Research Project: Enhanced Resistance of Maize to Aspergillus flavus Infection, Aflatoxin Accumulation, and Insect Damage

Location: Corn Host Plant Resistance Research

2020 Annual Report

Objectives
1. Identify new sources of maize germplasm with resistance to Aspergillus (A.) flavus infection and aflatoxin accumulation. We have developed and released maize germplasm lines with resistance to A. flavus infection and aflatoxin accumulation; however, germplasm with higher levels of resistance and different mechanisms of resistance are needed. We will evaluate germplasm obtained through the Germplasm Enhancement of Maize (GEM) project, the International Maize and Wheat Improvement Center (CIMMYT), and from other sources using methodologies developed in the research unit. Although we have developed effective procedures for inoculating plants with A. flavus and measuring aflatoxin, we will continue to evaluate other methods of quantifying resistance to increase efficiency and reduce costs. 2. Identify new sources of maize germplasm with resistance to fall armyworm, southwestern corn borer, and corn earworm. We have developed and released germplasm lines with excellent resistance to fall armyworm and southwestern corn borer leaf feeding damage using methodology for screening developed in the research unit. We will continue to screen germplasm from the GEM project, CIMMYT, and other sources to identify new sources of resistance to leaf feeding. We will also screen germplasm to identify sources of resistance to ear damage by fall armyworm, southwestern corn borer, and corn earworm. Because insect feeding provides a site for fungi to enter the developing ears of maize, resistance to ear-feeding insects should be reduced. 3. Develop and characterize genetic mapping populations, identify and elucidate functions of genes associated with resistance, and develop molecular markers for enhancing maize germplasm resistance to A. flavus/aflatoxin and insects. We have genotyped and phenotyped populations of F2:3 families to identify quantitative trait loci associated with resistance to aflatoxin accumulation and insect feeding, and additional populations are currently being developed. We have created near-isogenic lines (NILs) to validate the QTLs. We evaluated a 300-line association mapping panel for aflatoxin accumulation and ear damage by caused by insect feeding. From these studies we will identify and confirm additional QTL and candidate genes associated with resistance. We will investigate expression and function of these and other genes or groups of genes using a variety of techniques. 4. Develop and release maize germplasm with resistance to A. flavus infection, aflatoxin accumulation, and insect damage. Germplasm identified in Objectives 1 and 2 will be used in developing germplasm lines with resistance to aflatoxin accumulation and insect damage using conventional phenotypic selection. We will use information on molecular markers associated with resistance obtained from Objective 3 to enhance our efforts to produce better adapted lines with higher levels of pest resistance.

Approach
Objective 1: Identify new sources of maize germplasm with resistance to Aspergillus (A.) flavus infection and aflatoxin accumulation. Screen maize germplasm obtained from the Germplasm Enhancement of Maize project (GEM) project, International Center for Maize and Wheat Improvement (CIMMYT), and other sources for resistance to aflatoxin accumulation to identify new and potentially useful sources of resistance. Determine optimal combinations of A. flavus and A. parasiticus isolates for screening maize germplasm for resistance to aflatoxin accumulation under different environmental conditions. Objective 2: Identify new sources of maize germplasm with resistance to fall armyworm, southwestern corn borer, and corn earworm. Screen maize germplasm from CIMMYT, the GEM project, and other sources for resistance to leaf feeding by fall armyworm and southwestern corn borer. Screen maize germplasm for resistance to ear feeding by fall armyworm, southwestern corn borer, and corn earworm. Objective 3: Develop and characterize genetic mapping populations, identify and elucidate functions of genes associated with resistance, and develop molecular markers for enhancing maize germplasm with resistance to flavus/aflatoxin and insects. Identify genetic loci associated with resistance to A. flavus infection and aflatoxin accumulation via analysis of linkage and association mapping and sequencing data. Identify genetic loci associated with resistance to lepidopteran insect feeding via analysis of linkage and association mapping, pathway analysis, or sequencing data. Identify genes and elucidate functions of genes associated with resistance to A. flavus/aflatoxin and insects. Objective 4: Develop and release maize germplasm with resistance to A. flavus infection, aflatoxin accumulation, and insect damage. Develop and release maize germplasm lines with resistance to A. flavus infection and aflatoxin accumulation using conventional breeding methods, and develop and release lines with resistance to feeding by southwestern corn borer and fall armyworm using conventional breeding methods. Develop lines with resistance to A. flavus infection, aflatoxin accumulation, and insects using molecular markers and release together with marker information.

