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Fate of Escherichia coli O157 and detection of stx phage during fermentation of maize, an animal feedstuff.

AIMS: The fate of inoculated Escherichia coli O157, stx phages and the physico-chemical properties of maize were studied during laboratory-scale fermentation by naturally occurring lactic acid bacteria. METHODS AND RESULTS: Before fermentation, chopped maize was inoculated with 6.2 log(10) CFU g(-1) of a five-isolate mixture of E. coli O157. After fermentation, the silage contained 70.6 g kg(-1) dry matter (DM) lactic acid and 12.8 g kg(-1) DM acetic acid and was pH 4.0. Levels of E. coli O157 fell rapidly, and none of the five isolates could be recovered from the fermenting maize after 8 days. Using a resuscitation step did not consistently enhance recovery of E. coli O157. Stx phages were not isolated from the fermenting maize at any time. Pulsed-field gel electrophoresis (PFGE) genotyping showed that E. coli O157 2975 and 64a/01 survived better than the other three isolates studied. Escherichia coli O157 isolate 1474/00 was particularly sensitive to the laboratory procedures used to harvest the inocula and contaminate the maize. Some colonies recovered during the fermentation had one to three band alterations compared with the initial PFGE genotypes. SIGNIFICANCE AND IMPACT OF THE STUDY: None of the five different E. coli O157 genotypes survived maize fermentation. Fermentation of maize produces an animal feedstuff that is unlikely to contain E. coli O157 or stx phages.

Animal Feed↗

Flow sorting and microcloning of maize chromosome 1.

Flow sorting maize chromosome 1 and construction of the first chromosome 1 DNA Lambda library are described. Maize metaphase chromosome suspensions were prepared from synchronized seedling root tip cells of the maize hybrid line Seneca 60 and stained with propidium iodide for flow karyotyping and sorting. The observed flow karyotype was very similar to the predicted flow karyotype constructed based on published values for the relative chromosome sizes of Seneca 60. The estimated size of chromosomes from the peak for the chromosome 1 matched the expected size of maize chromosome 1. The peak for the chromosome 1 was well resolved from other peaks on the flow karyotype. An average of 7 x 10(3) chromosomes of chromosome 1 could be produced from 10 root tips. About 0.6 million chromosomes of maize chromosome 1 were sorted and pooled based on the cytogram of fluorescent pulse area Vs fluorescent pulse width and stored at -20 degrees C in the freezer. DNA isolated from sorted chromosomes was good quality of more than 100 kb in size. Chromosome 1 DNA was partially digested with BamHI, dephosphorylated and ligated with arms of BamHI digested Lambda Dash vector. A total of 1.2 x 10(5) independent recombinants with the average insert size 12.6 kb was obtained. This library covered approximately 90% of maize chromosome 1. Hybridization of cloned fragments with labeled maize genomic DNA showed that the high, middle, or low copy number DNA sequences presented in the different phage clones. PCR (polymerase chain reaction) using chromosome-specific primers confirmed the specificity of this library. The individual chromosome library is useful in plant genome mapping and gene isolation.

Chromosomes, Plant↗

GISHGenomic in situ hybridization reveals cryptic genetic differences between maize and its putative wild progenitor Zea mays subsp. parviglumis.

The aim of this paper is to test with genomic in situ hybridization the genomic affinities between maize and its putative progenitor Zea mays subsp. parviglumis. Blocking procedures were applied for the purpose of improving discrimination among chromosome regions. Unlabeled genomic DNA from Z. mays subsp. parviglumis as a blocking agent and labeled genomic DNA from maize were hybridized on maize chromosomes. On the other hand, mitotic metaphases from Z. mays subsp. parviglumis were blocked with unlabeled genomic DNA of maize and hybridized with labeled genomic DNA from Z. mays subsp. parviglumis. Both experiments showed that either maize or Z. mays subsp. parviglumis chromosomes have their own unique sequences. This means an unexpected degree of divergence if Z. mays subsp. parviglumis is the only progenitor of maize, a result that is discussed in relation to our previous genomic in situ hybridization observations and to the different scenarios proposed about the origin of maize.

