Reduced Aflatoxin Accumulation Research Articles

Performance of testers with contrasting provitamin A content to evaluate provitamin A maize for resistance to Aspergillus flavus infection and aflatoxin production.

In sub-Saharan Africa (SSA), millions of people depend on maize as a primary staple. However, maize consumers in SSA may be exposed to malnutrition due to vitamin A deficiency (VAD) and unsafe aflatoxin levels, which can lead to serious economic and public health problems. Provitamin A (PVA) biofortified maize has been developed to alleviate VAD and may have additional benefits such as reduced aflatoxin contamination. In this study, maize inbred testers with contrasting PVA content in grain were used to identify inbred lines with desirable combining ability for breeding to enhance their level of resistance to aflatoxin. Kernels of 120 PVA hybrids generated by crossing 60 PVA inbreds with varying levels of PVA (5.4 to 51.7 µg/g) and two testers (low and high PVA, 14.4 and 25.0 µg/g, respectively) were inoculated with a highly toxigenic strain of Aspergillus flavus. Aflatoxin had a negative genetic correlation with β-carotene (r= -0.29, p < 0.0001) and PVA (r = -0.23, p < 0.0001), indicating that hybrids with high PVA content accumulated less aflatoxin than those with low to medium PVA. Both general combining ability (GCA) and specific combining ability (SCA) effects of lines and testers were significant for aflatoxin accumulation, number of spores, PVA, and other carotenoids, with additive gene actions playing a prominent role in regulating the mode of inheritance (GCA/SCA ratio >0.5). Eight inbreds had combined significant negative GCA effects for aflatoxin accumulation and spore count with significant positive GCA effects for PVA. Five testcrosses had combined significant negative SCA effects for aflatoxin with significant positive SCA effects for PVA. The high PVA tester had significant negative GCA effects for aflatoxin, lutein, β-carotene, and PVA. The study identified lines that can be used as parents to develop superior hybrids with high PVA and reduced aflatoxin accumulation. Overall, the results point out the importance of testers in maize breeding programs to develop materials that can contribute to controlling aflatoxin contamination and reducing VAD.

Pre-harvest strategy for reducing aflatoxin accumulation during storage of maize in Argentina

Maize (Zea mays L.) is an important crop in Argentina. Aspergillus flavus may infect this crop at growing stage and the harvested kernels can be contaminated with aflatoxins (AFs), whose levels may increase during storage. In Argentina, silo bags, a hermetic type of storage system, are widely used. Biocontrol based on competitive exclusion by atoxigenic A. flavus strains is a useful tool for AFs management at pre-harvest stage. The aim of the present study was to evaluate the effect of pre-harvest biocontrol treatments on aflatoxin B1 (AFB1) accumulation in maize stored in silo bags during 3 and 6 months. Three bioformulations based on A. flavus AFCHG2 and ARG5/30 strains were applied during field trials as single and mixed inocula. Harvested kernels were stored in non-hermetic and hermetic silo bags. At initial time (t0), 3 and 6 months (t3 and t6) the following parameters were evaluated: percentage of damaged kernels, moisture content, water activity, Aspergillus section Flavi incidence, relative humidity, O2 and CO2 levels into the silo bags, and AFB1 levels. The biocontrol strains included in the 3 bioformulations were able to infect maize kernels during the field trial and displaced native toxigenic isolates. At t0 control plots showed 10.9 ± 0.4 μg/kg of AFB1 while no AFs were detected in all the treatments. Along the storage assay AFB1 levels varied from not detected (<1 μg/kg) to 20.1 ± 0.8 μg/kg. Hermetic bags were better than non-hermetic bags in preventing AFB1 accumulation. Both single and mixed inocula were effective to control AFB1 accumulation in maize kernels during 3 and 6 months. AFB1 was not detected in kernels from the treatment at field stage with AFCHG2 + ARG5/30 after 6 months of storage into hermetic bags. The application of the biocontrol agents at field stage is an appropriate tool to reduce AFB1 accumulation under storage in hermetic silo bags. This is the first report on biocontrol strategy based on native atoxigenic strains applied at pre-harvest stage to reduce AFB1 accumulation during storage in Argentina.

