A potential role of calcium in apoptosis and aberrant chromatin forms in porcine kidney PK15 cells induced by individual and combined ochratoxin A and citrinin.

The aim of this study was to establish the involvement of calcium signalling in genotoxicity, apoptosis and necrosis evoked by ochratoxin A (OTA) and citrinin (CTN) alone or in combination in porcine kidney PK15 cells. Cell proliferation test (MTT) and trypan blue assays (24 h) demonstrated that CTN (IC(50) = 73.5 ± 1.0, 75.4 ± 1.4 µM, respectively) was less toxic than OTA (IC(50) = 14.0 ± 2.4, 20.5 ± 1.0 µM, respectively). To test their cytotoxic interactions, two doses of single OTA (6 and 10 µM) and CTN (30 and 50 µM) and their combinations were applied. Combined treatment showed additive cytotoxic effects. OTA and CTN induced dose-dependent increase in cytosolic calcium level (assessed with Fura-2 AM). However, combined treatment did not provoke additional increase in calcium signal. The rate of apoptosis and necrosis (DAPI-antifade staining) was significantly higher after 12 h than 24 h, while the frequencies of micronuclei (MNs) and nuclear buds (NBs) were higher after 24 h than 12 h treatment. Combined exposure resulted in apoptotic and necrotic synergism, while genotoxic effects of OTA + CTN were noted as antagonistic or additive. Co-exposure of cells to calcium chelator BAPTA-AM significantly reduced CTN and OTA + CTN-evoked apoptosis. Twenty-four hour after co-exposure to BAPTA-AM and a single OTA and CTN, MNs significantly decreased while NBs dropped significantly after co-treatment with BAPTA-AM and OTA + CTN. In conclusion, disturbance of Ca(2+) homeostasis caused by OTA and CTN plays a significant role in cell genotoxicity and death.

Beauvericin and ochratoxin A genotoxicity evaluated using the alkaline comet assay: single and combined genotoxic action.

This study was aimed at investigating the genotoxic potential of single beauvericin (BEA) and ochratoxin A (OTA) as well as their interaction in porcine kidney epithelial PK15 cells and human leukocytes using the alkaline comet assay. IC(50) of BEA (5.0 +/- 0.6) and OTA (15.8 +/- 1.5) estimated by MTT reduction assay shows that BEA is three times more toxic than OTA. BEA (0.1 and 0.5 microM) and OTA (1 and 5 microM) were applied alone or in combination of these concentrations for 1 and 24 h in PK15 cells and human leukocytes. Genotoxicity of these toxins to PK15 cells was time- and concentration dependent. After 1 h, significant increase in tail length, tail intensity, tail moment, and abnormal sized tails (AST) was noted upon exposure to 1 muM of OTA alone and BEA + OTA combinations. Single BEA (0.5 microM) and OTA (1 and 5 microM) and their combinations evoked significant DNA damage in PK15 cells, considering all comet tail parameters measured after 24 h of treatment. Human leukocytes were slightly concentration but not time dependent. After 1 h of exposure, there were no significant changes in the tail length. Tail intensity, tail moment, and/or incidence of AST were significantly higher in cells treated with single OTA or BEA and their combinations than in control cells. DNA damage in leukocytes was significantly higher after 24 h of exposure to single toxins and their combinations, considering all comet tail parameters, but these changes were less pronounced than in PK15 cells. Combined toxins showed additive and synergistic effects in PK15 cells, while only additive effects were observed in human leukocytes. Combined prolonged exposure to BEA and OTA in subcytotoxic concentrations through food consumption could induce DNA damage contributing to the carcinogenicity in animals and humans.

Synthesis and biological evaluation of O-methyl and O-ethyl NSAID hydroxamic acids.

This paper reports the synthesis of O-methyl and O-ethyl NSAID hydroxamic acids, their antimicrobial activities, and their ability to inhibit urease and soybean lipoxygenase activities. Ibuprofen and fenoprofen hydroxamic acids with free hydroxy groups present the highest antimicrobial activity, while indomethacin and diclofenac analogs show significantly lower antimicrobial activity. Diclofenac hydroxamic acid 4e exerts the highest anti-urease activity. Indomethacin O-ethyl hydroxamic acid 3h and ibuprofen O-benzyl hydroxamic acid 4b exert significant inhibitory activities on soybean lipoxygenase. Fenoprofen and indomethacin O-ethyl hydroxamic acids 3b and 3h and diclofenac and indomethacin O-benzyl analogs 4g and 4i highly inhibit lipid peroxidation. The highest antioxidant activity was shown by fenoprofen derivative 3b.

