Quantification of trichothecene-producing Fusarium species in harvested grain by competitive PCR to determine efficacies of fungicides against Fusarium head blight of winter wheat.
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We developed a PCR-based assay to quantify trichothecene-producing Fusarium based on primers derived from the trichodiene synthase gene (Tri5). The primers were tested against a range of fusarium head blight (FHB) (also known as scab) pathogens and found to amplify specifically a 260-bp product from 25 isolates belonging to six trichothecene-producing Fusarium species. Amounts of the trichothecene-producing Fusarium and the trichothecene mycotoxin deoxynivalenol (DON) in harvested grain from a field trial designed to test the efficacies of the fungicides metconazole, azoxystrobin, and tebuconazole to control FHB were quantified. No correlation was found between FHB severity and DON in harvested grain, but a good correlation existed between the amount of trichothecene-producing Fusarium and DON present within grain. Azoxystrobin did not affect levels of trichothecene-producing Fusarium compared with those of untreated controls. Metconazole and tebuconazole significantly reduced the amount of trichothecene-producing Fusarium in harvested grain. We hypothesize that the fungicides affected the relationship between FHB severity and the amount of DON in harvested grain by altering the proportion of trichothecene-producing Fusarium within the FHB disease complex and not by altering the rate of DON production. The Tri5 quantitative PCR assay will aid research directed towards reducing amounts of trichothecene mycotoxins in food and animal feed. (+info)
Identification of deoxynivalenol- and nivalenol-producing chemotypes of Gibberella zeae by using PCR.
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Gibberella zeae, a major cause of cereal scab, may be divided into two chemotypes based on production of the trichothecenes deoxynivalenol (DON) and nivalenol (NIV). We cloned and sequenced the gene cluster for trichothecene biosynthesis from each chemotype. G. zeae H-11 is a DON producer isolated from corn, and G. zeae 88-1 is a NIV producer from barley. We sequenced a 23-kb gene cluster from H-11 and a 26-kb cluster from 88-1, along with the unlinked Tri101 genes. Each gene cluster contained 10 Tri gene homologues in the same order and transcriptional directions as those of Fusarium sporotrichioides. Between H-11 and 88-1 all of the Tri homologues except Tri7 were conserved, with identities ranging from 88 to 98% and 82 to 99% at the nucleotide and amino acid levels, respectively. The Tri7 sequences were only 80% identical at the nucleotide level. We aligned the Tri7 genes and found that the Tri7 open reading frame of H-11 carried several mutations and an insertion containing 10 copies of an 11-bp tandem repeat. The Tri7 gene from 88-1 carried neither the repeat nor the mutations. We assayed 100 G. zeae isolates of both chemotypes by PCR amplification with a primer pair derived from the Tri7 gene and could differentiate the chemotypes by polyacrylamide gel electrophoresis. The PCR-based method developed in this study should provide a simple and reliable diagnostic tool for differentiating the two chemotypes of G. zeae. (+info)
4-Acetyl-12,13-epoxyl-9-trichothecene-3,15-diol isolated from the fruiting bodies of Isariajaponica Yasuda induces apoptosis of human leukemia cells (HL-60).
(19/447)
The fruiting bodies of Isaria fungi have been traditionally used in Korea to treat cancer. An apoptosis-inducing compound, 4-acetyl-12,13-epoxyl-9-trichothecene-3,15-diol, was isolated from the methanol extract of fruiting bodies of Isaria japonica Yasuda by bioassay-guided fractionation. The apoptosis of the human leukemia cells (HL-60) by the compound was accessed by propidium iodide-staining flow cytometric analysis, and apoptosis-inducing activity at IC50 concentration (10 nmol/l) was further confirmed by a nuclear morphological change, a ladder pattern of internucleosomal DNA fragmentation, and an activation of caspase-3. (+info)
Assay and relationship of HT-2 toxin and T-2 toxin formation in liquid culture.
(20/447)
Both T-2 toxin and HT-2 toxin can be conveniently quantitated in crude extracts by using a combination of thin-layer chromatography and fluorodensitometry. This technique was used to follow the production of these toxins by liquid cultures of Fusarium poae (NRRL 3287). T-2 toxin was produced prior to HT-2 toxin and hexadeuterio-T-2 toxin was converted by the culture to trideuterio-HT-2 toxin. (+info)
Comparative studies on microbial and chemical modifications of trichothecene mycotoxins.
