Neosolaniol Research Articles

Metabolic pathways of trichothecenes

Trichothecenes are a group of mycotoxins mainly produced by the fungi of Fusarium genus. Consumers are particularly concerned over the toxicity and food safety of trichothecenes and their metabolites from food-producing animals. The metabolism of T-2 toxin, deoxynivalenol (DON), nivalenol (NIV), fusarenon-X (FX), diacetoxyscirpenol (DAS), 3-acetyldeoxy-nivalenol (3-aDON), and 15-acetyldeoxynivalenol (15-aDON) in rodents, swine, ruminants, poultry, and humans are reviewed in this article. Metabolic pathways of these mycotoxins are very different. The major metabolic pathways of T-2 toxin in animals are hydrolysis, hydroxylation, de-epoxidation, and conjugation. After being transformed to HT-2 toxin, it undergoes further hydroxylation at C-3' to yield 3'-hydroxy-HT-2 toxin, which is considered as an activation pathway, whereas transformation from T-2 to T-2 tetraol is an inactivation pathway in animals. The typical metabolites of T-2 toxin in animals are HT-2 toxin, T-2 triol, T-2 tetraol, neosolaniol (NEO), 3'-hydroxy-HT-2, and 3'-hydroxy-T-2, whereas HT-2 toxin is the main metabolite in humans. De-epoxidation is an important pathway for detoxification in animals. De-epoxy products, DOM-1, and de-epoxy-NIV are the main metabolites of DON and NIV in most animals, respectively. However, the two metabolites are not found in humans. Deacetyl can occur rapidly on the acetyl derivatives, 3-aDON, 15-aDON, and FX. DAS is metabolized in animals to 15-monoacetoxyscirpenol (15-MAS) via C-4 deacetylation and then transformed to scirpentriol (SCP) via C-15 deacetylation. Finally, the epoxy is lost, yielding de-epoxy-SCP. De-epoxy-15-MAS is also the main metabolite of DAS. 15-MAS is the main metabolite in human skin. The review on the metabolism of trichothecenes will help one to well understand the fate of these toxins' future in animals and humans, as well as provide basic information for the risk assessment of them for food safety.

Multitoxin extraction and detection of trichothecenes in cereals: an improved LC‐MS/MS approach

AbstractBACKGROUND: Many multiresidual methods to evaluate natural occurrence of Fusarium toxins are already reported in the scientific literature but a new rapid, reliable, cost‐efficient and high‐sensitivity method for the simultaneous determination of several fusariotoxins is always welcome. Nivalenol (NIV), deoxynivalenol, fusarenon‐X (FUS‐X), 3‐acetyldeoxynivalenol, diacetoxyscirpenol (DAS), HT‐2 toxin, T‐2 toxin, neosolaniol (NEO), zearalanone and zearalenone (ZON) belong to the most common mycotoxins in food matrix grains, e.g., wheat and maize. The proposed method is a multitoxin analytical method that combines high‐performance liquid chromatography (HPLC), atmospheric pressure chemical ionization (APCI), triple‐quadrupole tandem mass spectrometry (LC‐MS/MS) under the selected reaction monitoring (SRM) mode, and it is focused on the optimization of the sample preparation without the need for any cleanup.RESULTS: Three different methods for sample preparation and for the simultaneous extractions of the above‐mentioned fusariotoxins were tested: two of these were followed by a different cleanup step for comparison, while the extraction method proposed in this work, which uses an 84% (v/v) acetonitrile aqueous solution, sample homogenization and subsequent filtration, was validated without any further cleanup step.CONCLUSION: Calibration curves for all analytes are linear, except DAS, HT‐2 and ZON, over the working range of 10–1000 µg kg−1. The calibration curve of DAS was linear between 10 and 500 µg kg−1, although the curves of HT‐2 and ZON were linear in the range 10–250 µg kg−1. Squared correlation coefficients (R2) were in the range 0.995–0.998 for the all point calibration curves. The lowest limits of detection (LOD) were found for DON and ZAN with 0.5 and 0.2 µg kg−1, respectively, while the highest LODs were obtained for NIV, FUS‐X and NEO, with 3.3 µg kg−1 for each toxin. Copyright © 2009 Society of Chemical Industry

Cloning, expression, and characterization of a single-domain antibody fragment with affinity for 15-acetyl-deoxynivalenol

