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United States Department of Agriculture

Agricultural Research Service

Research Project: ENHANCING CORN WITH RESISTANCE TO AFLATOXIN CONTAMINATION AND INSECT DAMAGE

Location: Corn Host Plant Resistance Research

2012 Annual Report


1a.Objectives (from AD-416):
Identify and develop corn germplasm with resistance to Aspergillus flavus infection/aflatoxin contamination and ear-feeding insects and release this germplasm together with information on molecular markers and methodology that will expedite its deployment into commercially available corn hybrids. Specific objectives include the following: (1) determine the effects of indigenous fungal species and ear-feeding insects on A. flavus infection and aflatoxin accumulation in corn grain; (2) identify new sources of corn germplasm with resistance to A. flavus infection and aflatoxin accumulation and/or resistance to damage by southwestern corn borer, fall armyworm, and corn earworm; (3) identify quantitative trait loci, genes, and proteins associated with resistance in corn to A. flavus infection, aflatoxin accumulation, and insect damage; and (4) enhance corn germplasm with resistance to A. flavus infection, aflatoxin accumulation, and insect damage and release germplasm lines as sources of resistance.


1b.Approach (from AD-416):
Objective 1. Determine the effects of indigenous fungal species and ear-feeding insects on A. flavus infection and aflatoxin accumulation in corn grain. Colonization of corn grain is rarely by a single fungal species, but rather a mixture of fungi. Fusarium verticillioides (syn. F. moniliforme) is the most commonly reported fungus infecting corn in the USA, and it is frequently found together with A. flavus. Acremonium zeae is a common contaminant of preharvest corn in the Southeast. It has been reported to suppress growth of both A. flavus and F. verticillioides in laboratory experiments. The interactions of these fungi will be investigated to determine whether F. verticillioides and A. zeae affect A. flavus infection of corn grain and the subsequent accumulation of aflatoxin, and if so, whether these fungi are impediments to the identification of aflatoxin-resistant corn germplasm. The association between insect damage and aflatoxin accumulation in different corn genotypes will be investigated and the extent to which resistance to damage by southwestern corn borer, Diatraea grandiosella; fall armyworm, Spodoptera frugiperda; or corn earworm, Helicoverpa zea, reduces aflatoxin contamination will be determined. Objective 2. Identify new sources of corn germplasm with resistance to A. flavus infection and aflatoxin accumulation and/or resistance to damage by southwestern corn borer, fall armyworm, and corn earworm. Corn germplasm from diverse backgrounds will be screened for resistance to A. flavus/aflatoxin, southwestern corn borer, fall armyworm, and corn earworm. Information on the effects of other fungi or insects on A. flavus/aflatoxin accumulation (Objective.
1)will be used to refine and improve techniques for evaluating germplasm for resistance. Newly identified sources of resistance will be used to pursue Objectives 3 and 4. Objective 3. Identify quantitative trait loci, genes, and proteins associated with resistance in corn to A. flavus infection, aflatoxin accumulation, and insect damage. Populations of F2:3 families and recombinant inbred lines derived from crosses between aflatoxin or insect resistant inbred lines and susceptible lines will be used to identify quantitative trait loci (QTL) associated with resistance. Resistant and susceptible corn inbred lines and recombinant inbred lines will be used in complementary investigations to identify candidate genes and proteins associated with resistance. Molecular markers identified in these investigations will be used in developing improved germplasm lines (Objective 4). Objective 4. Enhance corn germplasm with resistance to A. flavus infection, aflatoxin accumulation, and insect damage and release germplasm lines as sources of resistance. Both breeding methods based on phenotypic performance and those based on molecular markers will be used to enhance germplasm with resistance to aflatoxin contamination and insect damage. The effectiveness of molecular markers based on QTL, genes, and proteins identified in Objective 3 in transferring resistance to A. flavus/aflatoxin and insect damage into germplasm lines with desirable agronomic qualities will be determined.


