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ARS Home Southeast Area New Orleans, Louisiana Southern Regional Research Center Food and Feed Safety Research Research





- Rajasekaran, Kanniah - Rajah
- Lebar, Matthew
- Gilbert, Matthew
- Mack, Brian
- Castano-Duque, Lina
- Sweany, Rebecca
- 2023
- 2022
- 2021
- Publications
- Food Safety, (Animal and Plant Products) (108)

Research Project: Development of Aflatoxin Resistant Corn Lines Using Omic Technologies **Location:
                            Food and Feed Safety Research** 2023 Annual Report **Objectives**
                                

1. Identify differentially expressed genes in resistant [R] and susceptible [S] corn lines that can serve as targets in controlling Aspergillus flavus and aflatoxin contamination.

2. Identify and characterize corn seed metabolites for enhancing resistance to aflatoxin contamination.

3. Develop and evaluate transgenic corn lines by over-expressing resistance-associated protein genes, or gene editing and silencing of Aspergillus flavus genes critical to growth and aflatoxin production.

4. Develop detection methods for pre- and post- harvest contamination in food supply and predictive modeling for mycotoxin contamination.

Subobjective 4.A: Develop and evaluate different spectral-based imaging instruments for non-destructive detection of AF contamination in corn. Advance and commercialize hyperspectral-based, rapid and non-invasive, imaging technology.

Subobjective 4.B: Develop methods to detect and prevent contamination of alternative proteins from plant-based sources with fungal toxins.

Subobjective 4.C: Modeling mycotoxin contamination in the US and developing a forecast system to forewarn US stakeholders of mycotoxin contamination in corn. **Approach**
                                

Aflatoxin (AF) contamination in food and feed crops such as corn, peanut, cottonseed, and tree nuts, caused by Aspergillus flavus, is a global concern that compromises food safety and marketability. Aflatoxins are potent carcinogens and their contamination in food are one of the major causes of liver cancer worldwide. The most efficient and practical approach to reduce pre-harvest AF contamination in corn is the development of resistant lines, the overall goal of this project plan. We will delineate the molecular basis of A. flavus resistance in corn seeds through “systems biology” approach that will involve a combination of transcriptomic, proteomic, and metabolomic analyses. Identification of novel regulatory genes and gene networks that play key roles in host plant resistance against the fungus will contribute to the development of robust markers for use in marker-assisted breeding and/or to the identification of candidate genes for editing. We will apply functional genomics to identify key metabolic pathways (polyamines, carotenoids, flavonoids) that contribute to resistance against A. flavus and AF production. Transgenic corn expressing antifungal proteins/peptides that strongly inhibit A. flavus growth will be generated. In addition, “host induced gene silencing” approach will be used to target fungal genes that are critical for growth, pathogenesis, and production of AFs. In addition to food crops, safety of alternative proteins of plant origin from fungal toxins will also be appraised. Using artificial intelligence (AI) and machine learning (ML), algorithmic models will be constructed to predict in advance possible mycotoxin breakout so that timely mitigation efforts can be employed. Finally, non-destructive, hyperspectral-based imaging systems for several platforms will be refined and commercialized to detect AF contamination in stored kernels. The knowledge and products generated from this research will be invaluable for the consumers, stakeholder groups, scientific community, and regulatory agencies to protect and preserve food safety in the United States and abroad. **Progress Report**
                                

The research objectives, which fall under National Program 108 Food Safety, Component 1, Foodborne Contaminants, are designed to understand the preharvest aflatoxin (a toxic and carcinogenic compound) contamination of important food and feed crops, such as corn, caused by the fungus, Aspergillus (A.) flavus, and develop effective mitigation strategies. To accomplish this, it is important to identify and evaluate native corn genes (Objective 1) and chemicals (Objective 2) that contribute to resistance to aflatoxin contamination. Key fungal genes responsible for fungal growth and aflatoxin production will be learnt from the results of the sister project 6054-41420-009-000D - “Aflatoxin control through identification of intrinsic and extrinsic factors governing the Aspergillus flavus-corn interaction.” Combined together, availability of candidate corn genes for resistance and production of key chemicals such as flavonoids (antioxidants), carotenoids (yellow and orange pigments), chlorophylls (green pigments), and polyamines (chemicals that control growth and development of plants and provide stress tolerance) and others will be vital to develop corn lines resistant to aflatoxin contamination through conventional or molecular (use of modern biotechnological methods) breeding (Objective 3) as detailed below.

