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ABSTRACT This study explores the dynamics of immune gene expression, ruminal metabolome, and gut microbiota in cows due to the duration of high-grain feeding, shedding light on host response and microbial dynamics in parallel. Cows consumed forage for a week, then gradually transitioned to a high-grain diet, which they consumed for 4 weeks. Immune response was evaluated in ruminal papillae by expression of genes related to the nuclear factor-kappaB (NFkB) pathway and correlated with the microbiota. Rumen metabolome was evaluated with high-performance liquid chromatography coupled with mass spectrometry and anion-exchange chromatography. Rumen and fecal microbiota were evaluated with 16S rRNA gene amplicon sequencing. In the rumen, expression of inflammation-associated genes increased with the duration on high grain, indicating activation of pro-inflammatory cascades; microbial diversity decreased with a high-grain diet but stabilized after week 3 on high grain. Changes in microbial relative abundance and metabolite enrichment were observed throughout the 4 weeks on high grain, with increments in propionogenic taxa (i.e., Succinivibrionaceae). Metabolite enrichment analysis showed that at the start of high-grain feeding, simple carbohydrates were enriched; then, these were substituted by their fermentation products. There were correlations between certain ruminal bacterial taxa (i.e., Ruminococcaceae UCG-005) and expression of genes of the NFkB pathway, suggesting the influence of these taxa on host immune response. In feces, microbial diversity and several Ruminococcaceae members initially declined but recovered by weeks 3 and 4. Overall, despite the stabilization of microbial diversity, changes in microbial relative abundance and proinflammatory genes were observed throughout high-grain feeding, suggesting that cows need more than 4 weeks to fully adjust once consuming a high-grain diet.IMPORTANCEDespite the stepwise diet transition typically assumed to serve for animal adaptation, expression of signaling receptors, mediators, and downstream targets of nuclear factor-kappaB pathway were found throughout the 4 weeks on high grain, which correlated with changes in the rumen microbial profile. In addition, although microbial diversity recovered in the feces and stabilized in the rumen in week 3 on high grain, we observed changes in microbial relative abundance throughout the 4 weeks on high grain, suggesting that cows need more than 4 weeks to adjust once consuming this diet. Findings are particularly important to consider when planning experiments involving dietary changes.

Fusarium verticillioides produces fumonisins, which are mycotoxins inhibiting sphingolipid biosynthesis in humans, animals, and other eukaryotes. Fumonisins are presumed virulence factors of plant pathogens, but may also play a role in interactions between competing fungi. We observed higher resistance to added fumonisin B1 (FB1) in fumonisin-producing Fusarium verticillioides than in nonproducing F. graminearum, and likewise between isolates of Aspergillus and Alternaria differing in production of sphinganine-analog toxins. It has been reported that in F. verticillioides, ceramide synthase encoded in the fumonisin biosynthetic gene cluster is responsible for self-resistance. We reinvestigated the role of FUM17 and FUM18 by generating a double mutant strain in a fum1 background. Nearly unchanged resistance to added FB1 was observed compared to the parental fum1 strain. A recently developed fumonisin-sensitive baker’s yeast strain allowed for the testing of candidate ceramide synthases by heterologous expression. The overexpression of the yeast LAC1 gene, but not LAG1, increased fumonisin resistance. High-level resistance was conferred by FUM18, but not by FUM17. Likewise, strong resistance to FB1 was caused by overexpression of the presumed F. verticillioides “housekeeping” ceramide synthases CER1, CER2, and CER3, located outside the fumonisin cluster, indicating that F. verticillioides possesses a redundant set of insensitive targets as a self-resistance mechanism.

The use of lactic acid bacteria as a low-cost sustainable management tool to prevent further build-up of Fusarium mycotoxins during grain storage is increasingly propagated. It has been reported that even deoxynivalenol contamination already formed in the field can be reduced by such treatment in unknown ways. An enigmatic deoxynivalenol derivative, 3-lactyl-deoxynivalenol, has been reported already in 1982 as the toxic principle of Fusarium-infected barley from China, but very little is known about this metabolite. Here, we show that the enzymatic machinery of Fusarium graminearum is sufficient for its biosynthesis. Similarly, when challenged with propionic acid, F. graminearum can form a novel modified mycotoxin, 3-propionyl-deoxynivalenol. Lactic acid and propionic acid are first converted into lactyl-CoA and propionyl-CoA, respectively. These acyl-CoA derivatives can subsequently be used by the 3-O-acyltransferase encoded by TRI101. We expressed the respective genes in E. coli and utilized the affinity-purified proteins for enzymatic synthesis of the reference substances 3-lactyl- and 3-propionyl-deoxynivalenol. The structures of the purified compounds were confirmed by nuclear magnetic resonance spectroscopy. Preliminary toxicological assessment using in vitro translation assays indicated residual toxicity, most likely due to reactivation of deoxynivalenol by de-acylation. In conclusion, this study reports a method to synthesize 3-lactyl- and 3-propionyl-deoxynivalenol reference substances, which will be highly useful to determine occurrences of these acylated deoxynivalenol-derivatives in cereal samples and to perform more detailed studies to evaluate their toxicological relevance.

