Location: Food and Feed Safety Research
2022 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 harvested seed from whole corn plants that had been infected with A. flavus to identify key genes and toxic compounds, including aflatoxins, expressed by the fungus during infection. Kernels collected at 3 and 7 days after infection are currently being analyzed to identify and quantify fungal and corn metabolites. Ribonucleic acid (RNA) isolated from the infected corn kernels has been isolated and used to see what genes were active (or expressed). Additional RNA studies have looked at the expression of genes in the fungus as it infects isolated corn kernels in the laboratory. From these studies, we have identified nine fungal genes whose activity was correlated with infection of corn. Using a gene modification tool called CRISPR-Cas9, we were able to knock out (inactivate) these genes to better study their biological function in the fungus. One of these genes was shown to be involved in production of fungal spores that play a role in survival and spread of the fungus in the field. We are now doing studies of fungal growth rate, aflatoxin production, stress response, and production of spores with A. flavus strains that have had all these nine genes mutated so they are non-functioning.
Also in support of Objective 1, we continued our research on A. flavus genes involved in the production of four different cyclic peptides (small proteins) and a terpene (a chemical associated with plant oils) upon infection of corn by the fungus. The four peptide genes and the terpene gene have been individually knocked out in the fungus to determine their role in the biology of the fungus. An A. flavus strain with a knocked out cyclic peptide gene and a normal, unmodified A. flavus strain have been grown on corn kernels and chemical extracts of both strains are being analyzed 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. We are now looking at the degree to which knock out and normal strains can infect corn kernels and produce aflatoxin. In other studies, we have continued investigating the properties of an iron-binding toxin produced by A. flavus known as aspergillic acid that helps the fungus to invade the host plant. We discovered that the aspergillic acid gene cluster (a closely grouped set of genes responsible for production of aspergillic acid) is present in other species of Aspergillus fungi and may contribute to their ability to infect plants as well. ARS researchers in New Orleans, Louisiana, in collaboration with scientists at Northern Illinois University (Agreement no. 58-6054-9-009) have identified several genes that appear to regulate diverse functions such as aflatoxin production as well as fungal development. When one of these genes was knocked out, the A. flavus strain demonstrated reduced aflatoxin production and conidiation compared to a normal strain.
In support of Objective 2, ARS scientists in New Orleans, Louisiana, are studying how increasing levels of atmospheric CO2 might impact the ability of naturally occurring, non-aflatoxin producing (AF-) A. flavus strains to reduce growth and aflatoxin production of aflatoxin-producing (AF+) A. flavus. Our research has established that when growing mixed cultures of AF+ and AF- strains under present day atmospheric CO2 levels (350 ppm) a reduction in aflatoxin is observed. However, it remains to be determined if reduced aflatoxin levels are due to rapid growth of the AF- strain compared to the AF+ strain or if some other inhibitory mechanism is at work. Currently, growth assays are underway as is aflatoxin analysis. ARS scientists in New Orleans, Louisiana, are also studying the impact of CO2 levels on the ability of A. flavus to infect corn and produce aflatoxins. We have completed preliminary studies using a large, walk-in growth chamber capable of supporting growth of corn plants under different CO2 atmospheres. Ears on corn plants growing at either low CO2 (350 ppm) or high CO2 levels (1000 ppm) were infected with A. flavus. Five days after infection, the ears were harvested and kernels collected. The kernels are being analyzed for levels of fungal growth and aflatoxin production at the different CO2 levels.
In support of Objective 3, ARS scientists in New Orleans, Louisiana, have completed studies on the impact of extrolites (secreted chemicals) produced by several AF- A. flavus isolates from different geographic regions of the U.S. on growth of AF+ strains. Inhibition of growth of AF+ strains ranged from significant to minimal depending on the geographic origin of the AF- strains. We are currently performing chemical analysis for changes in levels of toxin production in AF+ strains caused by exposure to AF- extrolites. We have also generated one-liter batches of spent growth solution (remaining broth after the fungus has been removed) from 16 Aspergillus strains, both AF+ and AF-, in order to obtain enough material to chemically identify unique extrolites. We can then determine if extrolites unique to AF- strains may be responsible for observed inhibition of growth and toxin production upon exposure to AF+ strains. Also in support of Objective 3, we have grown Aspergillus strains from different geographic regions of the U.S. on different types of artificial growth media and corn kernels. Gases produced by the fungal cultures were captured and subjected to chemical analysis to identify unique gaseous chemicals. Gases unique to AF- strains may be responsible for inhibition of AF+ strains. Data on the gaseous chemical profiles for each of the Aspergillus cultures is currently being analyzed and prepared for reporting.