Differential response of maize hybrids to field infection with Aspergillus flavus and aflatoxin accumulation in the Chaco Semi-arid region of Argentina

Maize is affected by Aspergillus flavus and by the contamination of its kernels with aflatoxins, of which AFB1 is the most important. The Chaco Semi-arid is a region of Argentina in which the climate conditions increase this problem and due to the use of resistant cultivars is the best available tactic to reduce aflatoxin accumulation, we analyzed six hybrids against artificial inoculation with native aflatoxin-producing isolates of A. flavus. A multifactorial experiment was conducted in three complete randomized blocks, under Inoculated and Non-Inoculated treatment, in two locations and during two growing seasons, in which yield, kernel infection, and AFB1 accumulation (using HPLC) were evaluated. P2089VYHR was outstanding during 2018/19 with 11,089 kg ha−1, coinciding with the growing season with the highest rainfall and 4% decrease in yield (395 kg ha−1) was observed between treatments. No yield differences were observed in the hybrids between localities, or in the hybrid × treatment interaction. The level of infection by A. flavus showed differences between localities and growing seasons but not between hybrids. The Inoculated treatment registered kernel infections above 50%, while natural infection was 35% during 2019/20, associated with rainfall below crop requirements during the flowering period. The AFB1 accumulation analysis identified P2089VYHR and P1366VYHR without AFB1 accumulation. Yield correlated negatively with the percentage of kernel infection, but not with AFB1 accumulation, while a positive correlation was observed between these last two parameters. The pathogen causes a decrease in yield in the region and there are hybrids that are resistant to AFB1.

Control of aflatoxin using atoxigenic strains and irrigation management is complicated by maize hybrid diversity

AbstractMycotoxins produced by the fungus Aspergillus flavus are harmful to humans and animals that feed on contaminated products. This study was conducted to determine if there are any interactions between applications of an atoxigenic A. flavus, irrigation regimes, locations, and hybrids that affect contamination of maize (Zea mays L.). Total genotypic variation was 3.8% for transformed aflatoxin data, suggesting that the diverse hybrids used were a minor to moderate driver of aflatoxin variation in this experiment. Location (Loc) × year × irrigated (Irr) × genotype (Gen) for transformed aflatoxin explained 3.3% variation, which is useful in selecting reduced aflatoxin‐susceptible genotypes in differing environments. Variation from atoxigenic inoculation (Inoc) explained 2.7% variation and reduced aflatoxin accumulation by 11% on average. Location × Inoc explained 2.1% of variation, supporting the effectiveness of atoxigenic applications dependent on different locations. All effects containing Inoc × Gen totaled .45%, suggesting that genotypes could have a small influence on effectiveness of atoxigenic applications. These are critical components to understand that can aid a breeder in selecting aflatoxin‐tolerant germplasm.

Aflatoxin Accumulation in a Maize Diallel Cross Containing Inbred Lines with Expired Plant Variety Protection

In-field infection of maize (Zea mays L.) ears by the fungus Aspergillus flavus Link:Fr causes pre-harvest aflatoxin contamination of maize grain. Germplasm lines with host-plant resistance to aflatoxin accumulation are available to breeders, but these lines often possess undesirable agronomic characteristics. Commercial lines with expired plant variety protection (ex-PVP lines) are a potential source of elite germplasm available to public maize breeders. A diallel cross containing three aflatoxin-accumulation-resistant germplasm lines and seven ex-PVP lines were evaluated in replicated trials for aflatoxin contamination after artificial inoculation and for yield. The resistant germplasm lines Mp313E, Mp715, and Mp717 were the only lines with significant general combining ability (GCA) for reduced aflatoxin accumulation. Of the ex-PVP lines evaluated, the Stiff-Stalk line F118 was the most promising line to use in breeding crosses. Based on its GCA, it was the only ex-PVP line that did not significantly increase aflatoxin and the only ex-PVP line that significantly increased yield. Second-cycle breeding lines derived from crosses between F118 and the resistant donor lines will be valuable if they combine the donor lines’ disease resistance with F118’s earlier maturity while introgressing the disease resistance into a genetic background that aligns with the industry’s well-defined heterotic groups.

