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A new high-performance liquid (HPLC) chromatographic method is described for cyclopiazonic acid (CPA) determination in fungal cultures on a propylamino-bonded stationary phase with a CH3CN/CH3COONH4 buffer as mobile phase. Retention of CPA on propylamino modified silica under acidic conditions (protonated amino groups and deprotonated CPA) is governed by a mixed ion-exchange-reversed-phase mechanism. In addition to non-polar (hydrophobic) interactions, polar interactions with the surface silanols are also possible and become important as the polarity of the mobile phase decreases. A detection limit of 25 pg of CPA standard is obtained that represents an improvement of more than two orders of magnitude compared to existing HPLC procedures. UV-detector response was linear to 200 ng of CPA. Fungal extracts can be analysed after a simple dilution step with UV diode array detection that provides peak identity/purity assessment. The suitability of the proposed method as a rapid confirmatory test to assess the toxigenic potential of different Aspergillus and Penicillium strains is demonstrated by the analysis of 54 fungal extracts.

A bacteriocin produced by Pseudomonas syringae pv. ciccaronei, used at different purification levels and concentrations in culture and in planta, inhibited the multiplication of P. syringae subsp. savastanoi , the causal agent of olive knot disease, and affected the epiphytic survival of the pathogen on the leaves and twigs of treated olive plants. Treatments with bacteriocin from P. syringae pv. ciccaronei inhibited the formation of overgrowths on olive plants caused by P. syringae subsp. savastanoi strains PVBa229 and PVBa304 inoculated on V-shaped slits and on leaf scars at concentrations of 10 5 and 10 8 CFU ml −1 , respectively. In particular, the application of 6,000 arbitrary units (AU) of crude bacteriocin (dialyzed ammonium sulfate precipitate of culture supernatant) ml −1 at the inoculated V-shaped slits and leaf scars resulted in the formation of knots with weight values reduced by 81 and 51%, respectively, compared to the control, depending on the strains and inoculation method used. Crude bacteriocin (6,000 AU ml −1 ) was also effective in controlling the multiplication of epiphytic populations of the pathogen. In particular, the bacterial populations recovered after 30 days were at least 350 and 20 times lower than the control populations on twigs and on leaves, respectively. These results suggest that bacteriocin from P. syringae pv. ciccaronei can be used effectively to control the survival of the causal agent of olive knot disease and to prevent its multiplication at inoculation sites.

The development of new maize hybrids with resistance to Fusarium infection is an effective means of minimizing the risk of mycotoxin contamination. Several maize hybrids have been investigated for Fusarium ear rot and accumulation of fumonisin B1 (FB1, fumonisin B2 (FB2), beauvericin (BEA) and fusaproliferin (FP) after artificial inoculation in the field with toxigenic strains of Fusarium verticillioides and Fusarium proliferatum. The year of inoculation had a significant influence on the disease severity and mycotoxin accumulation in maize kernels. Of all the hybrids tested, only Mona exhibited resistance to ear rot caused by F. verticillioides and produced low levels of fumonisins during three years of experiments. In Fusarium-damaged kernels (FDK), fumonisin Bl, fumonisin B2, beauvericin and fusaproliferin were detected at concentrations much higher (up to 10–20 times) than in healthylooking kernels (HLK). Animal and human exposure to these mycotoxins can be drastically reduced by removing mouldy and visibly damaged kernels from the commodity.

The occurrence of beauvericin has been investigated in corn kernel (Zea mays L.) samples collected in 1996 (105 samples) and 1997 (104 samples) from 14 corn-producing counties of Croatia. Corn sample extracts were cleaned up by silica gel minicolumns and analyzed for beauvericin by high performance liquid chromatography with UV diode array detector. Higher incidence of positive samples was found in the 1996 crop as compared to the 1997 crop. In particular, 18 samples (17.4 %) of the 1996 crop were found contaminated with a mean beauvericin content of 393 ng/g and the highest level at 1864 ng/g. Only 1 out of 104 samples collected in the 1997 crop was contaminated with 696 ng/g of the toxin.n Beauvericin co-occurred with fumonisins B1 and B2 and with ochratoxin A in 17 and 4 samples, respectively. The results of mycological analysis of corn samples for beauvericin producing Fusarium species were in agreement with results of chemical analysis. In particular, higher incidence of Fusarium verticillioides (Sacc.) Nirenberg (known as Fusarium moniliforme Sheldon) (3.7 %) and Fusarium subglutinans (Wollenweber & Reinking) Nelson, Toussoun & Marasas (5.3 %) was found in 1996 with respect to 1997 (1.9 % of F. verticillioides and 0.4 % of F. subglutinans). This is the first report on the occurrence of beauvericin in Croatia., Učestalost bovericina analizirana je u uzorcima kukuruza skupljenim tijekom 1996. (105 uzoraka) i 1997. godine (104 uzorka) iz 14 županija Republike Hrvatske. Ekstrakt uzoraka kukuruza pročišćen je na silicij-gelskim minikolonama i analiziran na bovericin s tekućinskim kromatografom velike učinkovitosti (HPLC) s UV-diodnim detektorom. Učestalija pojava pozitivnih uzoraka nađena je godine 1996. u usporedbi s 1997. godinom. Godine 1996. nađeno je 18 uzoraka (17,4 %) kontaminiranih bovericinom sa srednjom vrijednošću pozitivnih uzoraka od 393 ng/g, odnosno s najvećom količinom od 1864 ng/g. Samo 1 od 104 skupljena uzorka godine 1997. bio je onečišćen s 696 ng/g toksina. Bovericin je nađen zajedno s fumonizinom B1 i B2 u 17 uzoraka, a s okratoksinom A samo u 4 uzorka. Mikološkom analizom utvrđene Fusarium vrste slažu se s kemijskim analizama na mikotoksine. Veća pojava Fusarium verticillioides (Sacc.) Nirenberg (poznat i kao Fusarium moniliforme Sheldon) (3,7 %) i F. subglutinans (Wollenweber & Reinking) Nelson, Toussoun & Marasas (5,3 %) nađena je godine 1996. u usporedbi s 1997. godinom kada je ustanovljeno (1,9 %) F. verticillioides i (0,4 %) F. subglutinans. Ovo je prvo izvješće o prisutnosti bovericina u Republici Hrvatskoj.

