Identification and characterization of a novel Iraqi isolate of Fusarium pseudograminearum causing crown rot in wheat

Crown rot is one of the main important fungal diseases affecting wheat in many areas of the world, including Australia, USA, and Iran. Until now, there had been no report of this pathogen in Iraq. Plants displaying crown rot symptoms were observed in Shaat Alarab (Basra, Iraq); we investigated the causal agent of the disease. Samples were surface-sterilized in bleach (1% available chlorine) and cultured on quarter-strength potato dextrose agar plates. DNA was extracted from fungal mycelia, using a modified CTAB protocol. The ITS/5.8S regions were amplified using primer pair ITS1 and ITS4. PCR products purified using a gel extraction kit were sequenced. The sequence that was detected was used to BLAST against NCBI data. The most similar sequence was the ITS/5.8S rDNA region of Fusarium pseudograminearum (strain NRRL28062), showing 97.95% identity. This species normally causes crown rot, resulting in severe damage under dry spring conditions. A pathogenicity test employed to assess the disease-causing ability of the strain showed significant disease symptoms up to 57% infected spikelets. The results confirmed the presence of F. pseudograminearum as a causal agent of wheat crown rot in Iraq. The presence of this pathogen demands further investigations to develop resistant cultivars and/or mechanical control.     

A novel approach for efficient plant regeneration from long-term suspension culture of wheat

Summary Hexaploid wheat plants were easily regenerated from young embryo-derived callus for twelve genotypes tested. After a 2.5 years culture period, however, most of the callus cells lost their ability to regenerate into shoots, but not into roots.A novel approach was used to regenerate shoots from the long-term suspension cultured cells. In general, instead of selecting embryogenic callus as source material, this approach requires the inoculation of unselected callus into liquid medium followed by removing the free floating cell portion, selecting out non-root forming cell clumps from the root forming primary suspension culture, and growing the putative shoot-competent clumps in liquid medium with reduced auxin concentrations. We have successfully established shoot-competent wheat suspension cultures for cv. Mustang. High (>80%) frequencies of plant regeneration were observed from plating of 2.5 year suspension cultures. The suspension cultures established by this approach have been utilized to select for heat tolerant variants and will be an ideal source material for protoplast culture and transformation studies. This approach can also be applied to other cereal crops which form roots easily but are unstable in maintaining long term regenerable cultures and which are not easily adaptable to suspension culture.     

Molecular Genetic Diversity in Populations of Fusarium pseudograminearum from Tunisia

Fusarium pseudograminearum is one of the major pathogens causing crown rot of wheat in the semi‐arid and arid areas in Tunisia. In this study, the molecular diversity of 74 isolates of F. pseudograminearum representing three populations from Tunisia and a set of isolates from the world collection was investigated. The potential mycotoxin‐producing ability was tested by PCR using primer pairs specific for the Tri3, Tri7 and Tri13 genes. Results indicated that all the isolates are potentially DON and/or 3‐AcDON producers. The mating‐type idiomorphs were identified using diagnostic PCR primer for MAT1‐1 and MAT1‐2. Both mating types were recovered from the same region and in some cases from the same field. Restriction analysis of the nuclear ribosomal DNA (nrDNA) intergenic spacer region (IGS) revealed 11 haplotypes, five of which were identified in the world collection. The analysis of population structure using the combined IGS and MAT data revealed that the total gene diversity (H_T = 0.108) was mostly attributable to diversity within populations (H_S = 0.102) and that the genetic differentiation among the four populations was low (G_ST = 0.09). The analysis of molecular variance (amova ) showed that 15% of the variability was between the Tunisian populations and the world collection. These findings indicate that quarantine measures should be in place to limit the introduction of new populations of F. pseudograminearum into Tunisia.     

Molecular characterization of bla(IMP-5), a new integron-borne metallo-beta-lactamase gene from an Acinetobacter baumannii nosocomial isolate in Portugal