Isolation and cytotoxicity of low-molecular-weight metabolites of Candida albicans.

In this study, the low molecular weight lypophilic metabolites of C. albicans and C. dubliniensis strains produced in a synthetic medium with the addition of fetal calf serum were identified using LC/MS and MS/MS technique and quantified. All strains investigated produce a metabolite with a UV spectra maximum at 224 and 279 nm and minimum at 243 nm. Following comparison with ESI, MS/MS spectral data of a reference compound, the metabolite was identified as 3-indoleethanol (tryptophol). The concentration of extracellular tryptophol in the biosynthesis of C. albicans and C. dubliniensis ranged from 2.45 microg/mL to 191 microg/mL, respectively. Contrary to previously published data, gliotoxin or gliotoxin-like compounds were not detected, and all investigated C. albicans and C. dubliniensis strains have the same metabolite profile. Cytotoxic effects of tryptophol and 3-indolelactic acid (precursor of tryptophol biosynthesis) were cell-line-dependent. The EC50 of tryptophol ranged between 2 and 7 mM, with the EC50 of 3-indolelactic acid approximately double (between 4 and 8 mM). Tryptophol exhibited cell-type dependent cytotoxicity in relatively high concentrations, with domination of apoptosis.

Increased permeability of the blood-brain barrier following administration of glyceryl trinitrate in common carp (Cyprinus carpio L.).

Nitric oxide (NO) has been implicated in the pathogenesis of migraine and treatment with its exogenous donor glyceryl trinitrate (GTN) represents widely accepted experimental "migraine model". In this study, glyceryl trinitrate was administered intraperitoneally to carps, serum nitrite and nitrate levels were determined, permeability of blood-brain barrier was investigated, and histological changes of brain tissue were analyzed. Serum nitrite and nitrate levels displayed characteristic biphasic pattern with moderate initial increase and maximal terminal increase, suggesting the GTN-induced endogenous NO synthesis. Increased permeability of the blood-brain barrier in GTN-treated animals was determined based on Evans blue capillary leakage into the brain tissue. Histological analysis revealed changes consistent with vasodilatation and oedema. Our study strongly supports the importance of the NO role in the pathogenesis of migraine attacks and increase in blood-brain barrier permeability during the attack. The study has also provided evidence that this mechanism of action is conserved to the lower vertebrate.

Lipid peroxidation and glutathione levels in porcine kidney PK15 cells after individual and combined treatment with fumonisin B(1), beauvericin and ochratoxin A.

Individual and combined effects of the mycotoxins fumonisin B(1), beauvericin and ochratoxin A on cell viability, lipid peroxidation (TBARS) and intracellular glutathione (GSH) were studied on porcine kidney epithelial cells (PK15). Cells were treated with 0.05, 0.5 and 5 microg/ml of each mycotoxin or the combinations of two or all three applied in equal concentrations for 24 and 48 hr. Changes in cell viability, GSH and TBARS levels showed that the cytotoxic effects of these mycotoxins were concentration- and time-dependent. After 24 hr, cell viability was significantly decreased by the exposure to 5 microg/ml of fumonisin B(1) (25%), beauvericin (30%) and ochratoxin A (35%), as compared to controls. Only ochratoxin A (5 microg/ml) increased TBARS (56%), with further significant increase (85%) after 48 hr exposure. Fumonisin B(1) and beauvericin significantly increased TBARS (57% and 80%, respectively) only when the highest dose was applied for 48 hr. After 24 hr, GSH was significantly decreased (18%) by ochratoxin A (0.05 microg/ml), whereas fumonisin B(1) and beauvericin significantly decreased GSH at the concentration of 0.5 microg/ml. Combined treatment with fumonisin B(1), beauvericin and ochratoxin A resulted mostly in additive effects especially after a 24-hr exposure, although synergistic as well as antagonistic interactions could not be excluded depending on toxin concentrations and time of exposure. This is the first report on beauvericin-induced effects on lipid peroxidation and GSH in animal cells.

