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Research Project: Genetic and Environmental Factors Controlling Aflatoxin Biosynthesis

Location: Food and Feed Safety Research

2021 Annual Report

Objective 1. Identify key genes, using transcriptome analysis of Aspergillus flavus and Aspergillus flavus-crop interaction that are involved in fungal growth, morphogenesis, toxin production and virulence which can be used as targets for intervention strategies. Objective 2. Identify metabolites produced by predicted secondary metabolic gene clusters in Aspergillus flavus, characterize the molecular regulation of their biosynthesis, and determine if they contribute to the fungus’ ability to survive, colonizes host crops and produce aflatoxin. Objective 3. Examine the role of climatic and environmental pressures on the growth, virulence, toxigenic potential, geographical distribution and aflatoxin production by Aspergillus flavus.

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 the destruction of contaminated crops. While biosynthesis of these toxins has been extensively studied, much remains to be determined regarding regulatory factors, their interactions and gene networks that respond to environmental cues governing fungal development and aflatoxin production. Using an –omics approach (transcriptomics, interactomics, proteomics, metabolomics), fungal genes/proteins will be identified and functionally characterized that are critical for successful host plant colonization and aflatoxin production during interaction of A. flavus with the plant. Interactions of regulatory proteins involved in fungal growth and toxin production, such as AflR and other velvet (VeA)-dependent proteins with global regulators, will be examined to elucidate novel mechanisms governing aflatoxin production and fungal morphogenesis. We will also identify and characterize the biological roles of other secondary metabolites produced by A. flavus, their impact on aflatoxin production and food safety in general. Further, we will better define the molecular mechanisms affected by physiological stress (i.e. changing environmental conditions) to the fungus and plant. We expect to utilize the fundamental knowledge gained from the proposed studies for development of targeted strategies (biological control or host-resistance) to significantly reduce pre-harvest aflatoxin contamination of crops intended for consumption by humans or animals.

Progress Report
This is the final report for the Project 6054-41420-008-00D terminated in April 2021, which has been replaced by new Project 6054-41420-009-00D. For additional information, see the new project report. Significant progress was made by ARS scientists at New Orleans, Louisiana in all three objectives, which fall under National Program 108 Food Safety, Component 1, Foodborne Contaminants. To understand the preharvest aflatoxin (a toxic and carcinogenic compound) contamination process and develop effective aflatoxin mitigation strategies, it is important to understand the genetic make-up and the gene expression profile of the aflatoxin-producing fungus, Aspergillus (A.) flavus, under various environmental conditions (including changing climate), especially during interaction of the fungus with the host plant (for example, corn). Objective 1, ARS scientists in New Orleans, Louisiana, have made significant progress in identifying key genes from A. flavus that are involved in controlling the ability of the fungus to grow, infect crops and produce aflatoxins. ARS scientists used improved methods such as ribonucleic acid-sequencing (RNA-seq; a technique that is used to measure the level of activation of genes) and specifically looked at the level of expression of genes in the fungus during colonization of corn seed. These RNA-seq studies resulted in identification of several genes that regulate of A. flavus growth, virulence and aflatoxin production. ARS scientist will try to inactivate the A. flavus spermidine synthase gene (spds; needed to make a chemical involved in fungal virulence and aflatoxin production) using a strategy known as host-induced gene silencing. Evaluation of RNA-seq data led to the identification of a number of fungal genes whose expression correlated with active infection of corn seed. These included genes responsible for the production of proteins termed transcription factors that serve as key regulators of fungal growth and toxin production. Additionally, a number of genes involved in the production of peptide (a small protein) and terpene (a class of compounds often associated with plant essential oils) secondary metabolites (compounds not required for fungal growth but are needed for virulence and survival and can often be toxic to other organisms) were also shown to be highly expressed during infection of corn seed by the fungus and are believed to play a role in the ability of the fungus to successfully invade seed tissues. Genes responsible for production of two of the peptide compounds produced during infection of corn have been knocked out to better understand their biological role in the fungus. Using comparative RNA-seq of infected kernels from corn lines susceptible or resistant to A. flavus infection and aflatoxin contamination, ARS scientists have identified and are studying four fungal genes that are expressed at a higher level during infection of resistant lines compared to a susceptible line and therefore may be responsible in part for the ability of the fungus to infect corn. Objective 2, ARS scientists have performed a number of experiments to identify other chemicals (not aflatoxin) produced by A. flavus during infection of corn. These studies led to the identification of aspergillic acid and ferriaspergillin (both toxic compounds) which play a role in the ability of A. flavus to infect corn. Additional studies led to the development of a more sensitive assay for detection of aspergillic acid and similar compounds in plant tissues such as corn seed. Further, ARS scientists in New Orleans, Louisiana, have identified a gene cluster that so far has only been found in a single A. flavus strain whereas most gene clusters are commonly found in all A. flavus strains. In a collaboration with scientists at the University of California Los Angeles, ARS scientists want to determine if the presence of this unique gene cluster provides the fungus with the ability to compete more successfully for infection of corn. A number of small molecules that inhibit histone modifying enzymes (proteins that can modify amino acid residues associated with chromosomal DNA termed histones which in turn can impact gene expression) were analyzed to assess their effects on fungal secondary metabolism. Aflatoxin-producing A. flavus strains were grown in the presence of the inhibitors and metabolic extracts have been prepared. Data acquisition and analysis is being performed by ARS scientists to determine if addition of the inhibitors leads to production of novel secondary metabolites that normally are not produced by the fungus during growth on standard nutrient-based growth media. Objective 3, ARS scientists related to predicted climate change conditions such as low moisture, high temperature, and elevated CO2 levels on the ability of A. flavus to produce aflatoxins and other toxic metabolites during infection of corn kernels. The data indicate that a combination of dry conditions and high CO2 levels resulted in earlier than normal production of aflatoxins in A. flavus during infection of individual corn kernels germinating in an incubator. Further, analysis of gene expression data of A. flavus growing under differing CO2 levels identified several genes that are potentially responsible for controlling the expression of fungal genes associated with response to higher CO2 levels. To better understand the impact of altered CO2 levels on the ability of A. flavus to infect and produce aflatoxins during growth on corn cobs, ARS scientists modified a large walk-in plant growth chamber that now allows us to grow corn to maturity under altered temperature, moisture and CO2 levels. This enables scientists to conduct A. flavus infection of developing corn plants under environmental conditions simulating predicted climate change that cannot be simulated during infection of individual corn kernels. Using this chamber scientists have inoculated kernels present on ears of corn with A. flavus under conditions of 350 ppm CO2 (roughly the current global level of atmospheric CO2) or at 1000 ppm (levels predicted to be present at the end of this century). After 3 days of infection, individual seeds were harvested, flash frozen in liquid N2 and stored at -80 for later analysis of gene expression in the fungus as well as determine the level of aflatoxin being produced. The overall impact of the research arising from these three objectives is to support our biological control research and to transfer basic information to our Host Resistance project for application in the development of improved host plant aflatoxin resistance strategies. In collaboration with ARS scientists in Peoria, Illinois, Maricopa, Arizona, and scientists at Wageningen University, Food Safety Research (WFSR) in The Netherlands, ARS scientists at New Orleans, Louisiana will attempt to develop a mobile application with a predictive model and decision support system to aid farmers in determining if environmental conditions will be conducive to aflatoxin/fumonisin contamination of their corn in the field and decide on best practices that can be applied to mitigate contamination.

