Location: Food and Feed Safety Research
2021 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
This is the final report for the Project 6054-42000-026-00D terminated in April 2021, which has been replaced by new Project 6054-41420-009-00D.
In support of Objective 1, ARS scientists in New Orleans, Louisiana, showed the potential for secreted compounds (liquid or gaseous) from Aspergillus (A.) flavus biological control (biocontrol; utilizing natural field isolates of A. flavus that do not make the carcinogenic toxins known as aflatoxins) strains to greatly reduce the production of both aflatoxin and cyclopiazonic acid (CPA), by other toxin producing Aspergillus strains. Multiple experiments were conducted involving an aflatoxin- and CPA-non-producing (biocontrol) strain of A. flavus (LA1) and three aflatoxin- and CPA-producing strains (LA2-LA4) from Louisiana. The purpose of these experiments was to determine if secreted metabolites from the biocontrol strain were responsible for the observed decrease in toxin levels in toxic strains in the absence of physical contact between them. Experiments were conducted to determine the impact of liquid metabolites (extrolites) secreted by LA1 on growth and/or toxin production of individual strains (LA1, LA2, LA3 or LA4). Reduced growth (although minimal) was observed for toxic strains LA2-LA4, as well as greatly reduced production of aflatoxin (up to 78%) and CPA (up to 92%). A separate chemical analysis of liquid medium in which LA1 had been grown revealed the presence of a previously unidentified compound. Experiments are underway to isolate and characterize this compound since it could be the very chemical responsible for the observed reduction in toxin production.
The ability of gaseous metabolites (VOCs; volatile organic compounds) produced by A. flavus biocontrol strains to impact growth and/or toxin production was also tested by ARS scientists. ARS researchers conducted experiments involving strains LA1-LA4 that were exposed to individual VOCs. As with the extrolite experiments, growth was not impacted for any of the strains. However, toxin analysis showed that three of the tested VOCs (from A. flavus biocontrol strains) significantly reduced aflatoxin and CPA (a toxic compound) levels up to 100%. ARS scientists then tested various combinations of the three most effective VOCs to determine if there could be a combined effect on growth and toxin production. As with the individual VOCs, growth was not significantly impacted. Analysis of changes in production of aflatoxin, CPA and additional toxic metabolites are currently underway.
ARS scientists in New Orleans, Louisiana also captured and identified VOCs produced by strains LA1-LA4 while growing on each of three different types of synthetic media and two corn varieties (one susceptible and the other resistant to A. flavus infection). These VOCs were captured when the strains were grown individually, as well as when each toxic strain was grown close in proximity to the LA1 biocontrol strain. The goal of capturing VOCs from Louisiana strains was to expand the library of VOCs used for the study of their impact on fungal growth and toxin production. Captured VOCs found to be unique to each strain have now been identified. It appears that the type(s) of VOCs produced by these strains are substrate-dependent since some were only observed during growth on a particular substrate.
Another explored mechanism of biocontrol involves touch inhibition whereby an aflatoxin producing strain ceases to produce aflatoxin when it comes into physical contact with a biocontrol strain. ARS researchers conducted an RNA sequencing (RNA-seq; a technique that is used to measure the level of activation of genes) experiment involving the LA1 strain and an aflatoxin producing strain from Louisiana (KD53) previously shown to turn off its aflatoxin production upon physical contact with A. flavus biocontrol strains. ARS scientists looked at gene-level changes that occurred when these A. flavus strains touched. Analysis of the RNA-seq data revealed evidence of a gene (called a polyketide synthase or PKS) being activated in LA1 that is associated with the production of a secondary metabolite (a compound not required for fungal growth but needed for virulence and survival and can often be toxic to other organisms). Another activated gene has been associated with the process of moving metabolites out of fungal cells, which could relate to the uncharacterized extrolite secreted by LA1. The PKS gene is part of a group of genes known as “cluster 46”. This gene cluster had not yet been studied so its associated metabolite and biological functions are unknown. An A. flavus strain was generated with the cluster 46 PKS gene inactivated and metabolite extracts from this strain as well as the wild-type (normal strain with active PKS gene) strain were compared to identify cluster 46-associated metabolites. Comparative metabolite analyses have identified the presence of several potential chemical signals (metabolites) missing in the mutant strain that are present in the wild-type strain. Follow up analyses are planned to validate and identify these metabolites.
With respect to Objective 1, ARS scientists in New Orleans, Louisiana have shown that one likely mechanism of A. flavus biocontrol involves production of chemicals (in liquid or gas form) that cause toxin producing strains to reduce or turn off production of one or more of these harmful compounds.
In support of Objective 2, ARS scientists in New Orleans, Louisiana, showed that the ability of an A. flavus biocontrol strain to grow and compete against toxin producing strains is not linked to its ability to mate or survive unfavorable environmental conditions. A. flavus mutant strains, in which the gene required for mating (MAT) was inactivated or swapped with a different MAT gene from another A. flavus strain, were generated. These mutants were compared to their wild type (normal) selves in growth and mating studies. These mutants grew just as well as the normal strains, despite loss or alteration of their MAT genes. For the one aflatoxin producing strain examined, its MAT gene mutants were still able to produce aflatoxin indicating that toxin production is also not linked to the MAT gene. Mating experiments involving these MAT mutant strains proved that there is no way for a strain with a missing or altered MAT gene to undergo sex and produce offspring. This means that biocontrol strains lacking a functional MAT gene, and therefore unable to reproduce sexually, are able to survive in nature as well as toxin-producing A. flavus strains. It may soon be possible to test an engineered biocontrol strain (lacking a MAT gene) for effectiveness in the field if one with a naturally defective MAT gene cannot be found.
Also initiated by ARS scientists in New Orleans, Louisiana were comparative growth studies for multiple biocontrol strains and a few aflatoxin producing strains that were subjected to different climatic conditions such as low vs. high water availability, low vs. high CO2 levels, and preferred vs. non-preferred food sources. Data indicated that all strains experienced reduced growth under the more stressful conditions of low water availability, high CO2 and non-preferred food source. This trend was observed for both aflatoxin producers and non-producers. However, measurement of toxin levels was inconclusive, so these studies are being repeated.
Accomplishments
1. Touch inhibition as a mechanism of Aspergillus (A.) flavus biological control. ARS scientists in New Orleans, Louisiana, believe A. flavus biocontrol strains are good at preventing aflatoxin production by other Aspergillus strains. However, scientists still do not know the mechanism by which non-aflatoxin producing biocontrol strains inhibit aflatoxin production in A. flavus strains. One theory is that physical contact with a biocontrol strain turns off aflatoxin production. Using sophisticated molecular techniques, scientists looked at genes that are turned on in the biocontrol strain when it touches an aflatoxin producer. One gene of interest was inactivated, and this resulted in several compounds no longer being produced. Identifying these compounds will allow scientists to determine if they play a role in the mechanism of biocontrol. This knowledge will help improve biocontrol formulations leading to a safer and secure food and feed supply.
Review Publications
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.
Gebru, S.T., Mammel, M.K., Gangiredla, J., Tartera, C., Cary, J.W., Moore, G.G., Sweany, R.R. 2020. Draft genome sequences of 20 Aspergillus flavus isolates from corn kernels and cornfield soils in Louisiana. Microbiology Resource Announcements. 9(38):e00826-20. https://doi.org/10.1128/MRA.00826-20.
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.
