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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** 2022 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 and evaluate different spectral-based imaging instruments for non-destructive detection of aflatoxin contamination. Advance and commercialize hyperspectral-based, rapid and non-invasive, imaging technology. **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. 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 (AF, a toxic and carcinogenic compound) contamination process and develop effective AF mitigation strategies. To accomplish this, it is important to identify and evaluate native corn resistance genes (Objective 1) and corn secondary metabolites that are natural products not involved in normal growth and development (Objective 2). Key fungal genes responsible for AF production will be derived from the results of the sister project 6054-41420-009-00D - “Aflatoxin control through identification of intrinsic and extrinsic factors governing the Aspergillus (A.) flavus-corn interaction”. Combined together, availability of candidate genes and metabolites will be vital to develop corn lines resistant to AF contamination through conventional or molecular breeding (Objective 3) as detailed below.

Under Objective 1, ARS researchers at New Orleans, Louisiana, collected kernels from corn plants after infection with A. flavus at 3 and 7 days to identify corn genes highly activated during the plant-fungal interaction. Same kernel samples were also analyzed to identify and quantify levels of unique chemicals (or metabolites) in the kernels under Objective 2. Ribonucleic acid (RNA) isolated from the infected corn kernels has been used to see which genes were active. Several genes from both corn and A. flavus were found to be active. To further identify novel plant genes of interest, we incorporated gene expression and field data from a genome-wide association study (GWAS), which allowed us to compare the presence of flavonoids (compounds in corn that provide color and have antioxidant effects ) in correlation with A. flavus infections. We found that the flavonoids helped the corn fight off the infection. We are now conducting greenhouse experiments under controlled conditions with specific corn varieties that have different types and levels of flavonoid content. We expect to see differences between resistant and susceptible varieties with regard to fungal infection and flavonoid content, which will confirm the results of our previous study. Roles of other plant chemicals or metabolites such as provitaminA (or carotenoids) and polyamines (PAs), which are ubiquitous nitrogenous molecules that control growth and development of plants, are also being evaluated in reducing A. flavus contamination in Objective 2. Kernel samples from greenhouse or laboratory experiments have been sent to North Carolina State University for detection and identification of unique proteins in corn kernels after A. flavus infection. For precise analysis of these secondary metabolites produced by corn kernels in response to A. flavus infection, we recently bought and installed a sophisticated high resolution mass spectrometer. This sensitive instrument is now being calibrated by us to detect metabolites at very low levels. We have also procured kernels of several of corn varieties with various levels of carotenoids from collaborators at the International Institute for Tropical Agriculture (IITA) in Nigeria for further evaluation and analyses.

Using the information from Objectives 1 and 2 and from the sibling project 6054-41420-009-000D ARS continued to make significant progress in molecular breeding of corn for resistance to A. flavus and AF contamination under Objective 3. For example, (a) we screened ten antifungal synthetic peptides (small proteins) from a collaborating company [Agreement #58-6054-8-010], for effectiveness against toxin-producing A. flavus strains. Two of the peptides were highly effective relative to other peptides in inhibiting fungal growth. Transformation of corn lines with genes encoding these two peptides has been completed and transgenic lines are being regenerated. These two peptides demonstrated antifungal activity against several other microbial pathogens as well. (b) Several key A. flavus genes responsible for growth, infection and toxin production were shut down in corn plants using ribonucleic acid interference (RNAi, a technology that enables specific genes to be targeted for down-regulation or silencing). So far, we have successfully demonstrated this approach so that transgenic RNAi corn lines can shut down key fungal genes needed for growth and AF production resulting in significant AF reduction in transgenic kernels. Recently we conducted a field  test using corn lines with the capacity to silence a fungal alkaline protease (enzymes that break down proteins) gene (alk) and showed significant resistance to AF contamination under field conditions. We also demonstrated that the resistance to AF contamination is directly associated with the silencing of fungal alk gene. Additional transgenic corn lines capable of silencing other fungal genes have been self-pollinated to advance generations. (c) We also demonstrated the critical roles of polyamines in fungal growth and toxin production. First, inactivation of a key fungal PA gene, spermidine synthase (Spds), was demonstrated to reduce fungal growth, infection, and AF production in corn kernels. In addition, comparisons of maize genotypes susceptible or resistant to A. flavus identified a key gene (S-adenosylmethionine decarboxylase or SAMDC) that regulates the production of higher PAs, possibly contributing to fungal resistance. We have recently transformed corn with SAMDC gene for whole plant or seed-specific expression to evaluate its contribution towards resistance to fungal colonization and AF production.

To control AF in corn under pre- and post- harvest conditions, ARS scientists in New Orleans, Louisiana, collaborated with a collaborator [Agreement #58-6054-2-002], and showed that treatment with dead or live cells of a non-toxic marine bacterium, Vibrio gazogenes, can significantly reduce AF production by A. flavus. They have also shown that the active agent from the bacterium responsible for the reduction in AF production is a red pigment called prodigiosin. Growth of A. flavus on artificial nutrient medium supplemented with various levels of pure prodigiosin showed that AF production can be inhibited up to 99% at a very low concentration of 4μg/mL. Growth of A. flavus on corn seed imbibed prodigiosin resulted in significant inhibition of AF contamination; however, there was little to no significant reduction in growth of the fungus. In a recent greenhouse experiment we treated corn cobs with various concentrations of pure prodigiosin and then infected with A. flavus. Fungal growth and AF assays are currently underway on kernels isolated from the infected ears.

