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ARS Home » Southeast Area » New Orleans, Louisiana » Southern Regional Research Center » Food and Feed Safety Research » Research » Research Project #430862

Research Project: Use of Classical and Molecular Technologies for Developing Aflatoxin Resistance in Crops

Location: Food and Feed Safety Research

2021 Annual Report

Objective 1. Develop aflatoxin-resistant corn with enhanced resistance traits against other mycotoxins and drought tolerance. Identify gene regulatory factors, networks and pathways related to resistance-associated proteins (RAPs). These data are then transferred to others to assist in selection by marker-assisted breeding. Objective 2. Identify resistance associated protein (RAPs) genes from corn and cotton using transcriptomic analyses of the Aspergillus flavus-host plant interaction and evaluate for control of fungal growth and aflatoxin contamination. Objective 3. Develop and evaluate transgenic corn and cotton containing over-expressed identified RAP genes (Objectives 1 and 2) or with RNA interference (RNAi)-based silencing of Aspergillus flavus genes critical to growth and aflatoxin production. Objective 4. Advance and license the rapid, non-destructive hyperspectral imaging technology; develop and evaluate instruments suitable for different user platforms.

Aflatoxin contamination in crops such as corn, cottonseed, peanut, and tree nuts caused by Aspergillus (A.) flavus is a worldwide food safety problem. Aflatoxins are potent carcinogens and cause enormous economic losses from destruction of contaminated crops. Utilizing resistant germplasm against A. flavus growth and aflatoxin contamination is the most practical solution for pre-harvest control, the overall goal of this project plan. To this end, we plan to elucidate the complex, multi-genic resistance mechanisms in corn identified in resistant genotypes bred through a collaborative program. We will understand the molecular basis of seed-based resistance through transcriptomic analysis of the corn-A. flavus interaction allowing identification of genes and networks correlated with resistance for use in marker-assisted breeding. RNA interference technology will be used to a) determine the roles and contribution of selected corn genes to overall resistance; and b) to target genes critical to A. flavus growth and toxin production to generate corn varieties with enhanced resistance. Resistance genes identified from transcriptomic analysis of the A. flavus-cottonseed interaction, along with identified corn resistance genes will be over-expressed in cotton to achieve enhanced resistance. Finally, instrumentation for non-destructive, hyperspectral imaging detection will be refined and modified to address practical applications suitable for different user-specified platforms. The proposed research will result in development of cotton and corn germplasm with enhanced resistance to A. flavus growth and aflatoxin contamination. Information and material generated from this research will benefit the scientific community, stakeholder groups, food and feed safety regulatory agencies and consumers, both nationally and internationally.

