Occurrence of different trichothecenes and deoxynivalenol-3-β-d-glucoside in naturally and artificially contaminated Danish cereal grains and whole maize plants

Fusarium mycotoxins such as deoxynivalenol (DON) can occur in cereals conjugated to glucose and probably also to other sugars. These conjugates, which are often referred to as ??masked mycotoxins??, will not be detected with routine analytical techniques. Furthermore, it is suspected that the parent toxin may again be released after hydrolysis in the digestive tracts of animals and humans. Today, our knowledge of the occurrence of these compounds in cereal grains is limited. In this paper, a LC-MS/MS method for the simultaneous determination of DON, deoxynivalenol-3-??-d-glucoside (DON-3-glucoside), 3 acetyl-DON, nivalenol, fusarenon-X, diacetoxyscirpenol, HT-2 toxin, and T-2 toxin in naturally (n?=?48) and artificially (n?=?30) contaminated cereal grains (wheat, barley, oat, rye triticale) is reported. The method has also been applied to whole fresh maize plant intended for production of maize silage (n?=?10). The samples were collected from the harvest years 2006?C2010, The results show that DON-3-glucoside and DON co-occurred in cereal grains and, especially in several of the highly contaminated samples, the concentration of the glucoside can be relatively high, corresponding to over 37?% of the DON concentration. The DON-3-glucoside levels in both the naturally and in the artificially grain inoculated with Fusarium were second only to DON, and were generally higher than those of the other tested trichothecenes, which were found at low concentrations in most samples, in many cases even below the detection limit of the method. This argues for the importance of taking DON-3-glucoside into account in the ongoing discussion within the European Community concerning exposure re-evaluations for setting changed values for the tolerable intake for DON. Our results indicate that, in the naturally contaminated grains and in the Fusarium infested cereal grains (winter and spring wheat, oat, triticale), the concentration level of DON-3-glucoside is positively correlated to the DON content. When the DON concentration is high, then the content of DON-3-glucoside will most probably also be high and vice versa.     

Biotransformation of the Mycotoxin Deoxynivalenol in Fusarium Resistant and Susceptible Near Isogenic Wheat Lines

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

The ability to detoxify the mycotoxin deoxynivalenol colocalizes with a major quantitative trait locus for Fusarium head blight resistance in wheat

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

Fate of deoxynivalenol and deoxynivalenol-3-glucoside during cereal-based thermal food processing: a review study

Deoxynivalenol (DON), the most commonly occurring trichothecene in nature, may affect animal and human health through causing diarrhea, vomiting, gastrointestinal inflammation, and immunomodulation. DON-3-glucoside (DON-3G) as a major plant metabolite of the mycotoxin is another “emerging” food safety issue in recent years. Humans may experience potential health risks by consuming DON-contaminated food products. Thus, it is crucial for human and animal health to study also the degradation of DON and DON-3G during thermal food processing. Baking, boiling, steaming, frying, and extrusion cooking are commonly used during thermal food processing and have promising effects on the reduction of mycotoxins in food. For DON, however, the observed effects of these methods, as reported in numerous studies, are ambiguous and do not present a clear picture with regard to reduction or transformation. This review summarized the influence of thermal processing on the stability of DON and the formation of degradation/conversion products. Besides this, also a release of DON and DON-3G from food matrix as well as the release of DON from DON-3G during processing is discussed. In addition, some conflicting findings as reported from the studies on thermal processing as well as cause-effect relationships of the different thermal procedures are explored. Finally, the potential toxic profiles of DON degradation products are discussed as well when data are available.     

(−)-Epigallocatechin gallate suppresses the cytotoxicity induced by trichothecene mycotoxins in mouse cultural macrophages

Trichothecene mycotoxins are toxic secondary metabolites produced by a number of fungi including Fusarium species, which adversely affect lymphocytes. Deoxynivalenol (DON) and HT-2 toxin (HT-2) belong to the trichothecene group of mycotoxins and the occurrence of cereals and foodstuffs with these compounds are serious health problems. The aim of this study was to examine the effect of (−)-epigallocatechin gallate (EGCG), one of the main components in green tea catechins, on DON- or HT-2-induced cytotoxicity in mouse macrophages. EGCG had protective effects against the trichothecene-induced cytotoxicities of both mycotoxins. Additionally, EGCG suppressed the DON-induced activation of caspase-3/7, which is an indicator of apoptosis. These results indicate that EGCG might be useful in protection against DON- or HT-2-induced cell death, suggesting that EGCG could contribute to reducing the toxicities of trichothecenes.     

