Dietary exposure to aflatoxin from maize and groundnut in young children from Benin and Togo, West Africa

Aflatoxins are a family of fungal toxins that are carcinogenic to man and cause immunosuppression, cancer and growth reduction in animals. We conducted a cross-sectional study among 480 children (age 9 months to 5 years) across 4 agro-ecological zones (SS, NGS, SGS and CS) in Benin and Togo to identify the effect of aflatoxin exposure on child growth and assess the pattern of exposure. Prior reports on this study [Gong, Y.Y.,Cardwell, K., Hounsa, A., Egal, S., Turner, Hall, A.J., Wild, C.P., 2002. Dietary aflatoxin exposure and impaired growth in young children from Benin and Togo: cross sectional study. British Medical Journal 325, 20-21, Gong, Y.Y., Egal, S., Hounsa, A., Turner, P.C., Hall, A.J., Cardwell, K., Wild, C.P., 2003. Determinants of aflatoxin exposure in young children from Benin and Togo, West Africa: the critical role of weaning and weaning foods. International Journal of Epidemiology, 32, 556-562] showed that aflatoxin exposure among these children is widespread (99%) and that growth faltering is associated with high blood aflatoxin-albumin adducts (AF-alb adducts), a measure of recent past exposure. The present report demonstrates that consumption of maize is an important source of aflatoxin exposure for the survey population. Higher AF-alb adducts were correlated with higher A. flavus (CFU) infestation of maize (p=0.006), higher aflatoxin contamination (ppb) of maize (p<0.0001) and higher consumption frequencies of maize (p=0.053). The likelihood of aflatoxin exposure from maize was particularly high in agro-ecological zones where the frequency of maize consumption (SGS and CS), the presence of aflatoxin in maize (SGS) or the presence of A. flavus on maize (NGS and SGS) was relatively high. Socio-economic background did not affect the presence of A. flavus and aflatoxin in maize, but better maternal education was associated with lower frequencies of maize consumption among children from the northernmost agro-ecological zone (SS) (p=0.001). The impact of groundnut consumption on aflatoxin exposure was limited in this population. High AF-alb adduct levels were correlated with high prevalence of A. flavus and aflatoxin in groundnut, but significance was weak after adjustment for weaning status, agro-ecological zone and maternal socio-economic status (resp. p=0.091 and p=0.083). Ingestion of A. flavus and aflatoxin was high in certain agro-ecological zones (SS and SGS) and among the higher socio-economic strata due to higher frequencies of groundnut consumption. Contamination of groundnuts was similar across socio-economic and agro-ecological boundaries. In conclusion, dietary exposure to aflatoxin from groundnut was less than from maize in young children from Benin and Togo. Intervention strategies that aim to reduce dietary exposure in this population need to focus on maize consumption in particular, but they should not ignore consumption of groundnuts.     

LC-MS/MS method for the determination of the fungal pigment bikaverin in maize kernels as an indicator of ear rot

Bikaverin is a polyketide-derived pigment produced by multiple species of the fungus Fusarium, some of which can cause ear and kernel rot of maize. A method was developed for the analysis of bikaverin by high-performance liquid chromatography (LC) coupled to electrospray ionisation tandem mass spectrometry (MS/MS). The quantitative nature of the LC-MS/MS method was demonstrated over a range of concentrations of bikaverin in maize. For spike-recovery experiments utilising maize spiked with bikaverin to a level 5?µg?g^?1 of maize, the measured recovery (%) was 70.6?±?10.4. Based on the utilised method, the limit of detection (based on a signal-to-noise ratio (S/N)?>?3) was better than 0.5?µg?g^?1 from bikaverin spiked into uncontaminated ground maize. Further, the limit of quantitation (LOQ) was 3?µg?g^?1 (based on S/N?>?10) from bikaverin spiked into ground maize. The method was applied to assess contamination of maize with bikaverin following inoculation of developing maize ears with Fusarium verticillioides under agricultural field conditions.     

