Trichothecenes produced by Stachybotrys atra from Eastern Europe

A total of 17 isolates of Stachybotrys atra isolated from various parts of Hungary and Czechoslovakia were grown on rice, and the toxin production of each isolate was analyzed by high-performance liquid chromatography. Of the 17 isolates, 14 produced macrocyclic trichothecenes (satratoxins F, G, and H, roridin E, and verrucarin J) as well as trichoverrols A and B. Most isolates produced satratoxins G and H in higher quantities than the other trichothecenes. The yield (in milligrams) of trichothecenes produced by one isolate grown on 800 g of rice was as follows: roridin E, 12; satratoxin F, 10; satratoxin G, 75; satratoxin H, 100; trichoverrol A, 15; and trichoverrol B, 30.     

Macrocyclic trichothecenes produced by Stachybotrys isolated from Egypt and Eastern Europe

Twenty seven isolates of Stachybotrys chartarum, S. albipes, S. kampalensis and S. microspora from Egypt and Eastern Europe were tested for production of macrocyclic trichothecenes. Twenty of the 27 isolates, grown on rice seeds, were toxic to brine shrimp larvae. Based on TLC and HPLC analyses, 5 macrocyclic trichothecenes (verrucarin J, roridin E, satratoxins F, G & H) as well as trichoverrols were identified. When grown in liquid culture on rice extract medium, only 3 isolates were toxic and produced verrucarin J, roridin E and satratoxins G & H. Extracts from mycelial mats were more toxic than culture filterates of two isolates grown on rice extract and both contained the same macrocyclic trichothecenes (285.5 mg/4 L), in addition to trichoverrols A & B (31 mg/4 L) found in mycelial mats only. When grown on 3% sucrose Czapek's medium supplemented with peptone and yeast extract (still cultures), all isolates were non-toxic to brine shrimp and no trichothecenes could be detected in the extracts.     

Macrocyclic trichothecene toxins produced by Stachybotrys atra strains isolated in Middle Europe.

A total of 17 strains of Stachybotrys atra isolated in Hungary and Czechoslovakia were cultured on Sabouraud agar, and the toxins produced by them were chemically analyzed by gas-liquid chromatography, high-pressure liquid chromatography, and mass spectroscopy. Furthermore, brine shrimp (Artemia salina) bioassay was used for the determination of toxicity of the compounds examined. Macrocyclic trichothecenes (satratoxins H and G, roridin E, and verrucarin J as well as two other unidentified macrocyclic trichothecenes) were found in all of the cultures tested. The identities of satratoxins H and G, roridin E, and verrucarin J were qualitatively determined by high-pressure liquid chromatography and gas-liquid chromatography. The ratio of satratoxins H and G and roridin E was found to be similar in each of the strains tested, but the amount of verrucarin J found was different in each of them. One of the unidentified macrocyclic trichothecenes was equivalent to the compound isolated by Harrach et al. (Harrach et al., Appl. Environ. Microbiol. 41:1428-1433, 1981). The other one proved to be a newly isolated macrocyclic trichothecene toxin. Stachybotryotoxicosis, one of the oldest mycotoxicoses known, and a serious problem in Middle Europe (Gy. Danko, Magy. Allatorv. Lapja 31:226-232, 1976), is believed to be caused by macrocyclic trichothecene toxins produced by Stachybotrys atra (R. M. Eppley, in Rodricks et al., ed., Mycotoxins in Human and Animal Health, p. 285-293, 1977). Forty years ago, the death of animals in the Soviet Union was associated with this fungus (C. U. Ruhliada, in Proceedings of the All-Union Sci. and Tech. Conf., p. 47-51, 1980).(ABSTRACT TRUNCATED AT 250 WORDS)     

Production and characterization of monoclonal antibodies to the macrocyclic trichothecene roridin A