Trichothecene Content of Rye and Wheat Genotypes Inoculated with a Deoxynivalenol- and a Nivalenol-producing Isolate of Fusarium culmorum

Head blight caused by Fusarium culmorum may lead to yield reduction and the contamination of cereal grain with the mycotoxins deoxynivalenol (DON), 3-acetyl deoxynivalenol (3-ADON), nivalenol (NIV), fusarenone-X (FUS), and others. In this study, the covariation between DON and NIV accumulation of 12 rye and eight wheat genotypes that differed in resistance were analysed by inoculating them with a DON-and a NIV-producing isolate, respectively, in three locations. The resistance traits head blight rating and plot yield relative to the uninoculated plots of the same genotype were assessed and the contents of DON, 3-ADON, NIV, and FUS in the grain were analysed by gas chromatography with mass spectrometry. The NIV-producing isolate was significantly (P=0.05) less aggressive and led to a considerably lower mean NIV content in the grain compared with the aggressiveness and mean DON content of the DON-producing isolate (19.5 mg NIV/kg grain versus 48.4 mg DON/kg). Wheat and rye genotypes significantly differed in their DON and NIV accumulation. All genotypes reacted in a similar manner to both chemotypes of F. culmorum for the resistance traits and the respective mycotoxin contents with the exception of one wheat variety, that caused a change in rank order for mycotoxin content. In conclusion, resistance to head blight and tolerance to mycotoxin accumulation seems to be most likely the same for DON- and NIV-producing isolates of F. culmorum .     

Identification of a UDP-glucosyltransferase conferring deoxynivalenol resistance in Aegilops tauschii and wheat

Aegilops tauschii is the diploid progenitor of the wheat D subgenome and a valuable resource for wheat breeding, yet, genetic analysis of resistance against Fusarium head blight (FHB) and the major Fusarium mycotoxin deoxynivalenol (DON) is lacking. We treated a panel of 147 Ae. tauschii accessions with either Fusarium graminearum spores or DON solution and recorded the associated disease spread or toxin-induced bleaching. A k-mer-based association mapping pipeline dissected the genetic basis of resistance and identified candidate genes. After DON infiltration nine accessions revealed severe bleaching symptoms concomitant with lower conversion rates of DON into the non-toxic DON-3-O-glucoside. We identified the gene AET5Gv20385300 on chromosome 5D encoding a uridine diphosphate (UDP)-glucosyltransferase (UGT) as the causal variant and the mutant allele resulting in a truncated protein was only found in the nine susceptible accessions. This UGT is also polymorphic in hexaploid wheat and when expressed in Saccharomyces cerevisiae only the full-length gene conferred resistance against DON. Analysing the D subgenome helped to elucidate the genetic control of FHB resistance and identified a UGT involved in DON detoxification in Ae. tauschii and hexaploid wheat. This resistance mechanism is highly conserved since the UGT is orthologous to the barley UGT HvUGT13248 indicating descent from a common ancestor of wheat and barley.     

A Versatile Family 3 Glycoside Hydrolase from Bifidobacterium adolescentis Hydrolyzes β-Glucosides of the Fusarium Mycotoxins Deoxynivalenol,Nivalenol, and HT-2 Toxin in Cereal Matrices

Glycosylation plays a central role in plant defense against xenobiotics, including mycotoxins. Glucoconjugates of Fusarium toxins, such as deoxynivalenol-3-O-β-d-glucoside (DON-3G), often cooccur with their parental toxins in cereal-based food and feed. To date, only limited information exists on the occurrence of glucosylated mycotoxins and their toxicological relevance. Due to a lack of analytical standards and the requirement of high-end analytical instrumentation for their direct determination, hydrolytic cleavage of β-glucosides followed by analysis of the released parental toxins has been proposed as an indirect determination approach. This study compares the abilities of several fungal and recombinant bacterial β-glucosidases to hydrolyze the model analyte DON-3G. Furthermore, substrate specificities of two fungal and two bacterial (Lactobacillus brevis and Bifidobacterium adolescentis) glycoside hydrolase family 3 β-glucosidases were evaluated on a broader range of substrates. The purified recombinant enzyme from B. adolescentis (BaBgl) displayed high flexibility in substrate specificity and exerted the highest hydrolytic activity toward 3-O-β-d-glucosides of the trichothecenes deoxynivalenol (DON), nivalenol, and HT-2 toxin. A K_m of 5.4 mM and a V_max of 16 μmol min⁻¹ mg⁻¹ were determined with DON-3G. Due to low product inhibition (DON and glucose) and sufficient activity in several extracts of cereal matrices, this enzyme has the potential to be used for indirect analyses of trichothecene-β-glucosides in cereal samples.     