Chromosome Mapping↗

Genomic relationships between maize and its wild relatives

Recent molecular studies confirm the long-held theory that maize is a tetraploid, but the identity of the ancestral diploid species remains an enigma. The various hypotheses were investigated using genomic in situ hybridization (GISH). Total genomic DNA from 10 wild relatives of maize were used as probes onto maize chromosomes to see if this could identify the ancestral genome donors in maize. While none of the taxa hybridized to a subset of chromosomes, genomic DNA from Zea mays ssp. mexicana, Z. mays ssp. parviglumis, Z. diploperennis, Tripsacum dactyloides and Coix lacryma-jobi all showed a similar hybridization pattern consisting of a dispersed signal over all maize chromosomes. Moreover, the first four species also showed highly localized subtelomeric signal on the long arms of maize chromosomes 5, 6, 7, and 8. In contrast, three Sorghum species tested (S. bicolor, S. halapense, and S. versicolor) only showed hybridization at the nucleolar organizer region. In light of recent data on retrotransposon occurrence in maize, the results may provide insights into the timing of speciation of Zea, Tripsacum, and Coix. Data obtained from the tetraploid Z. perennis strongly supported its taxonomic separation from the diploid Z. diploperennis.

Journal Article↗

The genetics of maize evolution.

Maize and its closest wild relatives, the teosintes, differ strikingly in the morphology of their female inflorescences or ears. Despite their divergent morphologies, several studies indicate that some varieties of teosinte are cytologically indistinguishable from maize and capable of forming fully fertile hybrids with maize. Molecular analyses identified one form of teosinte (Zea mays ssp. parviglumis) as the progenitor of maize. Analyses of the inheritance of the morphological traits that distinguish maize and teosinte indicates that they are under the control of multiple genes and exhibit quantitative inheritance. Nevertheless, these analyses have also identified a few loci of large effect that appear to represent key innovations during maize domestication. Remaining challenges are to identify additional major and minor effect genes, the polymorphisms within these genes that control the phenotypes, and how the combination of the individual and epistatic effects of these genes transformed teosinte into maize.

Biological Evolution↗

SNP frequency, haplotype structure and linkage disequilibrium in elite maize inbred lines.

BACKGROUND: Recent studies of ancestral maize populations indicate that linkage disequilibrium tends to dissipate rapidly, sometimes within 100 bp. We set out to examine the linkage disequilibrium and diversity in maize elite inbred lines, which have been subject to population bottlenecks and intense selection by breeders. Such population events are expected to increase the amount of linkage disequilibrium, but reduce diversity. The results of this study will inform the design of genetic association studies. RESULTS: We examined the frequency and distribution of DNA polymorphisms at 18 maize genes in 36 maize inbreds, chosen to represent most of the genetic diversity in U.S. elite maize breeding pool. The frequency of nucleotide changes is high, on average one polymorphism per 31 bp in non-coding regions and 1 polymorphism per 124 bp in coding regions. Insertions and deletions are frequent in non-coding regions (1 per 85 bp), but rare in coding regions. A small number (2-8) of distinct and highly diverse haplotypes can be distinguished at all loci examined. Within genes, SNP loci comprising the haplotypes are in linkage disequilibrium with each other. CONCLUSIONS: No decline of linkage disequilibrium within a few hundred base pairs was found in the elite maize germplasm. This finding, as well as the small number of haplotypes, relative to neutral expectation, is consistent with the effects of breeding-induced bottlenecks and selection on the elite germplasm pool. The genetic distance between haplotypes is large, indicative of an ancient gene pool and of possible interspecific hybridization events in maize ancestry.

Alleles↗

The maize INDETERMINATE1 flowering time regulator defines a highly conserved zinc finger protein family in higher plants.