In Vitro Biological Control of Aspergillus flavus by Hanseniaspora opuntiae L479 and Hanseniaspora uvarum L793, Producers of Antifungal Volatile Organic Compounds.

Aspergillus flavus is a toxigenic fungal colonizer of fruits and cereals and may produce one of the most important mycotoxins from a food safety perspective, aflatoxins. Therefore, its growth and mycotoxin production should be effectively avoided to protect consumers’ health. Among the safe and green antifungal strategies that can be applied in the field, biocontrol is a recent and emerging strategy that needs to be explored. Yeasts are normally good biocontrol candidates to minimize mold-related hazards and their modes of action are numerous, one of them being the production of volatile organic compounds (VOCs). To this end, the influence of VOCs produced by Hanseniaspora opuntiae L479 and Hanseniaspora uvarum L793 on growth, expression of the regulatory gene of the aflatoxin pathway (aflR) and mycotoxin production by A. flavus for 21 days was assessed. The results showed that both yeasts, despite producing different kinds of VOCs, had a similar effect on inhibiting growth, mycotoxin biosynthetic gene expression and phenotypic toxin production overall at the mid-incubation period when their synthesis was the greatest. Based on the results, both yeast strains, H. opuntiae L479 and H. uvarum L793, are potentially suitable as a biopreservative agents for inhibiting the growth of A. flavus and reducing aflatoxin accumulation.

Mapping Quantitative Trait Loci Associated With Resistance to Aflatoxin Accumulation in Maize Inbred Mp719.

Aflatoxins are carcinogenic and toxic compounds produced principally by fungal species Aspergillus flavus (Link: Fries) and A. parasiticus (Speare), which are common contaminants of food and feed. Aflatoxins can be found at dangerously high levels and can readily contaminate pre-harvest maize (Zea mays L.) grain. Sources of resistance to aflatoxin accumulation in maize have been identified, however, the highly quantitative nature and complex inheritance of this trait have limited the introgression of aflatoxin accumulation resistance into agronomically desirable lines. Mapping of quantitative trait loci (QTL) was performed on a bi-parental population comprised of 241 F2:3 families derived from the cross of inbred lines Mp705 (susceptible) × Mp719 (resistant). The mapping population was phenotyped in replicated field trials in three environments for resistance to aflatoxin accumulation under artificial inoculation with an A. flavus spore suspension. The genetic linkage map was constructed with 1,276 single nucleotide polymorphism (SNP) and simple sequence repeat (SSR) molecular markers covering a total genetic distance of 1,642 cM across all ten maize chromosomes. Multiple interval mapping revealed that majority of the aflatoxin-reducing alleles and the source for the larger effect QTL identified in this study were contributed from Mp719, the resistant parent. Two QTL identified on chromosome 1 (bin 1.06–1.07) and chromosome 3 (bin 3.09) were the most stable across different environments and when combined, explained 24.6% of the total phenotypic variance across all three environments. Results from the study showed that these chromosomal regions harbor important QTL for influencing aflatoxin accumulation, which is consistent with previous reports with other different mapping populations. These stable QTL were the most promising for controlling aflatoxin accumulation in maize grain. Identifying beneficial alleles derived from Mp719 and closely linked molecular markers through QTL analysis for implementation of MAS could accelerate breeding efforts to reduce aflatoxin accumulation in maize.

Biocontrol Strains Differentially Shift the Genetic Structure of Indigenous Soil Populations of Aspergillus flavus.

