Fusarium DNA traceability along the bread production chain

Wheat grain contamination with toxigenic Fusarium spp. is of great economic concern to cereal producers and to the grain processing industry and is of great relevance for the quality and safety of the final products. In particular, the bread production chain can potentially be a vehicle for mycotoxin ingestion above the tolerable total daily intake. A quantitative polymerase chain reaction‐based analytical approach has been developed as a possible tool to estimate and control the risk of mycotoxins, particularly deoxynivalenol (DON). This DNA‐based analytical method has been applied to detect and quantify contamination levels of Fusarium graminearum and Fusarium culmorum in naturally infected wheat grain samples. The persistence of Fusarium contamination was also detected along the bread production chain in wholemeal, flour and bread. A significant correlation was found between Fusarium DNA and DON concentration in all samples.     

Molecular survey of trichothecene genotypes of Fusarium graminearum species complex from barley in southern Brazil

Fusarium head blight is a disease of primary concern to small-grain cereals of Brazil, including barley. Its main causal agent, Fusarium graminearum species complex (Fg complex)¸ is able to produce mycotoxins, especially deoxynivalenol (DON) and nivalenol (NIV), that usually contaminate grain. Strains that produce DON may also produce its acetylated derivatives: 3-acetyl-DON (3-ADON) and 15-acetyl-DON (15-ADON). Ninety two isolates were obtained from samplings of barley grain during three years (2007, 2008 and 2009) from several fields in both southern and northern production regions of Rio Grande do Sul state, Brazil. These isolates were examined for polymerase chain-reaction-based (PCR) trichothecene genotype based on the amplification of portions of Tri3 and Tri12. There was no effect of year or region on the proportion of trichothecene genotypes. Overall, 66% of the strains (61/92) were 15-ADON, 4.4% (4/92) were 3-ADON and 29.3% (27/92) were NIV. The overall NIV/DON ratio estimated (0.41) was five times higher than that found in previous studies with strains from wheat grown in the same region. Species identification of nine strains representing the trichothecene genotypes, based on comparisons of DNA sequences of portions of the PHO, RED and URA genes with sequences from curated reference isolates of Fusarium from GenBank, revealed that they belong to F. graminearum sensu stricto (four 15-ADON and one 3-ADON strain), F. meridionale (three NIV strains) and F. austroamericanum (one 3-ADON strain). These results add to the current regional knowledge of trichothecene genotypes and species within the Fg complex affecting barley in the region.     

Production of mycotoxins byFusarium species isolated in Germany

Summary Cultures ofFusarium tricinctum 434 formed large amounts of the trichothecene mycotoxins deoxynivalenol (DON) and 3-acetyldeoxynivalenol (AcDON), as well as the macrocyclic secondary metabolite zearalenone on moistened, autoclaved maize, rice and oats. The formation of zearalenone was low, with levels from 15 to 72 mg/kg as compared to the trichothecene production with maximum quantities of 917 mg/kg of AcDON on rice and 750 mg/kg DON on oats. In the cultures ofF. graminearum 183, total mycotoxin amounts found were lower, with maximum levels of zearalenone up to 150 mg/kg and AcDON up to 160 mg/kg on rice. DON, however, was produced in quantities of about 740 mg/kg on rice.

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Produktion von Mykotoxinen durch in Deutschland isolierte Fusarium-Arten1. Zeitverlauf der Deoxynivalenol-, 3-Acetyldeoxynivalenol-, und Zearalenon-Bildung auf festen Substraten

Zusammenfassung Kulturen vonFusarium tricinctum 434 bildeten auf feuchtem, autoklaviertem Mais, Reis bzw. Hafer relativ hohe Mengen der Trichothecen-Mykotoxine Deoxynivalenol (DON) und 3-Acetyldeoxynivalenol (AcDON) sowie das zu den makrocyclischen Lactonen zählende Mykotoxin Zearalenon. Die Zearalenonbildung war mit Werten von 15 bis 72 mg/kg gegenüber der Trichothecenproduktion mit maximal 917 mg/kg AcDON auf Reis und 750 mg/kg DON auf Hafer deutlich niedriger. In den Kulturen vonF. graminearum 183 wurden insgesamt geringere Toxinmengen gefunden mit maximalen Zearalenon-konzentrationen bis zu 150 mg/kg sowie AcDON Mengen bis zu 160 mg/kg auf Reis. Dagegen erreichte die DON-Bildung auf Reis 740 mg/kg.

