from 1972 to 1973. Sample FS 808 (stored in Indiana) contained 20 ppm of deoxynivalenol (20 micrograms/g), 16 ppm of 15-acetyl-deoxynivalenol, 5 ppm of zearalenone, and 0.2 ppm of alpha-zearalenol. Sample FS 362 (stored in Minnesota) contained 3 ppm of deoxynivalenol, 1 ppm of 15-acetyldeoxynivalenol, and 0.3 ppm of zearalenone. The presence of 15-acetyl-deoxynivalenol is significant because it is the first report of it occurring naturally in refusal factor corn, and it may account in part for the refusal that could not be solely attributed to deoxynivalenol.