Biocontrol using non-aflatoxigenic strains of Aspergillus flavus has the greatest potential to mitigate aflatoxin contamination in agricultural produce. However, factors that influence the efficacy of biocontrol agents in reducing aflatoxin accumulation under field conditions are not well-understood. Shifts in the genetic structure of indigenous soil populations of A. flavus following application of biocontrol products Afla-Guard and AF36 were investigated to determine how these changes can influence the efficacy of biocontrol strains in reducing aflatoxin contamination. Soil samples were collected from maize fields in Alabama, Georgia, and North Carolina in 2012 and 2013 to determine changes in the population genetic structure of A. flavus in the soil following application of the biocontrol strains. A. flavus L was the most dominant species of Aspergillus section Flavi with a frequency ranging from 61 to 100%, followed by Aspergillus parasiticus that had a frequency of <35%. The frequency of A. flavus L increased, while that of A. parasiticus decreased after application of biocontrol strains. A total of 112 multilocus haplotypes (MLHs) were inferred from 1,282 isolates of A. flavus L using multilocus sequence typing of the trpC, mfs, and AF17 loci. A. flavus individuals belonging to the Afla-Guard MLH in the IB lineage were the most dominant before and after application of biocontrol strains, while individuals of the AF36 MLH in the IC lineage were either recovered in very low frequencies or not recovered at harvest. There were no significant (P > 0.05) differences in the frequency of individuals with MAT1-1 and MAT1-2 for clone-corrected MLH data, an indication of a recombining population resulting from sexual reproduction. Population mean mutation rates were not different across temporal and spatial scales indicating that mutation alone is not a driving force in observed multilocus sequence diversity. Clustering based on principal component analysis identified two distinct evolutionary lineages (IB and IC) across all three states. Additionally, patristic distance analysis revealed phylogenetic incongruency among single locus phylogenies which suggests ongoing genetic exchange and recombination. Levels of aflatoxin accumulation were very low except in North Carolina in 2012, where aflatoxin levels were significantly (P < 0.05) lower in grain from treated compared to untreated plots. Phylogenetic analysis showed that Afla-Guard was more effective than AF36 in shifting the indigenous soil populations of A. flavus toward the non-toxigenic or low aflatoxin producing IB lineage. These results suggest that Afla-Guard, which matches the genetic and ecological structure of indigenous soil populations of A. flavus in Alabama, Georgia, and North Carolina, is likely to be more effective in reducing aflatoxin accumulation and will also persist longer in the soil than AF36 in the southeastern United States.

Characterization and competitive ability of non-aflatoxigenic Aspergillus flavus isolated from the maize agro-ecosystem in Argentina as potential aflatoxin biocontrol agents

Aspergillus flavus is an opportunistic pathogen and may produce aflatoxins in maize, one of the most important crops in Argentina. A promising strategy to reduce aflatoxin accumulation is the biological control based on competitive exclusion. In order to select potential biocontrol agents among isolates from the maize growing region in Argentina, a total of 512 A. flavus strains were isolated from maize kernels and soil samples. Thirty-six per cent of the isolates from maize kernels did not produce detectable levels of aflatoxins, while 73% of the isolates from soil were characterized as non-aflatoxin producers. Forty percent and 49% of the isolates from maize kernels and soil samples, respectively, were not producers of cyclopiazonic acid (CPA). Sclerotia morphology was evaluated using Czapek Dox media. Eighty-six per cent of the isolates from maize kernels and 85% of the isolates from soil samples were L sclerotia morphotype (average diameter > 400 μm). The remaining isolates did not produce sclerotia. All isolates had MAT 1-1 idiomorph. The competitive ability of 9 non aflatoxigenic strains, 4 CPA(+) and 5 CPA(−), was evaluated in co-inoculations of maize kernels with an aflatoxigenic strain. All evaluated strains significantly (p < 0.05) reduced aflatoxin contamination in maize kernels. The aflatoxin B1 (AFB1) reduction ranged from 6 to 60%. The strain A. flavus ARG5/30 isolated from maize kernels would be a good candidate as a potential biocontrol agent to be used in maize, since it was characterized as neither aflatoxin nor CPA producer, morphotype L, MAT 1-1 idiomorph, and reduced AFB1 content in maize kernels by 59%. This study showed the competitive ability of potential aflatoxin biocontrol agents to be evaluated under field trials in a maize agro-ecosystem in Argentina.