A single-domain variable heavy chain (V HH) antibody fragment specific to the mycotoxin 15-acetyldeoxynivalenol (15-AcDON) was obtained after immunization of a llama ( Llama glama) with the protein conjugate 15-DON-BSA plus TiterMax™ Classic adjuvant. After confirmation of a polyclonal response to DON toxin in both conventional (cIgG) and heavy chain antibody (HCAb) fractions, a V HH library was constructed from amplified cDNA by nested PCR. V HH fragments with binding affinity for the mycotoxin were selected by panning of the phagemid library against microtiter plates coated with 15-DON-OVA. The dominant clone (NAT-267) was expressed in E. coli and was purified as a V HH monomer (mNAT-267) at a final concentration of 1.3 mg mL −1. Isolated NAT-267 V HH DNA was fused to the homopentamerization domain of the B subunit of verotoxin to generate the pentabody format of single-domain antibody (sAb). The V HH pentamer (pNAT-267) was expressed in E. coli and was purified at a final concentration of 1.0 mg mL −1. Surface plasmon resonance (SPR) analysis of soluble mNAT-267 binding kinetics to immobilized 15-DON-Horse Radish Peroxidase (HRP) indicated a dissociation constant ( K D) of 5 μM. Competitive direct enzyme-linked immunosorbent assay (CD-ELISA) and fluorescence polarization assay (FPA) inhibition experiments with monomer and pentamer confirmed binding to 15-AcDON. Competitive inhibition FPAs with mNAT-267 and pNAT-267 determined IC 50 values of 1.24 and 0.50 μM, respectively, for 15-AcDON hapten. These values were similar to the IC 50 value of 1.42 μM for 15-AcDON given by polyclonal llama serum sampled 56 days after immunization. Competition formats for structurally related trichothecenes resulted in no cross-reactivity to: DON; 3-acetyldeoxynivalenol (3-AcDON); neosolaniol (NEO); diacetoxyscirpenol (DAS); and T-2 toxin. Our study confirmed that recombinant V HH fragments capable of binding low molecular weight haptens can be produced through the creation and panning of hyper-immunized single-domain (sdAb) libraries.

Occurrence of Fusarium species and trichothecenes in Nigerian maize

A total of 180 maize samples meant for human consumption from four maize-producing states of southwestern Nigeria were screened for twelve major Fusarium mycotoxins (trichothecenes). Mycological examination of the samples showed that Fusarium verticillioides was the most commonly isolated fungi (71%), followed by F. sporotrichioides (64%), F. graminearum (32%), F. pallidoroseum (15%), F. compactum (12%), F. equiseti (9%), F. acuminatum (8%), F. subglutinans (4%) and F. oxysporum (1%). The trichothecenes include deoxynivalenol (DON), 3, mono-acetyldeoxynivalenol (3-AcDON), 15, mono-acetyldeoxynivalenol (15-AcDON), nivalenol (NIV), HT-2 toxin (HT-2), neosolaniol (NEO), T-2 toxin (T-2), T-2 tetraol and T-2 triol, diacetoxyscirpenol (DAS), MAS-monoacetoxyscirpenol (MAS) and fusarenone-X. Quantification was by high performance liquid chromatography coupled with mass spectroscopy (HPLC/MS); the detection limits for each of the mycotoxins varied between 20 and 200 μg kg − 1 . Sixty six samples (36.3%) were contaminated with trichothecenes, DON (mean: 226.2 μg kg − 1 ; range: 9.6–745.1 μg kg − 1 ), 3-AcDON (mean: 17.3 μg kg − 1 ; range: 0.7–72.4 μg kg − 1 ) and DAS (mean: 16.0 μg kg − 1 ; range: 1.0–51.0 μg kg − 1 ) were detected in 22%, 17% and 9% of total samples respectively. There were no 15-AcDON, NIV, HT-2, NEO, T-2, T-2 tetraol, T-2 triol, MAS and fusarenone-X detected. This is the first comprehensive report about the natural occurrence of DON, AcDON and DAS in maize for direct human consumption in Nigeria.