In addition to developing crops resistant to AF contamination, ARS researchers in New Orleans, Louisiana, in collaboration with Geosystems Research Institute of Mississippi State University (MSU) in Mississippi State, Mississippi, based at the Stennis Space Center, Hancock county, Mississippi, developed a non-invasive, inexpensive and rapid hyperspectral and multispectral imaging technique under Objective 4 to detect post-harvest contamination of corn kernels. This imaging technique detects and quantifies AFs in corn kernels. Hyperspectral instruments have already demonstrated the ability to differentiate toxigenic and atoxigenic A. flavus strains. A spectral signature to detect AF-contaminated corn has been developed and licensed. To advance rapid and non-destructive spectral-based contamination detection, research was continued with shortwave near infrared (SWIR) hyperspectral imaging in the wavelength range of 1,000 – 2,500 nm and Raman hyperspectral imaging with a 785 nm line laser to further characterize the spectral signatures of AF contaminated corn kernels. A total of 900 kernels were inoculated with water (as controls), an AF-producing A. flavus strain, and a non-AF-producing A. flavus strain. Kernel fungal infection and AF contamination were analyzed with the SWIR and Raman imaging techniques. A tablet-based portable detection system equipped with a UV-LED light source and an in-house developed software app was completed. Contamination detection and sorting with the device has been completed using field corn in the US. Continuing work aims to improve the performance of the device and its portability for practical, real-world application. To improve its ability for deployment in remote regions where power supply is limited, the detection system has been upgraded to use battery power with solar charge capability. To improve manual sorting efficiency based on the detection results, the original sample tray has been replaced with a new gridded sample holder where each corn kernel can be easily identified and sorted. **Accomplishments**
                                

1. Gene silencing results in reduction in aflatoxin contamination. Aspergillus (A.) flavus is a mold that infects corn and other susceptible crops and subsequently contaminates them with aflatoxin. Aflatoxins are potent carcinogens that adversely impact human and animal health worldwide.Additionally, contamination of crops with aflatoxin costs tens of millions of dollars annually due to economic losses from the devaluation or destruction of contaminated crops. ARS scientist in New Orleans, Louisiana, had previously shown that an enzyme called alkaline protease is highly expressed by the fungus during the infection of corn kernels. We showed, in collaboration with university scientists, that reducing the level of this fungal enzyme using a technique called host-induced gene silencing, caused an 87% reduction in toxin production in corn. This resistant trait was also transferred to three cultivated corn varieties by hybridization. This work will result in breeding of resistant varieties that can defend themselves against A. flavus. **Review Publications**
                            *Castano-Duque, L.M., Gilbert, M.K., Mack, B.M., Lebar, M.D., Carter-Wientjes, C.H., Sickler, C.M., Cary, J.W., Rajasekaran, K. 2021. Flavonoids modulate the accumulation of toxins from Aspergillus flavus in maize kernels. Frontiers in Plant Science. 12:761446. https://doi.org/10.3389/fpls.2021.761446.*
                                *Omolehin, O., Raruang, Y., Hu, D., Han, Z.-Q., Wei, Q., Wang, K., Rajasekaran, K., Cary, J.W., Chen, Z.-Y. 2021. Resistance to aflatoxin accumulation in maize mediated by host-induced silencing of the Aspergillus flavus alkaline protease (alk) gene. The Journal of Fungi. 7(11):904. https://doi.org/10.3390/jof7110904.*
                                *Tao, F., Yao, H., Hruska, Z., Rajasekaran, K., Qin, J., Kim, M.S. 2021. Use of line-scan Raman hyperspectral imaging to identify corn kernels infected with Aspergillus flavus. Journal of Cereal Science. 102:103364. https://doi.org/10.1016/j.jcs.2021.103364.*
                                *Tao, F., Yao, H., Hruska, Z., Kincaid, R., Rajasekaran, K. 2022. Near-infrared hyperspectral imaging for evaluation of aflatoxin contamination in corn kernels. Biosystems Engineering. 221:181-194. https://doi.org/10.1016/j.biosystemseng.2022.07.002.*
                                *Kandel, S.L., Jesmin, R., Mack, B.M., Majumdar, R., Gilbert, M.K., Cary, J.W., Lebar, M.D., Gummadidala, P.M., Calvo, A.M., Rajasekaran, K., Chanda, A. 2022. Vibrio gazogenes inhibits aflatoxin production through downregulation of aflatoxin biosynthetic genes in Aspergillus flavus. PhytoFrontiers. 2(3):218-229. https://doi.org/10.1094/PHYTOFR-09-21-0067-R.*


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