Exposure of mouse oocytes to bisphenol A causes meiotic arrest but not aneuploidy

Mouse oocytes isolated from large antral follicles were exposed to a wide range of concentrations of bisphenol A (BPA) during maturation in vitro (50 ng/ml to 10 microg/ml BPA in medium). Exposure to high concentrations of BPA (10 microg/ml) affected spindle formation, distribution of pericentriolar material and chromosome alignment on the spindle (termed congression failure), and caused a significant meiotic arrest. However, BPA did not increase hyperploidy at meiosis II at any tested concentration. Some but not all meiosis I arrested oocytes had MAD2-positive foci at centromeres of chromosomes in bivalents, suggesting that they had failed to pass the spindle checkpoint control. In a second set of experiments prepubertal mice were exposed sub-chronically for 7 days to low BPA by daily oral administration, followed by in vitro maturation of the denuded oocytes to metaphase II in the absence of BPA, as this treatment protocol was previously reported to induce chromosome congression failure and therefore suspected to cause aneuploidy in oocytes. The sub-chronic exposure subtly affected spindle morphology and oocyte maturation. However, as with the exposure in vitro, there was no evidence that low BPA doses increased hyperploidy at meiosis II. In conclusion, the data suggest that mouse oocytes from mice respond to BPA-induced disturbances in spindle formation by induction of meiotic arrest. This response might result from an effective checkpoint mechanism preventing the occurrence of chromosome malsegregation and aneuploidy. Low chronic BPA exposure in vivo as such does not appear to pose a risk for induction of errors in chromosome segregation at first meiosis in mouse oocytes. Additional factors besides BPA may have caused the high rate of congression failure and the temporary increase in hyperploidy in mouse metaphase II oocytes reported previously.     

Elaboration of vomitoxin and zearalenone by Fusarium isolates and the biological activity of Fusarium-produced toxins

Sixteen Fusarium isolates belonging to F. graminearium Schw. and F. culmorum (W.G. Smith) Sacc. produced vomitoxin and zearalenone on cracked corn at 28 degrees C. Quantitation for vomitoxin was by gas-liquid chromatography. This toxin was produced in quantities of 5 to 236 microgram/g of fermented corn. Vomitoxin showed weak antibiotic activity against Penicillium digitatum Sacc., Mucor ramannianus M?ller, and Saccharomyces bayanus Sacc., but did not inhibit gram-positive, gram-negative, or acid fast bacteria. The two molds and the yeast were inhibited by T-2 toxin at 5 micrograms, and diacetoxyscirpenol inhibited the molds at 5 micrograms and the yeast at 50 micrograms.     

Monoclonal antibody-based enzyme-linked immunosorbent assay of Fusarium T-2 and zearalenone toxins in cereals.

Direct, competitive enzyme-linked immunosorbent assays (ELISAs) with monoclonal antibodies have been developed for quantitative determination of trichothecene T-2 toxin (T-2), and zearalenone (F-2) from different cereals: Among the several extraction solvents tried, 89% acetonitrile with additives was chosen. The extracts were then used without cleanup in the ELISA. With appropriate dilution of the samples (1:25 or 1:50), the matrix effects caused by lipid and/or protein content of the samples can be diminished to the extent that the assay is no longer impaired. The mean recoveries from cereals infected with 100 to 2,000 ng of T-2 and 50 to 500 ng of F-2 per g were 85 and 91%, respectively. The measuring range of the T-2 test is 100 to 2,000 ng/g, and that of the F-2 test is 25 to 400 ng/g. The mean within-assay and interassay coefficients of variation of standard curves are both less than 10%. According to recovery results with artificially infected cereals, our tests proved to be suitable for rapid screening of food and feed samples for the presence of T-2 and F-2 toxins.     

Elaboration of vomitoxin and zearalenone by Fusarium isolates and the biological activity of Fusarium-produced toxins.

