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Microdochium majusandMicrodochium nivalecause serious disease problems in grasses and cereal crops in the temperate regions. Both fungi can infect the plants during winter (causing pink snow mould) as well as under cool humid conditions during spring and fall. We conducted a pathogenicity test of 15 M. nivaleisolates and twoM. majusisolates from Norway at low temperature on four different grass cultivars ofLolium perenneandFestuloliumhybrids. Significant differences betweenM. nivaleisolates in the ability to cause pink snow mould were detected. TheM. nivalestrains originally isolated from grasses were more pathogenic than isolates from cereals. The genetic diversity ofM. nivaleandM. majusisolates was studied by sequencing four genetic regions; Elongation factor-1 alpha (EF-1α), β-tubulin, RNA polymerase II (RPB2)and the Internal Transcribed Spacer (ITS). Phylogenetic trees based on the sequences of these four genetic regions resolvedM. nivaleandM. majusisolates into separate clades. Higher genetic diversity was found amongM. nivaleisolates than amongM. majusisolates.M. nivaleisolates revealed genetic differences related to different host plants (grasses vs. cereals) and different geographic regions (Norway and UK vs. North America). Sequence results from theRPB2and β-tubulin genes were more informative than those from ITS andEF-1α. The genetic and phenotypic differences detected between NorwegianM. nivaleisolates from cereals and grasses support the assumption that host specialization exist withinM. nivaleisolates.

Leaf blotch diseases (LBD), such as Septoria nodorum bloch (Parastagnospora nodorum), Septoria tritici blotch (Zymoseptoria tritici) and Tan spot (Pyrenophora tritici-repentis) can cause severe yield losses (up to 50%) in Norwegian spring wheat (Triticum aestivum) and are mainly controlled by fungicide applications. A forecasting model to predict disease risk can be an important tool to optimize disease control. The association between specific weather variables and the development of LBD differs between wheat growth stages. In this study, a mathematical model to estimate phenological development of spring wheat was derived based on sowing date, air temperature and photoperiod. Weather factors associated with LBD severity were then identified for selected phenological growth stages by a correlation study of LBD severity data (17 years). Although information regarding host resistance and previous crop were added to the identified weather factors, two purely weather-based risk prediction models (CART, classification and regression tree algorithm) and one black box model (KNN, based on K nearest neighbor algorithm) were most accurate to predict moderate to high LBD severity (>5% infection). The predictive accuracy of these models (76–83%) was compared to that of two existing models used in Norway and Denmark (60 and 61% accuracy, respectively). The newly developed models performed better than the existing models, but still had the tendency to overestimate disease risk. Specificity of the new models varied between 49 and 74% compared to 40 and 37% for the existing models. These new models are promising decision tools to improve integrated LBD management of spring wheat in Norway.

To mitigate the risk of erosion and nutrient runoff, reduced tillage has become more prevalent in Norway. Within within recent decades, there have been some years with relatively high occurrence of Fusarium head blight and mycotoxins in Norwegian cereal grain. This is thought to have been caused by an increased inoculum potential (IP) of Fusarium spp. due to larger amount of crop residues remaining on the soil surface, in combination with weather conditions promoting fungal growth and infection of cereal plants. The objective of this work was to elucidate the influence of different tillage practices on the IP of Fusarium spp. and the subsequent Fusarium-infection and mycotoxin contamination of spring wheat grain at harvest. Tillage trials were conducted at two locations in southeast Norway (Solør and Toten) over three years, 2010-2012. Residues of wheat from the previous year were collected in spring. Fusarium avenaceum and Fusarium graminearum were the most common Fusarium species recorded on wheat straw residues. IP was calculated as the percentage of the residues infested with Fusarium spp. multiplied by the proportion of the soil surface covered with residues. The IP of Fusarium spp. was lower in ploughed plots compared to those tilled with harrowing only. Ploughing in spring resulted in a similarly low IP as autumn ploughing. In contrast, harrowing in autumn generally reduced IP more than did spring harrowing. The mycotoxin levels in the harvested wheat were generally low, except for deoxynivalenol at high levels in Solør 2011. Despite a lower IP of ploughed versus harrowed plots, this was not reflected in the content of Fusarium and mycotoxins in harvested grain. The Fusarium species that dominated in the residues examined in this study were the same as those detected in the harvested grain, supporting the finding that residues are an important source of inoculum.