烟草品种对镰刀菌根腐病的抗性鉴定

采用盆栽烟株带菌麦粒接种法测定12个烟草品种（系）对烟草镰刀菌根腐病的苗期抗病性,结果表明,中烟98、贵烟6号、云烟87、7478、NC89和豫烟13号对镰刀菌具有较高抗性,KRK26、中烟100和中烟205中等抗病,小黄金1025、豫烟10号和长脖黄对镰刀菌易感,在所有测试样品中,长脖黄抗性最低。长脖黄、小黄金1025和豫烟10号可作为对镰刀菌根腐病检测预警材料及镰刀菌根腐病的感病对照样品应用于相关研究中。      

Comparison of Aspergillus ear rot and aflatoxin contamination in grain of high-oil and normal-oil corn hybrids

High-oil corn (Zea mays L.) grain is a valuable component of feed for monogastric livestock. One method of increasing the concentration of oil in corn grain is the TopCross method. With TopCross, ears of a cytoplasmic male-sterile, normal-oil hybrid are pollinated by a male-fertile, high-oil synthetic hybrid. The concentration of oil in the resulting grain is increased because of xenia effects. Kernels of high-oil corn typically have a larger germ and a smaller endosperm than kernels of comparable normal hybrids. The growth of Aspergillus flavus Link:Fr within germ tissue has been reported to be more extensive than that on the whole corn kernel; therefore, the severity of Aspergillus ear rot could be more extensive and aflatoxin concentrations could be higher in high-oil grain produced by TopCross than in grain with a lower concentration of oil. The objective of this study was to compare Aspergillus ear rot severity levels and aflatoxin concentrations in the grains of hybrids crossed with high-oil or normal-oil pollinators. Fifteen hybrids were evaluated in 1998 and 1999 in Urbana, Ill. Primary ears were inoculated with A. flavus and evaluated for susceptibility to Aspergillus ear rot and aflatoxin production in grain. Concentrations of aflatoxin and oil in corn kernels were significantly higher for high-oil hybrids than for normal-oil hybrids; however, ear rot severity was unaffected by the type of pollinator. These results suggest that grain from high-oil hybrids is at greater risk for aflatoxin contamination during some growing seasons.     

Growth of an Aspergillus flavus transformant expressing Escherichia coli beta-glucuronidase in maize kernels resistant to aflatoxin production.

Kernels of a maize inbred that demonstrated resistance to aflatoxin production in previous studies were inoculated with an Aspergillus flavus strain containing the Escherichia coli beta-D-glucuronidase reporter gene linked to a beta-tubulin gene promoter and assessed for both fungal growth and aflatoxin accumulation. Prior to inoculation, kernels were pin-wounded through the pericarp to the endosperm, pin-wounded in the embryo region, or left unwounded. After 7 days incubation with the fungus, beta-glucuronidase activity (fungal growth) in the kernels was quantified using a fluorogenic assay and aflatoxin B content of the same kernels was analyzed. Kernels of a susceptible inbred, similarly treated, served as controls. Results indicate a positive relationship between aflatoxin levels and the amount of fungal growth. However, resistant kernels wounded through the pericarp to the endosperm before inoculation supported an increase in aflatoxin B over levels observed in nonwounded kernels, without an increase in fungal growth. Wounding kernels of the resistant inbred through the embryo resulted in both the greatest fungal growth and the highest levels of aflatoxin B1 for this genotype. Maintenance of resistance to aflatoxin B1 in endosperm-wounded kernels may be due to the action of a mechanism which limits fungal access to the kernel embryo.     

Relative severity of aflatoxin contamination of cereal crops in West Africa.

Aflatoxins are a common contaminant of cereals that can cause cancer, liver disease, immune suppression, retarded growth and development, and death, depending on the level and duration of exposure. Maize is an introduced crop to Africa and there have been efforts over the last 20 years or so to replace traditional cereal crops, such as sorghum (Sorghum bicolor) and pearl millet (Pennisetum glaucum), with maize. We found that maize was significantly more heavily colonized by aflatoxin-producing Aspergillus spp. than either sorghum or millet, with overall aflatoxin levels being correspondingly higher. On average, Nigerians consume 138 kg cereals annually. If the primary cereal is sorghum instead of maize, then the risk of aflatoxin-related problems is reduced 4-fold; if it is pearl millet, then the risks are reduced 8-fold. Development programs and other ventures to increase maize production in marginal cropping areas of Africa should be reconsidered and, instead, efforts to improve/maintain traditional crops encouraged.     

