Determination of six <Emphasis Type="Italic">Alternaria</Emphasis> toxins with UPLC-MS/MS and their occurrence in tomatoes and tomato products from the Swiss market

An ultra performance liquid chromatography (UPLC)-tandem mass spectrometry (MS/MS) method was developed for the determination of the Alternaria toxins tenuazonic acid, alternariol, alternariol monomethyl ether, altenuene, altertoxin I and tentoxin. Owing to its instability, altenusin could not be determined. The sample preparation includes an acidic acetonitrile/water/methanol extraction, followed by SPE clean-up step, before injection into the UPLC-MS/MS system. The separation was made on an Acquity UPLC column using a water/acetonitrile gradient with ammonium hydrogen carbonate as a modifier. Matrix compounds of real samples led to enhancement as well as suppression of the target compounds, depending on analyte and matrix. The recoveries were between 58 and 109% at a level of 10 μg/kg. Eighty-five tomato products, consisting of peeled and minced tomatoes, soup and sauces, tomato purées and concentrates, ketchup as well as dried and fresh tomatoes, were taken from the Swiss market in 2010. Tenuazonic acid was found most frequently (81 out of 85 samples) and in the highest levels of up to 790 μg/kg. Alternariol and alternariol monomethyl ether were found in lower concentrations, ranging from <1 to 33 μg/kg for alternariol and <5 to 9 μg/kg for alternariol monomethyl ether. Only a few samples were positive for altenuene and tentoxin. Altertoxin I was never detected.     

Antimicrobial fungal endophytes from the botanical medicine goldenseal (Hydrastis canadensis)

The potential of fungal endophytes to alter or contribute to plant chemistry and biology has been the topic of a great deal of recent interest. For plants that are used medicinally, it has been proposed that endophytes might play an important role in biological activity. With this study, we sought to identify antimicrobial fungal endophytes from the medicinal plant goldenseal (Hydrastis canadensis L., Ranunculaceae), a plant used in traditional medicine to treat infection. A total of 23 fungal cultures were obtained from surface-sterilized samples of H. canadensis roots, leaves and seeds. Eleven secondary metabolites were isolated from these fungal endophytes, five of which had reported antimicrobial activity. Hydrastis canadensis plant material was then analyzed for the presence of fungal metabolites using liquid chromatography coupled to high resolving power mass spectrometry. The antimicrobial compound alternariol monomethyl ether was detected both as a metabolite of the fungal endophyte Alternaria spp. isolated from H. canadensis seeds, and as a component of an extract from the H. canadensis seed material. Notably, fungi of the Alternaria genus were isolated from three separate accessions of H. canadensis plant material collected in a time period spanning 5 years. The concentration of alternariol monomethyl ether (991 mg/kg in dry seed material) was in a similar range to that previously reported for metabolites of ecologically important fungal endophytes. The seed extracts themselves, however, did not possess antimicrobial activity.     

Blue light inhibits mycotoxin production and increases total lipids and pigmentation in Alternaria alternata

Light inhibits production of the mycotoxins alternariol and alternariol monomethyl ether, both polyketids produced by Alternaria alternata. This effect seems to be general because seven isolates of A. alternata with different alternariol- and alternariol monomethyl ether-producing abilities all respond to continuous light with reduced levels of alternariol and alternariol monomethyl ether when the mycotoxins were calculated on a microgram-per-milligram (dry weight) basis. Blue light inhibited alternariol and alternariol monomethyl ether production 69 and 77%, respectively. Red light gave no reduction of toxin levels. Total lipids were increased 25% when mycelium was grown in blue light as compared with red light or darkness. In white or blue light, but not in red light or darkness, a red-brown pigment accumulated by the mycelium.     

Blue light inhibits mycotoxin production and increases total lipids and pigmentation in Alternaria alternata.

