Gary L. Windham

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.

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.

Comparison of Aflatoxigenic and Nonaflatoxigenic Isolates of Aspergillus flavus using DNA Amplification Fingerprinting Techniques

Aspergillus flavus is a filamentous fungus that produces mycotoxins in many food and feed crops, such as maize (Zea mays L.). Isolates were analyzed for toxin production by nucleic acid profiles in an attempt to differentiate aflatoxigenic from nonaflatoxigenic isolates. A total of 41 aflatoxigenic and 34 nonalfatoxigenic isolates were included in the study. The isolates were evaluated initially using DNA amplification fingerprinting (DAF) without clear resolution of the groups. A weak association of aflatoxigenic isolates was observed, as evidenced by their clustering in 18 of 81 trees recovered from maximum parsimony analysis of binary characters derived from arbitrary signatures from amplification profiles (ASAP) data; nonaflatoxigenic isolates exhibited a pattern of paraphyletic laddering. Up to five markers unambiguously supported the aflatoxigenic isolate grouping, but the presence of alternative conflicting topologies in equally parsimonious trees precluded the observation of meaningful statistical support. With additional markers for genome of A. flavus, this method could be used to resolve toxigenic from nontoxigenic strains. This additional work could resolve aflatoxigenic isolates of A. flavus present on maize plants using ASAP, which would reduce labor intense costs and potentially lead to faster determination of resistant cultivars in breeding efforts.

Inoculation techniques used to quantify aflatoxin resistance in corn

The development of Aspergillus flavus inoculation techniques has played an important part in developing corn (Zea mays L.) germplasm resistant to aflatoxin contamination. Corn genotypes evaluated for aflatoxin resistance in field studies must be artificially inoculated due to the sporadic nature of aflatoxin contamination from year to year. A number of different inoculation techniques are used by researchers in the South and Midwest. Field inoculation techniques either wound developing kernels or leave the kernels intact. Non‐wounding techniques apply A. flavus conidia to exposed silks or silks inside the husks without damaging kernels. Wounding techniques deliver A. flavus conidia onto kernels that have been mechanically damaged. Inoculation techniques utilizing ear feeding insects to vector conidia have also been used in field studies. Environmental conditions such as ambient temperature and drought stress appear to have a significant impact on artificial inoculations. Laboratory evaluation techniques have been developed to confirm aflatoxin resistance identified in corn genotypes in the field. Color mutants and transformants of Aspergillus spp. have been used in field and laboratory studies to identify resistant genotypes. More efficient, less labor intensive, and less costly inoculation techniques need to be developed to aid in the production of aflatoxin resistant corn hybrids.

Anti-fungal activity of maize silk proteins and role of chitinases in Aspergillus flavus resistance

Studies were conducted to identify proteins in maize silks that may be contributing to Aspergillus flavus resistance. We first performed bioassays using silk extracts collected from two A. flavus-resistant inbred lines and two susceptible inbred lines. Fungal biomass was quantified by measuring fluorescence of a green fluorescent protein (GFP)-tagged A. flavus and by measuring ergosterol levels. The silk extracts from resistant inbreds had greater anti-fungal activity compared to susceptible inbreds. Comparative proteomic analysis of the two resistant and susceptible inbreds led to the identification of several anti-fungal proteins. One of the anti-fungal proteins that we further investigated was chitinase. There were three chitinases that were differentially expressed in the resistant lines (PRm3 chitinase, chitinase I, and chitinase A). We conducted chitinase assays on silk proteins from extracts of resistant and susceptible inbred lines. Silk extracts from resistant inbred lines showed significantly higher activity in the resistant maize inbreds compared to the susceptible inbreds (P < 0.01). The differential expression of chitinases in maize resistant and susceptible inbred silks suggests that these proteins may contribute to A. flavus resistance.

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.

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.

Enhancing Maize Germplasm with Resistance to Aflatoxin Contamination

Preharvest kernel infection by Aspergillus flavus and the subsequent accumulation of aflatoxin in maize grain are chronic problems in the southeastern United States. Aflatoxin is a natural carcinogen, and its presence markedly reduces the value of grain. Losses to aflatoxin contamination reach devastating levels some years. Development and deployment of maize hybrids with resistance to aflatoxin contamination is generally considered the most feasible method of reducing or eliminating the problem. Research to address the aflatoxin problem was initiated by USDA–ARS at Mississippi State, MS, in the late 1970s. The goals of the research were to identify and develop aflatoxin‐resistant maize germplasm. First, reliable techniques for screening germplasm were developed. Then, germplasm from numerous sources was screened. The release of Mp313E in 1988 was the first release of maize germplasm with resistance to aflatoxin contamination. Two other germplasm lines, Mp420 and Mp715, were released in 1991 and 1999, respectively. Additional germplasm lines have been developed, but not yet released. Efforts are currently underway to identify other sources of resistance.When used in crosses with other lines, the aflatoxin‐resistant lines markedly reduce the level of aflatoxin contamination in the resulting hybrids. Analysis of a diallel cross indicated that general combining ability was a significant source of variation in the inheritance of resistance to aflatoxin contamination. Efforts to combine resistance to aflatoxin combination and agronomic qualities using both conventional breeding methods and molecular marker assisted selection have been initiated.

IDENTIFYING MAIZE GERMPLASM WITH RESISTANCE TO AFLATOXIN ACCUMULATION

Contamination of maize grain, Zea mays L., with aflatoxin, a toxin produced by the fungus Aspergillus flavus, reduces its value and marketability. Growing hybrids with resistance is generally considered a highly desirable way to reduce A. flavus infection and aflatoxin accumulation. Identifying maize germplasm with resistance is critical to the development and production of such hybrids. USDA-ARS scientists at Mississippi State, Mississippi; Tifton, Georgia; and Raleigh, North Carolina; have engaged in a multilocation approach to germplasm screening. A major component of this has been the evaluation of accessions obtained from the Germplasm Enhancement of Maize (GEM) project at both Mississippi State and Tifton. Selections from GEM accessions 250_01_XL370A_S11_F2S4_9214_Blk21/00-# and 2250_02_XL370A_S11_F2S4_3363_Blk03/00-# exhibited the highest levels of resistance both as lines per se and in testcrosses. Lines developed at the International Maize and Wheat Improvement Center (CIMMYT) and North Carolina State University also exhibited reduced levels of aflatoxin contamination. CML348, NC388, NC400, NC408, and NC458 were among those with low levels of aflatoxin contamination. The lines that displayed low levels of contamination should be useful in maize breeding programs for developing parental inbred lines and aflatoxin-resistant maize hybrids.

Identifying and developing maize germplasm with resistance to accumulation of aflatoxins

Efforts to identify maize germplasm with resistance to Aspergillus flavus infection and subsequent accumulation of aflatoxins were initiated by the US Department of Agriculture, Agricultural Research Service at several locations in the late 1970s and early 1980s. Research units at four locations in the south-eastern USA are currently engaged in identification and development of maize germplasm with resistance to A. flavus infection and accumulation of aflatoxins. The Corn Host Plant Resistance Research Unit, Mississippi State, MS, developed procedures for screening germplasm for resistance to A. flavus infection and accumulation of aflatoxins. Mp313E, released in 1990, was the first line released as a source of resistance to A. flavus infection. Subsequently, germplasm lines Mp420, Mp715, Mp717, Mp718, and Mp719 were released as additional sources of resistance. Quantitative trait loci associated with resistance have also been identified in four bi-parental populations. The Crop Protection and Management ...