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cDNA Clones for Corn Leaf NADH:Nitrate Reductase and Chloroplast NAD(P):Glyceraldehyde-3-Phosphate Dehydrogenase : Characterization of the Clones and Analysis of the Expression of the Genes in Leaves as Influenced by Nitrate in the Light and Dark.

cDNA clones were selected from a corn (Zea mays L.) leaf lambda gt11 expression library using polyclonal antibodies for corn leaf NADH:nitrate reductase. One clone, Zmnrl, had a 2.1 kilobase insert, which hybridized to a 3.2 kilobase mRNA. The deduced amino acid sequence of Zmnrl was nearly identical to peptide sequences of corn leaf NADH:nitrate reductase. Another clone, Zm6, had an insert of 1.4 kilobase, which hybridized to a 1.4 kilobase mRNA, and its sequence coded for chloroplastic NAD(P)(+):glyceraldehyde-3-phosphate dehydrogenase based on comparisons to sequences of this enzyme from tobacco and corn. When nitrate was supplied to N-starved, etiolated corn plants, nitrate reductase, and glyceraldehyde-3-phosphate dehydrogenase mRNA levels in leaves increased in parallel. When green leaves were treated with nitrate, only nitrate reductase mRNA levels were increased. Nitrate is a specific inducer of nitrate reductase in green leaves, but appears to have a more general effect in etiolated leaves. In the dark, nitrate induced nitrate reductase expression in both etiolated and green leaves, indicating light and functional chloroplast were not required for enzyme expression.

Journal Article↗

Effects and Absorption of Sethoxydim in Cell Cycle Progression of Corn (Zea mays) and Pea (Pisum sativum).

The mechanisms of selective herbicidal action of sethoxydim were investigated by using cultured root tips of corn (Zea mays L. cv Goldencrossbantam) and pea (Pisum sativum L. cv Alaska). Meristematic cells in the cultured roots were arrested in G(1) and G(2) of the cell division cycle by sucrose starvation and resumed growth and cell division (proliferation) when sucrose was provided. Corn root growth after sucrose addition was inhibited by sethoxydim at concentrations of 0.01 micromolar and greater when roots were treated in the presence of sucrose but was not inhibited at 10 micromolar sethoxydim when they were treated during sucrose starvation. Greater absorption of [(14)C]sethoxydim into the meristematic region of corn roots was observed when cells were in proliferative condition but not when they were arrested by sucrose starvation, whereas no greater absorption of the herbicide into pea meristems was observed in either growth condition. In the cell cycle study, greater absorption of [(14)C]sethoxydim into the corn root meristem was observed at a certain limited time before S (DNA synthesis) stage. The physiological effects and the greater absorption of sethoxydim clearly depended on cell cycle progression of corn root meristem, whereas fatty acid synthesis, as well as its inhibition by sethoxydim, was not associated with either cell cycle progression or greater absorption of the herbicide.

Journal Article↗

Preharvest aflatoxin contamination: effect of moisture and substrate variation in developing cottonseed and corn kernels.

Variations in moisture and substrate in preharvest corn kernels and cottonseed were linked with the ability of Aspergillus parasiticus to infect the seed and produce aflatoxin. Osmotic pressures and moisture content (MC) levels of developing starch-rich corn kernels and lipid-rich cottonseed were determined. For in vivo studies, corn kernels and cottonseed were inoculated with A. parasiticus conidia and retained on plants through maturation. For in vitro studies, samples of corn kernels and cottonseed were collected at various stages, sterilized, inoculated, incubated for 2 weeks, and assayed for toxin. Aflatoxin levels were highest in corn kernels inoculated at 28 days postflowering (52% MC) in both the in vivo and in vitro tests. Toxin concentrations in cottonseed were greatest with inoculation at 35 days postflowering (70% MC) in seed retained on the plant, but toxin accumulation continued to increase with the maturity of the seed inoculated in cottonseed used in the in vitro trials. Moisture and substrate conditions in the midrange of seed development provided optimum conditions for fungal development and toxin production in seed retained on the plant.