Biodiversity of complexes of mycotoxigenic fungal species associated with Fusarium ear rot of maize and Aspergillus rot of grape

Fusarium ear rot of maize and Aspergillus rot of grape are two examples of important plant diseases caused by complexes of species of mycotoxigenic fungi. These complexes of species tend to be closely related, produce different classes of mycotoxins, and can induce disease under different environmental conditions. The infection of maize and grape with multiple fungal species and the resulting production of large classes of mycotoxins is an example of mutual aggressiveness of microorganisms toward host species as well as to humans and animals that eat feed or food derived from the infected and contaminated plants. Infection of crop plant with a complex of microbial species certainly represents a greater threat to a crop plant and to human and animal health than infection of the plant with a single fungal species.     

Spread of Aspergillus flavus and aflatoxin accumulation in postharvested maize treated with biocontrol products

Maize is a major staple crop and calorie source for many people living in Sub-Saharan Africa. In this region, Aspergillus flavus causes ear rot in maize, contributing to food insecurity due to aflatoxin contamination. The biological control principle of competitive exclusion has been applied in both the United States and Africa to reduce aflatoxin levels in maize grain at harvest by introducing atoxigenic strains that out-compete toxigenic strains. The goal of this study was to determine if the efficacy of preharvest biocontrol treatments carry over into the postharvest drying period, the time between harvest and the point when grain moisture is safe for storage. In Sub-Sahara Africa, this period often is extended by weather and the complexities of postharvest drying practices. Maize grain was collected from fields in Texas and North Carolina that were treated with commercial biocontrol products and untreated control fields. To simulate moisture conditions similar to those experienced by farmers during drying in Sub-Sahara Africa, we adjusted the grain to 20% moisture content and incubated it at 28 °C for 6 days. Although the initial number of kernels infected by fungal species was high in most samples, less than 24% of kernels were infected with Aspergillus flavus and aflatoxin levels were low (<4 ppb). Both toxigenic and atoxigenic strains grew and spread through the grain over the incubation period, and aflatoxin levels increased, even in samples from biocontrol-treated fields. Our molecular analysis suggests that applied biocontrol strains from treated fields may have migrated to untreated fields. These results also indicate that the population of toxigenic A. flavus in the harvested grain will increase and produce aflatoxin during the drying period when moisture is high. Therefore, we conclude that preharvest biocontrol applications will not replace the need for better postharvest practices that reduce the drying time between harvest and storage.     

Impregnated bags for safer storage of legume grains in West and Central Africa

The use of jute bags impregnated with aqueous extracts from two insecticidal plants, Chenopodium ambrosioides and Lantana camara, to reduce damage to stored legume seeds by two major bruchid insect pests, Acanthoscelides obtectus and Callosobruchus maculatus, was compared with a direct seed-treatment method using plant powders. In the first method bags were treated with a 10% concentration of the aqueous plant extract and filled with bean or cowpea seeds, whereas in the second method seeds were thoroughly mixed with leaf powder from the same plants at the rate of 4% (w/w). The bag-treatment method significantly reduced seed damage compared with the untreated control after 6 months of storage (4- to 6-fold decrease in percentage seed damage). Even though relatively low (? 20%), damage by the two insect species controlled by this method was significantly higher than the damage recorded in the seed-treatment method. Orthogonal contrast analysis across test insects showed a significant seed damage difference between treatments with C. ambrosioides and those with L. camara, the latter having the lowest damage profile. The biosafety implication of the use of plant extract-impregnated bags for the protection of food grains against damage by storage bruchid pests is highlighted.     

