Location: Food and Feed Safety Research
2018 Annual Report
Objectives
Objective 1. Determine the mechanism by which atoxigenic strains of Aspergillus flavus reduce pre-harvest aflatoxin contamination by toxigenic strains.
Objective 2. Determine the role of mating-type genes and climatic (environmental) stressors on the ability of Aspergillus flavus biocontrol strains to compete, survive and recombine, thereby impacting the persistence and efficacy of these strains.
Approach
Aflatoxins are toxic and carcinogenic secondary metabolites that contaminate important agricultural commodities. One implemented strategy for prevention of aflatoxin contamination involves field application of a biocontrol agent, comprised of one or more nonaflatoxigenic Aspergillus (A.) flavus strains, to the soil and aerial parts of susceptible plants during the growing season. This strategy greatly reduces aflatoxin contamination by indigenous strains. However, the mechanism responsible for this reduction is unknown. In order to develop strategies that will increase the effectiveness of this approach and address unintended or unforeseen consequences, it is important to elucidate how introduced nonaflatoxigenic strains prevent native toxigenic strains from affecting crops. It is important to determine if the ability of the atoxigenic strain to outcompete the toxigenic strain is through chemo-regulation or simply by occupying the same niche. Examination of the transcriptomic and metabolomic profiles of biocontrol strains during interactions with toxigenic strains will allow us to better elucidate the molecular mechanisms controlling efficacy traits for generating improved biocontrol agents. Additionally, evidence for sexual recombination has been obtained in natural A. flavus populations, and laboratory pairing of sexually compatible A. flavus strains. However, it must be ascertained that such recombination does not occur at a high enough frequency to affect the stability of the biocontrol strains, especially under higher ecological stress. The proposed study will establish the conditions for long-term ecological stability of biocontrol strains and provide insights that will help improve the efficacy of pre-harvest biocontrol. Through our studies we hope to provide guidance for those who will use biocontrol and ensure they know: how to select a stable biocontrol strain, the absolute frequency of its application, and its measure to overcome any potential pitfalls.
Progress Report
Substantial progress has been made in both objectives of the project, all of which fall under National Program 108 Food Safety, Component 1, Foodborne Contaminants. Progress on this project focuses on Objective 1, the need to determine the mechanism by which atoxigenic strains of the fungus Aspergillus (A.) flavus reduce pre-harvest aflatoxin contamination by toxigenic strains, and Objective 2, the need to determine the role of mating-type genes and climatic (environmental) stressors on the ability of Aspergillus flavus biocontrol strains to compete, survive and recombine, thereby impacting the persistence and efficacy of these strains. In support of Objective 1, Agricultural Research Service (ARS) scientists at the Southern Regional Research Center (SRRC) in New Orleans, Louisiana, made significant progress in conducting extrolite (compounds excreted by the fungus, some of which have growth-inhibiting properties) and volatile organic compound (VOC, diffusible, gaseous compounds produced by one fungus that can affect growth and extrolite production by another) studies involving aflatoxin (a compound that is toxic and carcinogenic to humans and animals) producing strains vs a non-aflatoxigenic strain, all of which originated in Louisiana corn fields. The aflatoxin-producing (toxigenic) strains included: an Aspergillus flavus (A. flavus) L-strain named LA2 that produces B-type aflatoxins and another mycotoxin known as cyclopiazonic acid (CPA), an A. flavus S-strain named LA3 that produces only B-type aflatoxins (no CPA), and an Aspergillus parasiticus strain named LA4 that produces B- and G-type aflatoxins. The non-aflatoxigenic A. flavus strain (LA1) was also negative for CPA production (atoxigenic). ARS scientists used three different growth media during the course of these experiments. The completed split-plate VOC experiments were designed to determine if previously uncharacterized VOC compounds produced by atoxigenic LA1 could affect growth of toxigenic strains. ARS scientists did not observe noticeable growth, aflatoxin and/or CPA reductions from this set of experiments. There was also a series of solid medium + membrane experiments analyzing the ability of uncharacterized extrolites, produced by