Natural Occurrence of 16 Fusarium Toxins in Grains and Feedstuffs of Plant Origin from Germany

A total of 220 samples comprising cereals, cereal byproducts, corn plants and corn silage as well as non-grain based feedstuffs was randomly collected during 2000 and 2001 from sources located in Germany and analysed for 16 Fusarium toxins. The trichothecenes scirpentriol (SCIRP), 15-monoacetoxyscirpenol (MAS), diacetoxyscirpenol (DAS), T-2 tetraol, T-2 triol, HT-2 and T-2 toxin (HT-2, T-2), neosolaniol (NEO), deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivealenol (15-ADON), nivalenol (NIV) and fusarenon-X (FUS-X) were determined by gas chromatography/mass spectrometry. Zearalenone (ZEA) and alpha- and beta-zearalenol (alpha- and beta-ZOL) were analysed by high performance liquid chromatography with fluorescence and UV-detection. Detection limits ranged between 1 and 19 microg/kg. Out of 125 samples of a group consisting of wheat, oats, corn, corn byproducts, corn plants and corn silage only two wheat samples did not contain any of the toxins analysed. Based on 125 samples the incidences were at 2-11% for DAS, NEO, T-2 Triol, FUS-X, alpha- and beta-ZOL, at 20-22% for SCIRP, MAS, T-2 tetraol and 3-ADON, at 44-74% for HT-2, T-2, 15-ADON, NIV and ZEA, and at 94% for DON. Mean levels of positive samples were between 6 and 758 microg/kg. Out of 95 samples of a group consisting of hay, lupines, peas, soya meal, rapeseed meal and other oil-seed meals, 64 samples were toxin negative. DAS, T-2 triol, NEO and FUS-X were not detected in any sample. The incidences of DON and ZEA were at 14 and 23% respectively, those of the other toxins between 1-4%, mean levels of positive samples were between 5 and 95 microg/kg.

Morphological, chemical and molecular differentiation of Fusarium equiseti isolated from Norwegian cereals

The morphological variation, secondary metabolite profiles and restriction fragment length polymorphisms (RFLPs) of PCR amplified intergenic spacer (IGS) ribosomal DNA (rDNA) were studied in 27 isolates of Fusarium equiseti, 25 isolated from Norwegian cereals and 2 from soil obtained from the IBT culture collection (BioCentrum, Technical University of Denmark). All 27 isolates were tested for production of fusarochromanone (FUSCHR), zearalenone (ZEA) and the trichothecenes: 15-monoacetoxy-scirpentriol (MAS), diacetoxy-scirpenol (DAS), T-2 and HT-2 toxins, T2-triol, neosolaniol (NEO), deoxynivalenol (DON), nivalenol (NIV) and 4-acetylnivalenol (Fus-X). The trichothecenes were analysed by GC-MS in a selected ion monitoring mode, while FUSCHR was determined by ion pair HPLC with fluorometric detection and production of ZEA by TLC. For amplification of IGS rDNA primers CNL12 and CNS1 were applied. IGS rDNA was digested with the four restriction enzymes: AvaII, CfoI, EcoRI and Sau3A. In addition, we sequenced the IGS rDNA region of three of the Norwegian isolates. There were two morphological types among the Norwegian strains of F. equiseti, type I with short apical cells (dominating) and type II with long apical cells, with four haplotypes identified based on the RFLP data. Variation in secondary metabolite profiles within and between the morphological groups was observed and the levels of produced toxins were: FUSCHR 3000–42,500 and 25–30 ng/g, NIV 20–2500 and 120–700 ng/g, FUS-X 20–15,000 and 0 ng/g, DAS 30–7500 and 0–600 ng/g, and MAS 10–600 and 0–500 ng/g, for strains with short and long apical cells, respectively. NEO was detected in 16/27 strains tested (all morphotype I). All but four strains of type I (these four lacked a restriction site for EcoRI) had identical RFLP profiles. The isolates of type II had two haplotypes. The IGS sequence similarity data indicated differences between these morphotypes corresponding to two separate lineages apparently at the species level.

