Location: Food and Feed Safety Research
2024 Annual Report
Objectives
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.
Approach
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.
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 process and develop effective aflatoxin mitigation strategies. To accomplish this, 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.
In support of Objective 1, ARS researchers at New Orleans, Louisiana
expanded gene knockout technique based on CRISPR/Cas9 technology to remove multiple clustered genes at the same time. “Knocking out” a gene means that it can no longer function. This technique has allowed for rapid generation of mutants missing large sections of their chromosomes, which was previously quite difficult and time consuming to do. We continued our research on the chemical and biological characterization of A. flavus genes believed to be involved in the production of unknown peptides (small, robust proteins termed RiPPs) that are produced by the fungus upon infection of corn. Of the five core RiPPs genes investigated using the CRISPR technology, one was shown to produce four novel peptides during infection. Several of the genes surrounding this core RiPP gene we also knocked out using CRISPR to aid in determining the chemical structure of the associated peptides. The chemical structures of these new peptides have been partially determined and the expected amino acids coded by the RiPPs core gene are present in the structures. Further analysis using nuclear magnetic resonance (the same technology used in MRI machines) is ongoing to figure out the complicated novel chemical structures. Preliminary observations indicate that fungal growth and aflatoxin production are not significantly inhibited due to individual RiPPs genes being knocked out, however, multiple gene knock outs do show decreases in toxins and growth. This may indicate that peptide products from several RiPPs clusters may work in concert to assist the fungus to infect and contaminate corn. If production of one RiPP is disabled, other RiPPs may fill that role. Furthermore, we collaborated with scientists at Northern Illinois University (agreement no. 58-6435-4-015) and identified several genes that regulate diverse functions in A. flavus. When some of these genes were knocked out, the resulting A. flavus strains showed slower growth and reduction in toxin production, including aflatoxins and an unregulated toxin named cyclopiazonic acid. Our collaboration with scientists at NIU has also identified a bacterium (Pseudomonas fluorescens) that, when grown together with A. flavus, significantly reduces growth and toxin production of the fungus. Iron appears to play a key role in the bacteria-fungus interaction and iron-binding metabolites detected in the bacteria are likely responsible. Studies are underway to confirm the identity of these bacterial metabolites and determine if they alone are responsible for inhibition of growth and toxin production. We have also collaborated with scientists at Tulane University (agreement 6054-41420-009-004S) to investigate how parts of the chromosome are readily lost, or in some cases gained. These findings could help explain how the fungus can lose the ability to produce toxins and may aid in developing more effective biocontrol strains. We found that application of A. flavus biocontrol improved the microbiome of the treated corn. Occurrence of a plant pathogen (a microbe that harms plants) decreased in the treated corn plants, while several microbes beneficial to the plant were unaffected by treatment. We expanded this investigation in collaboration with scientists at Louisiana State University (agreement 6054-41420-009-005S) to assess the soil microbiome in corn fields and its potential to control aflatoxin contamination. Due adverse weather (severe heat and drought) in SE Louisiana the field experiment was halted. We have set up the field experiment again this year and are processes initial samples. Additionally, our collaboration with Mycologics (agreement 6054-41420-009-009C) continued and has resulted in a formulation of bacterial extract that will soon be tested in field studies to assess its effectiveness and practically for reducing aflatoxin contamination on food crops.
In support of Objective 2, we examined the impact of high and low atmospheric CO2 levels in conjunction with high and low soil nitrogen levels on the ability of A. flavus to infect corn plants and produce aflatoxins. We should gain insight into how predicted increases in CO2 affect the corn plant and how adjusting soil nitrogen levels in response can improve plant health. We conducted a small-scale experiment using a walk-in growth chamber capable of supporting growth of many corn plants while maintaining desired levels of CO2 while differing amounts of nitrogen fertilizer. Measurements taken include plant growth rates, and cob formation, and the timeline of plant development has been established. Tissues are being processed for protein, lipid, and mineral content. Post-inoculation kernels have been collected and are being assessed for their susceptibility to infection.
In support of Objective 3, we continued studies involving chemical compounds, produced and secreted by several aflatoxin-free A. flavus isolates from Louisiana, Georgia, Arizona, and Mississippi, to inhibit toxin producing strains from the same regions. The sequencing of RNA (ribonucleic acid) from a set of strains from Louisiana that had been exposed to an inhibitory compound is complete and data analysis is underway to assess the level of activation/inactivation of key genes that play important roles in toxin production in the fungus. Experiments have now been completed that involve exposure of toxin producing strains from Arizona, Georgia and Mississippi to known volatile organic compounds (VOCs, a class of gaseous compounds released by the growing fungus) produced by aflatoxin-free Aspergillus strains to assess their impacts on growth and toxin production. We are in the process of preparing for another RNA sequencing study involving the most inhibitory VOC(s) for most of the strains and assessing their impact at the gene level. In collaboration with researchers at Pennsylvania State University (agreement 6054-41420-009-010S) we have expanded our investigation of VOCs to include live plants grown in the greenhouse. Preliminary data has been collected from corn infected with A. flavus using the custom-made sampling equipment developed at Penn. State and sent to our collaborators for analysis.
Accomplishments
1. A marker-free chromosomal deletion technique disables toxin production in Aspergillus flavus. One strategy to ensure a safer food supply that is free of fungus-produced toxins like aflatoxins is to apply biocontrol fungus to outcompete local toxin producing fungi. However, many of the current biocontrol strains produce other toxins that are unregulated. Removal of large DNA segments from A. flavus, such as those responsible for toxin production, has been difficult and time-consuming using standard molecular biology techniques. Therefore, ARS researchers in New Orleans, Louisiana, developed a new technique called marker-free chromosomal deletion. Using this new technique, large DNA segments – including gene clusters for production of harmful metabolites such as aflatoxin and cyclopiazonic acid – were efficiently removed from common toxigenic isolates, effectively converting toxin producing strains to non-toxic A. flavus strains. This technique can be used to engineer biocontrol fungus strains with improved fitness that are incapable of producing any toxic substances.
Review Publications
Moore, G.G., Chalivendra, S., Mack, B.M., Gilbert, M.K., Cary, J.W., Rajasekaran, K. 2023. Microbiota of maize kernels as influenced by Aspergillus flavus infection in susceptible and resistant inbreds. Frontiers in Microbiology. 14. Article 1291284. https://doi.org/10.3389/fmicb.2023.1291284.
Chang, P. 2023. Creating large chromosomal segment deletions in Aspergillus flavus by a dual CRISPR/Cas9 system: Deletion of gene clusters for production of aflatoxin, cyclopiazonic acid, and ustiloxin B. Fungal Genetics and Biology. 170. Article 103863. https://doi.org/10.1016/j.fgb.2023.103863.
Calvo, A.M., Dabholkar, A., Wyman, E.M., Lohmar, J.M., Cary, J.W. 2024. Regulatory functions of homeobox domain transcription factors in fungi. Applied and Environmental Microbiology. https://doi.org/10.1128/aem.02208-23.
Castano-Duque, L.M., Winzeler, H.E., Blackstock, J.M., Cheng, L., Vergopolan, N., Focker, M., Barnett, K., Owens, P.R., Van Der Fels-Klerx, I., Vaughan, M.M., Rajasekaran, K. 2023. Dynamic geospatial modeling of mycotoxin contamination of corn in Illinois: unveiling critical factors and predictive insights with machine learning. Frontiers in Microbiology. 14. Article 1283127. https://doi.org/10.3389/fmicb.2023.1283127.