Lower fumonisin mycotoxin levels in the grain of Bt corn grown in the United States in 2000-2002

Fumonisins were monitored in corn grain collected from Bt hybrids grown in 107 locations across the United States in 2000-2002. Bt corn hybrids contain the Cry1Ab protein from Bacillus thuringiensis that controls European corn borers and other stalk-boring pests. Fumonisin levels were frequently lower in grain from Bt hybrids grown in field trials under conditions of natural (FACT trials) or manual insect infestation (university trials). Over three years of FACT trials, there were 126/210 comparisons when fumonisin levels in grain from control hybrids were >2 ppm, exceeding U.S. FDA guidance levels of 2 ppm for human food. Grain from Bt hybrids was at or below 2 ppm of fumonisins for 58 of the 126 comparisons. The use of Bt hybrids can increase the percentage of corn grain that would be suitable for use in food and feed.     

Role of maize hybrids and their chemical composition in Fusarium infection and fumonisin production

This study was designed to investigate the role of hybrids in maize Fusarium section Liseola interaction and fumonisin production, with particular emphasis on the occurrence and accumulation of hidden fumonisins in maize (masking phenomenon). In this 2 year study, naturally infected field crops were chosen with 10 maize hybrids, six of them grown in both years. Maize samples collected in 2010 showed a higher incidence of fungal infection as well as higher fumonisin contamination than those obtained in 2009 but a very similar incidence of F. section Liseola. Fumonisin masking was confirmed in raw maize, with a lower amount of hidden forms as compared to free fumonisins detected in the year with higher contamination. The chemical composition of the different hybrids was determined and correlated with the contamination data: the results obtained highlight the main role of fatty acids, with a higher fumonisin contamination in hybrids showing a higher linoleic acid content and a higher masking action in hybrids with higher oleic to linoleic ratio. These results represent a good basis to explain maize hybrid susceptibility to F. section Liseola infection, fumonisin contamination, and masking not related to a specific commercial hybrid but extendable to all hybrids.     

Occurrence of Fusarium species and mycotoxins in nepalese maize and wheat and the effect of traditional processing methods on mycotoxin levels

Maize (Zea mays) and wheat (Triticum aestivum) collected in the foothills of the Nepal Himalaya Mountains were analyzed for Fusarium species and mycotoxins: fumonisins, nivalenol (NIV), and deoxynivalenol (DON). Predominant species were Gibberella fujikuroi mating population A (F. moniliforme) in maize and F. graminearum in maize and wheat; G. fujikuroi mating population D (F. proliferatum), F. acuminatum, F. avenaceum, F. chlamydosporum, F. equiseti, F. oxysporum, F. semitectum, and F. torulosum were also present. Strains of G. fujikuroi mating population A produced fumonisins, and strains of F. graminearum produced NIV or DON. By immunoassay or high-performance liquid chromatography, fumonisins were >1000 ng/g in 22% of 74 maize samples. By immunoassay or fluorometry, NIV and DON were >1000 ng/g in 16% of maize samples but were not detected in wheat. Fumonisins and DON were not eliminated by traditional fermentation for producing maize beer, but Nepalese rural and urban women were able to detoxify contaminated maize by hand-sorting visibly diseased kernels.     

Fumonisin production by Fusarium verticillioides strains isolated from maize in Mexico and development of a polymerase chain reaction to detect potential toxigenic strains in grains

Fumonisins are mycotoxins produced by Fusarium verticillioides (Sacc. Nirenberg) in maize (Zea mays L.), a staple crop in Mexico. In this study, we report the isolation and identification of 67 Fusarium strains isolated from maize kernels collected in Northwest and Central Mexico. The strains were characterized regarding fumonisin B(1) production and the presence of the FUM1 gene. F. verticillioides was the predominant species isolated in both geographic regions, but the isolates from Northwest Mexico produced higher levels of fumonisin. A polymerase chain reaction (PCR)-based method, to detect a region of the FUM1 gene involved in fumonisin biosynthesis, was developed and employed to detect mycotoxigenic fungi in pure culture and in contaminated maize. The presence of the FUM1 gene was associated with fumonisin production in most isolates, except seven that did not synthesize fumonisin but contained the gene in their genome. The PCR method allowed the direct detection of fungal contamination in ground corn and could be employed to screen for the presence of potential mycotoxigenic fusaria.     