Aflatoxins↗

Fate of Fumonisin B(1) in Naturally Contaminated Corn during Ethanol Fermentation.

Two lots of corn naturally contaminated with fumonisin B(1) (15 and 36 ppm) and a control lot (no fumonisin B(1) detected) were used as substrates for ethanol production in replicate 8.5-liter yeast fermentations. Ethanol yields were 8.8% for both the control and low-fumonisin corn, while the high-fumonisin corn contained less starch and produced 7.2% ethanol. Little degradation of fumonisin occurred during fermentation, and most was recovered in the distillers' grains, thin stillage, and distillers' solubles fractions. No toxin was detected in the distilled alcohol or centrifuge solids. Ethanol fermentation of fumonisin-contaminated corn coupled with effective detoxification of distillers' grains and aqueous stillage is suggested as a practical process strategy for salvaging contaminated corn.

Journal Article↗

Simultaneous occurrence of fumonisin B1 and other mycotoxins in moldy corn collected from the People's Republic of China in regions with high incidences of esophageal cancer.

A total of 31 corn samples collected from households in the counties of Cixian and Linxian of the People's Republic of China, where high incidences of esophageal cancer have been reported, were analyzed for fumonisin B1 (FB1), aflatoxin, and total trichothecene mycotoxins. High levels of FB1 (18 to 155 ppm; mean, 74 ppm) were found in 16 of the samples that showed heavy mold contamination. FB1, at lower levels (20 to 60 ppm; mean, 35.3 ppm), was also found in 15 samples, collected from the same households, that did not show any visible mold contamination. The levels of aflatoxin in the samples were low (1 to 38.4 ppb; mean, 8.61 ppb). High levels of total type-A trichothecenes were also found in the moldy corn samples (139 to 2,030 ppb; mean, 627 ppb). Immunochromatography of selected samples revealed that these samples contained T-2 toxin, HT-2 toxin, iso-neosolaniol, monoacetoxyscirpenol, and several other type-A trichothecenes. The concentration of total type-B trichothecenes in 15 moldy corn samples was in the range of 470 to 5,826 ppb (mean, 2,359 ppb). High levels (3.7 to 5.0 mg/g) of FB1 were produced in corn in the laboratory by five Fusarium moniliforme strains isolated from the moldy corn. These fungi were also capable of forming various nitrosamines (5 to 16 micrograms per flask) in the presence of nitrate and precursor amines.(ABSTRACT TRUNCATED AT 250 WORDS)

China↗

Effect of corn and peanut cultivation on soil populations of Aspergillus flavus and A. parasiticus in southwestern Georgia.

The effect of corn and peanut cultivation on the proportion of Aspergillus flavus to A. parasiticus in soil was examined. Soil populations were monitored in three fields during three different years in southwestern Georgia. Each field was planted in both peanuts and corn, and soil was sampled within plots for each crop. A. flavus and A. parasiticus were present in similar proportions in plots from all fields at the beginning of the growing season. A. terreus, A. niger, and A. fumigatus were the other dominant aspergilli in soil. Fields A and B did not show drought stress in peanut or corn plants, and soil populations of A. flavus and A. parasiticus remained stable during the course of the year. In field C, drought stress in corn plants with associated A. flavus infection and aflatoxin contamination greatly increased soil populations of A. flavus relative to A. parasiticus upon dispersal of corn debris to the soil surface by a combine harvester. Colonization of organic debris after it has been added to the soil may maintain soil populations of A. parasiticus despite lower crop infection.

Arachis↗

Effects of pH on distribution of Listeria ribotypes in corn, hay, and grass silage.

