Determinación de perfiles de producción de metabolitos secundarios característicos de especies del género Alternaria aisladas de tomate

BackgroundMany Alternaria species have been studied for their ability to produce bioactive secondary metabolites, such as tentoxin (TEN), some of which have toxic properties. The main food contaminant toxins are tenuazonic acid, alternariol (AOH), alternariol monomethyl ether (AME), altenuene, and altertoxins i, ii and iii.AimsTo determine the profiles of secondary metabolites characteristic of Alternaria strains isolated from tomato for their chemotaxonomic classification.MethodsThe profiles of secondary metabolites were determined by HPLC MS.ResultsThe Alternaria isolates obtained from spoiled tomatoes belong, according to their morphological characteristics, to the species groups Alternaria alternata, Alternaria tenuissima and Alternaria arborescens, with A. tenuissima being the most frequent. The most frequent profiles of secondary metabolites belonging to the species groups A. alternata (AOH, AME, TEN), A. tenuissima (AOH, AME, TEN, tenuazonic acid) and A. arborescens (AOH, AME, TEN, tenuazonic acid) were determined, with some isolates of the latter being able to synthesize AAL toxins.ConclusionsSecondary metabolite profiles are a useful tool for the differentiation of small spored Alternaria isolates not easily identifiable by their morphological characteristics.     

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.     

Survey of <Emphasis Type="Italic">Alternaria</Emphasis> toxin contamination in food from the German market,using a rapid HPLC-MS/MS approach

A HPLC-MS/MS-based method for the quantification of nine mycotoxins produced by fungi of the genus Alternaria in various food matrices was developed. The method relies on a single-step extraction, followed by dilution of the raw extract and direct analysis. In combination with an analysis time per sample of 12 min, the sample preparation is cost-effective and easy to handle. The method covers alternariol (AOH), alternariol monomethyl ether (AME), tenuazonic acid (TeA), altenuene (ALT), iso-altenuene (isoALT), tentoxin (TEN), altertoxin-I (ATX-I), and the AAL toxins TA₁ and TA₂. Some Alternaria toxins which are either not commercially available or very expensive, namely AOH, AME, ALT, isoALT, and ATX-I, were isolated as reference compounds from fungal cultures. The method was extensively validated for tomato products, bakery products, sunflower seeds, fruit juices, and vegetable oils. AOH, AME, TeA, and TEN were found in quantifiable amounts and 92.1 % of all analyzed samples (n?=?96) showed low level contamination with one or more Alternaria toxins. Based on the obtained results, the average daily exposure to Alternaria toxins in Germany was calculated.     

Alternaria mycotoxins in black rot lesion of tomato fruit: Conditions and regulation of their production

Alternaria represents the most common decay organism of the post-harvest tomato fruit. The prevalent type of decay, black rot lesion, is caused byA. alternata which may invade tomato tissue damaged by sun scald.Aspergillus niger, A. flavus andRhizopus stolonifer come in the second count level and occupy high to moderate occurrence. The mainly natural mycotoxins produced in rotted tomato are alternariol (AOH), alternariol monomethyl ether (AME), and tenuazonic acid (TA). Altertoxin I & II (AT-I & AT-II), in addition to AOH, AME and TA were produced by localA. alternata in a synthetic medium. The optimum temperature for toxin production byA. alternata IMI 89344 was 28 °C for AOH and AME, 21 °C for TA, and 14 °C for AT-I and AT-II. The growth and toxin were produced in a noticeable amount at 7 °C but drop at 35 °C. Significant inhibition in these toxins was attained at 500 ppm cinnamon oil in YES-Czapeks medium and in a tomato homogenate.     

Effect of heat treatments on stability of altemariol, alternariol monomethyl ether and tenuazonic acid in sunflower flour

A study was carried out to evaluate the effect of heat treatment on the stability of alternariol (AOH), alternariol monomethyl ether (AME) and tenuazonic acid (TeA) in sunflower flour and the effectiveness of this treatment by a biological assay in rats. The concentrations of AOH and AME remained constant during heating at 100°C for up to 90 minutes while TeA concentration decreased with time to 50% after 90 minutes. The most effective treatment in reducing AOH and AME levels was heating at 121°C for 60 minutes. Histopathological evaluation in the biological assay in rats fed withAlternaria toxins showed marked atrophy and fusion of villi in the intestines and liver cell damage; these lesions were less severe in rats fed heat-treated sunflower flour in line with the reduced toxin content. However, a lower weight gain and a noticeable renal damage in rats were produced when they fed decontaminated flour.     

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.     

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.     

