Low-cost grain sorting technologies to reduce mycotoxin contamination in maize and groundnut.

The widespread contamination of foods by mycotoxins continues to be a public health hazard in sub-Saharan Africa, with maize and groundnut being major sources of contamination. This study was undertaken to assess the hypothesis that grain sorting can be used to reduce mycotoxin contamination in grain lots by removing toxic kernels. We tested a set of sorting principles and methods for reducing mycotoxin levels in maize and groundnut from a variety of genotypes and environments. We found that kernel bulk density (KBD) and 100-kernel weight (HKW) were associated with the levels of aflatoxins (AF) and fumonisins (FUM) in maize grain. A low-cost sorter prototype (the 'DropSort' device) that separated maize grain based on KBD and HKW was more effective in reducing FUM than AF. We then evaluated the effectiveness of DropSorting when combined with either size or visual sorting. Size sorting followed by DropSorting was the fastest method for reducing FUM to under 2 ppm, but was not effective in reducing AF levels in maize grain to under 20 ppb, especially for heavily AF-contaminated grain. Analysis of individual kernels showed that high -AF maize kernels had lower weight, volume, density, length, and width and higher sphericity than those with low AF. Single kernel weight was the most significant predictor of AF concentration. The DropSort excluded kernels with lower single kernel weight, volume, width, depth, and sphericity. We also found that visual sorting and bright greenish-yellow fluorescence sorting of maize single kernels were successful in separating kernels based on AF levels. For groundnut, the DropSort grouped grain based on HKW and did not significantly reduce AF concentrations, whereas size sorting and visual sorting were much more effective.

Tissue-specific components of resistance to Aspergillus ear rot of maize.

Aspergillus flavus and other Aspergillus spp. infect maize and produce aflatoxins. An important control measure is the use of resistant maize hybrids. There are several reports of maize lines that are resistant to aflatoxin accumulation but the mechanisms of resistance remain unknown. To gain a better understanding of resistance, we dissected the phenotype into 10 components: 4 pertaining to the response of silk, 4 pertaining to the response of developing kernels, and 2 pertaining to the response of mature kernels to inoculation with A. flavus. In order to challenge different tissues and to evaluate multiple components of resistance, various inoculation methods were used in experiments in vitro and under field conditions on a panel of diverse maize inbred lines over 3 years. As is typical for this trait, significant genotype-environment interactions were found for all the components of resistance studied. There was, however, significant variation in maize germplasm for susceptibility to silk and kernel colonization by A. flavus as measured in field assays. Resistance to silk colonization has not previously been reported. A significant correlation of resistance to aflatoxin accumulation with flowering time and kernel composition traits (fiber, ash, carbohydrate, and seed weight) was detected. In addition, correlation analyses with data available in the literature indicated that lines that flower later in the season tend to be more resistant. We were not able to demonstrate that components identified in vitro were associated with reduced aflatoxin accumulation in the field.

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.

Aspergillus flavus Biomass in Maize Estimated by Quantitative Real-Time Polymerase Chain Reaction Is Strongly Correlated with Aflatoxin Concentration.

Aspergillus flavus causes ear rot of maize and produces aflatoxins that can contaminate grain even in the absence of visible symptoms of infection. Resistance to aflatoxin accumulation and pathogen colonization are considered distinct traits in maize. Colonization of grain by fungi such as A. flavus has been difficult to quantify. We developed and validated two quantitative real-time polymerase chain reaction (qPCR) assays to estimate fungal biomass in maize tissues. In order to study the relationship between fungal biomass and aflatoxin accumulation, qPCR was conducted and aflatoxin concentrations were assayed in milled samples of mature maize kernels for two diverse sets of maize germplasm. The first was a set of hybrids that was inoculated with A. flavus in a conducive field environment in Mississippi. These hybrids, mainly early tropical and non-stiff-stalk genotypes adapted to local conditions, carry known sources of resistance among their progenitors. The second set, also tested in Mississippi, was a group of inbred lines representing a wider sample of maize genetic diversity. For both sets, our results showed a high correlation between fungal load and aflatoxin concentration in maize kernels. Our qPCR methodology could have a direct impact on breeding programs that aim to identify lines with resistance to aflatoxin accumulation, and set the stage for future studies on the genetic dissection of aflatoxin-related traits.

Aflatoxin in maize: a review of the early literature from "moldy-corn toxicosis" to the genetics of aflatoxin accumulation resistance.

