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

Location: Corn Host Plant Resistance Research

2022 Annual Report

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

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
In 2021, we evaluated 174 inbred lines for their response to Aspergillus (A.) flavus and to Fusarium (F.) verticillioides. The 174 lines included: (1) every available germplasm line registered and released as a source of resistance to A. flavus infection and aflatoxin accumulation; (2) commercial inbreds with expired Plant-Variety Protection (ex-PVPs); (3) Germplasm Enhancement of Maize (GEM) lines that had shown promise in previous years for resistance to either A. flavus or F. verticillioides; and (4) several inbreds from International Maize and Wheat Improvement Center (CIMMYT). Lines were evaluated for standard agronomic traits and ears were rated for ear-rot after inoculation with A. flavus and F. verticillioides. Aflatoxin was quantified for the A. flavus inoculated ears. Based on the 2021 evaluations, 80 lines were selected for further evaluation in 2022. These lines were planted in replicated trials at two locations in Mississippi. They are being evaluated for standard agronomic traits. Ear-rot and aflatoxin after A. flavus inoculation and ear-rot and fumonisin after F. verticillioides inoculation will be quantified after harvest. This data will be used to: (1) identify the most promising sources of resistance to use as donors in breeding crosses; (2) fully characterize the resistant germplasm so that strengths and weaknesses are identified; and (3) identify the promising lines to use as recurrent parents in breeding crosses that will combine resistance to aflatoxin accumulation with improved agronomic traits. In addition to these germplasm evaluations, we also screened 26 lines for the GEM project in 2021. We shared the results of our evaluations with the GEM coordinators and they were made public in the 2021 GEM cooperator’s meeting. GEMS-0050 and GEMS-0032 have shown promise as sources of resistance. The more recently released GEM lines with the lowest levels of aflatoxin in the 2021 trials were GEMS-0273, GEMS-0280, GEMS-0293, GEMS-0303, GEMS-0310. We are evaluating 30 GEM lines for the GEM project in 2022. These are being evaluated for standard agronomic traits, for ear-rot and aflatoxin accumulation after A. flavus inoculation, and ear-rot and fumonisin accumulation after F. verticillioides inoculation. Results will be shared with GEM coordinators and published at the GEM cooperator’s meeting for 2022. We evaluated 20 selections from the GEM project for resistance to fall armyworm leaf feeding damage in 2021: GEMN-0135, GEMN-0292, GEMN-0270, and GEMS-0294 sustained the least damage. We shared the results of our evaluations with GEM cooperators. Of the GEM selections evaluated in our 2022 trials, GEMS-0294, GEMN-0292, GEMS-0270, and GEMN-0135 sustained the least damage. Although these lines exhibited only moderate resistance to fall armyworm damage in our trials, they may be useful in producing breeding crosses with some of our highly resistant lines that lack other desirable agronomic qualities. Several lines developed by the CIMMYT, including CML71, CML122, CML286, CML370, and CML484, exhibited resistance comparable to some of the lines that we developed and released. In addition to our evaluations for fall armyworm leaf feeding damage to whorl stage corn, we initiated research to identify resistance to ear damage as well. We are comparing ear damage following infestation of developing ears with neonate or third instar fall armyworm or southwestern corn borer larvae at anthesis. Damage to husks, ears, ear shanks, and stalks is being assessed. Additionally, we conducted a study to compare damage sustained by single cross hybrids infested with fall armyworm or southwestern corn borer larvae at either the mid-whorl stage of growth or at anthesis. We conducted a preliminary study to evaluate a multiple-infestation strategy for identifying resistance to fall armyworm at various growth stages throughout the growing season. The third year of phenotyping of a 3-year Genome Wide Association Study (GWAS) for resistance to fall armyworm was concluded in 2021. This study provides additional information on molecular markers that can be used in marker-assisted breeding for enhanced resistance. A manuscript has been written and will soon be submitted for publication. From this 3-year study, germplasm lines that showed promise as sources of resistance to fall armyworm were further evaluated this year. These will be incorporated into our breeding program as appropriate. In 2021 a gene expression study for fall armyworm was initiated and repeated this year. Leaf tissues from fall armyworm infested and non-infested plants at different growth stages from resistant and susceptible corn germplasm lines were collected. The ribonucleic acid (RNA) extractions from the leaf tissues have been initiated and are being processed for further analysis for proteomic, metabolomic, and gene expression studies. In 2021 we crossed three resistant germplasm lines (Mp313E, Mp715, and Mp717) to a series of ex-PVP lines. The F1’s were selfed and backcrossed in the 2021-22 winter nursery, generating segregating breeding populations. In 2022, the F1’s are being evaluated in replicated trials planted in Mississippi and in Georgia for agronomic potential and resistance to A. flavus and aflatoxin accumulation. The corresponding breeding crosses were selfed and backcrossed in the 2022 summer nursery. The results of the 2022 F1 trial will be used to make selections between the breeding populations. Selected breeding populations will then be advanced through pedigree breeding. The goal is to use previously released germplasm lines in additional breeding cycles to develop lines that possess resistance to A. flavus infection and aflatoxin accumulation and improved agronomic characteristics. We developed germplasm lines that exhibited high levels of resistance to fall armyworm leaf feeding damage in both 2021 and 2022. These lines are being further evaluated for agronomic quality and as sources of resistance to ear damage by fall armyworm. A study was initiated to evaluate the effect of resistance to fall armyworm and southwestern corn borer feeding on aflatoxin accumulation in corn hybrids. The leaf damage, ear damage, plant heights and yield loss will be assessed. The same evaluation will be repeated next year. Marker-assisted selection was performed in the summer of 2022. Near-isogenic lines (NILs) possessing quantitative trait loci (QTL) for resistance to aflatoxin accumulation and QTL for resistance to fall armyworm are being developed. The aflatoxin NILs have been backcrossed 4 times and approximately 97% of the recurrent parent genome should have been recovered. Those NILs were selfed for the first time in 2022, and they need an additional generation of selfing. The fall armyworm NILs were backcrossed this summer. This was the second generation of backcrossing for those NILs. They require two more generations of backcrossing and then two generations of selfing.

