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Research Project: Molecular and Environmental Factors Controlling Aflatoxin Reduction by Non-Toxigenic Aspergillus Strains

Location: Food and Feed Safety Research

2017 Annual Report

1a. Objectives (from AD-416):
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.

1b. Approach (from AD-416):
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.

3. Progress Report:
Progress was made on both objectives. In support of Objective 1, Agricultural Research Service (ARS) scientists in New Orleans, Louisiana, initiated 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 were sampled in Louisiana. The aflatoxin-producing (toxigenic) strains included: an Aspergillus (A.) flavus L-strain that produces B-aflatoxins and cyclopiazonic acid (CPA, a toxic compound), an A. flavus S-strain that produces only B-aflatoxins (no CPA), and an A. parasiticus strain that produces B- and G-aflatoxins. The non-aflatoxigenic strain was also negative for CPA production (atoxigenic). We used three different growth media during the course of the experiments: Czapek’s (CZ), Yeast Extract Sucrose (YES), and Corn Meal Agar (CMA). VOC experiments were designed to determine if previously uncharacterized VOC compounds produced by the atoxigenic fungal strain could affect growth of toxigenic strains. We observed a reduction in growth rate for all toxigenic strains exposed to the atoxigenic fungus; however, the reduction was not substantial. Aflatoxin measurements are underway for the toxigenic strains from this set of VOC experiments. A series of experiments have been conducted to optimize protocols for analyzing the ability of extrolites, produced by an atoxigenic strain, to inhibit the growth of a toxigenic strain. The experiments required the atoxigenic strain to be inoculated onto a porous membrane placed on the surface of the growth medium. When the atoxigenic fungus had covered approximately two-thirds of the growth medium surface, the membrane was removed and the toxigenic strain was then inoculated directly onto the medium surface and its growth was measured for 3-5 days while being incubated at a specific temperature. Reduced growth of the toxigenic strains was observed during growth on media containing extrolites produced by the atoxigenic strain, and the reduction in growth was more pronounced than observed in the VOC study. Experiments to detect and measure extrolites secreted by both strains are underway using liquid chromatography-mass spectrometry (LC-MS, a specialized instrument that can detect and measure minute amounts of compounds such as extrolites). Once we identify growth-inhibiting extrolites produced by the atoxigenic strain, we can infuse them individually into growth media to determine their effectiveness at reducing growth and aflatoxin contamination by toxigenic strains. We can then screen potential biocontrol strains (atoxigenic fungal strains that can be used to inhibit the growth of toxigenic strains) for the production of these aflatoxin-antagonistic compounds as a means to identify improved biocontrol strains. Progress was also made to determine the impact of previously characterized VOC compounds on the growth of our Louisiana fungal strains. Previous studies have identified VOC compounds that are unique to toxigenic and non-aflatoxigenic strains. We used five known VOC compounds, unique to toxigenic A. flavus, to test their impact on the growth of the non-aflatoxigenic strain, as well as using five known VOC compounds, unique to non-aflatoxigenic A. flavus, to test their impact on the growth of each toxigenic strain. Analysis of fungal growth is ongoing. In support of Objective 2, ARS scientists in New Orleans, Louisiana, have made significant progress in creating fungal mutants for which the mating-type (MAT) gene (required for the fungus to reproduce sexually) has been inactivated. Successful mating in A. 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 our study, the MAT1-1 strain is a toxigenic A. flavus isolate while the MAT1-2 strain is an Environmental Protection Agency (EPA) approved, commercial-use, non-aflatoxigenic A. flavus biocontrol strain (developed by ARS scientists in New Orleans, Louisiana) designated as AF36. Both strains have been paired in previous mating tests that resulted in production of viable offspring. We have generated mutants of each of these strains, such that their MAT genes are no longer functional. Additionally, we are creating MAT gene swap mutants, whereby the MAT1-2 gene from the AF36 strain will be replaced with the MAT1-1 gene from the toxigenic strain; and conversely, the MAT1-1 gene from the toxigenic strain 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 for a primarily asexual organism.

4. Accomplishments

Review Publications
Moore, G.G., Mack, B.M., Beltz, S.B., Gilbert, M.K. 2016. Draft genome sequence of an aflatoxigenic Aspergillus species, A. bombycis. Genome Biology and Evolution. 8(11):3297-3300.

Uka, V., Moore, G.G., Arroyo-Manzanares, N., Nebija, D., De Saeger, S., Diana Di Mavungu, J. 2017. Unravelling the diversity of the cyclopiazonic acid family of mycotoxins in Aspergillus flavus by UHPLC Triple-TOF HRMS. Toxins. 9:35. doi:10.3390/toxins9010035.