Assay and Relationship of HT-2 Toxin and T-2 Toxin Formation in Liquid Culture

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.     

Comparative yields of T-2 toxin and related trichothecenes from five toxicologically important strains of Fusarium sporotrichioides.

The range and comparative yields of T-2 toxin and related trichothecenes from five toxicologically important strains of Fusarium sporotrichioides, i.e., NRRL 3299, NRRL 3510, M-1-1, HPB 071178-13, and F-38, were determined. Lyophilized cultures of the five strains maintained in the International Toxic Fusarium Reference Collection were used to inoculate autoclaved corn kernels. Corn cultures were incubated at 15 degrees C for 21 days and analyzed for trichothecenes by thin-layer chromatography and capillary gas chromatography. All five strains produced T-2 toxin, HT-2 toxin, T-2 triol, and neosolaniol. Two strains also produced T-2 tetraol, and two others produced diacetoxyscirpenol. The highest producer of T-2 toxin (1,300 mg/kg), HT-2 toxin (200 mg/kg), T-2 triol (1.9 mg/kg), and neosolaniol (170 mg/kg) was NRRL 3510, which was originally isolated from millet associated with outbreaks of alimentary toxic aleukia in the USSR. The second highest producer of T-2 toxin (930 mg/kg) was NRRL 3299. The other three strains produced T-2 toxin at levels ranging from 130 to 660 mg/kg. Thus, the five strains differed considerably in the amounts of T-2 toxin and other trichothecenes produced under identical laboratory conditions. These strains are being maintained under optimal conditions for the preservation of Fusarium cultures and are available from the Fusarium Research Center, The Pennsylvania State University, University Park.     

Comparative yields of T-2 toxin and related trichothecenes from five toxicologically important strains of Fusarium sporotrichioides

The range and comparative yields of T-2 toxin and related trichothecenes from five toxicologically important strains of Fusarium sporotrichioides, i.e., NRRL 3299, NRRL 3510, M-1-1, HPB 071178-13, and F-38, were determined. Lyophilized cultures of the five strains maintained in the International Toxic Fusarium Reference Collection were used to inoculate autoclaved corn kernels. Corn cultures were incubated at 15 degrees C for 21 days and analyzed for trichothecenes by thin-layer chromatography and capillary gas chromatography. All five strains produced T-2 toxin, HT-2 toxin, T-2 triol, and neosolaniol. Two strains also produced T-2 tetraol, and two others produced diacetoxyscirpenol. The highest producer of T-2 toxin (1,300 mg/kg), HT-2 toxin (200 mg/kg), T-2 triol (1.9 mg/kg), and neosolaniol (170 mg/kg) was NRRL 3510, which was originally isolated from millet associated with outbreaks of alimentary toxic aleukia in the USSR. The second highest producer of T-2 toxin (930 mg/kg) was NRRL 3299. The other three strains produced T-2 toxin at levels ranging from 130 to 660 mg/kg. Thus, the five strains differed considerably in the amounts of T-2 toxin and other trichothecenes produced under identical laboratory conditions. These strains are being maintained under optimal conditions for the preservation of Fusarium cultures and are available from the Fusarium Research Center, The Pennsylvania State University, University Park.     

Screeing of toxic isolates of Fusarium poae and Fusarium sporotrichiodes involved in causing alimentary toxic aleukia.

A total of 131 isolates of Fusarium poae and F. sporotrichioides from overwintered cereals, which were associated with the alimentary toxic aleukia toxicoses in the Soviet Union, were tested for their ability to produce T-2 toxin [4 beta, 15 diacetoxy-8alpha-(3-methylbutyryloxy)-12,13-epoxytrichothec-9-en 3alpha-ol]. The presence of T-2 toxin was determined by thin-layer chromatography, gas-liquid chromatography, spectroscopic analyses, and the rabbit skin test. A good correlation was demonstrated between T-2 toxin dectetion by thin-layer chromatography and inflammatory skin reactions of rabbits.     

