Biocontrol Strains Differentially Shift the Genetic Structure of Indigenous Soil Populations of Aspergillus flavus.

Abstract

Biocontrol using non-aflatoxigenic strains of Aspergillus flavus has the greatest potential to mitigate aflatoxin contamination in agricultural produce. However, factors that influence the efficacy of biocontrol agents in reducing aflatoxin accumulation under field conditions are not well-understood. Shifts in the genetic structure of indigenous soil populations of A. flavus following application of biocontrol products Afla-Guard and AF36 were investigated to determine how these changes can influence the efficacy of biocontrol strains in reducing aflatoxin contamination. Soil samples were collected from maize fields in Alabama, Georgia, and North Carolina in 2012 and 2013 to determine changes in the population genetic structure of A. flavus in the soil following application of the biocontrol strains. A. flavus L was the most dominant species of Aspergillus section Flavi with a frequency ranging from 61 to 100%, followed by Aspergillus parasiticus that had a frequency of <35%. The frequency of A. flavus L increased, while that of A. parasiticus decreased after application of biocontrol strains. A total of 112 multilocus haplotypes (MLHs) were inferred from 1,282 isolates of A. flavus L using multilocus sequence typing of the trpC, mfs, and AF17 loci. A. flavus individuals belonging to the Afla-Guard MLH in the IB lineage were the most dominant before and after application of biocontrol strains, while individuals of the AF36 MLH in the IC lineage were either recovered in very low frequencies or not recovered at harvest. There were no significant (P > 0.05) differences in the frequency of individuals with MAT1-1 and MAT1-2 for clone-corrected MLH data, an indication of a recombining population resulting from sexual reproduction. Population mean mutation rates were not different across temporal and spatial scales indicating that mutation alone is not a driving force in observed multilocus sequence diversity. Clustering based on principal component analysis identified two distinct evolutionary lineages (IB and IC) across all three states. Additionally, patristic distance analysis revealed phylogenetic incongruency among single locus phylogenies which suggests ongoing genetic exchange and recombination. Levels of aflatoxin accumulation were very low except in North Carolina in 2012, where aflatoxin levels were significantly (P < 0.05) lower in grain from treated compared to untreated plots. Phylogenetic analysis showed that Afla-Guard was more effective than AF36 in shifting the indigenous soil populations of A. flavus toward the non-toxigenic or low aflatoxin producing IB lineage. These results suggest that Afla-Guard, which matches the genetic and ecological structure of indigenous soil populations of A. flavus in Alabama, Georgia, and North Carolina, is likely to be more effective in reducing aflatoxin accumulation and will also persist longer in the soil than AF36 in the southeastern United States.