1. Common bacteria found in corn kernels may reduce levels of toxic compounds. ARS scientists at New Orleans, Louisiana, believe 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 costs stakeholders tens of millions of dollars annually. Management of aflatoxin contamination in corn is difficult so understanding what contributes to kernel resistance is of great importance in developing control strategies. Using sophisticated DNA sequencing technologies, ARS scientists in New Orleans, Louisiana, were able to identify groups of bacteria that were naturally present in greater numbers in kernels of resistant corn lines compared to non-resistant lines. Continued evaluation 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.

2. Genetic switches turn off production of a deadly fungal toxin. ARS scientists at New Orleans, Louisiana, suggests the fungus, Aspergillus (A.) flavus, produces a toxic and carcinogenic family of compounds known as aflatoxins during growth on crops such as corn. Consumption of aflatoxin contaminated corn can lead to adverse health effects in humans and animals such as immunosuppression, stunting of growth in children and liver cancer. It is important to understand the complex genetic mechanisms that govern the fungus’ ability to infect corn and produce aflatoxin to develop effective aflatoxin control strategies. Using sophisticated molecular techniques, ARS scientists have found several novel genes that function as key genetic “switches” capable of controlling A. flavus growth and aflatoxin production. Information gained from these studies is being used to develop corn with enhanced resistance to A. flavus infection and aflatoxin contamination which will positively impact our stakeholders and the general public in the form of increased food safety and security.

Review Publications
Chang, P.-K., Chang, T.D., Katoh, K. 2020. Deciphering the origin of Aspergillus flavus NRRL21882, the active biocontrol agent of Afla-Guard®. Letters in Applied Microbiology.
Chang, P.-K. 2021. Authentication of Aspergillus parasiticus strains in the genome database of the National Center for Biotechnology Information. BMC Research Notes. 14:111.
Gebru, S.T., Mammel, M.K., Gangiredla, J., Tartera, C., Cary, J.W., Moore, G.G., Sweany, R.R. 2020. Draft genome sequences of 20 Aspergillus flavus isolates from corn kernels and cornfield soils in Louisiana. Microbiology Resource Announcements. 9(38):e00826-20.
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.
Fountain, J.C., Clevenger, J.P., Nadon, B.D., Youngblood, R.C., Chang, P., Starr, D., Wang, H., Wiggins, R., Kemerait, R.C., Bhatnagar, D., Ozias-Akins, P., Varshney, R.K., Scheffler, B.E., Vaughn, J.N., Guo, B. 2020. Two new Aspergillus flavus reference genomes reveal a large insertion potentially contributing to isolate stress tolerance and aflatoxin production. Genes, Genomes, and Genomics. 10(9).