Survey of Fusarium toxins in foodstuffs of plant origin marketed in Germany

A total of 219 samples of foodstuffs of plant origin, consisting of grain-based food, pseudocereals and gluten-free food as well as vegetables, fruits, oilseeds and nuts, were randomly collected during 2000 and 2001 in food and health food stores. A spectra of 13 trichothecene toxins including diacetoxyscirpenol (DAS), 15-monoacetoxyscirpenol (MAS), scirpentriol (SCIRP), T-2 and HT-2 toxins (T-2, HT-2), T-2 triol, T-2 tetraol, neosolaniol (NEO) of the A-type as well as deoxynivalenol (DON), 3- and 15-acetyl-DON (3-, 15-ADON), nivalenol (NIV), and fusarenon-X (FUS-X) of the B-type were determined by gas chromatography/mass spectrometry. Analysis of zearalenone (ZEA), α- and β-zearalenol (α- and β-ZOL) was made by high-performance liquid chromatography with fluorescence and UV-detection. Detection limits ranged between 1 and 19 μg/kg. Out of 84 samples of cereal-based including gluten-free foods, 60 samples were positive for at least one of the toxins DON, 15-ADON, 3-ADON, NIV, T-2, HT-2, T-2 tetraol and ZEA, with incidences at 57%, 13%, 1%, 10%, 12%, 37%, 4% and 38%, respectively, whereas SCIRP and its derivatives MAS and DAS, T-2 triol, Fus-X as well as α- and β-ZOL were not detected in any sample of this subgroup. Contents of DON ranged between 8 and 389 μg/kg, for all other toxins determined concentrations were below 100 μg/kg. The pseudocereals amaranth, quinoa and buckwheat were free of the toxins investigated. Ten of 85 samples of vegetables and fruits were toxin positive. ZEA and the type A trichothecenes MAS, SCIRP, DAS, HT-2 were detected in 7, 3, 2, 1 and 1 samples, respectively. Out of 35 samples of oilseeds and nuts, 7 samples were toxin positive. HT-2, T-2 and ZEA were detected in 4, 3 and 4 samples, respectively. In vegetables and fruits as well as in oilseeds and nuts, toxin levels were below 50 μg/kg. None of the B-type trichothecenes analysed was found for both subgroups.

Diversity in metabolite production by Fusarium langsethiae, Fusarium poae, and Fusarium sporotrichioides

The production of mycotoxins and other metabolites by 109 strains of Fusarium langsethiae, Fusarium poae, Fusarium sporotrichioides, and F. kyushuense was investigated independently in four laboratories by liquid or gas chromatography analyses of cultural extracts with UV diode array, electron capture, or mass spectrometric detection systems. From the compiled results, it was found that F. langsethiae consistently produced the trichothecenes diacetoxyscirpenol (DAS), T-2 toxin (T-2), HT-2 toxin (HT-2), and neosolaniol (NEO) and, to a lesser extent, some additional trichothecene derivatives. F. langsethiae also produced culmorins, chrysogine (CHRYS), aurofusarin (AUF), and enniatin (EN). F. sporotrichioides showed a metabolite profile similar to that of F. langsethiae, while F. poae had a different profile as 41 of 49 strains produced nivalenol (NIV) and other 8-keto trichothecenes, in addition to DAS and derivatives of this metabolite. Only a trace amount of NIV was detected from one strain of F. kyushuense. In summary, all the three core taxa of this joint study were found to produce trichothecenes. Fusarin C (F-C) was not detected from F. langsethiae, but it was produced by F. poae and F. sporotrichioides. Aurofusarin was only detected from a few strains of F. langsethiae, while nearly all strains of F. poae and F. sporotrichioides produced this compound. In contrast, chrysogine was not detected from F. poae, but was produced by the other two taxa. Production of enniatins was scattered among the three main taxa of this study, whereas beauvericin (BEA) was produced by many strains of F. poae and F. sporotrichioides. Only one odd strain of F. langsethiae (IBT 9959) produced beauvericin. However, the status of this strain is uncertain. By a polyphasic approach using species-specific metabolite profiles, the fruity odour of F. poae, and morphological observations, it was concluded that F. langsethiae, F. poae, and F. sporotrichioides should be regarded as three significant taxa at a species level.

Tri16 is required for esterification of position C-8 during trichothecene mycotoxin production by Fusarium sporotrichioides.

We previously characterized Tri1, a gene required for hydroxylation of the C-8 position during trichothecene mycotoxin biosynthesis in Fusarium sporotrichioides NRRL 3299. Sequence analysis of the region surrounding Tri1 revealed a gene, named Tri16, which could encode an acyltransferase. Unlike the wild-type parent strain NRRL 3299, which accumulates primarily T-2 toxin along with low levels of diacetoxyscirpenol (DAS) and neosolaniol (NEO) and trace amounts of 8-propionyl-neosolaniol (P-NEO) and 8-isobutyryl-neosolaniol (B-NEO), mutants containing a disruption of Tri16 were blocked in the production of the three C-8 esterified compounds T-2 toxin, P-NEO, and B-NEO and accumulated the C-8-hydroxylated compound NEO along with secondary levels of DAS. These data indicate that Tri16 encodes an acyltransferase that catalyzes the formation of ester side groups at C-8 during trichothecene biosynthesis. We also report the presence of a Tri16 ortholog in Gibberella pulicaris R-6380 that is likely linked to a presumably inactive ortholog for Tri1.