Aflatoxin is a potent toxin produced by Aspergillus flavus Link:Fr, an opportunistic ear-rot pathogen of maize (Zea mays L. subsp. Mays). Prior to the discovery of aflatoxin, A. flavus was considered a minor pathogen and was not a priority for maize breeders or pathologists. Aflatoxin was discovered in England in 1961 following an epidemic in poultry. By the early 1970s, surveys of agricultural commodities in the USA found that maize produced in the Southeast was especially vulnerable to aflatoxin contamination. Aflatoxin contamination was initially treated as a post-harvest issue, but pre-harvest contamination was proven by 1975. Pre-harvest contamination meant that genetically based host-plant resistance was a possible solution. The potential magnitude of the problem became apparent in 1977 when the southeastern US maize crop suffered epidemic aflatoxin contamination. The first experiment demonstrating the heritability of host-plant resistance to aflatoxin accumulation was published in 1978. These events combined to make breeding for reduced aflatoxin contamination both a high priority and a rational breeding objective. This review surveys the early scientific literature in order to place research on the genetics of aflatoxin accumulation in maize into historical context. It tells the story of how multi-disciplinary research began with veterinary diseases of unknown etiology and resulted in host-plant resistance to a previously minor plant pathogen becoming a central public sector breeding objective.

Differential Expression of Signaling Pathway Genes Associated With Aflatoxin Reduction Quantitative Trait Loci in Maize (Zea mays L.).

The roles of signaling pathway genes related to the aflatoxin reduction trait in maize were studied for the improvement of maize resistance to the fungal pathogen Aspergillus flavus (A. flavus). In this study, 55 maize genes in plant-pathogen interaction signaling pathways were investigated among 12 maize near-isogenic lines (NILs) that carry maize quantitative trait loci (QTL) associated with aflatoxin reduction. These maize NILs were developed from maize inbred lines Mp313E (resistant donor parent) and Va35 (susceptible recurrent parent). The quantitative RT-PCR (qRT-PCR) technique was used to study the gene expression patterns. Seven calcium-dependent protein kinases and one respiratory burst oxidase displayed significant differential expression levels among the maize QTL-NILs. In addition, the gene expression profiles of WRKY transcription factors were also examined. Maize WRKY 52, WRKY 71, and WRKY83 genes displayed significantly differential expression levels among the QTL-NILs. The elucidation of differentially expressed signaling pathway genes involving maize resistance to A. flavus can provide insights into maize disease resistance and enhance maize molecular breeding.

A Histological Study of Aspergillus flavus Colonization of Wound Inoculated Maize Kernels of Resistant and Susceptible Maize Hybrids in the Field.

Aspergillus flavus colonization in developing kernels of maize single-cross hybrids resistant (Mp313E × Mp717) and susceptible (GA209 × T173) to aflatoxin accumulation was determined in the field over three growing seasons (2012-2014). Plants were hand pollinated, and individual kernels were inoculated with a needle dipped in a suspension of A. flavus conidia 21 days after pollination. Kernels were harvested at 1- to 2-day intervals from 1 to 21 days after inoculation (DAI). Kernels were placed in FAA fixative, dehydrated, embedded in paraffin, sectioned, and stained with toluidine blue. Kernels were also collected additional kernels for aflatoxin analyses in 2013 and 2014. At 2 DAI, A. flavus hyphae were observed among endosperm cells in the susceptible hybrid, but colonization of the endosperm in the resistant hybrid was limited to the wound site of the resistant hybrid. Sections of the scutellum of the susceptible hybrid were colonized by A. flavus by 5 DAI. Fungal growth was slower in the resistant hybrid compared to the susceptible hybrid. By 10 DAI, A. flavus had colonized a large section of the embryo in the susceptible hybrid; whereas in the resistant hybrid, approximately half of the endosperm had been colonized and very few cells in the embryo were colonized. Fungal colonization in some of the kernels of the resistant hybrid was slowed in the aleurone layer or at the endosperm-scutellum interface. In wounded kernels with intact aleurone layers, the fungus spread around the kernel between the pericarp and aleurone layer with minimal colonization of the endosperm. Aflatoxin B1 was first detected in susceptible kernel tissues 8 DAI in 2013 (14 µg/kg) and 2014 (18 µg/kg). The resistant hybrid had significantly lower levels of aflatoxin accumulation compared to the susceptible hybrid at harvests 10, 21, and 28 DAI in 2013, and 20 and 24 DAI in 2014. Our study found differential A. flavus colonization of susceptible and resistant kernel tissues, and that the aleurone and the outer layer of the scutellum slowed the rate of colonization by A. flavus.