Sixteen Fusarium isolates belonging to F. graminearium Schw. and F. culmorum (W.G. Smith) Sacc. produced vomitoxin and zearalenone on cracked corn at 28 degrees C. Quantitation for vomitoxin was by gas-liquid chromatography. This toxin was produced in quantities of 5 to 236 microgram/g of fermented corn. Vomitoxin showed weak antibiotic activity against Penicillium digitatum Sacc., Mucor ramannianus Möller, and Saccharomyces bayanus Sacc., but did not inhibit gram-positive, gram-negative, or acid fast bacteria. The two molds and the yeast were inhibited by T-2 toxin at 5 micrograms, and diacetoxyscirpenol inhibited the molds at 5 micrograms and the yeast at 50 micrograms.     

Effect of the volume of medium and number of oocytes during in vitro fertilization on embryo development in pigs

The present study was designed to determine the effect of the volume of medium (VM) and the number of oocytes (NOOC) during in vitro fertilization (IVF) on embryo development in pigs. Groups of 15, 30 and 50 in vitro matured oocytes were transferred to 2, 1 and 0.1 ml of modified Tris-buffered medium (mTBM) and inseminated with frozen-thawed spermatozoa (2000 spermatozoa/oocyte) in a 3 x 3 factorial experiment. A total of 2739 oocytes from four replicates were exposed to spermatozoa for 6 h and then cultured in embryo culture medium for 6 h (pronuclear formation) or 7 days (blastocyst formation: BF). The efficiency of fertilization (EF: number of monospermic oocytes/total number of inseminated oocytes) and BF decreased (P<0.03) as the VM increased (EF: 45.9+/-2.2, 43.8+/-2.6 and 36.9+/-1.6% and BF: 29.4+/-2.7, 23.2+/-1.8 and 19.9+/-2.1% for VM 0.1, 1 and 2 ml, respectively). The BF, but not EF, was also affected (P<0.04) by NOOC (19.8+/-1.6, 28.1+/-2.3 and 24.6+/-2.9% for groups of 15, 30 and 50 oocytes, respectively). The effect of the interaction VM x NOOC on EF and BF was not significant. These results indicate that when 2000 spermatozoa/oocyte were used, a low volume of IVF medium (0.1 ml) and the number of oocytes during IVF (30-50) can improve the in vitro embryo production in pigs.     

Prdm9 deficiency of rat oocytes causes synapsis among non-homologous chromosomes and aneuploidy

Mammalian Genome - Aneuploidy (abnormal chromosome number) accompanies reduced ovarian function in humans and mice, but the reasons behind this concomitance remain underexplored. Some variants in...     

Deoxynivalenol, acetyl deoxynivalenol, and zearalenone formation by Canadian isolates of Fusarium graminearum on solid substrates.

Three isolates of Fusarium graminearum (DAOM 180377, 180378, and 180379) were screened for their ability to produce mycotoxins on the solid substrates corn and rice. They all produced deoxynivalenol and zearalenone on corn. On rice, only DAOM 180378 and 180379 produced significant amounts of these mycotoxins, with levels of deoxynivalenol being much higher than those of zearalenone. The effects of the initial moisture content before autoclaving, incubation temperature, and time were studied with isolate DAOM 180378. At 19.5 degrees C the main product was zearalenone, whereas at 25 degrees C both deoxynivalenol and zearalenone were formed. Higher incubation temperatures (28 degrees C) favored deoxynivalenol formation, the maximum amount being 515 ppm (515 micrograms/g) formed after 24 days at an initial moisture content of 40%. The maximum level of zearalenone produced at the same temperature was 399 ppm, but at an initial moisture content of 35%. Other factors, such as pH, oxygen and carbon dioxide concentrations, and size of the culture flask also appeared to affect the production of mycotoxins.     