Effects of Azoxystrobin on Mycotoxin Production in a Carbendazim-Resistant Strain ofFusarium sporotrichioides

Carbendazim-resistant (RS) and control (CS) strains ofFusarium sporotrichioides Sherb., previously developed in our laboratory, were exposed to graded concentrations of azoxystrob in in broth media under shake-culture conditions for 2, 3, 4 and 8 days. Azoxystrobin concentrations were 0, 1, 10 and 100 mg 1-1 broth and cultures were incubated at a constant 25°C. Mycelial growth was significantly affected by strain (P 0.05) at all three doses of azoxystrobin. Significant (P<0.05 or better) strain-fungicide interactions were recorded in trichothecene production following exposure to azoxystrobin. At 4 and 8 days of incubation, the 10 mg 1-1 addition of azoxystrobin stimulated T-2 toxin synthesis (P<0.05) only in RS cultures. In contrast, T-2 toxin enhancement in CS cultures occurred only on day 8 but at a lower level of azoxystrobin (1 mg1-1). Thus, the stimulation of T-2 toxin synthesis depended upon strain and azoxystrobin level. Production of diocetoxyscirpenol (DAS) was affected by a more complex set of interactions. Overall means showed that, in comparison with initial values (on day 2 or 3), DAS output maximized significantly(P<0.05) on day 4 in RS cultures and on day 8 in CS. Marked strain effects were observed on exposure to the 10 mg 1-1 level of azoxystrobin. At this level, DAS production was enhanced in RS only after 4 (P<0.01 ) and 8 (P<0.05) days of incubation, while in contrast, CS reduced DAS production. As with T-2 toxin, DAS production in CS was stimulated (P<0.05 or better) only at low exposure levels of azoxystrobin. In the case of neosolaniol (NEO), however, the main effect of strain was significant (P<0.05), with CS producing consistently more of the mycotoxin than RS on day 4 of the experiment. At this point, the NEO:T-2 toxin ratio was also higher in CS (0.63) than in RS (0.12), a feature reported by us previously. In conclusion, the present investigation has shown for the first time that the development of resistance to one fungicide can affect trichothecene production inF. sporotrichioides on exposure to a second fungicide. These results have been incorporated into a new classification scheme for fungicide efficacy which is also presented in this paper.

Mycotoxin production in a carbendazim-resistant strain of fusarium sporotrichioides.

Mycelial yield and production of three trichothecenes, namely T-2 toxin, diacetoxyscirpenol (DAS) and neosolaniol (NEO) were compared in control (CS) and carbendazim-resistant strains (RS) ofFusarium sporotrichioides. Each strain was exposed to graded concentrations of carbendazim (0, 1, 2, and 4 μg/ml media) for 2, 5 and 7 days under shake-culture conditions at an incubation temperature of 25°C. Mycelial yield was significantly (P<0.001) affected by strain, carbendazim concentration and incubation time. The strain differences in mycelial mass at 2 days (P<0.05) became more pronounced at 5 and 7 days of incubation (P<0.001). However, mycelial growth differences between the two strains were greatest following exposure to carbendazim, with the effects becoming more divergent with time. Combined results for the three incubation times showed dose related effects in carbendazim inhibition of T-2 toxin production by CS isolates. In contrast, RS cultures exposed to the 2 μg/ml addition of carbendazim significantly increased T-2 toxin production (P<0.05 or better). At 1 and 4 μg/ml additions, T-2 toxin inhibition occurred but the effect was less marked than in the CS series. RS yielded more DAS than CS at 5 days (P<0.05) and at 7 days (P<0.01) of incubation. The major component of this strain difference arose from the effects of the 2 μg/ml addition of carbendazim (P<0.01). NEO production was also higher in RS than in CS, with the difference becoming progressively more pronounced from day 5 (P<0.05) to day 7 (P<0.01) of incubation. However, these differences reflected enhanced NEO output with carbendazim addition of 4 μg/ml (P<0.05) in day 5 extracts and of both 2 μg/ml (P<0.01) and 4 μg/ml additions (P<0.05) in day 7 samples. Moreover, the ratio of NEO to T-2 toxin production was affected by an interaction involving incubation time, strain and carbendazim dose (P<0.05 or better). On day 5, this ratio was greater in CS exposed to 2 μg/ml, but at 4 μg/ml, the ratio was higher in RS. It is concluded that carbendazim resistance induced genuine differences in the synthesis of T-2 toxin and NEO. It is suggested that the strain difference may reside in the conversion of NEO to T-2 toxin which may be sensitive to fungicide concentration. This would imply that carbendazim resistance induces changes in the terminal rather than initial phases of trichothecene biosynthesis.