Trichothecenes produced by Fusarium species are commonly detected in oats. However, the ratios of the concentrations of free trichothecenes and their conjugates and how they are impacted by different interacting environmental conditions are not well documented. This study aims to examine the effect of water activity (0.95 and 0.98 aw) and temperature (20 and 25 °C) stress on the production of T-2 and HT-2 toxins, deoxynivalenol and their conjugates, as well as diacetoxyscirpenol (DAS). Multiple mycotoxins were detected using liquid chromatography–tandem mass spectrometry from 64 contaminated oat samples. The highest concentrations of HT-2-glucoside (HT-2-Glc) were observed at 0.98 aw and 20 °C, and were higher than other type A trichothecenes in the natural oats’ treatments. However, no statistical differences were found between the mean concentrations of HT-2-Glc and HT-2 toxins in all storage conditions analysed. DAS concentrations were generally low and highest at 0.95 aw and 20 °C, while deoxynivalenol-3-glucoside levels were highest at 0.98 aw and 20 °C in the naturally contaminated oats. Emerging mycotoxins such as beauvericin, moniliformin, and enniatins mostly increased with a rise in water activity and temperature in the naturally contaminated oats treatment. This study reinforces the importance of storage aw and temperature conditions in the high risk of free and modified toxin contamination of small cereal grains.

Fusarium is a genus that mostly consists of plant pathogenic fungi which are able to produce a broad range of toxic secondary metabolites. In this study, we focus on a type A trichothecene-producing isolate (15-39) of Fusarium sporotrichioides from Lower Austria. We assessed the secondary metabolite profile and optimized the toxin production conditions on autoclaved rice and found that in addition to large amounts of T-2 and HT-2 toxins, this strain was able to produce HT-2-glucoside. The optimal conditions for the production of T-2 toxin, HT-2 toxin, and HT-2-glucoside on autoclaved rice were incubation at 12 °C under constant light for four weeks, darkness at 30 °C for two weeks, and constant light for three weeks at 20 °C, respectively. The HT-2-glucoside was purified, and the structure elucidation by NMR revealed a mixture of two alpha-glucosides, presumably HT-2-3-O-alpha-glucoside and HT-2-4-O-alpha-glucoside. The efforts to separate the two compounds by HPLC were unsuccessful. No hydrolysis was observed with two the alpha-glucosidases or with human salivary amylase and Saccharomyces cerevisiae maltase. We propose that the two HT-2-alpha-glucosides are not formed by a glucosyltransferase as they are in plants, but by a trans-glycosylating alpha-glucosidase expressed by the fungus on the starch-containing rice medium.

Microbial composition and activity in the gastrointestinal tract (GIT) of cattle has important implications for animal health and welfare, driving the focus of research toward ways to modify their function and abundance. However, our understanding of microbial adaption to nutritional changes remains limited. The aim of this study was to examine the progressive mechanisms of adaptation in the rumen and hindgut of cattle receiving increasing amounts of starch with or without dietary supplementation of a blended phytogenic feed additive (PFA; containing menthol, thymol and eugenol). We used 16S rRNA gene amplicon sequencing to assess the microbial composition and predicted metabolic pathways in ruminal solid and liquid digesta, and feces. Furthermore, we employed targeted liquid chromatography-mass spectrometry methods to evaluate rumen fluid metabolites. Results indicated a rapid microbial adaptation to diet change, starting on the second day of starch feeding for the particle associated rumen liquid (PARL) microbes. Solid rumen digesta- and feces-associated microbes started changing from the following day. The PARL niche was the most responsive to dietary changes, with the highest number of taxa and predicted pathways affected by the increase in starch intake, as well as by the phytogenic supplementation. Despite the differences in the microbial composition and metabolic potential of the different GIT niches, all showed similar changes toward carbohydrate metabolism. Metabolite measurement confirmed the high prevalence of glucose and volatile fatty acids (VFAs) in the rumen due to the increased substrate availability and metabolic activity of the microbiota. Families Prevotellaceae, Ruminococcaceae and Lachnospiraceae were found to be positively correlated with carbohydrate metabolism, with the latter two showing wide-ranging predicted metabolic capabilities. Phytogenic supplementation affected low abundant taxa and demonstrated the potential to prevent unwanted implications of feeding high-concentrate diet, such as reduction of microbial diversity. The inclusion of 50% concentrate in the diet caused a major shift in microbial composition and activity in the GIT of cattle. This study demonstrated the ability of microorganisms in various GIT niches to adjust differentially, yet rapidly, to changing dietary conditions, and revealed the potential beneficial effects of supplementation with a PFA during dietary adaptation.

Data on the evolution of blood metabolites and metabolic markers in neonatal piglets are scarce, although this information is vital to detect physiological aberrations from normal development. We aimed to characterize age- and nutrition-related changes in the plasma metabolome and serum biochemistry of suckling and newly weaned piglets and assess metabolite patterns as physiological markers for the two phases. In two replicate batches (n = 10 litters/group), piglets either received sow milk alone or were additionally offered creep feed from day 10 until weaning (day 28). Blood was collected from one piglet/litter on days 7, 14, 21, 28, 31 and 35 of life, totaling five females and five males/group/day. Signature feature ranking identified plasma triglycerides (TG) as discriminative for age and nutrition during the suckling phase. Influential TG 20:4_36:5, TG 17:0_34:2 and TG 18:2_38:6 were higher in creep-fed piglets on days 14, 21 and 28 of life, respectively, compared to only sow milk-fed piglets. Metabolites belonging to pathways within histidine, D-glutamine and D-glutamate metabolism as well as hippuric acid were distinctive for the postweaning compared to the suckling period. In conclusion, plasma lipid profiles especially corresponded to the type of nutrition in the suckling phase and showed a strong weaning effect.

