Production of trichothecenes and zearalenone by Fusarium species isolated from wheat

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 °C) during first week of incubation favoured yield of zearalenone. About 50% of zearalenone was produced by surface mycelium.     

Accumulation of type A trichothecenes in maize, wheat and rice by Fusarium sporotrichioides isolates under diverse culture conditions

Toxigenic isolates of Fusarium sporotrichioides were tested for the production of type A trichothecenes (T-2 toxin, HT-2 toxin, diacetoxyscirpenol and neosolaniol) when grown on three substrates (maize, rice and wheat) under various conditions of temperature and water activity in the laboratory for 3 weeks. Trichothecenes were determined by high-performance liquid chromatography with fluorescence detection, after derivatisation with coumarin-3-carbonyl chloride. This is the first time this analytical method has been applied to an extensive study of trichothecene accumulation. With minor exceptions, greater trichothecene production occurred when samples were incubated at 20 degrees C and moistened with 35% water (water activity 0.990) although incubation conditions affected the substrates studied in different ways. No correlation between the different pairs of trichothecenes was found except for neosolaniol and diacetoxyscirpenol (r=0.56). Principal component analysis results show that the data points can be grouped in three rough clusters related to cereal type, which points out that the composition of these cereals can influence the production of type A trichothecenes.     

Occurrence of Fusarium Head Blight species and Fusarium mycotoxins in winter wheat in the Netherlands in 2009

Most recent information on the occurrence of Fusarium Head Blight species and related mycotoxins in wheat grown in the Netherlands dates from 2001. This aim of this study was to investigate the incidence and levels of Fusarium Head Blight species and Fusarium mycotoxins, as well as their possible relationships, in winter wheat cultivated in the Netherlands in 2009. Samples were collected from individual fields of 88 commercial wheat growers. Samples were collected at harvest from 86 fields, and 2 weeks before the expected harvest date from 21 fields. In all, 128 samples, the levels of each of seven Fusarium Head Blight species and of 12 related mycotoxins were quantified. The results showed that F. graminearum was the most frequently observed species at harvest, followed by F. avenaceum and M. nivale. In the pre-harvest samples, only F. graminearum and M. nivale were relevant. The highest incidence and concentrations of mycotoxins were found for deoxynivalenol, followed by zearalenone and beauvericin, both pre-harvest and at harvest. Other toxins frequently found - for the first time in the Netherlands - included T-2 toxin, HT-2 toxin, and moniliformin. The levels of deoxynivalenol were positively related to F. graminearum levels, as well as to zearalenone levels. Other relationships could not be established. The current approach taken in collecting wheat samples and quantifying the presence of Fusarium Head Blight species and related mycotoxins is an efficient method to obtain insight into the occurrence of these species and toxins in wheat grown under natural environmental conditions. It is recommended that this survey be repeated for several years to establish inter-annual variability in both species composition and mycotoxin occurrence.     

Occurrence of Fusarium Head Blight species and Fusarium mycotoxins in winter wheat in the Netherlands in 2009

Most recent information on the occurrence of Fusarium Head Blight species and related mycotoxins in wheat grown in the Netherlands dates from 2001. This aim of this study was to investigate the incidence and levels of Fusarium Head Blight species and Fusarium mycotoxins, as well as their possible relationships, in winter wheat cultivated in the Netherlands in 2009. Samples were collected from individual fields of 88 commercial wheat growers. Samples were collected at harvest from 86 fields, and 2 weeks before the expected harvest date from 21 fields. In all, 128 samples, the levels of each of seven Fusarium Head Blight species and of 12 related mycotoxins were quantified. The results showed that F. graminearum was the most frequently observed species at harvest, followed by F. avenaceum and M. nivale. In the pre-harvest samples, only F. graminearum and M. nivale were relevant. The highest incidence and concentrations of mycotoxins were found for deoxynivalenol, followed by zearalenone and beauvericin, both pre-harvest and at harvest. Other toxins frequently found – for the first time in the Netherlands – included T-2 toxin, HT-2 toxin, and moniliformin. The levels of deoxynivalenol were positively related to F. graminearum levels, as well as to zearalenone levels. Other relationships could not be established. The current approach taken in collecting wheat samples and quantifying the presence of Fusarium Head Blight species and related mycotoxins is an efficient method to obtain insight into the occurrence of these species and toxins in wheat grown under natural environmental conditions. It is recommended that this survey be repeated for several years to establish inter-annual variability in both species composition and mycotoxin occurrence.     