Postbloom fruit drop (PFD) of citrus is caused by Colletotrichum acutatum. PFD isolates infect flower petals, induce abscission of small fruit and can cause severe yield loss on most citrus cultivars. Isolates from Key lime anthracnose (KLA) cause that disease on the Mexican lime, but also cause PFD on sweet orange. Both PFD and KLA isolates exhibited resistance to the common selection agents including hygromycin, bialaphos, benomyl and geneticin/G418. A genetic transformation system was developed for C. acutatum to confer resistance to sulfonylurea (chlorimuron ethyl) by expressing an acetolactate synthase gene (sur) cassette from Magnaporthe grisea. The protocol was tested on 11 different KLA and PFD isolates. The transformation frequencies were highly variable among isolates and among experiments (0-17.9 per microg circular DNA using 10(7) protoplasts). Southern blot analysis of transformants indicated that the plasmid vector was randomly integrated in multiple copies into the genome of C. acutatum. Addition of restriction enzymes or use of a vector with homologous sequences did not change the transformation frequencies, but tended to reduce the number integrated. Over 97% of the transformants retained the sulfonylurea resistance phenotype under non-selective conditions. Of 300 transformants tested, three were unable to cause necrotic lesions on detached Key lime leaves. The transformation method opens up opportunities for the genetic manipulation of C. acutatum.     

Production and fitness of Fusarium pseudograminearum inoculum at elevated carbon dioxide in FACE

Rising atmospheric carbon dioxide (CO₂) concentration is increasingly affecting food production but how plant diseases will influence production and quality of food under rising CO₂ is not well understood. With increased plant biomass at high CO₂ the stubble‐borne fungal pathogen Fusarium pseudograminearum causing crown rot (CR) of wheat may become more severe. We have studied inoculum production by Fusarium using fungal biomass per unit wheat stubble, stem browning from CR and the saprophytic fitness of Fusarium strains isolated from two wheat varieties grown in 2007 and 2008 at ambient and elevated CO₂ in free‐air CO₂ enrichment (FACE) with or without irrigation and once in a controlled environment. Fungal biomass, determined using primers for fungal ribosomal 18s and the TRI5 gene, increased significantly at elevated CO₂ in two of the three studies. Stem browning increased significantly at elevated CO₂ in the 2007 FACE study. At elevated CO₂ increased stem browning was not influenced by irrigation in a susceptible variety but in a resistant variety stem browning increased by 68% without irrigation. Wheat variety was significant in regression models explaining stem browning and Fusarium biomass but pathogen biomass at the two CO₂ levels was not significantly linked to stem browning. Fusarium isolates from ambient and elevated CO₂ did not differ significantly in their saprophytic fitness measured by the rate of colonization of wheat straw. We show that under elevated CO₂Fusarium inoculum in stubbles will be amplified from increased crop and pathogen biomass while unimpeded saprophytic fitness will retain its effectiveness. If resistant varieties cannot completely stop infection, Fusarium will rapidly colonize stubble to further increase inoculum once the crop is harvested. Research should move beyond documenting the influence of elevated CO₂ to developing disease management strategies from improved knowledge of pathogen biology and host resistance under rising CO₂.     

Regulation of cytokinin content in plant cells

Cytokinin levels in plant cells are dependent on cytokinin biosynthesis and/or uptake from extracellular sources, metabolic interconversions, inactivation and degradation. Cytokinin conversion to compounds differing in polarity seems to be decisive for their entrapment within the cell and intracellular compartmentation, which affects their metabolic stability. Increase in cytokinin levels, resulting either from their uptake or intracellular biosynthesis, may promote further autoinductive accumulation of cytokinins which may function in the induction of cytokinin-initiated physiological processes. Accumulated cytokinins are capable of inducing cytokinin oxidase which consequently decreases cytokinin levels. This seems to be the mechanism of re-establishment and maintenance of cytokinin homeostasis required for further development of physiological events induced by transient cytokinin accumulation. Auxin may influence cytokinin levels by down regulation of cytokinin biosynthesis and/or by promotion of cytokinin degradation. A model of the regulation of cytokinin levels in plant cells based on these phenomena is presented and its physiological role(s) is discussed.     

Plant remains from Maadi,a predynastic site in lower Egypt

The economy of predynastic Maadi, situated on a Pleistocene terrace, was largely based upon the exploitation of the Nile valley close by. Wheat and barley were the main crop plants. Melon (Cucumis melo) was also grown, but there is no firm evidence of local pulse crop and flax cultivation. The identification of wheat grains posed problems; it is not clear whether in addition to Triticum dicoccum, free-threshing wheat (T. durum/aestivum) is also represented. Both two-rowed barley (Hordeum distichum) and the six-rowed form (H. vulgare) could be demonstrated. The arable weed flora included Lolium (temulentum), Vicia spp., Rumex (dentatus), Phalaris (paradoxa), Lathyrus hirsuta and Medicago (tuberculata). A concentration of Acacia seeds including pod segments points to the use of Acacia pods for tanning. The Nile valley produced most of the timber used by the Maadi inhabitants: Acacia, Tamarix, Salix. Scarce remains of coniferous wood (Cedrus, Juniperus, Pinus) are evidence of foreign trade.     