Two murine monoclonal antibodies to the macrocyclic trichothecene roridin A are described. Screening for antibody production was performed on absorbed anti-mouse immunoglobulin serum as double-antibody solid phase, and further characterization was done on affinity-purified anti-mouse IgG serum. The antibodies, designated 5G11 and 4H10, had affinity constants for roridin A of 9.25 X 10(7) and 1.7 X 10(7) liters/mol, respectively. In monoclonal antibody-based direct enzyme immunoassays, these IgG1 antibodies had detection limits for roridin A of 0.4 ng/ml (0.02 ng per assay) and 1.8 ng/ml (0.09 ng per assay), respectively. Both antibodies were most specific for the tested macrocyclic trichothecenes. The relative cross-reactivities of antibody 5G11 with roridin A, roridin J, verrucarin A, satratoxin G, and satratoxin H were 100.0, 43.8, 16.7, 3.7, and 18.9%, respectively; for antibody 4H10 they were 100.0, 6.3, 64.0, 4.4, and 4.9%, respectively.     

Production and characterization of monoclonal antibodies to the macrocyclic trichothecene roridin A.

Two murine monoclonal antibodies to the macrocyclic trichothecene roridin A are described. Screening for antibody production was performed on absorbed anti-mouse immunoglobulin serum as double-antibody solid phase, and further characterization was done on affinity-purified anti-mouse IgG serum. The antibodies, designated 5G11 and 4H10, had affinity constants for roridin A of 9.25 X 10(7) and 1.7 X 10(7) liters/mol, respectively. In monoclonal antibody-based direct enzyme immunoassays, these IgG1 antibodies had detection limits for roridin A of 0.4 ng/ml (0.02 ng per assay) and 1.8 ng/ml (0.09 ng per assay), respectively. Both antibodies were most specific for the tested macrocyclic trichothecenes. The relative cross-reactivities of antibody 5G11 with roridin A, roridin J, verrucarin A, satratoxin G, and satratoxin H were 100.0, 43.8, 16.7, 3.7, and 18.9%, respectively; for antibody 4H10 they were 100.0, 6.3, 64.0, 4.4, and 4.9%, respectively.     

Enzyme immunoassay for the macrocyclic trichothecene roridin A: production, properties, and use of rabbit antibodies

Antisera against roridin A were prepared by using a roridin A-hemisuccinate derivative coupled to human serum albumin as the immunogen. Antibodies could be detected in the sera of the immunized rabbits as early as 4 weeks after the initial exposure. After one booster injection at week 14, high antibody titers were measured over a period of 21 weeks. The specificity and sensitivity of the antibodies were tested by using roridin A-hemisuccinate coupled to horseradish peroxidase as an enzyme-linked toxin in a competitive assay with a double-antibody solid phase. The assay was most specific for the tested macrocyclic trichothecenes, and the relative cross-reactivities with roridin A, roridin J, verrucarin A, satratoxin H, and satratoxin G were 1, 0.41, 0.15, 0.15, and 0.07, respectively. When 16 nonmacrocyclic trichothecenes were tested, only diacetylverrucarol (0.0015) and verrucarol (0.0005) showed minor cross-reactivity. The sensitivity of the enzyme immunoassay for the detection of roridin A was in the range of 5 to 50 ng/ml (0.16 to 1.6 ng per assay).     

Enzyme immunoassay for the macrocyclic trichothecene roridin A: production, properties, and use of rabbit antibodies.

Antisera against roridin A were prepared by using a roridin A-hemisuccinate derivative coupled to human serum albumin as the immunogen. Antibodies could be detected in the sera of the immunized rabbits as early as 4 weeks after the initial exposure. After one booster injection at week 14, high antibody titers were measured over a period of 21 weeks. The specificity and sensitivity of the antibodies were tested by using roridin A-hemisuccinate coupled to horseradish peroxidase as an enzyme-linked toxin in a competitive assay with a double-antibody solid phase. The assay was most specific for the tested macrocyclic trichothecenes, and the relative cross-reactivities with roridin A, roridin J, verrucarin A, satratoxin H, and satratoxin G were 1, 0.41, 0.15, 0.15, and 0.07, respectively. When 16 nonmacrocyclic trichothecenes were tested, only diacetylverrucarol (0.0015) and verrucarol (0.0005) showed minor cross-reactivity. The sensitivity of the enzyme immunoassay for the detection of roridin A was in the range of 5 to 50 ng/ml (0.16 to 1.6 ng per assay).     