Listeria app, isolated from 13 of 129 (10%) corn silage samples, 21 of 76 (28%) hay silage samples, and 3 of 5 (60%) grass silage samples during a previous Vermont survey were subjected to automated ribotype (RT) analysis. The 13 positive corn silage samples contained 3 Listeria monocytogenes isolated (three RTs, including one known clinical RT) and 10 L. innocua isolates (four RTs). Similarly, 2 L. monocytogenes isolates (two RTs) and 19 L. innocua isolates (three RTs) were identified in the 21 positive hay silage samples. The three positive grass silage samples contained two L. innocua isolates (two RTs) and one isolate of L. welshimeri. One hundred seven of 129 (83%) high-quality (pH < 4.0) corn silage samples accounted for 8 of 13 Listeria isolates from corn silage, including isolates belonging to one L. monocytogenes clinical RT. In contrast, low-quality hay silage (70 of 76 [92%] samples having a pH of > or = 4.0) harbored 20 of 21 isolates, including isolates belonging to two nonclinical L. monocytogenes RTs. Poor-quality silage is readily discernible by appearance; however, these findings raise new concerns regarding the safety of high-quality (pH < 4.0) corn silage, which can contain Listeria spp., including L. monocytogenes strains belonging to RTs of clinical importance in cases of food-borne listeriosis.

Animal Feed↗

Toxicity to rats of corn invaded by Chaetomium globosum.

Of 53 isolates of Chaetomium globosum Kunze isolated from corn, manufactured feed, and fiberboard, grown on autoclaved corn, and fed to rats, 25 were lethal within 4 to 6 days. Thirty-six single ascospore cultures of one isolate were uniformly lethal when similarly grown and fed to rats. Incubation of the fungus on corn for 6 to 8 weeks resulted in greater toxicity than shorter incubation times. Premortem symptoms indicated damage to the central nervous system of the affected animals, and postmortem lesions included hemoglobinuria, hemorrhagic enteritis, and subdural hemorrhaging. Corn invaded by C. globosum and toxic to rats had no detectable effects on swine when fed as their sole ration for 6 weeks, and appeared to be consumed with relish. The toxic substance was best obtained by defatting the fungus-invaded corn with petroleum ether (bp, 60 to 70 C), extraction with acetone, and further purification by elution from a silica gel column with acetone and 5% acetone in chloroform.

Animals↗

Radiation sterilization of prototype military foods: low-temperature irradiation of codfish cake, corned beef, and pork sausage.

"Screening" packs comprising 10 lots each of codfish cake, corned beef, and pork sausage, each lot containing about 10(6) spores of a different strain (five type A and five type B) of Clostridium botulinum per can, were irradiated at -30 +/- 10 C with a series of increasing doses (20 replicate cans/dose) of (60)Co gamma rays. The cans were incubated for 3 months at 30 C and examined for swelling, toxin, and recoverable botulinal cells. Based on the latter criterion of spoilage, median lethal dose (LD(50)) and D values were estimated for each strain in each food. The most resistant strain in codfish cake, corned beef, and pork sausage was, respectively, 53B, 77A, and 41B. There was no clear-cut trend in the comparative order of resistance between the two antigenic types among the three foods. LD(50) values gave essentially the same order of resistances as the D values and may be used interchangeably with the latter for the 10 test organisms. "Clearance" packs consisting of the most resistant strain (about 10(7) spores/can) with its respective food were irradiated with a variety of doses at -30 +/- 10 C, using 100 replicate cans/dose (about 10(9) spores/dose). These packs were incubated for 6 months at 30 C and assayed for the three types of spoilage. Based on recoverable cells, the experimental sterilizing doses (ESD) for codfish cake, corned beef, and pork sausage were 2.5< ESD </= 3.0, 2.0 < ESD </= 2.5, and 1.5 < ESD </= 2.0 Mrad, in that order. Assuming exponential spore death, the 12D values, or minimal radiation doses (MRD), were 3.24, 2.44, and 2.65 Mrad, respectively. Estimation of the MRD values by a method which assumes that spore death in the cans follows a normal distribution, yielded 3.09, 2.57, and 2.39 Mrad, respectively. Weibull analyis of the pooled 10-strain viable cell spoilage data of the screening packs for codfish cake or corned beef suggested that spore death in the cans follows a normal distribution yielded 3.09, 2.57, pooled data were not amenable to such analysis. Sublethal doses (0.5, 0.75 Mrad) increased the visible spoilage rate of corned beef over that of unirradiated controls. Apparently radiation-injured spores of C. botulinum were sensitized to the presence of food additives such as curing salts, NaCl, and spices.