Economic losses of dairy products due to spoilage by yeasts have been increasing in European companies because of the reduced use of preservatives, packaging in modified atmospheres, or new formulations that do not strictly control the growth of these organisms. This study reports the results of a survey of yeast species and populations in 145 samples of cow and buffalo dairy products collected in some regions of Southern Italy. Yeasts were isolated from 74% and 57% of cow and buffalo products, respectively. Candida inconspicua was the predominant species in unripened products from cow's milk, while C. famata was detected in medium and long-term ripened dairy products, mostly in association with other yeasts and with moulds belonging to the genus Penicillium . For dairy products produced from buffalo milk, C. inconspicua was the most important yeast frequently isolated from dairy products. Total yeast populations ranged from 5×10 2 to 5×10 5 cfu/g, indicating a good hygienic quality of the products. The isolation of C. albicans from one stracciatella sample is noteworthy, as this yeast represents a potential contamination by human. Even though yeasts are considered as environmental contaminants, the occurrence of some of them in dairy products at high levels could represent a risk for human health, in particular for immunocompromised patients.

Moulds parasites of livestock foodstuffs alter the quality of grains by synthesizing mycotoxins. Zearalenone (ZEA) and its derivatives (alpha- and beta-zearalenol, zeranol, taleranol and zearalanone) are produced by fungi of the genus Fusarium and, after ingestion via contaminated cereals, may lead to fertility disturbances and other reproductive pathologies. Zearalenone, alpha-zearalenol and zearalanone were tested, at levels ranging from 0.3 to 30 microg/ml, in order to evaluate the effect on the in vitro maturation (IVM) rate of bovine oocytes and on the formation of 17 beta-estradiol in supernatants of mural granulosa cells (GC) cultures. These compounds induced dose-dependent oocyte maturation delay and chromatin abnormalities. Maturation of oocytes to metaphase II (M II) was inhibited in oocytes cultured in the presence of 30 microg/ml ZEA, alpha-zearalenol or zearalanone, with a significant increase in chromatin abnormalities occurring in the presence of ZEA (P

The stability of fumonisin B1 and fumonisin B2 during processing of corn flakes was investigated with three different methods for analysis of the naturally contaminated raw material (corn flour), intermediate product (extruded, but not roasted corn flakes), and final product (roasted corn flakes). Only one method, using immunoaffinity column clean-up, provided reliable results in the determination of fumonisins in corn flake samples at the intermediate and final steps of processing. About 60 to 70% of the initial amount of fumonisins were lost during the entire cycle of corn flake processing, with less than 30% losses occurring during the intermediate extrusion step (70 to 170 degrees C for 2 to 5 min). The effect of different additives commonly present in commercial products (sodium chloride, sucrose, and ferrous sulfate heptahydrate) on the reliability of fumonisin analysis has also been investigated. The presence of sodium chloride strongly reduced fumonisin recovery when strong anion-exchange (SAX) columns were used for the clean-up step, whereas the other additives appeared to have little or no effect on the accuracy of fumonisin analysis. The use of reliable analytical methods that are effective for both raw materials and processed products is of paramount relevance for studying the effect of food processing on mycotoxin-contaminated commodities. Despite the fact that some effective fumonisin decontamination occurring during corn flake processing has been shown, more work is needed to identify the thermal breakdown products of fumonisins and their relevant toxicity.

The determination of fumonisins in cornflakes is a challenging matter as the actually available methods for the analysis of corn do not perform well when applied to this more complex matrix. After testing several factors that may affect the analytical performance, an accurate method for the determination of fumonisin B1 (FB1) and B2 (FB2) in cornflakes has been developed. The method uses immunoaffinity chromatography for clean-up and high performance liquid chromatography (HPLC) for quantification of the toxins. Samples were extracted twice with acetonitrile-methanol-water (25:25:50) and the combined extracts were diluted with phosphate buffered saline (PBS) and applied to a FumoniTest immunoaffinity column. After washing with PBS, fumonisins were eluted from the column with methanol and reacted with o-phthaldialdehyde/2-mercaptoethanol to form fluorescent derivatives. Fumonisin derivatives were analysed by reversed phase HPLC with fluorometric detection using methanol-0.1 M phosphate buffer (77:23; pH adjusted at 3.35) as mobile phase. The average recoveries for FB1 and FB2 spiked in the ranges of 0.33 2.80 microg/g and 0.17-1.40 microg/g were 102.6% and 95.1%, respectively, with average relative standard deviations of 9% and 8%. The limit of quantification for FB1 and FB2 was 0.005 microg/g based on a signal-to-noise ratio of 6.1 by using a sensitive fluorescence detector. The method was used to analyse 18 cornflakes and cornflake cereals samples/for FB1 and FB2 contamination. All but one sample were found to be contaminated, with maximum FB1 and FB2 concentrations of 1.092 microg/g and 0.235 microg/g, respectively. Mean FB1 and FB2 concentrations were 0.157 microg/(g and 0.036 microg/g, respectively.

