Earlier onset of DNA fragmentation in leaves of wheat compared to barley and rye

We have found extensive nucleosomal fragmentation of native DNA extracted from leaves of healthy cereal plants, as indicated by ladder patterns on agarose gels and TUNEL staining. The time of first appearance of fragmentation differed among cereals. Native DNA from the first leaf of 10-day-old plants formed a clear ladder pattern of multiples of 180 bp fragments in wheat and triticale but not in barley and oats. In one cultivar of rye a weak ladder pattern occurred but not in another. Freezing and thawing of samples before DNA extraction resulted in much more extensive DNA fragmentation in wheat but not in rye and barley, indicating that DNA-degrading enzymes are present in the cytoplasm of wheat, but not in barley and rye, at this stage. In barley, nucleosomal fragmentation was first detected in 25-day-old plants. These results indicate that programmed cell death takes place in developing leaves of young cereal plants, but that the time of onset differs among cereal species.     

<Emphasis Type="Italic">Penicillium verrucosum</Emphasis> occurrence and Ochratoxin A contents in organically cultivated grain with special reference to ancient wheat types and drying practice

This study addresses the relationship between the ochratoxigenic strains of Penicillium verrucosum and ochratoxin A (OTA) contents in organically cultivated grain. It included 37 combined, non-dried grain samples from farmers with no drying facilities as well as 19 non-dried and 22 dried samples from six farms with on-farm drying facilities (Case studies 1–6). The study focused on the ancient wheat type spelt but also included samples of wheat, rye, barley, oats, triticale, emmer, and einkorn. All 78 samples were analysed for moisture content (MC) and occurrence of P. verrucosum. The latter was assessed by plating non-disinfected kernels on DYSG agar and counting those contaminated by the fungus. Fifty-five samples were analysed for OTA. Most of the combine harvested samples (82%) were contaminated with P. verrucosum prior to drying. This was ascribed to difficult harvest conditions and many samples of spelt, which was significantly more contaminated by P. verrucosum than oats, wheat and barley. Though not statistically significant, the results also indicated that spelt was more contaminated than rye, which is usually regarded the most sensitive small grain cereal. No correlation was found between number of kernels contaminated by P. verrucosum and OTA content. Despite many non-dried samples being contaminated by P. verrucosum, only two exceeded the EU maximum limit for grain (5 ng OTA g^–1), both being spring spelt with 18 and 92 ng g^–1, respectively. The problems were most likely correlated to a late harvest and high MC of the grain. The case studies showed exceedings of the maximum limit in a batch of dried oats and spring wheat, respectively, probably to be explained by insufficient drying of late harvested grain with high MC. Furthermore, our results clearly indicate that OTA is not produced in significant amounts in samples with MCs below 17%. All dried samples with MCs above 18% exceeded the 5 ng OTA g^–1 limit in grain. However, no correlation between MC and the amount of OTA produced was found.     

II. Cereal disease: the interplay between resistance and pathogenicity: Inheritance of pathogenicity in Gaeumannomyces graminis var. tritici

Variation in host response of isolates of the eyespot pathogen from different sources was examined over a number of years. Pathogen types were found in intensively-cropped couch-infested cereal sites that were almost as virulent on Agropyron repens (couch) as on wheat or barley. The commonly occurring wheat (W) type isolates from couch-free cereal crops were virulent on wheat and barley but avirulent on couch. Couch (C) types were isolated not only from couch but also from wheat, barley and oat crops with couch infestation. In pathogenicity tests on rye, C. types did not differ in virulence from the more commonly occurring W types. Aegilops ventricosa was equally resistant to both types. W type isolates from wheat and barley were examined to assess differential pathogenicity on wheat and barley. Sequential cropping with single cereal crops was used to separate out possible specific types. Isolates from fourth wheat and fourth barley crops were more pathogenic on the original than on the alternative host. When comparisons were made between isolates from third and fifth consecutive wheat and barley crops only those from barley showed a preference for the original host. An experiment comparing isolates from third and seventh consecutive wheat and barley crops showed a decline in virulence from the short to the longer sequences on the alternative but not on the original host.     