Occurrence of trichothecenes in Argentinean beer: a preliminary exposure assessment

The presence of trichothecenes (deoxynivalenol, 3-acetyl deoxynivalenol, 15-acetyl deoxynivalenol, nivalenol, neosolaniol and diacetoxyscirpenol) was studied in 50 samples of Argentinean beer (nine different brands). Gas chromatography with electron capture detector was used for identification and quantification of these mycotoxins. The only mycotoxin detected was deoxynivalenol (DON). It was present in 44% of the samples, 18% were contaminated with more than 20ng/ml. Toxin levels ranged from 4 to 221ng/ml in positive samples. This is the first report on DON contamination of Argentinean beer. The estimate of probable daily intake (PDI) of DON from beer consumption in Argentina does not indicate a health hazard, but it has to be taken into account in calculations of total DON exposure in the population.     

Fusarium species (section Liseola) and its mycotoxins in maize harvested in northern Argentina

Maize and maize products harvested in small fields and stored by farmers in northern Argentina were assayed for Fusarium and fumonisin and beauvericin contamination. Fumonisins were present in six of the 18 samples. The levels of fumonisins ranged from 603 to 1888ng/kg. Fumonisin B₃ (FB₃) and beauvericin were not detected in the samples evaluated. Fusarium subglutinans was one of the most prevalent species isolated. Twenty-five strains of F. subglutinans isolated from maize kernels and belonging to Gibberella fujikuroi mating population E were beauvericin-producers in culture. Seven of these strains also produced moniliformin. This is the first report on beauvericin-production by maize isolates of F. subglutinans from Argentina.     

Occurrence of type A trichothecenes in conventionally and organically produced oats and oat products

Among cereals, oats are known to be very frequently contaminated with type A trichothecenes and so they can play a major role in the exposition of the consumer to these mycotoxins. Seventy representative oat samples of both conventional and organic production were drawn at mills and at wholesale stage according to Commissions Regulation (EC) No 401/2006 and analyzed for nine type A trichothecenes by LC-MS/MS. High contamination rates were found for most of the toxins in conventional as well as in organic products (e. g. 100% for T-2 toxin or 99% for HT-2 toxin). The mean concentration of T-2/HT-2 (sum of the toxins) was 17 +/- 18 microg/kg (mean +/- SD) in all samples, 27 +/- 21 microg/kg in conventional, and 7.6 +/- 4.6 microg/kg in organic products, respectively. The highest T-2/HT-2 level has been determined in conventionally produced oat flakes (85 microg/kg). The mean level of T-2 tetraol (9.5 +/- 7.7 microg/kg) in all samples was found to be even higher than that of T-2 (5.1 +/- 6.0 microg/kg), whereas levels of T-2 triol, 4,15-diacetoxyscirpenol, 15-monoacetoxyscirpenol, and neosolaniol were considerably lower. For oats and oat products from organic farming contamination levels of T-2, HT-2, T-2 triol, T-2 tetraol, and neosolaniol were significantly lower. The results are discussed with respect to possible health risks for the consumer.     