The identification of rye trisomics by translocations and Giemsa staining

By the Giemsa C-banding of six rye (Secale cereale) trisomics and by crossing them to translocation tester stocks it was possible to identify the trisomics and the tester stocks so that their correspondence to the wheat homoeologous groups could be established. The Heines Hellkorn trisomics 1/23, 4/11, 4/9, 1/19, 1/21 and 3/23 were found to correspond to Sears' Chinese Spring/Imperial additions E, G, C, A, F and D respectively. These additions most probably correspond to the wheat homoeologous groups 1, 3, 4, 5, 6 and 7.     

A ras GTPase-activating protein-binding protein, TaG3BP, associated with the modulation of male fertility in a thermo-sensitive cytoplasmic male sterile wheat line

Hybrid wheat development may contribute to higher, more stable yield and could result in greater food security for much of the world's growing population. YS type thermo-sensitive cytoplasmic male sterile (TCMS) wheat lines were developed for use in hybrid wheat breeding in China. To investigate the molecular mechanism of modulation of male fertility in the YS-type TCMS wheat lines, a ras GTPase-activating protein-binding protein (TaG3BP) was examined. The deduced amino acid sequence encoded by TaG3BP was conserved in the sequenced genomes of Embryophyte. TaG3BP expression in the anthers of YS-type TCMS lines taken at the critical fertility reversion stage of pollen development from male fertile anthers was higher than that from male-sterile anthers, either by quantitative real-time PCR or by western blot analysis. Sequence analysis on the cDNA and genomic DNA of TaG3BP in three kinds of K-type CMS wheat lines and their maintainers indicated that there were no significant difference between the genes or in their 5' flanking sequences. The TaG3BP expression revealed by quantitative real-time RT-PCR was lower in the young spikes of these CMS lines than that of their maintainers. This indicates that TaG3BP expression is associated with the modulation, from male-sterile to fertile, of the TCMS wheat line, and TaG3BP might be a key factor in the pathway responsible for the fertility reversion.     

Flow sorting of mitotic chromosomes in common wheat (Triticum aestivum L.)

The aim of this study was to develop an improved procedure for preparation of chromosome suspensions, and to evaluate the potential of flow cytometry for chromosome sorting in wheat. Suspensions of intact chromosomes were prepared by mechanical homogenization of synchronized root tips after mild fixation with formaldehyde. Histograms of relative fluorescence intensity (flow karyotypes) obtained after the analysis of DAPI-stained chromosomes were characterized and the chromosome content of all peaks on wheat flow karyotype was determined for the first time. Only chromosome 3B could be discriminated on flow karyotypes of wheat lines with standard karyotype. Remaining chromosomes formed three composite peaks and could be sorted only as groups. Chromosome 3B could be sorted at purity >95% as determined by microscopic evaluation of sorted fractions that were labeled using C-PRINS with primers for GAA microsatellites and for Afa repeats, respectively. Chromosome 5BL/7BL could be sorted in two wheat cultivars at similar purity, indicating a potential of various wheat stocks for sorting of other chromosome types. PCR with chromosome-specific primers confirmed the identity of sorted fractions and suitability of flow-sorted chromosomes for physical mapping and for construction of small-insert DNA libraries. Sorted chromosomes were also found suitable for the preparation of high-molecular-weight DNA. On the basis of these results, it seems realistic to propose construction of large-insert chromosome-specific DNA libraries in wheat. The availability of such libraries would greatly simplify the analysis of the complex wheat genome.     