atoxigenic LA1, to inhibit the growth of the toxigenic strains. The growth studies, aflatoxin B1 (AFB1) and CPA measurements are finished. Extrolite studies involving liquid YES medium have been initiated, whereby colony growth is determined by its weight instead of diameter. It may also be possible to determine that the observed reduction in growth, from the solid medium study, was not due to nutrient depletion by atoxigenic LA1. This can be achieved through supplementation of “infused” medium with a small amount of fresh medium. Detection and measurement of extrolites secreted by all strains were performed using liquid chromatography-mass spectrometry (LC-MS, a specialized instrument that can detect and measure minute amounts of compounds such as extrolites). ARS researchers are scaling-up media quantities for these experiments to better allow for identification of any extrolites that are secreted into both liquid and solid media. Further progress has been made to determine the impact of previously characterized VOC compounds on the growth of the Louisiana fungal strains. ARS scientists have completed growth studies for five known VOC compounds, reportedly unique to toxigenic A. flavus, to test their impact on the growth of the four Louisiana strains. The controls involved growing each strain without the presence of each VOC. Studies have now been initiated using five known VOC compounds, reportedly unique to non-aflatoxigenic A. flavus, to test their impact on the growth of the four Louisiana strains. Analyses of AFB1 and CPA levels are ongoing, but toxin data for three of the toxigenic VOCs have been completed. VOC headspace studies for each LA strain have been initiated so that specifically-identified compounds from each can be tested at various concentrations against the other strains. In collaboration with Louisiana State University in Baton Rouge, Louisiana, ARS scientists have formulated an experimental protocol for RNA-Sequencing (allows for determining which fungal genes are turned on/off during interaction of strains) to identify the mechanism by which touch inhibition reduces aflatoxin production during the interaction of toxigenic strains with atoxigenic strains. The fungal strains, named KD17 (atoxigenic) and KD53 (toxigenic) utilized for the RNA-Sequencing experiment were selected because these strains consistently resulted in complete inhibition of aflatoxin in a previous study. Mycelia was harvested for both strains individually, as well as when both strains were “touching” (i.e. when fungal mycelia from both strains were in contact). A number of biological replicates were required to acquire enough tissue for RNA extraction. Tissues were recently prepared for RNA extraction and will soon be sent to the North Carolina State University Sequencing Facility in Raleigh, North Carolina, for sequencing.
In support of Objective 2, ARS scientists at the SRRC in New Orleans, Louisiana, created fungal mutants for which the mating-type (MAT) gene (required for the fungus to reproduce sexually) has been inactivated. Successful mating in Aspergillus flavus requires one strain to have a unique MAT gene (designated MAT1-1) while the other strain (known as the opposite mating-type) must have a MAT gene designated MAT1-2. In this study, the MAT1-1 strain (SRRC 1582) is a toxigenic A. flavus isolate, while the MAT1-2 strain is a non-aflatoxigenic A. flavus biocontrol strain, approved for commercial use (developed by ARS scientists in New Orleans, Louisiana), named AF36. Both strains have been paired in previous mating tests that resulted in production of viable offspring. ARS researchers have generated mutants of each of these strains, such that their MAT genes are no longer functional. Additionally, MAT gene swap mutants have been created, whereby the MAT1-2 gene from the AF36 strain will be replaced with the MAT1-1 gene from SRRC 1582; and conversely, the MAT1-1 gene from SRRC 1582 will be replaced with the MAT1-2 gene from the AF36 strain. These MAT gene mutants and MAT swap strains will allow us to determine the contribution of MAT genes to the overall biology of the fungus, such as growth, survival and aflatoxin production, as well as their roles in generating diversity in populations, for a primarily asexual organism. Morphological comparisons between each mutant and its respective wild type are nearly complete. Aflatoxin assays are being conducted for each mutant, individually as well as comingled. Full metabolome (all primary and secondary metabolites being produced by an organism) investigations are being conducted by collaborators at Ghent University, Ghent, Belgium. Mating experiments are near completion, and results data are forthcoming.