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.

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.

Effect of different postharvest drying temperatures on Aspergillus flavus survival and aflatoxin content in five maize hybrids.

After harvest, maize is dried artificially to halt fungal growth and mycotoxin production while in postharvest storage. The process often limits harvest capacity and has been a frequent cause of seed injury. Higher drying temperatures could lead to shorter drying periods and faster turnover; however, there is often a deterioration of the physical grain quality, including increased breakage susceptibility and loss of viability. The goals of this study were to determine the effect of different postharvest drying temperatures on Aspergillus filavus and Fusarium verticillioides survival and aflatoxin content in maize and to determine the viability of the seed. Five corn hybrids varying in resistance to A. flavus were side needle-inoculated with A. flavus, harvested at physiological maturity, and dried at temperatures ranging from 40 to 70 degrees C. Kernels were evaluated for aflatoxin, stress cracks, germination, and kernel infection by A. flavus and a natural infestation of F. verticillioides. Drying temperature had no effects on aflatoxin concentration given the heat stability of the toxin. With increased temperatures from 40 to 70 degrees C, germination decreased significantly, from 96 to 27%, and stress cracks increased significantly (1.4 up to 18.7). At temperatures above 60 degrees C, F. verticillioides kernel infection was significantly reduced to less than 18%. At 70 degrees C, there was a significant reduction in A. flavus kernel infection, from 11 to 3%. This information is useful in determining a range of temperatures that can be used for drying seed when fungal infection, stress cracks, and seed viability are of interest.

Characterization of the Maize Chitinase Genes and Their Effect on Aspergillus flavus and Aflatoxin Accumulation Resistance.

Maize (Zea mays L.) is a crop of global importance, but prone to contamination by aflatoxins produced by fungi in the genus Aspergillus. The development of resistant germplasm and the identification of genes contributing to resistance would aid in the reduction of the problem with a minimal need for intervention by farmers. Chitinolytic enzymes respond to attack by potential pathogens and have been demonstrated to increase insect and fungal resistance in plants. Here, all chitinase genes in the maize genome were characterized via sequence diversity and expression patterns. Recent evolution within this gene family was noted. Markers from within each gene were developed and used to map the phenotypic effect on resistance of each gene in up to four QTL mapping populations and one association panel. Seven chitinase genes were identified that had alleles associated with increased resistance to aflatoxin accumulation and A. flavus infection in field grown maize. The chitinase in bin 1.05 identified a new and highly significant QTL, while chitinase genes in bins 2.04 and 5.03 fell directly beneath the peaks of previously published QTL. The expression patterns of these genes corroborate possible grain resistance mechanisms. Markers from within the gene sequences or very closely linked to them are presented to aid in the use of marker assisted selection to improve this trait.

Genome wide association study for drought, aflatoxin resistance, and important agronomic traits of maize hybrids in the sub-tropics.

The primary maize (Zea mays L.) production areas are in temperate regions throughout the world and this is where most maize breeding is focused. Important but lower yielding maize growing regions such as the sub-tropics experience unique challenges, the greatest of which are drought stress and aflatoxin contamination. Here we used a diversity panel consisting of 346 maize inbred lines originating in temperate, sub-tropical and tropical areas testcrossed to stiff-stalk line Tx714 to investigate these traits. Testcross hybrids were evaluated under irrigated and non-irrigated trials for yield, plant height, ear height, days to anthesis, days to silking and other agronomic traits. Irrigated trials were also inoculated with Aspergillus flavus and evaluated for aflatoxin content. Diverse maize testcrosses out-yielded commercial checks in most trials, which indicated the potential for genetic diversity to improve sub-tropical breeding programs. To identify genomic regions associated with yield, aflatoxin resistance and other important agronomic traits, a genome wide association analysis was performed. Using 60,000 SNPs, this study found 10 quantitative trait variants for grain yield, plant and ear height, and flowering time after stringent multiple test corrections, and after fitting different models. Three of these variants explained 5-10% of the variation in grain yield under both water conditions. Multiple identified SNPs co-localized with previously reported QTL, which narrows the possible location of causal polymorphisms. Novel significant SNPs were also identified. This study demonstrated the potential to use genome wide association studies to identify major variants of quantitative and complex traits such as yield under drought that are still segregating between elite inbred lines.

Aflatoxin and fumonisin contamination of commercial corn (Zea mays) hybrids in Mississippi.