     

Distribution of deoxynivalenol and 3‐acetyldeoxynivalenol in naturally contaminated and Fusarium culmorum infected triticale samples

Eight triticale genotypes, naturally contaminated (C) and collected from heads inoculated with two separate isolates Fusarium culmorum (I₁ and I₂) kernels were analysed for accumulation ofFusarium toxins. Triticale kernels were contaminated with deoxynivalenol (DON) and 3‐acetyldeoxynivalenol (3‐AcDON). Statistical analysis revealed significant differences between naturally contaminated samples (C) and those inoculated with isolate I₁ and I₂. The following accumulation of toxins in kernels (mean mg/kg) was found: C, I₁, I₂ – DON 0.06, 14.45, 8.09; 3‐AcDON – 0.01, 0.20, 0.49. Distribution of DON and 3‐AcDON was studied in four size fractions of kernels (> 2.8 mm; ≤ 2.8–2.5 mm, < 2.5–2.2 mm and < 2.2 mm).The highest concentration of DON and 3‐AcDON was found in the smallest fraction of damaged kernels. The percentage distribution of DON and 3‐AcDON in fraction < 2.2 mm was (C) 66%, 77%, (I₁) 38%, 27%, (I₂) 46%, 40%; and in fraction < 2.5 mm: (C) 85%, 94%, (I₁) 57%, 51%, (I₂) 80%, 76%. The naturally contaminated kernel fraction < 2.2 mm (although only 2% of all) contained 33% DON and 59% 3‐AcDON, for more toxigenic isolate I₁ 10%, 15%, 14%, less toxigenic isolate I₂ 4%, 13%, 11%, respectively, calculated on dry matter basis. No significant correlation between examined characteristics was found for either I₁ or I₂ isolate. The biosynthesis mechanism of toxic metabolites was to approximate (significantly correlated) to pair objects C/I₂.     

Multiplex Real‐Time PCR Method for Detection and Quantification of Mycotoxigenic Fungi Belonging to Three Different Genera

Abstract: Cereal crop plants are colonized by many fungal species such as Aspergillus ochraceus and Penicillium verrucosum, which produce ochratoxins, and Fusarium graminearum, which produces trichothecene mycotoxins. A multiplex real‐time PCR method using TaqMan probes was developed to simultaneously detect and quantify these mycotoxigenic Fusarium, Penicillium and Aspergillus species in cereal grains. Primers and probes used in this method were designed targeting the trichothecene synthase (Tri5) gene in trichothecene‐producing Fusarium, rRNA gene in Penicillium verrucosum, and polyketide synthase gene (Pks) in Aspergillus ochraceus. The method was highly specific in detecting fungal species containing these genes and was sensitive, detecting up to 3 pg of genomic DNA. These PCR products were detectable over five orders of magnitude (3 pg to 30 ng of genomic DNA). The method was validated by evaluating sixteen barley culture samples for the presence of deoxynivalenol (DON) and ochratoxin A (OTA) producing fungi. Among the barley culture samples tested, 9 were positive for Fusarium spp, 5 tested positive for Penicillium spp, and 2 tested positive for Aspergillus spp. Results were confirmed by traditional microbiological methods. These results indicate that DON‐ and OTA‐producing fungi can be detected and quantified in a single reaction tube using this multiplex real‐time PCR method. Practical Application: This method would be helpful in detecting and quantifying the mycotoxin producing fungi such as Fusarium, Aspergillus, and Penicillium in cereal grains and cereal‐based foods.     