Development of a UHPLC-FLD method for the analysis of ergot alkaloids and application to different types of cereals from Luxembourg

Ergot alkaloids are toxins produced by some species of fungi in the genus Claviceps, that may infect rye and triticale and, in a minor degree, other types of cereals. In this study, a new UHPLC-FLD method for the quantification of the six major ergot alkaloids as well as their corresponding epimers was developed. The sample preparation was done by a solid-liquid extraction with acetonitrile and clean-up via freeze-out. The method was fully validated and then applied to 39 samples (wheat, rye, triticale, and barley) harvested in Luxembourg in 2016. Samples were sieved (1.9?×?20 mm) prior to analysis in order to remove sclerotia, hosting the alkaloids. However, 23 samples still contained at least one ergot alkaloid >?LOQ and concentrations of the sum of the 6 ergot alkaloids ranged from 0.3 to 2530.1 μg/kg. Interestingly, the highest concentrations were measured in wheat and not in rye or triticale, suggesting that all kinds of cereals should be included in monitoring programs. The outcome of this study allowed giving a first overview of ergot alkaloid concentrations in cereals harvested in Luxembourg, and the measured concentrations were in similar ranges than in other parts of the world (e.g., Canada, France, Germany).     

Repeated sequence DNA relationships in four cereal genomes

The effect of DNA fragment size on the extent of hybridisation that occurs between repeated sequence DNAs from oats, barley, wheat and rye has been investigated. The extent of hybridisation is very dependent on fragment size, at least over the range of 200 to 1000 nucleotides. This is because only a fraction of each fragment forms duplex DNA during renaturation. From these results estimates of the proportions of repeated sequences of each of the cereal genomes that are homologous with repeated sequences in the other species have been determined and a phylogenetic tree of cereal evolution constructed on the basis of the repeated sequence DNA homologies. It is proposed that wheat and rye diverged after their common ancestor had diverged from the ancestor of barley. This was preceded by the divergence of the common ancestor of wheat, rye and barley and the ancestor of oats. Once introduced in Gramineae evolution most families of repeated sequences appear to have been maintained in all subsequently diverging species. — The repeated sequences of oats, barley, wheat and rye have been divided into Groups based upon their presence or absence in different species. Repeated sequences of related families are more closely related to one another within a species than between species. It is suggested that this is because repeated sequences have been involved in many rounds of amplification or quantitative change via unequal crossing over during species divergence in cereal evolution.     

Lacewings (Chrysopidae,Neuroptera) in cereal agrocenoses of the forest-steppe of Western Siberia

Twelve species of lacewings: Chrysopa altaica, Ch. commata, Ch. perplexa, Ch. phyllochroma, Ch. dasyptera, Ch. carnea, Ch. formosa, Ch. intima, Ch. perla, Ch. prasina, Ch. septempunctata, and Nineta inpunctata were found in cereal agroecosystems of the forest-steppe zone of Western Siberia. The dominant species were Ch. carnea and Ch. phyllochroma. The biological characteristics, seasonal dynamics of the abundance of lacewings in the agrocenoses of winter rye, spring wheat, and oats are given. The abundance of larval and adult lacewings in the spring wheat agrocenosis was not affected by the level of chemicalization (upon condition of the rational use of insecticides), tillage variant, and predecessor crop. In the years when the density of lacewings was low, no differences in their population were found between the agrocenoses of wheat (after fallow), winter rye, oats, vetch-oats, canola, barley, and barley with melilot. In the years characterized by relatively high abundance of lacewings, they occurred more frequently in the crop rotations with wheat after fallow and with oats. These plants were settled by cereal aphids to the greatest extent. In all the years studied, the density of lacewings on alfalfa was 2–2.8 times as great as that on wheat after fallow.     

Cytoplasmic DNA variation and relationships in cereal genomes

Summary Chloroplast (cp) and mitochondrial (mt) DNAs were isolated from four cereal genomes (cultivated wheat, rye, barley and oats) and compared by restriction nuclease analysis. Cleavage of cp and mt DNAs by Sal I, Kpn I, Xho I and EcoR I enzymes indicated that each cereal group contains specific cytoplasmic DNAs. A phylogenetic tree of cereal evolution has been obtained on the basis of cp DNA homologies. It is suggested that wheat and rye diverged after their common ancestor had diverged from the ancestor of barley. This was preceded by the divergence of the common ancestor of wheat, rye and barley and the ancestor of oats.The molecular weight of the different cp DNAs was determined from the Sal I and Kpn I patterns. cp DNAs from wheat, rye, barley and oats appeared to be characterized by a very similar molecular weight of about 80–82.10⁶ d.In the case of the mt DNAs, the great number of restriction fragments obtained with the restriction enzymes used prevented precise comparisons and determination of molecular weights.     