Light inhibits production of the mycotoxins alternariol and alternariol monomethyl ether, both polyketids produced by Alternaria alternata. This effect seems to be general because seven isolates of A. alternata with different alternariol- and alternariol monomethyl ether-producing abilities all respond to continuous light with reduced levels of alternariol and alternariol monomethyl ether when the mycotoxins were calculated on a microgram-per-milligram (dry weight) basis. Blue light inhibited alternariol and alternariol monomethyl ether production 69 and 77%, respectively. Red light gave no reduction of toxin levels. Total lipids were increased 25% when mycelium was grown in blue light as compared with red light or darkness. In white or blue light, but not in red light or darkness, a red-brown pigment accumulated by the mycelium.     

Production of alternarioi and alternariol monomethyl ether in natural substrates in comparison with semisynthetic culture medium

The comparison In toxins production and growth byAlternarla strains in liquid, solid culture media and natural substrates (rice and sunflower) was evaluated. Ground rice- corn steep liquor medium (GRCS) was the more suitable medium for production of alternariol (AOH) and alternariol monomethyl ether(AME). The maximum levels produced were 676 μg/50ml AOH and 1570/50ml AME. Rice was better than sunflower In supporting toxins production. Different ratios AOH/AME were found according to the substrate evaluated.     

<Emphasis Type="Italic">Alternaria</Emphasis> toxins in South African sunflower seeds: cooperative study

Sunflower seed samples (N = 80) from different sunflower cultivars originating from different localities in South Africa were analyzed for 15 toxins produced by fungi of the genus Alternaria by means of a simple one-step extraction dilute-and-shoot HPLC-MS/MS approach. References for valine-tenuazonic acid (Val-TeA), altenusin (ALTS), and altenuisol (ALTSOH) were isolated from fungal culture extracts and spectroscopically characterized. Additionally, valine-tenuazonic acid was tested regarding its cytotoxicity in comparison with tenuazonic acid (TeA) and showed less activity on HT-29 cells. Furthermore, alternariol monomethyl ether-3-O-ß-D-glucoside (AME-3G) was produced by fermentation of alternariol monomethyl ether (AME) with the fungus Rhizopus oryzae. The seed samples were analyzed both with and without hulls. The method covers the AAL toxins TA₁ and TA₂, altenuene (ALT) and iso-altenuene (iso-ALT), altenuisol, altenusin, altertoxin I (ATX-I) and altertoxin II (ATX-II), alternariol (AOH) and alternariol monomethyl ether, alternariol monomethyl ether-3-O-ß-D-glucoside, tenuazonic acid, allo-tenuazonic acid (allo-TeA) and valine-tenuazonic acid, and tentoxin (TEN). More than 80% of the samples were positive for one or more analytes above the respective limit of detection (0.2–23 μg/kg). Alternariol, its monomethyl ether, tentoxin, tenuazonic acid, altenuisol, and valine-tenuazonic acid were found in quantifiable amounts. The highest prevalences were found for tentoxin (73% positive, mean content 13.2 μg/kg, maximum level 130 ± 0.9 μg/kg) followed by tenuazonic acid (51% positive, mean content 630 μg/kg, maximum level 6300 ± 560 μg/kg). The obtained data were further analyzed statistically to identify quantitative or qualitative relationships between the levels of Alternaria toxin in the samples.     

链格孢霉醇和链格孢霉醇单甲醚产生菌株的筛选

本文对分离自小麦、马铃薯、番茄和茄子上链格孢霉属(Alternaria)2个种(链格孢和茄链格孢)的96个菌株,用枯草杆菌生长抑制试验筛选链格孢霉醇(AOH)和链格孢霉醇单甲醚(AME)的产生菌株,有48株产生毒性作用(占所测菌株的50％)。18株产强、中毒性菌用高效液相色谱分析,有13株产AOH和AME(占所测菌株的72.2％)。链格孢的产毒素菌株率比茄链格孢低。但产毒素含量却是前者明显高于后者。其中产AOH和AME的最高含量,链格孢菌株XA-8分别为280和5140mg/kg,而茄链格孢菌株SA-10分别为95.9和94.3mg/kg。     

