Location: Food and Feed Safety Research
2020 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
Progress has been made by ARS reseachers at New Orleans, Louisiana, in both objectives of the project, all of which fall under National Program 108 Food Safety, Component 1, Foodborne Contaminants. Progress on this project includes two Objectives. Objective 1 is to determine how some toxin-free strains of the fungus Aspergillus (A.) flavus stop other A. flavus strains from producing a dangerous toxin known as aflatoxin. This prevention is termed biocontrol. Objective 2 is to determine the role of (1) sex-related genes, and (2) environmental stress, on the ability of biocontrol strains to grow, survive and undergo genetic changes, thereby influencing their effectiveness. In support of Objective 1, ARS researchers in New Orleans, Louisiana, made substantial progress in studies on the effect of gases produced by the biocontrol fungus that can affect growth and aflatoxin production by other fungi. ARS reseachers at New Orleans, Louisiana, tested five gases from an aflatoxin-free A. flavus strain (not from Louisiana) against three aflatoxin-producing strains from Louisiana. Three of the gases, each tested by itself, significantly reduced aflatoxin production indicating the potential for these gases to contribute to biocontrol. ARS reseachers at New Orleans, Louisiana, are now using different combinations of these three gases to see if they can achieve 100% aflatoxin prevention. Since different toxin-free A. flavus strains may produce different gases, ARS researchers in New Orleans, Louisiana, are looking for gases produced by another aflatoxin-free strain (this one is from Louisiana) that could reduce or prevent aflatoxin production. The aflatoxin-free strain from Louisiana produced many volatile organic compounds (VOCs), and currently four are being tested for their ability to prevent aflatoxin production. ARS scientists in New Orleans, Louisiana, have also completed an RNA-sequencing experiment to determine which fungal genes are turned on or off when an aflatoxin-free A. flavus strain prevents aflatoxin production simply by touching an aflatoxin-producing strain (termed touch inhibition). ARS reseachers at New Orleans, Louisiana, have obtained and analyzed the raw RNA-sequencing data, and are selecting “turned on” genes in the aflatoxin-free strain to determine their roles in biocontrol via touch inhibition. Studies have begun to identify compounds that leak out of the aflatoxin-free strain during its contact with the aflatoxin-producing strain that may be responsible for preventing aflatoxin production.
In support of Objective 2, ARS researchers in New Orleans, Louisiana, have completed studies to determine the importance of two sex-related genes (termed mating-type (MAT) genes) to the appearance of two A. flavus strains. Strain SRRC 1582 is an aflatoxin producer with a type MAT1-1 gene, and AF36 is aflatoxin-free and has a MAT1-2 gene. First, the MAT genes were turned off in both strains. Then, the MAT genes were swapped in each strain, whereby the MAT gene from AF36 was replaced with the MAT gene from SRRC 1582 and vice-verse. Fungal strains with altered genes are termed mutants, and the original strains are termed wild types. Researchers looked for changes to physical appearance between each mutant and its original wild type self. Turning off the MAT genes did not affect the appearance of the mutant strains, nor did swapping them, so these genes are not linked to physical appearance of this fungus. The SRRC 1582 mutants produced the same amount of aflatoxin as the wild type, so there appears to be no link between MAT genes and aflatoxin production. Changes in the production of other toxic compounds are being investigated. Previous studies have proven that the wild types of the SRRC 1582 and AF36 can mate with each other and produce healthy offspring. Therefore, ARS researchers also tested the fertility of the MAT mutants to have sex (termed pairings). They found that only the wild type strains created healthy offspring since none of the pairings involving MAT mutants resulted in offspring. ARS scientists in New Orleans, Louisiana, are also simulating environmental stress while growing several A. flavus strains to test their adaptability. These stressors include alterations to the available water, carbon dioxide and temperature these strains will have. The goal is to see if aflatoxin-free strains will maintain the same degree of aggressiveness they do under normal conditions and still prevent contamination by aflatoxin producing A. flavus strains.
Accomplishments