Ergosterol has traditionally been used as a proxy to estimate fungal biomass as it is almost exclusively found in fungal lipid membranes. Ergosterol determination has been mostly used for fungal samples from terrestrial, freshwater, salt marsh- and mangrove-dominated environments or to describe fungal degradation of plant matter. In the open ocean, however, the expected concentrations of ergosterol are orders of magnitude lower than in terrestrial or macrophyte-dominated coastal systems. Consequently, the fungal biomass in the open ocean remains largely unknown. Recent evidence based on microscopy and -omics techniques suggests, however, that fungi contribute substantially to the microbial biomass in the oceanic water column, highlighting the need to accurately determine fungal biomass in the open ocean. We performed ergosterol extractions of an oceanic fungal isolate (Rhodotorula sphaerocarpa) with biomass concentrations varying over nine orders of magnitude. While after the initial chloroform-methanol extraction ~87% of the ergosterol was recovered, a second extraction recovered an additional ~10%. Testing this extraction method on samples collected from the open Atlantic Ocean, we successfully determined ergosterol concentrations as low as 0.12 pM. Thus, this highly sensitive method is well suited for measuring fungal biomass from open ocean waters, including deep-sea environments.

The Fusarium mycotoxin deoxynivalenol (DON) is a common contaminant of cereals and is often co-occurring with its modified forms DON-3-glucoside (D3G), 3-acetyl-DON (3ADON) or 15-acetyl-DON (15ADON). A stable-isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) based method for their determination in cereals was developed and validated for maize. Therefore, 13C-labelled D3G was enzymatically produced using 13C-DON and [13C6Glc]-sucrose and used as an internal standard (IS) for D3G, while uniformly 13C labelled IS was used for the other mycotoxins. Baseline separation was achieved for the critical peak pair DON/D3G, while 3ADON/15ADON could not be fully baseline separated after testing various reversed phase, fluorinated phase and chiral LC columns. After grinding, weighing and extracting the cereal samples, the raw extract was centrifuged and a mixture of the four 13C-labelled ISs was added directly in a microinsert vial. The subsequent analytical run took 7 min, followed by negative electrospray ionization and selected reaction monitoring on a triple quadrupole MS. Maize was used as a complex cereal model matrix for validation. The use of the IS corrected the occurring matrix effects efficiently from 76 to 98% for D3G, from 86 to 103% for DON, from 68 to 100% for 15ADON and from 63 to 96% for 3ADON.

The emerging mycotoxin fusaproliferin is produced by Fusarium proliferatum and other related Fusarium species. Several fungi from other taxonomic groups were also reported to produce fusaproliferin or the deacetylated derivative, known as siccanol or terpestacin. Here, we describe the identification and functional characterization of the Fusarium proliferatum genes encoding the fusaproliferin biosynthetic enzymes: a terpenoid synthase, two cytochrome P450s, a FAD-oxidase and an acetyltransferase. With the exception of one gene encoding a CYP450 (FUP2, FPRN_05484), knock-out mutants of the candidate genes could be generated, and the production of fusaproliferin and intermediates was tested by LC-MS/MS. Inactivation of the FUP1 (FPRN_05485) terpenoid synthase gene led to complete loss of fusaproliferin production. Disruption of a putative FAD-oxidase (FUP4, FPRN_05486) did not only affect oxidation of preterpestacin III to terpestacin, but also of new side products (11-oxo-preterpstacin and terpestacin aldehyde). In the knock-out strains lacking the predicted acetyltransferase (FUP5, FPRN_05487) fusaproliferin was no longer formed, but terpestacin was found at elevated levels. A model for the biosynthesis of fusaproliferin and of novel derivatives found in mutants is presented.

The mycotoxin zearalenone (ZEN) is produced by many plant pathogenic Fusarium species. It is well known for its estrogenic activity in humans and animals, but whether ZEN has a role in plant–pathogen interaction and which process it is targeting in planta was so far unclear. We found that treatment of Arabidopsis thaliana seedlings with ZEN induced transcription of the AtHSP90.1 gene. This heat shock protein (HSP) plays an important role in plant–pathogen interaction, assisting in stability and functionality of various disease resistance gene products. Inhibition of HSP90 ATPase activity impairs functionality. Because HSP90 inhibitors are known to induce HSP90 gene expression and due to the structural similarity with the known HSP90 inhibitor radicicol (RAD), we tested whether ZEN and its phase I metabolites α- and ß-zearalenol are also HSP90 ATPase inhibitors. Indeed, AtHSP90.1 and wheat TaHSP90-2 were inhibited by ZEN and ß-zearalenol, while α-zearalenol was almost inactive. Plants can efficiently glycosylate ZEN and α/ß-zearalenol. We therefore tested whether glucosylation has an effect on the inhibitory activity of these metabolites. Expression of the A. thaliana glucosyltransferase UGT73C6 conferred RAD resistance to a sensitive yeast strain. Glucosylation of RAD, ZEN, and α/ß-zearalenol abolished the in vitro inhibitory activity with recombinant HSP90 purified from Escherichia coli. In conclusion, the mycotoxin ZEN has a very prominent target in plants, HSP90, but it can be inactivated by glycosylation. This may explain why there is little evidence for a virulence function of ZEN in host plants.

Fusarium head blight (FHB), mainly caused by Fusarium graminearum, is a devastating disease of wheat that results in economic losses worldwide. During infection, F. graminearum produces trichothecene mycotoxins, including deoxynivalenol (DON), that increase fungal virulence and reduce grain quality. Transgenic wheat expressing a barley UDP-glucosyltransferase (HvUGT13248) were developed and evaluated for FHB resistance, DON accumulation, and the ability to metabolize DON to the less toxic DON-3-O-glucoside (D3G). Point-inoculation tests in the greenhouse showed that transgenic wheat carrying HvUGT13248 exhibited significantly higher resistance to disease spread in the spike (type II resistance) compared with nontransformed controls. Two transgenic events displayed complete suppression of disease spread in the spikes. Expression of HvUGT13248 in transgenic wheat rapidly and efficiently conjugated DON to D3G, suggesting that the enzymatic rate of DON detoxification translates to type II resistance. Under field conditions, FHB severity was variable; nonetheless, transgenic events showed significantly less-severe disease phenotypes compared with the nontransformed controls. In addition, a seedling assay demonstrated that the transformed plants had a higher tolerance to DON-inhibited root growth than nontransformed plants. These results demonstrate the utility of detoxifying DON as a FHB control strategy in wheat.