Progress Report
This is the final report for the Project 6054-42000-025-00D terminated in April 2021, which has been replaced by new Project 6054-42000-027-00D. For additional information, see the new project report. Significant progress has been made by ARS scientists in New Orleans, Louisiana in all four objectives of the project, all of which fall under National Program 108 Food Safety, Component 1, Foodborne Contaminants. One of the best means of combating aflatoxin contamination is through development of resistant corn lines through classical or molecular breeding. Objective 1, ARS researchers in New Orleans, Louisiana, have identified several proteins in corn kernels linked with enhanced resistance to infection by the aflatoxin (AF, a carcinogen)-producing fungus, Aspergillus (A.) flavus. These genes have been transferred into commercial varieties by classical breeding. In collaboration with the International Institute of Tropical Agriculture, Nigeria, ARS researchers produced six corn varieties that were deposited into the ARS repository and made available to other researchers. These six lines were used to develop new elite hybrids with AF resistance in several African countries and in the United States. They are available to all domestic and international breeders. Objective 2, ARS researchers in New Orleans, Louisiana, in collaboration with the J. Craig Venter Institute, La Jolla, California, used modern RNA-Sequencing (RNA-seq, a means of determining levels of activity of individual genes in both the fungus and corn) technology to study the expression of genes during the corn-A. flavus interaction. For this work, two resistant and one susceptible corn lines were evaluated. Comparative analysis indicated several novel corn genes and biological processes related to resistance to fungal infection and AF contamination. To further identify novel genes of interest, ARS scientists incorporated genomic and field data from a genome wide association analysis (GWAS) of corn varieties with gene expression data. These efforts identified significant association between flavonoid (plant nutrients with strong antioxidant and defense capabilities) genes and AF contamination. One of the resistant lines has a higher production rate of the flavonoid metabolite called naringenin than susceptible line suggesting the important roles flavones play in fungal growth and AF production. Non-coding small RNAs (ncRNAs, do not encode for proteins) present in corn lines were also sequenced and analyzed by ARS scientists in New Orleans, Louisiana in collaboration with researchers at Louisiana State University, Baton Rouge, Louisiana to identify microRNAs (miRNAs, a class of ncRNAs that can control the expression of specific genes) with potential roles in A. flavus resistance. Differences in miRNAs were observed between resistant and susceptible corn lines. In summary, candidate genes and small RNAs with positive roles in corn resistance against A. flavus have been identified. The value of these genes in marker-assisted breeding will be further investigated. Using the information from Objectives 1 and 2 and from the sibling project 6054-41420-008-00D ARS researchers in New Orleans, Louisiana, continued to make significant progress in molecular breeding of corn for resistance to A. flavus and AF contamination under Objective 3. a) Transgenic corn kernels expressing a synthetic peptide gene demonstrated a significant reduction in fungal growth and AF contamination reduction. Field evaluation of these corn lines in 2021 was discontinued because of excessive heat and rain in the test site. Corn was also transformed with a gene from another plant that encodes a novel antifungal protein. This antifungal protein inhibits a key enzyme (alpha-amylase) that is necessary for the fungus to grow and infect seed. (b) To understand the contribution of a corn kernel protein to A. flavus resistance, it was silenced using a ribonucleic acid interference (RNAi, a technology that enables specific genes to be targeted for down-regulation or silencing) approach in transgenic corn lines. This gene also affected the functioning of other genes involved in disease resistance. Similar RNAi-based approaches were carried out to silence fungal genes that are critical for the fungus to grow, infect and produce toxins. Several corn lines capable of shutting down fungal genes that control growth such as a-amylase and p2c (pectinase), AF biosynthetic genes or global regulators such as aflR, aflS, aflM, aflC, veA and nsdC showed significant AF reduction in transgenic kernels. Two of these lines (aflM and p2c) also showed significant resistance to AF contamination under field conditions. A significant reduction (up to 98%) was observed of AFs in select transgenic corn lines expressing foreign genes or that are capable of silencing fungal genes. Additional corn transformation experiments were not possible due to shut down of service provides during pandemics. (c) Unlike in corn, no resistance to A. flavus has been identified in cotton seed sock so far. ARS scientists in New Orleans, Louisiana, in collaboration with scientists at the University of Louisiana-Lafayette assayed several varieties of cotton to identify natural resistance to A. flavus and AF accumulation. Upon screening for any innate resistance to the fungus, old-world cotton varieties of Gossypium (G.) arboreum were found to be most resistant and commercially cultivated upland cotton varieties were all susceptible to A. flavus infection. (d) Fatty acid accumulation in developing cottonseed was well-correlated with the ability of an AF-producing fungal strain to grow and produce AFs. This study identified key factors controlling A. flavus infection and AF production in cottonseed. (e) Transgenic cotton lines expressing an antifungal synthetic peptide designated D4E1 demonstrated resistance to A. flavus in greenhouse studies or seedling pathogens under field conditions. They were field-tested by ARS scientists in collaboration with University of Arizona for resistance to AF contamination. Results from the small field experiment were inconclusive due to lack of natural A. flavus infection and AF contamination. (f) New experiments to produce transgenic corn lines expressing improved synthetic peptides with potency against A. flavus have also been initiated by ARS researchers in New Orleans, Louisiana under a cooperative agreement. ARS researchers, in collaboration with Louisiana State University, Baton Rouge, Louisiana, also identified a key gene that is overexpressed in cotton boll pericarp (the outer wall) called spot11 catalase from RNA-seq analyses (Objective 2). Due to pandemics, limited number of transgenic cotton lines expressing this gene were regenerated for further analyses on resistance to fungal infection and AF contamination. Several cultures were also lost during maximized telework. (g) ARS researchers have also demonstrated the critical roles in fungal growth and toxin production by polyamines (PAs), which are ubiquitous nitrogenous molecules that control growth and development of plants under biotic (pest) and abiotic (drought, salt) stress. First, inactivation of a key fungal gene, spermidine synthase (Spds), was demonstrated to reduce fungal growth, pathogenicity, and aflatoxin production in corn kernels. In addition, analysis of maize genotypes susceptible or resistant to Aspergillus flavus identified a key gene (S-adenosylmethionine decarboxylase or SAMDC) that regulates the production of higher PAs, possibly contributing to fungal resistance. The fungal and plant genes identified from this work provide potential targets for improvement of maize resistance to fungal colonization and aflatoxin production. 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, Mississippi, developed a non-invasive, inexpensive and rapid imaging technique that collects and processes information from across the light-spectrum under Objective 4 to detect and quantify AFs in corn kernels. These special cameras have already demonstrated the ability to differentiate toxigenic and atoxigenic Aspergillus flavus strains. A patented method to analyze different wavelengths was initially used. Subsequently, a joint effort between ARS researchers in New Orleans, Louisiana, Mississippi State University and a collaborator in Martin, Tennessee, was established to develop a novel rapid method to improve AF detection by a dual-camera imaging system. This system was successfully tested with fungal-inoculated commercial corn in the United States. With support from the United States Agency for International Development (USAID), ARS researchers and collaborators perfected the concept into a low-cost portable device called AflaGoggles to detect AF contamination in corn kernels for use in developing countries. The device has now been updated to a tablet-based system equipped with UV-LED light source. The device employs an in-house developed Android App for image acquisition and detection of contaminated kernels for manual removal by the user. Following successful experiments in the United States on corn kernels, work is being continued to improve the accuracy of detection and the device’s portability for field deployment.