Diallel analysis for aflatoxin accumulation and fall armyworm leaf-feeding damage in maize

ABSTRACTTwo major impediments to profitable maize, Zea mays L., production in the southern United States are from feeding by fall armyworm, Spodoptera frugiperda (J.E. Smith), and losses from the production and accumulation of aflatoxin in maize grain. A diallel cross was produced by making all possible crosses among five germplasm lines developed as sources of resistance to fall armyworm leaf feeding and five lines developed as sources of resistance to aflatoxin accumulation. For resistance to both leaf feeding and aflatoxin accumulation, general combining ability (GCA) was a significant source of variation. Specific combining ability (SCA) was significant for fall armyworm feeding only. Estimates of GCA effects for reduced aflatoxin accumulation were significant for Mp715 and Mp719, two lines selected for resistance to aflatoxin accumulation. The GCA effects for reduced fall armyworm damage were significant for all five lines selected for fall armyworm resistance: Mp707, Mp708, Mp713, Mp714, and Mp716.

Expression Profiling Coupled with In-silico Mapping Identifies Candidate Genes for Reducing Aflatoxin Accumulation in Maize.

Aflatoxin, produced by Aspergillus flavus, is hazardous to health of humans and livestock. The lack of information about large effect QTL for resistance to aflatoxin accumulation is a major obstacle to employ marker-assisted selection for maize improvement. The understanding of resistance mechanisms of the host plant and the associated genes is necessary for improving resistance to A. flavus infection. A suppression subtraction hybridization (SSH) cDNA library was made using the developing kernels of Mp715 (resistant inbred) and B73 (susceptible inbred) and 480 randomly selected cDNA clones were sequenced to identify differentially expressed genes (DEGs) in response to A. flavus infection and map these clones onto the corn genome by in-silico mapping. A total of 267 unigenes were identified and majority of genes were related to metabolism, stress response, and disease resistance. Based on the reverse northern hybridization experiment, 26 DEGs were selected for semi-quantitative RT-PCR analysis in seven inbreds with variable resistance to aflatoxin accumulation at two time points after A. flavus inoculation. Most of these genes were highly expressed in resistant inbreds. Quantitative RT-PCR analysis validated upregulation of PR-4, DEAD-box RNA helicase, and leucine rich repeat family protein in resistant inbreds. Fifty-six unigenes, which were placed on linkage map through in-silico mapping, overlapped the QTL regions for resistance to aflatoxin accumulation identified in a mapping population derived from the cross between B73 and Mp715. Since majority of these mapped genes were related to disease resistance, stress response, and metabolism, these should be ideal candidates to investigate host pathogen interaction and to reduce aflatoxin accumulation in maize.

Ameliorating Effects of Bacillus subtilis ANSB060 on Growth Performance, Antioxidant Functions, and Aflatoxin Residues in Ducks Fed Diets Contaminated with Aflatoxins.

Bacillus subtilis ANSB060 isolated from fish gut is very effective in detoxifying aflatoxins in feed and feed ingredients. The purpose of this research was to investigate the effects of B. subtilis ANSB060 on growth performance, body antioxidant functions, and aflatoxin residues in ducks fed moldy maize naturally contaminated with aflatoxins. A total of 1500 18-d-old male Cherry Valley ducks with similar body weight were randomly assigned to five treatments with six replicates of 50 ducks per repeat. The experiment design consisted of five dietary treatments labeled as C0 (basal diet containing 60% normal maize), M0 (basal diet containing 60% moldy maize contaminated with aflatoxins substituted for normal maize), M500, M1000, and M2000 (M0 +500, 1000 or 2000 g/t aflatoxin biodegradation preparation mainly consisted of B. subtilis ANSB060). The results showed that ducks fed 22.44 ± 2.46 μg/kg of AFB1 (M0) exhibited a decreasing tendency in average daily gain (ADG) and total superoxide dismutase (T-SOD) activity in serum, and T-SOD and glutathione peroxidase (GSH-Px) activities in the liver significantly decreased along with the appearance of AFB1 and AFM1 compared with those in Group C0. The supplementation of B. subtilis ANSB060 into aflatoxin-contaminated diets increased the ADG of ducks (p > 0.05), significantly improved antioxidant enzyme activities, and reduced aflatoxin accumulation in duck liver. In conclusion, Bacillus subtilis ANSB060 in diets showed an ameliorating effect to duck aflatoxicosis and may be a promising feed additive.

