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United States Department of Agriculture

Agricultural Research Service

Research Project: Developing Resistance to Aflatoxin Through Seed-Based Technologies

Location: Food and Feed Safety Research

2013 Annual Report


1a.Objectives (from AD-416):
1. Develop and implement marker-assisted corn breeding strategy. Identify and characterize novel markers associated with aflatoxin-resistance, e.g., resistance-associated proteins (RAPs), in developing and mature kernels through proteomic and genomic comparisons of resistant and susceptible corn genotypes. 2. Identify new sources of corn germplasm and develop new germplasm resistant to fungal infection and aflatoxin contamination with national and international collaboration, using laboratory and field inoculations of corn kernels with tester fungi designed for rapid resistance screening. 3. Evaluate the contribution of novel RAP genes from corn (see Objective.
1)for resistance to A. flavus growth and aflatoxin production and use these genes or others to develop transgenic cotton with enhanced resistance to aflatoxin contamination under greenhouse and field conditions. Identify and transfer resistant varieties to cooperating plant breeders for development of varieties for commercial application. 4. Develop rapid, non-destructive hyperspectral imaging methodology to: a) measure fungal growth and aflatoxin contamination in corn as a tool for use in enhancement of Homeland Security, and b) measure spectral signatures associated with traits for resistance to fungal infection and aflatoxin contamination in corn kernels.


1b.Approach (from AD-416):
Resistance to aflatoxin contamination will be enhanced in corn and cottonseed through marker assisted breeding and genetic engineering, respectively. In order to accomplish these goals, complex natural resistance mechanisms in corn kernels will be elucidated in resistant corn inbreds through identification of resistance associated proteins using proteomics and other resistance associated compounds through chemical analysis. Understanding the molecular basis of seed based resistances will lead to identification of biochemical factors correlated with resistance for use in marker assisted breeding and/or when pertinent resistance genes are identified and cloned, for use in enhancement of resistance in crops through genetic engineering. This strategy is especially pertinent to cottonseed, which does not possess practical levels of natural resistance to aflatoxin producing fungi in its germplasm base. Another goal is to assess resistance related biochemical products for their stability of expression in native and transgenic crops under environmental conditions (e.g. drought) known to be conducive to aflatoxin contamination. Also as a part of this project, rapid, non destructive detection methodology based upon hyperspectral imaging will be developed to measure fungal growth and aflatoxin in corn kernels and spectral signatures associated with traits for resistance to fungal infection and aflatoxin contamination in corn kernels, and also to measure physical and biochemical attributes in kernels potentially useful in resistance marker selection.


3.Progress Report:
Support of Objective 1, ARS scientists have correlated the expression of sets of selected resistance genes with the aflatoxin-resistance phenotype (observable characteritics) as an approach towards developing markers to transfer resistance to agronomically (adapability to varying conditions) desirable corn lines. Support of Objective 2, five of six inbred corn lines previously developed and released through a collaborative breeding program between International Institute of Tropical Agriculture (IITA) and Southern Regional Research Center (SRRC), New Orleans, LA, were tested in field trials in Starkville, MS and Baton Rouge, LA, to compare their levels of resistance with that shown by well-known resistant lines. Resistant corn inbreds (progeny from genetically similar parents) developed through the IITA-SRRC corn breeding collaboration were used to develop corn hybrids (progeny from genetically dissimilar parents) which are being tested in National Programs of Central and West Africa. Support of Objective 3, corn cells have transformed [foreign deoxyribonucleic acid (DNA) introduced] with DNA expressing a green fluorescent protein (GFP) gene or the antifungal D4E1 gene. Five ribonucleic acid interference (RNAi) constructs (vehicles to carry genes) targeting genes required for fungal growth or aflatoxin production have been introduced into corn cells and kernels containing the RNAi constructs are being analyzed for enhanced resistance to fungal growth and aflatoxin production. Using the GFP-producing Aspergillus (A.) flavus strain, ARS scientists at the Southern Regional Research Center in New Orleans, LA, visualized the mode of infection and spread of A. flavus in corn kernels lines that are resistant or susceptible to infection, providing a quick and reliable method to evaluate A. flavus resistance in undamaged corn kernels. To evaluate resistance to A. flavus of transgenic cotton lines carrying the D4E1 antifungal gene, field trials are being conducted at the University of Arizona station in Maricopa, AZ, a region known for chronic aflatoxin contamination of cotton. These studies include targeting of the D4E1 antifungal peptide to different compartments of the plant cell for greater resistance to fungal infection and toxin production. In collaboration with the University of Louisiana in Lafayette, LA, ARS scientists at the Southern Regional Research Center in New Orleans, LA, have screened a pool of 26 different cotton varieties to determine if any demonstrate natural resistance; a total of six demonstrated enhanced resistance. Seed from these plants are being subjected to additional screens for A. flavus resistance. Support of Objective 4, a whole corn ear hyperspectral (entire electromagnetic spectrum) imaging system was developed under a grant from Bill & Melinda Gates Foundation Grand Challenge Exploration (GCE round 8), awarded in 2012 to develop portable technology to detect aflatoxin contamination in single corn ears for farmers in the developing countries. This, our previous project is a joint effort between Mississippi State University and ARS-SRRC to extend knowledge from collaborative project to more practical applications.