Estimating mycotoxin contents of Fusarium-damaged winter wheat kernels

Winter wheat (Triticum aestivum L., cultivars Ritmo and Dekan) grain was sampled in Northern Germany between 2001 and 2006. Kernels damaged by fungi of the genus Fusarium were separated from sound grain by visual assessment. Samples containing 0%, 20%, 40%, 60%, 80% and 100% of Fusarium-damaged kernels were compiled and analyzed for the Fusarium type B trichothecenes deoxynivalenol (DON, 2001-2006), nivalenol (NIV, 2006), 3-acetyl-deoxynivalenol (3AcDON, 2006) and 15-acetyl-deoxynivalenol (15AcDON, 2006). The relationship between mycotoxin contents and the percentage of Fusarium-damaged kernels was calculated for each lot of grain. Apart from one exception, relationships between the percentage of Fusarium-damaged kernels and NIV, 3AcDON or 15AcDON were non-significant. In contrast, close relationships between the percentage of Fusarium-damaged kernels and the DON content were observed (r(2)=0.93-0.99). The y-axis intercepts were not significantly different from zero, but the DON content of the damaged kernels varied by a factor of 11.59 between years and by a factor of 1.87 between cultivars. Fusarium-damaged kernels contained between 0.21 and 2.39 microg DON kernel(-1). The overall average DON content of a Fusarium-damaged wheat kernel was 1.29 +/- 0.11 microg. The DON content of diseased kernels was affected by environment and wheat genotype but not by genotype x environment interaction. On average, Fusarium-damaged kernels contained 9.7-fold more DON than 15AcDON, 19.5-fold more DON than NIV, and 26.9-fold more DON than 3AcDON. 3AcDON and 15AcDON contents per wheat kernel were not significantly different between cultivars. On average, 4.27% of Fusarium-damaged kernels were sufficient to reach the 1.25 mg DON kg(-1) grain limit for unprocessed cereals in the EU. Given the low percentages of Fusarium-damaged kernels that are equivalent to current legal DON limits, grading accuracies >96% would be needed when using automatic grading systems for separating sound from damaged kernels.     

Influence of environmental factors on the biosynthesis of type B trichothecenes by isolates of Fusarium spp. from Spanish crops

Various species of Fusarium can produce trichothecene mycotoxins that contaminate food commodities and can represent a risk for human and animal health. In this paper, a full factorial design was applied to study the influence of incubation temperature, water activity (a(w)) and type of isolate on the production of deoxynivalenol (DON), nivalenol (NIV) and 3-acetyldeoxynivalenol (3-AcDON) in corn kernel cultures by three isolates of Fusarium graminearum and three isolates of Fusarium culmorum from crops grown in Spain. The tested temperatures were 15, 20, 28 and 32 degrees C. The a(w)-values were 0.960, 0.970 and 0.980. Moisture of cultures (within the studied range) did not affect significantly production of trichothecenes; however, the temperature affected significantly mycotoxin production and the optimal values were 28, 20 and 15 degrees C for DON, NIV and 3-AcDON, respectively. Four additional isolates of F. graminearum and two additional isolates of F. culmorum were examined for production of these mycotoxins at the optimal temperatures. Of the seven isolates of F. graminearum, four produced DON (0.88-3.97 microg/g), seven produced NIV (1.53-124 microg/g), and three produced 3-AcDON (0.65-10.6 microg/g). Of the five isolates of F. culmorum, four produced DON (1.20-4.93 microg/g), four produced NIV (6.94-701 microg/g), and four produced 3-AcDON (0.83-7.70 microg/g). Practically all isolates seem to belong to the NIV-chemotype. This is the first study done with regard to interaction between strain and ecological variables on type B trichothecene production by isolates of these two species from crops grown in Spain.     

Fusarium mycotoxins in various barley cultivars and their transfer into malt

BACKGROUND: Fusarium toxins, secondary metabolites of toxinogenic Fusarium species, are found in a range of cereal grains. In this study the occurrence of the most commonest Fusarium toxins, namely nivalenol (NIV), deoxynivalenol (DON), deoxynivalenol‐3‐glucoside, fusarenon‐X, 3‐ and 15‐acetyldeoxynivalenol, HT‐2 and T‐2 toxins and zearalenone, in various barley cultivars harvested in 2005–2008 was monitored. The impact of weather, locality, fungicide treatment and barley cultivar (hulless or covered) on contamination was evaluated. The transfer of these mycotoxins into malt was assessed. RESULTS: The most prevalent toxin was DON, which was found in 83% of samples (maximum level 180 µg kg^?1), while HT‐2 was detected in 62% of samples (maximum level 716 µg kg^?1). Using analysis of covariance, weather was found to be the key factor in all years (P < 0.001). A relationship between cultivar and contamination was confirmed only for HT‐2 (P < 0.001) and T‐2 (P = 0.037), with higher levels of these toxins being observed in hulless cultivars. With the exception of NIV (P = 0.008), no significant relationship was found between fungicide treatment and contamination. No distinct trend regarding DON levels in malt was found, with both decreases and increases occurring. CONCLUSION: The results show an inter‐annual variation in mycotoxin occurrence in barley cultivars as well as differences in contamination of malt produced from fungicide‐treated and untreated barley. Copyright © 2010 Society of Chemical Industry     