Cereal grain contaminated by Fusarium mycotoxins is undesirable in food and feed because of the harmful health effects of the mycotoxins in humans and animals. Reduction of mycotoxin content in grain by cleaning and size sorting has mainly been studied in wheat. We investigated whether the removal of small kernels by size sorting could be a method to reduce the content of mycotoxins in oat grain. Samples from 24 Norwegian mycotoxin-contaminated grain lots (14 from 2015 and 10 from 2018) were sorted by a laboratory sieve (sieve size 2.2 mm) into large and small kernel fractions and, in addition to unsorted grain samples, analyzed with LC-MS-MS for quantification of 10 mycotoxins. By removing the small kernel fraction (on average 15% and 21% of the weight of the samples from the two years, respectively), the mean concentrations of HT-2+T-2 toxins were reduced by 56% (from 745 to 328 &micro, g/kg) in the 2015 samples and by 32% (from 178 to 121 &micro, g/kg) in the 2018 samples. Deoxynivalenol (DON) was reduced by 24% (from 191 to 145 &micro, g/kg) in the 2018 samples, and enniatin B (EnnB) by 44% (from 1059 to 594 &micro, g/kg) in the 2015 samples. Despite low levels, our analyses showed a trend towards reduced content of DON, ADON, NIV, EnnA, EnnA1, EnnB1 and BEA after removing the small kernel fraction in samples from 2015. For several of the mycotoxins, the concentrations were considerably higher in the small kernel fraction compared to unsorted grain. Our results demonstrate that the level of mycotoxins in unprocessed oat grain can be reduced by removing small kernels. We assume that our study is the first report on the effect of size sorting on the content of enniatins (Enns), NIV and BEA in oat grains.

The bread-making quality of wheat depends on the viscoelastic properties of the dough in which gluten proteins play an important role. The quality of gluten proteins is influenced by the genetics of the different wheat varieties and environmental factors. Occasionally, a near complete loss of gluten strength, measured as the maximum resistance towards stretching (Rmax), is observed in grain lots of Norwegian wheat. It is hypothesized that the loss of gluten quality is caused by degradation of gluten proteins by fungal proteases. To identify fungi associated with loss of gluten strength, samples from a selection of wheat grain lots with weak gluten (n = 10, Rmax < 0.3 N) and strong gluten (n = 10, Rmax ≥ 0.6 N) was analyzed for the abundance of fungal operational taxonomic units (OTUs) using DNA metabarcoding of the nuclear ribosomal Internal Transcribed Spacer (ITS) region ITS1. The DNA quantities for a selection of fungal pathogens of wheat, and the total amount of fungal DNA, were analyzed by quantitative PCR (qPCR). The mean level of total fungal DNA was higher in grain samples with weak gluten compared to grain samples with strong gluten. Heightened quantities of DNA from fungi within the Fusarium Head Blight (FHB) complex, i.e. Fusarium avenaceum, Fusarium graminearum, Microdochium majus, and Microdochium nivale, were observed in grain samples with weak gluten compared to those with strong gluten. Microdochium majus was the dominant fungus in the samples with weak gluten. Stepwise regression modeling based on different wheat quality parameters, qPCR data, and the 35 most common OTUs revealed a significant negative association between gluten strength and three OTUs, of which the OTU identified as M. majus was the most abundant. The same analysis also revealed a significant negative relationship between gluten strength and F. avenaceum detected by qPCR, although the DNA levels of this fungus were low compared to those of M. majus. In vitro growth rate studies of a selection of FHB species showed that all the tested isolates were able to grow with gluten as a sole nitrogen source. In addition, proteins secreted by these fungi in liquid cultures were able to hydrolyze gluten substrate proteins in zymograms, confirming their capacity to secrete gluten-degrading proteases. The identification of fungi with potential to influence gluten quality can enable the development of strategies to minimize future problems with gluten strength in food-grade wheat.