Occurrence of alternariol, alternariol monomethyl ether and tenuazonic acid in Argentinean tomato puree

The occurrence ofAlternaria mycotoxins was investigated in 80 samples of tomato puree processed and sold in Argentina. Alternariol (AOH), alternariol monomethyl ether (AME) and tenuazonic acid (TA) were searched for by liquid chromatography. Thirty-nine of the 80 samples showed mycotoxin contamination. TA was found in 23 samples (39-4021 μg/kg), AOH in 5 samples (187-8756 μg/kg), and AME in 21 samples (84-1734 μg/kg). Co-occurrence of two of these toxins was detected in 10 samples. This is the first report of natural occurrence of AOH, AME and TA in tomato products in Argentina.     

Stability ofAlternaria toxins in fruit juices and wine

Alternaria alternata is a common fungal parasite on fruits and other plants and produces a number of mycotoxins, including alternariol (3,7,9-trihydroxy-1-methyl-6H-dibenzo [b,d]pyran-6-one), alternariol monomethyl ether (3,7-dihydroxy-9-methoxy-1-methyl-6H-dibenzo[b,d]pyran-6-one), and the mutagen altertoxin I {[1S-(1α,12aβ,12bα)] 1,2,11,12,12a, 12b-hexahydro-1,4,9,12a-tetrahydroxy-3,10-perylenedione}. Alternariol and alternariol monomethyl ether have previously been detected in some samples of fruit beverages. Stability studies of these toxins as well as altertoxin I added to fruit juices and wine (10–100 ng/mL) were carried out. To include altertoxin I in the analysis, cleanup with a polymer-based Varian Abselut solid phase extraction column was used, as recoveries from C-18 columns were low. The stabilities of alternariol and alternariol monomethyl ether in a low acid apple juice containing no declared vitamin C were compared with those in the same juice containing added vitamin C (60 mg/175 ml); there were no apparent losses at room temperature over 20 days or at 80°C after 20 min. in either juice. Altertoxin I was moderately stable in pH 3 buffer (75% remaining after a two week period). Furthermore, altertoxin I was stable or moderately stable in three brands of apple juice tested over 1–27 day periods and in a sample of red grape juice over 7 days. It is concluded that altertoxin I is sufficiently stable to be found in fruit juices and should be included in methods for alternariol and alternariol monomethyl ether.     

Inhibition of polyketide synthesis in Alternaria alternata by the fatty acid synthesis inhibitor cerulenin.

The fatty acid synthase inhibitor cerulenin (50 to 100 micrograms/ml) inhibited production of the polyketide mycotoxins alternariol (AOH) and alternariol monomethyl ether (AME) by the mold Alternaria alternata. The results suggested that AOH synthesis was inhibited by a direct mechanism by cerulenin, whereas production of AME was probably limited by a shortage of the precursor AOH.     

Inhibition of polyketide synthesis in Alternaria alternata by the fatty acid synthesis inhibitor cerulenin.

The fatty acid synthase inhibitor cerulenin (50 to 100 micrograms/ml) inhibited production of the polyketide mycotoxins alternariol (AOH) and alternariol monomethyl ether (AME) by the mold Alternaria alternata. The results suggested that AOH synthesis was inhibited by a direct mechanism by cerulenin, whereas production of AME was probably limited by a shortage of the precursor AOH.     

