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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

2012 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:
In support of Objective 1, ARS scientists have compared stored messenger ribonucleic acid (mRNA) and stored proteins in mature maize kernels of resistant and susceptible seed to gain more understanding of their role in resistance to Aspergillus (A.) flavus. In support of Objective 2, five of six inbred maize lines previously developed and released through a collaborative breeding program between International Institute of Tropical Agriculture and Southern Regional Research Center (SRRC), New Orleans, LA, were tested in field trials in 2011 as single-cross hybrids in Starkville, MS, Lubbock and College Station, TX, and Baton Rouge, LA, to compare their levels of resistance with that shown by well-known resistant lines. Also in support of Objective 2, screening of inbred lines (pure breeding lines) demonstrating dual resistance against the fungus A. flavus and Fusarium verticillioides was completed. In support of Objective 3, corn transformation capability (ability to insert genes) at SRRC has been initiated to evaluate candidate genes that play a role in aflatoxin contamination. The corn cells have also been transformed with deoxyribonucleic acid (DNA) expressing the antifungal D4E1 gene. A similar approach will be used for transformation with candidate genes deduced from differential proteomic (total protein profile) analysis of resistant/susceptible corn genotypes that demonstrate inherent resistance or susceptibility to aflatoxin contamination. For example, new RNA interference constructs (vehicles to carry genes) with anti-aflatoxin genes have been made and will be used for corn transformation. We have also documented the mode of infection and spread of A. flavus in corn kernels lines that are resistant or susceptible. To evaluate resistance to A. flavus of transgenic cotton lines carrying the D4E1 gene, field trials are being planned with collaborators in an area of Arizona endemic to aflatoxin contamination of cotton. New peptide genes, native and synthetic, with demonstrated antifungal activity against A. flavus are also being introduced into cotton cells. Screening of different corn and cotton varieties to identify natural resistance factors is being carried out in collaboration with scientists at University of Louisiana and North Carolina State University. In conjunction with scientists at Louisiana State University, analysis of RNA isolated from cotton bolls inoculated with A. flavus is being used to identify and isolate genes involved in resistance to the fungus. Twenty-eight genes were identified from infected boll pericarp (structure surrounding seed) and seeds. Nine of them will be studied in more detail. In support of Objective 4, studies were completed using both toxigenic and atoxigenic A. flavus fungal strains to confirm and further characterize the fluorescence peak shift phenomenon that was identified among groups of kernels with different aflatoxin contamination levels.

1. New maize inbred (pure breeding line) lines demonstrate resistance to aflatoxin contamination in field trials. Of the six inbreds previously developed and released by the International Institute of Tropical Agriculture and ARS scientist at Southern Regional Research Center, New Orleans, LA, aflatoxin-resistance maize breeding program, five were tested in 2011 as single-cross hybrids (combination of different varieties) in field trials held in Mississippi, Texas (College Station and Lubbock), and Louisiana. Aflatoxin resistance was demonstrated in at least two of the five on a consistent basis and at levels comparable to other well-known resistant lines. Promising yield numbers were also attained by two of the lines compared to other resistant lines and to two commercial hybrids (mixed between two type of parents). The development of new resistant corn lines in good agronomic backgrounds (suitable for desired environmental conditions) offers the possibility of enhancing resistance of commercial lines through marker-assisted breeding. New linkages of resistance genes may have also been facilitated through the breeding of these lines.

2. Novel synthetic peptides demonstrate significant antifungal activity against mycotoxigenic fungi including Aspergillus (A.) flavus. Each year millions of dollars are lost to crops contaminated with toxic and carcinogenic aflatoxins produced by A. flavus. ARS scientists at the Southern Regional Research Center in New Orleans, LA, have tested a number of novel antifungal peptides for control of mycotoxigenic fungal pathogens. Peptides were provided by collaborators at Tuskegee University and AgroMed LLC. The ß-sheet (form of secondary structure of protein) peptides D4E1, AGM181 and AGM182, and the a-helical (form of structure of protein) peptides AGM184 and RCJ-1, demonstrated potent in vitro inhibitory activity compared to natural peptides against A. flavus, Fusarium verticillioides, and Verticillium dahliae and showed little or no hemolytic (destructive to red blood cells) activity. Development of transgenic crops expressing genes encoding these antifungal peptides will provide an effective means of controlling aflatoxin contamination in susceptible crops such as corn, cotton, peanut, and tree nuts thus benefitting both producers and consumers.

Review Publications
Brown, R.L., Menkir, A., Chen, Z.-Y., Luo, M., Bhatnagar, D. 2011. Identification of gene markers in aflatoxin-resistant maize germplasm for marker-assisted breeding. In: Guevara-Gonzalez, R.G., editor. Aflatoxins - Biochemistry and Molecular Biology. Rijeka, Croatia: Intech Open Access Publishers. pp 91-106.

Rajasekaran, K., Cary, J.W., Chlan, C.A., Jaynes, J.M., Bhatnagar, D. 2012.Strategies for controlling plant diseases and mycotoxin contamination using antimicrobial synthetic peptides. In: Rajasekaran, K., Cary, J.W., Jaynes, J.M., Montesinos, E., editors. Small Wonders: Peptides for Disease Control. American Chemical Society Symposium Series. American Chemical Society, Washington, DC: Oxford University Press, Inc. p. 295-315.

Chettri, P., Calvo, A.M., Cary, J.W., Dhingra, S., Guo, Y., McDougal, R.L., Bradshaw, R.E. 2012. The veA gene of the pine needle pathogen Dothistroma septosporum regulates sporulation and secondary metabolism. Fungal Genetics and Biology. 49:141-151.

Rajasekaran, K., Cary, J.W., Jaynes, J.M., Montesinos, E. 2012. Small wonders: peptides for disease control. American Chemical Society Symposium Series 1095. American Chemical Society, Washington, DC: Oxford University Press, Inc. 472 p.

Rajasekaran, K., Cary, J.W., Jaynes, J.M., Montesinos, E. 2012. Preface. In: Rajasekaran, K., Cary, J.W., Jaynes, J.M., Montesinos, E., editors. Small Wonders: Peptides for Disease Control. American Chemical Society Symposium Series. American Chemical Society, Washington, DC: Oxford University Press, Inc. p. ix-xi.

Last Modified: 4/20/2014
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