BACKGROUND: The maize INDETERMINATE1 gene, ID1, is a key regulator of the transition to flowering and the founding member of a transcription factor gene family that encodes a protein with a distinct arrangement of zinc finger motifs. The zinc fingers and surrounding sequence make up the signature ID domain (IDD), which appears to be found in all higher plant genomes. The presence of zinc finger domains and previous biochemical studies showing that ID1 binds to DNA suggests that members of this gene family are involved in transcriptional regulation. RESULTS: Comparison of IDD genes identified in Arabidopsis and rice genomes, and all IDD genes discovered in maize EST and genomic databases, suggest that ID1 is a unique member of this gene family. High levels of sequence similarity amongst all IDD genes from maize, rice and Arabidopsis suggest that they are derived from a common ancestor. Several unique features of ID1 suggest that it is a divergent member of the maize IDD family. Although no clear ID1 ortholog was identified in the Arabidopsis genome, highly similar genes that encode proteins with identity extending beyond the ID domain were isolated from rice and sorghum. Phylogenetic comparisons show that these putative orthologs, along with maize ID1, form a group separate from other IDD genes. In contrast to ID1 mRNA, which is detected exclusively in immature leaves, several maize IDD genes showed a broad range of expression in various tissues. Further, Western analysis with an antibody that cross-reacts with ID1 protein and potential orthologs from rice and sorghum shows that all three proteins are detected in immature leaves only. CONCLUSION: Comparative genomic analysis shows that the IDD zinc finger family is highly conserved among both monocots and dicots. The leaf-specific ID1 expression pattern distinguishes it from other maize IDD genes examined. A similar leaf-specific localization pattern was observed for the putative ID1 protein orthologs from rice and sorghum. These similarities between ID1 and closely related genes in other grasses point to possible similarities in function.

Amino Acid Sequence↗

The maize duplicate genes narrow sheath1 and narrow sheath2 encode a conserved homeobox gene function in a lateral domain of shoot apical meristems.

The narrow sheath (ns) phenotype of maize is a duplicate factor trait conferred by mutations at the unlinked loci ns1 and ns2. Recessive mutations at each locus together confer the phenotypic deletion of a lateral compartment in maize leaves and leaf homologs. Previous analyses revealed that the mediolateral axis of maize leaves is comprised of at least two distinct compartments, and suggest a model whereby NS function is required to recruit leaf founder cells from a lateral compartment of maize meristems. Genomic clones of two maize homeodomain-encoding genes were isolated by homology to the WUSCHEL-related gene PRESSED FLOWER (PRS). PRS is required for lateral sepal development in Arabidopsis, although no leaf phenotype is reported. Co-segregation of the ns phenotype with multiple mutant alleles of two maize PRS homologs confirms their allelism to ns1 and ns2. Analyses of NS protein accumulation verify that the ns-R mutations are null alleles. ns transcripts are detected in two lateral foci within maize meristems, and in the margins of lateral organ primordia. Whereas ns1 and ns2 transcripts accumulate to equivalent levels in shoot meristems of vegetative seedlings, ns2 transcripts predominate in female inflorescences. Previously undiscovered phenotypes in the pressed flower mutant support a model whereby the morphology of eudicot leaves and monocot grass leaves has evolved via the differential elaboration of upper versus lower leaf zones. A model implicating an evolutionarily conserved NS/PRS function during recruitment of organ founder cells from a lateral domain of plant meristems is discussed.

Amino Acid Sequence↗

Influence of chemical treatments on glutathione S-transferases of maize with activity towards metolachlor and cinnamic acid.

The subcellular distribution of glutathione S-transferase (GST) activity extracted from shoots of 3-day-old etiolated seedlings of maize (Zea mays L., Northrup-King 9283 hybrid) and the induction of soluble and membrane-bound GST activity by the safener benoxacor, the herbicide metolachlor and their combination (CGA-180937) were investigated. GST activity extracted from maize shoots was detected in both cytosolic and microsomal fractions and utilized 1-chloro-2,4-dinitrobenzene (CDNB), metolachlor, and trans-cinnamic acid (CA) as substrates. Soluble GST activity extracted from maize shoots was greater than microsomal with CDNB or metolachlor as substrate. Membrane-bound GST activity was greater than soluble with cinnamic acid as substrate. Washing the microsomal preparations from maize shoots with Triton X-100 increased GST(CA) activity. Pretreatment with the safener benoxacor or a formulated combination of the herbicide metolachlor with benoxacor induced soluble GST(CDNB), GST(metolachlor) and GST(CA) activities in maize shoots. Benoxacor and CGA-180937 induced also membrane-bound GST(CDNB) and GST(CA) activities in maize shoots, but did not affect membrane-bound GST(metolachlor) activity. These results confirm that maize contains multiple GST isozymes that differ in their substrate specificity and inducibility by safeners or other chemicals.