Reaction of winter wheat (Triticum aestivum L.) cultivars to infection with Fusarium spp.: mycotoxin contamination in grain and chaff

This study compares the susceptibility of winter wheat (Triticum aestivum L.) cultivars to Fusarium head blight (FHB) and accumulation of mycotoxins in kernels and chaff under different climatic conditions in two locations–Cerekwica near Poznan (Central West Poland) and Sitaniec, near Zamosc, Lublin region (South East Poland). Very high variations were found in the concentrations of mycotoxins (zearalenone, ZEA; nivalenol, NIV; deoxynivalenol, DON; moniliformin, MON) in examined fractions: Fusarium-damaged kernels (FDK) and healthy looking kernels (HLK) and in chaff for individual cultivars in both locations. In most cases, significantly higher concentrations of investigated toxins were recorded in wheat from the area of Lublin than from Poznan (p < 0.05). The highest Fusarium infection rates and mycotoxin biosynthesis levels were observed in the Lublin location, with the percentage of the FDK fraction ranging 8.1–81.6. In this region, ZEA concentration (µg g^?1) after inoculation with F. culmorum and F. graminearum ranged from 0.02–0.48 and 0.32–1.04, respectively. In the Poznan area, the toxin concentrations were considerably lower, ranging 0.01–0.10 and 0.03–0.13 µg g^?1 for both isolates, respectively. The concentration of DON was significantly higher than ZEA or NIV levels. The levels of MON accumulation (µg g^?1) in the FDK fraction were between 0.14 and 1.73 (Poznan area) and ND (not detected) to 2.51 (Lublin area). F. avenaceum infection rate ranged 7–35% in samples where the toxin was detected.     

Relationship between Fusarium spp. diversity and mycotoxin contents of mature grains in southern Belgium

Over a 4-year period (2010–13), a survey aiming at determining the occurrence of Fusarium spp. and their relations to mycotoxins in mature grains took place in southern Belgium. The most prevalent species were F. graminearum, F. avenaceum, F. poae and F. culmorum, with large variations between years and locations. An even proportion of mating type found for F. avenaceum, F. culmorum, F. cerealis and F. tricinctum is usually a sign of ongoing sexual recombination. In contrast, an unbalanced proportion of mating type was found for F. poae and no MAT1-2 allele was present in the F. langsethiae population. Genetic chemotyping indicates a majority of deoxynivalenol (DON)-producing strains in F. culmorum (78%, all 3-ADON producers) and F. graminearum (95%, mostly 15-ADON producers), while all F. cerealis strains belong to the nivalenol (NIV) chemotype. Between 2011 and 2013, DON, NIV, enniatins (ENNs) and moniliformin (MON) were found in each field in various concentrations. By comparison, beauvericin (BEA) was scarcely detected and T-2 toxin, zearalenone and α- and β-zearalenols were never detected. Principal component analysis revealed correlations of DON with F. graminearum, ENNs and MON with F. avenaceum and NIV with F. culmorum, F. cerealis and F. poae. BEA was associated with the presence of F. tricinctum and, to a lesser extent, with the presence of F. poae. The use of genetic chemotype data revealed that DON concentrations were mostly influenced by DON-producing strains of F. graminearum and F. culmorum, whereas the concentrations of NIV were influenced by the number of NIV-producing strains of both species added to the number of F. cerealis and F. poae strains. This study emphasises the need to pay attention to less-studied Fusarium spp. for future Fusarium head blight management strategies, as they commonly co-occur in the field and are associated with a broad spectrum of mycotoxins.     