Animals↗

Influence of modified atmosphere storage on aflatoxin production in high moisture corn.

Samples of freshly harvested corn and remoistened corn were inoculated with Asphergillus flavus and stored for 4 weeks at about 27 C in air and three modified atmospheres. Aflatoxins and fat acidity were determined weekly. Corn stored in the modified atmospheres did not accumulate over 15 mug of total aflatoxins per kg. Corn from the high CO2 treatment (61.7 per cent CO2, 8.7 per cent O2, and 29.6 per cent N2) was visibly molded at 4 weeks and had a higher fat acidity than the other treatments. In the N2 (99.7 per cent N2 and 0.3 per cent O2) and controlled atmosphere (13.5 per cent CO2, 0.5 per cent O2, 84.5 per cent N2) treatments, a fermentation-like odor was detected. When the corn was removed from the modified atmospheres it deteriorated rapidly and was soon contaminated with aflatoxins.

Aflatoxins↗

Molecular mapping of QTLs for resistance to Gibberella ear rot, in corn, caused by Fusarium graminearum.

Gibberella ear rot, caused by the fungus Fusarium graminearum Schwabe, is a serious disease of corn (Zea mays) grown in northern climates. Infected corn is lower yielding and contains toxins that are dangerous to livestock and humans. Resistance to ear rot in corn is quantitative, specific to the mode of fungal entry (silk channels or kernel wounds), and highly influenced by the environment. Evaluations of ear rot resistance are complex and subjective; and they need to be repeated over several years. All of these factors have hampered attempts to develop F. graminearum resistant corn varieties. The aim of this study was to identify molecular markers linked to the genes for resistance to Gibberella ear rot. A recombinant inbred (RI) population, produced from a cross between a Gibberella ear rot resistant line (CO387) and a susceptible line (CG62), was field-inoculated and scored for Gibberella ear rot symptoms in the F4, F6, and F7 generations. The distributions of disease scores were continuous, indicating that resistance is probably conditioned by multiple loci. A molecular linkage map, based on segregation in the F5 RI population, contained 162 markers distributed over 10 linkage groups and had a total length of 2237 cM with an average distance between markers of 13.8 cM. Composite interval mapping identified 11 quantitative trait loci (QTLs) for Gibberella ear rot resistance following silk inoculation and 18 QTLs following kernel inoculation in 4 environments that accounted for 6.7%-35% of the total phenotypic variation. Only 2 QTLs (on linkage group 7) were detected in more than 1 test for silk resistance, and only 1 QTL (on linkage group 5) was detected in more than 1 test for kernel resistance, confirming the strong influence of the environment on these traits. The majority of the favorable alleles were derived from the resistant parent (CO387). The germplasm and markers for QTLs with significant phenotypic effects may be useful for marker-assisted selection to incorporate Gibberella ear rot resistance into commercial corn cultivars.

Chromosome Mapping↗

Characterization and genetic variability of Fusarium verticillioides strains isolated from corn and sorghum in Brazil based on fumonisins production, microsatellites, mating type locus, and mating crosses.