A new and accurate method to quantify ochratoxin A (OA) in table wine has been developed. The method uses commercial immunoaffinity columns for clean-up and high-performance liquid chromatography (HPLC) with fluorescence detection for quantification of the toxin. Wine was diluted with a solution containing 1% polyethylene glycol (PEG 8000) and 5% sodium hydrogencarbonate, filtered and applied to an OchraTest immunoaffinity column. The column was washed with a solution containing sodium chloride (2.5%) and sodium hydrogencarbonate (0.5%) followed by water. OA was eluted with methanol and quantified by reversed-phase HPLC with fluorometric detection (excitation wavelength 333 nm, emission wavelength 460 nm) using acetonitrile-water-acetic acid (99:99:2) as mobile phase. Average recoveries of OA from white, rose and red wine samples spiked at levels from 0.04 to 10 ng/ml ranged from 88% to 103%, with relative standard deviations (RSDs) between 0.2 and 9.7%. Detection limit was 0.01 ng/ml based on a signal-to-noise ratio of 3:1. The method was applied successfully to 56 samples of red (38), rose (8), white (9) and dessert (1) wine. The levels of OA ranged from

The ear rot severity of nine maize hybrids and the accumulation of fumonisin B(1) (FB(1)), fumonisin B(2) (FB(2)), beauvericin (BEA) and fusaproliferin (FP) after artificial inoculation in the field with a toxigenic strain of Fusarium proliferatum have been investigated. Different degrees of ear rot were observed in different hybrids. Inoculated ears contained 11-38% of Fusarium-damaged kernels (FDK). Mycotoxin analyses showed a pronounced contamination of FDK with concentrations ranging from 116 to 343 mgkg(-1) for FB(1), from 8 to 29 mgkg(-1) for FB(2), from 1 to 14 mgkg(-1) for BEA and from 2 to 10 mgkg(-1) for FP. Lower levels of contamination were found in healthy-looking kernels (up to 26, 2, 0.2 and 0.3 mgkg(-1) for FB(1), FE(2), BEA and FP respectively). A good correlation was observed between mycotoxin contamination and the Fusarium ear rot index, calculated on the basis of average ear infection with a scale ranging from 0 to 500 to represent healthy cobs and totally rotted cobs respectively. (C) 1999 Society of Chemical Industry.

The occurrence of ochratoxin A, fumonisin B1 and B2 has been investigated in maize samples collected in 1996 (105 samples) and 1997 (104 samples) in 14 counties of Croatia, including Brodsko-Posavska county, the main area of Balkan endemic nephropathy in Croatia. Ochratoxin A and fumonisins co-occurred in 21% of the examined samples. In particular, ochratoxin A (OTA) was found in 10 samples (10%) of the 1996 and 36 samples (35%) of the 1997 crops with mean concentrations of positive samples of 37.9 ng/g and 57.1 ng/g, and highest concentrations at 223.6 ng/g and 613.7 ng/g, respectively. Similar incidence of OTA contamination was observed in 1996 samples from both endemic and non endemic areas of Balkan nephropathy, whereas a significant difference (P

Thirty-seven Fusarium strains belonging to different species of the Sporotrichiella section, from different plant and geographic origins, were tested for production of trichothecene derivatives on autoclaved com kernels. The seventeen strains of F. sporotrichioides were able to produce T-2 toxin (up to 1600 mg/kg), HT-2 toxin (up to 500 mg/kg) and neosolaniol (up to 300 mg/kg). One of four strains of F. poae produced diacetoxyscirpenol (80 mg/kg). None of the nine strains of F. tricinctum and of the seven strains of F. chlamydosporum produced T-2 toxin derivatives. The toxicity of the culture extracts towards Artemia salina larvae was strictly related to the occurrence of the above trichothecenes, although two strains of F. tricinctum, non-producers of trichothecenes, showed toxicity related to unidentified compounds.

A rapid and reliable procedure has been developed for the determination of ochratoxin A in wheat and oats. The method consists of extraction of the sample with acidic chloroform, followed by defatting with n-hexane and finally, HPLC determination with fluorometric detection. Mean recoveries for wheat and oats spiked at levels between 1 and 100 micrograms/kg ranged from 80 to 104%. The limit of determination (field blank +6 sigma) was 0.8 micrograms/kg and the precision (within-laboratory relative standard deviation) ranged from 3 to 7%. The method was tested on 34 wheat and 34 oats samples. Ochratoxin A was confirmed in some positive samples by methyl ester formation and/or by clean-up of the extracts with immunoaffinity columns. The method was not appropriate for the analysis of barley (45 tested samples), rye (69 samples) or trout feed (13 samples). A false positive was recorded within the four positive barley samples and 18 false positives were recorded within the 21 positive rye samples whereas trout feed samples could not be analysed due to insufficient clean-up. The use of immunoaffinity columns made the analysis of trout feed and rye samples possible, providing excellent clean-up of the extracts with no false positive results and a good limit of determination (0.2 micrograms/kg).