Method for detecting production of zearalenone, zearalenol, T-2 toxin, and deoxynivalenol by Fusarium isolates

Three methods for detecting toxigenic fusaria in culture were compared by using known producers of zearalenone, zearalenol, T-2 toxin, and deoxynivalenol. Moist, autoclaved rice cultures of known toxigenic isolates grown in 20-ml tubes yielded oily extracts containing compounds which interfered with qualitative and quantitative analysis for the mycotoxins. Vermiculite moistened with nutrient broth in 20-ml tubes yielded a much cleaner extract. Growing the fungi on a liquid medium required a shorter incubation period, but yields of T-2 toxin and deoxynivalenol were low and variable, and the method required greater space in the incubator. Screening of the extracts by thin-layer chromatography with colorimetric spray reagents to detect the presence of these toxins permitted reduction in the number of extracts quantified by the more lengthy gas-liquid chromatographic method. Culturing in nutrient broth on vermiculite in tubes coupled to a qualitative screen before quantitation proved to be a convenient, inexpensive, and relatively rapid method that enabled reliable screening of a large number of Fusarium isolates for toxin production as compared with prior methods.     

In vitro assessment of adsorbents aiming to prevent deoxynivalenol and zearalenone mycotoxicoses

The high prevalence of the Fusarium mycotoxins, deoxynivalenol (DON) and zearalenone (ZON) in animal feeds in mild climatic zones of Europe and North America results in considerable economic losses, as these toxins affect health and productivity particularly of pigs from all age groups. The use of mycotoxin adsorbents as feed additives is one of the most prominent approaches to reduce the risk for mycotoxicoses in farm animals, and to minimise carry-over of mycotoxins from contaminated feeds into foods of animal origin. Successful aflatoxin adsorption by means of different substances (phyllosilicate minerals, zeolites, activated charcoal, synthetic resins or yeast cell-wall-derived products) has been demonstrated in vivo and in vitro. However, attempts to adsorb DON and ZON have been less encouraging. Here we describe the adsorption capacity of a variety of potential binders, including compounds that have not been evaluated before, such as humic acids. All compounds were tested at realistic inclusion levels for their capacity to bind ZON and DON, using an in vitro method that resembles the different pH conditions in the gastro-intestinal tract of pigs. Mycotoxin adsorption was assessed by chemical methods and distinct bioassays, using specific markers of toxicity as endpoints of toxicity in cytological assays. Whereas none of the tested substances was able to bind DON in an appreciable percentage, some of the selected smectite clays, humic substances and yeast-wall derived products efficiently adsorbed ZON (>70%). Binding efficiency was indirectly confirmed by the reduction of toxicity in the in vitro bioassays. In conclusion, the presented test protocol allows the rapid screening of potential mycotoxin binders. Like other in vitro assays, the presented protocol combining chemical and biological assays cannot completely simulate the conditions of the gastro-intestinal tract, and hence in vivo experiments remain mandatory to assess the efficacy of mycotoxin binders under practical conditions.     

Method for detecting production of zearalenone, zearalenol, T-2 toxin, and deoxynivalenol by Fusarium isolates.

Three methods for detecting toxigenic fusaria in culture were compared by using known producers of zearalenone, zearalenol, T-2 toxin, and deoxynivalenol. Moist, autoclaved rice cultures of known toxigenic isolates grown in 20-ml tubes yielded oily extracts containing compounds which interfered with qualitative and quantitative analysis for the mycotoxins. Vermiculite moistened with nutrient broth in 20-ml tubes yielded a much cleaner extract. Growing the fungi on a liquid medium required a shorter incubation period, but yields of T-2 toxin and deoxynivalenol were low and variable, and the method required greater space in the incubator. Screening of the extracts by thin-layer chromatography with colorimetric spray reagents to detect the presence of these toxins permitted reduction in the number of extracts quantified by the more lengthy gas-liquid chromatographic method. Culturing in nutrient broth on vermiculite in tubes coupled to a qualitative screen before quantitation proved to be a convenient, inexpensive, and relatively rapid method that enabled reliable screening of a large number of Fusarium isolates for toxin production as compared with prior methods.     

