Ochratoxin A Defective Aspergillus carbonarius Mutants as Potential Biocontrol Agents

Abstract

Aspergillus carbonarius is one of the main species responsible for wine, coffee and cocoa toxin contamination. The main mycotoxin produced by this fungus, ochratoxin A (OTA), is a secondary metabolite categorized as a possible carcinogen because of its significant nephrotoxicity and immunosuppressive effects. A polyketide synthase gene (otaA) encodes the first enzyme in the OTA biosynthetic pathway. It is known that the filamentous fungi, growth, development and production of secondary metabolites are interconnected processes governed by global regulatory factors whose encoding genes are generally located outside the gene clusters involved in the biosynthesis of each secondary metabolite, such as the veA gene, which forms part of the VELVET complex. Different fungal strains compete for nutrients and space when they infect their hosts, and safer non-mycotoxigenic strains may be able to outcompete mycotoxigenic strains during colonization. To determine the possible utility of biopesticides based on the competitive exclusion of mycotoxigenic strains by non-toxigenic ones, we used A. carbonarius ΔotaA and ΔveA knockout mutants. Our results showed that during both in vitro growth and infection of grapes, non-mycotoxigenic strains could outcompete the wild-type strain. Additionally, the introduction of the non-mycotoxigenic strain led to a drastic decrease in OTA during both in vitro growth and infection of grapes.

Keywords: VELVET complex; competence; mycotoxin; outcompete; polyketide synthase; secondary metabolism.

Figure 1

Phenotypic traits of the wild-type strain of A. carbonarius ITEM 5010 (denoted as ‘wt’, black bars), two ectopic mutants (denoted as ‘E’, gray bars) and three knockout mutants (color-filled bars) of genes otaA (B–D) and veA (E–G), denoted as Δpks and ΔveA, respectively. (A) The front and back colony views of the different strains point-inoculated on PDA plates in the dark at 7 days post-inoculation. Growth area (B,E), conidiation (C,F) and OTA production (D,G) were tested on the PDA plates centrally point-inoculated with 5 uL of 1 × 10⁵ conidia/mL. After incubation at 28 °C for 5 days, colonies were scanned to determine the growth area with the ImageJ software. Three plugs were collected from the center, middle and inner parts of the colony for conidia determination and OTA extraction purposes. Values were normalized to those of the wt growing under the same conditions. Error bars represent the standard error of the mean of at least three biological replicates. The bars with different letters in the same panel are statistically different as determined by the one-way ANOVA and Tukey’s test (p < 0.05). nd. not detected under the assayed conditions.

Figure 2

Growth of the wild-type strain A. carbonarius ITEM 5010 (■) and two Δpks mutants (△ represents Δpks8a, and ▽ denotes Δpks27c) in the presence of different H₂O₂ (A), CFW (B), NaCl (C), SDS (D) and sorbitol (E) concentrations and at distinct pHs (F). Growth was determined as the area under the curve (AUC) after 7 days of incubation at 24 °C. The two-way ANOVA, followed by Tukey’s test (p < 0.05), was performed to determine the significant growth of the Δpks8a (denoted as *) and Δpks27c (denoted as Φ) knockout mutants compared to the wild type. Values represent the mean ± standard error of the mean of three biological replicates. The experiment was repeated twice.

Figure 3

Growth of the wt strain A. carbonarius ITEM 5010 (■) and two ΔveA (△ represents ΔveA10b, and ▽ denotes ΔveA12a) in the presence of different H₂O₂ (A), CFW (B), NaCl (C), SDS (D) and sorbitol (E), concentrations and at distinct pHs (F). Growth was determined as the area under the curve (AUC) after 7 days of incubation at 24 °C. The two-way ANOVA, followed by Tukey’s test (p < 0.05, indicated as *), was performed to establish whether significant differences existed between the knockout mutants and the wild type. Values represent the mean and standard error of the mean of three biological replicates. The experiment was repeated twice.

Figure 4

Competitiveness of the Δpks (A) and Δvea (B) knockout mutants against the mycotoxigenic wild-type (wt) strain A. carbonarius ITEM 5010 on potato dextrose broth (PDB). Competition assays were performed at the 1:0, 10:1, 1:1, 1:10 and 0:1 (wt:Δ) ratios. The expected values for the wt strain are indicated as gray dashed lines. The percentage of each strain was estimated by qPCR. Values are the mean of at least three biological replicates and error bars represent the standard error of the mean (SEM). OTA production during the competition assays of (C) the wt strain vs. the Δpks mutant, and the (D) wt vs. Δvea mutant. Values are normalized based on the OTA production by the wt strain.

Figure 5

OTA production (A,B) during the infection of grape berries. Co-inoculation of the mycotoxigenic wil-type (wt) strain A. carbonarius ITEM 5010 and the Δpks mutant (A,C), and the wt and ΔveA (B,D). Competitive assays were performed at the 1:0, 10:1, 1:1, 1:10, and 0:1 ratios (wt:Δ). Grape berries were incubated at 28 °C for up to 10 days. Values are the mean of three biological replicates and error bars represent the standard error of the mean (SEM). Values are normalized based on the production of OTA by the wt strain.