Characterization of the Aspergillus flavus Population from Highly Aflatoxin-Contaminated Corn in the United States

Abstract

Aflatoxin contamination of corn is a major threat to the safe food and feed. The United States Federal Grain Inspection Service (FGIS) monitors commercial grain shipments for the presence of aflatoxin. A total of 146 Aspergillus flavus were isolated from 29 highly contaminated grain samples to characterize the visual phenotypes, aflatoxin-producing potential, and genotypes to explore the etiological cause of high aflatoxin contamination of US corn. Five of the isolates had reduced sensitivity (43-49% resistant) to the fungicide azoxystrobin, with the remainder all being over 50% resistant to azoxystrobin at the discriminating dose of 2.5 µg/mL. Only six isolates of the highly aflatoxigenic S morphotype were found, and 48 isolates were non-aflatoxigenic. Analysis of the mating type locus revealed 45% MAT 1-1 and 55% MAT 1-2. The A. flavus population originating from the highly aflatoxin contaminated grain samples was compared to a randomly selected subset of isolates originating from commercial corn samples with typical levels of aflatoxin contamination (average < 50 ppb). Use of simple sequence repeat (SSR) genotyping followed by principal component analysis (PCoA) revealed a similar pattern of genotypic distribution in the two populations, but greater diversity in the FGIS-derived population. The noticeable difference between the two populations was that genotypes identical to strain NRRL 21882, the active component of the aflatoxin biocontrol product Afla-Guard™, were ten times more common in the commercial corn population of A. flavus compared to the population from the high-aflatoxin corn samples. The other similarities between the two populations suggest that high aflatoxin concentrations in corn grain are generally the result of infection with common A. flavus genotypes.

Keywords: fungicide resistance; maize; mating type; mycotoxin; population genetics.

Figure 1

Visual phenotypes of A. flavus isolates from US corn samples with high aflatoxin contamination. (A,B) present the pigmentation and fluorescence of the growth medium (potato dextrose agar amended with β-cyclodextrin). The colony morphology in (C) indicates an S-type phenotype with numerous small sclerotia and limited sporulation or an L-type phenotype with abundant sporulation and either a few large sclerotia or no sclerotia present.

Figure 2

Resistance to azoxystrobin fungicide in isolates of A. flavus isolates from US corn samples with high aflatoxin contamination. All isolates were grown on potato dextrose agar amended with a discriminatory dose of azoxystrobin (2 µg/mL) and salicylhydroxamic acid (100 µg/mL). The criteria of Ali et al. (2020) were used to characterize each isolate as sensitive (0–24% resistant), reduced sensitivity (25–49% resistant), moderately resistant (50–85% resistant), or resistant (86–100% resistant).

Figure 3

Mating type distribution of A. flavus isolates from US corn samples with high aflatoxin contamination. Mating types were determined by multiplex amplification of the MAT locus.

Figure 4

Distribution of non-aflatoxigenic (zero), low (1–19 ng/mL)-, medium (20–300 ng/mL)-, and high AF (>300 ng/mL)-producing isolates, separated by MAT genotype.

Figure 5

Principal coordinate analysis of A. flavus SSRs. Points designated as commercial corn isolates are based on the genotypes of isolates from a random subsample of A. flavus isolates from US commercial corn samples. Here, 21882 and 18543 are reference points based on the genotypes of two commercial biocontrol isolates. Principle coordinates 1 and 2 explain 31% and 9% of the variance, respectively. FGIS Zero, Low, Med, and High indicate isolates that are non-aflatoxigenic or that produced 1–19, 20–300, or >300 ng/mL aflatoxin, respectively.

Figure 6

Comparisons of diversity between A. flavus isolates from highly aflatoxin contaminated (>50 ppb) corn (FGIS samples) and from commercial corn samples with typical levels of aflatoxin contamination (average <50 ppb). Na is the number of alleles. Ne is the number of effective alleles. Haploid genetic diversity is given as h. Shannon’s information index is given as I. Tukey’s mean separation test results given for each series with a capital letter (h), a lowercase letter (I), asterisks (Na), and § or ‡ (Ne). FGIS Zero, Low, Med, and High indicate isolates that are non-aflatoxigenic or produced 1–19, 20–300, or >300 ng/mL aflatoxin, respectively.