小麦品种间感染纹枯病的差异及普遍率与严重度的关系

通过人工接菌方法比较了６个小麦主栽品种间感染纹枯病的差异,结果表明,高感类型有阜阳８６１、温麦４号和皖麦１９,中感类型有扬麦１５８、豫麦１８和豫麦２１．各品种苗期病株率反映不出品种感病程度的差异,以灌浆后期的病情指数为标准比较品种间抗感染程度较为适宜．认为寄主生育阶段影响小麦纹枯病的ＩＳ关系．回归分析表明,按内茎和外茎发病程度分级可减少田间调查误差,且省时省工．     

Genetic mapping of the wheat dimeric α-amylase inhibitor multi-gene family using allele-specific primers based on intergenomic SNPs

Single nucleotide polymorphisms (SNPs) identified in EST sequences can be used to map expressed genes. Though SNPs are useful markers for genetic mapping, SNP mapping of genes in common wheat is challenging because the genetic complement of wheat consists of three closely related genomes (designated A, B, and D), and most genes are present in triplicate sets. Mapping multi-gene family members is further complicated by the fact that it is difficult to distinguish SNP differences between the various paralogs from those between the different genomes. We have developed a PCR-based method for assigning wheat EST sequences to their proper genetic loci by first identifying and mapping SNPs that distinguish the three genomes. To develop this method, we focused on EST sequences encoding the dimeric α-amylase inhibitors (WDAI), The WDAI coding regions of hexaploid wheat were aligned. The sequences were classified into three groups based on nucleotide variations. Twenty-two SNPs were identified that distinguish the three groups. Group-specific primers based on these SNPs were designed to permit selective amplification of each group. The chromosomal location of each group was then determined using Group 3 ditelosomic lines of Chinese Spring. Groups 1 and 2 were assigned to chromosome locations 3DS and 3BS, respectively, whereas no sequence could be assigned to 3AS. A remarkable feature of this method is the ability to discriminate the location of homoeologous multigenes in the three genomes of wheat. This strategy can be useful for assigning unknown wheat EST sequences to specific chromosomes.     

外源化合物诱导后小麦对麦长管蚜和粘虫的抗虫性研究

喷施外源化合物和昆虫取食均可诱导小麦的防御反应,如挥发物组成发生改变、某些次生物质含量增加或减少,进而影响昆虫和天敌的行为反应。本实验中小麦苗经茉莉酸或几丁质喷施诱导,可显著提高小麦中丁布的含量,但茉莉酸甲酯的诱导作用不明显。喷施茉莉酸及麦长管蚜或粘虫取食诱导小麦,会导致小麦挥发物的种类及含量与对照相比有显著不同,3种处理间小麦的挥发物也存在显著差异。用经茉莉酸处理的麦苗饲养麦长管蚜和粘虫,可显著抑制二者的生长发育速度,使体重减轻,并导致麦长管蚜繁殖力下降。经茉莉酸处理的麦苗的挥发物对粘虫寄生蜂有吸引作用,表明茉莉酸在诱导小麦产生直接抗虫性的同时,还能诱导其释放吸引天敌的挥发性化合物从而产生间接抗性。     

α-amylase inhibitors in Triticum aestivum: Purification and physical-chemical properties

Two α-amylase inhibitors in aqueous extracts of wheat flour have been resolved by DEAE-Sephadex chromatography. α-Amylase inhibitor I, the major inhibitor, was homogeneous by disc gel electrophoresis. It had a MW of 20 000 daltons and an isoelectric point of 6·7. α-Amylase inhibitor II had two minor contaminants when analysed by electrophoresis. These inhibitors were classified as typical wheat albumin proteins. A third α-amylase inhibitor was discovered when it was observed that an albumin protein which is found only in Triticum aestivum varieties of wheat could also inhibit pancreatic α-amylase. All three of these inhibitors could be distinguished by their characteristic electrophoretic mobilities.     

小麦根系三维形态建模及可视化

作物三维形态建模和可视化技术是数字植物研究的重要组成部分,本文旨在构建基于形态特征参数的小麦根系三维形态模型,并实现小麦根系生长的可视化.基于小麦根系生长的可视化技术框架,首先构建了小麦根轴的三维显示模型,包括根轴生长模型、分枝几何模型和根轴曲线模型;然后结合根系拓扑结构,确定相应的图元,利用根系形态模型输出的形态特征参数,对整个小麦根系进行三维重构;最后基于OpenGL图形平台,综合纹理映射、光照渲染、碰撞检测等真实感处理手段,实现了小麦根系生长的三维可视化表达.结果表明：模型输出的根系真实感较强,能较好地实现不同品种、水分和氮素条件下小麦根系的三维可视化表达.研究结果为进一步建立完整的可视化小麦生长系统奠定了技术基础.     