Interaction between certain moulds and aflatoxin B1 producer Aspergillus flavus NRRL 3251.

The objective of this study was to evaluate biotic interaction between some mould species and active producer of aflatoxin B1 Aspergillus flavus NRRL 3251, co-cultured in yeast-extract sucrose (YES) broth. Twenty-five mould strains of Alternaria spp., Cladosporium spp., Mucor spp., A. flavus and A. niger, used as biocompetitive agents, were isolated from outdoor and indoor airborne fungi, scrapings of mouldy household walls, and from stored and post-harvest maize. Aflatoxin B1 was extracted from mould biomasses with chloroform and detected using the multitoxin TLC method. The results confirm antagonistic interaction between all strains tested. With Alternaria spp. and Cladosporium spp., aflatoxin B1 production decreased 100%, compared to detection in a single culture of A. flavus NRRL 3251 (Cmean=18.7 microg mL-1). In mixed cultures with Mucor spp., aflatoxin B1 levels dropped to (5.6-9.3) microg mL-1, and the inhibition was from 50% to 70%. Four of five aflatoxin non-producing strains of A. flavus interfered with aflatoxin production in mixed culture, and reduced AFB1 productivity by 100%. One strain showed a lower efficacy in inhibiting AFB1 production (80%) with a detectable amount of AFB1 3.7 microg mL-1 when compared to control. A decrease in toxin production was also observed in dual cultivation with A. niger strains. It resulted in 100% reduction in three strains), 90% reduction in one strain (Cmean=1.9 microg mL-1) and 80% reduction in one strain (Cmean=3.7 microg mL-1) inhibition.

A year-round aeromycological study in Zagreb area, Croatia.

A 1-year aeromycological study was conducted in the area of Zagreb, the capital of Croatia, in order to establish seasonal variations in the composition and concentration of aeromycota. Sampling was carried out at 3 locations: the city centre, the Pharmaceutical botanical garden and the mountain of Medvednica, at regular intervals using the Mas 100 Eco Air-sampler with Sabouraud-dextrose agar. Airborne fungi peaked during spring and summer (110-284 cfu/m3), while lower levels were detected in autumn and winter at each of the 3 sampling sites (6-128 cfu/m3). Significantly lower concentrations were found in Medvednica region (p < 0.01) during most sampling periods. Yeasts were present in higher concentrations in autumn and winter (11-46 cfu/m3) than during spring and summer (9-11 cfu/m3) in the city centre and botanical garden. In the Medvednica region, yeasts were found at significantly lower concentrations than at other locations only during the autumn and winter (1-16 cfu/m3). The dominant fungi contributing to these differences were species of Cladosporium, Penicillium and Alternaria. These genera comprised between 30.4-79.5% of the samples. Other stable components of aeromycota were Fusarium, Aspergillus and sterile mycelia (11.1-44.0%). Total counts of airborne fungi as well as individual counts of Cladosporium and Alternaria showed significant positive correlations with temperature and solar radiation (p < 0.05). Alternaria also showed a significant correlation with wind speeds while Cladosporium was negatively correlated with atmospheric pressure (p < 0.05). Yeasts showed a significant positive correlation with relative humidity, yet were negatively correlated with temperature and solar radiation in the city centre and the botanical garden. In contrast, a significant positive correlation in the case of yeasts was observed in the Medvednica region with respect to temperature and solar radiation (p < 0.05).

Mycotoxigenicity of clinical and environmental Aspergillus fumigatus and A. flavus isolates.

Clinical isolates of fifty strains of A. fumigatus and 30 strains of A. flavus from immmunocompromised patients from the hematological unit were analyzed for mycotoxin production and compared with the same number of environmental isolates (from soil, compost, and air). Only 9 (18%) strains of A. fumigatus produced gliotoxin in a mean concentration 2.22 mg mL-1 (range 0.5-5 mg mL-1). Aflatoxin B1 was detected in 7 (23%) isolates (range from 0.02 to 1.2 mg L-1) and aflatoxin G1 in one (3%) of clinical A. flavus isolates (0.12 mg L-1). In the group of environmental isolates, 11 (37%) were positive for aflatoxin B1 production (range from 0.02 to 1.2 mg L-1) and one for aflatoxin G1 (0.02 mg L-1). Bioautoantibiogram ("bioassay in situ") on TLC plates against Bacillus subtilis NCTC 8236 showed that only gliotoxin-producing strains have bactericidal activity of Rf values corresponding to gliotoxin. The secondary-metabolite profiles of clinical and environmental A. fumigatus and A. flavus isolates were homogeneous, except for gliotoxin production, which was detected only in the group of clinical isolates of A. fumigatus (18%).