Concentration of ergosterol in small-grained naturally contaminated and inoculated cereals

Ergosterol (ERG) is a major sterol constituent of most fungi. Its concentration is negligible in higher plants, but can be used as a chemical marker of the presence of fungal contaminations. In this study, ERG concentration was assessed in randomly collected samples of naturally contaminated grain (wheat, barley and oat) and in samples of grain (wheat, barley, triticale and oat) harvested after inoculation of heads with conidia of different Fusarium species. Wheat samples were analysed at three stages of grain development. The lowest ERG concentration was found in non-inoculated samples at the first stage of grain development. This concentration was increasing with grain ripening. In naturally contaminated samples collected after harvest, ERG concentration was lower in wheat than in barley and oat. ERG concentrations in inoculated samples varied significantly, but were always significantly higher than in naturally contaminated samples. In the above cereal samples it was much lower than the levels assayed in laboratory cultures inoculated with fungi from genus Fusarium. The content of ERG was also analyzed in milling products of small-grained cereals and other foodstuffs, where a considerable variation was observed. The lowest ERG amounts were assayed in flours with a high degree of purification, while the highest ones in case of flours and products with a low purification rate. The results indicate the potential application of HPLC combined with microwave-assisted extraction both when assaying samples with low ERG concentrations (naturally contaminated) and those characterized with high contents of fungal biomass (strongly infected, artificially inoculated). It also facilitates analyses of fungal biomass in technological processes, where results may be expected to vary considerably.     

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.     

Distribution of ergot alkaloids and ricinoleic acid in different milling fractions

The sclerotia of the fungus Claviceps sp. are still a challenge for the milling industry. Ergot sclerotia are a constant contamination of the rye crop and have to be removed by modern milling technologies. Changing sizes and coloration of the sclerotia make it difficult to separate them from the grain. Ergot sclerotia are a problem when cleaning is insufficient and non-separated specimens or sclerotia fragments get into the milling stream and thus ergot alkaloids are distributed into the different cereal fractions. In model milling experiments, the residues of ergot in rye flour and the distribution of ergot into different milling fractions were investigated. Rye grains were mixed with whole ergot sclerotia and in another experiment with ergot powder and cleaned afterwards before milling. The ergot alkaloids ergometrine, ergosine, ergotamine, ergocornine, ergocryptine, ergocristineand their related isomeric forms (-inine-forms), and additionally ricinoleic acid as a characteristic component of ergot, were quantified in the different milling fractions. From the first experiment, it can be shown that after harvesting even simple contact of sclerotia with bulk grains during ordinary handling or movement of bulk grain in the granary is sufficient to contaminate all the healthy or sound rye grains with ergot alkaloids. Thereby, the amount of ergot residue correlates with the amount of peripheral layers of rye grains in the flour. In an additional experiment without sclerotia specimens, bulk rye grains were loaded with powder of sclerotia. After subsequent cleaning, aconcentration of ergot alkaloids was detected, which was tenfold higher than the ergot alkaloidconcentration of the experiment with intact ergot sclerotia.     

Pathogenicity of Fusarium graminearum Schwabe isolates from Poland towards seedlings of cereal species

Ten isolates ofFusarium graminearum Schwabe originating from diseased cereal plants and kernels were tested for pathogenicity to various cultivars of wheat, rye, triticale and oats. The isolates varied greatly in their pathogenicity to the seedlings of the species, and were most pathogenic to rye and triticale, less pathogenic to barley and wheat and least pathogenic to oats.     

Determination of ergot alkaloids in feed by HPLC

A HPLC method for the determination of ergometrine, ergotamine, ergocristine, α-ergocryptine and ergocornine in cereals for animal feed and in mixed feed with high cereal content was developed. Samples were extracted under acidic conditions using a mixture of phosphoric acid and acetonitrile, the extract purified with solid phase extraction cartridges (strong cation exchange), and ergot alkaloids detected after gradient elution on a C18 column by HPLC with fluorescence detection. Detection and determination limits for each individual alkaloid were at 5 (μ/kg and 10 (μg/kg, respectively. With this method, high recovery (82–120%) and good reproducibility was achieved for wheat, rye and mixed feeds, at a sum of total determined alkaloids of < 500 (μg/kg. This method was used to analyse Bavarian feeds (n=124) over three years (2005–2007), and ergot alkaloids were detected in 91 % of the samples. The majority of positive samples had ergot alkaloid contents of < 250 μg/kg, the median alkaloid level was at 70 (μg/kg. The maximum sum of total determined alkaloids exceeded 1000 (μg/kg in wheat, triticale, rye, and mixed feeds, the highest result was obtained for mixed feed (4880 (μg/kg). Parts presented at the Feed Safety Conference, Namur, Belgium, Nov 27–28, 2007     

Composition of guttation fluid from rye, wheat, and barley seedlings

This work was undertaken to determine the kinds and amount of substances that would account for the previously demonstrated differential growth of Claviceps purpurea on guttation fluids from Rosen rye, Genesee wheat, and Traill barley seedlings. Chromatographic methods were used for determining amino acids and sugars, spot tests and spectrometric methods for inorganic materials, and microbiological methods for vitamins.