Quantitative trait loci (QTL) for reducing aflatoxin accumulation in corn

Aflatoxin produced by Aspergillus flavus in corn poses significant health risks to both humans and livestock. Exploitation of host-plant resistance in breeding programs is a sustainable way to minimize aflatoxin contamination. Identification of quantitative trait loci (QTL) associated with resistance to aflatoxin accumulation in kernels can accelerate development of aflatoxin-resistant corn using marker-assisted selection. An F2:3 mapping population, developed from a cross involving a resistant inbred Mp715 and a susceptible inbred B73, was evaluated in replicated field trials with developing ears artificially inoculated with A. flavus for 2 years to identify QTL for reduced aflatoxin accumulation. Using composite interval mapping, 6 to 7 QTL for aflatoxin content were identified in both years with contribution of individual QTL ranging from <1 to 10% of phenotypic variation. More QTL were detected for husk coverage with phenotypic variance range of <1 to 16% explained by individual QTL. Both B73 and Mp715 alleles at these QTL loci contributed toward resistance. Husk coverage and aflatoxin levels were significantly correlated in both years. Our findings were further supported by overlapping of QTL for husk coverage ratings in four genomic regions on chromosomes 4, 8, and 10, where aflatoxin resistance QTL were reported in previous studies. Since most of the QTL were of low to moderate effects, pyramiding of these QTL may lead to enhanced resistance to aflatoxin accumulation in corn.

Evaluation of African-Bred Maize Germplasm Lines for Resistance to Aflatoxin Accumulation

Aflatoxins, produced by the fungus Aspergillus flavus, contaminate maize grain and threaten human food and feed safety. Plant resistance is considered the best strategy for reducing aflatoxin accumulation. Six maize germplasm lines, TZAR101–TZAR106, were released by the International Institute of Tropical Agriculture-Southern Regional Research Center (IITA-SRRC) maize breeding collaboration for use in African National Programs and U.S. maize breeding programs. The present investigation was conducted to evaluate aflatoxin reduction by these lines in a U.S. environment. As germplasm lines, resistance was demonstrated by the lines tested in 2010 and 2014 trials. In 2010, TZAR106 was among the lines with the lowest toxin accumulation, and in 2014, along with TZAR102, supported low aflatoxin. When evaluated as single cross hybrids in 2012, 2013 and 2014, several crosses involving IITA-SRRC lines accumulated low toxin. In 2012, TZAR103 × HBA1 was one of 4 lines with the lowest concentration of aflatoxin. In 2014, five IITA-SRRC hybrids were among the lowest with TZAR102 × Va35 and TZAR102 × LH132 being the two lowest. Results demonstrate significant aflatoxin reduction by IITA-SRRC lines in a U.S. aflatoxin-conducive environment (at Mississippi State University). Further testing in different locations and environments is needed to further evaluate the potential usefulness of these germplasm lines.

Identification and Quantification of a Toxigenic and Non-Toxigenic Aspergillus flavus Strain in Contaminated Maize Using Quantitative Real-Time PCR.

