2013 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.
Corn germplasm accessions obtained from the Germplasm Enhancement of Maize (GEM) project were evaluated for resistance to Aspergillus flavus infection and the subsequent accumulation of aflatoxin in the grain. Potential new sources of resistance were identified, and germplasm lines with high levels of resistance to aflatoxin accumulation have been developed and are being further evaluated in field tests. Germplasm lines Mp718 and Mp719 were developed from Mp715 × Va35 and released as sources of resistance to aflatoxin contamination. These lines are currently being used in gene expression studies conducted by a cooperator at Mississippi State University. A panel of 300 diverse corn inbred lines from U.S. breeding programs and international research centers were evaluated as testcrosses with Va35 for resistance to Aspergillus flavus infection and aflatoxin accumulation in eight environments in Mississippi and Texas. The 300 lines were genotyped via high-throughput sequencing in cooperation with ARS scientists in Ithaca, NY. This yielded a data file consisting of thousands of single nucleotide polymorphisms (SNPs) for each line. Analysis of this data together with phenotypic information involved associating each allele of each SNP with a change in resistance to aflatoxin accumulation. As a part of a USAID project, seed of the 30 most resistant lines from this investigation were provided to research scientists at the International Maize and Wheat Improvement Center (CIMMYT) in Mexico and the International Institute of Tropical Agriculture (IITA) in Nigeria to advance efforts to reduce aflatoxin accumulation of corn in developing countries. An analysis of quantitative trait loci (QTL) associated with resistance to aflatoxin accumulation in bi-parental populations provided information on molecular markers that is being used to develop near-isogenic lines (NILs) from backcrosses of Mp313E and Mp715 to B73, T173, and Va35. In 2012, field tests of NILs developed from Mp313E × Va35 indicated selection for the QTLs identified in Mp313E reduced aflatoxin contamination. Efforts to identify and confirm the effects of other QTLs on resistance to aflatoxin accumulation are underway. Although Mp715 and Mp717 were released as sources of resistance to Aspergillus flavus infection and aflatoxin accumulation, they also exhibited resistance to Fusarium verticillioides infection and fumonisin accumulation. Aspergillus flavus biomass, determined by quantitative polymerase chain reaction (q-PCR), and aflatoxin accumulation in corn grain were highly correlated in corn hybrids inoculated with toxin-producing Aspergillus flavus strain NRRL3357. Corn hybrids with genetic resistance to aflatoxin accumulation used in combination with a non-toxin producing strain of Aspergillus flavus as a bio-control agent showed promise as a means of reducing aflatoxin contamination in corn. Resistance to southwestern corn borer, fall armyworm, and corn earworm was also effective in reducing aflatoxin accumulation in environments where insect populations levels were high.
Deoxyribonucleic acid (DNA) sequences associated with resistance to aflatoxin contamination identified. Aflatoxin contamination of corn is a serious food and feed safety problem in the southern United States and many developing countries. Corn hybrids with genetic resistance are a key component in the strategy to eliminate or reduce contamination, and molecular markers are essential to the successful development and deployment into farmers’ fields of high quality corn hybrids with resistance to aflatoxin contamination. ARS scientists in the Crop Science Research Laboratory at Mississippi State, Mississippi, developed a protocol for evaluating DNA sequences from any corn germplasm line for its role in resistance to Aspergillus flavus infection and subsequent aflatoxin accumulation. Five genes have been found to be highly associated with resistance, and perfect markers have been developed for the genes with large effects. These markers will be used to rapidly and economically transfer resistance to aflatoxin contamination into agronomically desirable, but susceptible, parental inbred lines of corn. Evaluation of additional DNA sequences will continue in a search for other genes with even greater effects on resistance.
Improved method for inoculating corn with Aspergillus flavus to screen for resistance to aflatoxin contamination. Aflatoxin contamination of corn is a serious food and feed safety problem in the southern United States and in developing countries where corn is a primary dietary staple. Efficient methods for inoculating corn plants with Aspergillus flavus are essential to identifying and developing corn with genetic resistance to aflatoxin accumulation. ARS scientists in the Crop Science Research Laboratory at Mississippi State, Mississippi, used a hand-held applicator designed for infesting plants with insect larvae to dispense Aspergillus flavus infected wheat kernels into plant whorls at 5 and 7 weeks after planting. This method of inoculating with Aspergillus flavus was compared with the widely used side-needle technique in which a spore suspension is injected underneath the husks into the side of the developing ear. Results of a 2-year investigation indicated that although aflatoxin concentration in the grain was lower with the new inoculation method, the methods were equally effective in differentiating between resistant and susceptible corn hybrids. Because the developing ear is not wounded with the new inoculation method, it permits expression of resistance mechanisms that were heretofore masked by the side-needle technique. Inoculating plants with infected wheat will reduce the time and labor required for conducting evaluations and increase the number of germplasm lines evaluated.
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Henry, W.B., Windham, G.L., Rowe, D.E., Blanco, M.H., Murray, S.C., Williams, W.P. 2013. Diallel analysis of diverse maize germplasm lines for resistance to aflatoxin accumulation. Crop Science. 53:394-402.
Windham, G.L., Williams, W.P. 2012. Comparison of different inoculating methods to evaluate the pathogenicity and virulence of Aspergillus niger on two maize hybrids. Phytoparasitica. 40(4):305-310.
Narro, L., Franco, J.D., George, M.L., Arcos, A.L., Osorio, K., Warburton, M.L. 2013. Comparison of the performance of synthetic maize varieties created based on genetic distance or general combining ability of the parent. Maydica. 57:83-91.
Willcox, M., Davis, G., Warburton, M.L., Windham, G.L., Abbas, H.K., Betran, J., Holland, J.B., Williams, W.P. 2013. Confirming quantitative trait loci for aflatoxin resistance from Mp313E in different genetic backgrounds. Molecular Breeding. 32(1):15-26.
Pechanova, O., Pechan, T., Rodriguez, J., Williams, W.P., Brown, A. 2013. A two-dimensional proteome map of the aflatoxigenic fungus Aspergillus flavus. Proteomics. 13:1513-1518. DOI 10.1002/pmic.201100659.
Ankala, A., Kelley, R.Y., Rowe, D.E., Williams, W.P., Luthe, D.S. 2013. Foliar herbivory triggers local and long distance defense responses in maize. Plant Science. 200:103-112. http://www.sciencedirect.com/science/article/pii/S0168945212002257.