Stability of T-2 mycotoxin in aqueous media

3H-labeled T-2 mycotoxin was dissolved in various aqueous media and stored for up to 3 weeks at 4, 22, and 37 degrees C. At periods ranging from 1 to 21 days, aliquots were assayed by thin-layer chromatography. Thin-layer chromatography plates were scanned for breakdown products by use of a radioisotope scanner, and breakdown products were identified based on their comigration with known standards. Results indicated that T-2 toxin was more stable in tissue culture medium with or without serum, than in Hanks balanced salt solution (HBSS), at all temperatures. The metabolites HT-2, T-2 triol, and T-2 tetraol were detected as early as 1 day (HBSS; 37 degrees C) and as late as 3 weeks (HBSS; 4 degrees C) after storage. Stability of the toxin in aqueous media decreased with increasing temperature.     

Stability of T-2 mycotoxin in aqueous media.

3H-labeled T-2 mycotoxin was dissolved in various aqueous media and stored for up to 3 weeks at 4, 22, and 37 degrees C. At periods ranging from 1 to 21 days, aliquots were assayed by thin-layer chromatography. Thin-layer chromatography plates were scanned for breakdown products by use of a radioisotope scanner, and breakdown products were identified based on their comigration with known standards. Results indicated that T-2 toxin was more stable in tissue culture medium with or without serum, than in Hanks balanced salt solution (HBSS), at all temperatures. The metabolites HT-2, T-2 triol, and T-2 tetraol were detected as early as 1 day (HBSS; 37 degrees C) and as late as 3 weeks (HBSS; 4 degrees C) after storage. Stability of the toxin in aqueous media decreased with increasing temperature.     

Metabolism of T-2 toxin in Curtobacterium sp. strain 114-2.

The metabolic pathway of T-2 toxin in Curtobacterium sp. strain 114, one of the T-2 toxin-assimilating soil bacteria, was investigated by thin-layer and gas-liquid chromatographic analyses. T-2 toxin added to the basal medium as a single carbon and energy source was biotransformed into HT-2 toxin and an unknown metabolite. Infrared, mass spectrum, proton magnetic resonance, and other physico-chemical analyses identified this new metabolite as T-2 triol. T-2 toxin was first deacetylated by the bacterium into HT-2 toxin, and this metabolite was then biotransformed into T-2 triol without formation of neosolaniol and T-2 tetraol. No trichothecenes remained in the culture medium after prolonged culture. Some properties of T-2 toxin-hydrolyzing enzymes were observed with whole cells, the cell-free soluble fraction, and the culture filtrate. Besides T-2 toxin, trichothecenes such as diacetoxyscirpenol, neosolaniol, nivalenol, and fusarenon-X were also assimilated by this bacterium.     

Structures of deepoxytrichothecene metabolites from 3'-hydroxy HT-2 toxin and T-2 tetraol in rats.

3'-Hydroxy HT-2 toxin and T-2 tetraol, in vivo metabolites of T-2 toxin, were orally administered to Wistar rats, and four metabolites having a trichothec-9,12-diene nucleus, which were termed deepoxytrichothecenes, were newly found in the excreta. Their structures were confirmed as 3'-hydroxy-deepoxy HT-2, 3'-hydroxy-deepoxy T-2 triol, 15-acetyl-deepoxy T-2 tetraol, and deepoxy T-2 tetraol on the basis of mass and nuclear magnetic resonance spectroscopy. Resolution of T-2 metabolites and corresponding deepoxytrichothecenes by gas-liquid and thin-layer chromatography was also described.     