Determination of type B trichothecenes and macrocyclic lactone mycotoxins in field contaminated maize

A sensitive, reliable liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for determining some commonly found mycotoxins produced by Fusarium strains in maize was evaluated and applied to field samples. The selected substances were: trichothecenes B (nivalenol, deoxynivalenol, fusarenon X, 3- and 15-acetyldeoxynivalenol) and some macrocyclic lactones (zearalenone, α- and β-zearalenol, zearalanone, α- and β-zearalanol). Analytes were extracted from a 1 g sample by homogenization with acetonitrile/water (75:25, v/v, 25 mL final volume). 5 mL of crude extracts was cleaned-up on Carbograph-4 cartridges. Two fractions were obtained and were analyzed by HPLC-electrospray ionization (ESI) in negative mode. Recoveries for spiked maize samples were in the range 79–106% and method detection limits (MDLs) were ⩽6 ng/g for all compounds, except fusarenon X (12 ng/g). 25 random maize samples were analyzed both by the ELISA-based methods specific for deoxynivalenol and zearalenone and by this method for trichothecenes B and macrocyclic lactones. Results were comparable for zearalenone (R² = 0.982), but disagreed for deoxynivalenol. Finally, a total of 78 freshly harvested maize samples, collected from central and northern Italy during 2002, and divided in two different experiments, were analyzed by the developed method. Data show that there exists a phenomenon of random contamination from the target fusariotoxins just before harvest and an increase of trichothecene B and zearalenone abundance on field crop possibly related to damp climate, temperature range and delayed harvest period. Deoxynivalenol was the most abundant (up to 3430 ng/g) and frequent mycotoxin (40%) detected, followed by acetyldeoxynivalenol. Derivatives of zearalenone were present in traces and β-zearalanol was never found.     

Occurrence of zearalenone on Fusarium graminearum infected wheat and maize fields in crop organs, soil, and drainage water

The mycotoxin zearalenone (ZON) is a very potent natural endocrine disrupting chemical, produced by Fusarium graminearum fungi growing on crops such as wheat and maize. Although it is well-investigated in food and feed, very little is known about its environmental fate and behavior. Here, we report the occurrence of ZON on F. graminearum infected wheat and maize fields in crop organs and soil and its emission via drainage water. ZON amounts in the investigated crops and topsoil were between 6.1 and 25.0 and up to 5.6 g/ha, respectively. ZON concentrations in drainage water were in the low nanogram per liter range with a maximum of 35 ng/ L. Cumulated ZON amounts emitted via drainage water ranged from 0.1 to 4.3 mg/ha, depending on the crop cultivated in the respective period. This corresponds to fractions between 0.001 and 0.070% of the initially present ZON amount in the plants. Because of the low concentrations emitted via drainage water, it can be assumed that ZON contributes little if at all to the overall estrogenicity of major surface water bodies. However, in small creeks, mainly fed by agricultural runoff, ZON might be present in environmentally critical concentrations at times of F. graminearum infections.     

Occurrence of free and conjugated Fusarium mycotoxins in cereal-based food

A collection of 84 cereal-based food products in 25 composites, including beer, was screened for the presence of deoxynivalenol, zearalenone, and their respective metabolites deoxynivalenol-3-glucopyranoside, 3-acetyl-deoxynivalenol, zearalenol-4-glucopyranoside, α-zearalenol, β-zearalenol, α-zearalenol-4-glucopyranoside, β-zearalenol-4-glucopyranoside, and zearalenone-4-sulfate. The most abundant analyte was zearalenone-4-sulfate, which was found in 13 composites, albeit in low concentrations. Furthermore, deoxynivalenol was detected in eight, zearalenone in seven, and deoxynivalenol-3-glucopyranoside in two composites. None of the remaining six analytes was found in any matrices, which suggests that, if at all present, the concentrations of these latter metabolites are very low and, hence, do not impose any danger to consumers. The highest mycotoxin content was found in bran flakes with 254 ng g^?1 deoxynivalenol, 6 ng g^?1 zearalenone-4-sulfate, and 44 ng g^?1 zearalenone.     