Under Objective 1, we identified several corn genes linked to resistance to aflatoxin contamination by comparing levels of gene activation in susceptible and resistant corn varieties following infection with A. flavus. Gene activation analysis revealed that many of the corn genes, linked to aflatoxin resistance, were differentially activated. One of the genes that was highly activated upon infection in a resistant corn line was identified as nepenthesin-1, which is responsible for the production of an enzyme capable of degrading proteins. This gene was not activated during A. flavus infection of a susceptible corn line. Therefore, it is a promising candidate resistance gene against A. flavus infection and aflatoxin contamination.

Also in support of Objective 1 and in collaboration with scientists at Louisiana State University, Baton Rouge, Louisiana, we used a comprehensive set of genetic analyses to identify specific genes and gene regions in corn DNA that are linked to the ability to respond to A. flavus infection. These genetic analyses identified 14 corn genes that are activated only under A. flavus infection. Genes involved in cell wall biosynthesis, degradation and production of seed-specific proteins were found to be significantly associated with aflatoxin resistance. Results from these analyses led to the identification of corn genes and DNA makers. These can be used to develop aflatoxin resistant maize varieties demonstrating enhanced resistance to A. flavus growth and aflatoxin contamination.

In support of Objective 1 and in collaboration with scientists at Louisiana State University, Baton Rouge, Louisiana, we isolated small RNAs (ribonucleic acids) from kernels of an aflatoxin resistant, a moderately resistant, and a susceptible corn lines at different time points following infection with A. flavus. Analysis of the small RNAs identified 39 microRNAs (miRNAs; 19-25 nucleotide long RNAs that target specific genes and regulate their activation), and these were differentially activated in the three corn lines in response to the fungal infection. Of the 39 miRNAs identified, nine had not been previously reported in corn and thus were considered new and unique. Analysis of the level of activation of miRNAs between the three corn lines suggested the involvement of nine miRNAs that were specifically activated in the resistant corn line. These miRNAs targeted corn genes that are mostly involved in pathogen recognition and defense mechanisms. The miRNAs and target genes identified through this effort will be used for further experimental evaluation of their specific role in aflatoxin resistance. 

In Objective 2, we continued to evaluate the roles of native corn chemicals such as, flavonoids, provitamin A (or carotenoids) and polyamines, in reducing A. flavus growth and aflatoxin contamination. We demonstrated that flavonoids produced by plants at low concentrations can alter the composition and integrity of the fungal cell wall. Flavonoids were also found to be taken up into the fungus in the cell wall resulting in reduced aflatoxin production, by a yet to be determined mechanism. We have previously determined that the role of polyamines in plant disease resistance depends on the relative abundance of complex polyamines (such as, spermidine and spermine versus simpler diamines and breakdown products). Experiments designed to increase the levels of complex polyamines in corn by over-activation of the gene responsible for making the enzyme that produces the polyamine are in progress. We anticipate that this will result in corn variety that is resistant to A. flavus and aflatoxin contamination.

Progress in Objectives 1 and 2 and from the sibling project 6054-41420-009-000D ARS paves the way for making continued, significant progress in molecular breeding of corn for resistance to A. flavus and aflatoxin contamination under Objective 3. Under the Objective 3, we continued our corn transformation (introducing foreign genes into corn) work that inserts a known antifungal gene(s) in corn. For example, genes for two antifungal synthetic peptide (small proteins), which demonstrated broad spectrum antimicrobial activity, were introduced into corn.  This work is being performed in collaboration with Genvor LLC (Agreement #58-6054-8-010). Resistance to aflatoxin contamination will be determined in these corn lines. In another example, several key A. flavus genes responsible for growth, infection, and toxin production were selectively turned off in corn plants using a technique called ribonucleic acid interference (RNAi). Here, we have successfully generated transgenic RNAi corn lines with the innate ability to turn off key fungal genes needed for growth and aflatoxin production resulting in significant aflatoxin reduction in transgenic kernels. Recently in collaboration with scientists in Louisiana State University, Baton Rouge, Louisiana, we conducted a field test using RNAi corn lines capable of silencing a fungal polygalacturonase gene (p2c; also known as pectin depolymerase or pectinase). The pectinase is needed by the fungus to break down plant pectin (a major component of plant cell walls) during colonization of the plant. Transgenic RNAi plants with inactive fungal pectinase demonstrated significant resistance to aflatoxin contamination under field conditions. The resistance trait was transferred to elite corn lines by breeding and the progeny showed a significant reduction (up to 94%) in aflatoxin contamination. In the third example, we demonstrated previously the critical roles of corn polyamines in resistance to fungal growth and toxin production. To engineer corn for greater production of polyamines for resistance to aflatoxin contamination, we introduced a gene responsible for the over production of an enzyme that makes polyamines. These transgenic corn lines will be evaluated after greenhouse multiplication.

