Impact of Environmental Conditions on the Concentrations of Trichothecenes, Their Glucosides, and Emerging Fusarium Toxins in Naturally Contaminated, Irradiated, and Fusarium langsethiae Inoculated Oats.

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 sporotrichioides Produces Two HT-2-α-Glucosides on Rice.

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.

Comparison of LC-MS-based methods for the determination of carboxylic acids in animal matrices.

Carboxylic acids (CAs) are key players in human and animal metabolism. As they are hardly retained under reversed-phase liquid chromatography (RP-LC) conditions in their native form, derivatization is an option to make them accessible to RP-LC and simultaneously increase their response for mass spectrometric detection. In this work, two RP-LC tandem mass spectrometry-based methods using aniline or 3-nitrophenylhydrazine (3-NPH) as derivatization agents were compared with respect to several factors including completeness of derivatization, apparent recoveries (RAs) in both cow feces and ruminal fluid, and concentrations obtained in feces and ruminal fluid of cows. Anion exchange chromatography coupled to high-resolution mass spectrometry (AIC-HR-MS) served as reference method. Derivatization efficiencies were close to 100% for 3-NPH derivatization but variable (20-100%) and different in solvent solutions and matrix extracts for aniline derivatization. Likewise, average RAs of 13C-labeled short-chain fatty acids as internal standards were around 100% for 3-NPH derivatization but only 45% for aniline derivatization. Quantification of CAs in feces and ruminal fluid of cows initially fed a forage-only diet and then transitioned to a 65% high-grain diet which yielded similar concentrations for 3-NPH derivatization and AIC-HR-MS, but concentrations determined by aniline derivatization were on average five times lower. For these reasons, derivatization with aniline is not recommended for the quantitative analysis of CAs in animal samples.

Integrated microbiota-host-metabolome approaches reveal adaptive ruminal changes to prolonged high-grain feeding and phytogenic supplementation in cattle.

Diets rich in readily fermentable carbohydrates primarily impact microbial composition and activity, but can also impair the ruminal epithelium barrier function. By combining microbiota, metabolome, and gene expression analysis, we evaluated the impact of feeding a 65% concentrate diet for 4 weeks, with or without a phytogenic feed additive (PFA), on the rumen ecosystem of cattle. The breaking point for rumen health seemed to be the second week of high grain (HG) diet, with a dysbiosis characterized by reduced alpha diversity. While we did not find changes in histological evaluations, genes related with epithelial proliferation (IGF-1, IGF-1R, EGFR, and TBP) and ZO-1 were affected by the HG feeding. Integrative analyses allowed us to define the main drivers of difference for the rumen ecosystem in response to a HG diet, identified as ZO-1, MyD88, and genus Prevotella 1. PFA supplementation reduced the concentration of potentially harmful compounds in the rumen (e.g. dopamine and 5-aminovaleric acid) and increased the tolerance of the epithelium toward the microbiota by altering the expression of TLR-2, IL-6, and IL-10. The particle-associated rumen liquid microbiota showed a quicker adaptation potential to prolonged HG feeding compared to the other microenvironments investigated, especially by the end of the experiment.

Development of a fumonisin-sensitive Saccharomyces cerevisiae indicator strain and utilization for activity testing of candidate detoxification genes.

IMPORTANCE: Fumonisins can cause diseases in animals and humans consuming Fusarium-contaminated food or feed. The search for microbes capable of fumonisin degradation, or for enzymes that can detoxify fumonisins, currently relies primarily on chemical detection methods. Our constructed fumonisin B1-sensitive yeast strain can be used to phenotypically detect detoxification activity and should be useful in screening for novel fumonisin resistance genes and to elucidate fumonisin metabolism and resistance mechanisms in fungi and plants, and thereby, in the long term, help to mitigate the threat of fumonisins in feed and food.

UDP-glucosyltransferase HvUGT13248 confers type II resistance to Fusarium graminearum in barley.

Fusarium head blight (FHB) of barley (Hordeum vulgare) causes yield losses and accumulation of trichothecene mycotoxins (e.g. deoxynivalenol [DON]) in grains. Glucosylation of DON to the nontoxic DON-3-O-glucoside (D3G) is catalyzed by UDP-glucosyltransferases (UGTs), such as barley UGT13248. We explored the natural diversity of UGT13248 in 496 barley accessions and showed that all carried potential functional alleles of UGT13248, as no genotypes showed strongly increased seedling sensitivity to DON. From a TILLING population, we identified 2 mutant alleles (T368I and H369Y) that, based on protein modeling, likely affect the UDP-glucose binding of UGT13248. In DON feeding experiments, DON-to-D3G conversion was strongly reduced in spikes of these mutants compared to controls, and plants overexpressing UGT13248 showed increased resistance to DON and increased DON-to-D3G conversion. Moreover, field-grown plants carrying the T368I or H369Y mutations inoculated with Fusarium graminearum showed increased FHB disease severity and reduced D3G production. Barley is generally considered to have type II resistance that limits the spread of F. graminearum from the infected spikelet to adjacent spikelets. Point inoculation experiments with F. graminearum showed increased infection spread in T368I and H369Y across the spike compared to wild type, while overexpression plants showed decreased spread of FHB symptoms. Confocal microscopy revealed that F. graminearum spread to distant rachis nodes in T368I and H369Y mutants but was arrested at the rachis node of the inoculated spikelet in wild-type plants. Taken together, our data reveal that UGT13248 confers type II resistance to FHB in barley via conjugation of DON to D3G.