Progress Report
We evaluated 50 maize germplasm accessions obtained from the Germplasm Enhancement of Maize (GEM) project for resistance to aflatoxin accumulation in field trials conducted in 2019, and we will evaluate 50 additional accessions in 2020. Ten accessions that appeared to be potentially useful sources of resistance to aflatoxin accumulation in the 2019 field tests are being re-evaluated in 2020 for use in the breeding program. We evaluated 40 advanced generation breeding lines developed through multiple cycles of selection for resistance to aflatoxin accumulation in 2019. We are re-evaluating the best of these in 2020, and we are increasing seed in the breeding nursery in 2020. We evaluated near isogenic lines developed from the cross Mp313E × Va35 through multiple cycles of molecular marker assisted selection for resistance to aflatoxin accumulation. Near-isogenic lines selected for four of the quantitative trait loci (QTL) identified in the Mp313E × Va35 population exhibited significantly lower levels of aflatoxin accumulation than the recurrent (susceptible) parent, Va35, but more than Mp313E, the resistant parent. In collaboration with a commercial seed corn company, QTL identified in the Mp313E × Va35 population were introgressed into proprietary inbred lines representing both the male and female heterotic groups. Using these lines, we will develop hybrids that possess both resistance to aflatoxin accumulation and good agronomic qualities. Forty-five maize hybrids developed by scientists in Texas, Georgia, and Mississippi, were evaluated for resistance to aflatoxin accumulation and yield in five locations in 2019. In another investigation, we compared aflatoxin accumulation when developing ears of 10 maize germplasm lines with varying degrees of resistance to aflatoxin accumulation were inoculated with either Aspergillus (A.) flavus NRRL 3357, A. flavus K54, or A. parasiticus NRRL 2999. Aflatoxin accumulation did not differ significantly between the NRRL 3357 and 2999 isolates, but aflatoxin accumulation was significantly lower for K54. We compared results of a kernel-screening assay (KSA) for resistance to A. flavus infection and accumulation of aflatoxin in the laboratory with results of a field evaluation in which developing ears were inoculated with an A. flavus spore suspension. Although germplasm lines that exhibited resistance in the field evaluations generally showed resistance with the KSA, there were exceptions. Some lines that exhibited resistance with KSA exhibited high levels of aflatoxin accumulation in the field tests, and some lines that accumulated high levels of aflatoxin with KSA were consistently resistant in the field tests. We concluded that the KSA is not a useful method for identifying maize germplasm lines with resistance to aflatoxin accumulation; however, it may be useful for some laboratory studies. A collaborator at Mississippi State University (6064-21000-015-05S) completed whole genome sequencing of parental lines, Mp313E and Va35, and several of their near-isogenic lines to identify genes associated with resistance to aflatoxin accumulation and to generate molecular markers that will enhance efforts to develop superior lines with resistance to aflatoxin accumulation. We evaluated 300 lines for resistance to fall armyworm leaf feeding in 2019 and 2020. We found that lines developed by ARS scientists at Mississippi State, Mississippi, exhibited consistently high levels of resistance to leaf feeding by fall armyworm and southwestern corn borer. We will share these results with cooperators at the International Maize and Wheat Improvement Center (CIMMYT) in Nairobi, Kenya (6064-21000- 015-01R). Data from these evaluations along with additional data that we collect in 2021 will be included in a Genome Wide Association Study (GWAS) to identify genes associated with resistance to fall armyworm damage. We will analyze these data using a new technique for identifying metabolic pathways associated with fall armyworm resistance that we developed. The use of GWAS to identify genes affecting traits of interest in crop plants is widespread, but results are sometimes disappointing. GWAS frequently identifies many genes with small effects. This situation that is not helpful if plant breeders are trying to choose the offspring that have the best genes for that trait of interest. We have been working on the genetic mapping of maize for resistance to aflatoxin accumulation, a serious public health problem and economic concern for farmers worldwide. After running into the problems of finding many genes of small effect, in order to bring greater clarity to this confusion, we and our collaborators created a new analysis tool that takes the output of a GWAS mapping study and identifies genes and the metabolic pathways to which these genes belong. This higher order analysis identifies a handful of pathways that can be studied in depth to understand how the plant creates the trait and helps identify genes that are truly important for selection by plant breeders for in-depth study or by breeders to create improved new crop varieties. The tool, called The Pathway Association Studies Tool (PAST), will work on any organism, and is easy to use. We created two methods of running this analysis. One allows the user to work online with an easy-to-use, point-and-click graphical interface, and the other allows the user to download a unified set of scripts and work offline, changing scripts and parameters as desired. Researchers in private industry, universities, U.S. government and international agriculture research institutions have already downloaded the program and article describing it over a thousand times. The PAST tool will allow faster and more efficient creation of new cultivars or breeds of plants and livestock by identifying targets for marker assisted selection or gene editing.