This study aims to understand the environmental factors, focusing on rain and fungal infection, affecting the assembly of glutenin polymers during grain maturation. Spring wheat was grown in the field and grains were sampled from 50% grain moisture until maturity. Grain moisture content, protein content, size of glutenin polymers, the presence of proteases, and the amount of DNA from common wheat pathogenic fungi were analysed. Rain influenced the rate of grain desiccation that occurred parallel to the rate of glutenin polymer assembly. Rapid desiccation contributed to faster glutenin polymer assembly than gradual desiccation. Severe reduction in the glutenin polymer size coincided with increased grain moisture due to rain. Furthermore, increased fungal DNA followed by presence of gluten-degrading proteases was observed in the grain after humid conditions. The presence of gluten-degrading proteases was presumably involved in reducing the size of glutenin polymers in grain. Our study gave new insight into how environmental conditions could be associated with the assembly of glutenin polymers during grain maturation. The results suggest that rain and/or fungal proteases play an important role in reducing the molecular size of glutenin polymers.

The natural occurrence of fungi, mycotoxins and fungal metabolites was investigated in 100 samples of maize grains collected from south and southwestern Ethiopia in 2015. The maize samples were contaminated by Fusarium, Aspergillus and Penicillium species. Using liquid chromatography tandem mass spectrometry 127 secondary metabolites were analysed. Zearalenone was the most prevalent mycotoxin, occurring in about 96% of the samples. Zearalenone sulfate was the second most prevalent, present in 81% of the samples. Fumonisin B1 was detected in 70% of the samples with a mean level of 606 μg kg-1 in positive samples, while FB2, FB3 and FB4 were detected in 62%, 51% and 60% of the maize samples with mean levels of 202, 136 and 85 μg kg-1, respectively. Up to 8% of the samples were contaminated with aflatoxins, with a maximum level of aflatoxin B1 of 513 μg kg-1. Results were higher than earlier reports for maize from Ethiopia.

High concentrations of the mycotoxin deoxynivalenol (DON), produced by Fusarium graminearum have occurred frequently in Norwegian oats recently. Early prediction of DON levels is important for farmers, authorities and the Cereal Industry. In this study, the main weather factors influencing myco-toxin accumulation were identified and two models to predict the risk of DON in oat grains in Norway were developed: (1) as a warning system for farmers to decide if and when to treat with fungicide, and (2) for authorities and industry to use at harvest to identify potential food safety problems. Oat grain samples from farmers’ fields were collected together with weather data (2004–2013) A mathematical model was developed and used to esti- mate phenology windows of growth stages in oats (til- lering, flowering etc.). Weather summarisations were then calculated within these windows, and the Spearman rank correlation factor calculated between DON- contamination in oats at harvest and the weather summarisations for each phenological window. DON contamination was most clearly associated with the weather conditions around flowering and close to har- vest. Warm, rainy and humid weather during and around flowering increased the risk of DON accumulation in oats, as did dry periods during germination/seedling growth and tillering. Prior to harvest, warm and humid weather conditions followed by cool and dry conditions were associated with a decreased risk of DON accumu- lation. A prediction model, including only pre-flowering weather conditions, adequately forecasted risk of DON contamination in oat, and can aid in decisions about fungicide treatments.