Plant-oriented microbiome inoculum modulates age-related maturation of gut-mucosal expression of innate immune and barrier function genes in suckling and weaned piglets.

In the immediate time after weaning, piglets often show symptoms of gut inflammation. The change to a plant-based diet, lack of sow milk, and the resulting novel gut microbiome and metabolite profile in digesta may be causative factors for the observed inflammation. We used the intestinal loop perfusion assay (ILPA) to investigate jejunal and colonic expression of genes for antimicrobial secretion, oxidative stress, barrier function, and inflammatory signaling in suckling and weaned piglets when exposed to "plant-oriented" microbiome (POM) representing postweaning digesta with gut-site specific microbial and metabolite composition. Two serial ILPA were performed in two replicate batches, with 16 piglets preweaning (days 24 to 27) and 16 piglets postweaning (days 38 to 41). Two jejunal and colonic loops were perfused with Krebs-Henseleit buffer (control) or with the respective POM for 2 h. Afterward, RNA was isolated from the loop tissue to determine the relative gene expression. Age-related effects in jejunum included higher expression of genes for antimicrobial secretions and barrier function as well as reduced expression of pattern-recognition receptors post- compared to preweaning (P < 0.05). Age-related effects in the colon comprised downregulation of the expression of pattern-recognition receptors post- compared to preweaning (P < 0.05). Likewise, age reduced the colonic expression of genes encoding for cytokines, antimicrobial secretions, antioxidant enzymes, and tight-junction proteins post- compared to preweaning. Effect of POM in the jejunum comprised an increased the expression of toll-like receptors compared to the control (P < 0.05), demonstrating a specific response to microbial antigens. Similarly, POM administration upregulated the jejunal expression of antioxidant enzymes (P < 0.05). The POM perfusion strongly upregulated the colonic expression of cytokines and altered the expression of barrier function genes, fatty acid receptors and transporters, and antimicrobial secretions (P < 0.05). In conclusion, results indicated that POM signaled via altering the expression of pattern-recognition receptors in the jejunum, which in turn activated the secretory defense and decreased mucosal permeability. In the colon, POM may have acted pro-inflammatory via upregulated cytokine expression. Results are valuable for the formulation of transition feeds for the immediate time after weaning to maintain mucosal immune tolerance towards the novel digesta composition.

After weaning, piglets often show symptoms of gut inflammation and reduced performance. The plant-based diet, lack of sow milk, and the resulting novel gut microbiome and metabolite composition in digesta may be causative. However, the acute response of the gut mucosa when exposed to the novel digesta composition has not been fully elucidated. Here, we used the intestinal loop perfusion assay to characterize the immediate effect of a plant-oriented microbiome inoculum (POM) representing postweaning digesta composition on gene expression related to innate immune pathways and barrier function at the jejunal and colonic mucosa in suckling and weaned piglets. Results showed that the recognition of microbial components and barrier function changed in the jejunal and colonic mucosa from pre- to postweaning, indicating age-related maturation and priming by digesta compounds prior to the intestinal loop perfusion assay. In the jejunum, exposure to POM increased expression of receptors recognizing microbial components. In the colon, POM exposure upregulated the expression of genes for pro-inflammatory cytokines and other components of the first line of defense. Results have implications for the formulation of transition feeds for the immediate time after weaning. Inclusion of bioactive porcine milk components may help maintain mucosal immune tolerance towards the novel digesta composition.

Comparison of chromatographic conditions for the targeted tandem mass spectrometric determination of 354 mammalian metabolites.

