Two new trichothecenes produced by Fusarium sp.

Two new 12,13-epoxytrichothecenes have been isolated from the culture filtrate of Fusarium sp. strain K-5036 and characterized as 4β,15-diacetoxy     

Trichothecene mycotoxins from Fusarium sulphureum

Four mycotoxins isolated from moulded maize cultures of Fusarium sulphureum have been characterized as 3α,4β,15-triacetoxy-12,13-epoxytrichothec-9-ene, 4β,15-diacetoxy-3α-hydroxy-12,13-epoxytrichothec-9-ene, 15-acetoxy-3α,4β-dihydroxy-12,13-epoxytrichothec-9-ene and 4β-acetoxy-3α,15-dihydroxy-12,13-epoxytrichothec-9-ene.     

A new mycotoxin from Aspergillus candidus Link isolated from rough rice

A new mycotoxin (AcT₁) was obtained from the mycelium of Aspergillus candidus Link isolated from rough rice stored under tropical conditions. AcT₁ with the mol-formula C₂₈H₄₀O showed bright greenish blue fluorescence under uv (254 nm). The spectral and other characteristics indicated that the compound was a new one. The LD₅₀ of the toxin on white rats was found to be 4.5 mg/Kg when injected intraperitoneally.     

Phylogenecity and sequence alignment of Fusarium mycotoxin gene (Fum 5) with other mycotoxin producing fungi

The phylogenecity of the four Fusarium mycotoxin gene (Fum 5) that has already been deposited in National Center for Biotechnology Information (NCBI) gene bank was studied with a comparative sequence alignment with other fungi like Aspergillus sps. Cochiliobolus sps. These results clearly indicated that the Fum 5 genes of Fusarium species were originated from Aspergillues fumigates and Cochliobolus heterostrophus. Fum 5 gene closer identity was also observed clearly with other fungal Fusarium species of Aspergillus and Gibberella species.     

Chlamydosporol,a new metabolite from Fusarium chlamydosporum

Extracts of rice on which an isolate of Fusarium chlamydosporum had been cultured were toxic to brine shrimps. The toxic fraction was purified by flash chromatography to give two compounds which were identified by UV, IR, NMR and mass spectroscopy at the 6 and 6 isomers of 5-hydroxy-4-methoxy-6, 8a-dimethyl-6,7-dihydro-2H,8aH-pyrano[2,3-b]pyran-2-one. These lactones for which the name chlamydosporol is proposed have not been reported previously. When tested in brine shrimp and HeLa cell assays, the LC₅₀ concentration for a mixture of the isomers was approximately 400 g/ml in both systems.     

Reduced susceptibility to Fusarium head blight in Brachypodium distachyon through priming with the Fusarium mycotoxin deoxynivalenol

The fungal cereal pathogen Fusarium graminearum produces deoxynivalenol (DON) during infection. The mycotoxin DON is associated with Fusarium head blight (FHB), a disease that can cause vast grain losses. Whilst investigating the suitability of Brachypodium distachyon as a model for spreading resistance to F. graminearum, we unexpectedly discovered that DON pretreatment of spikelets could reduce susceptibility to FHB in this model grass. We started to analyse the cell wall changes in spikelets after infection with F. graminearum wild‐type and defined mutants: the DON‐deficient Δtri5 mutant and the DON‐producing lipase disruption mutant Δfgl1, both infecting only directly inoculated florets, and the mitogen‐activated protein (MAP) kinase disruption mutant Δgpmk1, with strongly decreased virulence but intact DON production. At 14 days post‐inoculation, the glucose amounts in the non‐cellulosic cell wall fraction were only increased in spikelets infected with the DON‐producing strains wild‐type, Δfgl1 and Δgpmk1. Hence, we tested for DON‐induced cell wall changes in B. distachyon, which were most prominent at DON concentrations ranging from 1 to 100 ppb. To test the involvement of DON in defence priming, we pretreated spikelets with DON at a concentration of 1 ppm prior to F. graminearum wild‐type infection, which significantly reduced FHB disease symptoms. The analysis of cell wall composition and plant defence‐related gene expression after DON pretreatment and fungal infection suggested that DON‐induced priming of the spikelet tissue contributed to the reduced susceptibility to FHB.     

