Location: Food and Feed Safety Research
2019 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 A. 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) researchers in New Orleans, Louisiana, made significant progress in conducting volatile organic compound (VOC, diffusible, gaseous compounds produced by one fungus that can affect growth and extrolite production by another) studies involving three A. flavus strains that produce aflatoxin (a compound that is toxic and carcinogenic to humans and animals) when grown in the presence of a non-aflatoxigenic strain, all of which originated in Louisiana corn fields. The aflatoxin-producing (toxigenic) strains included: an 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 also produces B-type aflatoxins and CPA, and an Aspergillus parasiticus-like strain named LA4 that produces B- and G-type aflatoxins and CPA. The non-aflatoxigenic A. flavus strain (LA1) was also negative for CPA production (atoxigenic). VOC headspace studies for LA strains grown in close proximity (in vial slants, but not touching) have been completed, and soon specifically-identified compounds from this paired study will be compared to VOC headspace compounds detected when each strain was grown individually. Unique VOCs will be tested at various concentrations against the other strains (similar to the known VOC experiments from 2018). The thigmoregulation (touch-inhibition) experiments, RNA (ribonucleic acid) extractions, and RNA-Sequencing (allows for determining which fungal genes are turned on/off during interaction of strains) have recently been completed for strains KD17 (atoxigenic) and KD53 (toxigenic). The raw data has been acquired and continues to be analyzed. Genes associated with possible aflatoxin reduction through thigmoregulation or extrolite production are currently being selected for knock-out and overexpression studies.
In support of Objective 2, ARS researchers in New Orleans, Louisiana, have completed fungal mutant studies in which the mating-type (MAT) gene (required for the fungus to reproduce sexually) has been inactivated in two A. flavus strains (SRRC 1582, aflatoxigenic, and MAT1-1; and AF36, non-aflatoxigenic, and MAT1-2). Additionally, MAT gene swap mutants were created, whereby the MAT1-2 gene from the AF36 strain was replaced with the MAT1-1 gene from SRRC 1582; and conversely, the MAT1-1 gene from SRRC 1582 was replaced with the MAT1-2 gene from the AF36 strain. Morphological comparisons between each mutant and its respective wild type are complete, whereby some changes were observed. For example, the placement of the AF36 MAT1-2 gene in SRRC 1582 resulted in severely stunted conidiophore stipes (fungal stems on which the asexual spores form). Mating experiments are complete, and only the wild-type strains created healthy, mature fruiting bodies (physical evidence of successful mating due to production of sexual spores). Aflatoxin assays showed no significant changes in the mutants, indicating that the MAT genes are not integral to mycotoxin production. Full metabolome (all primary and secondary metabolites being produced by an organism) investigations are still being conducted by collaborators at Ghent University, Ghent, Belgium. Altered climate impact studies on atoxigenic and toxigenic A. flavus strains are near completion. From a biocontrol perspective, the experiments involve growing the atoxigenic strain under conditions of low vs. high water availability (substrate-mediated), carbon dioxide and temperature to ascertain if any combination inhibits their ability to grow aggressively. Tests are underway involving competition and mating studies to determine their impacts on the stability of biocontrol strains.
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, so experiments have been performed to determine the role that previously characterized atoxigenic A. flavus VOCs (volatile organic compounds emitted by the fungus that may inhibit competing fungi) may have on three toxigenic strains tested. It has been determined by Agricultural Research Service researchers in New Orleans, Louisiana, that growth of A. flavus and aflatoxin production are not directly correlated, since reduction in aflatoxin was not always observed with decreased growth. VOCs (tested individually), unique to atoxigenic A. flavus, proved effective at significantly reducing aflatoxin and/or cyclopiazonic acid (CPA) production. In particular, 3-Octanone, Decane and 2-3-Dihydrofuran significantly reduced aflatoxin levels, and both Decane and 2-3-Dihydrofuran completely inhibited CPA production by strains LA3 and LA4, respectively. By elucidating the impact of VOC exposure on these strains, it may be possible to enhance post-harvest biocontrol (i.e., in storage) by exposure of the commodity to one or more of the VOCs tested.
Review Publications
Aduroja, E.D., Moore, G.G., Beltz, S.B., Soares, C., Lima, N., Fapohunda, S.O. 2018. Induction of biodeterioration on vegetables by three fungal species. Journal of Plant Pathology. 101(2):243-250. https://doi.org/10.1007/s42161-018-0175-y.
Moore, G.G., Lebar, M.D., Carter-Wientjes, C.H. 2018. The role of extrolites secreted by nonaflatoxigenic Aspergillus flavus in biocontrol efficacy. Journal of Applied Microbiology. 126:1257-1264. https://doi.org/10.1111/jam.14175.
Uka, V., Moore, G.G., Arroyo-Manzanares, N., Nebija, D., De Saeger, S., Diana Di Mavungu, J. 2019. Secondary metabolite dereplication and phylogenetic analysis identify various emerging mycotoxins and reveal the high intra-species diversity in Aspergillus flavus. Frontiers in Microbiology. 10:667. https://doi.org/10.3389/fmicb.2019.00667.
Gilbert, M.K., Mack, B.M., Moore, G.G., Downey, D.L., Lebar, M.D., Joarder, V., Losada, L., Yu, J., Nierman, W.C., Bhatnagar, D. 2018. Whole genome comparison of Aspergillus flavus L-morphotype strain NRRL 3357 (type) and S-morphotype strain AF70. PLoS One. 13(7):e0199169. https://doi.org/10.1371/journal.pone.0199169.
Fapohunda, S.O., Moore, G.G., Aroyeun, O.S., Ayeni, K.I., Aduroja, E.D., Odetunde, S.K. 2018. Isolation and characterization of fungi isolated from Nigerian cocoa samples. Current Life Sciences. 4(3):46-52. https://doi.org/10.5281/zenodo.1405083.