Recently, deoxynivalenol-3-sulfate (DON-3-sulfate) was proposed as a major DON metabolite in poultry. In the present work, the first LC-MS/MS based method for determination of DON-3-sulfate, deepoxy-DON-3-sulfate (DOM-3-sulfate), DON, DOM, DON sulfonates 1, 2, 3, and DOM sulfonate 2 in excreta samples of chickens and turkeys was developed and validated. To this end, DOM-3-sulfate was chemically synthesized and characterized by NMR and LC-HR-MS/MS measurements. Application of the method to excreta and chyme samples of four feeding trials with turkeys, chickens, pullets, and roosters confirmed DON-3-sulfate as the major DON metabolite in all poultry species studied. Analogously to DON-3-sulfate, DOM-3-sulfate was formed after oral administration of DOM both in turkeys and in chickens. In addition, pullets and roosters metabolized DON into DOM-3-sulfate. In vitro transcription/translation assays revealed DOM-3-sulfate to be 2000 times less toxic on the ribosome than DON. Biological recoveries of DON and DOM orally administered to broiler chickens, turkeys, and pullets were 74%–106% (chickens), 51%–72% (roosters), and 131%–151% (pullets). In pullets, DON-3-sulfate concentrations increased from jejunum chyme samples to excreta samples by a factor of 60. This result, put into context with earlier studies, indicates fast and efficient absorption of DON between crop and jejunum, conversion to DON-3-sulfate in intestinal mucosa, liver, and possibly kidney, and rapid elimination into excreta via bile and urine.

Climate change is primarily manifested by elevated temperature and carbon dioxide (CO2) levels and is projected to provide suitable cultivation grounds for pests and pathogens in the otherwise unsuitable regions. The impacts of climate change have been predicted in many parts of the world, which could threaten global food safety and food security. The aim of the present work was therefore to examine the interacting effects of water activity (aw) (0.92, 0.95, 0.98 aw), CO2 (400, 800, 1200 ppm) and temperature (30, 35 °C and 30, 33 °C for Fusarium verticillioides and F. graminearum, respectively) on fungal growth and mycotoxin production of acclimatised isolates of F. verticillioides and F. graminearum isolated from maize. To determine fungal growth, the colony diameters were measured on days 1, 3, 5, and 7. The mycotoxins produced were quantified using a quadrupole-time-of-flight mass spectrometer (QTOF-MS) combined with ultra-high-performance liquid chromatography (UHPLC) system. For F. verticillioides, the optimum conditions for growth of fumonisin B1 (FB1), and fumonisin B2 (FB2) were 30 °C + 0.98 aw + 400 ppm CO2. These conditions were also optimum for F. graminearum growth, and zearalenone (ZEA) and deoxynivalenol (DON) production. Since 30 °C and 400 ppm CO2 were the baseline treatments, it was hence concluded that the elevated temperature and CO2 levels tested did not seem to significantly impact fungal growth and mycotoxin production of acclimatised Fusarium isolates. To the best of our knowledge thus far, the present work described for the first time the effects of simulated climate change conditions on fungal growth and mycotoxin production of acclimatised isolates of F. verticillioides and F. graminearum.

Deoxynivalenol (DON) is a protein synthesis inhibitor produced by the Fusarium species, which frequently contaminates grains used for human or animal consumption. We treated a wheat suspension culture with DON or one of its acetylated derivatives, 3-acetyl-DON (3-ADON), 15-acetyl-DON (15-ADON) and 3,15-diacetyl-DON (3,15-diADON), and monitored the metabolization over a course of 96 h. Supernatant and cell extract samples were analyzed using a tailored LC-MS/MS method for the quantification of DON metabolites. We report the formation of tentatively identified DON-15-O-β-D-glucoside (D15G) and of 15-acetyl-DON-3-sulfate (15-ADON3S) as novel deoxynivalenol metabolites in wheat. Furthermore, we found that the recently identified 15-acetyl-DON-3-O-β-D-glucoside (15-ADON3G) is the major metabolite produced after 15-ADON challenge. 3-ADON treatment led to a higher intracellular content of toxic metabolites after six hours compared to all other treatments. 3-ADON was exclusively metabolized into DON before phase II reactions occurred. In contrast, we found that 15-ADON was directly converted into 15-ADON3G and 15-ADON3S in addition to metabolization into deoxynivalenol-3-O-β-D-glucoside (D3G). This study highlights significant differences in the metabolization of DON and its acetylated derivatives.

In the scientific field, there is a progressive awareness about the potential implications of food processing on mycotoxins especially concerning thermal treatments. High temperatures may cause, in fact, transformation or degradation of these compounds. This work is aimed to study the fate of mycotoxins during bakery processing, focusing on deoxynivalenol (DON) and deoxynivalenol-3-glucoside (DON3Glc), along the chain of industrial rusk production. Starting from naturally contaminated bran, we studied how concentrations of DON and DON3Glc are influenced by modifying ingredients and operative conditions. The experiments were performed using statistical Design of Experiment (DoE) schemes to synergistically explore the relationship between mycotoxin reduction and the indicated processing transformation parameters. All samples collected during pilot plant experiments were analyzed with an LC-MS/MS multimycotoxin method. The obtained model shows a good fitting, giving back relevant information in terms of optimization of the industrial production process, in particular suggesting that time and temperature in baking and toasting steps are highly relevant for minimizing mycotoxin level in rusks. A reduction up to 30% for DON and DON3Glc content in the finished product was observed within an acceptable technological range.