Natural deoxynivalenol occurrence and genotype and chemotype determination of a field population of the Fusarium graminearum complex associated with soybean in Argentina

Soybean (Glycine max L.), the main source of protein throughout the world, is used both as a food and a feedstuff. Currently, limited information about the occurrence of Fusarium species and mycotoxins in soybean grain and by-products is available. The aims of the present study were: (1) to identify toxigenic Fusarium species associated with soybean during crop reproductive stages; (2) to determine the occurrence of deoxynivalenol (DON) and nivalenol (NIV) in soybean seeds; (3) to determine the genotype and chemotype of selected Fg complex strains using molecular and chemical analysis, respectively; and (4) to characterize the strains using AFLP_s markers. One soybean field located at Córdoba Province, Argentina, was monitored and samples of soybean tissue were harvested at three reproductive stages: flowering (R2), full seed (R6) and full maturity (R8). A total of 389 Fusarium strains F. equiseti (40%) was the most frequently species recovered followed by F. semitectum (27%) and F. graminearum (Fg) (11%). From the 40 soybean samples analysed, only two presented detectable DON levels. Based on DON occurrence on soybean seeds at ripening stages, the toxigenic ability of Fg complex strains isolated from soybean seeds, pods and flowers were analysed. The trichothecene genotype was determined by a multiplex PCR using primers based on Tri3, Tri5 and Tri7 toxin genes and then the chemotype was verified by chemical analysis. Most Fg complex strains showed 15-ADON genotype and five strains presented a DON/NIV; these also produced both toxins under in vitro culture. Neither the NIV nor the 3-ADON genotypes were detected among the members of the population evaluated. All the 15-ADON genotype strains were characterized as F. graminearum sensu stricto (lineage 7), while the strains presented a DON/NIV genotype were characterized as F. meridionale (lineage 2). The present study contributes new information on the occurrence of Fusarium species and trichothecenes toxins on soybean at the pre-harvest stages. Also, this is the first report on the chemotype, genotype and lineages among Fg complex isolated from soybean.     

Less Fusarium infestation and mycotoxin contamination in organic than in conventional cereals

A total of 602 samples of cereals, consisting of organically and conventionally produced barley, oats and wheat, were collected at harvest during 2002–2004 in Norway. Organic and conventional cereals were sampled in comparable numbers regarding cereal species, localisation and harvest time, and analysed for Fusarium mould and mycotoxins. Fusarium infestation and mycotoxin content were dependent on cereal species and varied year-by-year. However, in all cereal species, Fusarium infestation and levels of important mycotoxins were significantly lower when grown organically than conventionally. Concerning the most toxic trichothecenes, HT-2 and T-2 toxin, lower concentrations were found in organic oats and barley. Wheat was not contaminated by HT-2 and T-2, but lower concentrations of deoxynivalenol (DON) and moniliformin (MON) were found when organically produced. For mycotoxins considered to constitute the main risk to humans and animals in Norwegian cereals, i.e. HT-2 in oats and DON in oats and wheat, the median figures (mean levels in brackets) were as follows: HT-2 in organic and conventional oats were <20 (80) and 62 (117) µg/kg, DON in organic and conventional oats were 24 (114) and 36 (426) µg/kg, and DON in organic and conventional wheat were 29 (86) and 51 (170) µg/kg, respectively. Concentrations of HT-2 and T-2 in the samples were strongly correlated (r = 0.94). Other mycotoxins did not show a significant correlation to each other. Both HT-2 and T-2 concentrations were significantly correlated with infestation of F. langsethiae (r = 0.65 and r = 0.60, respectively). Concentrations of DON were significantly correlated with F. graminearum infestation (r = 0.61). Furthermore, nivalenol (NIV) was significantly correlated with infestation of F. poae (r = 0.55) and MON with F. avenaceum (r = 0.37). As lower Fusarium infestation and mycotoxin levels were found in organic cereals, factors related to agricultural practice may reduce the risk of contamination with Fusarium mycotoxins. Studies of these issues will be presented separately.     