Quantification of Fusarium graminearum and Fusarium culmorum by real-time PCR system and zearalenone assessment in maize

Zearalenone (ZEA) is a mycotoxin produced by some species of Fusarium, especially by Fusarium graminearum and F. culmorum. ZEA induces hyperoestrogenic responses in mammals and can result in reproductive disorders in farm animals. In the present study, a real-time PCR (qPCR) assay has been successfully developed for the detection and quantification of Fusarium graminearum based on primers targeting the gene PKS13 involved in ZEA biosynthesis. A standard curve was developed by plotting the logarithm of known concentrations of F. graminearum DNA against the cycle threshold (Ct) value. The developed real time PCR system was also used to analyze the occurrence of zearalenone producing F. graminearum strains on maize. In this context, DNA extractions were performed from thirty-two maize samples, and subjected to real time PCR. Maize samples also were analyzed for zearalenone content by HPLC. F. graminearum DNA content (pg DNA/ mg of maize) was then plotted against ZEA content (ppb) in maize samples. The regression curve showed a positive and good correlation (R2=0.760) allowing for the estimation of the potential risk from ZEA contamination. Consequently, this work offers a quick alternative to conventional methods of ZEA quantification and mycological detection and quantification of F. graminearum in maize.     

Naturally occurring Fusarium toxins in New Zealand maize

Twenty samples of maize collected from healthy growing crops and at harvest time and during storage were screened for four Fusarium toxins (deoxynivalenol, diacetoxyscirpenol, T-2 toxin and zearalenone) by gas chromatography-mass spectrometry and thin-layer chromatography. Seventeen samples (85%) contained one or more of these toxins. Zearalenone was present in 15 samples at levels ranging between 0.1 and 16 ppm. Deoxynivalenol, diacetoxyscirpenol and T-2 toxin were found in 11, 6 and 13 samples respectively, all at levels below 1 ppm. This work documents the first reported natural occurrence of Fusarium toxins in New Zealand maize, and it is concluded that all four of the mycotoxins studied are prevalent in apparently healthy standing crops as well as in stored maize.     

Investigations on Fusarium spp. and their mycotoxins causing Fusarium ear rot of maize in Kosovo

After wheat, maize (Zea mays L.) is the second most important cereal crop in Kosovo and a major component of animal feed. The purpose of this study was to analyse the incidence and identity of the Fusarium species isolated from naturally infected maize kernels in Kosovo in 2009 and 2010, as well as the mycotoxin contamination. The disease incidence of Fusarium ear rot (from 0.7% to 40% diseased ears) on maize in Kosovo is high. The most frequently Fusarium spp. identified on maize kernels were Fusarium subglutinans, F. verticillioides/F. proliferatum and F. graminearum. Maize kernel samples were analysed by LC-MS/MS and found to be contaminated with deoxynivalenol (DON), DON-3-glucoside, 3-acetyl-DON, 15-acetyl-DON, zearalenone, zearalenone-14-sulphate, moniliformin, fumonisin B₁ and fumonisin B₂. This is the first report on the incidence and identification of Fusarium species isolated from naturally infected maize as well as the mycotoxin contamination in Kosovo.     

Ecophysiology of Fusarium temperatum isolated from maize in Argentina

The effect of water activity (a_w = 0.95, 0.98 and 0.995), temperature (15, 25 and 30°C), incubation time (7, 14, 21 and 28 days), and their interactions on growth and moniliformin (MON), beauvericin (BEA), fusaproliferin (FUS) and fumonisin B₁ (FB₁) production by two strains of Fusarium temperatum isolated from Argentinean maize were determined in vitro on sterile layers of maize grains. The results showed that there was a wide range of conditions for growth and mycotoxins production by F. temperatum. Both strains were found to grow faster with increasing a_w and at 30°C. In relation to mycotoxin production, the two strains produced more FUS than the other mycotoxins regardless of a_w or temperature evaluated (maximum = 50 000 μg g^?1). For FUS, MON and BEA, the maximum levels were observed at 0.98 a_w and 30°C (50 000, 5000 and 2000 μg g^?1 respectively). The lowest levels for these three mycotoxins were detected at 15°C and 0.95 a_w (1700 and 100 μg g^?1 for FUS and MON respectively), and at 0.98 a_w (400 μg g^?1 for BEA). The maximum levels of FB₁ were produced at 15°C and 0.98 a_w (1000 μg g^?1). At all a_w and temperatures combinations evaluated there was an increase in toxin concentrations with time incubation. The maximum levels were detected at 21 days. Statistical analyses of a_w, temperature, incubation time, and the two- and three-way interactions between them showed significant effects on mycotoxins production by F. temperatum. For its versatility on growth and mycotoxin production, F. temperatum represents a toxicological risk for maize in the field and also during grain storage.     