A rapid and accurate method to quantify zearalenone in corn is described. The method uses immunoaffinity chromatography for purification and high-performance liquid chromatography (HPLC) for detection and quantification of the toxin. Corn samples were extracted with acetonitrile-water (90:10, v/v) and the extract was diluted with water (1:10, v/v) and applied to a Vicam Zearala Test immunoaffinity column. The column was washed with water and zearalenone was eluted with methanol and quantified by reversed-phase HPLC with fluorometric detection (lambda(ex) = 274 nm, lambda(em) = 440 nm) using acetonitrile-water-methanol (46:46:8, v/v) as mobile phase. Zearalenone recoveries from the ZearalaTest column were higher than 95%, and the column can hold a maximum of 4.0 mu g of toxin. Average recoveries of zearalenone from corn spiked at levels of 0.1-10 mu g/g ranged from 93 to 99.5%, with relative standard deviations of

The widespread occurrence of fumonisins in maize and maize-based foods and feeds demands the development of rapid and reliable methods for the analysis of suitable biomarkers in biological fluids in order to assess human and animal exposure to these important mycotoxins. The increase in the ratio of free sphinganine/sphingosine (SA/SO) in urine has been recently proposed as a biomarker to evaluate exposure to fumonisins. The presently available method for the determination of SA and SO in biological samples is labor intensive, time consuming and insufficiently accurate. A new method has been proposed for the determination of SA and SO in human and animal urine which is more precise and accurate, and drastically reduces the number of steps during extraction and clean-up. The method is essentially based on the use of silica minicolumn clean-up of the chloroform extract from alkalinized urine. The final extract is derivatized with o-phthaldialdehyde reagent and SO and SA are determined by reversed-phase HPLC with fluorimetric detector. Urine samples spiked with SO, SA standards at concentrations ranging from 1.5 to 15 ng/ml have given mean recoveries higher than 80% and precision (coefficient of variation) lower than 10%. Detection limit for SO and SA was 0.1 ng/ml.

The accumulation of fumonisin B 1 (FB 1 ) and fumonisin B 2 (FB 2 ) after inoculation of 14 maize hybrids with Fusarium moniliforme has been examined in Poland during the seasons 1992-1995. Fumonisins have been detected in all 14 hybrids in amounts ranging from 0.09 mg kg -1 FB 1 + 0.02 mg kg -1 FB 2 to 93.2 mg kg -1 FB 1 + 15.8 mg kg -1 FB 2 . Inoculated ears contained from

The availability of biomarkers that display mycotoxin ingestions is important for a number of reasons. They can be used to conduct epidemiological studies to evaluate human exposure to mycotoxins, to prevent mycotoxicoses through an early detection of mycotoxin ingestions and to check the effectiveness of decontamination process for mycotoxins with appropriate in vivo studies. The increase of urinary sphinganine/sphingosine ratio (SA/SO) as biomarker for fumonisins has the potential to respond to these needs. Performance characteristics of this biomarker have been evaluated in a trial with rats fed with diets containing increasing doses of fumonisins. Contaminated diets were prepared by incorporating adequate amounts of maize culture of toxigenic Fusarium moniliforme in blank control rat diet, in order to prepare poertions containing fumonisins (FB1 + FB2) at level of 0.50, 1.0, 2.0, 7.0 and 15.0 μg/g. Each portion was analysed to check fumonisin concentration and than given to rats for 12-13 days, starting from the lower to the higher contaminated diet. The animals were housed in metabolic cages with free access to water and diet. Urine samples were collected at approximately two day intervals. Free SA and SO were extracted, purified on silica gel minicolumn and measured by HPLC/fluorescence detector as OPA-mercaptoethanol derivatives. Mean values ⊕ 1SD of SA and SO in urine of control rats were 1.29 4 ⊕ 0.67 ng/ml and 4.45 ⊕ 2.90 ng/ml, respectively. The relevant mean value of SA/SO ratio was 0.30 ⊕ 0.17. The minimum effective dose of fumonisins in the diet causing a significant increase of the SA/SO ratio in the urines of treated rats was 2.0 μg/g. Rats fed for 10 days with a diet containing 2.0 μg/g showed urinary mean value of the SA/SO ratio of 2.10 ⊕ 0.14 and SA and SO levels of 8.12 ⊕ 1.72 ng/ml and 3.84 ⊕ 1.0 ng/ml respectively. Higher values, both for SA/SO ratio and SA concentrations, were found in urines of rats fed with diets containing 7.0 and 15.0 μg/g of fumonisins, whereas the concentrations of SO remained roughly the same. A positive correlation between fumonisin intake and SA/SO values in rat urines was observed with a correlation coefficient value of 0.99.

Data on the occurrence of fumonisins B1 and B2 in maize genotypes (inbred lines and hybrids) cultivated in several countries from three continents will be presented. Samples originated at different times (from 1990 to 1994) from experimental stations in Argentina, Benin, Croatia, Poland, Portugal, Italy, Romania and Zambia. Fumonisin contamination was negligible in samples from eastern Europe (Croatia, Romania and Poland), whereas it was quite relevant and widespread in samples from Argentina, western Europe and Africa. A general trend of higher fumonisin levels was observed in Argentina, Zambia (limited to 1993 crop), Portugal, and Italy (mainly 1990 and 1991 crop years), with a significant percentage of samples showing more than 1000 ng/g, a level of concern. The data from the present study suggest that the environmental conditions in the specific area of cultivation play an important role in the accumulation of fumonisins in maize.