Accomplishments
1. Quantitative trait loci (QTL) associated with resistance to aflatoxin accumulation and fall armyworm leaf feeding damage identified. Pre-harvest accumulation of aflatoxin, a toxin produced by the fungus Aspergillus flavus, in corn grain is a major impediment to profitable corn production in the southern United States. Consumption of corn contaminated with aflatoxin poses a serious threat to wildlife, livestock, pets, and humans. Fall armyworm, a major insect pest of corn in the southern United States, feeds on all above ground portions of the corn plant causing yield losses and providing sites for fungi to enter developing ears. ARS researchers at Mississippi State, Mississippi, developed and released corn germplasm lines with resistance to aflatoxin accumulation and lines with resistance to fall armyworm damage. From a cross between Mp705, released as a source of resistance to fall armyworm, and Mp719, released as a source of resistance to aflatoxin accumulation, they produced a population of 243 segregating families. They identified three quantitative trait loci (QTL) on Chromosomes 1, 4, and 9 of Mp705 that accounted for 45% of the phenotypic variance associated with fall armyworm damage, and three QTL on Chromosomes 1 and 3 of Mp719 that accounted for 30% of the phenotypic variance associated with aflatoxin accumulation. By selecting for molecular markers associated with the target QTL and performing a series of crosses and backcrosses, researchers can produce lines with genetic resistance to both aflatoxin accumulation and fall armyworm damage. Using these lines as parents, plant breeders can develop high-yielding hybrids with genetic resistance to aflatoxin accumulation and fall armyworm damage for farmers in the United States and other countries where aflatoxin accumulation and fall armyworm damage are problems.

Review Publications
Thrash, A., Tang, J.D., DeOrnellis, M., Peterson, D.G., Warburton, M.L. 2020. PAST - pathway association studies tool to infer biological meaning from GWAS datasets. Plants. 9:58. https://doi.org/10.3390/plants9010058.
Womack, E.D., Williams, W.P., Smith, J.S., Warburton, M.L., Bhattramakki, D. 2020. Mapping quantitative trait loci for resistance to fall armyworm (Lepidoptera: Noctuidae) leaf-feeding damage in maize inbred Mp705. Journal of Economic Entomology. 113(2):956-963. https://doi.org/10.1093/jee/toz357.
Parish, F., Williams, W.P., Windham, G.L., Shan, X. 2019. Differential expression of signaling pathway genes associated with aflatoxin reduction quantitative trait loci in maize (Zea mays L.). Frontiers in Microbiology. 10(2683):1-10. https://doi.org/10.3389/fmicb.2019.02683.
Womack, E.D., Williams, W.P., Windham, G.L., Xu, W. 2020. Mapping quantitative trait loci associated with resistance to aflatoxin accumulation in maize inbred Mp719. Frontiers in Microbiology. 11(11):1-8. https://doi.org/10.3389/fmicb.2020.00045.
Liu, H., Wang, X., Xiao, Y., Luo, J., Qiao, F., Yang, W., Zhang, R., Meng, Y., Sun, J., Yan, S., Peng, Y., Niu, L., Jian, L., Song, W., Yan, J., Li, C., Zhao, Y., Liu, Y., Warburton, M.L., Zhao, J., Yan, J. 2020. CUBIC: an atlas of genetic architecture promises directed maize improvement. Genome Biology. 21(20):1-17. https://doi.org/10.1186/s13059-020-1930-x.
Oppong, A., Appiah-Kubi, D., Ifie, B.E., Abrokwah, L.A., Ofori, K., Offei, S.K., Adu-Dappah, H., Mochiah, M.B., Warburton, M.L. 2020. Analyzing combining abilities and heterotic groups among Ghanaian maize landraces for yield and resistance/tolerance to Maize Streak Virus Disease. Maydica. 6(3):1-10. Available: https://journals-crea.4science.it/index.php/maydica/article/view/1985.
Kakar, N., Jumaa, S.H., Redona, E.D., Warburton, M.L., K. Raja, R. 2019. Evaluating rice for salinity using pot-culture provides a systematic tolerance assessment at the seedling stage. Rice. 12:57. https://doi.org/10.1186/s12284-019-0317-7.
Hussain, M.M., Rauf, S., Warburton, M.L. 2019. Development of drought-tolerant breeding lines derived from Helianthus annuus × H. argophyllus interspecific crosses. Plant Breeding. 138(6):862-879. https://doi.org/10.1111/pbr.12731.
Aoun, M., Stafstrom, W., Priest, P., Fuchs, J., Windham, G.L., Williams, W.P., Nelson, R.J. 2020. Low-cost grain sorting technologies to reduce mycotoxin contamination in maize and groundnut. Food Control. 118:1-15. https://doi.org/10.1016/j.foodcont.2020.107363.