Location: Food and Feed Safety Research2021 Annual Report
Objective 1: Identify key genes and metabolites involved in fungal growth, toxin production and virulence during the Aspergillus flavus-corn interaction that can be used as targets for intervention strategies. Subobjective 1.A: Identify secondary metabolites produced by Aspergillus flavus during interaction with corn and characterize their structure, biosynthesis and contribution to the fungus’ ability to survive, colonize the crop and produce toxins. Subobjective 1.B: Identify key genes and gene networks using transcriptomic analysis of Aspergillus flavus and Aspergillus flavus-crop interaction that are involved in fungal growth, development, toxin production and virulence. Objective 2: In situ and in planta analysis of the impact of environmental stresses associated with predicted climate change on Aspergillus flavus biology and biocontrol. Subobjective 2.A: Analysis and functional characterization of genes differentially expressed in situ under altered environmental conditions. Subobjective 2.B: In planta assessment of fungal virulence and aflatoxin production. Objective 3: Identify volatile organic compounds (VOCs) and extrolites produced by non-aflatoxigenic Aspergillus flavus strains that reduce growth and/or toxin production in aflatoxigenic aspergilli and characterize their mechanism of action.
Aflatoxin contamination in crops such as corn, cottonseed, peanut, and tree nuts caused by Aspergillus flavus is a worldwide food safety problem. Aflatoxins are potent carcinogens and cause enormous economic losses from reduced value of contaminated crops. Biosynthesis of these toxins has been extensively studied, but much remains to be determined regarding how gene regulatory networks respond to the complex nutritional and environmental cues perceived by the fungus during colonization of the host crop. While transcriptomics has provided some insights into genes and gene networks that govern A. flavus development and aflatoxin production, very little is known about the role that fungal metabolites play in the infection process or during interactions with competing microbes in the field or on the crop. To address these knowledge gaps, we will use transcriptomics, metabolomics and bioassay to identify and functionally characterize fungal genes, gene networks and metabolites that are critical for fungal host colonization and aflatoxin production during interaction of A. flavus with corn. These analytical techniques will also be used to define how physiological stress (i.e. changing environmental conditions) affects fungal virulence and survival and how introduced non-aflatoxigenic A. flavus strains prevent native, aflatoxigenic strains from contaminating crops thus increasing the effectiveness of A. flavus biological control. We expect to utilize the fundamental knowledge gained from the proposed studies for the development, validation and implementation of targeted strategies (biological control and host-resistance) to significantly reduce pre-harvest aflatoxin contamination of crops intended for consumption by humans or animals.
This report documents progress for Project 6054-41420-009-00D, which started in April 2021. Due to the current project plan’s start date of April 2021, a period of only three months had passed at the time of writing of this annual report resulting in limited progress on research 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. Objective 1, ARS scientists in New Orleans, Louisiana, harvested seed from corn plants that had been infected with A. flavus to determine changes in fungal growth and gene expression as well as aflatoxin production. Kernels collected at 3 and 7 days are currently being analyzed to identify and quantify fungal and corn metabolites. The same ground samples will be used to analyze both fungal and corn gene expression. Using RNA-sequencing (RNA-seq; a technique that is used to measure the level of activation of genes) data, ARS scientists in New Orleans, Louisiana, have identified a number of genes that appear to play a significant role in A. flavus infection and aflatoxin contamination of corn. RNA-seq data showed that A. flavus genes involved in the production of four different cyclic peptides (small proteins) and a terpene (a chemical associated with plant oils) were activated upon infection of corn by the fungus. The four peptide genes and the terpene gene have been knocked out to determine role in the biology of the fungus. Additionally, results of RNA-seq analysis of the A. flavus-corn seed interaction has identified four genes from A. flavus that appear to be strongly associated with infection of corn. These four genes have been knocked out and will be further analyzed by ARS scientists at New Orleans, Louisiana for impact on the ability of the fungus to infect corn and produce aflatoxins. ARS scientists in New Orleans, Louisiana, in collaboration with scientists at Northern Illinois University have identified several genes that appear to be involved in diverse functions such as regulation of aflatoxin production as well as fungal development. These genes have been knocked out and assays will be performed to determine how they control fungal growth and aflatoxin production. Objective 2, experiments to determine novel changes in fungal gene expression, toxin and chemical production were performed by ARS scientists at New Orleans, Louisiana using corn plants grown in a greenhouse. Corn plant cobs were purposely contaminated with A. flavus and kernels will be collected at 3 and 7 days post-contamination for analyses. Additionally, preliminary corn plant experiments were done by growing corn in a chamber using 300 or 1000 ppm carbon dioxide (CO2). This preliminary study allowed determination of optimal growth and seed production conditions for the plants under the two CO2 treatments. Cobs of plants grown under the two CO2 treatments were infected with A. flavus and kernels were collected and kept at -80°C for future analyses. Objective 3, ARS scientists in New Orleans, Louisiana, have obtained several A. flavus isolates from Georgia, Arizona, Mississippi and Louisiana in preparation for studies on the role of fungal volatile organic compounds (gaseous compounds) and extrolites (secreted chemicals) on the ability of non-aflatoxin producing A. flavus strains to inhibit growth of aflatoxin producing strains.