Maize (Zea mays L.) genetic factors for preventing fumonisin contamination

Fusarium moniliforme and Fusarium proliferatum are the most frequently isolated fungi from maize (Zea mays L.) in Spain. Both Fusarium species produce toxins potentially dangerous for animals and humans, the fumonisins being the most significant of those toxins. White maize is preferred for human consumption, and extra care should be taken to avoid kernel mycotoxin contamination. The objectives of this study were to identify and quantify kernel infection by Fusarium spp. and contamination by fumonisin on white maize hybrids, to search for white maize sources of resistance to infection by Fusarium spp. and mycotoxin contamination, and to preliminarily study the genetics involved in such resistances. Ten F(1) single crosses derived from a diallel mating design among five white maize inbreds were evaluated in a randomized complete block design with three replications in 2002 at two locations. Fusarium verticilloides and F. proliferatum were detected on kernels of white maize hybrids cultivated in northwestern Spain. No differences in fungal infection were found among maize genotypes, but differences in fumonisin contamination were significant and could be related, in part, to differences in husk tightness. Among the genotypes studied, general combining ability (GCA) effects were the most important for resistance to fumonisin contamination. Inbreds EP10 and EC22 showed the most favorable GCA effects for husk tightness and fumonisin content, and the cross between them, EP10 x EC22, had the most favorable specific combining ability (SCA) effect for husk tightness. Inbreds EP10 and EC22 showed favorable GCA effects for fumonisin contamination and husk tightness, and the cross EP10 x EC22 was the only one with an average fumonisin level below 1 mug/g. Although this should be confirmed with more extensive studies, white maize inbreds developed from white maize landraces could be sources of resistance to fumonisin contamination.     

转Bt基因玉米自交系及其杂交种的抗螟性鉴定

采用基因枪法将Bt基因(GFM CrylA)导入东北春玉米自交系铁7922的幼胚中,获得转基因植株。经过连续3年的田间接卵、室内饲喂玉米螟鉴定,获得稳定抗虫转基因系。以此为亲本与常规自交系配制了四密25Bt、通单24Bt、吉单209Bt3个杂交种。转基因自交系和杂交种的抗螟性鉴定结果表明：转基因系间抗虫性差异极显著,同一转基因系单株间和配制的3个杂交种间抗虫性也存在差异;与对照相比,转基因系配制的杂交种抗虫性明显提高,差异显著。农艺性状鉴定结果显示,转基因系配制的杂交种与对照在株高、穗长、穗行数、行粒数、百粒重上无显著差异。本研究认为,GFM CrylA(Bt)基因可用于玉米的抗螟性改良,也为转Bt基因玉米自交系能够直接用于育种提供了依据。     

Fumonisins: probable role as effectors in the complex interaction of susceptible and resistant maize hybrids and Fusarium verticillioides

Fusarium verticillioides is best known for its worldwide occurrence on maize resulting in highly variable disease symptoms, ranging from asymptomatic to severe rotting and wilting and fumonisin production. The aim of this study was to investigate the influence of hybrid genotypes in the early stages of F. verticillioides infection, and the role of fumonisins as effectors in the outcome of this complex interaction. Disease symptoms, growth parameters, root morphology, and fungal colonization were evaluated at 7, 14, and 21 days after planting in seedlings from maize seeds of resistant (RH) and susceptible (SH) hybrids inoculated with F. verticillioides or watered with solutions of fumonisins. F. verticillioides induced growth enhancement or retardation depending on the plant genetic background and the fungal colonization rate, while fumonisins caused severe reduction in biomass and fitness. Seedlings watered with high fumonisin concentrations displayed lesions similar to those seen in F. verticillioides maize seedling disease, and also elicited inhibitory effects on root growth and morphology and on functional properties. In summary, these data strongly suggest a dual role for fumonisins in the F. verticillioides-maize interaction, acting as pathogenic factors at high concentrations, or triggering the plant detoxification mechanisms at low levels.     

