Location: Food and Feed Safety Research
2023 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, we developed a highly efficient gene knockout technique termed CRISPR/Cas9 to expedite planned gene function studies in A. flavus. To knock out a gene means that it no longer active. This technique is not specific to the type of A. flavus so it can be applied to any A. flavus found during field studies. We showed that multiple genes can be knocked out in a very short time. We also demonstrated its utility in several Aspergillus species other than A. flavus. We continue our research on the chemical and biological characterization a specific set of five A. flavus genes believed to be involved in the production of five different cyclic peptides (small proteins termed RiPPs) that are produced by the fungus upon infection of corn. The five RiPPs genes have been individually knocked out in the fungus using the CRISPR technology. We grew A. flavus strains with a knocked out RiPPs peptide gene and a normal, unmodified A. flavus strain on corn kernels. We are now analyzing chemical extracts of the infected corn tissues using a highly sensitive mass spectrometer (an instrument used to identify extremely small quantities of chemical compounds) to determine the presence and structure of the cyclic peptide. At the same time, we measured fungal growth and aflatoxin production for the RiPPs knockout strains and the normal strain. Preliminary observations indicate that fungal growth and aflatoxin production are not significantly inhibited due to the RiPPs genes being knocked out. This indicates that these cyclic peptides may not play a significant role assisting the fungus to infect and contaminate corn. We collaborated with scientists at Northern Illinois University (agreement no. 58-6054-9-009) and we identified several genes that regulate diverse functions in A. flavus. When some of these genes were knocked out, the resulting A. flavus strains demonstrated a number of biological differences from the normal strain including changes in growth and development, as well as reduced aflatoxin production. Additional biological assays are being performed with these various knockout strains to further characterize the function of the gene of interest. Our collaboration with scientists at NIU has also identified a bacterium (Pseudomonas fluorescens) that, when grown together with A. flavus, significantly inhibits the growth the fungus. The degree of inhibition was found to be dependent on iron availability in the environment. Studies are currently underway to determine the genetic and chemical mechanisms by which the bacterium cause the inhibition. Also in support of Objective 1, we continued studies to determine the biological functions of nine A. flavus genes that were previously indicated by gene activity assays to play a role in the fungus’ ability to infect isolated corn kernels in the laboratory. Biological assays have been performed using A. flavus strains carrying CRISPR-based knocked out versions of these nine regulator genes. Some of the fungal gene mutants demonstrated characteristics such as reduced growth capacity, response to environmental stress, and decreased aflatoxin accumulation following infection of kernels. This is consistent with the genes of interest being involved in fungal pathogenicity.
In support of Objective 2, we examined the impact of high (1000 ppm, parts per million) and low (350 ppm) atmospheric CO2 levels on the ability of A. flavus to infect ears on corn plants and produce aflatoxins. This experiment will provide a better understanding of the potential impacts of predicted increases in CO2 levels due to climate change on fungal pathogenicity and toxin production. 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. For initial analysis, ears from corn plants growing at either low or high CO2 levels were infected with A. flavus. Five days after infection, we harvested the ears and collected kernels. The kernel samples are currently being analyzed for levels of fungal growth and aflatoxin production at the different CO2 levels. Additionally, the activity of several genes shown to be responsive to alterations in CO2 levels from an earlier study of corn seed infected in the laboratory, is also being analyzed in the current whole corn plant study.
We are also examining the impact of atmospheric CO2 levels on the ability of naturally occurring, non-aflatoxin producing (AF-) A. flavus biocontrol strains to reduce growth and aflatoxin production of aflatoxin-producing (AF+) A. flavus. Previous data from our unit indicated that growing AF- strains together with AF+ strains using incubators set at low CO2 levels results in reduction of aflatoxin levels. Over the past year we have measured growth rates of AF+ and AF- strains and have conducted interactive growth assays using incubators set at 3 distinct CO2 levels (350 ppm, 650 ppm, and 1000 ppm). Understanding how and why CO2 affects A. flavus gene activity will have implications for future development of AF- biocontrol strains and help predict overall climate effects on A. flavus pathogenicity.