(21/447)
The microbial modification of several trichothecene mycotoxins by trichothecene-producing strains of Fusarium nivale and F. solani was studied. These results were compared with the corresponding chemical modifications. The growing mycelia of Fusarium spp. did not convert 4beta-acetoxy-3alpha,7alpha, 15-trihydroxy-12, 13-epoxytrichothec-9-en-8-one (fusarenon) into 3alpha,4beta, 7alpha,15-tetrahydroxy-12,13-epoxy-trichothec-9-en-8-one (nivalenol), whereas 3alpha,4beta,7alpha,15-tetracetoxy-12,13-epoxytrichothec-9-en-8-one (tetraacetylnivalenol) was deacetylated to yield 3alpha-hydroxy-4beta,7alpha,15-triacetoxy-12,13-epoxytrichothec-9-en-8-one (4,7,15-triae-tylnivalenol), which was resistant to further deacetylation. T-2 toxin was transformed intoHT-2 toxin, and 8alpha-(3-methylbutyryloxy)-3alpha,4beta,-15-triacetoxy-12,13-epoxytrichothec-9-e n-8-one (T-2 acetate) was transformed into HT-2 toxin via T-2 toxin. Chemical modification with ammonium hydroxide converted tetraacetylnivalenol into fusarenon via 4,7,15-triacetylnivalenol. 3alpha-7alpha,15-Triacetoxy-12,13-epoxytrichothec-9-en-8-one (triacetyldeoxynivalenol) gave deacetylation products lacking the C-7 or c-15 acetyl group in addition to 7alpha,15- diacetoxy-3alpha-hydroxy-12, 13-epoxytrichothec-9-en-8-one (7,15-diacetyldeoxynivalenol). These results demonstrate the regio-selectivity in microbial modification of trichothecenes. Based on the results and available knowledge concerning the transformation of trichothecenes, mechanisms for biological modifications of these mycotoxins are postulated. (+info)
Production of trichothecene mycotoxins by Fusarium species in shake culture.
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Twelve T-2 toxin-producing isolates and four fusarenon-X-producing isolates of Fusarium species were examined for their ability to produce trichothecene mycotoxins in shake culture and jar fermentation. T-2 toxin producers such as Fusarium solani, F. sporotrichiodes, and F. tricinctum produced T-2 toxin and neosolaniol in semisynthetic medium. F. solani M-1-1 produced the largest amount of the mycotoxins in a nutrient medium consisting of 5% glucose (or sucrose), 0.1% peptone, and 0.1% yeast extract in either shake culture or jar fermentation at 24 to 27 C for 5 days. None of the isolates produced significant amount of fusarenon-X in shake cultures. (+info)
Skeletal deformities induced by the intraperitoneal administration of deoxynivalenol (vomitoxin) in mice.
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The contamination of drinking water by organic acids, selenium deficiency and the ingestion of fungal mycotoxins are the three main aetiological factors in the development of Kashin-Beck disease. An avian tibial chondrodysplasia induced by mycotoxins has been reported. Deoxynivalenol (DON) is one of many mycotoxins produced by the most common contaminating species of fungi. The pattern of skeletal malformations induced by its administration intraperitoneally to pregnant mice is reported. Costo-vertebral segmentation abnormalities were the main deformities observed. The chondrodysplasia previously described was not seen. (+info)
A novel regulatory gene, Tri10, controls trichothecene toxin production and gene expression.
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We report here the characterization of Tri10, a novel regulatory gene within the trichothecene gene cluster. Comparison of Tri10 genomic and mRNA sequences revealed that removal of a single 77-bp intron provided a 1,260-bp open reading frame, encoding a 420-amino-acid protein. Disruption of Tri10 in Fusarium sporotrichioides abolished T-2 toxin production and dramatically decreased the transcript accumulation for four trichothecene genes (Tri4, Tri5, Tri6, and Tri101) and an apparent farnesyl pyrophosphate synthetase (Fpps) gene. Conversely, homologous integration of a disruption vector by a single upstream crossover event significantly increased T-2 toxin production and elevated the transcript accumulation of the trichothecene genes and Fpps. Further analysis revealed that disruption of Tri10, and to a greater extent the disruption of Tri6, increased sensitivity to T-2 toxin under certain growth conditions. Although Tri10 is conserved in Fusarium graminearum and Fusarium sambucinum and clearly plays a central role in regulating trichothecene gene expression, it does not show any significant matches to proteins of known or predicted function or to motifs except a single transmembrane domain. We suggest a model in which Tri10 acts upstream of the cluster-encoded transcription factor TRI6 and is necessary for full expression of both the other trichothecene genes and the genes for the primary metabolic pathway that precedes the trichothecene biosynthetic pathway, as well as for wild-type levels of trichothecene self-protection. We further suggest the presence of a regulatory loop where Tri6 is not required for the transcription of Tri10 but is required to limit the expression of Tri10. (+info)