Fusarium mycotoxins in cereals harvested from Hungarian fields

The Fusarium mycotoxins deoxynivalenol (DON), zearalenone (ZEN) and T-2 frequently contaminate grain crops in Middle and Eastern Europe. In this survey, 116 cereal samples (maize, wheat, barley and oat) were examined for DON, ZEN and T-2 mycotoxins. Samples were collected from different areas in two Hungarian regions (North and South Transdanubia). The method of analysis was indirect competitive ELISA. Maize was the most contaminated grain regarding DON (86%), ZEN (41%) and T-2 (55%) toxins. The average results of the deoxynivalenol and zearalenone tests of maize proved to be significantly higher than those of barley or oat. DON was the most represented Fusarium mycotoxin followed by T-2 and ZEN. The examination of these mycotoxins would be necessary at a larger scale as to re-evaluate permissible levels, so increase of the monitoring programme would be advisable for the future.     

Genotyping and phenotyping of Fusarium graminearum isolates from Germany related to their mycotoxin biosynthesis

Fusarium graminearum is the most important pathogen causing Fusarium head blight (FHB) of small cereal grains worldwide responsible for quantitative and qualitative yield losses. The presence in crops is often associated with mycotoxin contamination of foodstuff limiting its use for human and animal consumption. A collection of isolates of F. graminearum from Germany was characterized genetically and chemically for their potential to produce the B trichothecenes deoxynivalenol (DON) and nivalenol (NIV). Molecular methods with eight PCR assays were implemented based on functional Tri7 and Tri13 genes and on the tri5-tri6 intergenic region to differentiate between chemotaxonomic groups DON and NIV, resulting in a marked majority (61/63) of DON chemotypes. Mycotoxins produced on rice kernels were quantified by means of LC-MSMS including DON, NIV, 3-acetyl-DON (3-ADON), 15-acetyl-DON (15-ADON), DON-3-glucoside, fusarenon X, as well as zearalenone; all of them proving to be present in high concentration among the isolates. All DON-chemotype isolates also produced lower amounts of NIV with the amount being positively correlated (R² = 0.89) to the DON amount. 15-ADON and 3-ADON are reported to be produced simultaneously by the isolates, the former dominating over the latter in all but one isolate. Fungal biomass, was quantified via ergosterol amount on rice. It was used to calculate specific mycotoxin production per biomass of isolates, ranging from 0.104 to 1.815 mg DON mg-1 ergosterol, presenting a Gaussian distribution. Genotype and phenotype characterization revealed discrepancies with respect to mycotoxin production potential of the fungi, i.e. isolates from one chemotype were able to produce mycotoxins from other chemotypes in considerable amounts.     

Updated survey of Fusarium species and toxins in Finnish cereal grains

The aim of the project was to produce updated information during 2005–14 on the Fusarium species found in Finnish cereal grains, and the toxins produced by them, as the last comprehensive survey study of Fusarium species and their toxins in Finland was carried out at the turn of the 1960s and the 1970s. Another aim was to use the latest molecular and chemical methods to investigate the occurrence and correlation of Fusarium species and their mycotoxins in Finland. The most common Fusarium species found in Finland in the FinMyco project 2005 and 2006 were F. avenaceum, F. culmorum, F. graminearum, F. poae, F. sporotrichioides and F. langsethiae. F. avenaceum was the most dominant species in barley, spring wheat and oat samples. The occurrence of F. culmorum and F. graminearum was high in oats and barley. Infection by Fusarium fungi was the lowest in winter cereal grains. The incidence of Fusarium species in 2005 was much higher than in 2006 due to weather conditions. F. langsethiae has become much more common in Finland since 2001. F. graminearum has also risen in the order of importance. A highly significant correlation was found between Fusarium graminearum DNA and deoxynivalenol (DON) levels in Finnish oats, barley and wheat. When comparing the FinMyco data in 2005–06 with the results of the Finnish safety monitoring programme for 2005–14, spring cereals were noted as being more susceptible to infection by Fusarium fungi and the formation of toxins. The contents of T-2 and HT-2 toxins and the frequency of exceptionally high DON concentrations all increased in Finland during 2005–14. Beauvericin (BEA), enniatins (ENNs) and moniliformin (MON) were also very common contaminants of Finnish grains in 2005–06. Climate change is leading to warmer weather, and this may indicate more changes in Finnish Fusarium mycobiota and toxin contents and profiles in the near future.     