Macrocyclic trichothecenes are undetectable in kudzu (Pueraria montana) plants treated with a high-producing isolate of Myrothecium verrucaria.

Myrothecium verrucaria was found to be an effective pathogen against kudzu grown in the greenhouse and the field. M. verrucaria produced large amounts of macrocyclic trichothecenes when cultured on solid rice medium, including epiroridin E (16.8 mg/g crude extract), epiisororidin E (1 mg/g), roridin E (8.7 mg/g), roridin H (31.3 mg/g), trichoverrin A (0.6 mg/g), trichoverrin B (0.1 mg/g), verrucarin A (37.4 mg/g), and verrucarin J (2.2 mg/g). Most of these toxins were also isolated from M. verrucaria spores and mycelia grown on potato dextrose agar medium, including epiroridin E (32.3 mg/g), epiisororidin E (28.6 mg/g), roridin E (0 mg/g), roridin H (60 mg/g), trichoverrin A (1.3 mg/g), trichoverrin B (1.8 mg/g), verrucarin A (13.8 mg/g), and verrucarin J (131 mg/g). When M. verrucaria was cultured on liquid media, the numbers but not the amounts of toxins decreased. Only epiroridin E (28.3 mg/g), epiisororidin E (29.6 mg/g), verrucarin B (195 mg/g) and verrucarin J (52.6 mg/g) were measured when the fungus was cultured on cornsteep medium. On soyflour-cornmeal broth M. verrucaria produced several toxins, including epiroridin E (58.1 mg/g), epiisororidin E (5.8 mg/g), verrucarin B (29.9 mg/g) and verrucarin J (32 mg/g). In contrast, no macrocyclic trichothecenes were detected by HPLC analysis of plant tissues of kudzu, sicklepod, and soybean treated with aqueous suspensions of M. verrucaria spores formulated with a surfactant. Chloroform-methanol extracts of kudzu leaves and stems treated with M. verrucaria spores were less cytotoxic to four cultured mammalian cell lines than the corresponding extracts from control plants. Purified macrocyclic trichothecenes (verrucarin A and T-2 toxin) were very cytotoxic to the same cell lines (< or = 2 ng/ml). These results show that neither intact macrocyclic trichothecenes nor toxic metabolites could be detected in plant tissues after treatment with M. verrucaria spores. These results argue for both safety and efficacy for the use of M. verrucaria in biological control of kudzu and other noxious weeds, and support proceeding to animal feeding trials for further evaluation of safety.     

Production of zearalenone,moniliformin and trichothecenes in intact sugar beets under laboratory conditions

Five toxigenic isolates of Fusarium species were tested for the production of zearalenone, moniliformin and trichothecenes (deoxynivalenol, 15-acetyldeoxynivalenol, T-2, HT-2 and neosolaniol) when grown on solid sugar beet slices in the laboratory for thirty days. The isolates were also grown on a solid rice medium for comparison. High zearalenone and trichothecene-producing isolates originally obtained from corn and corn-based feedstuff were compared with isolates obtained from sugar beets. One moniliformin-producing isolate from wheat was included in the study. With the exception of moniliformin, all toxins were produced on both substrates; however, the rice medium yielded the greater concentrations except for HT-2 which was produced on sugar beets in equal or greater concentrations. Zearalenone production on rice reached 729–1943 gmg/g whereas on sugar beet it reached 72–193 gmg/g. The moniliformin-producing isolate grew well on both substrates; however, moniliformin was produced only on the rice substrate. This study demonstrates for the first time that Fusarium species can produce both zearalenone and the trichothecenes on a sugar beet substrate.     

Mass Spectrometry-Based Strategy for Direct Detection and Quantification of Some Mycotoxins Produced by Stachybotrys and Aspergillus spp. in Indoor Environments