Mycoflora isolation and molecular characterization of Aspergillus and Fusarium species in Tunisian cereals

Wheat, barley and maize are the mainly consumed cereals in Tunisia. This study aimed to determine the mycoflora of these cereals with special focus on the mycotoxigenic Aspergillus and Fusarium species. Freshly harvested samples and other stored samples of each type of cereal (31 and 34 samples, respectively) were collected in Tunisia and cultured for fungal isolation and identification. Identification of fungal genera was based on morphological features. Aspergillus and Fusarium species were identified by species specific PCR assays complemented with DNA sequencing. Alternaria (70.83%), Eurotium (62.50%), Aspergillus (54.17%) and Penicillium (41.67%) were the most frequent fungi isolated from wheat. Penicillium (75%), Aspergillus (70%), Eurotium (65%) and Alternaria (65%) were the most frequently recovered genera from barley. The predominant genera in maize were Aspergillus (76.19%), Eurotium (42.86%), and Penicillium (38.09%). Aspergilllus, Penicillium, Fusarium and Alternaria were detected in both stored and freshly harvested grain samples. The frequencies of contamination with Aspergillus, Fusarium and Alternaria were higher in freshly harvested samples, whereas Penicillium species were more frequent in stored samples. The predominant Aspergillus species detected were A. flavus and A. niger. The Fusarium species detected were F. equiseti, F. verticillioides, F. nygamai, and F. oxysporum. This study suggested the potential risk for Aflatoxins and, to a lesser extent, for Ochratoxin A in Tunisian cereals. This is the first survey about mycoflora associated with wheat, barley and maize in Tunisia.     

Starch branching enzyme IIb in wheat is expressed at low levels in the endosperm compared to other cereals and encoded at a non-syntenic locus

Studies of maize starch branching enzyme mutants suggest that the amylose extender high amylose starch phenotype is a consequence of the lack of expression of the predominant starch branching enzyme II isoform expressed in the endosperm, SBEIIb. However, in wheat, the ratio of SBEIIb and SBEIIa expression are inversely related to the expression levels observed in maize and rice. Analysis of RNA at 15 days post anthesis suggests that there are about 4-fold more RNA for SBE IIa than for SBE IIb. The genes for SBE IIa and SBE IIb from wheat are distinguished in the size of the first three exons, allowing isoform-specific antibodies to be produced. These antibodies were used to demonstrate that in the soluble fraction, the amount of SBE IIa protein is two to three fold higher than SBIIb, whereas in the starch granule, there is two to three fold more SBE IIb protein amount than SBE IIa. In a further difference to maize and rice, the genes for SBE IIa and SBE IIb are both located on the long arm of chromosome 2 in wheat, in a position not expected from rice–maize–wheat synteny.     

Map-based cloning identifies velvet A as a critical component of virulence in Fusarium pseudograminearum during infection of wheat heads

Although better known as a pathogen of wheat stem bases, Fusarium pseudograminearum also causes Fusarium head blight. A natural isolate of F. pseudograminearum was identified that showed severely reduced virulence towards wheat heads and a map-based cloning approach was undertaken to identify the genetic basis of this phenotype. Using a population of 95 individuals, a single locus on chromosome 1 was shown to be responsible for the low virulence. Fine mapping narrowed the region to just five possible SNPs of which one was in the F. pseudograminearum homologue of velvet A. Knockout mutants of velvet A, which were non-pathogenic towards wheat, confirmed that velvet A regulates virulence in this pathogen. The mutation in velvet A was only found in a single field isolate and the origin of the mutation is unknown.     

In vitro cytokinin binding to a particulate fraction of tobacco cells

At least two types of cytokinin-binding sites are present in a particulate fraction of tobacco (Nicotiana tabacum L.) cells that sediments at 80,000 x g. The major binding component has a low affinity towards cytokinins, is resistant to heating at 100°C, and is not specific for biologically active cytokinin analogues. The second site occurs in much lower frequency, is heat labile, shows high affinity towards cytokinins, and is specific for biologically active analogs of the hormone. The testing for binding specificity was mainly performed with a series of halogenated benzyladenine derivatives having a wide range of biological activities. The low-affinity binding site shows some of the same features as talcum powder, a non-biological material which binds cytokinins in a non-specific fashion. The properties of the high-affinity binding site are consistent with the expected characteristics of a cytokinin receptor. However, the role of the observed high-affinity binding site with regard to the biological action of cytokinins is not yet known.Abbreviations BA N ⁶-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - K_d equilibrium dissociation constant - R_t total concentration of binding sites In partial fulfillment of the requirements for the Ph.D. degree in the Department of Botany and Plant Pathology, Michigan State University     