Resistance to mycotoxin contamination was compared in field samples harvested from 45 commercial corn (maize) hybrids and 5 single-cross aflatoxin-resistant germplasm lines in years with high and moderate heat stress. In high heat stress, mycotoxin levels were (4.34 +/- 0.32) x 10(3) microg/kg [(0.95-10.5 x 10(3) microg/kg] aflatoxins and 11.2 +/- 1.2 mg/kg (0-35 mg/kg) fumonisins in commercial hybrids and 370 +/- 88 microg/kg (140-609 microg/kg) aflatoxins and 4.0 +/- 1.3 mg/kg (1.7-7.8 mg/kg) fumonisins in aflatoxin-resistant germplasm lines. Deoxynivalenol was detected (one-fourth of the samples, 0-1.5 mg/kg), but not zearalenone. In moderate heat stress, mycotoxin levels were 6.2 +/- 1.6 microg/kg (0-30.4 microg/kg) aflatoxins and 2.5 +/- 0.2 mg/kg (0.5-4.8 mg/kg) fumonisins in commercial hybrids and 1.6 +/- 0.7 microg/kg (0-7 microg/kg) aflatoxins and 1.2 +/- 0.2 mg/kg (0.5-3.0 mg/kg) fumonisins in aflatoxin-resistant germplasm lines. The results are consistent with heat stress playing an important role in the susceptibility of corn to both aflatoxin and fumonisin contamination, with significant reductions of both aflatoxins and fumonisins in aflatoxin-resistant germplasm lines.

Southwestern corn borer (Lepidoptera: Crambidae) damage and aflatoxin accumulation in maize.

Aflatoxin, a potent carcinogen, is produced by the fungus Aspergillus flavus Link: Fr. Drought, high temperatures, and insect damage contribute to increased levels of aflatoxin contamination in corn, Zea mays L. Plant resistance is widely considered a desirable method of reducing aflatoxin contamination. Germplasm lines with aflatoxin resistance have been developed. This investigation was undertaken to determine whether crosses among these lines exhibited resistance to southwestern corn borer, Diatraea grandiosella Dyar, and to assess the effects of southwestern corn borer feeding on aflatoxin accumulation. Differences in ear damage among southwestern corn borer infested hybrids were significant. Estimates of general combining ability effects indicated that the lines Mp80:04, Mp420, and Mp488 contributed to reduced ear damage, and SC213 and T165 contributed to greater damage when used in hybrids. Mean aflatoxin levels were 254 ng/g for hybrids infested with southwestern corn borer larvae and 164 ng/g for noninfested hybrids in 2000 when environmental conditions were conducive to aflatoxin production. In contrast, the overall mean aflatoxin level for southwestern corn borer infested hybrids was only 5 ng/g in 1999 when environmental conditions did not favor aflatoxin accumulation. Crosses that included lines selected for aflatoxin resistance as parents (Mp80:04 and Mp313E) exhibited lower levels of aflatoxin contamination both with and without southwestern corn borer infestation in 2000. Only the experimental line Mp80:04 contributed significantly to both reduced southwestern corn borer damage and reduced aflatoxin contamination.

Relating significance and relations of differentially expressed genes in response to Aspergillus flavus infection in maize.

Aspergillus flavus is a pathogenic fungus infecting maize and producing aflatoxins that are health hazards to humans and animals. Characterizing host defense mechanism and prioritizing candidate resistance genes are important to the development of resistant maize germplasm. We investigated methods amenable for the analysis of the significance and relations among maize candidate genes based on the empirical gene expression data obtained by RT-qPCR technique from maize inbred lines. We optimized a pipeline of analysis tools chosen from various programs to provide rigorous statistical analysis and state of the art data visualization. A network-based method was also explored to construct the empirical gene expression relational structures. Maize genes at the centers in the network were considered as important candidate genes for maize DNA marker studies. The methods in this research can be used to analyze large RT-qPCR datasets and establish complex empirical gene relational structures across multiple experimental conditions.

Identification of maize genes associated with host plant resistance or susceptibility to Aspergillus flavus infection and aflatoxin accumulation.

