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.     

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.     

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).     

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.     

Influence of dietary fibre on plasma and urinary levels of zearalenone and metabolites in swine

The genusAlternaria is responsible for different plant diseases such as tobacco brown spot, tomato blight, and citrus seedling chlorosis but can also be present during storage of grain. The objective of the present paper is to summarize the knowledge concerning regulation of secondary metabolism inAlternaria, particularA alternata (A tenuis). The paper mainly deals with regulation of polyketide biosynthesis, one of the major pathways leading to the biosynthesis of mycotoxins inAlternaria. The mostly studiedAlternaria mycotoxins are dibenzopyrones such as alternariol (AOH) and alternariol monomethyl ether (AME) and altenuene along with the tetramic acid tenuazonic acid. The biosynthesis ofAlternaria mycotoxins has been reviewed by Stinson (12). Most information is available for the biosynthesis of the polyketides AOH / AME while a few biosynthetic studies have been accomplished for tenuazonic acid (11).     

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, 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.     

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.     

Natural occurrence of aflatoxin andAlternaria mycotoxins in oilseed rape from Catalonia (Spain): Incidence of toxigenic strains

During an investigation of the mycoflora on oilseed rape, the predominant fungal species present in 20 samples collected from Catalonia (Spain) wereAlternaria alternata (Fries) Keissler,Penicillium spp. andAspergillus flavus. None of the 20 samples analyzed presented contamination byAlternaria mycotoxins (tenuazonic acid, alternariol, alternariol methyl ether, altertoxin I and altertoxin II). Only aflatoxin B₁ was detected in 1 of the 20 samples analyzed, with a concentration of 0.25 ppb. Of the 40Aspergillus flavus strains isolated from oilseed rape samples, only 3 revealed aflatoxigenic capacity. None of thePenicillium spp. isolated from oilseed rape samples revealed mycotoxigenic capacity (citreoviridin, griseofulvin, citrinin, patulin and penicillic acid).     

Mycoflora and mycotoxin contamination of Roundup Ready soybean harvested in the Pampean Region, Argentina

A total of 89 freshly harvested soybean seed samples (Roundup Ready [transgenic] soybean cultivars) from the 2010/2011 crop season were collected from five locations in the Northern Pampean Region II, Argentina. These samples were analyzed for internal mycoflora, toxin production of isolated fungi, and for a range of mycotoxins. Mycotoxin analysis of aflatoxins (AFs), zearalenone (ZEA), fumonisins (FBs) and ochratoxin A (OTA) was done by HPLC-FLD (high performance liquid chromatography with postcolumn fluorescence derivatization), alternariol and alternariol monomethyl ether with HPLC-UV (HPLC with UV detection), trichothecenes (deoxynivalenol, nivalenol, T-2 toxin, HT-2 toxin, fusarenon X, 3-acetyldeoxynivalenol and 15-acetyldeoxynivalenol were analyzed by GC-ECD (gas chromatography with electron capture detector). Fungal colonization was more frequently found for samples from América, Saladillo and Trenque Lauquen than for samples from General Villegas and Trenel; a total of 1,401 fungal isolates were obtained from the soybean seeds. The most commonly identified fungal genera were Alternaria, Sclerotinia, Chaetomium, Cladosporium, Aspergillus, Penicillium, Phomopsis and Fusarium. Alternaria alternata, A.tenuissima, Aspergillus flavus, Penicillium citrinum, Fusarium verticillioides and F.semitectum were the predominant toxigenic fungal species. Mycotoxin production was confirmed for several isolates of toxigenic species, including Aspergillus flavus, A. parasiticus, Alternaria alternata, A.tenuissima, Fusarium graminearum, F semitectum and F. verticillioides. In particular, the percentage of mycotoxigenic Alternaria alternata (100 %), A.tenuissima (95 %) and aflatoxigenic strains of A. flavus (57 %) were remarkably high. Although none of the mycotoxins, AFs, ZEA, FBs, trichothecenes and OTA, were directly detected in samples of soybean seeds, the frequent presence of toxigenic fungal species indicates the risk of multiple mycotoxin contamination.     

Mycotoxigenic potential of fungi isolated from freshly harvested Argentinean blueberries

Alternaria alternata, A. tenuissima, Fusarium graminearum, F. semitectum, F. verticillioides, Aspergillus flavus, and Aspergillus section Nigri strains obtained from blueberries during the 2009 and 2010 harvest season from Entre Ríos, Argentina were analyzed to determine their mycotoxigenic potential. Taxonomy status at the specific level was determined both on morphological and molecular grounds. Alternariol (AOH), alternariol monomethyl ether (AME), aflatoxins (AFs), zearalenone (ZEA), fumonisins (FBs), and ochratoxin A (OTA) were analyzed by HPLC and the trichotecenes deoxynivalenol (DON), nivalenol (NIV), HT-2 toxin (HT-2), T-2 toxin (T-2), fusarenone X (FUS-X), 3-acetyl-deoxynivalenol (3-AcDON), and 15-acetyl-deoxynivalenol (15-AcDON) by GC. Twenty-five out of forty two strains were able to produce some of the mycotoxins analyzed. Fifteen strains of Aspergillus section Nigri were capable of producing Fumonisin B₁ (FB₁); two of them also produced Fumonisin B₂ (FB₂) and one Fumonisin B₃ (FB₃). One of the F. graminearum isolated produced ZEA, HT-2, and T-2 and the other one was capable of producing ZEA and DON. Two A. alternata isolates produced AOH and AME. Four A. tenuissima were capable of producing AOH and three of them produced AME as well. One Aspergillu flavus strain produced aflatoxin B₁ (AFB₁), aflatoxin B₂ (AFB₂), and aflatoxin G₁ (AFG₁). To our knowledge, this is the first report showing mycotoxigenic capacity of fungal species isolated from blueberries that include other fungi than Alternaria spp.     