In vitro cytokinin binding to a particulate cell fraction from protonemata of Funaria hygrometrica

Cytokinin-induced bud formation in moss protonemata is specific for cytokinin bases, their ribosides being relatively inactive. Binding of [³H]benzyladenine (BA) to a 13,000–80,000 x g subcellular fraction from extracts of Funaria hygrometrica (L.) Sibth. was measured by a centrifugation assay. Increasing concentrations of non-radioactive BA decreased the binding proportionally to the logarithm of the BA concentration between 3×10^-8 and 10^-4M. [³H]Zeatin also bound to these fractions, although the extent of binding was not as great as with [³H]BA. Biologically active cytokinins, including BA, zeatin, 6-(3-methyl-2-enylamino)purine (IPA) and kinetin, competed for the binding of [³H]BA, whereas the ribosides of BA, zeatin and IPA competed poorly. Other biologically inactive compounds, such as adenine and 9-methyl-BA, were also ineffective as competitors. The ability to bind BA by the 13,000–80,000 x g fraction was greatly reduced by treatment with 1% Triton X-100, and heat treatment eliminated more than one-half of the binding activity. Competitive binding appeared to be pH-dependent, with maximal activity between pH 6.0 and 6.5. After fractionation by differential centrifugation, the ability to bind cytokinins was not correlated with the RNA content of the fraction and thus probably did not represent binding to ribosomes which has been reported in other plant tissues. Cytokinins also exhibited competitive binding to non-biological materials, e.g., talc. The detailed characteristics of the binding of BA to talc were different from those to the biological fractions. However, the problem remains, in all studies of cytokinin binding, to distinguish between binding that is biologically meaningful, and biological (biologically) non-meaningful physical adsorption.Abbreviations BA N⁶-benzyladenine - IPA 6-(3-methyl-2-enylamino)purine - 9-MeBA N⁶-benzyl-9-methyladenine     

Biosynthesis of lectin in roots of germinating and adult cereal plants

Root tips of wheat, rye, barley and rice seedlings contain lectins which are identical to the respective embryo lectins with respect to their molecular weight, sugar-specificity and serological properties. Using in vivo labelling techniques, it could be demonstrated that lectin is synthesized de novo in these tissues. The presence of lectin mRNA in seedlings was confirmed by in-vitro synthesis of lectin in root-tip extracts. Lectin synthesis occurs both in primary and first adventitious roots and is confined to the apical part (2mm) of the root. As seedling development proceeds, lectin synthesis in root tips gradually decreases. Adventitious roots of adult (five to six months old) wheat, rye and barley, but not rice, plants also contain lectins which are indistinguisable from the embryo lectins by the above-mentioned criteria. These lectins are synthesized in vivo in isolated root tips (5 mm) with labelled cysteine and in vitro in cell-free extracts prepared from root tips. Synthesis of lectin in roots of adult plants is also confined to the apical (2 mm) tip of the roots. At the molecular level, root lectin synthesis is very similar to that in embryos. All root lectins are synthesized as 23 000-M_r precursors which are post-translationally converted into the mature 18 000-M_r polypeptides. The observation that seedling roots and adventitious roots of six-month-old plants actively synthesize lectins strongly indicates that lectin genes are expressed in these tissues. In addition, since the root lectins are indistinguishable from the embryo lectins, we postulate that the same lectin genes are expressed.Abbreviations ABA abscisic acid - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - WGA wheat-germ agglutinin     

Interactions of temperature and water potential in displacement of Fusarium pseudograminearum from cereal residues by fungal antagonists

