Genes and genetic mechanisms contributing to fall armyworm resistance in maize.

Maize (Zea mays L.) is a crop of major economic and food security importance globally. The fall armyworm (FAW), Spodoptera frugiperda, can devastate entire maize crops, especially in countries or markets that do not allow the use of transgenic crops. Host-plant insect resistance is an economical and environmentally benign way to control FAW, and this study sought to identify maize lines, genes, and pathways that contribute to resistance to FAW. Of the 289 maize lines phenotyped for FAW damage in artificially infested, replicated field trials over 3 years, 31 were identified with good levels of resistance that could donate FAW resistance into elite but susceptible hybrid parents. The 289 lines were genotyped by sequencing to provide single nucleotide polymorphism (SNP) markers for a genome-wide association study (GWAS), followed by a metabolic pathway analysis using the Pathway Association Study Tool (PAST). GWAS identified 15 SNPs linked to 7 genes, and PAST identified multiple pathways, associated with FAW damage. Top pathways, and thus useful resistance mechanisms for further study, include hormone signaling pathways and the biosynthesis of carotenoids (particularly zeaxanthin), chlorophyll compounds, cuticular wax, known antibiosis agents, and 1,4-dihydroxy-2-naphthoate. Targeted metabolite analysis confirmed that maize genotypes with lower levels of FAW damage tend to have higher levels of chlorophyll a than genotypes with high FAW damage, which tend to have lower levels of pheophytin, lutein, chlorophyll b and ß-carotene. The list of resistant genotypes, and the results from the genetic, pathway, and metabolic study, can all contribute to efficient creation of FAW resistant cultivars.

Survey of Candidate Genes for Maize Resistance to Infection by Aspergillus flavus and/or Aflatoxin Contamination.

Many projects have identified candidate genes for resistance to aflatoxin accumulation or Aspergillus flavus infection and growth in maize using genetic mapping, genomics, transcriptomics and/or proteomics studies. However, only a small percentage of these candidates have been validated in field conditions, and their relative contribution to resistance, if any, is unknown. This study presents a consolidated list of candidate genes identified in past studies or in-house studies, with descriptive data including genetic location, gene annotation, known protein identifiers, and associated pathway information, if known. A candidate gene pipeline to test the phenotypic effect of any maize DNA sequence on aflatoxin accumulation resistance was used in this study to determine any measurable effect on polymorphisms within or linked to the candidate gene sequences, and the results are published here.

Characterization of the maize lipoxygenase gene family in relation to aflatoxin accumulation resistance.

Maize (Zea mays L.) is a globally important staple food crop prone to contamination by aflatoxin, a carcinogenic secondary metabolite produced by the fungus Aspergillus flavus. An efficient approach to reduce accumulation of aflatoxin is the development of germplasm resistant to colonization and toxin production by A. flavus. Lipoxygenases (LOXs) are a group of non-heme iron containing dioxygenase enzymes that catalyze oxygenation of polyunsaturated fatty acids (PUFAs). LOX derived oxylipins play critical roles in plant defense against pathogens including A. flavus. The objectives of this study were to summarize sequence diversity and expression patterns for all LOX genes in the maize genome, and map their effect on aflatoxin accumulation via linkage and association mapping. In total, 13 LOX genes were identified, characterized, and mapped. The sequence of one gene, ZmLOX10, is reported from 5 inbred lines. Genes ZmLOX1/2, 5, 8, 9, 10 and 12 (GRMZM2G156861, or V4 numbers ZM00001D042541 and Zm00001D042540, GRMZM2G102760, GRMZM2G104843, GRMZM2G017616, GRMZM2G015419, and GRMZM2G106748, respectively) fell under previously published QTL in one or more mapping populations and are linked to a measurable reduction of aflatoxin in maize grains. Association mapping results found 28 of the 726 SNPs tested were associated with reduced aflatoxin levels at p ≤ 9.71 x 10-4 according to association statistics. These fell within or near nine of the ZmLOX genes. This work confirms the importance of some lipoxygenases for resistance to aflatoxin accumulation and may be used to direct future genetic selection in maize.

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.

Integrated database for identifying candidate genes for Aspergillus flavus resistance in maize.

BACKGROUND: Aspergillus flavus Link:Fr, an opportunistic fungus that produces aflatoxin, is pathogenic to maize and other oilseed crops. Aflatoxin is a potent carcinogen, and its presence markedly reduces the value of grain. Understanding and enhancing host resistance to A. flavus infection and/or subsequent aflatoxin accumulation is generally considered an efficient means of reducing grain losses to aflatoxin. Different proteomic, genomic and genetic studies of maize (Zea mays L.) have generated large data sets with the goal of identifying genes responsible for conferring resistance to A. flavus, or aflatoxin. RESULTS: In order to maximize the usage of different data sets in new studies, including association mapping, we have constructed a relational database with web interface integrating the results of gene expression, proteomic (both gel-based and shotgun), Quantitative Trait Loci (QTL) genetic mapping studies, and sequence data from the literature to facilitate selection of candidate genes for continued investigation. The Corn Fungal Resistance Associated Sequences Database (CFRAS-DB) (http://agbase.msstate.edu/) was created with the main goal of identifying genes important to aflatoxin resistance. CFRAS-DB is implemented using MySQL as the relational database management system running on a Linux server, using an Apache web server, and Perl CGI scripts as the web interface. The database and the associated web-based interface allow researchers to examine many lines of evidence (e.g. microarray, proteomics, QTL studies, SNP data) to assess the potential role of a gene or group of genes in the response of different maize lines to A. flavus infection and subsequent production of aflatoxin by the fungus. CONCLUSIONS: CFRAS-DB provides the first opportunity to integrate data pertaining to the problem of A. flavus and aflatoxin resistance in maize in one resource and to support queries across different datasets. The web-based interface gives researchers different query options for mining the database across different types of experiments. The database is publically available at http://agbase.msstate.edu.

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.

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.