Natural Occurrence of Alternaria Toxins in Wheat-Based Products and Their Dietary Exposure in China

A total of 181 wheat flour and 142 wheat-based foods including dried noodle, steamed bread and bread collected in China were analyzed for alternariol (AOH), alternariol monomethyl ether (AME), tentoxin (TEN) and tenuazonic acid (TeA) by ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry. TeA was the predominant toxin found in 99.4% wheat flour samples at levels ranging from 1.76 μg/kg to 520 μg/kg. TEN was another Alternaria toxin frequently detected in wheat flour samples (97.2%) at levels between 2.72 μg/kg and 129 μg/kg. AOH and AME were detected in 11 (6.1%) samples at levels ranging from 16.0 μg/kg to 98.7 μg/kg (AOH) and in 165 (91.2%) samples with a range between 0.320 μg/kg and 61.8 μg/kg (AME). AOH was quantified at higher levels than AME with the ratio of AOH/AME ranging from 1.0 to 3.7. Significant linear regressions of correlation in toxin concentrations were observed between AOH and AME, AME and TeA, TEN and TeA, AOH+AME and TeA. At an average and 95th percentile, dietary exposure to AOH and AME in the Chinese general population and different age subgroups exceeded the relevant threshold value of toxicological concern (TTC), with the highest exposure found in children which deserves human health concern. TEN and TeA seem unlikely to be health concerns for the Chinese via wheat-based products but attention should be paid to synergistic or additive effects of TeA with AOH, AME, TEN and a further assessment will be performed once more data on toxicity-guided fractionation of the four toxins are available. It is necessary to conduct a systemic surveillance of Alternaria toxins in raw and processed foods in order to provide the scientific basis for making regulations on these toxins in China.     

Mycotoxins of Alternaria alternata produced on ceiling tiles

The production of mycotoxins by Alternaria alternata in cellulosic ceiling tiles was examined with thin-layer chromatography and high-performance liquid chromatography procedures. Alternariol and alternariol monomethyl ether were found in ceiling tile extracts, whereas extracts of control rice cultures of all three isolates produced these mycotoxins plus altenuene and altertoxin I. Extensive fungal growth and mycotoxin production occurred in the ceiling tiles at relative humidities of 84–89% and 97%. Received 28 May 1997/ Accepted in revised form 06 October 1997     

Precise determination of the <Emphasis Type="Italic">Alternaria</Emphasis> mycotoxins alternariol and alternariol monomethyl ether in cereal,fruit and vegetable products using stable isotope dilution assays

Cereal, fruit and vegetable products were analyzed for contamination with the Alternaria mycotoxins alternariol (AOH) and alternariol monomethyl ether (AME) using stable isotope dilution assays (SIDAs). Both toxins were practically not detected in cereals and cereal products: AOH—one out of 13 samples at a content of 4.1 μg/kg; AME—two out of 13 samples at contents ranging between 0.2 and 0.6 μg/kg. However, if cereals for animal nutrition were analyzed, much higher values were found: AOH—five out of six samples (13–250 μg/kg); AME—six out of six samples (3–100 μg/kg). This finding may pose a potential problem concerning animal health. AOH and AME were frequently detected in vegetable products: AOH—5 out of 10 samples (2.6–25 μg/kg); AME—6 out of 10 samples (0.1–5 μg/kg). Tomato products were affected, especially. The highest content of AOH (25 μg/kg) and AME (5 μg/kg) were found in triple concentrated tomato paste. Special wines like “Trockenbeerenauslese” or “Spätlese” (affected by noble rot in the vineyard) contained AOH (4/6 samples; 1.2–4.9 μg/kg) and AME (4/6 samples; 0.1–0.3 μg/kg), but the values did not exceed the values of both toxins that were found generally in wines.     

Light inhibits the production of alternariol and alternariol monomethyl ether in Alternaria alternata.

Alternaria alternata (Fr.) Keissler, grown in drop culture, produced alternariol and alternariol monomethyl ether in late growth phase. Production was almost completely inhibited when the fungal cultures were exposed to white light (180 W/m2), although mycelial dry weight was not significantly affected. The fungus was most sensitive to light during the exponential growth phase. Twelve hours of light exposure was sufficient to decrease significantly the production of the secondary metabolites. In light the fungus produced a red-brown pigment of unknown nature.     