Responses and genetics of maize germplasm resistant to the maize weevil Sitophilus zeamais Motschulsky in West Africa

The maize weevil, Sitophilus zeamais Motschulsky causes considerable damage to maize grain during storage in the tropics. The objectives of this study were to determine through two experiments the genetic basis of resistance to S. zeamais in African maize germplasm of open-pollinated cultivars, lines and hybrids. The first experiment tested for resistance of 20 maize cultivars (10 white and 10 yellow-grained) developed for lowland Africa grown in three relatively weevil-free savanna locations. The second experiment tested two sets of diallel crosses without reciprocals of 66 F₁ progeny from 12 inbred lines and 78 F₁ crosses from 13 inbred lines of subtropical and temperate origins. Variables studied for the first experiment included husk cover, grain texture, number of egg plugs, number of weevils produced, and number of grains damaged by the weevil. Results showed highly significant differences among the 20 maize cultivars tested. Significant cultivar×location interactions for egg plug and weevil production indicate the importance of environmental effects and complexity in weevil resistance. Of the 20 cultivars tested, Ikenne 83-TZSR-W-1 (open pollinated) and 8329-15 (hybrid) were the most promising for restricted weevil production and minimal grain damage. The results of genetic studies in the second experiment with 144 crosses indicated the presence of genetic differences in maize lines to weevil attack. General combining ability (GCA) and specific combining ability mean squares were highly significant, suggesting that the maize weevil resistance was controlled by both additive and non-additive gene actions and the inheritance was quantitative and polygenic. Among the lines used in the crosses, the two most weevil resistant lines with high GCA effects were TZi 30 (International Institute of Tropical Agriculture) and FLA2BT 106 (Florida).     

Resistance of selected Mozambican local and improved maize genotypes to maize weevil,Sitophilus zeamais (Motschulsky)

Maize weevil Sitophilus zeamais (Motschulsky) is one of the most important pests of on-farm stored maize in thetropics, particularly where there is no control of moisture content and application of pesticides. This study aimed todetermine the level of resistance among Mozambican maize genotypes against S. zeamais. A total of 17 genotypes,composed of seven experimental hybrids developed from S5 inbred lines from five populations, one released hybrid,two improved open pollinated varieties (OPV), three landraces from Mozambique, two resistant and two susceptiblechecks from Kenya were screened for their resistance to S. zeamais. Four hybrids were screened at their F1 and F2generations. The genotypes were evaluated in the post-harvest laboratory, Kiboko, Kenya in a completely randomizeddesign, replicated 4 times. Data was collected on the number of emerged insects, seed weight loss, seed damage,median development period; and protein, starch and oil contents. The Dobie index of susceptibility was used to groupthe genotypes. Six of the of the eight hybrids at the F1 generation were resistant while two were moderately resistant.Three of the four hybrids evaluated at the F2 generation were moderately resistant while one was susceptible. Two ofthe five OPVs evaluated (EV8430DMRSR and landrace Kandjerendjere) were resistant. Genotypes with high proteincontent showed resistance while those with high starch contents showed susceptibility. Weevils fed on the resistantgenotype produced low number of F1 progeny, had a high median developmental time, caused low seed damage andlow seed weight loss. The resistant genotypes can be either used as cultivars or as sources of resistance in a breedingprogram for weevil resistance.     

Investigations on Fusarium spp. and their mycotoxins causing Fusarium ear rot of maize in Kosovo

After wheat, maize (Zea mays L.) is the second most important cereal crop in Kosovo and a major component of animal feed. The purpose of this study was to analyse the incidence and identity of the Fusarium species isolated from naturally infected maize kernels in Kosovo in 2009 and 2010, as well as the mycotoxin contamination. The disease incidence of Fusarium ear rot (from 0.7% to 40% diseased ears) on maize in Kosovo is high. The most frequently Fusarium spp. identified on maize kernels were Fusarium subglutinans, F. verticillioides/F. proliferatum and F. graminearum. Maize kernel samples were analysed by LC-MS/MS and found to be contaminated with deoxynivalenol (DON), DON-3-glucoside, 3-acetyl-DON, 15-acetyl-DON, zearalenone, zearalenone-14-sulphate, moniliformin, fumonisin B₁ and fumonisin B₂. This is the first report on the incidence and identification of Fusarium species isolated from naturally infected maize as well as the mycotoxin contamination in Kosovo.     

Fumonisin contamination of maize in Burkina Faso, West Africa.