Toxicity of mycotoxins produced by four Alternaria species toArtemia salina larvae

22 isolates ofAlternaria alternata, A raphani, A consortiale, andA chartarum were examined for the production of alternariol (AOH), alternariol methyl ether (AME), altenuene (ALT), altertoxin I (ATX I), and tenuazonic acid (TA) on wheat grain and for toxicity of culture extracts toArtemia salina larvae. The total amount of 5 toxins produced under laboratory conditions ranged from 5 mg/kg to 11.112mg/kg. The toxic extracts showed EC50 values in the range of 3.3 to 144.5 mg/mL. There was no correlation between toxicity of extracts toArtemia salina and the amount of mentioned mycotoxins in culture.     

Mycotoxin production by some IndianAlternaria species

A total of seven species ofAlternaria: A. alternata (Fr.) Keissler;A. capsici-annui Savul & Sandu;A. citri Ellis & Pierce;A. porri (Ellis) Clfferi;A. radicina Meler, Drechsler & Eddy;A. tenuissima (Kunze: Pers) Wiltshire andA. tomato (Cooke) Jones were screened on rice culture medium for their ability to elaborate five majorAlternaria mycotoxins viz. tenuazonlc acid (TA), alternariol (AOH), alternariol methyl ether (AME), altenuene (ALT) and altertoxin-l (ATX-I). All the species produced mycotoxins in varying concentrations. A.capsici-annul was recorded as the mycotoxin producer for the first time. ALT byA. citri andA. tomato; ALT, and ATX-I byA. tenuissima; ALT, TA and AME byA. porri and TA byA. radicina are the new additions to the list of mycotoxins produced by the respective species ofAlternaria.     

Alternaria mycotoxins associated with grape berries in vitro and in situ

The aim of this study was to assess the production of secondary metabolites of Alternaria species isolated from grape berries and determined the occurrence of Alternaria mycotoxins in vitro and in dried berries. Direct morphological examination on different media was used for identification and HPLC/MS method for quantification of Alternaria mycotoxins. We isolated A. alternata and A. tenuissima and randomly selected strains in vitro and in dried berries. In vitro we identified the production of Alternaria metabolites, included mycotoxins: alternariol, alternariol methylether, tenuazonic acid. Beside that one in dry berries were in measurable concentrations: macrosporin A, tentoxin and altenuete (I, II).     

Alternatiol-O-methyltransferase fromAlternaria alternata: Partial purification and relation to polyketide synthesis

Alternaria alternata produces the polyketide mycotoxins alternariol (AOH) and alternariol monomethylether (AME) during the stationary growth phase when cultured in darkness. AME is formed by methylation of AOH by an alternariol-O-methyltransferase (AOH-MT). This methyltransferase was purified to near homogeneity from dark grown cultures ofA. alternata resulting in a 240-fold purification. The major protein in the enriched fraction of AOH-MT had a mass of 43,000 Da and was shown to bind the cofactorS-adenosyl-[³H]methionine by photoaffinity labeling, suggesting that this polypeptide contained the active site. WhenA. alternata was cultured in white light, the accumulation of AOH and AME was reduced to less than 4% of the production in darkness which is in agreement with earlier results. This reduction in polyketide content was accompanied by a reduced AOH-MT activity in extracts from light grown cultures. However, the activity of AOH-MT in mycelia grown in light was only reduced to 30% of the activity in dark grown cultures. Thus, it seems that the main target for light suppression of polyketide accumulation inA. alternata is either the activity or formation of the enzyme synthesizing AOH or the precursor availability for AOH synthesis.     

Incidence and mycotoxin production by Alternaria tenuis in decayed apples

Alternaria tenuis was the main species of Alternaria which produced post-harvest decay in apples. Alternariol (AOH) and alternariol monomethyl ether (AME) were the major mycotoxins produced in Alternaria -decayed apples at 25°C and 2°C. Only the strain Alternaria tenuis CMTA 65 at 25°C produced tenuazonic acid (TA). Altertoxin-I (ATX-I) and altertoxin-II (ATX-II) were not detected in any of the apple samples.
A large percentage of strains of A. tenuis studied produced TA (97%) and AT-I (82·3%) when grown in a yeast extract sucrose medium (YES) at 25°C. A much smaller percentage of strains produced AOH and AME and none were found to produce detectable levels of ATX-II.     

Occurrence of alternariol and alternariol monomethyl ether in beverages from the Entre Rios Province market, Argentina