This paper presents peer-reviewed studies comparing the content of deoxynivalenol (DON), HT-2+T-2 toxins, zearalenone (ZEA), nivalenol (NIV), ochratoxin A (OTA) and fumonisins in cereal grains, and patulin (PAT) in apple and apple-based products, produced in organically and conventionally grown crops in temperate regions. Some of the studies are based on data from controlled field trials, however, most are farm surveys and some are food basket surveys. Almost half of the studies focused on DON in cereals. The majority of these studies found no significant difference in DON content in grain from the two farming systems, but several studies showed lower DON content in organically than in conventionally produced cereals. A number of the investigations reported low DON levels in grain, far below the EU limits for food. Many authors suggested that weather conditions, years, locations, tillage practice and crop rotation are more important for the development of DON than the type of farming. Organically produced oats contained mainly lower levels of HT-2+T-2 toxins than conventionally produced oats. Most studies on ZEA reported no differences between farming systems, or lower concentrations in organically produced grain. For the other mycotoxins in cereals, mainly low levels and no differences between the two farming systems were reported. Some studies showed higher PAT contamination in organically than in conventionally produced apple and apple products. The difference may be due to more efficient disease control in conventional orchards. It cannot be concluded that any of the two farming systems increases the risk of mycotoxin contamination. Despite no use of fungicides, an organic system appears generally able to maintain mycotoxin contamination at low levels. More systematic comparisons from scientifically controlled field trials and surveys are needed to clarify if there are differences in the risk of mycotoxin contamination between organically and conventionally produced crops.

Pink snow mould caused by Microdochium nivale is a serious cereal and grass disease in several temperate regions. In this study, resistance to snow mould was evaluated under controlled conditions i...

High concentrations of the mycotoxins HT-2 and T-2 (HT2 + T2), primarily produced by Fusarium langsethiae, have occasionally been detected in Norwegian oat grains. In this study, we identified weather variables influencing accumulation of HT2 + T2 in Norwegian oat grains. Oat grain samples from farmers’ fields were collected together with weather data (2004–2013). Spearman rank correlation coefficients were calculated between the HT2 + T2 contamination in oats at harvest and a range of weather summarisations within estimated phenological windows of growth stages in oats (tillering, flowering etc.). Furthermore, we developed a mathematical model to predict the risk of HT2 + T2 in oat grains. Our data show that adequate predictions of the risk of HT2 + T2 in oat grains at harvest can be achieved, based upon weather data observed during the growing season. Humid and cool conditions, in addition to moderate temperatures during booting, were associated with increased HT2 + T2 accumulation in harvested oat grains, whereas warm and humid weather during stem elongation and inflorescence emergence, or cool weather and absence of rain during booting reduced the risk of HT2 + T2 accumulation. Warm and humid weather immediately after flowering increased the risk, while moderate to warm temperatures and absence of rain during dough development, reduced the risk of HT2 + T2 accumulation in oat grains. Our data indicated that HT2 + T2 contamination in oats is influenced by weather conditions both pre- and post-flowering. These findings are in contrast with a previous study examining the risk of deoxynivalenol contamination in oat reporting that toxin accumulation was mostly influenced by weather conditions from flowering onwards.

As has been observed in several European countries, the frequency of Fusarium head blight (FHB) caused by members of the Fusarium graminearum species complex (FGSC) has increased in Norwegian cereals in recent years, resulting in elevated levels of deoxynivalenol in cereal grains. The objective of this study was to determine if this increase was associated with changes in FGSC composition within Norway. FGSC isolates collected from wheat, oats and barley in Norway during two periods, mainly 1993–1998 and 2004–2007, were characterized to determine species and trichothecene genotype composition and to assess levels of genetic variation and population structure. In vitro growth rates at different temperatures and aggressiveness in spring wheat were further characterized for a sub-selection of isolates. All Norwegian isolates were identified as F. graminearum. The 3-acetyl-deoxynivalenol (3-ADON) trichothecene type was dominant. However, isolates with the 15-ADON chemotype were detected in Norway for the first time and may represent a recent introduction of this trichothecene type. Bayesian-model based clustering and analyses of genetic differentiation indicated the persistence over the last 20 years of two sympatric and partially admixed populations of F. graminearum in Norway. Significant differences in average in vitro growth rates and aggressiveness were observed between these two populations. Our results demonstrate that the recent increase in prevalence of the FGSC in Norwegian cereals do not correspond to any dramatic changes in FGSC species or trichothecene chemotype composition. However, significant changes in population frequencies were observed among Norwegian F. graminearum.