Structures of deepoxytrichothecene metabolites from 3'-hydroxy HT-2 toxin and T-2 tetraol in rats

3'-Hydroxy HT-2 toxin and T-2 tetraol, in vivo metabolites of T-2 toxin, were orally administered to Wistar rats, and four metabolites having a trichothec-9,12-diene nucleus, which were termed deepoxytrichothecenes, were newly found in the excreta. Their structures were confirmed as 3'-hydroxy-deepoxy HT-2, 3'-hydroxy-deepoxy T-2 triol, 15-acetyl-deepoxy T-2 tetraol, and deepoxy T-2 tetraol on the basis of mass and nuclear magnetic resonance spectroscopy. Resolution of T-2 metabolites and corresponding deepoxytrichothecenes by gas-liquid and thin-layer chromatography was also described.     

Uptake of aflatoxin B1 and T-2 toxin by two mycotoxin bioassay microorganisms: Kluyveromyces marxianus and Bacillus megaterium

Uptake of aflatoxin B1 (AFB1) and trichothecene T-2 toxin from growth medium by mycotoxin bioassay strains of Klutyveomyces marxianus and Bacillus megaterium was assessed by incubating, washing, and sonicating the cells, extracting samples with chloroform, and analysing the extracts by a combination of high-performance thin-layer chromatography (HPTLC) and fluorescence densitometry. Using cultures of K. marxianus, the entire AFB1 dose was recovered and no AFB1 metabolites were detected. Less than 1% of the AFB1 was recovered from the cells, and AFB1 did not inhibit growth. Methanol in the incubation medium had no significant effect on the levels of AFB1 associated with K. marxianus cells. The entire dose of T-2 toxin was also recovered from K. marxianus cultures, and no metabolites were detected; again, less than 1% of T-2 toxin was cell-associated, but growth was completely inhibited. AFB1 partially inhibited the growth of B. megaterium; approximately 12% of the dose could not be recovered, and no AFB1-related metabolites were detected. Methanol increased the levels of recoverable AFB1 associated with B. megaterium cells. In the case of T-2 toxin, around 8% of the dose was not recovered, and no metabolites were detected; growth of B. megaterium was stimulated. These results suggest irreversible binding of both toxins, or derivatives of them, to the cells of B. megaterium.     

Identification of the Toxic Principle in a Sample of Poaefusarin

A sample of poaefusarin (a mycotoxin suspected of being one of the toxins involved in alimentary toxic aleukia in the U.S.S.R.) was received from a Soviet scientist for evaluation and comparison with other mycotoxins. Although poaefusarin is presumed to be a steroid, analyses by thin-layer chromatography, gas-liquid chromatography, and infrared, ultraviolet, and mass spectrometry could not confirm the presence of a steroid structure. However, 2.5% of the sample was made up of the trichothecene T-2 toxin, an amount sufficient to explain the toxicity found in the rat and rabbit skin toxicity tests. In addition, neosolaniol (0.14%), T-2 tetraol (0.6%), and zearalenone (F-2) (0.43%) were present in the sample. Since the toxicity was found to be associated only with T-2 toxin, no attempt was made to determine the nature of the other nontoxic components of the sample.     

Method for detecting production of zearalenone, zearalenol, T-2 toxin, and deoxynivalenol by Fusarium isolates.

Three methods for detecting toxigenic fusaria in culture were compared by using known producers of zearalenone, zearalenol, T-2 toxin, and deoxynivalenol. Moist, autoclaved rice cultures of known toxigenic isolates grown in 20-ml tubes yielded oily extracts containing compounds which interfered with qualitative and quantitative analysis for the mycotoxins. Vermiculite moistened with nutrient broth in 20-ml tubes yielded a much cleaner extract. Growing the fungi on a liquid medium required a shorter incubation period, but yields of T-2 toxin and deoxynivalenol were low and variable, and the method required greater space in the incubator. Screening of the extracts by thin-layer chromatography with colorimetric spray reagents to detect the presence of these toxins permitted reduction in the number of extracts quantified by the more lengthy gas-liquid chromatographic method. Culturing in nutrient broth on vermiculite in tubes coupled to a qualitative screen before quantitation proved to be a convenient, inexpensive, and relatively rapid method that enabled reliable screening of a large number of Fusarium isolates for toxin production as compared with prior methods.     