Total sugar content is about equal in rye and barley fluids, but lower in wheat. Glucose is the principal sugar component of the rye and barley fluids and galactose highest in wheat. Most of the amino acid in all 3 fluids is aspartic acid or asparagine. Barley fluid is far higher than the other 2 in total amino acids, with wheat the lowest. Most inorganic elements are found to be highest in barley and lowest in wheat, with the exception of iron where rye is highest and barley lowest. Barley fluid is highest in choline, p-aminobenzoic acid, thiamine, and uracil, while rye is highest in inositol and pyridoxine. Wheat is much lower than the other 2 in choline and inositol.

     

Variation in chemical composition and physical characteristics of cereal grains from different genotypes

Genotypes of cereal grains, including winter barley (n = 21), maize (n = 27), oats (n = 14), winter rye (n = 22), winter triticale (n = 21) and winter wheat (n = 29), were assayed for their chemical composition and physical characteristics as part of the collaborative research project referred to as GrainUp. Genotypes of one grain species were grown on the same site, except maize. In general, concentrations of proximate nutrients were not largely different from feed tables. The coefficient of variation (CV) for the ether extract concentration of maize was high because the data pool comprised speciality maize bred for its high oil content. A subset of 8 barley, 20 rye, 20 triticale and 20 wheat samples was analysed to differ significantly in several carbohydrate fractions. Gross energy concentration of cereal grains could be predicted from proximate nutrient concentration with good accuracy. The mean lysine concentration of protein was the highest in oats (4.2 g/16 g N) and the lowest in wheat (2.7 g/16 g N). Significant differences were also detected in the concentrations of macro elements as well as iron, manganese, zinc and copper. Concentrations of arsenic, cadmium and lead were below the limit of detection. The concentration of lower inositol phosphates was low, but some inositol pentaphosphates were detected in all grains. In barley, relatively high inositol tetraphosphate concentration also was found. Intrinsic phytase activity was the highest in rye, followed by triticale, wheat, barley and maize, and it was not detectable in oats. Substantial differences were seen in the thousand seed weight, test weight, falling number and extract viscoelasticity characteristics. The study is a comprehensive overview of the composition of different cereal grain genotypes when grown on the same location. The relevance of the variation in composition for digestibility in different animal species will be subject of other communications.     

A direct repeat sequence associated with the centromeric retrotransposons in wheat.

A high-density BAC filter of Triticum monococcum was screened for the presence of a centromeric retrotransposon using the integrase region as a probe. Southern hybridization to the BAC digests using total genomic DNA probes of Triticum monococcum, Triticum aestivum, and Hordeum vulgare detected differentially hybridizing restriction fragments between wheat and barley. The fragments that hybridized to genomic DNA of wheat but not to that of barley were subcloned. Fluorescence in situ hybridization (FISH) analysis indicated that the clone pHind258 hybridized strongly to centromeric regions in wheat and rye and weakly to those in barley. The sequence of pHind258 was homologous to integrase and long terminal repeats of centromeric Ty3-gypsy retrotransposons of cereal species. Additionally, pHind258 has a pair of 192-bp direct repeats. FISH analysis indicated that the 192-bp repeat probe hybridized to centromeres of wheat and rye but not to those of barley. We found differential FISH signal intensities among wheat chromosomes using the 192-bp probe. In general, the A-genome chromosomes possess strong FISH signals, the B-genome chromosomes possess moderate signals, and the D-genome chromosomes possess weak signals. This was consistent with the estimated copy numbers of the 192-bp repeats in the ancestral species of hexaploid wheat.     

Food analysis within the<Emphasis Type="Italic">Fusarium</Emphasis> toxin monitoring programme of Saxony-Anhalt

A 3 yearFusarium andFusarium toxin monitoring programme was established within the food and feed control authorities of Saxony-Anhalt in 2001. The first year’s results of the analysis of deoxynivalenol in cereals and cereal products with assured origin in this federal state, showed a contamination rate of 24% for wheat and wheat products. The contamination incidence reached only 8% in rye and rye products, whereas it was 17% for barley and its products including beer. Zearalenone could be detected only in 2 of 162 analysed samples.     