Frequency of aneuploidy related to age in porcine oocytes

It is generally accepted that mammalian oocytes are frequently suffering from chromosome segregation errors during meiosis I, which have severe consequences, including pregnancy loss, developmental disorders and mental retardation. In a search for physiologically more relevant model than rodent oocytes to study this phenomenon, we have employed comparative genomic hybridization (CGH), combined with whole genome amplification (WGA), to study the frequency of aneuploidy in porcine oocytes, including rare cells obtained from aged animals. Using this method, we were able to analyze segregation pattern of each individual chromosome during meiosis I. In contrast to the previous reports where conventional methods, such as chromosome spreads or FISH, were used to estimate frequency of aneuploidy, our results presented here show, that the frequency of this phenomenon was overestimated in porcine oocytes. Surprisingly, despite the results from human and mouse showing an increase in the frequency of aneuploidy with advanced maternal age, our results obtained by the most accurate method currently available for scoring the aneuploidy in oocytes indicated no increase in the frequency of aneuploidy even in oocytes from animals, whose age was close to the life expectancy of the breed.     

Urinary deoxynivalenol (DON) and zearalenone (ZEA) as biomarkers of DON and ZEA exposure of pigs

Four diets contaminated with 1.1 to 5.0 mg/kg deoxynivalenol (DON) and 0.4 to 2.4 mg/kg zearalenone (ZEA) were fed to four groups of six growing Large White pigs. Urine samples were collected after 3 to 4 days and again after 6 to 7 days on the diets. On each sampling day, half of the animals were sampled in the morning, after an 8-h fast, and the other half were sampled in the afternoon, after 7 h of ad libitum access to feed. The urinary concentrations of DON, DON-glucuronide, DON-3-sulphate, de-epoxy-DON, as well as of ZEA, ZEA-14-glucuronide, α-zearalenol and α-zearalenol-14-glucuronide, analysed using LC-MS/MS, were used to calculate urinary DON and ZEA equivalent concentrations (DONe and ZEAe). The urinary concentration of DONe (P?<?0.001), but not of ZEAe (P?=?0.31), was lower in the fasted than that in the fed animals. The urinary DONe/creatinine and ZEAe/creatinine ratios were highly correlated with DON and ZEA intake per kg body weight the day preceding sampling (r?=?0.76 and 0.77; P?<?0.001). The correlations between DON intake during the 7 h preceding urine sampling in the afternoon and urinary DONe/creatinine ratio (r?=?0.88) as well as between mean ZEA intake during 3 days preceding urine sampling and urinary ZEAe/creatinine ratio (r?=?0.84) were even higher, reflecting the plasma elimination half-time of several hours for DON and of more than 3 days for ZEA. ZEAe analysed in enzymatically hydrolysed urine using an ELISA kit was highly correlated with the LC-MS/MS data (r?=?0.94). The urinary DONe and ZEAe to creatinine ratios, analysed in pooled urine samples of several pigs fed the same diet, can be used to estimate their exposure to DON and ZEA.     

Aflatoxin B1, zearalenone and deoxynivalenol production by Aspergillus parasiticus and Fusarium graminearum in interactive cultures on irradiated corn kernels

The influence of inoculum size in the production of aflatoxin B1 (AFB1), zearalenone (ZEN) and deoxynivalenol (DON) was determined when Aspergillus parasiticus NRRL 3000 and Fusarium graminearum ITEM 124 were cultured alone and in pairs on irradiated corn kernels at 28 °C and 0.97 water activity (aw). The highest levels of AFB1 produced by A. parasiticus were produced at the lowest levels of the inoculum (103 spores/ml). No significant differences were observed in ZEN and DON production at any inoculum level during the experimental period. When A. parasiticus was co-inoculated with F. graminearum both to the same inocula (106 spores/ml), AFB1 inhibition percentage were 60, 72 and 56% at 10, 20 and 35 days of incubation respectively, while at 106 spores/ml the percentages of inhibition were 34, 84 and 93% at 10, 20 and 35 days. In the mixture cultures A. parasiticus 103 × F. graminearum 106 spores/ml the percentage of inhibition of AFB1 oscillated in 99% during all the incubation. In the interaction A. parasiticus 106 spores/ml × F. graminearum 103 spores/ml the accumulation of AFB1 decreased in 80, 94 and 86% at 10, 20 and 35 days of incubation respectively. In single culture F. graminearum was inoculated with 10³ or 106 spores/ml and the highest levels of ZEN and DON were detected at 35 days of incubation. The levels oscillated in 538–622 μg/kg for ZEN and 870–834 μg/kg for DON respectively. In paired cultures there were no significant differences in the levels regardless of the spore concentrations during the incubation time. This revised version was published online in June 2006 with corrections to the Cover Date.     

