Controlling aflatoxin contamination and propagation of Aspergillus flavus by a soy-fermenting Aspergillus oryzae strain

Abstract

Aflatoxins (AFs) are a group of carcinogenic and immunosuppressive mycotoxins that threaten global food safety. Globally, over 4.5 billion people are exposed to unmonitored levels of AFs. Aspergillus flavus is the major source of AF contamination in agricultural crops. One approach to reduce levels of AFs in agricultural commodities is to apply a non-aflatoxigenic competitor, e.g., Afla-Guard, to crop fields. In this study, we demonstrate that the food fermenting Aspergillus oryzae M2040 strain, isolated from Korean Meju (a brick of dry-fermented soybeans), can inhibit aflatoxin B1 (AFB1) production and proliferation of toxigenic A. flavus in lab culture conditions and peanuts. In peanuts, 1% inoculation level of A. oryzae M2040 could effectively displace the toxigenic A. flavus and inhibit AFB1 production. Moreover, cell-free culture filtrate of A. oryzae M2040 effectively inhibited AFB1 production and A. flavus growth, suggesting A. oryzae M2040 secretes inhibitory compounds. Whole genome-based comparative analyses indicate that the A. oryzae M2040 and Afla-Guard genomes are 37.9 and 36.4 Mbp, respectively, with each genome containing ~100 lineage specific genes. Our study establishes the idea of using A. oryzae and/or its cell-free culture fermentate as a potent biocontrol agent to control A. flavus propagation and AF contamination.

Figure 1

Schematic presentation summarizing the major AFB1 and AFM1 contamination/exposure routes and adverse health effects to human.

Figure 2

Inhibitory effects of A. oryzae M2040 on AFB1 production by A. flavus. (A) Experimental design. (B) Levels of AFB1 accumulation in a liquid co-culture media. *P < 0.05; **P < 0.01. (C) HPLC chromatograms of AFB1 at 3-day incubation of A. flavus vs dead and live M2040. Note the differences in the AFB1 peak size. (D) AFB1 accumulation and in peanut co-inoculated with M2040 and Afla-Guard and A. flavus NRRL3357 at different ratios.

Figure 3

Quantitation of A. flavus displacement by A. oryzae M2040 and Afla-Guard on peanuts. (A) The GFP construct and 5 day old culture of AF-GFP showing highly fluorescent mycelia, hyphae, and conidial suspension. (B) Fluorescence (FL) and non-fluorescence images representing inoculation of control groups observed at 5 days of incubation. (C) AF-GFP conidial count and AFB1 accumulation in peanut samples co-inoculated with varying ratios of M2040 or Afla-Guard. *P < 0.05; **P < 0.01. (D) Fluorescence (FL) and non-fluorescence images of peanuts representing the treatment groups observed at 5 days of incubation. Photographs were taken in a dark room with a 1-2s exposure time.

Figure 4

Effects of cell-free culture filtrate of A. oryzae M2040. (A) Experimental design. (B) Time course of the AFB1 accumulation in a mixed liquid of M2040 cell-free culture fermentate and A. flavus mycelial cells. *P < 0.05. **P < 0.01. (C) Conidial numbers and AFB1 production in HT and non HT M2040 fermentate inoculated with different conidial numbers of A. flavus. **P < 0.05. **P < 0.01. (D) Conidial count and AFB1 production in different concentrations of HT and non HT A. oryzae fermentate inoculated with 5 × 105 A. flavus conidia/ml. *P < 0.05. **P < 0.01. ND: Conidia were not detected under a microscope. Fermentate was diluted in sterile distilled water. (E) Conidial count and AFB1 production in different concentrations of HT and non HT M2040 fermentate inoculated with 5 × 10⁵ A. flavus conidia/ml. **P < 0.05. **P < 0.01. Fermentate was diluted in fresh PDB.

Figure 5

Comparative genome analyses of A. oryzae M2040 and Afla-Guard. (A) Phylogenetic relationship of A. oryzae and A. flavus isolates. An unrooted phylogeny was generated using the Maximum Likelihood method from 305,543 SNPs across the entire genome. Branch lengths represent the number of substitutions per site. All bootstrap values were ≥94%. Blue and green taxa labels represent A. oryzae and A. flavus, respectively. (B) Deletion profiles in the AF gene cluster. The chromosomal architecture of the AFB1 gene cluster relative to the A. flavus NRRL 3357 genome is shown above the heatmap, where arrows represent genes, and their orientations represents the direction of transcription. The heatmap represents copy number estimates for each non overlapping 100 bp bin across the AF gene cluster. Black and white represent copy numbers of 0 and ≥1, respectively. Bottom bar shows the Afla-Guard heatmap depicting deletions relative to the AF gene cluster containing A. flavus NRRL 3357 EQ963478 scaffold. Windows represent copy number estimates for each non-overlapping 10 kb bin across the scaffold. The chromosomal region containing the AF cluster is outlined with a red box. (C) Genome architecture of examples M2040 lineage specific genes clusters. Microsynteny of regions covering a three gene (top) and 17 gene (bottom) cluster unique to the M2040 genome in comparison to A. oryzae RIB 40, A. flavus NRRL 3357 and Afla-Guard. For each cluster arrows represent genes, and their orientations represents the direction of transcription. Genes colored black are conserved in at least 2 isolates, while genes colored light blue are unique to the M2040 genome. Gray blocks represent genomic regions exhibiting sequence similarity between isolates. Chromosome, or scaffold identifiers containing these loci are listed under each isolate. Gene identifiers are listed for each gene in panel A, and for the range of genes in panel.