Dampness in buildings has been linked to adverse health effects, but the specific causative agents are unknown. Mycotoxins are secondary metabolites produced by molds and toxic to higher vertebrates. In this study, mass spectrometry was used to demonstrate the presence of mycotoxins predominantly produced by Aspergillus spp. and Stachybotrys spp. in buildings with either ongoing dampness or a history of water damage. Verrucarol and trichodermol, hydrolysis products of macrocyclic trichothecenes (including satratoxins), and trichodermin, predominately produced by Stachybotrys chartarum, were analyzed by gas chromatography-tandem mass spectrometry, whereas sterigmatocystin (mainly produced by Aspergillus versicolor), satratoxin G, and satratoxin H were analyzed by high-performance liquid chromatography-tandem mass spectrometry. These mycotoxin analytes were demonstrated in 45 of 62 building material samples studied, in three of eight settled dust samples, and in five of eight cultures of airborne dust samples. This is the first report on the use of tandem mass spectrometry for demonstrating mycotoxins in dust settled on surfaces above floor level in damp buildings. The direct detection of the highly toxic sterigmatocystin and macrocyclic trichothecene mycotoxins in indoor environments is important due to their potential health impacts.     

Method for small routine laboratories for the detection of satratoxins in straw samples

A method is described for thin-layer chromatographic analysis of field samples for satratoxins G and H, theStachybotrys toxins most soluble under physiological circumstances and representing the majority ofStachybotrys atra produced toxins. Besides brine shrimp bio-assay, the conversion of these macrocyclic trichothecenes to verrucarol is used to support their identification.     

Trichothecene-induced cytotoxicity on human cell lines

Trichothecene cytotoxicity of type A (T-2 toxin and HT-2 toxin), type B (deoxynivalenol, DON, and nivalenol, NIV), and type D (satratoxins G and H) compounds was determined comparatively by using eight permanent human cell lines (Hep-G2, A549, CaCo-2, HEp-2, A204, U937, RPMI 8226, and Jurkat). Viability of cells was measured by a water-soluble tetrazolium (WST-1) reagent cell proliferation assay assessing mitochondrial metabolic activity. Toxicity was expressed as the toxin concentration inhibiting 50% of cell viability (IC₅₀). Depending on the chemotype of the tested trichothecenes, relative cytotoxic activity differed by a factor of 100–1,000, and the corresponding IC₅₀ values were in the range from 2.2 nmol/l (satratoxin H on Jurkat and U937 cells) to 4,900 nmol/l (deoxynivalenol on HEp-2 cells). In contrast, the specific toxicity of each individual mycotoxin towards different cell lines was within remarkable close limits, and between-cell line differences were much smaller than previously reported. For the cell lines tested, IC₅₀ values were 4.4–10.8 nmol/l for T-2 toxin, 7.5–55.8 mol/l for HT-2 toxin, 600–4,900 nmol/l for DON, 300–2,600 nmol/l for NIV, and 2.2–18.3 nmol/l for satratoxins G/H. In addition, for the first time, the toxic activity of trichothecenes on primary cell culture of human endothelial cells (HUVEC) was tested. The susceptibility of this cell line was comparable to the other cell lines tested, with IC₅₀ values ranging from 16.5 nmol/l (T-2 toxin) to 4,500 nmol/l (DON). The results suggest that the current focus of cytotoxicological studies on trichothecenes on lymphoid cell lines may lead to an underestimate of their potential on other target cell systems.     

Growth and nutrient uptake of sorghum cultivated with vesicular-arbuscular mycorrhiza isolates at varying pH

This study was conducted to determine the effects of different pH regimes on root colonization with four vesicular-arbuscular mycorrhiza (VAM) isolates, and VAM effects on host plant growth and nutrient uptake. Sorghum [Sorghum bicolor (L.) Moench] was grown at pH 4.0, 5.0, 6.0 and 7.0 (±0.1) in hydroponic sand culture with the VAM isolates Glomus etunicatum UT316 (isolate E), G. intraradices UT143 (isolate I), G. intraradices UT126 (isolate B), and an unknown Glomus isolate with no INVAM number (isolate A). Colonization of roots with the different VAM isolates varied differentially with pH. As pH increased, root colonization increased with isolates B and E, remained unchanged with isolate I, and was low at pH 4.0 and high at pH 5.0, 6.0, and 7.0 with isolate A. Isolates E and I were more effective than isolates A and B in promoting plant growth irrespective of pH. Root colonization with VAM appeared to be independent of dry matter yields or dry matter yield responsiveness (dry matter produced by VAM compared to nonmycorrhizal plants). Dry matter yield responsiveness values were higher in plants whose roots were colonized with isolates E and I than with isolates A and B. Shoot P concentrations were lower in plants colonized with isolates E and I than with isolates A and B or nonmycorrhizal plants. This was probably due to the dilution effect of the higher dry matter yields. Neither the VAM isolate nor pH had an effect on shoot Ca, Mg, Zn, Cu, and Mn concentrations, while the VAM isolate affected not only P but also S, K, and Fe concentrations. The pH x VAM interaction was significant for shoot K, Mg, and Cu concentrations.     