Light inhibits the production of alternariol and alternariol monomethyl ether in Alternaria alternata

Alternaria alternata (Fr.) Keissler, grown in drop culture, produced alternariol and alternariol monomethyl ether in late growth phase. Production was almost completely inhibited when the fungal cultures were exposed to white light (180 W/m2), although mycelial dry weight was not significantly affected. The fungus was most sensitive to light during the exponential growth phase. Twelve hours of light exposure was sufficient to decrease significantly the production of the secondary metabolites. In light the fungus produced a red-brown pigment of unknown nature.     

Influence of fungicides on mycotoxin production by Alternaria mali

Extracts of fungicide induced variants ofAlternaria mali were tested with mice and bacteria. Both the living fungi and their crude chloroform extracts inhibited growth ofStaphylococcus aureaus, Sarcina lutea, Bacillus mycoides, andB. subtilis. B. megaterium was not sensitive to most of the extracts and was only slightly so to the remainder. The LD₅₀ in mice when injected intraperitoneally ranged from 300 mg/kg to 2400 mg/kg; however, in some cases there were no lethal effects. The toxicity of the wild type was greatly reduced when grown in the presence of fungicide decomposition products. Altenuene, alternariol, and alternariol monomethyl ether were not found in any of the extracts.     

Effect of water activity and temperature on Tenuazonic acid production byAlternaria alternata on sunflower seeds

Alternaria alternata is isolated in high frequency in sunflower seeds both in the field and storage. This species produces several toxic metabolites among them alternariol, alternariol monomethyl ether and tenuazonic acid. The accumulation of mycotoxins is regulated by physical, chemical and biological factors and for their production in many commodities nothing is known with regards to these conditions. In sunflower seeds the optimal conditions of temperature and water activity for tenuazonic acid production are unknown. The aim of this work was to investigate the effect of temperature and water activity on tenuazonic acid production byAlternaria alternata ITEM 539 in sunflower seeds. The temperature conditions evaluated were: 20, 25, and 30 °C and the water activities were 0.97, 0.90, 0.87, 0.80. The tenuazonic acid determinations were carried out during the incubation period at intervals of 7 days. Under conditions of constant temperature 25 °C) and variable water activities, 0.90 was the optimal value for tenuazonic acid production. At this water activity of the optimal temperature for tenuazonic acid production was 25 °C.     

Effect of pesticides inAlternaria alternata mycotoxins production

The effect of pesticides onAlternaria mycotoxins was evaluated both in culture media and in sunflower seeds. Different behaviour was observed depending on whether insecticides or fungicides were considered. When Captan, Lindaphor and Dichlorvos were evaluated in sunflower seeds their effects were dependent on the toxin being evaluated (alternariol, alternariol monomethyl ether and tenuazonic acid). A full spectrum of effects was observed, ranging from no effect, stimulation, to inhibition.     

Natural occurrence of alternariol and alternariol monomethyl ether in soya beans

The natural occurrence of alternariol (AOH) and alternariol monomethyl ether (AME) in soya beans harvested in Argentina was evaluated. Both toxins were simultaneously detected by using HPLC analysis coupled with a solid phase extraction column clean-up. Characteristics of this in-house method such as accuracy, precision and detection and quantification limits were defined by means of recovery test with spiked soya bean samples. Out of 50 soya bean samples, 60% showed contamination with the mycotoxins analyzed; among them, 16% were only contaminated with AOH and 14% just with AME. Fifteen of the positive samples showed co-occurrence of both mycotoxins analyzed. AOH was detected in concentrations ranging from 25 to 211?ng/g, whereas AME was found in concentrations ranging from 62 to 1,153?ng/g. Although a limited number of samples were evaluated, this is the first report on the natural occurrence of Alternaria toxins in soya beans and is relevant from the point of view of animal public health.