Deoxynivalenol and zearalenone in Fusarium-contaminated wheat in Mexico City

Fusarium spp. invasion causes head blight, a destructive disease in the world's main wheat-growing areas, and deoxynivalenol (DON) and zearalenone (ZEA) contamination in cereal-based products. No data are available on the relationship between Fusarium spp. on commercial wheat samples in Mexico City and the presence of mycotoxins. A total of 30 wheat samples were subject to a PCR method involving genes of the trichothecene and zearalenone biosynthesis pathways to detect the presence of Fusarium. Detection and quantification of DON and ZEA was performed using liquid chromatography coupled to UV detection. PCR indicated the presence of the Tri5 and PKS4 genes in 16.7 and 23.3% of samples, respectively. DON and ZEA contamination was found in 51.2 and 71.4% of samples, respectively, where a positive amplification was obtained. This work presents up-to-date information on mycotoxin contamination in Mexico, where improved contamination/exposure data and firm control/monitoring measures are needed.     

Analysis of deoxynivalenol and deoxynivalenol-3-glucosides content in Canadian spring wheat cultivars inoculated with Fusarium graminearum

Contamination of wheat grains with Fusarium mycotoxins and their modified forms is an important issue in wheat industry. The objective of this study was to analyse the deoxynivalenol (DON) and deoxynivalenol-3-glucosides (D3G) content in Canadian spring wheat cultivars grown in two locations, inoculated with a mixture of 3-acetyldeoxynivalenol (3-ADON)-producing Fusarium graminearum strains and a mixture of 15-acetlyldeoxynivalenol (15-ADON)-producing F. graminearum strains. According to the analysis of variance, significant differences were observed among the cultivars for Fusarium head blight (FHB) disease index, Fusarium-damaged kernel percentage (%FDK), DON content and D3G content. When the effect of chemotype was considered, significant differences were observed for FHB disease index, FDK percentage and DON content. The D3G content and D3G/DON ratio were not significantly different between the chemotypes, except for D3G content at the Winnipeg location. The Pearson correlation coefficient between DON and D3G was 0.84 and 0.77 at Winnipeg and Carman respectively. The highest D3G/DON ratio was observed in cultivars Carberry (44%) in Carman and CDC Kernen (63.8%) in Winnipeg. The susceptible cultivars showed lower D3G/DON ratio compared with the cultivars rated as moderately resistant and intermediate. The current study indicated that Canadian spring cultivars produce D3G upon Fusarium infection.     

Transfer of Fusarium mycotoxins and ‘masked’ deoxynivalenol (deoxynivalenol-3-glucoside) from field barley through malt to beer

The fate of five Fusarium toxins — deoxynivalenol (DON), sum of 15- and 3-acetyl-deoxynivalenol (ADONs), HT-2 toxin (HT-2) representing the main trichothecenes and zearalenone (ZON) during the malting and brewing processes — was investigated. In addition to these ‘free’ mycotoxins, the occurrence of deoxynivalenol-3-glucoside (DON-3-Glc) was monitored for the first time in a beer production chain (currently, only DON and ZON are regulated). Two batches of barley, naturally infected and artificially inoculated with Fusarium spp. during the time of flowering, were used as a raw material for processing experiments. A highly sensitive procedure employing high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was validated for the analysis of ‘free’ Fusarium mycotoxins and DON-conjugate in all types of matrices. The method was also able to detect nivalenol (NIV), fusarenon-X (FUS-X) and T-2 toxin (T-2); nevertheless, none of these toxins was found in any of the samples. While steeping of barley grains (the first step in the malting process) apparently reduced Fusarium mycotoxin levels to below their quantification limits (5–10 µg kg^?1), their successive accumulation occurred during germination. In malt, the content of monitored mycotoxins was higher compared with the original barley. The most significant increase was found for DON-3-Glc. During the brewing process, significant further increases in levels occurred. Concentrations of this ‘masked’ DON in final beers exceeded ‘free’ DON, while in malt grists this trichothecene was the most abundant, with the DON/DON-3-Glc ratio being approximately 5:1 in both sample series. When calculating mass balance, no significant changes were observed during brewing for ADONs. The content of DON and ZON slightly decreased by a maximum of 30%. Only traces of HT-2 were detected in some processing intermediates (wort after trub removal and green beer).     