Method for detecting production of zearalenone, zearalenol, T-2 toxin, and deoxynivalenol by Fusarium isolates

Three methods for detecting toxigenic fusaria in culture were compared by using known producers of zearalenone, zearalenol, T-2 toxin, and deoxynivalenol. Moist, autoclaved rice cultures of known toxigenic isolates grown in 20-ml tubes yielded oily extracts containing compounds which interfered with qualitative and quantitative analysis for the mycotoxins. Vermiculite moistened with nutrient broth in 20-ml tubes yielded a much cleaner extract. Growing the fungi on a liquid medium required a shorter incubation period, but yields of T-2 toxin and deoxynivalenol were low and variable, and the method required greater space in the incubator. Screening of the extracts by thin-layer chromatography with colorimetric spray reagents to detect the presence of these toxins permitted reduction in the number of extracts quantified by the more lengthy gas-liquid chromatographic method. Culturing in nutrient broth on vermiculite in tubes coupled to a qualitative screen before quantitation proved to be a convenient, inexpensive, and relatively rapid method that enabled reliable screening of a large number of Fusarium isolates for toxin production as compared with prior methods.     

Variability in the production of trichothecenes by Fusarium sporotrichioides

Production of trichothecenes as with all secondary metabolites, will vary from one producing strain to another. If a particular metabolite is produced at all it may be assumed that the part of the genome involved is intact and it would seem most likely that the quantitative variation observed from strain to strain might arise from modifications in metabolic control. An opportunity to study this phenomenon is presented by the existence of cultures of Fusarium sporotrichioides descended from the original T-2 toxin producing strain isolated in 1965 from French corn by W.C. Snyder and originally referred to as F. tricinctum and since then maintained independently by different culture collections. These strains continue to be T-2 toxin producers but are now morphologically distinct on media such as potato sucrose agar.Preliminary results are reported in studies designed to detail the differences in toxigenicity between these strains.     

Development and evaluation of multiplex PCR for detection of T-2 and zearalenone producing Fusarium spp

The study aimed to develop and evaluate a multiplex polymerase chain reaction assay (mPCR) for the concurrent detection of three major mycotoxin metabolic pathway genes, namely tri8 (T-2 toxin), tri6 (trichothecene) and pks4 (zearalenone), along with competitive internal amplification control. Specific primers for each of the aforementioned genes were optimized and validated using 14 reference strains and 10 pure culture isolates. The optimized mPCR assay detected the three metabolic pathway genes in artificially contaminated maize samples with a sensitivity of 2 × 10³ CFU per g for tri6 and pks4 positive Fusarium strains, whereas 2 × 10⁴ CFU per g for tri8 positive Fusarium strains. Application of the developed mPCR assay to 30 cereal and 20 feed samples revealed 24% (12 of 50) contamination with either one or more mycotoxins. The results of mPCR assay were further evaluated with high performance liquid chromatography (HPLC), and both methods provided unequivocal results. This mPCR assay might be a supplementary tool to conventional mycotoxin analytical techniques like thin-layer chromatography, HPLC, etc. The current mPCR assay is a rapid and reliable tool for simultaneous, sensitive and specific detection of T-2, zearalenone and trichothecene producing Fusarium spp. from naturally contaminated foods and to monitor them during the processing steps of food and feed commodities.     

Comparative study of the yield of T-2 toxic produced by Fusarium poae,F. sporotrichioides and F. sporotrichioides var. tricinctum strains from different sources

Eleven strains of Fusarium poae and F. sporotrichioides from the U.S.S.R. and 7 strains of these species and one of F. sporotrichioides var. tricinctum from U.S.A. and France have been compared as to their capacity to yield T-2 toxin. The presence of this toxin was confirmed by thin layer chromatography, gas liquid chromatography and mass spectroscopy. The strains which came from the U.S.S.R. and were originally involved in causing Alimentary Toxic Aleukia (ATA), produced several times more T-2 toxin than the others. We assume that the ability of the U.S.S.R. strains to produce much more T-toxin is due to the different ecological conditions under which they were first isolated. The remarkable maintenance of so high a level of toxin production over more than 30 years of culturing is worthy of note.This work was partly supported by the Joint Research Fund of the Hebrew University and Hadassah.     