Maize throughout the world is frequently contaminated with a family of mycotoxins, the fumonisins, produced by species of Fusaria. The study investigated the level of fumonisin contamination of maize samples from village farms and large market traders in Burkina Faso, West Africa. Maize samples (5 kg) from each of five to six large storage barns from farms in five villages in the district of N'Dorola, Kénédougou province, western Burkina Faso, were sampled (n = 26) in Jan 1999 (> 1 year storage), and a further 26 maize samples from the same farms were collected directly from the field in October 1999. In addition, 72 maize samples were obtained in July 1999 from large markets in Bobo Dioulasso. Fumonisins were extracted from dried maize, derivatized with o-phthaldialdehyde and quantified by reversed-phase high-performance liquid chromatography with fluorescence detection. All 26 samples from the first (mean 1170 microg kg(-1), range 110-3120 microg kg(-1)) and from the second (mean 130 microg kg(-1), range 10-450 microg kg(-1)) village collection were fumonisin positive. All 72 maize samples from the large markets were also positive for fumonisins, and had the highest levels of contamination (mean 2900 microg kg(-1), range 130-16,040 microg kg(-1)). As fumonisins were a ubiquitous contaminant of maize and given that this crop is a dietary staple in this region, chronic exposure is likely.     

A survey of pre-harvest ear rot diseases of maize and associated mycotoxins in south and central Zambia

Maize ear rots reduce grain yield and quality with implication on food security and health. Some of the pathogenic fungi produce mycotoxins in maize grain posing a health risk to humans and livestock. Unfortunately, the levels of ear rot and mycotoxin infection in grain produced by subsistence farmers in sub-Saharan countries are not known. A survey was thus conducted to determine the prevalence of the ear rot problem and levels of mycotoxins in maize grain. A total of 114 farmsteads were randomly sampled from 11 districts in Lusaka and southern provinces in Zambia during 2006. Ten randomly picked cobs were examined per farmstead and the ear rot disease incidence and severity were estimated on site. This was followed by the standard seed health testing procedures for fungal isolation in the laboratory. Results indicated that the dominant ear rots were caused by Fusarium and Stenocarpella. Incidence of Fusarium verticillioides ranged from 2 to 21%, whereas that of Stenocarpella maydis reached 37% on ear rot diseased maize grain. In addition, 2-7% F. verticillioides, and 3-18% Aspergillusflavus, respectively, were recovered from seemingly healthy maize grain. The mean rank of fungal species, from highest to lowest, was F. verticillioides, S. maydis, A.flavus, Fusarium graminearum, Aspergillus niger, Penicillium spp., Botrydiplodia spp., and Cladosporium spp. The direct competitive ELISA-test indicated higher levels of fumonisins than aflatoxins in pre-harvest maize grain samples. The concentration of fumonisins from six districts, and aflatoxin from two districts, was 10-fold higher than 2 ppm and far higher than 2 ppb maximum daily intake recommended by the FAO/WHO. The study therefore suggested that subsistence farmers and consumers in this part of Zambia, and maybe also in similar environments in sub-Saharan Africa, might be exposed to dangerous levels of mycotoxins due to the high levels of ear rot infections in maize grain.     

Silk maysin content and resistance of commercial corn [maize] hybrids to kernel contamination by aflatoxin

Several phenols and related compounds have been shown to have detrimental effects on insects while others have antibiotic activity against fungi which attack higher plants. Insects have also been implicated as contributors to preharvest contamination of corn [maize], Zea mays L, by aflatoxin. The objectives, therefore, were to determine (a) if commercial corn hybrids vary in their silk maysin content and aflatoxin contamination of the grain, and (b) if grain aflatoxin contamination is correlated with maysin and related compounds. During 4 years of testing, 16 corn hybrids varied significantly for silk maysin content and grain aflatoxin contamination. Based on correlations, grain aflatoxin content of the hybrids tested was not significantly associated with maysin, chlorogenic acid, and 3′-methoxymaysin contents. It was concluded that other untested phenolic compounds in the category of compounds analyzed in the present study could be involved in resistance to aflatoxin formation, and that other classes of compounds should also now be assessed to locate major chemical resistance components.     