Fusarium species identification and fumonisin production in maize kernels from Shandong Province,China, from 2012 to 2014

A total of 225 maize kernel samples were collected from Shandong Province in China from 2012 to 2014 and analysed for contamination with Fusarium spp. and fumonisins (FBs) using molecular methods and high-performance liquid chromatography with fluorescence detection. The results showed that the average incidences of Fusarium spp. in 2012, 2013 and 2014 were 23.3%, 37.1% and 36.5%, respectively, Fusarium verticillioides being the predominant species. In 2012, the average contamination level of FBs was 3071 ng g^?1, which was higher than that in 2014 (2913 ng g^?1) and 2013 (2072 ng g^?1). Of all samples, 13% and 19% had FB contamination levels higher than 2000 and 4000 ng g^?1, which are the maximum limits as set by the Food and Drug Administration of the United States and the European Commission, respectively. Therefore, efforts should be taken to minimise the potential risk of FBs to the health of humans and animals.     

Relationship between environmental factors, dry matter loss and mycotoxin levels in stored wheat and maize infected with Fusarium species

This study examined the relationship between storage environmental factors (water activity (a(w)) (0.89-0.97) and temperature (15°C-30°C)), colonisation of wheat and maize by Fusarium graminearum and F. verticillioides respectively and the dry matter losses (DMLs) caused and quantified by contamination with deoxynivalenol (DON), zearalenone (ZEA) and fumonisins (FUMs) during storage. Fungal growth was assessed by the amount of CO(2) produced under different interacting conditions of a(w) and temperature. DMLs were quantified using the cumulative CO(2) data, and these were shown to increase as temperature and a(w) increased. The amount of DON, ZEA (wheat for human consumption) and FUMs (feed maize) produced was significantly affected by the storage conditions. The three toxins however showed different patterns of production. Optimum for DON was at the wettest conditions (0.97a(w)) and the highest temperature assessed (30°C), whereas for ZEA this shifted to 25°C. FUMs were produced in higher amounts in maize at 30°C and 0.97a(w); however, at intermediate a(w) levels (0.955a(w)), the highest production occurred at 25°C followed by 20°C. Polynomial models were developed for the effect of the storage factors on DMLs and toxin production. DMLs under different environmental conditions were significantly correlated with DON and FUMs. DON contamination was above the EU limits in at least 80% of the wheat samples with DMLs >1%, whereas at least 70% of the same samples contained ZEA above the respective EU legislative limits. Similarly, at least 75% of the maize samples with DMLs ≥ 0.9% exceeded the EU limits for the sum of FUMs in feed. These results show that it may be possible to use temporal CO(2) production during storage of grains as an indicator of the level of contamination of the grain with mycotoxins.     

Relationship between environmental factors,dry matter loss and mycotoxin levels in stored wheat and maize infected with Fusarium species

This study examined the relationship between storage environmental factors (water activity (a _w) (0.89–0.97) and temperature (15°C–30°C)), colonisation of wheat and maize by Fusarium graminearum and F. verticillioides respectively and the dry matter losses (DMLs) caused and quantified by contamination with deoxynivalenol (DON), zearalenone (ZEA) and fumonisins (FUMs) during storage. Fungal growth was assessed by the amount of CO₂ produced under different interacting conditions of a _w and temperature. DMLs were quantified using the cumulative CO₂ data, and these were shown to increase as temperature and a _w increased. The amount of DON, ZEA (wheat for human consumption) and FUMs (feed maize) produced was significantly affected by the storage conditions. The three toxins however showed different patterns of production. Optimum for DON was at the wettest conditions (0.97a _w) and the highest temperature assessed (30°C), whereas for ZEA this shifted to 25°C. FUMs were produced in higher amounts in maize at 30°C and 0.97a _w; however, at intermediate a _w levels (0.955a _w), the highest production occurred at 25°C followed by 20°C. Polynomial models were developed for the effect of the storage factors on DMLs and toxin production. DMLs under different environmental conditions were significantly correlated with DON and FUMs. DON contamination was above the EU limits in at least 80% of the wheat samples with DMLs >1%, whereas at least 70% of the same samples contained ZEA above the respective EU legislative limits. Similarly, at least 75% of the maize samples with DMLs?≥?0.9% exceeded the EU limits for the sum of FUMs in feed. These results show that it may be possible to use temporal CO₂ production during storage of grains as an indicator of the level of contamination of the grain with mycotoxins.     