Fluorescence polarization for mycotoxin determination

Over the last few years several laboratories have reported fluorescence polarization (FP) immunoassays for mycotoxins. These have included assays for fumonisins, deoxynivalenol and acetylated derivatives, aflatoxins, ochratoxin A, and zearalenone and related metabolites. Sensitivity in the FP assays may change dramatically over time, depending upon the antibody/tracer combination used. An important aspect of these homogeneous assays is the time required to reach an equilibrium endpoint. Although it is counterintuitive, the sensitivity of FP assays can actually be improved with shorter incubation times. However, the need for sensitivity must often be balanced against the need for the analyst to reproducibly time the incubation. The technical acumen of the analyst would be relatively more important in assays where measurements are taken before the system reaches equilibrium. In many cases the desired assays are those which reach equilibrium (and therefore give a stable endpoint) quickly, which may occur at the expense of sensitivity. It is for this reason the FP immunoassays are frequently not as sensitive as traditional ELISAs. Nevertheless, for many of the major mycotoxins rapid FP immunoassays can be developed, provided the appropriate combinations of antibody and tracer are used. Presented at the EU-USA Bilateral Workshop on Toxigenic Fungi & Mycotoxins, New Orleans, USA, July 5–7, 2005 Financial support: United States Department of Agriculture     

Experimental acute poisoning in mice induced by emestrin,a new mycotoxin isolated from Emericella species

The effects of emestrin (EMS), a secondary metabolite of the Emericella species, on male ICR mice were examined. The intraperitoneal LD₅₀ values of EMS were 17.7 and 13.0 mg/kg at 24 and 48 hr, respectively. The target organs of EMS were the heart, liver and thymus. In doses over 30 mg/kg the experimental animals died from cardiac failure shortly after the injections. Several survivors that were given EMS in doses under 20 mg/kg showed severe centrilobular necrosis in the liver at 24 hr. Marked degeneration of mitochondria was seen in electron micrographs of both cardiac muscle cells and hepatocytes. In the degenerated hepatocytes, prominent proliferation of RER, membrane-limited inclusions containing both ribosome-like granules and RER, and fenestrated lamella-like structures were observed. Massive necrosis of lymphocytes was always observed in the cortical layer of the thymus of the survivors within 24 hr, while bilateral adrenalectomized mice showed no discernible pathomorphological changes in the lymphoid tissues. Pretreatment of mice with diethyl maleate increased the incidence and severity of hepatic necrosis, whereas that with either cysteine or CoCl₂ reduced the severity of centrilobular necrosis of the liver. Pretreatment with phenobarbital had no significant effect on EMS-induced hepatic lesions.     

Novel developments in rapid mycotoxin detection

Rapid antibody-based mycotoxin screening techniques are designed to be used outside a laboratory environment, at the place of sampling. Results are expected immediately, so that commodities can be further processed without delay. Because they are used for mycotoxin analysis, very low levels (ppb and ppt range) should be detected. A further requirement is that the obtained results are accurate with a false negative rate of <5% at the level of interest. At first, plastic microtiter plates were used as solid phase materials for immobilizing antibodies (enzyme-linked immunosorbent assays). However, to increase speed and user-friendliness, plastics were replaced by microporous membranes. As an example a flow-through enzyme immunoassay for the detection of fumonisins in cornflakes with a cut-off value of 275 μg/kg is described. No false negative results were observed and the false positive rate was 18%. However, enzyme labels, used to enable visual evaluation of results, did not seem to be completely satisfactory in terms of stability and repeatability of the generated signal. Therefore microparticle labels such as colloidal gold particles are used more and more,e.g. in a lateral flow dipstick immunoassay. When applied to the detection of aflatoxin B₁ in pig feed a cut-off value of 5 μg/kg could be reached with no false negative results and a false positive rate of only 10%. Sample pretreatment for screening techniques should be rapid and simple. Preferably a simple solvent extraction is used, followed by a filtration and dilution step. However, for strongly coloured or complex food matrices, this did not seem to work. The combination of clean-up and detection in one single test device is a new approach. When using this clean-up tandem assay column for the detection of ochratoxin A in roasted coffee, a cut-off value of 6 μg/kg was reached. No false positive results were obtained, however, the false negative rate was 8%. Presented at the EU-USA Bilateral Workshop on Toxigenic Fungi & Mycotoxins, New Orleans, USA, July 5–7, 2005 Financial support: Belgian Federal Science Policy Office (SPSDII-CPAA 15 project), IWT-Flanders (research project 020448/Toxi-Test) and Bijzonder Onderzoeksfonds Ghent University (01 1D02803)     

Sambutoxin, a new mycotoxin produced by toxic Fusarium isolates obtained from rotted potato tubers.