Visoltricin (VIS), acuminatopyrone (ACP), clamydosporol (CL), isochlamydosporol (ICL) and chlamydospordiol (DIOL), recently characterized Fusarium metabolites, were separated on a polymeric RP-18 column eluted with acetonitrile-0.01% ammonia solution (35:65) at 1 ml/min and detected with a diode-array UV detector. The presence of ammonia in the mobile phase improved the shape of the CL and VIS peaks. The use of a polymeric column was required owing to the basic pH of the mobile phase. Maize cultures of several strains of F. tricinctum and F. chlamydosporum were analysed with this procedure after extraction with aqueous methanol, partitioning with methylene chloride and clean-up with a C 18 minicolumn. VIS was produced only by F. tricinctum, whereas ACP and chlamydosporols were produced by both Fusarium species.

International audience; The natural occurrence of fumonisin B-1 (FB1) and fumonisin B-2 (FB2) has been investigated in 26 maize inbred lines grown in Italy and in 72 maize hybrids grown in Croatia (19), Poland (7), Portugal (9), Romania (6), Benin (9), and Zambia (20). The incidence and levels of fumonisin contamination resulted in two major groups of countries. The first with high contamination included Italy, Portugal, Zambia, and Benin, with incidence of 100, 100, 100, and 82%, and fumonisin (FB1 + FB2) levels up to 2850, 4450, 1710, and 3310 ng/g, respectively. The second group, including Croatia, Poland, and Romania, showed very low levels of contamination (less than or equal to 70 ng/g) with 50% incidence of positive samples. A general trend for higher contamination levels was observed in maize genotypes with higher FAO maturity class or dent-type endosperm. Although the environmental conditions of the specific area of cultivation seem to play a role in the formation of fumonisin in maize, further investigations are needed to thoroughly establish the genotype-area-season interaction.

The stability of fumonisins B1 and B2 (FB1 and FB2) during 6 week storage was studied in acetonitrile:water (1:1) and methanol at four storage temperatures (-18, 4, 25 and 40 degrees C). Samples were analysed by HPLC with fluorescence detection after derivatization with o-phthaldialdehyde. Decomposition of fumonisins was evident in a methanol solution after 6 week storage at 4, 25 and 40 degrees C (signals for both toxins decreased by 5, 35 and 60%, respectively), whereas no losses were observed in samples stored at -18 degrees C. Both fumonisins were quite stable in acetonitrile:water solution at any of the four storage temperatures. The stability of fumonisins in acetonitrile:water (1:1) was confirmed over a period of 6 months at three temperatures (-18, 4 and 25 degrees C) in two solutions at different mass concentrations. No significant difference (t0.95-test) was observed between solutions stored in the dark at different temperatures and for different times (3 and 6 months). The overall coefficient of variation (n = 24, four measurements at each storage time and temperature) in each solution was < 3% for both toxins. Acetonitrile:water (1:1), unlike methanol, is a suitable solvent for fumonisin reference solutions.

A T-2 toxin specific monoclonal antibody, IgGl K, with a low level of ELISA cross-reactivity to Acetyl T-2, HT-2, and iso T-2 toxins has been produced. The ability of this monoclonal antibocly to neutralize the cytotoxicity of T-2 toxin in PHA stimulated cultures of human lymphocytes was determined by the MTT method. The complete neutralization of the toxic effect of 0.02 μM T-2 toxin was obtained with 0.03 μM of MoAb, whereas the 50% neutralizing dose (ND50) was observed at 0.009 of MoAb. Partial neutralization was observed with Acetyl T-2 toxin (ND, = 0.038 μM) and HT-2 (ND50 = 0.94 μM). These results could represent a rational for clinical use of T-2 toxin specific monoclonal antibody in prophylaxis and therapy of T-2 toxemia. © 1994 Wiley-Liss, Inc.

An in vitro peripheral lymphocyte blastogenesis system was used to investigate the biological activities of the fungal toxin fusarochromanone (TDP-1) and its monoacetyl derivative TDP-2. Briefly, cultures of human or bovine peripheral lymphocytes were exposed to TDP-1 or TDP-2 and a mitogen (PHA, Con A or PWM). After a standard incubation time, cell proliferation was quantified using the MTT bioassay. Human and bovine lymphocyte proliferation was inhibited by high concentrations of TDP-1; however, bovine lymphocyte proliferation was significantly increased at low concentrations of TDP-1. TDP-2 has similar but less pronounced effects on lymphocyte proliferation. © 1992 Wiley-Liss, Inc.

The relationship between the toxicities of crude extracts and purified toxins of Fusarium spp. belonging to the section Sporotrichiella has been assessed. Toxicity was determined on the basis of death of Artemia salina larvae and of viability and blastogenic response of bovine and human lymphocytes. Trichothecene-producing strains of Fusarium sporotrichioides and Fusarium poae were toxic to A. salina and to lymphocyte blastogenesis. A strain of Fusarium tricinctum, producing visoltricin and chlamydosporol, induced differentiated activity in different bioassays (toxicity to A. salina but only minor activity against lymphocyte blastogenesis). Other, non-toxin-producing strains of Fusarium chlamydosporum, F. poae, and F. tricinctum were not active in the tested biosystems.

A selective method for the determination of altertoxin-I and altertoxin-II by high-performance liquid chromatography with electrochemical detection is described. Altertoxins were separated on a reversed-phase column with methanol-water containing 0.1 M sodium nitrate and 1 mM nitric acid (60:40) as eluent and detected with dual in-series electrodes operating in the "redox" mode (generator electrode +1.0 V, indicator electrode -0.1 V). The method was applied successfully to the determination of sub-ppm levels of altertoxins in samples of maize, rice and tomatoes infected by Alternaria alternata.