We present the complete genome sequence of Luteibacter rhizovicinus type strain LJ96T, a yellow-pigmented gammaproteobacterium isolated from the rhizosphere of barley (Hordeum vulgare) Johansen et al. (2005) , a species with numerous potential applications. The genome sequence was deposited to NCBI GenBank with the accession number CP017480.

The aim of this study was to evaluate the performance and usefulness of three rapid test kits for analysis of HT-2 and T-2 toxins (HT-2 and T-2), two of the most potent trichothecenes commonly found in European oats. Concentrations of these two toxins combined (HT-2+T-2) were analysed in naturally contaminated oat samples (n=68) using the following test kits: Ridascreen® FAST T-2 Toxin (‘Fast ELISA’), DRAFT Ridascreen® HT-2/T-2 (‘Standard ELISA’, not commercially available), and the lateral flow device ROSA® HT-2-T-2 (‘Rosa LFD’). Mycotoxin analysis by LC-MS/MS was used as a reference method. Rosa LFD offered the best reliability, achieving detection that was stable across toxin levels, whereas detection by both ELISA kits differed significantly among toxin levels (P2=0.94), and the corresponding R2 values for Fast and Standard ELISA were 0.61 and 0.83, respectively. Rosa LFD was well suited for on-site detection. Standard ELISA allows simultaneous testing of several samples that are useful for centralised laboratories.

During the last ten years, Norwegian cereal grain industry has experienced large challenges due to Fusarium spp. and Fusarium mycotoxin contamination of small-grained cereals. To prevent severely contaminated grain lots from entering the grain supply chain, it is important to establish surveys for the most prevalent Fusarium spp. and mycotoxins. The objective of our study was to quantify and calculate the associations between Fusarium spp. and mycotoxins prevalent in oats and spring wheat. In a 6-year period from 2004-2009, 178 grain samples of spring wheat and 289 samples of oats were collected from farmers’ fields in South East Norway. The grains were analysed for 18 different Fusarium-mycotoxins by liquid chromatography – mass spectrometry. Generally, the median mycotoxin levels were higher than reported in Norwegian studies covering previous years. The DNA content of Fusarium graminearum, Fusarium culmorum, Fusarium langsethiae, Fusarium poae and Fusarium avenaceum were determined by quantitative PCR. We identified F. graminearum as the main deoxynivalenol (DON) producer in oats and spring wheat, and F. langsethiae as the main HT-2 and T-2-toxins producer in oats. No association was observed between quantity of F. graminearum DNA and quantity of F. langsethiae DNA nor for their respective mycotoxins, in oats. F. avenaceum was one of the most prevalent Fusarium species in both oats and spring wheat. The following ranking of Fusarium species was made based on the DNA concentrations of the Fusarium spp. analysed in this survey (from high to low): F. graminearum = F. langsethiae = F. avenaceum > F. poae > F. culmorum (oats); F. graminearum = F. avenaceum > F. culmorum > F. poae = F. langsethiae (spring wheat). Our results are in agreement with recently published data indicating a shift in the relative prevalence of Fusarium species towards more F. graminearum versus F. culmorum in Norwegian oats and spring wheat.

Lack of resistance to pink snow mould (Microdochium nivale) is a major constraint for adaptation of perennial ryegrass (Lolium perenne L.) to continental regions with long-lasting snow cover at higher latitudes. Almost all investigations of genetic variation in resistance have been performed using cold acclimated plants. However, there may be variation in resistance mechanisms that are functioning independently of cold acclimation. In this study our aim was to identify candidate genes involved in such resistance mechanisms. We first characterized variation in resistance to M. nivale among non-acclimated genotypes from the Norwegian cultivar ‘Fagerlin’ based on relative regrowth and fungal quantification by real-time qPCR. One resistant and one susceptible genotype were selected for transcriptome analysis using paired-end sequencing by Illumina Hiseq 2000. Transcriptome profiles, GO enrichment and KEGG pathway analysis indicate that defense response related genes are differentially expressed between the resistant and the susceptible genotype. A significant up-regulation of defense related genes, as well as genes involved in cell wall cellulose metabolic processes and aryl-alcohol dehydrogenase (NADP+) activity, was observed in the resistant genotype. The candidate genes identified in this study might be potential molecular marker resources for breeding perennial ryegrass cultivars with improved resistance to pink snow mould.