Ninety-nine isolates of Fusarium species were obtained from rotted potato tubers from various parts of Korea. Of these isolates, 80 were identified as Fusarium oxysporum, F. solani, or F. sambucinum. The isolates of these species were grown on autoclaved wheat grains and examined for toxicity in a rat-feeding test. A total of 8 of 57 F. oxysporum isolates, 3 of 14 F. solani isolates, and 5 of 9 F. sambucinum isolates caused the death of the rats. Of the 16 toxic isolates, 1 isolate of F. oxysporum produced a substantial amount of moniliformin, which could account for its toxicity. None of the other 15 isolates produced trichothecenes, moniliformin, fusarochromanone, fumonisin B1, or wortmannin. F. sambucinum PZF-4 produced an unknown toxin in wheat culture. This new toxin, given the trivial name sambutoxin, caused toxic effects in rats, including body weight loss, feed refusal, hemorrhage in the stomach and intestines, and, finally, death when rats were fed diets supplemented with 0.05 and 0.1% sambutoxin. The toxin was also toxic to chicken embryos, and the 50% lethal concentration was 29.6 micrograms per egg. Sambutoxin formed as white crystals that turned purple when combined with reagents such as sulfuric acid and p-anisaldehyde. It exhibited a green color immediately after treatment with potassium ferricyanide-ferric chloride. Its UV spectrum had absorption maxima at 213, 233, and 254 nm, and its infrared spectrum showed an amide group at 1,650 and 1,560 cm-1 and a hydroxy group at 3,185 cm-1. Mass spectrometry showed that the molecular weight of the toxin was 453 and the molecular formula was C28H39NO4.(ABSTRACT TRUNCATED AT 250 WORDS)     

A molecular imprinted polymer with recognition properties towards the carcinogenic mycotoxin ochratoxin A

A molecularly imprinted polymer which recognises the mycotoxin ochratoxin A was prepared using the mimic N-(4-chloro-1-hydroxy-2-naphthoylamido)-(L) -phenylalanine as a template. The polymer was obtained by dissolving the template, methacrylic acid and ethylendimethacrylate in chloroform and polymerising the mixture by thermal treatment at 60°C. The monolith obtained was crushed, sieved to 30–90 m and extensively washed till the template could no longer be found in the washing solution. The binding properties towards the template, ochratoxin A and several related molecules were measured by eluting with acetonitrile and chloroform a HPLC column packed with the imprinted polymer. The experimental results show that the polymer recognises not only the template well, but also the ochratoxin A. The specific molecular recognition effect is due to hydrogen bond interactions but in order to assure the full recognition effect adjunctive steric factors are necessary. The magnitude of these interactions can be controlled by the use of limited amounts of acetic acid in the mobile phase.From the measurement of the relative selectivity it was found that only the simultaneous presence of the carboxyl, the phenolic hydroxyl and certain peculiar substructures such as the chlorine atom assures the whole recognition of the template.     

The mitochondrial membrane protein FgLetm1 regulates mitochondrial integrity,production of endogenous reactive oxygen species and mycotoxin biosynthesis in Fusarium graminearum

Deoxynivalenol (DON) is a mycotoxin produced in cereal crops infected with Fusarium graminearum. DON poses a serious threat to human and animal health, and is a critical virulence factor. Various environmental factors, including reactive oxygen species (ROS), have been shown to interfere with DON biosynthesis in this pathogen. The regulatory mechanisms of how ROS trigger DON production have been investigated extensively in F. graminearum. However, the role of the endogenous ROS‐generating system in DON biosynthesis is largely unknown. In this study, we genetically analysed the function of leucine zipper‐EF‐hand‐containing transmembrane 1 (LETM1) superfamily proteins and evaluated the role of the mitochondrial‐produced ROS in DON biosynthesis. Our results show that there are two Letm1 orthologues, FgLetm1 and FgLetm2, in F. graminearum. FgLetm1 is localized to the mitochondria and is essential for mitochondrial integrity, whereas FgLetm2 plays a minor role in the maintenance of mitochondrial integrity. The ΔFgLetm1 mutant demonstrated a vegetative growth defect, abnormal conidia and increased sensitivity to various stress agents. More importantly, the ΔFgLetm1 mutant showed significantly reduced levels of endogenous ROS, decreased DON biosynthesis and attenuated virulence in planta. To our knowledge, this is the first report showing that mitochondrial integrity and endogenous ROS production by mitochondria are important for DON production and virulence in Fusarium species.     