Accomplishments
1. Mechanism(s) by which Aspergillus (A.) flavus biocontrol strains reduce aflatoxin contamination. Significant control of aflatoxin contamination of crops has been achieved through biocontrol using atoxigenic strains of the fungus. However, the mechanism of biocontrol is not known for atoxigenic A. flavus strains. One mode may be through chemical compounds produced by the biocontrol strain. Experiments have been performed to determine the role that fungal extrolites (compounds excreted by the fungus, many of which may have growth-inhibiting properties) may play in the efficacy of biocontrol strains. It has been determined by Agricultural Research Service researchers in New Orleans, Louisiana, that at least one uncharacterized extrolite compound secreted by the atoxigenic strain included in this study reduced growth of toxigenic A. flavus and A. parasiticus strains on two types of growth medium (4-45%), as well as aflatoxin B1 by 60-78%. Once we identify and characterize the inhibitory extrolite(s) secreted by the atoxigenic strain, they will be synthesized and tested as a spray treatment, or will be used as a screen for future biocontrol strains that readily produce the inhibiting compound(s).
2. Mechanism(s) by which Aspergillus (A.) flavus biocontrol strains reduce aflatoxin contamination. Significant control of aflatoxin contamination of crops has been achieved through biocontrol using atoxigenic strains of the fungus. However, the mechanism of biocontrol is not known for atoxigenic A. flavus strains. One mode may be through chemical compounds produced by the biocontrol strain. So far, experiments have been performed to determine the role that previously characterized toxigenic A. flavus VOCs (volatile organic compounds emitted by the toxigenic fungus that may inhibit growth of competing fungi) may have on four Louisiana strains tested. It has been determined by Agricultural Research Service researchers in New Orleans, Louisiana, that benzaldehyde may be an important VOC since its exposure to all strains resulted in reduced growth as volume of the VOC increased, including the atoxigenic strain. Aflatoxin B1 reduction, consistent with increased VOC, was observed for only one strain, LA4 (Aspergillus parasiticus), when exposed to benzaldehyde and eucalyptol, which may mean these compounds are not naturally produced by A. parasiticus. By elucidating the impact of benzaldehyde exposure on the atoxigenic strain, future biocontrol strains should be screened for competitive resistance to production of this VOC by toxigenic A. flavus, which could enhance their effectiveness.
Review Publications
Moore, G.G., Olarte, R.A., Horn, B.W., Elliott, J.L., Singh, R., O'Neal, C.J., Carbone, I. 2017. Global population structure and adaptive evolution of aflatoxin-producing fungi. Ecology and Evolution. 7:9179-9191.
Moore, G.G., Mack, B.M., Beltz, S.B., Puel, O. 2018. Genome sequence of an aflatoxigenic pathogen of Argentinian peanut, Aspergillus arachidicola. BMC Genomics. 19:189. https://doi.org/10.1186/s12864-018-4576-2.
Carvajal-Campos, A., Manizan, A.L., Tadrist, S., Akaki, D.K., Koffi-Nevry, R., Moore, G.G., Fapohunda, S.O., Bailly, S., Montet, D., Oswald, I.P., Lorber, S., Brabet, C., Puel, O. 2017. Aspergillus korhogoensis, a novel aflatoxin producing species from the Côte d’Ivoire. Toxins. 9:353. https://doi.org/10.3390/toxins9110353.
Ozturk, I.K., Chettri, P., Dupont, P-Y., Barnes, I., McDougal, R.L., Moore, G.G., Sim, A., Bradshaw, R.E. 2017. Evolution of polyketide synthesis in a Dothideomycete forest pathogen. Fungal Genetics and Biology. 106:42-50.