1. Corn plants shut down fungal genes and toxin production. Corn is an important food and feed crop, and it is highly susceptible to A. flavus infection and aflatoxin (a carcinogen) contamination. Transgenic corn plants were generated by ARS researchers in New Orleans, Louisiana, in collaboration with scientists at Louisiana State University, Baton Rouge, Louisiana, to selectively shut down one of the fungal genes (called p2c-polygalacturonase or pectinase) necessary for its growth, infection and spread. The transgenic corn lines with the capacity to silence p2c were tested in the laboratory and field for three years. Aflatoxin production was significantly reduced (60- 95%) corresponding to reduced levels of fungal growth (up to 40%). This technology, made possible by the short-lived silencing RNA molecules, does not require expression of a foreign protein in the plant so food produced from resistant transgenic lines of corn should be more acceptable to regulatory agencies and consumers. Corn plants carrying this gene will also serve as an excellent parent material to transfer the resistance trait to other commercial varieties. Aflatoxin-resistant corn lines improve food safety and security while minimizing economic losses borne by farmers.

2. Bacteria naturally found in corn kernels are associated with reduced toxin levels. ARS scientists in New Orleans, Louisiana, suggest a toxin, called aflatoxin, produced by the fungus Aspergillus (A.) flavus during growth on corn is a worldwide food safety problem. Aflatoxins are potent carcinogens that adversely impact human and animal health and contamination of crops with aflatoxins costing stakeholders tens of millions of dollars annually. Management of aflatoxin contamination in corn is complex so identification of any factors that contribute to kernel resistance is of great importance in developing control strategies. Using sophisticated DNA sequencing technologies, ARS researchers in New Orleans, Louisiana were able to identify specific groups of bacteria that were present in greater numbers in kernels of resistant corn lines compared to susceptible lines. Many of the identified bacteria are known to produce compounds that can stop the growth of toxin-producing fungi. Continued evaluation by ARS scientists in New Orleans, Louisiana of bacteria predicted to possess antifungal and anti-aflatoxigenic properties will aid in their development as effective agents for enhanced resistance to A. flavus infection and aflatoxin contamination thus ensuring a safer and more secure food supply.

Review Publications
Tao, F., Yao, H., Hruska, Z., Kincaid, R., Rajasekaran, K., Bhatnagar, D. 2020. A novel hyperspectral-based approach for identification of maize kernels infected with diverse Aspergillus flavus fungi. Biosystems Engineering. 200:415-430.
Majumdar, R., Kandel, S.L., Cary, J.W., Rajasekaran, K. 2021. Changes in bacterial endophyte community following aspergillus flavus infection in resistant and susceptible maize kernels. International Journal of Molecular Sciences. 22(7). Article 3747.