Possible role of plant phenolics in the production of trichothecenes by Fusarium graminearum strains on different fractions of maize kernels

Four trichothecene-producing strains of Fusarium graminearum were grown on three maize grain fractions, whole grain, degermed grain, and the germ, to determine the effect of natural substrates on mycotoxin production. Monitoring the ergosterol content after 25 days of incubation indicated that fungal growth on all grain fractions was comparable. Trichothecene (TCT) production was highest on degermed grain, less on whole grain, and very low or nondetectable on the germ; similar results were found with four different strains. It was concluded that inhibitor(s) of TCT biosynthesis were present in maize germ. The presence of phenolic compounds was investigated in the different fractions. The hydroxamate 4-acetylbenzoxazolin-2-one (4-ABOA), a known inhibitor of mycotoxin production, was found in the degermed and whole grain fractions but not in the germ. Therefore, the TCT inhibition observed on the maize germ fraction used in our study is clearly not linked to 4-ABOA. Other soluble phenolic compounds were found at a much higher concentration in the germ than in the two other fractions. The inhibition property of the soluble ester-bound extracts was tested in liquid culture. A possible role for these compounds is discussed.     

Phenolics in maize genotypes differing in susceptibility to Gibberella stalk rot (Fusarium graminearum Schwabe)

The relationship between phenolic compounds and maize pith resistance to Fusarium graminearum, the causal agent of Gibberella stalk rot, was investigated. The phenolic acid profiles in the stalks of six maize inbred lines of varying susceptibility were evaluated from silking to grain maturity. Four different fractions of phenolic compounds were extracted from inoculated and non-inoculated (control) pith tissues: insoluble cell-wall-bound, free, soluble ester-bound, and soluble glycoside-bound phenolics. Analysis by HPLC revealed that p-coumaric acid and ferulic acid were the most abundant compounds in the soluble and cell-wall-bound fractions. The quantity of free, glycoside-bound, and ester-bound phenolics in the pith was lower than the level required for the inhibition of Fusarium growth or mycotoxins production; however, significant negative correlations between diferulic acid contents in the cell walls and disease severity ratings 4 days after inoculation were found. The results indicated that future studies should focus on the levels of diferulic acids during the early infection process. Diferulates may play a role in genotypic resistance of maize to Gibberella stalk rot as preformed barriers to infection.     

Assessment of nitrogen uptake and utilization in drought tolerant and Striga resistant tropical maize varieties

Maize (Zea mays L.) is an important food crop in the Guinea savannas of Nigeria. Despite its high production potential, drought, Striga hermonthica parasitsim, and poor soil fertility particularly nitrogen deficiency limit maize production in the savannas. Breeders at IITA have developed drought- and Striga-tolerant cultivars for testing, dissemination, and deployment in the region. Information on the response of these cultivars to N fertilization is, however, not available. This study evaluated grain yield, total N uptake (TNU), N uptake (NUPE), N utilization (NUTE), and N use efficiency (NUE) of selected maize cultivars along with a widely grown improved maize cultivar at two locations in the Guinea savannas of northern Nigeria. Maize grain yield increased with N application. The average grain yield of the maize cultivars was 76% higher at 30, 156% higher at 60, and 203% higher at 120 kg N ha^?1 than at 0 kg N ha^?1. This suggests that N is a limiting nutrient in the Nigerian savannas. Five drought-tolerant cultivars produced consistently higher yields when N was added at all levels. These cultivars had either high NUPE or NUTE confirming earlier reports that high N uptake or NUTE improves maize grain yield. The study also confirms earlier reports that maize cultivars that are selected for tolerance to drought are also efficient in uptake and use of N fertilizer. This means that these cultivars can be grown with application of less N fertilizer thereby reducing investment on fertilizers and reduction in environmental pollution.     