Glycosylation is an important plant defense mechanism and conjugates of Fusarium mycotoxins often co-occur with their parent compounds in cereal-based food and feed. In case of deoxynivalenol (DON), deoxynivalenol-3-O-β-D-glucoside (D3G) is the most important masked mycotoxin. The toxicological significance of D3G is not yet fully understood so that it is crucial to obtain this compound in pure and sufficient quantities for toxicological risk assessment and for use as an analytical standard. The aim of this study was the biochemical characterization of a DON-inactivating UDP-glucosyltransferase from rice (OsUGT79) and to investigate its suitability for preparative D3G synthesis. Apparent Michaelis constants (Km) of recombinant OsUGT79 were 0.23 mM DON and 2.2 mM UDP-glucose. Substrate inhibition occurred at DON concentrations above 2 mM (Ki = 24 mM DON), and UDP strongly inhibited the enzyme. Cu2+ and Zn2+ (1 mM) inhibited the enzyme completely. Sucrose synthase AtSUS1 was employed to regenerate UDP-glucose during the glucosylation reaction. With this approach, optimal conversion rates can be obtained at limited concentrations of the costly co-factor UDP-glucose. D3G can now be synthesized in sufficient quantity and purity. Similar strategies may be of interest to produce β-glucosides of other toxins.

The aim of this review is to provide a brief overview of analytical methods used for the determination of deoxynivalenol and its modified forms deoxynivalenol-3-β-D-glucoside, 3-acetyl-deoxynivalenol and 15-acetyl-deoxynivalenol in beer. The analytical methods discussed involve gas chromatography coupled with flame ionization detection, electron capture detection and mass spectrometry as well as liquid chromatography hyphenated to ultra-violet detection and mass spectrometry. Special attention was paid to sample preparation. Immunochemical methods such as enzyme-linked immunosorbent assays (ELISAs) which represent efficient tools for fast screening of beer with no sample purification are also discussed.

Currently beer is booming, mainly due to the steady rise of craft breweries worldwide. Previous surveys for occurrence of mycotoxins in beer, were mainly focussed on industrial produced beer. The present survey reports the presence of mycotoxins in craft beer and how this compares to industrial produced beer. More than 1000 beers were collected from 47 countries, of which 60% were craft beers. A selection of 1000 samples were screened for the presence of aflatoxin B1, ochratoxin A (OTA), zearalenone (ZEN), fumonisins (FBs), T-2 and HT-2 toxins (T-2 and HT-2) and deoxynivalenol (DON) using a mycotoxin 6-plex immunoassay. For confirmatory analysis, a liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed and applied. The 6-plex screening showed discrepancies with the LC-MS/MS analysis, possibly due to matrix interference and/or the presence of unknown mycotoxin metabolites. The major mycotoxins detected were DON and its plant metabolite deoxynivalenol-3-β-D-glucopyranoside (D3G). The 6-plex immunoassay reported the sum of DON and D3G (DON+D3G) contaminations ranging from 10 to 475 μg/L in 406 beers, of which 73% were craft beers. The popular craft beer style imperial stout, had the highest percentage of samples suspected positive (83%) with 29% of all imperial stout beers having DON+D3G contaminations above 100 μg/L. LC-MS/MS analysis showed that industrial pale lagers from Italy and Spain, predominantly contained FBs (3-69 μg/L). Besides FBs, African traditional beers also contained aflatoxins (0.1-1.2 μg/L). The presence of OTA, T-2, HT-2, ZEN, β-zearalenol, 3/15-acetyl-DON, nivalenol and the conjugated mycotoxin zearalenone 14-sulfate were confirmed in some beers. This study shows that in 27 craft beers, DON+D3G concentrations occurred above (or at) the Tolerable Daily Intake (TDI). Exceeding the TDI, may have a health impact. A better control of brewing malts for craft beer, should be put in place to circumvent this potential problem.

Deoxynivalenol (DON), a frequent contaminant of flour, can be partially degraded by baking. It is not clear: (i) How the choice of processing parameter (i.e., ingredients, leavening, and baking conditions) affects DON degradation and thus (ii) how much DON can be degraded during the large-scale industrial production of bakery products. Crackers, biscuits, and bread were produced from naturally contaminated flour using different processing conditions. DON degradation during baking was quantified with the most accurate analytical methodology available for this Fusarium toxin, which is based on liquid chromatography tandem mass spectrometry. Depending on the processing conditions, 0−21%, 4−16%, and 2−5% DON were degraded during the production of crackers, biscuits, and bread, respectively. A higher NaHCO3 concentration, baking time, and baking temperature caused higher DON degradation. NH4HCO3, yeast, vinegar, and sucrose concentration as well as leavening time did not enhance DON degradation. In vitro cell viability assays confirmed that the major degradation product isoDON is considerably less toxic than DON. This proves for the first time that large-scale industrial baking results in partial detoxification of DON, which can be enhanced by process management.

The authors wish to make the following corrections to their paper [...]