Mycotoxins in crude building materials from water-damaged buildings

We analyzed 79 bulk samples of moldy interior finishes from Finnish buildings with moisture problems for 17 mycotoxins, as well as for fungi that could be isolated using one medium and one set of growth conditions. We found the aflatoxin precursor, sterigmatocystin, in 24% of the samples and trichothecenes in 19% of the samples. Trichothecenes found included satratoxin G or H in five samples; diacetoxyscirpenol in five samples; and 3-acetyl-deoxynivalenol, deoxynivalenol, verrucarol, or T-2-tetraol in an additional five samples. Citrinine was found in three samples. Aspergillus versicolor was present in most sterigmatocystin-containing samples, and Stachybotrys spp. were present in the samples where satratoxins were found. In many cases, however, the presence of fungi thought to produce the mycotoxins was not correlated with the presence of the expected compounds. However, when mycotoxins were found, some toxigenic fungi usually were present, even if the species originally responsible for producing the mycotoxin was not isolated. We conclude that the identification and enumeration of fungal species present in bulk materials are important to verify the severity of mold damage but that chemical analyses are necessary if the goal is to establish the presence of mycotoxins in moldy materials.     

Isolation of macrocyclic and non-macrocyclic trichothecenes (stachybotrys and fusarium toxins) from the Environment of 200 III Sport Horses

Satratoxins H and G, verrucarin J, and roridin E were isolated from the bedding straw of 200 sport horses exhibiting typical symptoms of stachybotryo-toxicosis. At the same time, the oat feed consumed by the horses contained non-macrocyclicFusarium trichothecenes: T-2 toxin and diacetoxyscirpenol.     

Veratryl alcohol as an inducer of laccase by an ascomycete, Botryosphaeria sp., when screened on the polymeric dye Poly R-478

A.M. BARBOSA, R.F.H. DEKKER AND G.E. ST HARDY. 1996. Forty fungi isolated from diverse environments in Western Australia were screened for ligninolytic activity based on in-vivo decolorization of the polymeric dye Poly R-478. Three isolates identified as Aspergillus, Botryosphaeria and Coniochaeta species were selected for further studies. The Botryosphaeria and Coniochaeta isolates were found to produce laccase constitutively in submerged culture when grown on glucose, or on ryegrass seed by solid state fermentation. A comparison of the three isolates grown on glucose in the presence of 3,4-dimethoxybenzyl (veratryl) alcohol (40 mmol 1⁻¹) showed that only the Botryosphaeria isolate produced laccase, with laccase activities 115-fold higher than when grown on glucose alone.     

Growth hormone-like activities of macrocyclic trichothecenes in in vitro callus induction and growth of four Baccharis species

The ability of two plant-produced macrocyclic trichothecenes (baccharinoid B4 and roridin E) to induce callus growth of two trichothecene-producing Baccharis species (B. coridifolia and B. megapotamica) and two nontrichothecene-producing species (B. halimifolia and B. neglecta) was investigated. Roridin E had no effect in the induction of callus of B. coridifolia, a roridin-producing plant, but induced callus of nonroridin-producing plants (B. megapotamica, B. halimifolia, and B. neglecta). Baccharinoid B4 stimulated callus growth of B. megapotamica, a baccharinoid-producing plant, and inhibited growth of B. coridifolia, B. halimifolia, and B. neglecta callus tissues. The ability of roridin E to induce callus was most effective at concentrations of 10^–8 and 10^–6 M and when synergistically coupled with auxin, 2,4-dichlorophenoxyacetic acid (2,4-D). The ability of baccharinoid B4 to stimulate callus growth appeared to increase with increased concentration in the culture medium. Analysis of callus cultures grown in medium amended with roridin E showed that B4, roridin E, and 8-hydroxyroridin E and verrucarols were formed in the tissues but not in the medium. The results of this study indicated that while the callus-inducing ability of roridin E seemed to be nonspecies-specific in nature, the ability of B4 to stimulate callus was a highly species-specific phenomena. Callus-inducing activity of roridin E may depend on the capacity of plant species to transform exogenous roridin E into baccharinoids or other macrocyclic trichothecene derivatives.     