4.Accomplishments
1. A rapid and reliable method to evaluate resistance to aflatoxin contamination in kernels of maize genotypes using a green fluorescent protein (GFP)-expressing Aspergillus flavus strain. In addition to the identification of resistance genes, successful corn breeding programs must also elucidate the etiology (study of causes) of the fungus so that the breeding objectives include the expression of relevant tissue-or organ-specific resistance genes. ARS scientists at the Southern Regional Research Center in New Orleans, LA, have shown that a GFP-expressing Aspergillus (A.) flavus’ entry into undamaged kernels occurs invariably through the rachilla and pedicel (parts of seed). ARS scientists at the Southern Regional Research Center in New Orleans, LA, have also established a close relationship between GFP fluorescence and aflatoxin levels for this strain of A. flavus. In view of these results, corn breeders should place an emphasis on developing resistance to this saprophytic fungus in the pedicel and basal endosperm region in developing kernels.

2. Successful transformation of corn. As researchers identify potential fungal/aflatoxin resistance genes in corn it will be necessary to determine the contribution of these genes to the plant’s overall resistance. This will require the ability to transform corn with ribonucleic acid (RNA) interference (RNAi) constructs (vehicles to carry genes) that target these putative resistance genes for downregulation thus allowing determination of any alterations in resistance in the transformed corn due to decreased expression of the targeted resistance gene. Using a green fluorescent protein (GFP)-based plasmid construct, scientists in the Food and Feed Safety Research Unit, Southern Regional Research Center, New Orleans, LA have demonstrated successful insertion of a GFP gene into the corn genome. This now paves the way for experiments to introduce non-native antifungal genes or RNAi constructs into corn. The ability to routinely transform corn will aid in the identification of novel corn antifungal/anti-aflatoxin genes that will provide corn breeders with much needed information on genes involved in resistance and it will also provide a means to quickly introduce resistance genes from other sources into corn.


Review Publications
Rajasekaran, K. 2013. Biolistic transformation of cotton zygotic embryo meristem. In: Zhang, B-H. (editor). Transgenic Cotton: Methods and Protocols, Methods in Molecular Biology. Humana Press. 958:47-57.

Rajasekaran, K. 2013. Biolistic transformation of cotton embryogenic cell suspension cultures. In: Zhang, B-H. (editor). Transgenic Cotton: Methods and Protocols, Methods in Molecular Biology. Humana Press. 958:59-70.

Rajasekaran, K., Sickler, C.M., Brown, R.L., Cary, J.W., Bhatnagar, D. 2013. Evaluation of resistance to aflatoxin contamination in kernels of maize genotypes using a GFP-expressing Aspergillus flavus strain. World Mycotoxin Journal. 6(2):151-158.

Brown, R.L., Bhatnagar, D., Cleveland, T.E., Chen, Z.-Y., Menkir, A. 2013. Development of maize host resistance to aflatoxigenic fungi. In: Prof. Mehdi Razzaghi-Abyaneh (Ed.). Aflatoxins - Recent Advances and Future Prospects. ISBN: 978-953-51-0904-4. InTech, Available: http://www.intechopen.com/books/aflatoxins-recent-advances-and-future-prospects/development-of-maize-host-resistance-to-aflatoxigenic-fungi.

Yao, H., Hruska, Z., Kincaid, R., Brown, R.L., Bhatnagar, D., Cleveland, T.E. 2013. Detecting corn inoculated with toxigenic and atoxigenic fungal strains with fluorescence hyperspectral imagery. Biosystems Engineering. 115:125-135.

Last Modified: 9/3/2014
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