Plant small-molecule UDP-glycosyltransferases (UGT) glycosylate a vast number of endogenous substances but also act in detoxification of metabolites produced by plant-pathogenic microorganisms. The ability to inactivate the Fusarium graminearum mycotoxin deoxynivalenol (DON) into DON-3-O-glucoside is crucial for resistance of cereals. We analyzed the UGT gene family of the monocot model species Brachypodium distachyon and functionally characterized two gene clusters containing putative orthologs of previously identified DON-detoxification genes from Arabidopsis thaliana and barley. Analysis of transcription showed that UGT encoded in both clusters are highly inducible by DON and expressed at much higher levels upon infection with a wild-type DON-producing F. graminearum strain compared with infection with a mutant deficient in DON production. Expression of these genes in a toxin-sensitive strain of Saccharomyces cerevisiae revealed that only two B. distachyon UGT encoded by members of a cluster of six genes homologous to the DON-inactivating barley HvUGT13248 were able to convert DON into DON-3-O-glucoside. Also, a single copy gene from Sorghum bicolor orthologous to this cluster and one of three putative orthologs of rice exhibit this ability. Seemingly, the UGT genes undergo rapid evolution and changes in copy number, making it difficult to identify orthologs with conserved substrate specificity.

Trichothecenes are a major group of toxins produced by phytopathogenic fungi, including Fusarium graminearum. Trichothecenes inhibit protein synthesis in eukaryotic cells and are toxicologically relevant mycotoxins for humans and animals. Because they promote plant disease, the role of host responses to trichothecene accumulation is considered to be an important aspect of plant defense and resistance to fungal infection. Our overall objective was to examine the barley response to application of the type B trichothecene deoxynivalenol (DON). We found that DON is diluted by movement from the application site to acropetal and basipetal florets. A susceptible barley genotype converted DON to DON-3-O-glucoside, indicating that UDP-glucosyltransferases capable of detoxifying DON must exist in barley. RNA profiling of DON-treated barley spikes revealed strong upregulation of gene transcripts encoding ABC transporters, UDP-glucosyltransferases, cytochrome P450s, and glutathione-S-transferases. We noted that transcripts encoding cysteine synthases were dramatically induced by DON, and that toxin-sensitive yeast on glutathione- or cysteine-supplemented media or carrying a gene that encodes a cysteine biosynthetic enzyme exhibit DON resistance, suggesting that preventing glutathione depletion by increasing cysteine supply could play a role in ameliorating the impact of DON. Evidence for nonenzymatic formation of DON-glutathione adducts in vitro was found using both liquid chromatography–mass spectrometry and nuclear magnetic resonance analysis, indicating that the formation of DON-glutathione conjugates in vivo may reduce the impact of trichothecenes. Our results indicate that barley exhibits multiple defense mechanisms against trichothecenes.

Resistance to the virulence factor deoxynivalenol (DON) due to formation of DON-3-O-glucoside (D3G) is considered to be an important component of resistance against Fusarium spp. which produce this toxin. Multiple candidate UDP-glycosyltransferase (UGT) genes from different crop plants that are either induced by Fusarium spp. or differentially expressed in cultivars varying in Fusarium disease resistance have been described. However, UGT are encoded by a very large gene family in plants. The study of candidate plant UGT is highly warranted because of the potential relevance for developing Fusarium-spp.-resistant crops. We tested Arabidopsis thaliana genes closely related to a previously identified DON-glucosyltransferase gene by heterologous expression in yeast and showed that gene products with very high sequence similarity can have pronounced differences in detoxification capabilities. We also tested four candidate barley glucosyltransferases, which are highly DON inducible. Upon heterologous expression of full-length cDNAs, only one gene, HvUGT13248, conferred DON resistance. The conjugate D3G accumulated in the supernatant of DON-treated yeast transformants. We also present evidence that the product of the TaUGT3 gene recently proposed to encode a DON-detoxification enzyme of wheat does not protect yeast against DON.

Trichothecene toxins are confirmed or suspected virulence factors of various plant-pathogenic Fusarium species. Plants can detoxify these to a variable extent by glucosylation, a reaction catalyzed by UDP-glucosyltransferases (UGTs). Due to the unavailability of analytical standards for many trichothecene-glucoconjugates, information on such compounds is limited. Here, the previously identified deoxynivalenol-conjugating UGTs HvUGT13248 (barley), OsUGT79 (rice) and Bradi5g03300 (Brachypodium), were expressed in E. coli, affinity purified, and characterized towards their abilities to glucosylate the most relevant type A and B trichothecenes. HvUGT13248, which prefers nivalenol over deoxynivalenol, is also able to conjugate C-4 acetylated trichothecenes (e.g., T-2 toxin) to some degree while OsUGT79 and Bradi5g03300 are completely inactive with C-4 acetylated derivatives. The type A trichothecenes HT-2 toxin and T-2 triol are the kinetically preferred substrates in the case of HvUGT13248 and Bradi5g03300. We glucosylated several trichothecenes with OsUGT79 (HT-2 toxin, T-2 triol) and HvUGT13248 (T-2 toxin, neosolaniol, 4,15-diacetoxyscirpenol, fusarenon X) in the preparative scale. NMR analysis of the purified glucosides showed that exclusively β-D-glucosides were formed regio-selectively at position C-3-OH of the trichothecenes. These synthesized standards can be used to investigate the occurrence and toxicological properties of these modified mycotoxins.