乳酸菌对真菌毒素的脱毒作用研究进展

真菌毒素广泛存在于农业产品中,对人和动物的健康构成巨大威胁。乳酸菌作为一种公认安全的微生物,在食品生物减毒方面具有巨大的应用潜力,成本低廉且不会对食品品质及生态环境造成不良影响。文章主要根据近年来国内外研究进展,阐述乳酸菌对食品和饲料中几种常见真菌毒素的脱毒作用(抑制真菌生长、毒素的吸附和降解),关注乳酸菌在生物脱毒方面的实际应用,为乳酸菌在食品保鲜领域的应用提供理论指导。     

真菌毒素玉米赤霉烯酮生物降解的研究进展

玉米赤霉烯酮(Zearalenone,ZEN)及衍生物是一类主要由镰刀菌属的真菌产生的非甾体雌激素类真菌毒素,广泛存在于玉米、大麦、小麦和高粱等谷物饲料及其副产品中,严重危害牲畜及人类健康,迫切需要相关的技术对ZEN进行降解脱毒。传统的物理化学方法不能有效去除谷物中的毒素,并会破坏谷物的营养成分,影响食物口感,甚至造成二次污染,因此利用生物工程技术对ZEN及其衍生物进行脱毒是未来解决这一问题的主要方法。文中简要介绍了ZEN及衍生物和降解ZEN的微生物种类、降解特性,然后详细介绍了目前研究的ZEN降解酶种类、解析唯一的蛋白结构及其异源表达和应用情况,以期为通过分子酶工程和发酵工程等生物工程技术降低ZEN降解酶的成本提供指导,从而提高食品安全。     

Comparative study of Aspergillus mycotoxin production on enriched media and construction material

Isolates of Aspergillus flavus and Aspergillus fumigatus from indoor air were compared with a known mycotoxin producer for their capacity to produce mycotoxins on a variety of enrichment media and with growth on indoor substrates such as ceiling tile and wall board. In enrichment media, four of seven isolates of A. flavus produced at least one aflatoxin and both isolates of A. fumigatus produced mycotoxins. The spectrum of mycotoxins and their concentrations varied with the strain and medium. When the mycotoxin-positive strains were grown to a dense concentration on indoor construction and finishing materials such as ceiling tile and wall boards, mycotoxins were not detected in extracts of the materials. Colonization of indoor surfaces by mycotoxin-producing strains of A. flavus and A. fumigatus may not necessarily expose inhabitants to mycotoxins or result in production of mycotoxins. Received 09 February 1999/ Accepted in revised form 28 June 1999     

Patterns of mycotoxin production by Fusarium gmminearum isolated from Argentine wheat

Fusarium graminearum was isolated from several wheat samples of the 1985/86 Argentine crop, taken from lots that had suffered extensive invasion by this fungus. Previous chemical analysis of the cereal, had revealed contamination with deoxynivalenol (DON), but the presence of the other mycotoxins could not be excluded with certainty, due to the low sensitivity of the analytical methodology employed.Twenty four F. graminearum isolates were grown on white corn with 50% water, for 21 days at 28 °C, or in liquid medium (Sucrose 3%, Peptone 0,1% and Yeast Extract 0,1%) for 7 days at 28 °C without shaking, and tested for the production of mycotoxins. Eight isolates (33% of the total) were found to produce toxins in one or both media. Toxins detected were: DON (6 isolates), 15-AcetylDON (5), 3-AcetylDON (2) and Zearalenone (3). No traces of Nivalenol, Fusarenon-X or other trichothecenes were found.These results suggest that strains of F. graminearum, prevailing in Argentine wheat-growing regions, might belong to the DON/AcetylDON chemotype, since no organisms of the Nivalenol/Fusarenon-X chemotype were detected in this study.