Aflatoxins, which are produced by Aspergillus flavus, are toxic to humans, livestock, and pets. The value of maize (Zea mays) grain is markedly reduced when contaminated with aflatoxin. Plant resistance and biological control using non-toxin producing strains are considered effective strategies for reducing aflatoxin accumulation in maize grain. Distinguishing between the toxin and non-toxin producing strains is important in determining the effectiveness of bio-control strategies and understanding inter-strain interactions. Using polymorphisms found in the fungal rRNA intergenic spacer region (IGS) between a toxigenic strain of A. flavus (NRRL 3357) and the non-toxigenic strain used in the biological control agent Afla-Guard® (NRRL 21882), we developed a set of primers that allows for the identification and quantification of the two strains using quantitative PCR. This primer set has been used to screen maize grain that was inoculated with the two strains individually and co-inoculated with both strains, and it has been shown to be effective in both the identification and quantification of both strains. Screening of co-inoculated ears from multiple resistant and susceptible genotypic crosses revealed no significant differences in fungal biomass accumulation of either strain in the field tests from 2010 and 2011 when compared across the means of all genotypes. Only one genotype/year combination showed significant differences in strain accumulation. Aflatoxin accumulation analysis showed that, as expected, genotypes inoculated with the toxigenic strain accumulated more aflatoxin than when co-inoculated with both strains or inoculated with only the non-toxigenic strain. Furthermore, accumulation of toxigenic fungal mass was significantly correlated with aflatoxin accumulation while non-toxigenic fungal accumulation was not. This primer set will allow researchers to better determine how the two fungal strains compete on the maize ear and investigate the interaction between different maize lines and these A. flavus strains.

Genome‐Wide Association Mapping of Aspergillus flavus and Aflatoxin Accumulation Resistance in Maize

ABSTRACTContamination of maize (Zea mays L.) with aflatoxin, produced by the fungus Aspergillus flavus Link, has severe health and economic consequences. Efforts to reduce aflatoxin accumulation in maize have focused on identifying and selecting germplasm with natural host resistance factors, and several maize lines with significantly reduced aflatoxin accumulation have been identified. Past linkage mapping studies have identified quantitative trait loci (QTL) that consistently reduce aflatoxin levels in maize. In addition, an association mapping panel of 300 maize inbred lines was previously created specifically for the dissection of aflatoxin accumulation resistance. Here we report the results of a genome‐wide association study (GWAS) using this panel of testcrossed maize hybrids. Each of the inbred parents of the testcrossed hybrids was genotyped by sequencing to generate 261,184 robust single nucleotide polymorphisms (SNPs), and the entire panel was phenotyped for aflatoxin accumulation following inoculation with A. flavus in multilocation, replicated field trials. Results uncovered 107 SNPs associated with aflatoxin accumulation in one or more environments in the association panel at a probability level between 9.78 × 10−6 and 2.87 × 10−10. Eight SNP trait associations were found with a false discovery rate (FDR) of less than 10% (p &lt; 3.83 × 10−7). These SNPs occur within the sequence of three uncharacterized genes. Variants in 25 other genomic regions showing high association values over more than one environment are also presented. These genomic regions are undergoing validation studies and will be of use to dissect the resistance to aflatoxin accumulation and improve this trait.

Aflatoxin Accumulation in a Maize Diallel Cross

Aflatoxins, produced by the fungus Aspergillus flavus, occur naturally in maize. Contamination of maize grain with aflatoxin is a major food and feed safety problem and greatly reduces the value of the grain. Plant resistance is generally considered a highly desirable approach to reduction or elimination of aflatoxin in maize grain. In this investigation, a diallel cross was produced by crossing 10 inbred lines with varying degrees of resistance to aflatoxin accumulation in all possible combinations. Three lines that previously developed and released as sources of resistance to aflatoxin accumulation were included as parents. The 10 parental inbred lines and the 45 single crosses making up the diallel cross were evaluated for aflatoxin accumulation in field tests conducted in 2013 and 2014. Plants were inoculated with an A. flavus spore suspension seven days after silk emergence. Ears were harvested approximately 60 days later and concentration of aflatoxin in the grain determined. Parental inbred lines Mp717, Mp313E, and Mp719 exhibited low levels (3–12 ng/g) of aflatoxin accumulation. In the diallel analysis, both general and specific combining ability were significant sources of variation in the inheritance of resistance to aflatoxin accumulation. General combining ability effects for reduced aflatoxin accumulation were greatest for Mp494, Mp719, and Mp717. These lines should be especially useful in breeding for resistance to aflatoxin accumulation. Breeding strategies, such as reciprocal recurrent selection, would be appropriate.