Acetamides↗

Structure and expression of maize phytochrome family homeologs.

To begin the study of phytochrome signaling in maize, we have cloned and characterized the phytochrome gene family from the inbred B73. Through DNA gel blot analysis of maize genomic DNA and BAC library screens, we show that the PhyA, PhyB, and PhyC genes are each duplicated once in the genome of maize. Each gene pair was positioned to homeologous regions of the genome using recombinant inbred mapping populations. These results strongly suggest that the duplication of the phytochrome gene family in maize arose as a consequence of an ancient tetraploidization in the maize ancestral lineage. Furthermore, sequencing of Phy genes directly from BAC clones indicates that there are six functional phytochrome genes in maize. Through Northern gel blot analysis and a semiquantitative reverse transcriptase polymerase chain reaction assay, we determined that all six phytochrome genes are transcribed in several seedling tissues. However, expression from PhyA1, PhyB1, and PhyC1 predominate in all seedling tissues examined. Dark-grown seedlings express higher levels of PhyA and PhyB than do light-grown plants but PhyC genes are expressed at similar levels under light and dark growth conditions. These results are discussed in relation to phytochrome gene regulation in model eudicots and monocots and in light of current genome sequencing efforts in maize.

Base Sequence↗

Nearly identical paralogs: implications for maize (Zea mays L.) genome evolution.

As an ancient segmental tetraploid, the maize (Zea mays L.) genome contains large numbers of paralogs that are expected to have diverged by a minimum of 10% over time. Nearly identical paralogs (NIPs) are defined as paralogous genes that exhibit > or = 98% identity. Sequence analyses of the "gene space" of the maize inbred line B73 genome, coupled with wet lab validation, have revealed that, conservatively, at least approximately 1% of maize genes have a NIP, a rate substantially higher than that in Arabidopsis. In most instances, both members of maize NIP pairs are expressed and are therefore at least potentially functional. Of evolutionary significance, members of many NIP families also exhibit differential expression. The finding that some families of maize NIPs are closely linked genetically while others are genetically unlinked is consistent with multiple modes of origin. NIPs provide a mechanism for the maize genome to circumvent the inherent limitation that diploid genomes can carry at most two "alleles" per "locus." As such, NIPs may have played important roles during the evolution and domestication of maize and may contribute to the success of long-term selection experiments in this important crop species.

Arabidopsis↗

Control of Aspergillus flavus in maize with plant essential oils and their components.

The effects of 11 plant essential oils for maize kernel protection against Aspergillus flavus were studied. Tests were conducted to determine optimal levels of dosages for maize protection, effects of combinations of essential oils, and residual effects and toxicity of essential oils to maize plants. Principal constituents of eight essential oils were tested for ability to protect maize kernels. Essential oils of Cinnamomum zeylanicum (cinnamon), Mentha piperita (peppermint), Ocimum basilicum (basil), Origanum vulgare (origanum), Teloxys ambrosioides (the flavoring herb epazote), Syzygium aromaticum (clove), and Thymus vulgaris (thyme) caused a total inhibition of fungal development on maize kernels. Thymol and o-methoxycinnamaldehyde significantly reduced maize grain contamination. The optimal dosage for protection of maize varied from 3 to 8%. Combinations of C. zeylanicum with the remaining oils gave efficient control. A residual effect of C. zeylanicum was detected after 4 weeks of kernel treatment. No phytotoxic effect on germination and corn growth was detected with any of these oils.