Accomplishments

Review Publications
Smith, J.S., Williams, W.P., Warburton, M.L., Windham, G.L., Xu, W., Bhattramakki, D. 2022. Mapping QTL for aflatoxin accumulation resistance in two populations containing resistant maize inbred Mp717. Crop Science. 62(2):780-791. https://doi.org/10.1002/csc2.20695.
Smith, J.S., Williams, W.P. 2021. Aflatoxin accumulation in a maize diallel cross containing inbred lines with expired Plant Variety Protection. Agronomy. 11(11):2285. https://doi.org/10.3390/agronomy11112285.
Bertagna, F.B., Kuki, M.C., Neto, H.Z., Tessmann, D.J., Pinto, R.B., Scapim, C.A., Williams, W.P., Warburton, M.L. 2021. Association mapping and pathway analysis of ear rot disease caused by Aspergillus flavus in a panel of tropical maize germplasm. Crop Science. 61(6):4128-4138. https://doi.org/10.1002/csc2.20629.
Ogunola, O.F., Smith, J.S., Xu, W., Bhattramakki, D., Jeffers, D., Williams, W.P., Warburton, M.L. 2021. Characterization of a source of resistance to aflatoxin accumulation in maize. Agrosystems, Geosciences & Environment. 4(3):e20203. https://doi.org/10.1002/agg2.20203.
Gouda, A.C., Warburton, M.L., Djedatin, G.L., Kpeki, S.B., Wambugu, P.W., Gnikoua, K., Ndjiondjop, M.N. 2021. Development and validation of diagnostic Kompetitive allele-specific PCR (KASP) markers for quality control (QC) genotyping in a collection of four rice species. Scientific Reports. 11:18617. https://doi.org/10.1038/s41598-021-97689-3.
Oppong, A., Dadzie, A.M., Ifie, B.E., Asante, M.D., Prempeh, R.A., Abrokwah, L.A., Kubi, Z.A., Marfo, E.A., Ananng, E.A., Warburton, M.L. 2021. Genetic analysis of new maize hybrids for yield and resistance to aflatoxin accumulation. Journal of Agricultural Science. 13(10):15-30. https://doi.org/10.5539/jas.v13n10p15.
Blanco, C., Conover, K., Hernandez, G., Valentini, G., Portilla, M., Abel, C.A., Williams, W.P., Nava-Camberos, U., Huschison, W., Dively, G. 2022. Grain yield is not impacted by early defoliation of maize: implications for Fall armyworm action thresholds. Southwestern Entomologist. 47(2):335-344. https://doi.org/10.3958/059.047.0209.
Willcox, M.C., Burgueño, J.A., Jeffers, D., Rodriguez-Chanona, E., Guadarrama-Espinoza, A., Kehel, Z., Chepetla, D., Shrestha, R., Swarts, K., Hearne, S., Buckler IV, E.S., Chen, N.C. 2022. Mining alleles for tar spot complex resistance from CIMMYT's maize germplasm bank. Frontiers in Sustainable Food Systems. 6:937200. https://doi.org/10.3389/fsufs.2022.937200.
Pingault, L., Basu, S., Vellichirammal, N.N., Williams, W.P., Sarath, G., Louis, J. 2022. Co-transcriptomic analysis of the maize-western corn rootworm interaction. Plants. https://doi.org/10.3390/plants11182335.
Aoun, M., Siegel, C., Windham, G.L., Williams, W.P., Nelson, R.J. 2022. Application of reflectance spectroscopy to identify maize genotypes and aflatoxin levels in single kernels. Food Control. 15(4):324-342. https://doi.org/10.3920/WMJ2021.2750.