Antifungal activity of fluid extract and essential oil from anise fruits (Pimpinella anisum L., Apiaceae).

Antifungal activities of fluid extract and essential oil obtained from anise fruits Pimpinella anisum L. (Apiaceae) were tested in vitro on clinical isolates of seven species of yeasts and four species of dermatophytes. Diffusion method with cylinders and the broth dilution method were used for antifungal activity testing. Anise fluid extract showed antimycotic activity against Candida albicans, C. parapsilosis, C. tropicalis, C. pseudotropicalis and C. krusei with MIC values between 17 and 20% (v/v). No activity was noticed against C. glabrata, and anis fruits extracts showed growth promotion activity on Geotrichum spp. Anise fruits extract inhibited the growth of dermatophyte species (Trichophyton rubrum, T. mentagrophytes, Microsporum canis and M. gypseum) with MIC values between 1.5 and 9.0% (V/V). Anise essential oil showed strong antifungal activity against yeasts with MIC lower than 1.56% (V/V) and dermatophytes with MIC lower than 0.78% (V/V). Significant differences in antifungal activities were found between anise fluid extract and anise essential oil (p<0.01). Anise essential oil exhibited stronger antifungal activities against yeasts and dermatophytes with MIC values between 0.10 and 1.56% (V/V), respectively.

Antimicrobial activity of some hydroxamic acids.

Several hydroxamic acids, viz., N-benzyl-N'-hydroxysuccinamide (BHS), poly[alpha,beta-(N-hydroxy)-DL-aspartamide] (PHA), poly[alpha,beta-(N-hydroxy-N-methyl-DL-aspartamide)] (PMHA) and poly[alpha,beta-(N-hydroxy)-DL- aspartamide]/poly[alpha,beta-(N-2-hydroxyethyl)-DL-aspartamide] (PHA-PHEA 1:1) were prepared and screened for their antimicrobial activity. Ten Gram-positive and 7 Gram-negative species of bacteria, 5 Candida species, 4 dermatophyte species and 3 mould species were used in tests. Compound showed no antimicrobial activity on any of the tested microorganisms. Other compounds showed a narrow spectrum of antibacterial activity, but no antifungal activity.

Investigation of antimicrobial activity of Pelargonium radula (Cav.) L'Hérit.

Antimicrobial activities of two ethanolic extracts, made from fresh and dried leaves of Pelargonium radula (Cav.) L'Hérit, were tested against fourteen species of bacteria and fifteen species of fungi. The well-diffusion method indicated the strongest activity against Pseudomonas aeruginosa. The broth dilution method revealed that the most sensitive microorganisms were Bacillus pumilus, Bacillus subtilis, Escherichia coli and Serratia marcescens. Extract prepared from fresh leaves showed significantly higher antimicrobial activity than the extract prepared from dried leaves.

Antimicrobial activity of flavonoids from Pelargonium radula (Cav.) L'Hérit.

Flavonoids from Pelargonium radula (Cav.) L'Hérit were purified by column chromatography. Two fractions were obtained: F1 (main flavonoid isoquercitrin) and F2 (main flavonoid rutin). In vitro antimicrobial activity of F1 and F2 were tested against eleven species of bacteria and eleven species of fungi. Both fractions demonstrated strong inhibitory activity against Staphylococcus aureus, Proteus rettgeri, Candida tropicalis and Microsporum gypseum. Staphylococcus sp. (coagulase-negative) and Candida lusitaniae were strongly inhibited only by fraction F1 and Fusarium graminearum only by fraction F2.

Toxic effects of Ustilago maydis and fumonisin B1 in rats.