Acrolein↗

A home-based method to reduce phytate content and increase zinc bioavailability in maize-based complementary diets.

This study aimed to develop and assess the feasibility of a home-based method to reduce the phytate content of maize and improve zinc bioavailability from maize-based complementary diets in rural Malawi. A method of extracting phytate through the soaking of pounded maize was developed, and found to reduce phytate content to 49% of unrefined maize. An educational program was used to teach the processing method to mothers of children receiving complementary foods in rural Malawian communities. Samples of maize flour prepared by this process by participants were collected and analysed for phytate and zinc content. Of these, 70% of samples were found to be adequately prepared; mean phytate content of these samples was 48% of unprocessed, unrefined maize flour controls. Most participants found the cooked product to have an acceptable taste (99%) and texture (68%), and felt the processing method took little or no extra time (86%) and was culturally acceptable (96%). The phytate and zinc content of the processed maize flour samples analysed from community prepared samples was substituted into the dietary analysis of complementary foods for 9- to 11-month-old children (n = 31). The bioavailability of zinc from the complementary diet would predict an increase from low (24%) to moderate (33%) levels.

Biological Availability↗

Understanding yields in alley cropping maize (Zea mays L.) and Cassia siamea Lam. under semi-arid conditions in Machakos, eastern Kenya.

Six seasons of experiments in Machakos, Kenya, revealed that above about 150 mm of rainfall, maize yields per row in alley cropped "replacement" agroforestry (AF) plots, of Cassia siamea Lam. and maize (Zea mays, cv. Katumani Composite B), may be expected to exceed those in the control (sole maize) plots. Such yields were insufficient to compensate for the area "lost" to the hedgerows. Below about 150 mm the control plots may be expected to perform better. This result was due to competition for water. Greater association of the fine roots of Cassia and maize was observed in the middle of the alleys than near the hedgerows. Photosynthetic consequences of shading were insignificant relative to other factors. In the alleys, reductions of soil temperature due to shade in the western and eastern maize rows were higher than in the middle row. Soil moisture extraction was higher in the AF than in the control plots. In the AF plots, moisture extraction was greater under the central maize rows than under those nearest the Cassia. Yield patterns followed such soil temperature and soil moisture patterns. Maize transpiration and photosynthetic rates were significantly higher in the control than in the AF plots during a below-average rainy season but not during above-average rainy seasons. It is concluded that alley cropping under semi-arid conditions should be approached differently from the system worked on. It must at least provide strong physical protection of crops and/or soils and have a strong economic incentive to be of interest to the farmers.

Agriculture↗

Nutritional and sensory evaluation of tempe-fortified maize-based weaning foods.

Maize-based weaning foods prepared from whole maize meal or dehydrated fermented maize (ogi) flour fortified with soybean, cowpea or groundbean tempe, with or without the addition of melon seed flour were fed to 40 albino weanling rats. The nutrient quality of the weaning foods was monitored by the measurement of protein efficiency ratio (PER), net protein ratio (NPR), true digestibility (TD), biological value (BV) and net protein utilization (NPU). Amino acid composition and amino acid scores were determined. The weaning foods were also evaluated for sensory properties and acceptability by nursing mothers and their children. Protein efficiency ratio, net protein retention and net protein utilization of the fortified weaning foods were not significantly different (P > 0.05) when compared to casein diet (control). True digestibility of tempe-fortified maize-based weaning foods averaged 85.4 +/- 3.4% compared to 94.4%, for casein diet. Results from sensory analysis showed that relative to tempe-fortified maize-based weaning foods, most mothers preferred Cerelac, a commercial weaning food, and its porridge because of its vanillic flavour and sweet taste. Despite the low scores recorded for aroma and taste of tempe-fortified maize-based weaning foods, most mothers and their children found porridges prepared from these foods acceptable, with a preference for ogi fortified with cowpea tempe. Tempe-fortified maize-based weaning foods had high protein quality and can support the growth of infants in developing countries especially during the critical weaning period (6-12 months). They were comparable to Cerelac, a commercial baby food in colour and texture.