One hundred and eighty five samples of red, white and rosé wines and different juices purchased in Entre Rios, Argentina, were analyzed for the Alternaria mycotoxins alternariol (AOH) and alternariol methyl ether (AME). White wines were analyzed after removal of alcohol by a nitrogen stream and concentrated. AOH in red wines was cleaned up by solid-phase extraction columns in series (octadecyl and amino propyl modified silica) and AME quantified directly on the sample. The juices were filtered and concentrated, and then all sample extracts were quantified by high performance liquid chromatography with photodiode array detector that allows confirmation through UV spectra. Method validation revealed a good sensitivity with adequate LOD and LOQ for AME and less sensitivity for AOH (i.e. white wine: AME 0.8 and 1.4 ng/mL, AOH 2 and 3.3 ng/mL; red wine: AME 0.1 and 0.2 ng/mL, AOH 4.5 and 7.5 ng/mL; apple juice: AME 1.7 and 2.8 ng/mL, AOH 5 and 9 ng/mL; other juices: AME 2.0 and 3.1 ng/mL, AOH 6 and 10 ng/mL). Recoveries in all cases were greater than 80 %. Four of 53 white wine samples were contaminated with AOH with a maximum level of 18 ng/mL, 6 of 56 samples of red wine had a maximum of 13 ng/mL, and 3 of 68 samples of juices had traces of AOH. AME was less frequently detected than AOH, and the LOD and LOQ for AME are smaller than for AOH. Only three samples of white wine and one of red wine were contaminated, but in only one white wine sample (AME 225 ng/mL) did the toxin level exceed the LOQ.     

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.     

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.     

Analysis of wines, grape juices and cranberry juices forAlternaria toxins

Sixty six samples of red and white wine from Ontario (VQA), British Columbia (VQA), Québec (“vins artisanaux”), imported wines (from Italy, South America and USA) and Canadian and US grape and cranberry juices were analysed for theAlternaria mycotoxins alternariol (AOH) and alternariol monomethyl ether (AME). After cleanup on aminopropyl SPE columns, AOH and AME were initially determined by reversed phase LC with UV detection. Positive sample extracts were re-analysed by LC-tandem negative ion electrospray mass spectrometry (MS/MS) in multiple reaction mode. Overall mean method recoveries measured by LC-UV were 93% for AOH and 81% for AME. Limits of detection in wine (and juice) by LC-UV for AOH were 0.8 (0.4) ng/ml and for AME were 0.5 (0.4) ng/ml; they were below 0.01 ng/ml by LC-MS/MS. As determined by LC-MS/MS, AOH was found in 13/17 Canadian red wines at levels of 0.03 to 5.02 ng/ml and in 7/7 imported red wines at 0.27–19.4 ng/ml, usually accompanied by lower concentrations of AME. Red grape juices (5 positive/10 samples) contained only sub ng/ml levels of AOH or AME except for one sample (39 ng AME/ml). White wines (3/23 samples), white grape juices (0/4 samples) and cranberry juices (1/5 samples) contained little AOH/AME (≤1.5 ng/ml). Presented at the World Mycotoxin Forum, Noordwijk, The Netherlands, November 10–11, 2005     

Putative mycotoxin-producing fungi isolated from alpataco (Prosopis flexuosa) fruits

Fungi contaminant of alpataco (Prosopis flexuosa) fruits from La Pampa province (Argentina) were identified. Alternaria alternata and Sphaeropsis sapinea were the dominant species. Phoma sp., Nigrospora sp., Preussia minima, Cladosporium sp., Pithomyces chartarum, Epicoccum nigrum, Aspergillus niger and Aspergillus speluneus were also isolated but with less frequency. Twelve strains of Alternaria alternata, the toxigenic species with higher incidence, were screened for alternariol (AOH), alternariol monomethyl ether (AME) and tenuazonic acid (TA) production. Since one isolate was able to produce AME, six isolates produced AOH and AME and two isolates produced AOH, AME and TA, these results indicate a potential risk of contamination with Alternaria toxins in this substrate.     

Alternaria toxins alternariol and alternariol monomethyl ether in grain foods in Canada

Alternaria alternata has been reported to be the most common fungus on Canadian Western wheat. The Alternaria toxins alternariol (AOH) and alternariol monomethyl ether (AME) are mutagenic in vitro and there is also limited evidence for carcinogenic properties. They have been found in wheat from Europe, Argentina, China and Australia, but they have not been looked for in Canadian grains or grain foods. In the present study, 83 samples of grain-based food sold in Canada, including flour, bran, breakfast cereals, infant cereals and bread, were analysed for AOH and AME using extraction with methanol, clean-up on combined aminopropyl/C18 solid phase extraction (SPE) columns, and liquid chromatography (LC) with tandem mass spectrometric (MS/MS) determination. The overall average recoveries of AOH and AME from a variety of spiked cereal foods (n?=?13) were 45?±?9?% and 53?±?9?%, which could be attributed mainly to MS matrix effects The instrumental limits of detection (LOD) were 0.34?ng/g and 0.13?ng/g for AOH and AME, respectively, and the instrumental limits of quantitation (LOQ) were 1.1 and 0.43?ng/g. Of 83 samples analysed, 70 were positive for AOH (up to 63?ng/g, in a soft wheat bran) and 64 contained AME (up to 12?ng/g in a bran-based breakfast cereal). Of particular interest was the presence of AOH and/or AME in 27 out of 30 infant foods (up to 4.4?ng/g and 9.0?ng/g, respectively, in a sample of multigrain cereal).