Mycoflora and multimycotoxin detection in corn silage: experimental study

Agricultural activities involve the use of crop preservation such as "trench-type" silo, which can sometimes be contaminated by fungi. To investigate the exposure of livestock and farm workers to fungal spores and mycotoxins, a multimycotoxin analysis method has been developed. Six mycotoxins (aflatoxin B1, citrinin, deoxynivalenol, gliotoxin, ochratoxin A, and zearalenone) were quantified by high-performance liquid chromatography coupled to mass spectrometry after solid-phase extraction. An experimental study of fungal species and mycotoxins was conducted in corn silage (Normandy, France) during 9 months of monitoring. The results indicated the recurrence of around 20 different species, with some of them being potentially toxigenic fungi such as Aspergillus fumigatus, Aspergillus parasiticus, Fusarium verticillioides, and Monascus ruber, and the detection of aflatoxin B1 (4-34 ppb), citrinin (4-25 ppb), zearalenone (23-41 ppb), and deoxynivalenol (100-213 ppb). This suggested a possible chronic exposure to low levels of mycotoxins.     

Fumonisin content in masa and tortillas from Mexico

Fumonisins, a family of mycotoxins produced by Fusarium moniliforme and Fusarium proliferatum, are found in maize worldwide and have been associated with animal diseases. There is concern that high dietary intake of a maize-based diet may expose people in Mexico and Central America to fumonisins. Nixtamalized maize products from Mexico and the United States were examined to evaluate methods for quantitation of the different forms of fumonisins. The chelating reagent EDTA (exceeding the calcium concentration by a factor of 1. 36) was added to enhance extraction of fumonisins because calcium remained in the samples as a result of processing. It was expected that the majority of the fumonisin detected would be in the hydrolyzed form, yet the highest level of hydrolyzed fumonisin B(1) detected was 0.1 ppm. The amount of fumonisin B(1) was significantly higher in Mexican samples (mean = 0.79 ppm) than in samples purchased in the United States (mean = 0.16 ppm).     

Fumonisin production and bioavailability to maize seedlings grown from seeds inoculated with Fusarium verticillioides and grown in natural soils

The fungus Fusarium verticillioides infects maize and produces fumonisins. The purpose of this study was to determine the ability of F. verticillioides to produce fumonisins in synthetic and natural soils and their biological availability to maize roots. Maize seeds were inoculated with a pathogenic strain of F. verticillioides (MRC826) and planted in synthetic and three different natural soils. There were statistically significant reductions in stalk weight and root mass and increased leaf lesions in the MRC826-treated seedlings in all soil types. Fumonisins were detected in all of the soils of seedlings grown from MRC826-inoculated seeds. The fumonisin produced in the soils was biologically available to seedlings as demonstrated by the statistically significant elevation of free sphingoid bases and sphingoid base 1-phosphates in their roots. These results indicate that F. verticillioides produced fumonisins in the autoclaved synthetic and natural soils and that the fumonisin produced is biologically available on the basis of evidence of inhibition of ceramide synthase.     

Changes in root length at the reproductive stage of maize plants grown in the field and quartz sand

The aims of this work were to investigate possible reasons for root mortality of maize plants at the reproductive stage and relationships between root mortality and internal sugar and external nitrogen (N) supply. Maize (Zea mays L.) plants were grown in the field in fertile soil and in a greenhouse in quartz sand with sufficient or deficient N supply. Deficient N supply reduced plant growth and total N uptake by 38% and 52%, respectively. The lengths of the seminal roots and of the early initiated adventitious roots of the first two whorls declined after reaching their maximum values before silking, no matter whether the plants were grown in the field or in quartz sand in the greenhouse. The lengths of the adventitious roots from higher nodes of plants grown in quartz sand, irrespective of N supply, did not decrease at the reproductive stage despite of decreasing sugar concentrations. In contrast, under field conditions, the length of adventitious roots from higher nodes decreased during grain filling. Total activity of all roots of greenhouse‐grown plants as deduced from translocation of N and cytokinins in the xylem exudate reached peak values at the end of the growing period, whereas in field‐grown plants N translocation decreased and cytokinin translocation did not change toward the end of the growing period. The results indicate that the pattern of root growth and mortality of maize plants in the reproductive stage was not affected by external N supply. Differences between glasshouse‐ and field‐grown plants are possibly due to effects of soil biota, which have to be further studied.     

Surveillance of fumonisins in maize-based feeds and cereals from spain.