The aim of this study was to evaluate the performance of rapid test kits for analysis of deoxynivalenol (DON), a mycotoxin commonly found in small-grain cereals. Concentrations of DON and its acetylated derivative 3-acetyldeoxynivalenol (3-ADON) were analysed in naturally contaminated wheat (n=56) and oats (n=81) using two enzyme-linked immunosorbent assays, i.e. Ridascreen® DON (‘Standard ELISA’) and Ridascreen® FAST DON (‘Fast ELISA’), and two lateral flow devices, i.e. Rida® QUICK DON (‘Quick LFD’) and Rosa® DON (‘Rosa LFD’). Analysis by the LC-MS/MS reference method showed a median DON + 3-ADON concentration of 1,168 μg/kg (90th percentile 3,264 μg/kg) in wheat and 2,250 μg/kg (90th percentile of 9,008 μg/kg) in oats. Rosa LFD offered the best reliability, achieving detection that was stable across toxin levels in both matrices. In addition, Rosa LFD had the highest accuracy (i.e. proportion of correct results) at a DON level of 1,250 μg/kg in wheat (0.83) and 8,000 μg/kg in oats (0.96). Standard ELISA achieved the highest accuracy at a DON level of 1,750 μg/kg in oats (0.85). Detection by Quick LFD in wheat and Fast ELISA in both wheat and oats differed significantly among toxin levels (P≤0.05), with recoveries outside European Union requirements (70-120%) in some cases. The correlation with results from LC-MS/MS analysis in oats was best for Rosa LFD (R2=0.79). In wheat, however the correlation was best for Fast ELISA (R2=0.81). Rosa LFD was regarded as well-suited for on-site detection. Standard ELISA allowed simultaneous testing of several samples and achieved detection that was stable across toxin levels in both matrices, making it a useful alternative in laboratories. To our knowledge, this is the first report describing the performance of rapid test kits for detection of DON in naturally contaminated oats.

We present the complete genome sequence of Luteibacter rhizovicinus type strain LJ96T, a yellow-pigmented gammaproteobacterium isolated from the rhizosphere of barley (Hordeum vulgare) Johansen et al. (2005) , a species with numerous potential applications. The genome sequence was deposited to NCBI GenBank with the accession number CP017480.

The effect of plant age and cold hardening on resistance to pink snow mould caused by Microdochium nivale was studied in perennial ryegrass. Resistance to M. nivale was estimated as relative regrowth after inoculation and incubation under artificial snow cover at 2°C. Resistance increased with increasing plant age. Cold hardened and unhardened plants of the same age displayed identical resistance. Preliminary studies indicate that expression of genes coding for the PR proteins chitinase and PR-1a increased during incubation of inoculated perennial ryegrass, but no clear difference in expression of these genes was found between plants of different ages, or in hardened versus unhardened plants.

Pink snow mould is a serious disease on grasses and winter cereals in cold and temperate zones during winter. To better understand the basis for the variation in pathogenicity between different isolates of Microdochium nivale and M. majus and to simplify selection of highly pathogenic isolates to use when screening for resistance to pink snow mould in perennial ryegrass, we sought traits correlated with pathogenicity. Isolates of M. nivale were more pathogenic on perennial ryegrass than isolates of M. majus, as measured by survival and regrowth of perennial ryegrass after infection and incubation under simulated snow cover. Pathogenicity as measured by relative regrowth was highly correlated with fungal growth rate on potato dextrose agar (PDA) at 2°C. Measuring fungal growth on PDA therefore seems to be a relatively simple method of screening for potentially highly pathogenic isolates. In a study of a limited number of isolates, highly pathogenic isolates showed an earlier increase and a higher total specific activity of β-glucosidase, a cell wall-degrading enzyme, compared with less pathogenic isolates. None of the M. majus isolates was highly pathogenic on perennial ryegrass. Our results indicate biological differences between M. nivale and M. majus and thus strengthen the recently published sequence-based evidence for the elevation of these former varieties to species status.