The detection of aflatoxin B1 in peanut kernels,peanut butter and maize using a monoclonal antibody based enzyme immunoassay

A simple procedure was validated for the routine immunochemical analysis of AFB₁ in peanut kernels, peanut butter and maize. The specificity, affinity and sensitivity of the monoclonal antibody (McAb) employed was such that minimal sample preparation was required. The enzyme immunoassay (EIA) had a sensitivity for standard AFB₁ of 0·2 ng ml⁻¹ with a working range up to 30 ng ml⁻¹ and was not significantly affected by matrix interference of samples. Essential protocol features were (1) blending substrate with methanol: water; (2) filtering blended sample; and (3) analysis of filtrate by EIA after dilution with buffer. Average recoveries of AFB₁ spiked samples at levels of 6–400 ppb were 90–112·5%. Using laboratory prepared samples contaminated with Aspergillus flavus there was high positive correlation (r = 0·97) when EIA results were compared with thin layer chromatography (TLC) techniques.     

Application of essential oils in maize grain: impact on Aspergillus section Flavi growth parameters and aflatoxin accumulation

The antifungal activity of Pimpinella anisum L. (anise), Pëumus boldus Mol (boldus), Hedeoma multiflora Benth (mountain thyme), Syzygium aromaticum L. (clove), and Lippia turbinate var. integrifolia (griseb) (poleo) essential oils (EOs) against Aspergillus section Flavi was evaluated in sterile maize grain under different water activity (a_w) condition (0.982, 0.955, and 0.90). The effect of EOs added to maize grains on growth rate, lag phase, and aflatoxin B₁ (AFB₁) accumulation of Aspergillus section Flavi were evaluated at different water activity conditions. The five EOs analyzed have been shown to influence lag phase and growth rate. Their efficacy depended mainly on the essential oil concentrations and substrate water activity conditions. All EOs showed significant impact on AFB₁ accumulation. This effect was closely dependent on the water activity, concentration, and incubation periods. Important reduction of AFB₁ accumulation was observed in the majority of EO treatments at 11 days of incubation. Boldus, poleo, and mountain thyme EO completely inhibited AFB₁ at 2000 and 3000 μg g⁻¹. Inhibition of AFB₁ accumulation was also observed when aflatoxigenic isolates grew with different concentration of EOs during 35 days.     

Distribution of aflatoxin in whole peanut kernels, sampling plans for small samples

It is well known that the distribution of aflatoxin in a lot of whole peanut kernels is extremely heterogeneous. Several different statistical distribution models have been proposed, fitting the experimental data reasonably well as long as the samples are very large, but differing considerably when applied to small samples. Therefore, it is important to know the real distribution between single kernels for the evaluation of the effectiveness of sampling plans for small samples. It is shown by the analysis of 368 samples of 1-10,000 kernels from the same lot of peanuts that the negative binomial distribution represents a good statistical model. The variance can be estimated from the mean concentration of the analysed samples, as confirmed by the comparison of data from several independent investigations. Decisions based on small samples are especially unfavourable to the consumer, as even a lot with a high mean concentration will tend to give negative results. A reasonably small risk of a false decision, both to the consumer and to the producer, can be reached only if very large samples are analysed.     

Delivery systems for biological control agents to manage aflatoxin contamination of pre-harvest maize

While soil application of a competitive non-toxigenic Aspergillus flavus strains is successful in reducing aflatoxin contamination in certain crops, direct application to aerial reproductive structures could be more effective for maize. A sprayable, clay-based water-dispersible granule formulation was developed to deliver non-toxigenic A. flavus strain K49 directly to maize ears. The efficacy of the K49 water-dispersible granule in mitigating aflatoxin in maize (Zea mays L.) was evaluated. Field studies were conducted to compare K49 colonization and effectiveness in reducing aflatoxin contamination when applied either as a soil inoculant or as a directed spray in plots infested with toxigenic strain F3W4. Fifty percent of non-toxigenic A. flavus was recovered from non-treated controls and from plots soil inoculated with K49 on wheat. In spray treatments with formulated or unformulated K49 conidia, over 90% of A. flavus recovered was non-toxigenic. Soil-applied K49 reduced aflatoxin contamination by 65% and spray applications reduced contamination by 97%. These findings suggest direct spray application of non-toxigenic A. flavus strains may be better than soil inoculation at controlling maize aflatoxin contamination and that a water-dispersible granule is a viable delivery system for maintaining viability and efficacy of the biological control agent, K49.