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 ...

Genome‐wide nucleotide variation distinguishesAspergillus flavusfromAspergillus oryzaeand helps to reveal origins of atoxigenicA. flavusbiocontrol strains

“…The places of their isolation may reflect respective ecological niches preferable for growth and development. A recent report on the application of AF36 and NRRL21882 to maize fields in Alabama, Georgia and North Carolina, where AF36 is not registered for use, shows that A. flavus individuals belonging to the 21882‐type classified by multilocus haplotypes are the most dominant in the soils, but AF36‐type individuals are recovered only in very low frequencies (Lewis et al ). Although NRRL21882 also can be found in above‐ground tissues, consistent with the observed niche adaption NRRL21882‐type isolates have been found in soils of many southern states in the United States, and it is estimated that 16·3% of the total A. flavus isolates collected from those soil samples belong to this type (Horn and Dorner ; Chang et al ).…”

The Frequency of Sex: Population Genomics Reveals Differences in Recombination and Population Structure of the Aflatoxin-Producing Fungus Aspergillus flavus

“…However, high genotypic diversity is suggestive of some recombination between lineages ( 18 , 19 ). Recently, mating between A. flavus isolates has been achieved in field experiments ( 20 , 21 ) using extremely high population densities that better reflect artificial biocontrol conditions; i.e., higher densities of nonaflatoxigenic A. flavus propagules have been applied to agricultural fields than would normally exist under agricultural or natural conditions. The idea that A. flavus may be predominantly sexual ( 21 ) is difficult to reconcile with a large body of evidence about the predominantly asexual nature of this fungus.…”

“…Recently, mating between A. flavus isolates has been achieved in field experiments ( 20 , 21 ) using extremely high population densities that better reflect artificial biocontrol conditions; i.e., higher densities of nonaflatoxigenic A. flavus propagules have been applied to agricultural fields than would normally exist under agricultural or natural conditions. The idea that A. flavus may be predominantly sexual ( 21 ) is difficult to reconcile with a large body of evidence about the predominantly asexual nature of this fungus. Importantly, these field studies occurred on very short timelines and thus do not capture whether the fitness of recombinant progeny is reduced by the disassociation of coadapted traits (i.e., recombination load), and thus sexual reproduction makes little contribution to the overall population.…”

Biological Control and Mitigation of Aflatoxin Contamination in Commodities

“…Hruska et al [ 33 ] demonstrated via competitive exclusion that the decreased AF level was positively correlated to the low population of the toxigenic A. flavus under treatment, where the biocontrol A. flavus strain showed increased propagation and colonization. Recent studies demonstrated that biocontrol non-aflatoxigenic strains reduced AF concentrations in treated crops by more than 80% under both field and storage conditions [ 34 , 35 , 36 ]. In Argentina, a native atoxigenic A. flavus strain showed a remarkable biocontrol potential on peanuts, and the pre-harvest application of the biocontrol agent had a carry-over effect and protected peanuts under storage conditions [ 37 ].…”