Fusarium pseudograminearum (Fp) is a stubble-borne fungus that causes crown rot in wheat and barley. Displacement of Fp from stubble by other fungi was explored. Fungi were isolated from field collections of stubble and identified using morphological and molecular methods. The most abundant species were Alternaria sp., Sterile sp. 1 and Fp. Representative isolates of all species were screened in dual culture with Fp to select candidates for detailed experiments on displacement. Trichoderma harzianum (Th), Alternaria infectoria (Ai), Fusarium equiseti (Fe) and Fusarium nygamai (Fn) were chosen as representing the range of interaction types found. The effects of temperature (5–35 °C) and water potential (?0.3 to ?5 MPa) on growth rates, displacement of Fp from infested barley straw and dual culture interaction with Fp were determined. Th was the strongest antagonist overall, but displaced Fp very poorly at the lowest temperatures and water potentials. It was more antagonistic than expected from effects on growth at high temperature and less antagonistic than expected at low temperature. Fe and Fn showed consistent mutual antagonism with Fp on contact in dual culture and gave moderate displacement of Fp from straw, with Fe being the most effective antagonist under cool dry conditions. Ai gave very poor displacement of Fp from straw, despite being the most common straw fungus, confirming that displacement was due to antagonism and not just the presence of other fungi. The type of interaction in dual culture was consistent with degree of displacement from straw and could be used to indicate which environmental conditions were most limiting for an antagonist. The ability to displace Fp under cool dry conditions appears to be critical.     

Regulation of cytokinin biosynthesis, compartmentalization and translocation

Cytokinins, a group of mobile phytohormones, play an important role in plant growth and development, and their activity is finely controlled by environmental factors in the control of morphogenic and metabolic adaptations. Inorganic nitrogen sources, such as nitrate, are a major factor regulating gene expression of adenosine phosphate-isopentenyltransferase (IPT), a key enzyme of cytokinin biosynthesis. Modulation of IPT and macronutrient transporter gene expression in response to nitrate, sulphate and phosphate, and cytokinin-dependent repression of the transporter genes suggest that cytokinins play a critical role in balancing acquisition and distribution of macronutrients. Biased distribution of trans-zeatin (tZ)-type cytokinins in xylem and N(6)-(Delta(2)-isopentenyl)adenine (iP)-type cytokinins in phloem saps suggest that, in addition to acting as local signals, cytokinins communicate acropetal and systemic long-distance signals, and that structural side chain variations mediate different biological messages. The compartmentalization of tZ- and iP-type cytokinins implies the involvement of a selective transport system. Recent studies have raised the possibility of subsets of the purine permease family as a transporter of cytokinin nucleobases and equilibrative nucleoside transporters (ENT) for cytokinin nucleosides. These biochemical and transgenic data suggest that AtENT6, an Arabidopsis ENT, could also participate in cytokinin nucleoside transport with a preference for iP riboside in vascular tissue.     

Identification of 11 polymorphic simple sequence repeat loci in the phytopathogenic fungus Fusarium pseudograminearum as a tool for genetic studies

Simple sequence repeat (SSR) markers for Fusarium pseudograminearum with 2 to 3 bp repeat motifs were identified by screening the genome database of the related species Fusarium graminearum. Twelve SSRs amplified single loci in both F. graminearum and F. pseudograminearum. Forty F. pseudograminearum and six F. graminearum individual isolates were screened to determine levels of polymorphism, with all SSRs displaying three to 14 alleles across all isolates. Eleven SSRs were polymorphic across F. pseudograminearum isolates tested proving the usefulness of genome databases of closely related species in identifying genetic markers.     

Dynamics of expression and distribution of cytokinin oxidase/dehydrogenase in developing maize kernels

Maturing maize kernels are a rich source of cytokinins and cytokinin oxidase/dehydrogenase activity, but the relationship between kernel development, cytokinin levels, the induction of cytokinin oxidase/dehydrogenase and the control of cell division is not known. Using polyclonal antibodies raised against recombinant maize cytokinin oxidase/dehydrogenase, we investigated the appearance of cytokinin oxidase/dehydrogenase (ZmCKX1) in both hybrid and inbred maize kernels as a function of time after pollination. Cytokinin oxidase/dehydrogenase was detected by five days after pollination (5 DAP) in a hybrid line, but significantly later in inbred lines. The bulk of the cytokinin oxidase/dehydrogenase detected was associated with the embryo and placental/chalazal region of the kernels rather than with the endosperm. We identified additional maize sequences in the database that appear to encode cytokinin oxidase/dehydrogenase gene family members and correspond closely with a subset of the ten cytokinin oxidase/dehydrogenase genes identified in the rice genome. Gene expression of Zmckx1 was examined by RT-PCR in immature kernels and compared with that of three putative maize cytokinin oxidase/dehydrogenase homologs. We conclude that the manipulation of kernel cytokinin levels to increase endosperm cell division will require a more detailed understanding of specific expression patterns and localization of multiple cytokinin oxidase/dehydrogenases within kernels.