Fast methods for screening of trichothecenes in fungal cultures using GC-MS/MS

Fast methods for screening for production of simple and macrocyclic trichothecenes, produced on solid substrates were developed. Crude extracts fromFusarium cultures grown on yeast extract sucrose agar (approx. 0.3 cm(2) culture) were derivatised using pentafluoropropionic anhydride and analysed by GC - tandem mass spectrometry using a Finnigan GCQ(+) ion trap. The MS was operated in the EI(+) multiscan mode allowing simultaneous full scan and MS/MS of 3-4 parent ions. Production of acetyl T-2 toxin (AT-2), T-2 toxin, HT-2 toxin (HT-2), T-2 triol (T-2TR), T-2 tetraol (T-2TE), neosolaniol (NEO), iso-neosolaniol (I-NEO), scripentriol (SCR), 4,15-diacetoxyscirpenol (DAS), 15-acetoxyscripentriol (15MAS), 4-acetoxyscripentriol (4MAS), nivalenol (NIV), fusarenon-X (F-X), deoxynivalenol (DON), 15-acetoxy-DON (15DON) and 3-acetoxy-DON (3DON) was studied for severalFusarium species. In hydrolysed crude extracts ofStachybotrys albipes, Trichoderma harzianum, andMemnoniella echinata trichodermol was detected, in cultures ofS. chartarum both verrucarol and trichodermol were detected as the heptafluorobuturyl esters after derivatisation with a imidazole based reagent.

Toxicity, of extracts of barley kernels inoculated withFusarium spp. toArtemia salina

Toxicity toA. salina, of the Fusarium metabolites: deoxynivalenol (DON), its acetylated derivatives (3- and 15-AcDON), zearalenone (ZON), neosolaniol (NEO), nivalenol (NIV), T-2, HT-2 toxins, has been examined and compared with toxicity of extracts of barley kernels (8 cultivars and 4 lines) inoculated withFusarium culmorum, F. graminearum andF. sporotrichioides respectively. Estimated LC50 values were expressed as relative toxicity (RT) in mg DON/kg for samples inoculated withF. culmorum, F. graminearum or in mg T-2/kg forF. sporotrichioides inoculations.Toxicity of extracts of the same genotype/line kernels was compared among different pathogens used for inoculation and differences in Fusarium head blight susceptibility of different genotypes/lines inoculated with the sameFusarium strain were found. Significant correlation between toxicity of extracts (LC50, RT) and toxic metabolites concentration was found ([Formula: see text]; P = 0.01).Bioassays withA. Salina offer a fast, easy and inexpensive method to examine cereal genotypes susceptibility to Fusarium head blight and mycotoxins accumulation in kernels.

Production of neosolaniol by Fusarium tumidum.

Extracts from autoclaved maize culture of Fusarium tumidum strain R-5823 were toxic towards Artemia salina. Bioassay-guided fractionation of the organic extract led to the isolation of the toxic compound that was identified as the trichothecene toxin neosolaniol (NEOS) by 1H, 13C nuclear magnetic resonance spectroscopy and low-resolution electronic impact mass spectrometry. The amount of NEOS produced by the strain R-5823 was 300 mg/kg maize culture. NEOS was also detected by HPLC in cultures of four out of seven additional strains of F. tumidum and Gibberella tumida with different origin, in amounts ranging from 1 to 311 mg/kg. This is the first report on the production of a trichothecene toxin by F. tumidum.