The toxicity of Ustilago maydis and the possible synergism with fumonisin B1 (FB1) were studied in Fischer rats by evaluating pathological changes and biochemical parameters in blood serum (LDH, ALT, GGT, ChE) and tissue homogenate of brain and liver (AChE, ChE, GGT, ALP). One experimental group (US) consumed diet with 70% of U. maydis galls and the other group (US+FB1) was fed pellets containing 70% of U. maydis galls and 1 mg of FB1 per kg of diet for 17 days. Control group (C) consumed standard pellets. During the trial, experimental animals were more excited, showing hyperactivity. Body mass gains slightly increased in both groups compared to the control. Gross pathological changes in liver, lungs, uterus and ovaries were more pronounced in the US+FB1 than in the US group. Specific catalytic activities of AChE decreased by 61% and by 63% in the liver and brain homogenate of the US group (p<0.05) compared to the control, indicating neurotoxic activity of U. maydis. Also, specific catalytic concentration of AChE and ALP was significantly decreased in the liver of the US+FB(1) group (p<0.05). Activity of LDH in the blood serum was increased up to 166% and 165% in the US+FB1 group (p<0.05) compared to the control and US group values, respectively, which indicates that FB1 was responsible for the disruption of cell membrane integrity. These findings suggest that Ustilago maydis and FB1 showed neurotoxicity in Fischer rats, which could be related to the alkaloids of U. maydis and disruption of sphingolipid metabolism by FB1 activity.

Verruculogen production in airborne and clinical isolates of Aspergillus fumigatus Fres.

Among airborne aspergilli sampled in outdoor air of the Zagreb area (2002/2003), Aspergillus niger (v. Teigh.) and A. fumigatus (Fres.) were the most abundant species (20-30%), with low mean annual concentrations (0.21-1.04 CFU m-3). Higher concentrations of A. fumigatus were observed in autumn and winter (0.5-1.05 CFU m-3) than in spring and summer (0-0.4 CFU m-3). On the other hand, A. fumigatus was found to be the most frequent isolate from upper and/or lower respiratory tracts of imunocompromised patients in many studies. This species produces several mycotoxins, including the tremorgenic mycotoxin verruculogen that can be found in spores and during myceliar growth. Verruculogen production ability was tested on 30 airborne and 33 clinical isolates of A. fumigatus. In both groups, high percentage of verruculogen-producing strains was noticed (84% of airborne and 91% of clinical isolates). Verruculogen production was not significantly different in the groups of airborne isolates (0.34+/-0.16 mg mL-1), and clinical isolates (0.26+/-0.19 mg mL-1).

Antimicrobial activity of juniper berry essential oil (Juniperus communis L., Cupressaceae).

Juniper essential oil (Juniperi aetheroleum) was obtained from the juniper berry, and the GC/MS analysis showed that the main compounds in the oil were alpha-pinene (29.17%) and beta-pinene (17.84%), sabinene (13.55%), limonene (5.52%), and mircene (0.33%). Juniper essential oil was evaluated for the antimicrobial activity against sixteen bacterial species, seven yeast-like fungi, three yeast and four dermatophyte strains. Juniper essential oil showed similar bactericidal activities against Gram-positive and Gram-negative bacterial species, with MIC values between 8 and 70% (V/V), as well as a strong fungicidal activity against yeasts, yeast-like fungi and dermatophytes, with MIC values below 10% (V/V). The strongest fungicidal activity was recorded against Candida spp. (MIC from 0.78 to 2%, V/V) and dermatophytes (from 0.39 to 2%, V/V).

Flavonoid analysis and antimicrobial activity of commercially available propolis products.

Propolis ethanolic solutions are the most used propolis products on the market for the treatment of minor ulcers in the mouth, angina, thrush or skin infections. Since it is still an unofficial drug in pharmacy, we analyzed the contents of flavonoids in ten commercially available ethanolic solutions of propolis from the Croatian market using two complementary colorimetric methods. Antimicrobial activities, determined with the diffusion method, against six bacterial species (Bacillus subtilis NCTC 8236, Staphylococcus aureus ATCC 25923, Streptococcus pyogenes ATCC 12204, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 10536, Pseudomonas aeruginosa ATCC 27853, and one yeast-like fungus Candida albicans ATCC 10231 were compared. Results of flavonoids analysis suggested that the contents of flavones and flavonols in the products were uniform and ranged from 0.14 to 0.41%, but the content of flavanones varied greatly from 0.43 to 18.78%. Total flavonoid content, as the sum of two colorimetric methods, in propolis products was between 0.78 and 18.92%, and most products had the flavonoids content below 9%. All products with the total flavonoids content above 1% showed antimicrobial activity against the four Gram-positive bacterial species tested, and against P. aeruginosa and the yeast-like fungus C. albicans. Total flavonoids contents, expressed as the sum of two colorimetric methods, could be useful methods for estimating the flavonoid contents of propolis products. Our results indicate that the quality of commercially available propolis products requires verification.