ARS scientists in New Orleans, Louisiana, working with a collaborator (Agreement no. 58-6054-2-002) have identified a chemical produced by a bacterium that can significantly reduce aflatoxin production in A. flavus. The bacterium, Vibrio gazogenes, produces a red pigment known as prodigiosin. Growth of A. flavus on artificial nutrient medium supplemented with various levels of pure prodigiosin showed that aflatoxin production can be inhibited up to 99%. Growth of A. flavus on corn seed imbibed with prodigiosin resulted in 65% inhibition of aflatoxin contamination however there was little to no significant reduction in growth of the fungus. Corn ears on whole plants growing in a greenhouse were treated with various concentrations of pure prodigiosin and then infected with A. flavus. Fungal growth and aflatoxin assays are currently underway on kernels isolated from the infected ears.
Accomplishments
1. A bacterial compound can significantly inhibit aflatoxin (AF) production by the mold Aspergillus (A.) flavus. Aflatoxin contamination in crops such as corn, cottonseed and peanut caused by A. flavus is a worldwide food safety problem as aflatoxins are potent carcinogens. Additionally, contamination of crops with aflatoxins costs tens of millions of dollars annually due to economic losses from the devaluation or destruction of contaminated crops. Now, ARS scientists in New Orleans, Louisiana, together with collaborators have identified a bacterium that when growing in the presence of A. flavus can significantly inhibit aflatoxin production. The mechanism appears to be related to production of a red pigment by the bacterium known as prodigiosin. The researchers showed that when A. flavus was grown in an artificial solution with prodigiosin, aflatoxin production was reduced by 99%. The finding is important, and ARS researchers at New Orleans, Louisiana, are working with the collaborators to see if the bacterium and prodigiosin can be used as biological control agents that can be applied to the crop before or after harvest to reduce aflatoxin contamination.
Review Publications
Wang, P., Xu, J., Chang, P.-K., Liu, Z., Kong, Q. 2022. New insights of transcriptional regulator AflR in Aspergillus flavus physiology. Microbiology Spectrum. 10(1). Article e00791-21.
Kandel, S.L., Jesmin, R., Mack, B.M., Majumdar, R., Gilbert, M.K., Cary, J.W., Lebar, M.D., Gummadidala, P.M., Calvo, A.M., Rajasekaran, K., Chanda, A. 2022. Vibrio gazogenes inhibits aflatoxin production through downregulation of aflatoxin biosynthetic genes in Aspergillus flavus. PhytoFrontiers. 2(3):218-229. https://doi.org/10.1094/PHYTOFR-09-21-0067-R.
Luis, J.M., Carbone, I., Mack, B.M., Lebar, M.D., Cary, J.W., Gilbert, M.K., Bhatnagar, D., Carter-Wientjes, C.H., Payne, G.A., Moore, G.G., Ameen, Y.O., Ojiambo, P.S. 2022. Development of sexual structures influences metabolomic and transcriptomic profiles in Aspergillus flavus. Fungal Biology. 126:187-200.
Sweany, R.R., Mack, B.M., Moore, G.G., Gilbert, M.K., Cary, J.W., Lebar, M.D., Rajasekaran, K., Damann Jr, K.E. 2021. Genetic responses and aflatoxin inhibition during co-culture of aflatoxigenic and non-aflatoxigenic Aspergillus flavus. Toxins. 13:794. https://doi.org/10.3390/toxins13110794.
Chang, P. 2022. Aspergillus flavus La3279, a component strain of the Aflasafe biocontrol product, contains a partial aflatoxin biosynthesis gene cluster followed by a genomic region highly variable among A. flavus isolates. International Journal of Food Microbiology. 366:109559. https://doi.org/10.1016/j.ijfoodmicro.2022.109559.
Moore, G.G., Lebar, M.D., Carter-Wientjes, C.H. 2022. Cumulative effects of non-aflatoxigenic Aspergillus flavus volatile organic compounds to abate toxin production by mycotoxigenic aspergilli. Toxins. 14:340. https://doi.org/10.3390/toxins14050340.
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. https://doi.org/10.1186/s13104-021-05527-6.
Moore, G.G. 2021. Practical considerations will ensure the continued success of pre-harvest biocontrol using non-aflatoxigenic Aspergillus flavus strains. Critical Reviews in Food Science and Nutrition. 1-18. https://doi.org/10.1080/10408398.2021.1873731.
Moore, G.G., Lebar, M.D., Carter-Wientjes, C.H., Gilbert, M.K. 2021. The potential role of fungal volatile organic compounds in Aspergillus flavus biocontrol efficacy. Biological Control. 160:104686. https://doi.org/10.1016/j.biocontrol.2021.104686.