In addition to developing corn plants resistant to aflatoxin contamination, we have also developed a non-invasive, inexpensive and rapid hyperspectral (a technique that analyzes a wide spectrum of light) and multispectral (capturing the reflection of light energy in the environment) imaging techniques that detect and quantify pre- and post-harvest aflatoxin contamination in corn kernels under Objective 4. This work is in collaboration with Geosystems Research Institute of Mississippi State University in Mississippi State, Mississippi, (Agreement No. 58-6054-8-009). A spectral signature (the variation of reflectance or emittance with respect to wavelengths) to detect aflatoxin -contaminated corn kernels has been developed and licensed. We also employed shortwave near infrared (SWIR) hyperspectral imaging (wavelength range of 1,000 – 2,500 nm) and Raman hyperspectral imaging with a 785 nm line laser to characterize the spectral signatures of aflatoxin contaminated corn kernels. Using these methods, we were able to distinguish corn kernels inoculated with aflatoxin -producing or non- aflatoxin -producing strains of A. flavus under both laboratory and field conditions. Recently, a low-cost, tablet-based portable detection system (costing less than $200) equipped with a UV light source was developed and successfully tested with contaminated, field-grown corn samples. The detailed description of the detection system has been published. Recently, the detection system has been upgraded to use battery power with solar charge capability for use in remote regions. **Accomplishments**
                                

