Toxigenic profile of Alternaria alternata and Alternaria radicina occurring on umbelliferous plants.

Alternaria alternata and Alternaria radicina are fungal species that occur in several food crops and may produce mycotoxins and phytotoxins. The toxigenic profile of A. alternata and A. radicina isolated from carrot and other umbelliferous plants was determined by growing the fungus on rice and carrot discs. Most of the tested isolates of A. alternata produced the mycotoxins tenuazonic acid, alternariol, alternariol methyl ether and altertoxin-I on rice. Only alternariol and alternariol methyl ether were produced on carrot discs. When cultured on rice, none of the isolates of A. alternata from umbelliferous plants produced AAL toxins and fumonisins. AAL toxins, but not fumonisins, were instead produced by A. alternata f. sp. lycopersici isolate NRRL 18822 isolated from tomato. A. radicina produced the phytotoxic compounds radicinin, epi-radicinol and radicinol on carrot discs, whereas it produced radicinin and radicinol on rice. Although A. alternata has been frequently found in organic carrots, none of the above mycotoxins was detected in carrot roots or in carrot commercial products. The reduction of alternariol and alternariol methyl ether during carrot juice processing at laboratory scale was estimated to be >98%. Based on these findings and previous reports, it can be concluded that Alternaria mycotoxins in carrots do not represent a hazard for consumers.

Fusariotoxins in kernels of winter wheat cultivars field samples collected during 1993 in Poland.

In South-Eastern region of Poland (near Lublin), where frequency of scab (fusariosis) is much higher than in other parts of the country, during harvest of 1993 kernels of 25 winter wheat cultivars were collected. On the basis of morphological studies Fusarium graminearum was found in 42% of investigated samples while other fungi appeared less frequently: F. nivale and F. poae (35%), F. avenaceum (31%) and F. culmorum (12%). Chemical analysis (by HPLC) revealed that the tested cultivars were contaminated with deoxynivalenol (96% of investigated samples), its acetyl derivatives (48%), nivalenol (76%) and moniliformin (28%). The average levels of the metabolite concentrations were as follows: 104; 16; 97; and 63 micrograms/kg, respectively. Co-occurrence of 2 toxic metabolites was found in the following percentage of the positive samples: deoxynivalenol and nivalenol (72%), deoxynivalenol and moniliformin, as well as nivalenol and moniliformin (24%). Usually (71-83% of contaminated samples) mycotoxins were accumulated in the concentration range > or = 10, < 100 micrograms/kg.

Detection of T-2 toxin in different cereals by flow-through enzyme immunoassay with a simultaneous internal reference.

In most previously described membrane-based immunoassays a separate negative control assay is always carried out to evaluate the performance of the assay. To overcome this problem, a membrane-based flow-through enzyme immunoassay with an internal control has been developed for the detection of T-2 toxin in cereals (patent pending). An Immunodyne ABC membrane was coated with 2 microL of goat anti-horseradish peroxidase (HRP) (internal control spot) (1:1000) and 2 microL of rabbit anti-mouse (test spot) (undiluted) immunoglobulins, and the free binding sites were blocked. In addition to the antibody-coated Immunodyne ABC membrane, the assay also comprises a plastic snap-fit device, absorbent cotton wool, mouse anti-T-2 monoclonal antibodies (Mab), and T-2-HRP conjugate. The color intensity (Delta) of the internal control and that of the negative sample showed no difference (P > 0.05), whereas there was a significant difference between the internal control and positive samples (P < 0.05). The minimum detectable limit that could be visually detected with confidence was 50 ng of T-2 per gram of cereal sample.

Deoxynivalenol accumulation and other scab symptoms in six romanian wheat genotypes inoculated withFusarium graminearum.

At anthesis, under field conditions at Fundulea, each of 6 Romanian winter wheat genotypes was inoculated with 3Fusarium graminearum isolates used individually.Fusarium head blight (FHB) was assessed according to the following traits: relative weight of spikes (RWS), percentage of Fusarium damaged kernels (FDK), relative weight of kernels per head (RWKH), area under the disease progress curve (AUDPC) and deoxynivalenol (DON) content in total sample of kernels. Correlations between these traits and parameters revealed important differences between examined wheat genotypes in: DON accumulation, progress of FHB development, yield reduction, and models of host - pathogen interactions in theTriticum - Fusarium pathosystem. Significant correlations between different attributes of FHB were found forFusarium isolate 1 which is a moderate producer of DON (0.89 µg g(-1)). Weight of spike was significantly correlated with weight of kernels per spike (r = 0.93**) and with percentage of damaged kernels (r = - 0.87**), while FDK was highly correlated with RWKH (r = - 0.85*) and with DON content (r = 0.82*). Area under the disease progress curve was also found to be significantly correlated with DON content (r = 0.86*).

Ochratoxin A in porcine blood and in consumed feed samples.