Occurrence of deoxynivalenol and zearalenone in brewing barley grains from Brazil

Barley (Hordeum vulgare L.) is an important cereal crop for food and represents one of the main ingredients in beer production. Considering the importance of barley and its derived products, the knowledge about the mycotoxin contamination in the barley production is essential in order to assess its safety. In this study, the levels of deoxynivalenol (DON) and zearalenone (ZEN) in brewing barley were determined using a LC-MS/MS method. A survey was conducted in 2015 to estimate the mycotoxin levels in these products (n?=?76) from four crop regions in Brazil. The results showed high levels of DON and ZEN in the analyzed samples, with contamination levels of 94 and 73.6%, respectively. The mean levels of DON and ZEN ranged from 1700 to 7500 μg/kg and from 300 to 630 μg/kg, respectively. Barley samples from regions 1 and 2 presented higher levels of ZEN and DON, respectively, and those from region 4 presented lower levels of both. Co-occurrence of DON and ZEN was seen in the majority of the barley grain samples, and the mycotoxin content was above the maximum levels established by the Brazilian and European regulations.     

Transient effect of single or repeated acute deoxynivalenol and zearalenone dietary challenge on fecal microbiota composition in female finishing pigs

Mycotoxins are a major contaminant of pig feed and have negative effects on health and performance. The present study investigated the impact of single or repeated acute challenges with a diet naturally contaminated with deoxynivalenol (DON) and zearalenone (ZEN) on growth performances of finishing pigs and their fecal microbiota composition. A total of 160 pigs (castrated males and females) in two successive batches were randomly divided into four experimental groups of 40 pigs each. The control group received a control finisher diet from 99 to 154 days of age. Challenged groups were subjected to a 7-day acute challenge by being fed a DON- and ZEN-contaminated diet (3.02 mg DON/kg feed and 0.76 mg ZEN/kg feed) at 113 days (group DC), 134 days (group CD) or both 113 and 134 days (group DD). Microbiota composition was analyzed via 16S rRNA sequencing from fecal samples collected from the 80 females at 99, 119, 140 and 154 days. Challenged pigs (i.e. groups DC, CD and DD) reduced their average daily feed intake by 25% and 27% (P < 0.001) and feed efficiency by 34% and 28% (P < 0.05) during the first and second mycotoxin exposure, respectively. Microbiota composition was affected by mycotoxin exposure (P = 0.07 during the first exposure and P = 0.01 during the second exposure). At the family level, mycotoxin exposure significantly (P < 0.05) decreased the relative abundances of Ruminococcaceae, Streptococcaceae and Veillonellaceae and increased that of Erysipelotrichaceae at both 119 and 140 days of age. After the 7-day DON/ZEN challenge, the relative abundance of 6 to 148 operational taxonomic units (OTUs) differed among the treatment groups. However, none of these OTUs changed in all treatment groups. Using 27 functional pathways, pigs exposed to DON/ZEN challenges could be distinguished from control pigs using sparse partial least squares discriminant analysis, with a 15% misclassification rate. Regarding the functionality of these predictors, two pathways were involved in detoxifying mycotoxins: drug metabolism and xenobiotic metabolism by cytochrome P450. In challenged pigs, microbiota composition returned to the initial state within 3 weeks after the end of a single or repeated DON/ZEN challenge, highlighting the resilience of the gut microbiome. The feeding and growth performances of the pigs during challenge periods were significantly correlated with biological pathways related to health problems and modifications in host metabolism. To conclude, short-term DON/ZEN challenges resulted in transient modifications in the composition and functions of fecal microbiota.     