[Beauvericin: chemical and biological aspects and occurrence]. / Bovericin: kemizam, bioloski aspekti i rasirenost.

Beauvericin (BEA) is a cyclic hexadepsipeptide produced by Beauveria bassiana, Paecilomyces fumosoroseus, Paecilomyces tenuipes, Polyporus sulphurous, and a variety of Fusarium species. This mycotoxin shows antimicrobial, insecticidal, cytotoxic, and apoptotic activity. It is the most potent specific inhibitor of cholesterol acyltransferase and possesses ionophoric properties. BEA increases ion permeability in biological membranes by forming a complex with essential cations (Ca2+, Na+, K+), which may affect the ionic homeostasis. BEA has been frequently found in maize samples in Europe, USA and Africa and co-contamination with other Fusarium toxins such as fumonisins, and moniliformin was also found. There is only one report of BEA occurrence and co-occurrence with fumonisin B1, fumonisin B2 and ochratoxin A in Croatia. Biological activity of BEA may increase the toxicity of other mycotoxins that co-occur with BEA in food. The role of BEA in the development of human and animal mycotoxicosis is still unknown.

Influence of media and temperature on gliotoxin production in Aspergillus fumigatus strains.

Gliotoxin is a secondary metabolite of the epipolythiodioxopiperazine family with biologically active internal disulfide bridge. It is produced by many fungal species, including Aspergillus fumigatus and A. terreus. A. fumigatus, which produces gliotoxin and more than twenty other secondary metabolites, is the leading cause of invasive aspergillosis. Gliotoxin production in situ influence the development of aspergillosis. This study investigated the in vitro production of gliotoxin in nine A. fumigatus isolates from the upper respiratory tract of immunocompromised patients. The effects of media composition and incubation temperature were studied. Gliotoxin was extracted from biomass and its concentration was semi-quantitatively analysed using thin-layer chromatography. Gliotoxin production was higher in the yeast-extract liquid medium (YES) than in the synthetic Czapek-Dox liquid medium (CZA). Incubation at 37 degrees C resulted in higher gliotoxin production than at 25 degrees C, probably because higher temperatures favour expansive growth of the mycelium. Gliotoxin could be detected after three days of incubation at concentrations 4.06 mg mL(-1) (in YES at 37 degrees C) and 1.07 mg mL(-1) (in CZA at 25 degrees C). YES broth as a medium containing 4% sucrose and 2% of yeast extract is a very rich substrate for the production of gliotoxin in vitro.

Toxigenic potential of Fusarium species isolated from non-harvested maize.

The objective of this study was to determine the frequencies of distribution and the toxigenic potential of Fusarium species isolated from non-harvested maize left in field over winter in 1999 (N = 56) and 2003 (N = 56) in northern Croatia. Zearalenone and trichothecenes (DON, DAS, T-2) were isolated and detected using multitoxin extraction and TLC method. Modified TLC method was applied to detect fumonisin B1. Fusarium species were the most frequent fungi found in maize with 78.6% in 1999 and 85.0% in 2003. Among fusaria F. verticillioides was dominant species found in 12.5% (1999) and 35.7% (2003) of maize samples. Other determined fusaria were F. graminearum (8.9% in 2003), F. poae and F. sporotrichoides (2.0% to 3.6%), F. tricinctum and F. tabacinum (2.0% in 1999). Production of FB1 was established for all F. verticillioides (7/7) isolated in 1999 in concentration range from 280 mg L(-1) to 918 mg L(-1), and for 11 of 20 strains found in 2003 (48 mg L(-1) to 400 mg L(-1)). Three strains also produced zearalenone: one strain in 1999 produced 3.80 mg L(-1) and 2 strains in 2003 produced 20.0 mg L(-1) and 70.0 mg L(-1). In addition, four strains of F. graminearum isolated in 2003 produced higher amounts of zearalenone (60.0 mg L(-1) to 180.0 mg L(-1)). T-2 production was found in F. tricinctum (1.55 mg L(-1)) isolated in 1999.