Characterization of Variability in Some Pathogenic Species of Alternaria Based on the Nucleotide Sequence of Ribosomal DNA

The internal transcribed spacer (ITS) sequences within the ribosomal DNA (rDNA) region were targeted to delineate genetic variability among eight Alternaria species that cause economically important diseases in crops. The rDNA regions of Alternaria species comprising of rRNA genes and the ITS regions were cloned and sequenced. Phylogenetic relationship based on the rDNA sequences and PCR-RFLP of amplified rDNA sequences clustered eight species of Alternaria into three major groups. A. macrospora and A. helianthi accumulated wide genetic variations and are distantly related to rest of the six species which formed two major groups. Group I comprised of three species viz., A. dianthicola, A. brassicae and A. citri, while group II had A. longipes, A. porri and A. alternata. Incorporation of unique stretches of nucleotides and single nucleotide substitutions within relatively conserved ITS1 and ITS2 regions led to clustering of the members of Alternaria species in each group. The divergent sequences within the ITS regions can be employed to design species-specific PCR primer for use in molecular diagnostics.     

<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.     

Activities of human recombinant cytochrome P450 isoforms and human hepatic microsomes for the hydroxylation ofAlternaria toxins

TheAlternaria toxins alternariol (AOH), alternariol-9-methyl ether (AME), altenuene (ALT) and isoaltenuene (iALT) undergo extensive oxidative metabolism, but the cytochrome P450 (CYP) isoforms responsible for the reported hydroxylation reactions are yet unknown. In the present study, the activities of twelve human CYP isoforms for the hydroxylation of AOH, AME, ALT and iALT at different positions have been determined. The most active monooxygenase for AOH and AME was CYP1A1, and lower activities were observed for CYP1A2, 2C19 and 3A4. Hydroxylation at C-2 of AOH and AME was the preferred reaction of most isoforms. For ALT and iALT, CYP2C19 had the highest activity, followed by 2C9 and 2D6. The dominating metabolite of all active isoforms was the 8-hydroxylated ALT and iALT. The activities of the CYP isoforms are consistent with the pattern of metabolites of theAlternaria toxins obtained with pooled human hepatic microsomes. Based on the activities of the CYP isoforms, a significant extrahepatic hydroxylation must be expectede.g. in the lung and esophagus for AOH and AME, and in the intestine and ovaries for ALT and iALT. As all major hydroxylation products are catechols, the extrahepatic metabolism ofAlternaria toxins may be of toxicological relevance. Presented at the 30^th Mycotoxin Workshop, Utrecht, Netheriands, April 28–30. 2008. Financial support: State of Baden-Württemberg (Research Program “Mycotoxins” as part of the Research Initiative “Food and Health”)     

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.     

Effect of different foods on development,reproduction and survival ofPhytoseiulus longipes [Acarina: Phytoseiidae]

The effect of different prey mite species on development of the immature stages and on survival and oviposition of adult predator femalePhytoseiulus longipes Evans was studied.P. longipes completed its development onTetranychus pacificus McGregor,Oligonychus punicae (Hirst), andPanonychus citri (McGregor), but not onPetrobia harti (Ewing). Females oviposited while feeding onT. pacificus andO. punicae, but not onP. citri orP. harti. The highest percent survival was observed onT. pacificus, followed byO. punicae andP. citri. The longevity ofP. longipes onP. harti (vial arenas) and on pollen (leaf arenas) was the same as that on leaves with no food. The provision of 10% sucrose solution and clover honey doubled the longevity ofP. longipes females compared with no food.     

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.     

Incidence of Alternaria Species Associated with Watermelon Leaf Blight in Korea

Alternaria leaf blight is one of the most common diseases in watermelon worldwide. In Korea, however, the Alternaria species causing the watermelon leaf blight have not been investigated thoroughly. A total of 16 Alternaria isolates was recovered from diseased watermelon leaves with leaf blight symptoms, which were collected from 14 fields in Korea. Analysis of internal transcribed spacer (ITS) region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and RNA polymerase II second largest subunit (RPB2) were not competent to differentiate the Alternaria isolates. On the contrary, analysis of amplicon size of the histone H3 (HIS3) gene successfully differentiated the isolates into three Alternaria subgroups, and further sequence analysis of them identified three Alternaria spp. Alternaria tenuissima, A. gaisen, and A. alternata. Representative Alternaria isolates from three species induced dark brown leaf spot lesions on detached watermelon leaves, indicating that A. tenuissima, A. gaisen, and A. alternata are all causal agents of Alternaria leaf blight. Our results indicate that the Alternaria species associated watermelon leaf blight in Korea is more complex than reported previously. This is the first report regarding the population structure of Alternaria species causing watermelon leaf blight in Korea.     

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.