Capacity of adult and prepubertal mouse oocytes to undergo embryo development in the presence of cysteamine

The present study was carried out to study de novo glutathione (GSH) synthesis and to evaluate the effect of stimulating GSH synthesis during in vitro maturation (IVM) of adult and prepubertal mouse oocytes on the embryo developmental rate. Adult (8 weeks old) and prepubertal mice (24-26 days old) were primed with 5 IU of PMSG and oocytes were retrieved from the ovary 48 hr later for IVM. After IVM (18 hr) Cumulus oocyte complexes (COC) were in vitro fertilized (IVF) and in vitro culture (IVC) in order to observe embryo development. The IVM medium was supplemented with: 0, 25, 50, 100, or 200 microM of cysteamine. To study the novo GSH synthesis, 5 mM BSO was added during IVM of adult or prepubertal oocyte. Developmental rates up to blastocyst were recorded for each group. Experiments also included a group of ovulated oocytes (in vivo matured) after priming with PMSG and HCG. After IVM of adult mice oocytes, an improvement was observed on embryo development in all the supplemented groups when compared with the untreated group (P < 0.05). No differences were observed in blastocyst rate among IVM oocytes with cysteamine and ovulated oocytes. Prepubertal IVM mouse oocytes had a lower cleavage rate compared with ovulated oocytes (P < 0.05). Cysteamine failed to improve prepubertal oocytes developmental rates (P > 0,05). 2-cell embryos, coming from IVM prepubertal oocytes and ovulated oocytes had the same preimplantation developmental rate up to the blastocyst stage. In prepubertal, and adult oocytes an inhibition of embryo development was observed when buthionine sulfoximide (BSO), a specific inhibitor of the gamma-glutamylcysteine synthetase, was added during oocyte maturation (P < 0.01). In conclusion, an improvement in mouse embryo development was observed when cysteamine was added to the IVM medium of adult mice oocytes. In prepubertal oocytes cysteamine addition during oocyte maturation failed to improve embryo developmental rates. The presence of BSO lowered or completely blocked blastocyst development. This proves that, de novo GSH synthesis during oocyte maturation of adult and prepubertal oocytes undoubtedly plays an important role in embryo development. The improvement on oocyte competence observed in adult mice oocytes is probably related to intracellular GSH synthesis stimulated by cysteamine. Nevertheless the reason why cysteamine failed to improve prepubertal oocytes competence remains as an open question.     

Elevated intracellular pH appears in aged oocytes and causes oocyte aneuploidy associated with the loss of cohesion in mice

Increases in the aneuploidy rate caused by the deterioration of cohesion with increasing maternal age have been well documented. However, the molecular mechanism for the loss of cohesion in aged oocytes remains unknown. In this study, we found that intracellular pH (pH_i) was elevated in aged oocytes, which might disturb the structure of the cohesin ring to induce aneuploidy. We observed for the first time that full-grown germinal vesicle (GV) oocytes displayed an increase in pH_i with advancing age in CD1 mice. Furthermore, during the in vitro oocyte maturation process, the pH_i was maintained at a high level, up to ～7.6, in 12-month-old mice. Normal pH_i is necessary to maintain protein localization and function. Thus, we put forward a hypothesis that the elevated oocyte pH_i might be related to the loss of cohesion and the increased aneuploidy in aged mice. Through the in vitro alkalinization treatment of young oocytes, we observed that the increased pH_i caused an increase in the aneuploidy rate and the sister inter-kinetochore (iKT) distance associated with the strength of cohesion and caused a decline in the cohesin subunit SMC3 protein level. Young oocytes with elevated pH_i exhibited substantially the increase in chromosome misalignment.     