A survey has been carried out to determine the levels of fumonisins in 171 samples of maize-based feeds and cereals available in Spain. Also, the samples were examined for mold count and fungal species. Aspergillus, Penicillium, and Fusarium were the most frequent genera, and Fusarium and Aphanocladium had the highest individual percentage counts. Regarding Fusarium species, F. moniliforme (47. 4%) was the predominant species; F. proliferatum (5.3%) and F. subglutinans (7.0%) were isolated at low frequency. The high-performance liquid chromatography-o-phthaldialdehyde fluorescence method was used for the analysis of fumonisins. The highest levels of fumonisins were detected in maize. Overall, fumonisin B(1) (FB(1)) and fumonisin B(2) (FB(2)) were detected in 79.5 and 14.6%, of samples respectively, with average FB(1) levels of 3.3 microg/g and average FB(2) levels of 1.7 microg/g. Low levels of fumonisins in wheat, barley, and soybean were detected. This would appear to be the first report of concentrations of fumonisins in these commodities.     

Natural variability of metabolites in maize grain: differences due to genetic background

Understanding the impact of genetic diversity on crop biochemical composition is a prerequisite to the interpretation and potential relevance of biochemical differences experimentally observed between genotypes. This is particularly important in the context of comparative safety assessments for crops developed by new technologies such as genetic engineering. To interrogate the natural variability of biochemical composition, grain from seven maize hybrids grown at four geographically distinct sites in Europe was analyzed for levels of proximates (fat, protein, moisture, ash, and carbohydrates), fiber, amino acids, fatty acids, four vitamins, nine minerals, and secondary metabolites. Statistical evaluation of the compositional data at the p < 0.05 level compared each hybrid against every other hybrid (head-to-head) for all analytes at each site and then across all sites to understand the factors contributing to variability. Of the 4935 statistical comparisons made in this study, 40% (1986) were found to be significant. The magnitude of differences observed, as a percent, ranged between 0.84 and 149% when all individual sites and the combined sites were considered. The large number of statistically significant differences in the levels of these analytes between seven commercial hybrids emphasizes the importance of genetic background and environment as determinants of the biochemical composition of maize grain, reflects the inherent natural variability in those analytes across a representative sampling of maize hybrids, and provides a baseline of the natural range of these nutritional and antinutritional components in maize for comparative compositional assessments.     

Characterization of protein fractions from Bt-transgenic and non-transgenic maize varieties using perfusion and monolithic RP-HPLC. Maize differentiation by multivariate analysis

Protein fractions from transgenic Bt and non-transgenic maize varieties, extracted by the Osborne solvent fraction procedure, were characterized for the first time by perfusion and monolithic RP-HPLC in very short analysis times. Albumins and globulins from different transgenic Bt maizes as well as from their non-transgenic isogenic varieties were eluted in four peaks using perfusion RP-HPLC, whereas prolamins and glutelins were separated in seven peaks. Monolithic RP-HPLC enabled the separation of maize proteins in a large number of peaks showing 6 and 10 main peaks for albumins and globulins, respectively. Prolamins migrated at retention times higher than 5 min as seven peaks, whereas glutelins were separated in three main peaks appearing at retention times higher than 6.0 min. Moreover, chromatograms of the whole protein extracts showed 8 and 11 components for perfusion and monolithic RP-HPLC, respectively. A comparison of the chromatograms of the whole protein extracts relative to transgenic and non-transgenic varieties evidenced quantitative differences on the percentages of area, mainly for peaks 2 and 3 by perfusion RP-HPLC and for peaks 3 and 7 by monolithic RP-HPLC. A discriminant analysis based on these proteic profiles was carried out to classify and predict transgenic Bt maize lines, achieving 100% correct classification using perfusion RP-HPLC.     

Association of a specific cationic peroxidase isozyme with maize stress and disease resistance responses, genetic identification, and identification of a cDNA coding for the isozyme

The presence of a pI 9.0 cationic peroxidase isozyme from milk stage pericarp of six susceptible and five resistant inbreds was correlated significantly with previously reported field data on percentage infection by Aspergillus flavus in the inbreds and their hybrids. The isozyme was constitutively expressed in some additional maize tissues and lines examined, and frequently induced by mechanical damage, heat shock, Fusarium proliferatum, and/or Bacillus subtilis in other lines tested. Native/IEF two-dimensional electrophoresis identified the isozyme as the previously genetically identified px5. A cDNA clone expressed in black Mexican sweet (BMS) maize cell cultures produced the pI 9.0 isozyme. In addition to potential use in marker-assisted breeding, enhanced expression of this cationic peroxidase through breeding or genetic engineering may lead to enhanced disease or insect resistance.     