Background The mycotoxin zearalenone (ZEA) and its derivatives, alpha and beta-zearalenol (alpha and beta-ZOL), synthesized by genera Fusarium, often occur as contaminants in cereal grains and animal feeds. The importance of ZEA on reproductive disorders is well known in domestic animals species, particularly in swine and cattle. In the horse, limited data are available to date on the influence of dietary exposure to ZEA on reproductive health and on its in vitro effects on reproductive cells. The aim of this study was to evaluate the effects of ZEA and its derivatives, alpha and beta-ZOL, on granulosa cells (GCs) from the ovaries of cycling mares. Methods The cell proliferation was evaluated by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test after 3 days exposure at different concentrations of ZEA and its derivatives (from 1 × 10-7 to 0.1 microM). The apoptosis induction was evaluated after 1 day exposure, by DNA analysis using flow cytometry. Results An increase in cell proliferation with respect to the control was observed in the presence of ZEA at 1 × 10-3 and 1 × 10-4 microM and apoptosis was induced by all mycotoxins at different concentrations. Conclusion The simultaneous presence of apoptosis and proliferation in GC cultures treated with zearalenones could indicate that these mycotoxins could be effective in inducing follicular atresia. These effects of zearalenones may result from both direct interaction with oestrogen-receptors as well as interaction with the enzymes 3alpha (beta)-hydroxysteroid dehydrogenase (HSD), involved in the synthesis and metabolism of endogenous steroid hormones. These cellular disturbances, described for the first time in equine GCs cultured in vitro, could be hypothesized as referred to reproductive failures of unknown ethiology in the mare.

Compounds with molecular weights 14 a.m.u. lower than their corresponding parent compounds have been detected in trichothecene standards during gas chromatography-mass spectrometry analysis. The positive ion chemical ionization mass spectra indicated that these compounds were trichothecene homologs lacking a methyl group on the isovaleroxy substituent at the C-8 position. Demethylated derivatives have been found in the standards of T-2, HT-2, and T-2 triol prepared in our laboratory and those obtained from a commercial source.

Samples of wheat naturally infected in the field byFusarium culmorum (W.G.Sm.) Sacc. andFusarium graminearum Schwabe were analyzed for deoxynivalenol, 3-acetyldeoxynivalenol, and zearalenone. No zearalenone was detected at levels higher than 0.5mg/kg. Deoxynivalenol was present in 100% and 3-acetyldeoxynivalenol in 80% of the examined samples at levels of 0.21 to 30.4 mg/kg and 0.54 to 29.54 mg/kg, respectively. The mycotoxin levels in the chaff were 5 to 50 times higher than in the kernels. This is the first report on natural occurrence of 3-acetyldeoxynivalenol in wheat. This toxin, in addition to deoxynivalenol, was highly correlated with wheat head fusariosis. These findings suggest that more attention should be given to the occurrence of 3-acetyldeoxynivalenol in cereal grains during the growth as well as during storage.

Culture extracts of eight strains F. acuminatum isolated in Italy from soil and corn stalk or ear contained considerable amounts of trichothecenes, were highly toxic to Artemia salina larvae, and inhibited the growth of tomato seedlings. The trichothecene production pattern of these islates (mainly related to 8-acetoxyneosolaniol) was different from that of other isolates of the same species reported in the literature, producing mainly T-2 toxin and its derivatives

Fusarium avenaceum isolates from wheat, rye, barley, triticale, corn and potato formed substance with yellow fluorescence with properties similar to citrinin. Pathogenicity of 17 isolates tested against cereals seedlings was weaker than F. culmorum isolates; one isolate only was strong, two medium and the remaining 82 % were very weak or non pathogenic.

The polarographic behaviour of representative trichothecenes is described. Compounds characterized by an α,β-unsaturated carbonyl group or by a macrocyclic lactonic ring were found to be electroactive in a methanolic Britton—Robinson buffer. Differential pulse polarography provides detection limits of the order of 40 nmol l−1 with linear ranges extending up to 9 μmol l−1. The application of differential pulse polarography is described for the determination of nivalenol (3α, 4β, 7α, 15-tetrahydroxy-12,13-epoxytrichotec-9-en-8-one), deoxynivalenol (3α, 7α, 15-trihydroxy-12,13-epoxytrichothec-9-en-8-one) and fusarenone-X (3α, 7α, 15-trihydroxy-4 β-acetoxy-12,13-epoxytrichothec-9-en-8-one) in infected maize. A suitable liquid/liquid extraction and chromatographic cleanup procedure is reported. Detection limits for the overall procedure and for a sample size of 50 g are of the order of 50 ng g−1 while average recoveries range from 20 to 70% at the 1 μg g−1 level. Polarographic results are compared with those obtained by a gas-chromatographic method.

The occurrence of wheat head fusariosis in various regions of Poland was observed in 1985 and 1986. The incidence of fusariosis was usually low — about 0,01% — only on South — East in some localities reached 5 – 20%. The most important species isolated from infected heads were :Fusarium culmorum F.grami nearurn,F.ni vale andF.avenaceum, in addition to whichF.crookwel1ense,F.eqiseti,F.subqluti nans andF.tricinctum (sensu Nelson et al.1983) were observed. Deoxynivalenol was present in 100% examined kernels subsamples at level 5–18 mg/kg and 3-acetyl deoxyn i val enol in 70% at level 1–3 mg/kg. The mycotoxins amount in chaff was 1,4 to 2,6 and 1,4 to 11 times higher (DON and 3AcD0N resp.) than in kernels.