In support of Objective 3, we continued studies testing the ability of chemical compounds, produced and secreted by several aflatoxin-free (AF-) A. flavus isolates from Louisiana, Georgia, Arizona, and Mississippi, to inhibit toxin producing strains (AF+) from the same regions. Preliminary chemical analyses of extracts of several AF- strains have identified compounds that can inhibit aflatoxin production in AF+ fungal isolates by up to 75%. Experiments are underway to confirm the anti-aflatoxin potential of these chemicals and determine the mechanism of inhibition. We also finished the analysis of volatile organic compounds (VOCs, a class of gaseous compounds released by the growing fungus) produced by 12 Aspergillus strains from the same four geographic regions mentioned above. While the VOC data from the Louisiana strains has been published, data obtained from the other strains/regions are still being evaluated. Additionally, we have begun experiments to analyze levels of gene activation in AF+ Aspergillus strains exposed to VOCs from AF- strains that have been shown to inhibit growth and toxin production in A. flavus. RNA (ribonucleic acid) is being isolated from the VOC-treated AF+ strains and will be used to analyze the level of activation of key genes that play important roles in toxin production and formation of reproductive structures in the fungus.
We continued our work with Mycologics, LLC (agreement no. 58-6054-3-016) and identified a chemical produced by a bacterium that can significantly reduce aflatoxin production by A. flavus. The bacterium, Vibrio gazogenes, produces a red pigment known as prodigiosin. A method for extraction of “crude” prodigiosin from the V. gazogenes bacteria was optimized which resulted in greater than 90% recovery of the compound. Preliminary fungal growth and aflatoxin assays have shown that addition of the crude prodigiosin extract to growth nutrients resulted in significant (= 95%) inhibition of aflatoxin production while having very little impact on fungal growth.
Accomplishments
1. Development of a technique to efficiently inactivate genes in the aflatoxin-producing fungus, Aspergillus (A.) flavus. To prevent aflatoxin (a toxic and carcinogenic compound) contamination and to ensure worldwide food safety and supply, understanding A. flavus gene functions is a prerequisite for curbing its infection of important crops. However, currently used molecular techniques to study gene function are labor-intensive and time-consuming. The lack of a simple and readily adopted gene editing technique in A. flavus has greatly hampered progress in research on aflatoxin biosynthesis and the fungus’ interaction with the host plant. Now, ARS scientists in New Orleans, Louisiana, have developed an improved gene editing technique to study gene function in Aspergillus species whose efficacy surpasses all previously available methods. The technique can disable A. flavus genes in a very short time. It also can delete large segments of an A. flavus genome with precision. Development of this gene editing technique will allow targeted genetic modification of any field strain of A. flavus. ARS researchers will now be able to use gene editing to develop A. flavus strains genetically equivalent to or better than current field isolated, non-aflatoxin producing biological control strains that in the future may be applied to crops before harvest to reduce aflatoxin contamination.
Review Publications
Lebar, M.D., Mack, B.M., Carter Wientjes, C.H., Wei, Q., Mattison, C.P., Cary, J.W. 2022. Small NRPS-like enzymes in Aspergillus sections Flavi and Circumdati selectively form substituted pyrazinone metabolites. Frontiers in Fungal Biology. 3:1029195. https://doi.org/10.3389/ffunb.2022.1029195.
Gilbert, M.K., Mack, B.M., Lebar, M.D., Chang, P.-K., Gross, S.R., Sweany, R.R., Cary, J.W., Rajasekaran, K. 2023. Putative core transcription factors affecting virulence in Aspergillus flavus during infection of maize. The Journal of Fungi. 9(1):118. https://doi.org/10.3390/jof9010118.
Chang, P.-K. 2023. A simple CRISPR/Cas9 system for efficiently targeting genes of Aspergillus section Flavi species, Aspergillus nidulans, Aspergillus fumigatus, Aspergillus terreus and Aspergillus niger. Microbiology Spectrum. 11(1):1-18. https://doi.org/10.1128/spectrum.04648-22.
Chang, P.-K., Scharfenstein, L.L., Mahoney, N., Kong, Q. 2023. Kojic acid gene clusters and the transcriptional activation mechanism of Aspergillus flavus KojR on expression of clustered genes. The Journal of Fungi. 9:259. https://doi.org/10.3390/jof9020259.
Sweany, R.R., Breunig, M., Opoku, J., Clay, K., Spatafora, J.W., Drott, M.T., Baldwin, T.T., Fountain, J.C. 2022. Why do plant-pathogenic fungi produce mycotoxins? Potential roles for mycotoxins in the plant ecosystem. Phytopathology. 112(10):2044-2051. https://doi.org/10.1094/PHYTO-02-22-0053-SYM.
Chang, P.-K., Sheng T. Hua, S. 2023. Are current Aspergillus sojae strains originated from native aflatoxigenic Aspergillus species population also present in California? Mycobiology. https://doi.org/10.1080/12298093.2023.2217495.