In this study, a total of nine different biotransformation products of the Fusarium mycotoxin deoxynivalenol (DON) formed in wheat during detoxification of the toxin are characterized by liquid chromatography-high resolution mass spectrometry (LC-HRMS). The detected metabolites suggest that DON is conjugated to endogenous metabolites via two major metabolism routes, namely 1) glucosylation (DON-3-glucoside, DON-di-hexoside, 15-acetyl-DON-3-glucoside, DON-malonylglucoside) and 2) glutathione conjugation (DON-S-glutathione, "DON-2H"-S-glutathione, DON-S-cysteinyl-glycine and DON-S-cysteine). Furthermore, conjugation of DON to a putative sugar alcohol (hexitol) was found. A molar mass balance for the cultivar 'Remus' treated with 1 mg DON revealed that under the test conditions approximately 15% of the added DON were transformed into DON-3-glucoside and another 19% were transformed to the remaining eight biotransformation products or irreversibly bound to the plant matrix. Additionally, metabolite abundance was monitored as a function of time for each DON derivative and was established for six DON treated wheat lines (1 mg/ear) differing in resistance quantitative trait loci (QTL) Fhb1 and/or Qfhs.ifa-5A. All cultivars carrying QTL Fhb1 showed similar metabolism kinetics: Formation of DON-Glc was faster, while DON-GSH production was less efficient compared to cultivars which lacked the resistance QTL Fhb1. Moreover, all wheat lines harboring Fhb1 showed significantly elevated D3G/DON abundance ratios.

We investigated the hypothesis that resistance to deoxynivalenol (DON) is a major resistance factor in the Fusarium head blight (FHB) resistance complex of wheat. Ninety-six double haploid lines from a cross between ‘CM-82036’ and ‘Remus’ were examined. The lines were tested for DON resistance after application of the toxin in the ear, and for resistances to initial infection and spread of FHB after artificial inoculation with Fusarium spp. Toxin application to flowering ears induced typical FHB symptoms. Quantitative trait locus (QTL) analyses detected one locus with a major effect on DON resistance (logarithm of odds = 53.1, R2 = 92.6). The DON resistance phenotype was closely associated with an important FHB resistance QTL, Qfhs.ndsu-3BS, which previously was identified as governing resistance to spread of symptoms in the ear. Resistance to the toxin was correlated with resistance to spread of FHB (r = 0.74, P < 0.001). In resistant wheat lines, the applied toxin was converted to DON-3-O-glucoside as the detoxification product. There was a close relation between the DON-3-glucoside/DON ratio and DON resistance in the toxintreated ears (R2 = 0.84). We conclude that resistance to DON is important in the FHB resistance complex and hypothesize that Qfhs.ndsu-3BS either encodes a DON-glucosyltransferase or regulates the expression of such an enzyme.

Based on prior observations that deoxynivalenol (DON) toxicity is sex-dependent, we compared metabolism and clearance of this toxin in male and female mice. Following intraperitoneal challenge with 1 mg/kg bw DON, the dose used in the aforementioned toxicity study, ELISA and LC–MS/MS analyses revealed that by 24 h, most DON and DON metabolites were excreted via urine (49–86%) as compared to feces (1.2–8.3%). Females excreted DON and its principal metabolites (DON-3-, DON-8,15 hemiketal-8-, and iso-DON-8-glucuronides) in urine more rapidly than males. Metabolite concentrations were typically 2 to 4 times higher in the livers and kidneys of males than females from 1 to 4 h after dosing. Trace levels of DON-3-sulfate and DON-15-sulfate were found in urine, liver and kidneys from females but not males. Fecal excretion of DON and DON sulfonates was approximately 2-fold greater in males than females. Finally, decreased DON clearance rates in males could not be explained by glucuronidation activities in liver and kidney microsomes. To summarize, increased sensitivity of male mice to DON’s toxic effects as compared to females corresponds to decreased ability to clear the toxin via urine but did not appear to result from differences in toxin metabolism.

The Fusarium mycotoxin zearalenone (ZEN) can be conjugated with polar molecules, like sugars or sulfates, by plants and fungi. To date, the fate of these modified forms of ZEN has not yet been elucidated in animals. In order to investigate whether ZEN conjugates contribute to the total ZEN exposure of an individual, ZEN (10 µg/kg b.w.) and equimolar amounts of two of its plant metabolites (ZEN-14-O-β-glucoside, ZEN-16-O-β-glucoside) and of one fungal metabolite (ZEN-14-sulfate) were orally administered to four pigs as a single bolus using a repeated measures design. The concentrations of ZEN, its modified forms and its mammalian metabolites ZEN-14-glucuronide, α-zearalenol (α-ZEL) and α-ZEL-14-glucuronide in excreta were analyzed by high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) based methods. The biological recovery of ZEN in urine was 26% ± 10%, the total biological recovery in excreta was 40% ± 8%. Intact ZEN-14-sulfate, ZEN-14-O-β-glucoside and ZEN-16-O-β-glucoside were neither detected in urine nor in feces. After ZEN-14-sulfate application, 19% ± 5% of the administered dose was recovered in urine. In feces, no ZEN metabolites were detected. The total biological recoveries of ZEN-14-O-β-glucoside and ZEN-16-O-β-glucoside in the form of their metabolites in urine were 19% ± 11% and 13% ± 7%, respectively. The total biological recoveries in urine and feces amounted to 48% ± 7% and 34 ± 3%. An explanation for the low biological recoveries could be extensive metabolization by intestinal bacteria to yet unknown metabolites. In summary, ZEN-14-sulfate, ZEN-14-O-β-glucoside, and ZEN-16-O-β-glucoside were completely hydrolyzed in the gastrointestinal tract of swine, thus contributing to the overall toxicity of ZEN.