The effects of chitosan on resistance to pink snow mould (Microdochium nivale) were studied in young winter wheat (Triticum aestivum L.) and perennial ryegrass (Lolium perenne L.) under controlled environmental conditions. In perennial ryegrass, the putative defence activator Bion was also tested. Resistance was measured as regrowth of plants after inoculation with M. nivale and incubation in darkness at 2°C. In winter wheat, pre-treatment with chitosan at 1000 μg per plant increased resistance to subsequent infection by M. nivale, but this effect was less significant in a replicate experiment. Chitosan-treated winter wheat plants expressed the gene for the pathogenesis-related protein chitinase at higher levels than non-treated plants. Chitinase gene expression was also stimulated by M. nivale infection in winter wheat. Perennial ryegrass pre-treated with Bion or chitosan and inoculated with M. nivale did not display better regrowth after incubation than non-treated, inoculated plants. Rather, regrowth was reduced in some of the Bion-treated plants after incubation. We speculate that the cost or the mechanism of induced resistance makes Bion non-effective in plants that are not actively growing. Bion at concentrations of 10, 100 and 1000 μg active ingredient per ml, and the highest concentration of chitosan used (2000 μg per ml) reduced in vitro growth of the pathogen, suggesting that both defence activators possess antifungal activity.

Wintering ability in the field and resistance to different winter-stress factors under controlled environmental conditions were studied in a full-sib family of perennial ryegrass (Lolium perenne L.). Significant variation in tolerance to freezing and ice encasement, resistance to pink snow mould (Microdochium nivale) and also in winter survival and spring growth were found between the different genotypes. No strong correlations were found between the resistances to the different stress factors. These results indicate that resistance to different winter-stress factors is controlled by separate genes in perennial ryegrass. A low but significant positive correlation was found between spring growth of plants in the field after the first winter and both freezing tolerance and M. nivale resistance measured in controlled environments. Cold hardening seemed to influence freezing tolerance and M. nivale resistance differently in the different genotypes, since no distinct correlation in tolerance to freezing or resistance to M. nivale was found between unhardened and hardened plants. Tolerance or resistance to most of the winter stress factors measured was positively correlated with plant size.

A glutathione (GSH) adduct of the mycotoxin 4-deoxynivalenol (DON), together with a range of related conjugates, has recently been tentatively identified by LC-MS of DON-treated wheat spikelets. In this study, we prepared samples of DON conjugated at the 10- and 13-positions with GSH, Cys, CysGly, γ-GluCys and N-acetylcysteine (NAC). The mixtures of conjugates were used as standards for LC-HRMS analysis of one of the DON-treated wheat spikelet samples, as well as 19 Norwegian grain samples of spring wheat and 16 grain samples of oats that were naturally-contaminated with DON at concentrations higher than 1 mg/kg. The artificially-contaminated wheat spikelets contained conjugates of GSH, CysGly and Cys coupled at the olefinic 10-position of DON, whereas the naturally-contaminated harvest-ripe grain samples contained GSH, CysGly, Cys, and NAC coupled mainly at the 13-position on the epoxy group. The identities of the conjugates were confirmed by LC-HRMS comparison with authentic standards, oxidation to the sulfoxides with hydrogen peroxide, and examination of product-ion spectra from LC-HRMS/MS analysis. No γ-GluCys adducts of DON were detected in any of the samples. The presence of 15-O-acetyl-DON was demonstrated for the first time in Norwegian grain. The results indicate that a small but significant proportion of DON is metabolized via the GSH-conjugation pathway in plants. To our knowledge, this is the first report of in vivo conjugation of trichothecenes via their epoxy group, which has generally been viewed as unreactive. Because conjugation at the 13-position of DON and other trichothecenes has been shown to be irreversible, this type of conjugate may prove useful as a biomarker of exposure to DON and other 12,13-epoxytrichothecenes. View Full-Text