Fusarium verticillioides (Gibberella fujikuroi mating population A) is a producer of fumonisins and one of the main contaminants of corn grains. In Brazil, some studies analyzing strains isolated from corn have demonstrated high levels of fumonisins, whereas the levels for strains isolated from sorghum have been found to be low. In the present study, we investigated the genetic diversity of 22 F. verticillioides strains isolated from corn and 21 strains isolated from sorghum cultivated in the State of São Paulo, Brazil. Differences in the genetic profile were observed between the strains isolated from the two substrates using single primer amplification reaction by polymerase chain reaction (SPAR-PCR). Fumonisins levels were higher in strains isolated from corn than in those isolated from sorghum. The MAT-1 and MAT-2 alleles were identified by PCR, and the isolates were subsequently crossed with Fusarium thapsinum (G. fujikuroi mating population F) reference strains because this species is morphologically similar to F. verticillioides and produces low levels of fumonisins. The SPAR haplotypes of some strains isolated from sorghum were similar to the F. thapsinum reference strain haplotypes, but there was no fertile mating between the strains isolated from the two substrates and the F. thapsinum references strains. The MAT-1:MAT-2 proportion was 5:17 and 14:7 for isolates from corn and sorghum, respectively.

Brazil↗

Development and use of an ELISA test to detect IgE antibody to Cry9c following possible exposure to bioengineered corn.

BACKGROUND: Starlink(TM), a variety of corn genetically engineered to contain the insecticidal protein Cry9c, had not been approved for human consumption because it possessed some characteristics associated with allergenic proteins. However, in the fall of 2000 CRY9C DNA was detected in several corn-containing products, suggesting that Starlink corn had entered the human food supply. Subsequently, consumers, following consumption of corn products, reported a number of adverse health events, possibly consistent with allergic reaction. METHODS: To investigate the possibility of allergic reactions due to Cry9c in these consumers an ELISA test was developed for the purpose of detecting IgE antibodies to Cry9c and blood samples were taken from a total of 18 people who self-reported allergic reactions. Sera collected prior to the 1996 development of Starlink were used as negative controls. RESULTS: None of the adverse event sera were found to be reactive with recombinant Cry9c antigen, based on comparison with normal controls. Although a known human positive control serum containing IgE specific for Cry9c was not available, other controls were incorporated into the ELISA protocol, including the use of sera from subjects allergic to other allergens and their homologous antigens (cat, grass, peanut) to validate the IgE detection reagents. CONCLUSIONS: While the results do not support the likely occurrence of allergic reactions to Cry9c, such reactions cannot be ruled out, nor can the possibility that sera might react with unique glycosylated epitopes of Cry9c that may be expressed in the corn plant/seed.

Bacillus thuringiensis Toxins↗

Comparative effects of non-gelatinized corn and rice starches on the life-span of ICR mice.

We compared the effects of non-gelatinized rice and corn starches on the life-span of ICR mice. Six groups of male ICR mice consisting of 30 animals each were maintained on purified experimental diets containing either corn or rice starch and different amounts of soybean oil (6, 12 or 24%) throughout their life-time. Plots of the survival rates of the mice indicate that rice compared to corn starch conferred a longer life-span to ICR mice, although a significant difference due to the starch type was only observed in the mice fed on the 24% fat diet (p=0.012). A divergent effect of rice and corn starches on the survival rate was apparent when observations were combined with respect to the starch type regardless of the dietary fat level (p=0.005). In addition, two-way ANOVA data indicate that the mean survival time was longer for the mice given rice starch (593-645 days) than for those fed corn starch (538-580 days) (p=0.011). However, no significant difference in these parameters due to dietary fat levels was observed. The results of our study indicate that starch type is one of the determinants of longevity in mice.

Animals↗

Effects of temperature and moisture on dilute-acid steam explosion pretreatment of corn stover and cellulase enzyme digestibility.