The aim of the study was to estimate occurrence of ochratoxin A (OA) in feeds and the metabolite residues in porcine blood serum in Poland. Samples were collected in the period from February to May, 1999, in the southern Wielkopolska region. Altogether 40 and 45 samples of feed and porcine blood serum, respectively, were analyzed for OA. Percentage of samples contaminated with OA, both in case of feeds and blood, collected in the winter season was considerably higher than that for the spring season. The percentages for feeds were as follows: 47.6 and 26.3 %, while for porcine serum: 66.7 and 50.0 %, respectively winter and spring. In 25 % of cases ochratoxin A was present in both types of investigated material (feed, blood), whereas in 27.5 % of samples this metabolite was detected in blood only, or in 7.5 % only in the feed. The presence of OA was found neither in the feed nor in the serum in 40 % of all cases. In subgroups (feed, blood) the concentration in the whole collective of positive samples were in the range 0.3-13.5 ng/g and 0.3-69.5 ng/ml, respectively, while median values were 2.3 ng/g and 6.0 ng/ml. Only one feed and three porcine serum samples, were found to be contaminated at concentration levels higher than 10 ng/g or 10 ng/ml.

Biosynthesis of gibberellic acid (GA3) and mycotoxins by F. moniliforme sheldon and other species of Liseola section.

F. moniliforme and other species of Liseola section, F. culmorum, F. dlamini, and F. nygamai, were examined for their ability to produce gibberellic acid (GA3), fumonisins, trichothecenes, zearalenone, moniliformin, and bikaverin (TLC method). Gibberellic acid was produced by F. moniliforme strains in liquid medium and on rice kernels with a maximum concentration level of 470 mg/dm3 and 1 g/kg, respectively. No strain isolated in Poland produced GA3. High-yielding gibberellic acid strains produced neither trichothecenes and fumonisins nor other tested compounds. Also the rest of strains of examined species did not produce trichothecenes and other mycotoxins except for fumonisins which were found in rice cultures of F. moniliforme, F. proliferatum, and F. subglutinans. Bikaverin was produced by F. moniliforme always together with fumonisins. Filtrates of liquid cultures of gibberellin producing strains were tested for their toxicity to brine shrimps larvae (Artemia salina). It was found that GA3 presence does not increase toxicity of these filtrates.

Distribution of trichothecene mycotoxins in maize ears infected withF. graminearum andF. crookwellense.

Maize ears naturally infected withF. graminearum (6 samples) andF. crookwellense (6 samples) were collected in 1990 and 1991. All samples exhibited symptoms of severe ear rot with high percentage ofFusarium damaged kernels. Thousand kernels weight ranged 108.5 - 235.0g and was significantly lower than in control, not infested kernels (330g). All ears were contaminated withFusarium toxins but DON and NIV were the most frequently analyzed metabolites. Distribution of trichothecene mycotoxins in different parts of ear as well as toxicity of extracts toArtemia salina is reported in this paper.

Decomposition of ochratoxin A by heat and gamma-irradiation.

Up to 50% of initial amounts of 2 and 100 ng/ml ochratoxin A were decomposed after gamma-irradiation of solutions in water, in 2% aqueous NaCl or an aqueous solution of 2% NaCl and NaNO2. Ochratoxin A in these solutions was not decomposed, however, after heating at 20, 121 or 135 degrees C for 15 min.

Possible sources of ochratoxin A in human blood in Poland.

Samples of plant origin and human and porcine blood samples were screened over a long period for the presence of ochratoxin A. Of 1353 cereal samples, 11.7% contained the mycotoxin; of 1372 samples of feed, 1.5%; of 368 bread samples, 17.2%; of 215 flour samples, 22.3%; of 894 porcine serum samples, 37.4%; and of 1065 human serum samples, 7.2%. Seasonal variations in the natural occurrence of ochratoxin A were observed, with an increased percentage of positive samples in the spring. Individual daily intake of the mycotoxin, estimated on the basis of residues in human serum, was found to be 0.4 ng/g of food consumed.

Biosynthesis and preparation of fiveAlternaria metabolites.

Metabolites ofAlternaria alternata were produced on rice as a solid substrate, chosen out of 6 substrates as the most useful. Optimal methods of extraction, purification, and separation of 5 metabolites were elaborated, using liquid - liquid partition, column chromatography, and preparative TLC. Alternariol, alternariol methyl ether, and copper salt of tenuazonic acid were obtained as crystals, altertoxin and altenuene as a film.

[A test for determining daily doses of consumption of ochratoxin A by humans in Poland]. / Próba okreslenia dawki dziennego spozycia ochratoksyny A przez czlowieka w polsce.

A trial was undertaken for determination of the average daily amount of ochratoxin A consumed with contaminated food by adults in Poland. Contamination of food with ochratoxin A was estimated to be 0.448 ng/g, and the mean level of this metabolite in human serum was 0.27 ng/cm3. These values showed a considerable similarity when they were analysed by simplified conversion factors of Mortensen et al.