Optimization of Yeast Bioassay for Trichothecene Mycotoxins

An improved disc diffusion type bioassay was developed for T-2 toxin (T-2), HT-2 toxin (HT-2), diacetoxyscirpenol (DAS), deoxynivalenol (DON), nivalenol (NIV), neosolaniol (NSL), fusarenon-X (FUS-X), trichothecin (TIN), roridin A (RDA) and verrucarin A (VCA) using the yeast, Kluyveromyces marxianus. Factors such as type of medium, agar volume per plate, preincubation time and temperature, incubation temperature, inoculum size and pH had variable, and in some cases a dramatic effect on the sensitivity of the assay. The effect of pH of the assay medium was particularly pronounced. The highest sensitivity was obtained when 6 ml per plate of a tryptic-soy-agar (TSA) medium (pH 7.5) containing 105 CFU inoculum per ml was incubated at 38°C for 18 h. All of the trichothecenes (TNN) were able to inhibit the growth of the yeast with the detection limit being 0.005, 0.01, 0.02, 0.02, 0.1, 0.5, 10, 10, 10 and 50 μg/disc for VCA, RDA, T-2, TTN, DAS, HT-2, DON, NSL, FUS-X and NIV, respectively. The detection limits for corn and wheat spiked with T-2 were 0.1 and 0.2 μg/g, respectively. Non-TNN mycotoxins that did not inhibit yeast at a concentration of 200 μg/disc were aflatoxin B1 (AFB1), ochratoxin A (OA), citrinin (CT), penicillic acid (PA), cyclopiazonic acid (CPA), penitrem-A (PTA) and zearalenone (ZEE). These results indicate that disc diffusion assay using K. marxianus under optimized conditions provides a sensitive method for the detection of low concentrations of several TNN.

Immunochemical analysis of trichothecenes produced by various fusaria

The production of type A trichothecene mycotoxins by 19 Fusaria, including 12Fusarium sporotrichioides, 4F. chlamydosporum and 3F. graminearum at 15°C and 25°C over a 35-day period was analyzed by ELISA using antibodies cross-reactive with most type A trichothecenes after conversion to T-2 tetraol tetraacetate. The toxin production peaked at 20–25 days of incubation with maximum yield between 4–6 mg type A trichothecene/ml of culture medium for 5F. sporotrichioides cultures and between 1 to 2 mg/ml for 6F. sporotrichioides cultures. OneF. sporotrichioides produced 700 µg type A trichothecenes/ml of culture medium. Detectable type A trichothecene was also found in the culture extracts ofF. chlamydosporum andF. graminearum, but the yield was very low (less than 100 µg/ml). Quantitative determination of individual trichothecenes was achieved by separation of different toxin in HPLC and followed by ELISA analysis. Eight to 10 immunoreactive peaks, corresponding to various type A trichothecenes, were detected in all the fungal extracts. T-2 tetraol (T-2-4ol), 4-acetyl-T-2 tetraol (4-Ac-T-2-4ol), neosolaniol (NEOS), diacetoxyscirpenol (DAS), HT-2 and T-2 toxin accounted for more than 85% of the total toxins. In general, low temperature was preferred for total type A trichothecene production. More T-2-4ol, 4-Ac-T-2-4ol, HT-2 and DAS were produced at 25°C. In contrast, more T-2 toxin and NEOS were produced at 15°C. Transformation of T-2 toxin and NEOS to polar metabolites such as T-2-4ol, 4-acetyl-T-2-4ol and HT-2 by various strains were observed at both temperatures after 25 days incubation.

High-performance liquid chromatographic screening method for mycotoxins using new retention indexes and diode array detection.

A reversed-phase high-performance liquid chromatographic (RP-HPLC) screening method for the determination of mycotoxins has been developed. The toxins were characterized by retention indexes and on-line UV spectra produced with a diode array detector (DAD). Retention indexes of mycotoxins exhibiting a wide range of polarities and chemical structures were determined during linear gradient elution with an acetonitrile/water solvent system. The retention index scale was based on the use of a new homologous series of 1-[4-(2,3-dihydroxypropoxy)phenyl]-1-alkanones (D-compounds) as internal standards. The mycotoxins studied were: five trichothecenes of group A: T-2 toxin (T-2), HT-2 toxin (HT-2), diacetoxyscirpenol (DAS), neosolaniol (NEO), and an isomer of neosolaniol (NEO isomer); three trichothecenes of group B: nivalenol (NIV), deoxynivalenol (DON), and fusarenon-X (FUS-X); four aflatoxins: B1, B2, G1, and G2; and five other mycotoxins: sterigmatocystin (STE), zearalenone (ZEA), ochratoxin A (OCH A), citrinin (CIT), and patulin (PAT).