Taxonomy, toxinogenicity and pathogenicity of 26 isolates of Fusarium-Sporotricbiella section were studied. Strains of Fusarium sporotrichioides Sherb. isolated in Poland from cereals have been found severely pathogenic towards cereal seedlings. They produced significant amounts of T-2 toxin (up to 600 mg/kg) and other trichothecenes under laboratory conditions. The investigated strains of F. tricinctum (Corda) Sacc. and F. poae (Peck) Wollenw. proved to be nonpathogenic and nontoxigenic under the same conditions. Zusammenfassung Fusarium sporotrichoides Sherb., F. tricinctum (Corda) Saco und F. poae (Peck) Wollenw. — Kulturmerkmale, Toxinogenitat und Pathogenitat gegenuber Getreide Untersucht wurde die Taxonomie, Toxinogenitat und Pathogenitat von 26 Fusarium-lsolaten aus der Sporotrichiella-Sektion. Fusarium sporotnchoides-Summe, die von Getreide in Polen isoliert wurden, zeigten sich als sehr pathogen gegenuber Getreidesamlingen. Unter Laborbedingungen produzierten diese Stamme bedeutende Mengen an T-2 Toxin (bis zu 600 mg/kg) und anderen Trichothecenen. Die untersuchten F. tricinctum- und F. poae-Samme zeigten sich unter den gleichen Bedingungen als nicht pathogen und nicht toxinogen.

All 20 strains of Fusarium graminearum Schwabe isolated from cereals in Poland formed perithecia of the teleomorph Gibberella zeae (Schw.) Petch; all except two produced zearalenone at concentrations of up to 90 mg/kg and deoxynivalenol (= vomitoxin) up to 77 mg/kg under laboratory conditions. The ten strains tested proved to be strong pathogens of wheat, rye, triticale and barley seedlings in infection experiments. Strains were pathogenic to a similar extent, irrespective of the cereal species from which they had been isolated. However, triticale seedlings were most susceptible to infection. Zusammenfassung Fusarium graminearum Schwabe (Teleomorph Gibberella zeae (Schw.) Petch — Kulturmerkmale, Pathogenitat gegenuber Getreidesamlingen und Fahigkeit der Mykotoxinproduktion Unter Laborbedingungen bildeten alle 20 Fusarium graminearum Schwabe-Stamme, die von Getreide in Polen isoliert worden waren, Perithezien der Hauptfruchtform Gibberella zeae (Schw.) Petch, sowie, mit Ausnahme von zwei Stammen, Zearalenon und Deoxynivalenol (= Vomitoxin) in Konzentrationen bis zu 90 bzw. 77 mg/kg. Die zehn getesteten Stamme zeigten sich als starke Pathogene an Weizen-, Roggen-, Triticale- und Gerstensamlingen in Infektionsversuchen. Die Pathogenitatsstarke der Stamme war gleich und mit der Wirtspflanze nicht gekoppelt. Die Triticalesamlinge erwiesen sich jedoch als am meisten anfallig.

A highly toxic strain ofFusarium sporotrichioides Sherb. (P-11) isolated from wheat in Poland produced on rice culture up to 11 trichothecenes, which are: T-2 toxin (750 ppm), neosolaniol (300 ppm), HT-2 toxin (75 ppm), acetyl T-2 toxin (35ppm), 3'-hydroxy-T-2 (20ppm), T-2 triol (12.5ppm), 3'-hydroxy-HT-2 (1.2ppm), 4-acetoxy-T-2 tetraol (1.1 ppm), 15-acetoxy-T-2 tetraol (0.65 ppm), 8-acetoxy-T-2 tetraol (0.45 ppm), and T-2 tetraol (0.2 ppm). The presence of most of these trichothecenes, including the 3'-hydroxy-derivatives, in the excreta of animals treated with T-2 toxin indicates the existence of some correlation between T-2 toxin metabolism in animals and microorganisms, respectively.

37 Fusarium isolates (F. culmorum 20, F. graminearum 4, F. avenaceum 3, F. solani 4, and F. equiseti 6 from wheat, rye and triticale) were examined for pathogenicity and tested for zearalenone (F-2) and trichothecenes production in vitro. Strong pathogens (F. culmorum and F. graminearum) produced deoxynivalenol and 3-acetyl-deoxynivalenol and zearalenone in considerable quantities.

Zearalenol was extracted from Fusarium -infected stems of corn from southern Italy. The toxin, which appeared as a single compound in various thin-layer chromatography systems, was resolved by high-pressure liquid chromatography into two components. A gas chromatography-mass spectrometry examination of a purified fraction confirmed the natural occurrence of zearalenol as a diastereomeric mixture and led to the identification of alpha (56 ng/g) and beta (27 ng/g) isomers. Among nine Fusarium species found associated with stalk rot in corn, only Fusarium culmorum (F. roseum `Culmorum') and F. equiseti (F. roseum `Gibbosum') produced zearalenol and always produced it in a diastereomeric mixture of alpha and beta isomers.