Diallel Analysis of Diverse Maize Germplasm Lines for Agronomic Characteristics

ABSTRACTDiverse, novel maize (Zea mays L.) germplasm from the Genetic Enhancement of Maize (GEM) Project and aflatoxin‐resistant and aflatoxin‐susceptible maize lines were used as parents to produce a diallel cross. This manuscript documents the agronomic performance of the progeny of the diallel cross. Nine GEM germplasm lines and six known resistant and susceptible lines were selected as parents of a diallel cross that was evaluated over 2 yr at Mississippi State, MS, and 1 yr at College Station, TX. Mp317, Mp494, NEI9008, and GEMN‐0130 had positive and significant GCA effects for yield in at least two of the three environments. This is especially useful because prior research suggested that both Mp494 and GEMN‐0130 also reduce aflatoxin accumulation. Of the GEM lines evaluated, NEI9008 was the best source for enhanced yield with significant and positive GCA values across all three environments. Mp313E × GEMN‐0157 yielded well across all environments with an additional benefit of relatively low aflatoxin accumulation in the Mississippi environments. Because the GEM lines evaluated in this diallel cross have a distinctly different genetic background than the aflatoxin‐resistant Mississippi inbreds, NEI9008 and GEMN‐0130 (improved yield), GEMS‐0002 and GEMS‐0074 (earlier maturity by 3 to 4 d), CUBA117, GEMS‐0030, GEMN‐0071, GEMS‐0100 (reduced plant height 0.1 to 0.2 m), GEMN‐0157, NEI9008, GEMS‐0002, and GEMS‐0074 (reduced height by 0.3 to 0.4 m) should be useful sources of alleles for pyramiding improved yield, earlier maturity, and shorter plant heights into improved breeding lines.

Maize (Zea mays L.) yield and aflatoxin accumulation responses to exogenous glycinebetaine application

Exogenously applied glycinebetaine (GB) accumulates at high levels in maize (Zea mays L.). Under water deficit and high temperature conditions GB application produces yield benefits. These sub-optimum conditions often result in high levels of aflatoxin accumulation which reduces grain quality. A 3-year (2008, 2009 and 2010) field experiment was conducted to determine the effects of GB on maize yield and aflatoxin accumulation. Weekly and alternate weekly GB application increased plant biomass by 10 and 13%, respectively. Net photosynthesis increased by 6% with GB application; however, stomatal conductance, transpiration rate and electron transport rate were not significantly affected. Grain yield increased by 6 and 13% with GB applied alternate weekly and weekly, respectively, over control plots averaged over years. GB application resulted in a trend of reduced aflatoxin accumulation in inoculated ears compared with non-inoculated controls in 2009 and 2010; however, inherent field and sampling variation did not allow us to conclude statistically any advantage attributable to GB application. We can conclude that GB did not significantly reduce aflatoxin production in the inoculated treatments.

Development of a gene-based marker correlated to reduced aflatoxin accumulation in maize

Aflatoxins are carcinogenic and toxic metabolites produced by the fungus Aspergillus flavus during infection of maize (Zea mays L.) and other seed oil crops. Climatic conditions in the southeastern United States favor A. flavus infection and aflatoxin contamination in maize, making it a major issue for farmers in the region. One of the most promising avenues to combat aflatoxin contamination is the development of resistant maize lines. However, this has proven difficult due to a lack of reliable markers for resistance. Previous studies have identified candi- date genes that were differentially expressed in response to A. flavus infection. One gene, encoding a chloroplast precursor, was found to contain multiple polymorphisms that were used to design a marker designated Mississippi Marker 1 (MpM1). The marker differentiates between the ''resistance'' and ''suscep- tible'' forms of the allele. This marker was used to screen three populations of F2:3 mapping families, where it was found to map to chromosome 4 and was associated with a significant effect for resistance to aflatoxin accumulation in the Mp313E 9 B73 map- ping population. Furthermore, the marker MpM1 identified a previously unknown quantitative trait loci for resistance to aflatoxin accumulation on maize chromosome 4. MpM1 is the first gene-based marker developed specifically for resistance to aflatoxin accumulation in maize and can now be integrated into existing marker assisted selection programs aimed at incorporating resistance into elite maize breeding lines.