BACKGROUND: Aspergillus flavus infection and aflatoxin contamination of maize pose negative impacts in agriculture and health. Commercial maize hybrids are generally susceptible to this fungus. Significant levels of host plant resistance have been observed in certain maize inbred lines. This study was conducted to identify maize genes associated with host plant resistance or susceptibility to A. flavus infection and aflatoxin accumulation. RESULTS: Genome wide gene expression levels with or without A. flavus inoculation were compared in two resistant maize inbred lines (Mp313E and Mp04:86) in contrast to two susceptible maize inbred lines (Va35 and B73) by microarray analysis. Principal component analysis (PCA) was used to find genes contributing to the larger variances associated with the resistant or susceptible maize inbred lines. The significance levels of gene expression were determined by using SAS and LIMMA programs. Fifty candidate genes were selected and further investigated by quantitative RT-PCR (qRT-PCR) in a time-course study on Mp313E and Va35. Sixteen of the candidate genes were found to be highly expressed in Mp313E and fifteen in Va35. Out of the 31 highly expressed genes, eight were mapped to seven previously identified quantitative trait locus (QTL) regions. A gene encoding glycine-rich RNA binding protein 2 was found to be associated with the host hypersensitivity and susceptibility in Va35. A nuclear pore complex protein YUP85-like gene was found to be involved in the host resistance in Mp313E. CONCLUSION: Maize genes associated with host plant resistance or susceptibility were identified by a combination of microarray analysis, qRT-PCR analysis, and QTL mapping methods. Our findings suggest that multiple mechanisms are involved in maize host plant defense systems in response to Aspergillus flavus infection and aflatoxin accumulation. These findings will be important in identification of DNA markers for breeding maize lines resistant to aflatoxin accumulation.

Occurrence of aflatoxin in three maize (Zea mays L.) hybrids over 5 years in Northern Mississippi.

Aflatoxins are produced as secondary metabolites under conducive climatic conditions by Aspergillus flavus. The incidence of aflatoxin varies with environmental conditions, genotype, and location. An expanded understanding of the interaction of the plant, fungus, and weather conditions is needed to further elucidate the field infection process of maize by A. flavus and subsequent aflatoxin contamination. One of the problems in evaluating maize hybrids for resistance to kernel infection and aflatoxin contamination is identifying a time period and environmental conditions that are most advantageous. Three maize genotypes (Pioneer Brand 3223, Mo18W x Mp313E, and Mp313E x Mp420) were evaluated from 1998 to 2002 in response to A. flavus inoculation and aflatoxin contamination and weather conditions favorable for aflatoxin contamination were identified. The highest aflatoxin levels were observed in 1998 and 2000 (1186 and 901 ng g(-1); P < 0.0001); while the lowest levels were detected in 1999 (39 ng g(-1)). Pioneer 3223 had significantly higher levels (1198 ng g(-1)) than Mp313E x Mp420 (205 ng g(-1)), and Mo18W xMp313E (161 ng g(-1); P < 0.0001). The hybrids had six weather-related variables in common that were positively correlated with aflatoxin accumulation. Four of these occurred during 65-85 days after planting and were temperature-related. These results suggest that regardless of the hybrid's maturity or physiological development, the time from 65 to 85 days after planting may be indicative of a period of stress which leads to greater aflatoxin accumulation at harvest.

Systemic infection of stalks and ears of corn hybrids by Aspergillus parasiticus.

This study was conducted to explore systemic infection by the Aspergillus flavus group into corn ears via the stalk. An A. parasiticus mutant which produces norsolorinic (NOR) acid (a visible orange intermediate of the aflatoxin biosynthetic pathway) was used in field studies to monitor systemic infection of corn stalk and ear tissues. Corn hybrids resistant and susceptible to aflatoxin contamination were grown in the field and inoculated prior to tasseling by inserting A. parasiticus infested toothpicks into stalks between the 5th and 6th node below the lowest ear shoot. Beginning 2 weeks after inoculation, systemic infection by the NOR mutant was assessed weekly by collecting ear shank tissue and stalk tissue from the nodes between the infection sites and the developing ears. Ears were collected at the end of the growing season to determine the level of kernel infection by the NOR mutant. In two separate studies, the A. parasiticus NOR mutant was isolated from stalk tissues at all of node positions and ear shank tissue from several susceptible corn hybrid plants at the first harvest date 2 weeks after inoculation. The NOR mutant was also isolated from stalk and ear tissue of a resistant hybrid. The NOR mutant was only isolated from kernels of susceptible hybrids in 2003 and 2004. Infection rates of kernels in infected ears were very low (<1%). In 2005, the fungus was found in only one kernel from an ear of the resistant hybrid. The NOR mutant was not isolated from stalks, ears, or kernels from control (uninoculated) plants grown in the plots with inoculated plants. Although infection levels of corn kernels were low, systemic movement of the A. parasiticus up the stalk appears to be another possible route to infection of developing corn ears.