Surveys of corn (infected plants and commercial kernels) for Fusarium species and their mycotoxins were carried out on samples collected all over Italy and from some European and mediterranean countries. Investigations on samples of corn stalk and ear rot standing in the field, mainly collected in southern Italy, proved to be contaminated with zearalenone (ZON), zearalenols (ZOL), and deoxynivalenol (DON). The Fusarium species most frequently isolated, and their recorded toxigenic capability (in parentheses), were: F. moniliforme; F. culmorum (ZON, ZOL, DON, 3AcDON); F. equiseti (ZON, ZOL); and F. proliferatum (MF). Along with these species, F. graminearum group 2 (ZON, DON and/or 3AcDON or 15AcDON); F. chlamydosporum; F. acuminatum (type-A trichothecene derivatives); and F. semitectum were often found to be associated. F. heterosporum (ZON, ZOL); F. solani; F. crookwellense (ZON, ZOL, FUS, NIV); F. oxysporum (MF); F. avenaceum (MF); F. sporotrichioides (T-2 toxin and derivatives); and F. poae (DAS, MAS) were occasionally isolated.

Isoaltenuene, a previously unknownAlternaria metabolite has been isolated from a rice culture ofAlternaria alternata and purified by semipreparative HPLC. The assigned structure, elucidated by UV, IR, MS, and NMR spectroscopy, was 2′, 3′, 4′, 5′-tetrahydro-3, 4′β, 5’α-trihydroxy-5-methoxy-2′α-methyldibenzo (α)-pyrone and corresponded to a diasteroisomer of altenuene with inverted configuration at C-2′. Isoaltenuene showed a minor phytotoxic activity on tomato leaves at level of 20 μg/spot and no antifungal activity onGeotrichum candidum (up to 20 μg/disk).

A limited survey of the natural occurrence of the major Alternaria mycotoxins, i.e. alternariol (AOH), alternariol methyl ether (AME), altenuene (ALT), altertoxin-I (ATX-I), and tenuazonic acid (TA) has been carried out on olives and related processing products (oil and husks). The toxigenicity of Alternaria strains isolated from olives and the possible mycotoxin transfer into the oil have also been investigated. Four out of 13 olive samples were contaminated by 2 to 4 Alternaria mycotoxins. The highest contamination was found in a badly damaged sample containing 2.9, 2.3, 1.4 and 0.3 mg/kg of AME, AOH, ALT and TA, respectively. No mycotoxins were detected in olive-oil destined for human consumption (6 samples) or olive-husks (3 samples) collected from oil-mills after the first pressing of olives. An oil sample produced in our laboratory by processing the most contaminated olive sample contained AME (0.79 mg/kg) and AOH (0.29 mg/kg). The estimated mycotoxin amount transferred into the oil was 4% for AME, 1.8% for AOH, and zero for ALT and TA (considering 15% the oil yield). Although Alternaria species, mostly A. alternata (Fr.) Keissler, were present at various extent on all the examined olive samples, mycotoxins were only detected in samples of physically damaged olives. The production of mycotoxins by A. alternata isolated from olives was much higher (up to 3 order of magnitude for TA) on rice cultures than on olive cultures.

A survey on the occurrence of aflatoxin M1 was carried out in the province of Bari (Italy) on a total of 106 samples of raw milk from local farms, commercially heattreated milk, and dried milk. About 72% of the samples were contaminated with aflatoxin M1 within the concentration range 4–280ng/Kg. The incidence of aflatoxin M1 was much higher in commercial milk (91%) than in farm milk (26%), and all the dried milk samples contained aflatoxin Ml (10 to 280ng per Kg of powder milk).

Fusaria isolates from wheat were tested for ability to produce trichothecenes and zearalenone. Four isolates of F. culmorum out of 13 produced vomitoxin (DON) and 3 Ac-DON, one produced diacetoxysirpenol and 12 zearalenone. Particularly high yield of zearalenone was observed in cultures of sever pathogenic isolates. Higher temperature (20 degrees C) during first week of incubation favoured yield of zearalenone. About 50% of zearalenone was produced by surface mycelium.

The main toxic metabolites of a strain ofFusarium poae, isolated from oats, were diacetoxyscirpenol (DAS) and a monoacetoxyscirpendiol. An accurate(1)H and 13C NMR analysis allowed to identify the monoacetoxyscirpendiol as the 15-acetoxy-3α, 4 β - di hydroxy -12,13 - epoxy trichothec - 9 - ene (15-acetoxyscirpendiol). This is the first report on the production of 15 - acetoxyscirpendiol byFusarium poae.

Fusarium sporotrichioides was found to be the predominant fungus in approximately 2 % of corn ears damaged byFusarium species, before harvest during 1984 and 1985 in Poland. Concentrations of up to 1,714.9 mg/kg of total type-A trichothecenes (T-2 Toxin, HT-2 Toxin, Neosolaniol, T-2 Triol, and T-2 Tetraol) were found in hand-selected, heavily damaged kernels obtained fromF. sporotrichioides-molded ears.

With reference to the mainstream technology, the most relevant failure mechanisms that affect reliability of Flash memory are reviewed, showing the primary role played by tunnel oxide defects. The degradation of device. performance induced by program/erase cycling is discussed, specifically for what concern the leakage that affect a very small fraction of memory cells after cycling. The dependence of the leakage on tunnel oxide thickness, number of cycles, and temperature is analyzed. The leakage current is explained by trap-assisted tunneling involving one, two or more traps, with decreasing occurrence probability. Finally, data are presented showing the robustness of scaled Flash memory to alpha particles and electromagnetic radiation.

A new method for characterizing the SILC in flash memories is presented. The role of the SILC distribution in determining the failure statistics of flash cell arrays is first pointed out. The new technique is then explained, and results from a test array are shown. The description in terms of a simplified formula for the leakage current provides evidence for the bimodal distribution of the SILC distribution in the memory array under test. The bimodal character of the SILC distribution is finally verified by directly comparing characteristics measured on real cells with results of the new method on the same memory chip.