Metabolomics is becoming increasingly popular in livestock research, but no single analytical method can cover the entire metabolome. As such, we compared similar and complementary chromatographic methods with respect to analyte coverage and chromatographic properties of mammalian metabolites. We investigated 354 biologically relevant primary metabolites from 19 compound classes including amino acids, bile acids, biogenic amines, carboxylic acids, lipids, nucleotides and sugars. A total of 2063 selected reaction monitoring transitions were optimized on a triple quadrupole mass spectrometer. We then determined the retention profiles and peak parameters of our compounds using an anion exchange chromatography (AIC), three reversed-phase (RP) and three hydrophilic interaction liquid chromatography (HILIC) methods. On average, HILIC methods covered 54% of all metabolites with retention factors >1, while average RP coverage was 41%. In contrast to RP, HILIC methods could also retain polar metabolites such as amino acids and biogenic amines. Carboxylic acids, nucleotides, and sugar related compounds were best separated by AIC or zwitterionic pHILIC with alkaline eluents. Combining two complementary HILIC and RP methods increased the library coverage to 92%. By further including important short chain fatty acids, a combination of HILIC, RP and AIC methods achieved a coverage of 97%. The resulting dataset of LC and MS/MS parameters will facilitate the development of tailor-made quantitative targeted LC-MS/MS methods to investigate the mammalian metabolome.

Creep Feeding and Weaning Influence the Postnatal Evolution of the Plasma Metabolome in Neonatal Piglets.

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.

Exposure to plant-oriented microbiome altered jejunal and colonic innate immune response and barrier function more strongly in suckling than in weaned piglets.

Weaning often leaves the piglet vulnerable to gut dysfunction. Little is known about the acute response of a gut mucosa primed by a milk-oriented microbiome before weaning to a plant-oriented microbiome (POM) after weaning. We evaluated the epithelial structure, secretory response and permeability in the small and large intestines of piglets receiving a milk-based (i.e., preweaning) or plant-based diet (i.e., postweaning) to POM inocula using intestinal loop perfusion assays (ILPA). The POM were prepared from jejunal and colonic digesta of four 7 week-old weaned (day 28 of life) piglets, having gut-site specific microbial and metabolite composition. Two consecutive ILPA were performed in 16 piglets pre- (days 24 to 27) and 16 piglets postweaning (days 38 to 41) in two replicate batches. Two jejunal and colonic loops per piglet were perfused with Krebs-Henseleit buffer (control) or the respective POM. The outflow fluid was analyzed for antimicrobial secretions. Jejunal and colonic loop tissue were collected after each ILPA for histomorphology and electrophysiology using Ussing chambers. ANOVA was performed using the MIXED procedure in SAS. The POM stimulated the secretory response by increasing mucin in the jejunal and colonic outflow by 99.7% and 54.1%, respectively, and jejunal IgA by 19.2%, whereas colonic lysozyme decreased 25.6% compared to the control (P < 0.05). Fittingly, the POM raised the number of goblet cells by 96.7% in jejunal and 56.9% in colonic loops compared to control loops (P < 0.05). The POM further flattened jejunal villi by 18.3% and reduced crypt depth in jejunal and colonic loops by 53.8% and 9.0% compared to the control (P < 0.05); observations typically made postweaning and indicative for mucosal recognition of 'foreign' compounds. The POM altered the jejunal and colonic net ion flux as indicated by 22.7% and 59.2% greater short-circuit current compared to control loops, respectively; the effect being stronger postweaning (P < 0.05). Colonic barrier function improved with age (P < 0.05), whereas POM perfusion compromised the mucosal barrier as suggested by 17.7% and 54.1% greater GT and mucosal-to-serosal flux of fluorescein-isothiocyanate dextran, respectively, compared to the control (P < 0.05). In conclusion, results demonstrated that the preweaning gut epithelium acutely responds to novel compounds in postweaning digesta by upregulating the first line of defense (i.e., mucin and lysozyme secretion) and impairment of the structural integrity.

Creep feed is offered during the suckling period to prepare the piglet's gut for the dietary transition from a milk- to a plant-based diet at weaning. Nevertheless, the discontinuation of sow milk consumption after weaning can lead to disturbed interactions between the host mucosa and the gut microbiota. Little information is available on the immediate mucosal response towards the altered microbial and metabolite composition in digesta. Therefore, the main objective of this study was to evaluate the immediate effect of the exposure of the jejunal and colonic mucosa to a plant-oriented microbiome (POM), prepared from intestinal digesta of weaned pigs, on the mucosal structure, secretory response, and permeability in piglets before and after weaning using the intestinal loop perfusion assay. The perfusion with POM stimulated the host's secretory response, altered the gut structure and decreased the epithelial integrity before and after weaning. Effects were less strong postweaning, indicating that adaptation processes at the gut epithelium occurred from pre- to postweaning which increased the tolerance towards the POM inoculum.

Progressive microbial adaptation of the bovine rumen and hindgut in response to a step-wise increase in dietary starch and the influence of phytogenic supplementation.

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.

Characterization of microbial intolerances and ruminal dysbiosis towards different dietary carbohydrate sources using an in vitro model.