Corn stover is emerging as a viable feedstock for producing bioethanol from renewable resources. Dilute-acid pretreatment of corn stover can solubilize a significant portion of the hemicellulosic component and enhance the enzymatic digestibility of the remaining cellulose for fermentation into ethanol. In this study, dilute H2SO4 pretreatment of corn stover was performed in a steam explosion reactor at 160 degrees C, 180 degrees C, and 190 degrees C, approx 1 wt % H2SO4, and 70-s to 840-s residence times. The combined severity (Log10 [Ro] - pH), an expression relating pH, temperature, and residence time of pretreatment, ranged from 1.8 to 2.4. Soluble xylose yields varied from 63 to 77% of theoretical from pretreatments of corn stover at 160 and 180 degrees C. However, yields >90% of theoretical were found with dilute-acid pretreatments at 190 degrees C. A narrower range of higher combined severities was required for pretreatment to obtain high soluble xylose yields when the moisture content of the acidimpregnated feedstock was increased from 55 to 63 wt%. Simultaneous saccharification and fermentation (SSF) of washed solids from corn stover pretreated at 190 degrees C, using an enzyme loading of 15 filter paper units (FPU)/ g of cellulose, gave ethanol yields in excess of 85%. Similar SSF ethanol yields were found using washed solid residues from 160 and 180 degrees C pretreatments at similar combined severities but required a higher enzyme loading of approx 25 FPU/g of cellulose.

Animal Feed↗

Measurement of rheological properties of corn stover suspensions.

Corn stover is currently being evaluated as a feedstock for ethanol production. The corn stover suspensions fed to reactors typically range between 10 and 40% solids. To simulate and design bioreactors for processing highly loaded corn stover suspensions, the rheologic properties of the suspension must be measured. In systems with suspended solids, rheologic measurements are difficult to perform owing to settling in the measurement devices. In this study, viscosities of corn stover suspensions were measured using a helical ribbon impeller viscometer. A calibration procedure is required for the impeller method in order to obtain the shear rate constant, k, which is dependent on the geometry of the measurement system. The corn stover suspensions are described using a power law flow model.

Animal Feed↗

Ammonia fiber explosion treatment of corn stover.

Optimizing process conditions and parameters such as ammonia loading, moisture content of biomass, temperature, and residence time is necessary for maximum effectiveness of the ammonia fiber explosion process. Approximate optimal pretreatment conditions for corn stover were found to be temperature of 90 degrees C, ammonia:dry corn stover mass ratio of 1:1, moisture content of corn stover of 60% (dry weight basis), and residence time (holding at target temperature), of 5 min. Approximately 98% of the theoretical glucose yield was obtained during enzymatic hydrolysis of the optimal treated corn stover using 60 filter paper units (FPU) of cellulase enzyme/g of glucan (equal to 22 FPU/g of dry corn stover). The ethanol yield from this sample was increased up to 2.2 times over that of untreated sample. Lowering enzyme loading to 15 and 7.5 FPU/g of glucan did not significantly affect the glucose yield compared with 60 FPU, and any differences between effects at different enzyme levels decreased as the treatment temperature increased.

Ammonia↗

Frequency of resistance to Bacillus thuringiensis toxin Cry1Ab in an Iowa population of European corn borer (Lepidoptera: Crambidae).

The refuge plus high-dose strategy for resistance management assumes that the frequency of resistance alleles is low. We used an F2 screen to estimate the frequency of resistance to transgenic corn that produces Bacillus thuringiensis Berliner Cry1Ab toxin (Bt corn) in an Iowa population of European corn borer, Ostrinia nubilalis (Hübner). We also proposed a modification to the statistical analysis of the F2 screen that extends its application for nonuniform prior distributions and for repeated sampling of a single population. Based on a sample of 188 isofemale lines derived from females caught at light traps during the 2nd flight of 1997, we show with 95% confidence that the frequency of resistance to Bt corn was <3.9 x 10(-3) in this Iowa population. These results provide weak evidence that the refuge plus high-dose strategy may be effective for managing resistance in O. nubilalis to Bt corn. Partial resistance to Cry1Ab toxin was found commonly. The 95% CI for the frequency of partial resistance were [8.2 x 10(-4), 9.4 x 10(-3)] for the Iowa population. Variable costs of the method were 14.90 dollars per isofemale line, which was a reduction of 25% compared with our initial estimate.

Alleles↗