Comparison of in vitro cytotoxicity of Fusarium mycotoxins,deoxynivalenol, T-2 toxin and zearalenone on selected human epithelial cell lines

Three human epithelial cell lines (CaCo-2, HEp-2 and HeLa) implicated as potential targets for three Fusarium toxins were tested for the extent of survival on exposure to increasing toxin concentration and incubation periods. Cytotoxicity assay using 3(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) was carried out with deoxynivalenol (DON), T-2 toxins and zearalenone (ZON) on CaCo-2, HEp-2 and HeLa cell lines. Of the three cell lines used, HeLa was the most sensitive, eliciting cell death after 2 days exposure at 100 ng ml^–1with T-2 toxin. HeLa was the only cell line to exhibit cytotoxicity towards ZON showing cell death at 1000 ng ml^–1after 2 days which increased to 4 days, showing substantial cell death at 200 ng ml^–1. HEp-2 was sensitive to DON showing cell death after 2 days (100 ng ml^–1) with complete cell death occurring at 200 ng ml^–1 after 4 days of exposure. Substantial cytoxicity of T-2 towards HEp-2 occurred after 2 days at 1000 ng ml^–1 and complete cell death occurred with 100 ng ml^–1 at day 4. The CaCo-2 cell line was generally resistant to the mycotoxins tested between 100 and 1000 ng ml^–1. This study shows that cytotoxicity of Fusarium toxins to epithelium cell lines is concentration- and time- dependant and results from ZON–HeLa interaction indicate possible cell type-mycotoxin specificity.     

Reduced susceptibility to Fusarium head blight in Brachypodium distachyon through priming with the Fusarium mycotoxin deoxynivalenol

The fungal cereal pathogen Fusarium graminearum produces deoxynivalenol (DON) during infection. The mycotoxin DON is associated with Fusarium head blight (FHB), a disease that can cause vast grain losses. Whilst investigating the suitability of Brachypodium distachyon as a model for spreading resistance to F. graminearum, we unexpectedly discovered that DON pretreatment of spikelets could reduce susceptibility to FHB in this model grass. We started to analyse the cell wall changes in spikelets after infection with F. graminearum wild‐type and defined mutants: the DON‐deficient Δtri5 mutant and the DON‐producing lipase disruption mutant Δfgl1, both infecting only directly inoculated florets, and the mitogen‐activated protein (MAP) kinase disruption mutant Δgpmk1, with strongly decreased virulence but intact DON production. At 14 days post‐inoculation, the glucose amounts in the non‐cellulosic cell wall fraction were only increased in spikelets infected with the DON‐producing strains wild‐type, Δfgl1 and Δgpmk1. Hence, we tested for DON‐induced cell wall changes in B. distachyon, which were most prominent at DON concentrations ranging from 1 to 100 ppb. To test the involvement of DON in defence priming, we pretreated spikelets with DON at a concentration of 1 ppm prior to F. graminearum wild‐type infection, which significantly reduced FHB disease symptoms. The analysis of cell wall composition and plant defence‐related gene expression after DON pretreatment and fungal infection suggested that DON‐induced priming of the spikelet tissue contributed to the reduced susceptibility to FHB.     

The effect of combinations of Fusarium mycotoxins (deoxynivalenol, zearalenone and fumonisin B1) on growth of brewing yeasts

The interactive effect of combinations of the Fusarium mycotoxins deoxynivalenol (DON), zearalenone (ZEA) and fumonisin B1 (FB1) on growth of brewing yeasts was examined. Yeast growth was assessed by measurement of dry weight or relative growth, cell number, viability and conductance change of the growth medium using direct and indirect methods. The interactive effect of a combination of these mycotoxins was subject to the ratio of toxins in the mixture and the toxicity of individual toxins on yeast growth. When a combination of mycotoxins at low concentration was added into the growth medium, no significant inhibitory effect on growth was observed compared to controls. However, when a combination of high concentrations of DON and ZEA which individually inhibited yeast growth was examined, the interactive effect was shown to pass from antagonism to synergism depending on the ratio of the toxins in the mixture. As a synergistic interaction between these Fusarium mycotoxins was observed only at high concentrations, which were far higher than would be expected in good quality grain, they are not a concern when related to yeast growth under the brewing conditions studied.