3.Progress Report:
Two corn germplasm lines, Mp718 and Mp719, were released as sources of resistance to aflatoxin accumulation in 2011. These lines are under investigation by USDA-ARS and a cooperator at Mississippi State University (6406-21000-011-11S) to identify and determine the functions of genes associated with resistance to aflatoxin accumulation. Aspergillus (A.) flavus biomass, as determined by quantitative polymerase chain reaction (q-PCR), and aflatoxin accumulation in corn grain were highly and positively correlated in corn hybrids inoculated with toxin-producing A. flavus strain NRRL3357. The application of A. flavus strains that do not produce aflatoxin to corn fields as a bio-control method to reduce losses to aflatoxin contamination is gaining acceptance in many areas. Polymorphisms have been identified and primers developed that permit differentiation between and quantification of fungal biomass of A. flavus strains NRRL 3357 (toxin producing) and NRRL 21882 (non-toxin-producing) using q-PCR. When susceptible and resistant corn hybrids were inoculated with a toxin-producing A. flavus strain, aflatoxin contamination was 10× higher in the susceptible hybrids than the resistant hybrids. When inoculated with a combination of toxin-producing and non-producing strains, toxin production in both susceptible and resistant hybrids was significantly reduced. Using hybrids with genetic resistance in combination with a non-toxin-producing strain of A. flavus as a bio-control agent to reduce aflatoxin contamination is a promising and potentially useful means of reducing aflatoxin contamination in corn. Results of two years indicate that use of non-toxin-producing A. flavus strains as a bio-control method and corn hybrids with genetic resistance to aflatoxin accumulation are compatible strategies in reducing aflatoxin contamination in corn. Other non-toxin-producing strains such as K49 are also being evaluated in field trials. An association mapping project was initiated in 2008: 300 germplasm lines were phenotyped as testcrosses for resistance to A. flavus infection and aflatoxin accumulation in replicated field tests at two locations in Texas (6406-21000-011-09S and 6406-21000-011-010S) and two locations in Mississippi in 2009 and 2010. Seed of the 30 lines with lowest levels of aflatoxin accumulation as testcrosses across all environments have been sent to the International Maize and Wheat Improvement Center (CIMMYT) in Mexico and International Institute for Tropical Agriculture (IITA) in Nigeria as a part of a United States Agency for International Development (USAID) project to reduce aflatoxin contamination of corn in developing countries. The 300 germplasm lines were genotyped via high-throughput sequencing in cooperation with USDA-ARS at Cornell University. This investigation yielded a file consisting of tens of thousands of single nucleotide polymorphisms (SNPs) for each line. Analysis of this data set together with the corresponding phenotypic information will involve associating each allele of each SNP with a change in the phenotype over the panel of 300 lines.


4.Accomplishments
1. Corn germplasm lines evaluated for aflatoxin accumulation. Genetic resistance is generally considered a highly desirable means of reducing aflatoxin contamination of corn. To identify alleles associated with reduced levels of aflatoxin accumulation, an association mapping project was conducted by USDA-ARS scientists at Mississippi State, Mississippi. Three hundred corn germplasm lines were evaluated as testcrosses for resistance to Aspergillus flavus infection and aflatoxin accumulation in replicated field tests conducted for two years at two locations in Mississippi and two locations in Texas (6406-21000-011-09S and 6406-21000-011-10S). The 300 germplasm lines were genotyped via high-throughput sequencing in cooperation with USDA-ARS researchers at Ithaca, New York. This investigation yielded a file consisting of tens of thousands of single nucleotide polymorphisms (SNPs) for each line. Analysis of this data set together with the corresponding phenotypic information will involve associating each allele of each SNP with a change in the phenotype over the panel of 300 lines and determine its importance in influencing phenotype. As an additional benefit of this effort, seed of the 30 germplasm lines with lowest levels of aflatoxin accumulation as testcrosses across all environments have been sent to the International Maize and Wheat Improvement Center (CIMMYT) in Mexico and the International Institute of Tropical Agriculture (IITA) in Nigeria to initiate a United States Agency for International Development (USAID) project to reduce aflatoxin contamination of corn in developing countries.


Review Publications
Smith, W.E., Shivaji, R., Williams, W.P., Luthe, D.S., Sandoya, G.V., Smith, C.L., Sparks, D.L., Brown, A.E. 2012. A maize line resistant to herbivory constitutively releases (E)-B-caryophyllene. Journal of Economic Entomology. 105:120-128.

McDaniel, A., Holmes, W.E., Williams, P., Armbrust, K.L., Brown, A.E. 2011. Effect of matrix clean-up for alfatoxin analysis in corn and dried distillers grains. Scientific Research and Essays. 2:1-8.

Williams, W.P., Windham, G.L. 2012. Registration of Mp718 and Mp719 germplasm lines of maize. Journal of Plant Registrations. 6:200-202.

Henry, W.B., Windham, G.L., Blanco, M.H. 2012. Evaluation of maize germplasm for resistance to aflatoxin accumulation. Agronomy. 2:28-39.

Kelley, R.Y., Williams, W.P., Mylroie, J.E., Boykin, D.L., Harper, J.W., Windham, G.L., Ankala, A., Shan, X. 2012. Identification of maize genes associated with host plant resistance and susceptibility to Aspergillus flavus infection and aflatoxin accumulation. PLoS One. 7:1-12. doi:10.1371/journal.pone.0036892

Warburton, M.L., Williams, W.P., Hawkins, L., Bridges, S., Gresham, C., Harper, J., Ozkan, S., Mylroie, J.E., Shan, X. 2011. A public platform for the verification of the phenotypic effect of candidate genes for resistance to aflatoxin accumulation and Aspergillus flavus infection in maize. Toxins. 3:754-765.

Ni, X., Chen, Y., Hibbard, B.E., Wilson, J.P., Williams, W.P., Buntin, G., Ruberson, J.R., Li, X. 2011. Foliar resistance to fall armyworm in corn germplasm lines that confer resistance to root- and ear-feeding insects. Florida Entomologist. 94(4):971-981.

Mideros, S.X., Windham, G.L., Williams, W.P., Nelson, R.J. 2012. Tissue-specific components of resistance to Aspergillus ear rot of maize. Phytopathology. 102:787-793.

Last Modified: 10/1/2014
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