AIM: This study aimed to characterize the critical points for determining the development of dysbiosis associated with feed intolerances and ruminal acidosis. METHODS AND RESULTS: A metabologenomics approach was used to characterize dynamic microbial and metabolomics shifts using the rumen simulation technique (RUSITEC) by feeding native cornstarch (ST), chemically modified cornstarch (CMS), or sucrose (SU). SU and CMS elicited the most drastic changes as rapidly as 4 h after feeding. This was accompanied by a swift accumulation of d-lactate, and the decline of benzoic and malonic acid. A consistent increase in Bifidobacterium and Lactobacillus as well as a decrease in fibrolytic bacteria was observed for both CMS and ST after 24 h, indicating intolerances within the fibre degrading populations. However, an increase in Lactobacillus was already evident in SU after 8 h. An inverse relationship between Fibrobacter and Bifidobacterium was observed in ST. In fact, Fibrobacter was positively correlated with several short-chain fatty acids, while Lactobacillus was positively correlated with lactic acid, hexoses, hexose-phosphates, pentose phosphate pathway (PENTOSE-P-PWY), and heterolactic fermentation (P122-PWY). CONCLUSIONS: The feeding of sucrose and modified starches, followed by native cornstarch, had a strong disruptive effect in the ruminal microbial community. Feed intolerances were shown to develop at different rates based on the availability of glucose for ruminal microorganisms. SIGNIFICANCE AND IMPACT OF THE STUDY: These results can be used to establish patterns of early dysbiosis (biomarkers) and develop strategies for preventing undesirable shifts in the ruminal microbial ecosystem.

Adapting an Ergosterol Extraction Method with Marine Yeasts for the Quantification of Oceanic Fungal Biomass.

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.

Development and Validation of an LC-MS/MS Based Method for the Determination of Deoxynivalenol and Its Modified Forms in Maize.

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.

Identification and Functional Characterization of the Gene Cluster Responsible for Fusaproliferin Biosynthesis in Fusarium proliferatum.

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 acyltransferase PMAT1 malonylates brassinolide glucoside.

Brassinosteroids (BRs) are steroid hormones of plants that coordinate fundamental growth and development processes. Their homeostasis is controlled by diverse means, including glucosylation of the bioactive BR brassinolide (BL), which is catalyzed by the UDP-glycosyltransferases (UGTs) UGT73C5 and UGT73C6 and occurs mainly at the C-23 position. Additional evidence had suggested that the resultant BL-23-O-glucoside (BL-23-O-Glc) can be malonylated, but the physiological significance of and enzyme required for this reaction had remained unknown. Here, we show that in Arabidopsis thaliana malonylation of BL-23-O-Glc is catalyzed by the acyltransferase phenolic glucoside malonyl-transferase 1 (PMAT1), which is also known to malonylate phenolic glucosides and lipid amides. Loss of PMAT1 abolished BL-23-O-malonylglucoside formation and enriched BL-23-O-Glc, showing that the enzyme acts on the glucoside. An overexpression of PMAT1 in plants where UGT73C6 was also overexpressed, and thus, BL-23-O-Glc formation was promoted, enhanced the symptoms of BR-deficiency of UGT73C6oe plants, providing evidence that PMAT1 contributes to BL inactivation. Based on these results, a model is proposed in which PMAT1 acts in the conversion of both endogenous and xenobiotic glucosides to adjust metabolic homeostasis in spatial and temporal modes.

Effect of Temperature, Water Activity and Carbon Dioxide on Fungal Growth and Mycotoxin Production of Acclimatised Isolates of Fusarium verticillioides and F. graminearum.

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.

Novel analytical methods to study the fate of mycotoxins during thermal food processing.

Food processing can lead to a reduction of contaminants, such as mycotoxins. However, for food processing operations where thermal energy is employed, it is often not clear whether a reduction of mycotoxins also results in a mitigation of the toxicological impact. This is often due to the reason that the formed degradation products are not characterized and data on their toxicity is scarce. From the perspective of an analytical chemist, the elucidation of the fate of a contaminant in a complex food matrix is extremely challenging. An overview of the analytical approaches is given here, and the application and limitations are exemplified based on cases that can be found in recent literature. As most studies rely on targeted analysis, it is not clear whether the predetermined set of compounds differs from the degradation products that are actually formed during food processing. Although untargeted analysis allows for the elucidation of the complete spectrum of degradation products, only one such study is available so far. Further pitfalls include insufficient precision, natural contamination with masked forms of mycotoxins and interferences that are caused by the food matrix. One topic that is of paramount importance for both targeted and untargeted approaches is the availability of reference standards to identity and quantity the formed degradation products. Our vision is that more studies need to be published that characterize the formed degradation products, collect data on their toxicity and thereby complete the knowledge about the mycotoxin mitigating effect during food processing.

Zearalenone and ß-Zearalenol But Not Their Glucosides Inhibit Heat Shock Protein 90 ATPase Activity.

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.