Natural occurrence ofAlternaria mycotoxins in the grain and chaff of cereals.

Samples of wheat and rye heads with evident black discoloration were collected in 1986 and 1987. Four fungal genera colonized such heads:Alternaria, Cladosporium, Drechslera, andEpicoccum. In chaff of wheat (19%) and rye (10%) samples alternariol was present in amounts up to 1.8mg/kg, and alternariol methyl ether up to 0.51 mg/kg. In 1 out of 21 wheat samples alternariol was also present in kernels (0.59mg/kg).

Mycotoxins produced by Discolor section Fusarium species.

An improved TLC method ofFusaria metabolites detection and quantitation has been elaborated. A total 92 isolates of Discolor sectionFusaria from cereals and potato have been examined from the point of view of cultures morphology and ability to produce characteristic mycotoxins. Low nutrient media (CLA, SNA) were found as suitable for production of uniform and typical macroconidia in studied cultures. All 26 isolates ofF. sambucinum Fuckel (=F.sulphureum, Schlecht) formed diacetoxyscirpenol in amount 20-1000 mg/kg and all 17.F. crookwellense Burgess N. & T. produced zearalenone (16-602 mg/kg).F. graminearum Schwabe produced: zearalenone 14/14 isolates, deoxynivalenol 11/14 isolates, both up to 77 mg/kg. Out of 26F. culmorum cultures originating from Poland 22 produced zearalenone up to 675 mg/kg, 17/26 3 acetyldeoxynivalenol up to 280 mg/kg and 16/26 deoxynivalenol up to 220 mg/kg. The difference in metabolism agrees with the difference in morphology of those species.

Spontaneous occurrence of ochratoxin A residues in porcine kidney and serum samples in Poland.

During the period 1 April 1983 to 31 July 1984, 214,700 swine were processed in a slaughterhouse in Poznan, Poland. Of these pigs, 122 (0.057%) exhibited macroscopical kidney changes typical for mycotoxic porcine nephropathy. Ochratoxin A was found in kidneys from 52 of these pigs. Porcine serum samples not biased for nephropathy were collected at random in the same slaughterhouse. Of 388 samples, 148 exhibited ochratoxin A residues from 1 to 520 ng/ml. Significant increases in nephropathy and ochratoxin A frequencies were observed during the spring of 1984.

Mycotoxins in cereal grain. Part XI. Simple multidetection procedure for determination of 11 mycotoxins in cereals.

A simple method for the simultaneous detection of the 11 mycotoxins aflatoxins (B1, B2, G1, G2), ochratoxin A, zearalenone, sterigmatocystin, citrinin, penicillic acid, T-2 toxin and rubratoxin B is reported. The elaborated method was tested for all cited mycotoxins extracted from 5 cereal species (rye, barley, wheat, oats and corn) spiked with mycotoxins standards. Different chromatoplates, developing solvents and spraying reagents were tested. New tests and modification of known confirmatory tests, recovery and detection limits are reported.

Chemical confirmatory tests for ochratoxin A, citrinin, penicillic acid, sterigmatocystin, and zearalenone performed directly on thin layer chromatographic plates.

Published tests have been improved and a new procedure is described for chemical confirmation of mycotoxins directly on thin layer plates. After extraction and preliminary cleanup chromatography with n-hexane or chloroform, the mycotoxins ochratoxin A, citrinin, penicillic acid, sterigmatocystin, and zearalenone were easily separated by thin layer chromatography (TLC) using toluene-ethyl acetate-90% formic acid (6 + 3 + 1) developing solvent. In chemical confirmatory methods, the developed chromatogram was exposed to vapors of pyridine, acetic anhydride, or a mixture, or the mycotoxins were over-spotted. With this treatment, ochratoxin A, citrinin, penicillic acid, and zearalenone were converted to new fluorescent compounds, and observed under 365 nm light after re-chromatography with the same developing solvent. Sterigmatocystin was confirmed chemically using TLC plates impregnated with 0.6N H2SO4 or 10% oxalic acid in methanol. The described procedures are satisfactory for confirming mycotoxins present in standards, artificially contaminated grain samples (barley, corn, oat, rye, and wheat), and extracts from both fungal cultures and naturally contaminated grain samples.

Mycotoxic porcine nephropathy and spontaneous occurrence of ochratoxin A residues in kidneys and blood of Polish swine.

Kidneys showing renal changes characteristic for mycotoxic porcine nephropathy were collected during the period 1 April 1982 to 31 March 1983 from 225,000 swine processed in a large slaughterhouse in the district of Poznan, Poland. Of 113 kidneys suspected of mycotoxic porcine nephropathy, 27 exhibited ochratoxin A levels from traces to 23 ng/g. In 17 kidneys the level of the toxin was lower than 2 ng/g. Increased frequency of ochratoxin A presence and its level in kidneys were observed during the spring. Of 195 porcine blood samples collected at random, 36 exhibited toxin levels from 3 to 270 ng/ml.