Trichothecene genotypes and chemotypes in Fusarium graminearum strains isolated from wheat in Argentina

Argentina is the fourth largest exporter of wheat in the world. The main pathogen associated with Fusarium Head Blight (FHB) of wheat in Argentina is Fusarium graminearum lineage 7 also termed F. graminearum sensu stricto in the F. graminearum species complex, which can produce the Type B trichothecenes, usually deoxynivalenol (DON) and its acetylated forms (3-ADON and 15-ADON) or nivalenol (NIV). We used a multiplex PCR assay of Tri3, Tri7, and Tri13 to determine the trichothecene genotype of 116 strains F. graminearum collected from three locations in Argentina and then verified the chemotype by chemical analysis. PCR assays and chemical analyses gave the same results for all strains that produced trichothecenes. Most strains (> 92%) had the 15-ADON genotype, with the remaining strains having the DON/NIV genotype. We observed neither the NIV nor the 3-ADON genotypes amongst the strains evaluated. The nine strains with the DON/NIV genotype produced DON when analyzed chemically. Thus, the Argentinean populations of F. graminearum are similar to those from wheat elsewhere in the world, in that all the strains produced DON/15-ADON and belong to lineage 7. However approximately 8% of the strains tested were incorrectly diagnosed as DON/NIV producers with the current multiplex PCR and were only DON producers by chemical analysis.     

Production of mycotoxins byFusarium species isolated in Germany 2. Time course of deoxynivalenol and 3-acetyldeoxynivalenol formation byFusarium graminearum in different liquid media

Summary Several semisynthetic liquid media were examined for the large-scale production of deoxynivalenol (DON) und 3-acetyldeoxynivalenol (AcDON) byFusarium graminearum 183. Only in three of the eight media used could high toxin yields of DON and AcDON be detected. The maximum levels of DON in a medium according to Miller were 3 mg/l and of AcDON 32 mg/1. In glucose-yeast extract-peptone (GYEP) medium containing 1 % glucose, the AcDON concentrations reached 33 mg/1 and the DON yields were 19 mg/1. In a rice flour liquid medium, however, the mean levels of AcDON and DON increased to 170 mg/l and 9 mg/l, respectively. The maximum amounts observed were 480 mg/1 for AcDON and 65 mg/1 for DON. The addition of trifluoracetic acid sodium salt or malonic acid, which are suggested to cause an accumulation of acetyl-CoA by inhibiting enzymes of the tricarboxylic acid cycle, did not stimulate the toxin formation.

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Bildung von Mykotoxinen durch in Deutschland isolierteFusarium-Arten. 2. Zeitverlauf der Deoxynivalenol und 3-Acetyldeoxynivalenolbildung durchFusarium graminearum in verschiedenen Flüssigmedien

Zusammenfassung Zur Herstellung präparativer Mengen von Deoxynivalenol (DON) und 3-Acetyldeoxynivalenol (AcDON) durchFusarium graminearum 183 wurden verschiedene semisynthetische Flüssigme-dien untersucht. Nur in drei von acht eingesetzten Methen konnten hohe Toxingehalte an DON und AcDON nachgewiesen werden. Im Medium nach Miller betrugen die maximal erreichten Konzentrationen von DON 3 mg/1, von AcDON 32 mg/1. In GYEP-Medium mit einem Glucosegehalt von 1 % stieg die AcDON Konzentration bis 33 mg/1, die von DON bis 19 mg/1 an. Dagegen konnten in einem auf Reismehl basierenden Flüssigmedium durchschnittlich 170 mg/l AcDON und 9 mg/1 DON und maximal 480 mg/l AcDON und 65 mg/l DON erzielt werden. Der Zusatz von Natriumtrifluoracetat oder Malonsäure, die aufgrund einer Hemmung von Enzymen des Citratcyclus zu einer Anreicherung von Acetyl-CoA und damit zur vermehrten Bildung von Sekundärmetaboliten führen sollten, brachte keine Steigerung der Toxinbildung.