Biodiversity of complexes of mycotoxigenic fungal species associated with Fusarium ear rot of maize and Aspergillus rot of grape

Fusarium ear rot of maize and Aspergillus rot of grape are two examples of important plant diseases caused by complexes of species of mycotoxigenic fungi. These complexes of species tend to be closely related, produce different classes of mycotoxins, and can induce disease under different environmental conditions. The infection of maize and grape with multiple fungal species and the resulting production of large classes of mycotoxins is an example of mutual aggressiveness of microorganisms toward host species as well as to humans and animals that eat feed or food derived from the infected and contaminated plants. Infection of crop plant with a complex of microbial species certainly represents a greater threat to a crop plant and to human and animal health than infection of the plant with a single fungal species.     

Occurrence of Fusarium mycotoxins in rice and its milling by-products in Korea

A total of 201 samples of brown rice, polished rice, and two types of by-products, blue-tinged rice and discolored rice, were collected from rice stores maintained at 51 rice processing complexes in Korea. These samples were analyzed for the presence of Fusarium mycotoxins such as deoxynivalenol (DON), nivalenol (NIV), and zearalenone (ZEA). Contaminants (and their ranges) found in discolored rice samples were DON (59 to 1,355 ng g(-1)), NIV (66 to 4,180 ng g(-1)), and ZEA (25 to 3,305 ng g(-1)); those found in blue-tinged (less-ripe) rice were DON (86 to 630 ng g(-1)), NIV (50 to 3,607 ng g(-1)), and ZEA (26 to 3,156 ng g(-1)). Brown rice samples were contaminated mostly with NIV and ZEA (52 to 569 ng g(-1) and 47 to 235 ng g(-1), respectively). Polished rice samples were largely free from mycotoxins, although one sample was contaminated with NIV (77 ng g(-1)). When the fungal flora associated with each rice sample was investigated, blue-tinged rice was the most often contaminated with Fusarium graminearum (3.8%), followed by the discolored rice (2.4%) and brown rice (1.6%) samples. Using PCR, toxin genotyping of 266 isolates of F. graminearum revealed that most isolates (96%) were NIV producers. In conclusion, this survey is the first report of the cocontamination of Korean rice and its by-products with trichothecenes and ZEA. Importantly, it also provides new information on the natural contamination of rice by Fusarium mycotoxins.     

Immunological detection of Fusarium species in cornmeal

An indirect enzyme-linked immunosorbent assay (ELISA) was developed to detect Fusarium species in foods. Antibodies to proteins extracted from the mycelia of Fusarium graminearum and Fusarium moniliforme (verticillioides) were produced in New Zealand white rabbits. These antibodies detected 13 Fusarium species in addition to the producer strains. Levels of Fusarium semitectum and Fusarium tricinctum strains were below the detection threshold. The specificity of the assay was tested against 70 molds and yeasts belonging to 23 genera. One strain of Monascus species and one strain of Phoma exigua were detected; however, these two molds are not common contaminants of cereal grains or foods and should not interfere with the assay. The indirect ELISA's detection limits for F. graminearum and F. moniliforme were 0.1 and 1 microg of mold mycelium per ml of a cornmeal mixture, respectively. When spores of each mold were added individually to cornmeal mixtures (at ca. 10 spores per g) and incubated at 25 degrees C, these spores were detected by the indirect ELISA when they reached levels of 10(2) to 10(3) CFU/ml after 24 to 36 h. The indirect ELISA developed here shows promise for the detection of Fusarium species in grains or foods.