1. Silencing a fungal gene to reduce aflatoxin contamination in corn kernels. The fungus, Aspergillus (A.) flavus produces a toxic and potent carcinogen known as aflatoxin during growth on corn and other food crops resulting in hundreds of millions of dollars in crop loss annually, and imposes globally a severe health risk to vulnerable populations. The consumption of aflatoxin-tainted food and feed crops can lead to liver cancer, stunted growth in children, and eventually death. Currently no single method of aflatoxin remediation has proven fully successful, thus the development of pre-harvest technologies is vital in reducing the effects of this toxigenic fungus. ARS scientists at New Orleans, Louisiana, demonstrated it is possible to reduce A. flavus growth and aflatoxin contamination in corn using a technology known as RNA-interference (RNAi). RNAi-mediated process can selectively turn off specific fungal gene(s) during infection so that the fungal growth and/or toxin production is destroyed. In collaboration with scientists at Louisiana State University, Baton Rouge, Louisiana, we exploited this natural plant defense system by generating corn plants that produces RNAi designed to target and degrade fungal RNAs necessary to produce a key enzyme, p2C (pectinase or polygalacturonase). The p2C enzyme is needed for fungal colonization of corn kernels and corn plants with the RNAi to turn off p2C resulted in significant reduction of fungal growth and aflatoxin levels (up to 94%). Further, since this method involves action at RNA level and does not result in production of foreign proteins in crops. As such, public concerns regarding GMOs is greatly reduced. The resistance trait due to silencing of fungal p2C has been successfully transferred to elite corn lines which also demonstrated reduced aflatoxin levels under field conditions. 2. A low cost portable device to detect aflatoxin contamination. The fungus, Aspergillus flavus (A. flavus) produces a toxic and potent carcinogen known as aflatoxin during growth on corn and other food crops. Aflatoxin contamination of corn imposes a severe health risk to vulnerable populations around the world. The consumption of aflatoxin-contaminated crops can lead to liver cancer, stunted growth in children, and eventually death. ARS researchers in New Orleans, Louisiana, in collaboration with scientists at Mississippi State University, Starkville, Mississippi have developed a table-top or tablet-based, low-cost (less than $200) portable detection system. This device was successfully tested with A. flavus infected and aflatoxin-contaminated field-grown corn samples. The detection system uses battery power with solar charge capability thus improving its utility in remote regions. This portable device will be extremely useful for small farmers and households in Africa or Asia to screen their stored kernels (or peanuts) for detection and elimination of contaminated kernels prior to cooking or consuming. 3. Prediction models for mycotoxin outbreaks using artificial intelligence (AI) and machine learning (ML). In response to suggestions from the National Corn Growers Association, ARS researchers in New Orleans, Louisiana, in collaboration with the National Program Leader, Food and Feed Safety, Beltsville, Maryland, ARS scientists in Peoria, Illinois, and State Department of Agriculture, Illinois have employed artificial intelligence and machine learning models to predict mycotoxin contamination levels in corn, including aflatoxin (a toxin produced by the fungus-Aspergillus) and fumonisin (a toxin produced by the fungus-Fusarium). Computational, mathematic models were developed using extensive correlation analyses of more than twenty years of mycotoxin contamination data as influenced by the then prevailing historic weather parameters, geospatial and soil physical properties, and satellite-derived vegetation data and land usage. The prediction models, when applied to Illinois-grown corn, demonstrated an accuracy of 95% in forecasting mycotoxin contamination in corn. Currently, in response to requests from other corn-growing states, the models are being trained to suit their respective states as well, one at a time. These models will be extremely useful to predict possible mycotoxin outbreak based on live feed of prevailing influential factors in any given year and help the farmers and stakeholders in the corn industry an advance warning on impending contamination so that timely execution of suitable mitigation measures is possible. **Review Publications**
                            *Castano-Duque, L., Lebar, M.D., Carter-Wientjes, C., Ambrogio, D., Rajasekaran, K. 2022. Flavonoids modulate Aspergillus flavus proliferation and aflatoxin production. The Journal of Fungi. 8(1):1211. https://doi.org/10.3390/jof8111211.*
                                *Yao, H., Hruska, Z., Kincaid, R., Tao, F., Rajasekaran, K. 2023. Effect of Aspergillus flavus fungi infection and aflatoxin contamination on single corn kernel mechanical strength. Applied Engineering in Agriculture. 39(2):197-205. https://doi.org/10.13031/aea.15266.*
                                *Castano-Duque, L., Vaughan, M., Lindsay, J., Barnett, K., Rajasekaran, K. 2022. Gradient boosting and bayesian network machine learning models predict aflatoxin and fumonisin contamination of maize in Illinois – First USA case study. Frontiers in Microbiology. 13. Article 1039947. https://doi.org/10.3389/fmicb.2022.1039947.*
                                *Sengupta, S., Pehlivan, N., Mangu, V., Rajasekaran, K., Baisakh, N. 2022. Characterization of a stress-enhanced promoter from the grass halophyte, Spartina alterniflora L. Biology. 11(12):1828. https://doi.org/10.3390/biology11121828.*
                                *Yao, H., Zhu, F., Kincaid, R., Hruska, Z., Rajasekaran, K. 2023. A low-cost, portable device for detecting and sorting aflatoxin-contaminated maize kernels. Toxins. 15:197. https://doi.org/10.3390/toxins15030197.*
                                *Raruang, Y., Omolehin, O., Hu, D., Wei, Q., Promyou, S., Parekattil, L.J., Rajasekaran, K., Cary, J.W., Wang, K., Chen, Z.-Y. 2023. Targeting the Aspergillus flavus p2c gene through host-induced gene silencing reduces A. flavus infection and aflatoxin contamination in transgenic maize. Frontiers in Plant Science. 14:1150086. https://doi.org/10.3389/fpls.2023.1150086.*
                                *Prasad, K., Yogendra, K., Sanivarapu, H., Rajasekaran, K., Cary, J.W., Sharma, K.K., Bhatnagar-Mathur, P. 2023. Multiplexed host-induced gene silencing of Aspergillus flavus genes confers aflatoxin resistance in groundnut. Toxins. 15:319. https://doi.org/10.3390/toxins15050319.*
                                *Baisakh, N., Da Silva, E.A., Pradhan, A.K., Rajasekaran, K. 2023. Comprehensive meta-analysis of QTL and gene expression studies identify candidate genes associated with Aspergillus flavus resistance in maize. Frontiers in Plant Science. 14:1214907. https://doi.org/10.3389/fpls.2023.1214907.*


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