Evaluation of trichothecene and nontrichothecene mycotoxins produced byFusarium in soybeans

Each of 12 cultures ofFusarium, comprising four species, isolated from moldy soybeans suspected of being involved in illness of wild geese, were grown separately in autoclaved moist rice, in autoclaved moist soybeans, and in surface sterilized-disinfected soybeans, assayed for various mycotoxins, and fed to rats. Four additional cultures that produced known toxins on rice were also grown on soybeans as controls. All isolates, except one ofF moniliforme, grown in rice resulted in weight loss of rats, and that one resulted in weight gain; 12 of the isolates caused death. One isolate ofF poae grown in soybeans caused death when consumed by rats, but none of the other 15 resulted in weight loss or overt injury. Much larger amounts of zearalenone, deoxynivalenol (DON), T-2 toxin, neosolaniol, T-2 tetraol, wortmannin, and moniliformin were produced by the cultures on rice than on soybeans, but more HT-2 toxin was produced by one isolate ofF poae grown on soybeans than when grown on rice. Soybeans appear to be a poor substrate for elaboration of most of the toxins produced by the isolates tested.     

Trichothecene production and the relationship to vegetative compatibility groups in shape Fusarium poae

Twenty-two Norwegian and two Polish isolates of Fusarium poae were cultured on rice and in two liquid media, MOSS and MYRO. All samples were analysed for trichothecenes by gas chromatography mass-spectrometer. Association of trichothecene production with vegetative compatibility groups was studied in the F. poae isolates. Twenty of the isolates produced the type A trichothecenes diacetoxyscirpenol or monoacetoxyscirpenol in at least one of the media, in the concentration range of 0.01 to 65 μg ml^-1 in the liquid culture and 0.1 to 67 μg g^-1 on rice. The other group of trichothecenes detected were of type B; nivalenol and its two acetyl-derivatives 4-acetylnivalenol and diacetylnivalenol. Twelve of the strains produced at least one of these metabolites in the concentration range of 0.01 to 7 μg ml^-1 in the liquid culture and 0.1 to 18 μg g^-1 on rice (sum of nivalenol and its acetylated derivatives). A significant correlation was observed between the two groups of toxins (logarithm). None of the isolates produced T-2 or HT-2 toxin. In a previous study these isolates were divided into 13 vegetative compatibility groups. Significant variation in the trichothecene production was observed between different vegetative compatibility groups, but also to some extent within the same group. This revised version was published online in August 2006 with corrections to the Cover Date.     

Mycotoxins in Crude Building Materials from Water-Damaged Buildings

We analyzed 79 bulk samples of moldy interior finishes from Finnish buildings with moisture problems for 17 mycotoxins, as well as for fungi that could be isolated using one medium and one set of growth conditions. We found the aflatoxin precursor, sterigmatocystin, in 24% of the samples and trichothecenes in 19% of the samples. Trichothecenes found included satratoxin G or H in five samples; diacetoxyscirpenol in five samples; and 3-acetyl-deoxynivalenol, deoxynivalenol, verrucarol, or T-2-tetraol in an additional five samples. Citrinine was found in three samples. Aspergillus versicolor was present in most sterigmatocystin-containing samples, and Stachybotrys spp. were present in the samples where satratoxins were found. In many cases, however, the presence of fungi thought to produce the mycotoxins was not correlated with the presence of the expected compounds. However, when mycotoxins were found, some toxigenic fungi usually were present, even if the species originally responsible for producing the mycotoxin was not isolated. We conclude that the identification and enumeration of fungal species present in bulk materials are important to verify the severity of mold damage but that chemical analyses are necessary if the goal is to establish the presence of mycotoxins in moldy materials.