Adult↗

Risks and benefits of genetically modified maize donations to southern Africa: views from Malawi.

In 2001 and 2002, many countries in the Southern African Development Community (SADC) have suffered from severe food shortages resulting in an estimated 14 million people facing starvation due to inadequate quantities of the staple maize. The international community's response has been the donation of foodstuffs, including genetically modified maize. Reactions of the recipient countries of Zambia, Zimbabwe, and Malawi have been different. Zambia appealed to the donors not to send genetically modified maize, whereas Malawi accepted the maize donations. Malawi is currently facing many public health challenges because 10% of its 10-million population is HIV-positive, maternal mortality rate has almost doubled between 1992 and 2000, and there are also an estimated 1 million orphans due to HIV/AIDS. In the European Union, genetically modified maize falls under "Novel Foods" and its marketing and distribution are strictly regulated by law. This has never been the case in the southern African countries. In this article, we discuss the ethical challenges associated with genetically modified maize donations to southern Africa. Although genetically modified food offers a way to avoid many adverse effects of food shortages, we believe that some of the ethical questions of genetically modified food donations should be solved first, under the leadership of the donor countries and partnership of the developing countries. There are fears that consummation of genetically modified maize could have adverse health effects. These fears must be addressed if the confidence of developing countries in the donor community is to be maintained.

Bioethical Issues↗

Excision of a transposable element from a viral vector introduced into maize plants by agroinfection.

The geminivirus maize streak virus (MSV) was used as a vector to introduce the maize transposable element Dissociation (Ds) and to study its excision in maize plants. MSV carrying Ds1 in its genome was introduced into maize plants by agroinfection. Excision of the Ds1 element from the MSV genome was detected only when functions from the transposable element Activator (Ac) were supplied in trans, either endogenously by the recipient maize plant or by co-transformation with Agrobacterium carrying a genomic Ac clone. The excision of Ds1 could easily be visualized by the appearance of viral symptoms induced by the revertant virus. The junction sequences left on the MSV genome after excision revealed 'footprints' typical of transposition as described for maize. From these results, we conclude that transposition functions in our system and that the use of the MSV replicon provides a rapid and simple tool for the investigation of the excision of transposable elements in maize plants.

Agrobacterium tumefaciens↗

[Effect of plastic-film mulch on water and nitrogen use by spring maize and on fate of applied nitrogen in the southern Loess Plateau].

The relationships between nitrogen and yield and between water and yield, and the fate of nitrogen under plastic film mulching for spring maize were studied with field plots and microplots in the southern Loess Plateau. The results showed that plastic-film mulching (N120C) could increase maize yield by 46.7%, comparing with no-mulching(N120UC) in same rate of fertilizer. The maize yield potentiality of mulching might be played fully, comparing with CK (no nitrogen), treatments N120 (urea nitrogen 120 kg.hm-2), N180 (urea nitrogen 180 kg.hm-2) and N120 M (urea nitrogen 120 kg.hm-2 + organic manure nitrogen 60 kg.hm-2) could raise grain yield by 41.8%, 43.9% and 34.7%, respectively. Mulching planting raised water use efficiency (WUE) by 57.9%, and raised rainfall use efficiency (RUE) by 54.5%. N120, N180 and N120M improved WUE by 38.4%, 47.4% and 32.4%, respectively, and improved RUE by 42.3%, 43.9% and 34.7%, respectively. Because the organic manure applied was not fully rotted cattle dung, which supplied available nitrogen slowly than urea, it raised yield and WUE to a low extent. Meanwhile, the experiment showed that 73.0%-83.7% of water used by maize from rainfall, which indicated that the key water to determine maize yield was rainfall in spring maize growth period. Mulching planting for maize had little impact on the fate of nitrogen fertilizer. The total recovery of nitrogen had little difference, NUE decreased 7.3%, and residual N in soil raised 6.4%, comparing with N120UC. A great part of the residual N was in 0-20 cm of soil, and no risk occurred for nitrogen leaching and accumulation to deeper soil layers in the first growth season.

Crops, Agricultural↗