Analysis of T-2 and HT-2 toxins in oats and other cereals by means of HPLC with fluorescence detection

Bearing in mind the high toxicity of T-2 and HT-2 toxins which occur in cereals (mainly in oats) EU plans legal limits for these mycotoxins. The occurrence data are insufficient because reliable and sensitive analysis methods are not available. A sensitive HPLC gradient method was developed which is applicable with common HPLC equipment (HPLC with fluorescence detection). After extraction of the toxins from sample matrix with methanol/water the diluted extracts were cleaned-up using immunoaffinity columns and then derivatized with 1-anthroylnitrile/DMAP. The T-2 and HT-2 toxins were separated from peaks of the cereal matrix and derivatization reagent by means of a relatively complex HPLC gradient method. The method was validated for oats, wheat, rye, barley, and maize. The recovery rates were in the range of 70–99%, the precision (RSD_R) of 3–8%. The limits of detection of T-2 and HT-2 toxins were 1 μg/kg. A total of 119 samples of cereals and cereal products was analyzed according to the optimized method. The analyses of 54 samples of dehulled oats and of 11 samples of processed oat products from food industry had a contamination frequency of 100%. The contents (sum of T-2 and HT-2 toxins) amounted to 3 to 174 μg/kg for the dehulled oats and to 4 to 48 μg/kg for the processed oat products. 29 samples of maize and maize products had a contamination frequency of 80% (2–106 μg/kg in the sum of T-2 and HT-2 toxins). In the samples of wheat and barley the toxins were detected only occasionally (contents: 1–10 μg/kg), in rye not at all.     

DON- und ZEA-Gehalte in Weizen, Roggen und Gerste: Besondere Ernteermittlung

In the annual national survey (‘Besondere Ernteermittlung’) the quality of German cereals was analysed. The samples for this approach were collected from statistically selected fields. One of the analysed parameters was the content of mycotoxins. Thus, it was possible to get an area-wide survey of the content of mycotoxins under natural growing conditions. In 2003 a total number of 974 samples were analysed consisting of wheat, rye and barley samples. A special mode of choosing the wheat samples ensured also a statistically validated result for the federal states of Germany. In the year 2003 the content of the mycotoxins DON and ZEA in wheat and rye samples was lower than in 2002 referred to the median. On the other hand the frequency of detection of DON was not reduced. In barley, which was analysed for the first time in this kind of study, the level of DON was in between the levels of wheat and rye.     

The occurrence of vesicular-arbuscular mycorrhiza in agro-ecosystems

In a long-lasting field experiment at the Research Station Hanninghof at Dülmen (Westphalia), laid out in 1971, the inoculum potential (IP) of vesicular-arbuscular mycorrhizal (VAM) fungi of soil from winter barley in a continuous monoculture (CM) and in a four-years cereal crop rotation (CR: winter wheat, winter barley, winter rye, oats) was estimated from 1980–1982. The influence of green manure and increased mineral N-fertilization (100 and 200 kg ha^–1) on VAM-IP was also assessed. VAM-IP was high at both levels of N-application, although the higher N-fertilization resulted in a reduction of VAM-IP in winter barley — in CR as well as in CM. No influence of N-fertilization on VAM-IP was observed with oats and winter rye, which were only cultivated in CR. Green manure had a negative effect on IP in CM barley, but not in CR barley. This may be due to the annual applications of green manure in CM, while green manure in CR was only applied prior to the cultivation of oats and winter wheat, but not to winter barley. Except for the effects of green manure remarkable differences in VAM-IP were found neither in CR nor in CM of winter barley. This is also reflected in the yields of barley in CM and indicates the relative self-tolerance of winter barley.     

Brotgetreide

Bread cereals Wheat and spelt are primarily used as bread cereals together with rye. To increase the worldwide wheat production and achieve cultivation goals faster, the very large bread wheat genome is currently analyzed intensively. Wheat is hexaploid and contains three genomes side by side which do not hybridize. The progenitors of einkorn (diploid) and emmer (tetraploid) have been the ancestors of today's wheat and spelt. Spelt is less demanding than wheat but requires an extra stage of husk removal before milling. In Germany, spelt is nowadays a modern bread cereal again. As it has a higher ratio of essential amino acids, the protein part of rye is more valuable for nutrition than that of wheat. Climatic conditions as well as poorer soils in Northern Germany are more suitable for rye than for wheat. Therefore rye has been a typical German bread cereal since medieval times.