Zearalenone (ZEN) is an estrogenic mycotoxin occurring in Fusarium-infected cereals. Glucosylation is an important plant defense mechanism and generally reduces the acute toxicity of mycotoxins to humans and animals. Toxicological information about ZEN-glucosides is limited due to the unavailability of larger amounts required for animal studies. HvUGT14077, a recently-validated ZEN-conjugating barley UDP-glucosyltransferase was expressed in Escherichia coli, affinity purified, and characterized. HvUGT14077 possesses high affinity (Km = 3 µM) and catalytic efficiency (kcat/Km = 190 s−1·mM−1) with ZEN. It also efficiently glucosylates the phase-I ZEN-metabolites α-zearalenol and β-zearalenol, with kcat/Km of 40 and 74 s−1·mM−1, respectively. HvUGT14077 catalyzes O-glucosylation at C-14 and C-16 with preference of 14-glucoside synthesis. Furthermore, relatively slow consecutive formation of 14,16-di-glucosides was observed; their structures were tentatively identified by mass spectrometry and for ZEN-14,16-di-glucoside confirmed by nuclear magnetic resonance spectroscopy. Recombinant HvUGT14077 allowed efficient preparative synthesis of ZEN-glucosides, yielding about 90% ZEN-14-glucoside and 10% ZEN-16-glucoside. The yield of ZEN-16-glucoside could be increased to 85% by co-incubation with a β-glucosidase highly selective for ZEN-14-glucoside. Depletion of the co-substrate UDP-glucose was counteracted by a sucrose synthase based regeneration system. This strategy could also be of interest to increase the yield of minor glucosides synthesized by other glucosyltransferases.

Fungal secondary metabolite biosynthesis genes are of major interest due to the pharmacological properties of their products (like mycotoxins and antibiotics). The genome of the plant pathogenic fungus Fusarium graminearum codes for a large number of candidate enzymes involved in secondary metabolite biosynthesis. However, the chemical nature of most enzymatic products of proteins encoded by putative secondary metabolism biosynthetic genes is largely unknown. Based on our analysis we present 67 gene clusters with significant enrichment of predicted secondary metabolism related enzymatic functions. 20 gene clusters with unknown metabolites exhibit strong gene expression correlation in planta and presumably play a role in virulence. Furthermore, the identification of conserved and over-represented putative transcription factor binding sites serves as additional evidence for cluster co-regulation. Orthologous cluster search provided insight into the evolution of secondary metabolism clusters. Some clusters are characteristic for the Fusarium phylum while others show evidence of horizontal gene transfer as orthologs can be found in representatives of the Botrytis or Cochliobolus lineage. The presented candidate clusters provide valuable targets for experimental examination.

The Fusarium mycotoxins HT-2 toxin (HT2) and T-2 toxin (T2) are frequent contaminants in oats. These toxins, but also their plant metabolites, may contribute to toxicological effects. This work describes the use of 13C-assisted liquid chromatography–high-resolution mass spectrometry for the first comprehensive study on the biotransformation of HT2 and T2 in oats. Using this approach, 16 HT2 and 17 T2 metabolites were annotated including novel glycosylated and hydroxylated forms of the toxins, hydrolysis products, and conjugates with acetic acid, putative malic acid, malonic acid, and ferulic acid. Further targeted quantitative analysis was performed to study toxin metabolism over time, as well as toxin and conjugate mobility within non-treated plant tissues. As a result, HT2-3-O-β-d-glucoside was identified as the major detoxification product of both parent toxins, which was rapidly formed (to an extent of 74% in HT2-treated and 48% in T2-treated oats within one day after treatment) and further metabolised. Mobility of the parent toxins appeared to be negligible, while HT2-3-O-β-d-glucoside was partly transported (up to approximately 4%) through panicle side branches and stem. Our findings demonstrate that the presented combination of untargeted and targeted analysis is well suited for the comprehensive elucidation of mycotoxin metabolism in plants.

Trichothecenes are a class of structurally diverse mycotoxins with more than 200 naturally occurring compounds. Previously, a new compound, pentahydroxyscirpene (PHS), was reported as a byproduct of a nivalenol producing Fusarium strain, IFA189. PHS contains a hydroxy group at C-8 instead of the keto group of type B trichothecenes. In this work, we demonstrate that IFA189 belongs to the species Fusarium kyushuense using molecular tools. Production of PHS in vitro was also observed for several isolates of other Fusarium species producing nivalenol. Furthermore, we report the formation of 4-acetyl-PHS by F. kyushuense on inoculated rice. Wheat ears of the variety Remus were infected with IFA189 and the in planta production of PHS was confirmed. Natural occurrence of PHS was verified in barley samples from the Czech Republic using a liquid chromatographic-tandem mass spectrometric method validated for this purpose. Toxicity of PHS to wheat ribosomes was evaluated with a coupled in vitro transcription and translation assay, which showed that PHS inhibits protein biosynthesis slightly less than nivalenol and deoxynivalenol.

Increasing frequencies of 3-acetyl-deoxynivalenol (3-ADON)-producing strains of Fusarium graminearum (3-ADON chemotype) have been reported in North America and Asia. 3-ADON is nearly nontoxic at the level of the ribosomal target and has to be deacetylated to cause inhibition of protein biosynthesis. Plant cells can efficiently remove the acetyl groups of 3-ADON, but the underlying genes are yet unknown. We therefore performed a study of the family of candidate carboxylesterases (CXE) genes of the monocot model plant Brachypodium distachyon. We report the identification and characterization of the first plant enzymes responsible for deacetylation of trichothecene toxins. The product of the BdCXE29 gene efficiently deacetylates T-2 toxin to HT-2 toxin, NX-2 to NX-3, both 3-ADON and 15-acetyl-deoxynivalenol (15-ADON) into deoxynivalenol and, to a lesser degree, also fusarenon X into nivalenol. The BdCXE52 esterase showed lower activity than BdCXE29 when expressed in yeast and accepts 3-ADON, NX-2, 15-ADON and, to a limited extent, fusarenon X as substrates. Expression of these Brachypodium genes in yeast increases the toxicity of 3-ADON, suggesting that highly similar genes existing in crop plants may act as susceptibility factors in Fusarium head blight disease.