Identification of mycotoxins produced by species of Fusarium and Stachybotrys obtained from Eastern Europe.

Isolates of Fusarium and Stachybotrys spp. and crude extracts from these fungi were obtained from Hungary and the U.S.S.R. and used for the evaluation of the mycotoxins they produced. The cultures were grown on millet and oats and extracted in Budapest, Hungary (Veterinary Medical Research Institute) and chemically analyzed at the University of Minnesota using thin-layer chromatography (TLC), gas-liquid chromatography (GLC), gas chromatograph-mass spectrometry (GC-MS), and the rat skin bioassay. Zearalenone was found in most of the Fusarium cultures, T-2 toxin, neosolaniol, T-2 tetraol, and HT-2 toxin were found in extracts of Fusarium poae and F. sporotrichioies. A special effort was made to isolate the steroid-like toxins reported in the early Russian literature as sporofusarin and poaefusarin. None of the extracts from the Fusarium species yielded poaefusarin or sporofusarin when analyzed by our chemical methods or by those of L.E. Olifson, S.M. Kenina, and V.L. Kartashova, 1972. We therefore accounted for the toxicity of the Fusarium extracts as due to the 12,13,epoxytrichothecenes. One culture of Stachybotrys alternans yielded a macrocyclic ester of 12,13-epoxytrichothecene which, upon hydrolysis, yielded verrucarol; a steroid-like molecule (SB-3) was also isolated. The former had skin-irritant activity but SB-3 did not; the latter exhibited cardiac activity on the heart of the cockroach.     

一株产T-2毒素镰孢菌的分离、鉴定及其产毒条件的研究

【目的】从青海大骨节病区小麦麦穗中分离的内生真菌中筛选产T-2毒素的菌株,并研究影响其合成该毒素的条件。【方法】采用种子胚芽抑制试验和抑菌试验从分离所得的菌株中筛选产毒菌株;利用薄层层析和高效液相检测待测菌株产物,复筛出产T-2毒素的菌株。通过显微形态学观察及ITS序列分析对筛选出的菌株5-5m-1进行鉴定。应用单因素筛选方案研究固体培养时间、温度以及液体培养转速、初始p H等对其产T-2毒素的影响,并采用正交试验进一步优化。【结果】菌株5-5m-1的显微形态与梨孢镰孢菌(Fusarium poae)相似;ITS序列分析显示,该菌株与F.poae的相似度也较高。其产T-2毒素的最佳条件为:玉米固体培养基、日温25°C/夜温15°C、光暗交替。【结论】5-5m-1菌株为梨孢镰孢菌,培养条件对其产T-2毒素能力有很大影响。实验结果将为进一步研究T-2毒素产生的机制和防止真菌毒素污染提供参考。     

Survey for Fusaria That Elaborate T-2 Toxin

Of the 136 strains of Fusarium examined, T-2 toxin was confirmed by thinlayer chromatography in 13 of the 21 extracts that inhibited either Rhodotorula rubra or Pencillium digitatum.     

Identification of mycotoxins in keratomycosis-derived Fusarium isolates by gas chromatography—mass spectrometry

The production of mycotoxins from Fusarium species has been demonstrated in isolates cultured from patients suffering from keratomycosis. The method employed a combination of thin-layer chromatography directly performed on gel plugs taken from the growth medium, cartridge column chromatography, silylation and gas chromatography on a non-polar stationary phase capillary column linked to mass spectrometry. The sensitivities of detection obtained for a signal-to-noise ratio of 33:1, were 200 pg for single stage GC-MS and 20 pg using tandem GC-MS-MS. Two mycotoxins, diacetoxyscirpenol and T-2 toxin were identified in three cultures.