Phytohormonal regulation of S-adenosylmethionine synthetase by gibberellic acid in wheat aleurones.

Gibberellic acid (GA3) brought about a 3-fold stimulation of AdoMet synthetase activity in wheat aleurones. At the qualitative level, three isozymes of AdoMet synthetase were observed by DE-52 chromatography in GA3-treated wheat aleurones. In contrast, the control wheat aleurones showed a single isozyme. Thus the phytohormone (GA3, 1 microM) induced two additional isozymes of AdoMet synthetase in wheat aleurones. The activity of all the three isozymes in GA3-treated aleurones was considerably decreased by the simultaneous presence of abscisic acid (ABA, 10 microM). Cycloheximide (20 micrograms/ml) also significantly lowered the levels of the three isozymes of AdoMet synthetase in Ga3-treated aleurones, thereby suggesting the requirement of de-novo protein synthesis for the complete induction of isozymes. However, wheat aleurones excised from embryonated wheat seeds, did not require the application of GA3 for the induction of two additional isozymes of AdoMet synthetase. Apparently, the transport of GA3 from the embryo to aleurones induced two new isozymes of AdoMet synthetase. Three isozymes of AdoMet synthetase were also observed in wheat embryos excised from germinated wheat grains, without exogenous application of GA3. The molecular weight of all the three isozymes of AdoMet synthetase in wheat system is 181,000. The molecular weight of the subunit of the enzyme is 84,000. The dimeric nature of AdoMet synthetase was established by SDS-PAGE analysis of the purified enzyme. In-vitro hybridization of two flanking isozymic peaks I and III by NaCl-freeze-thaw method resulted in the appearance of an additional middle activity peak (isozyme II). However, no additional isozymic peaks were generated when isozymic peaks I and III were individually given a freeze-thaw treatment. Thus the flanking isozymic peaks I and III represent homodimers that differed in their net charge. In contrast, the middle isozymic activity peak II, when subjected to NaCl-freeze-thaw treatments yielded two additional isozymic peaks, I and III, thereby suggesting its heterodimeric nature. We envisage that the three isozymes in GA3-treated wheat aleurone layers are formed by the random dimerization of two classes of enzyme subunits. The two enzyme subunits which differ in their net charge could be the product of two genes of AdoMet synthetase (SAM1 and SAM2). Based on this assumption, we propose that a single isozyme I in water imbibed control wheat aleurones is the product of SAM1 gene of AdoMet synthetase. The occurrence of three isozymes in GA3-treated aleurones could be ascribed to the expression of an alternate gene of AdoMet synthetase (SAM2 gene).(ABSTRACT TRUNCATED AT 400 WORDS)     

供磷水平对间套作物根系酸性磷酸酶活性的影响

选择小麦／大豆和小麦／玉米２种模式,用盆栽法和根系栽培法研究了不同供磷水平对间套作物根系酸性磷酸酶（ＡＰase)活性的影响,试验得出：间套种植种植根系Ａpase的分泌量,套作大表兄弟经单作大豆平均提高３５．９％,而小麦和玉米在间套种植时也有不同程度地提高,说明间套种植有利于土壤有机磷向有效化方向转化,大豆不论单作还是间套作其根系ＡＰase都远高于相应小麦,而小麦又高于玉米,说明大豆利用土壤的潜在的能力大于小麦,而小麦又高于玉米,可见,禾谷类的小麦与大豆间套后不但能改善小麦的氮素营养状况还使磷素营养也得以好转。     

The physiological characteristics of the low canopy temperature wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) genotypes under simulated drought condition

Crop canopy temperature reflects the interactions among plants, soil and atmosphere. Through a long-term investigation into physiological characteristics of different canopy temperature wheat, it is found that some wheat exist a high canopy temperature while others a low one, which was closely correlated with their corresponding performance. Under simulated drought conditions, the physiological aspects of different canopy temperature wheat, such as leaf functional duration, chlorophyll content, activities of superoxide dismutase, protein content, transpiration rate, net photosynthesis rate, etc., were investigated, the result showed that wheat with low canopy temperature could maintain superiority to wheat with high canopy temperature in those physiological traits. Therefore, the low canopy temperature in wheat could be used as an index to evaluate physiological capacities of wheat under drought conditions and also as a useful marker for wheat breeding for drought tolerance.