Earthworm populations in a northern U.S. Cornbelt soil are not affected by long-term cultivation of Bt maize expressing Cry1Ab and Cry3Bb1 proteins

Earthworms, which play a key role in biogeochemical processes in soil ecosystems, could be negatively affected by the cultivation of transgenic Bt crops. Studies to date have found few effects of Bt maize on earthworm species. If adverse effects occur, they are likely to be chronic or sub-lethal and expressed over large spatial and temporal scales. Our objective in the present study was to investigate potential effects on earthworm populations in soil cultivated with Bt maize in a large multiple-year field study. We surveyed the earthworm populations in 0.16-ha experimental field plots of two varieties of Cry1Ab Bt maize, one variety of Cry3Bb1 Bt maize, and three non-transgenic control varieties cultivated for four years. Four earthworm species were found in our sample: Aporrectodea caliginosa, Aporrectodea trapezoides, Aporrectodea tuberculata (collectively, the A. caliginosa species complex), and Lumbricus terrestris. We found no significant differences in the biomass of juveniles and adults for all four species between Bt and non-Bt maize varieties. From this and previous studies, we conclude that the effects of Cry1Ab and Cry3Bb1 Bt maize on the A. caliginosa species complex and L. terrestris are small. Nonetheless, general conclusions about the effects of Bt maize on earthworm populations are not warranted due to the small number of species tested. In future laboratory studies, earthworm species should be selected according to their association with a Bt crop and the impact of that species to valued soil ecosystem processes.     

Neither transgenic Bt maize (MON863) nor tefluthrin insecticide adversely affect soil microbial activity or biomass: A 3-year field analysis

Laboratory and greenhouse studies on transgenic Bacillus thuringiensis (Bt) maize have drawn attention to the persistence and activity of the Cry proteins in soil and their potential effects on soil microorganisms, but there have been few field assessments that evaluate the effects of Bt maize with those of insecticides on soil microbial populations. This study was conducted to determine the effects of Cry3Bb Bt maize with those of the insecticide tefluthrin on soil microbial biomass and activity in the field over a 3-year cropping cycle. The recently commercialized maize variety YieldGard^® Rootworm (MON863), which produces the Cry3Bb protein, was grown along with a non-Bt isoline with and without tefluthrin applied at planting. Microbial biomass, nitrogen (N) mineralization potential, short-term nitrification rate, and respiration rate were measured in rhizosphere and bulk soil samples collected from three replicate field plots just before planting, at anthesis, and at harvest in each year. There were clear seasonal effects on microbial biomass and activity in the field soils—as represented by the consistent changes in all measured variables across years and sampling times. Differences in the measured variables were also sometimes observed between bulk and rhizosphere soil. However, there were no adverse effects of either the Bt or non-Bt maize with insecticide applied compared to the non-Bt controls; on the contrary, microbial biomass and soil respiration data suggested a stimulatory effect of the Bt genotype, particularly in comparison to the non-Bt isoline. Although ‘higher’ does not necessarily mean ‘better’, the higher microbial biomass and respiration rates observed in the Bt and insecticide-applied soils compared to non-Bt soils does allay concerns that either the Bt protein or the tefluthrin typically used to control the corn rootworm reduce microbial biomass or its respiratory activity in field soils. Similarly, the higher N mineralization potential and nitrification rates observed in some soil samples from the Bt and tefluthrin-treated plots indicate higher activity of N-mineralizing microorganisms, a potentially positive consequence as both ammonium and nitrate are effective N sources for maize during grain filling. Our data suggest that cropping MON863 Bt maize is unlikely to adversely affect soil ecology in the short term. Longer-term monitoring of transgenic cropping systems should assure that the biotic functioning of the soil is maintained as a part of studies on overall ecosystem integrity.