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Article

Mutations and Metabolic Factors Affecting Interaction of Toxigenic and Atoxigenic Aspergillus flavus

by
Szilvia Kovács
1,
Barbara Brendzsák
1,
Walter P. Pfliegler
2,
Lajos Nagy
3,
Kamirán Áron Hamow
4,
István Pócsi
2 and
Tünde Pusztahelyi
1,*
1
Central Laboratory of Agricultural and Food Products, Faculty of Agricultural and Food Sciences and Environmental Management, University of Debrecen, Böszörményi Str. 138, H-4032 Debrecen, Hungary
2
Department of Molecular Biotechnology and Microbiology, Institute of Biotechnology, Faculty of Science and Technology, University of Debrecen, Egyetem Tér 1, H-4032 Debrecen, Hungary
3
Department of Applied Chemistry, Institute of Chemistry, Faculty of Science and Technology, University of Debrecen, Egyetem Tér 1, H-4032 Debrecen, Hungary
4
Department of Plant Physiology and Metabolomics, HUN-REN Centre for Agricultural Research, Brunszvik Str. 2, H-2462 Martonvásár, Hungary
*
Author to whom correspondence should be addressed.
Microorganisms 2026, 14(2), 473; https://doi.org/10.3390/microorganisms14020473
Submission received: 21 December 2025 / Revised: 8 February 2026 / Accepted: 11 February 2026 / Published: 14 February 2026
(This article belongs to the Special Issue Fungal Biology and Interactions—3rd Edition)
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Abstract

Aflatoxin B1 (AFB1) produced by Aspergillus flavus poses severe food safety risks. Competitive exclusion using atoxigenic A. flavus strains offers a promising biological control approach to managing agricultural contamination by reducing populations of toxigenic strains and aflatoxin levels. However, reliable identification of atoxigenic strains remains challenging, and the mechanisms underlying competitive interactions between toxigenic and atoxigenic strains require clarification for effective implementation. Therefore, this study systematically analysed A. flavus strains for aflatoxin gene clusters and AFB1 production to address these critical gaps. Our analysis revealed that atoxigenic strains had intron losses and high-impact mutations in several genes, particularly aflL and aflLa, which affect aflatoxin biosynthesis. Key genes norA/aflE, verA/aflN, and omtA/aflP emerged as mutation hotspots, sometimes causing false-negative PCR results that complicate strain identification. Also, AFB1 production was inversely related to spore concentration on MEA medium, with fewer spores resulting in higher toxin levels. Interaction tests demonstrated that toxigenic and atoxigenic strains exhibited morphological changes only when co-cultured without physical separation, suggesting that this was mediated by diffusible molecules. Furthermore, differences in the levels of linoleic acid reduction products distinguished toxigenic from atoxigenic strains. These findings thus illuminate the complex genetic and metabolic factors influencing aflatoxin production and fungal interactions.

1. Introduction

Competitive exclusion is a biological phenomenon in which one organism outcompetes another for resources, thereby suppressing or eliminating the less competitive species in a shared environment. Regarding Aspergillus flavus, a filamentous fungus that produces aflatoxins (potent carcinogenic mycotoxins harmful to humans and animals), competitive exclusion is a critical strategy for controlling its growth and toxin production in crops.
For A. flavus, the known competitive-exclusion-based biocontrol materialised through the application of non-aflatoxin-producing (mainly atoxigenic) A. flavus strains against aflatoxigenic filamentous fungi, e.g., Aspergillus flavus and Aspergillus parasiticus. Atoxigenic traits are usually caused by mutations and have been proven to be naturally stable and irreversible [1]. Aflatoxigenic and non-aflatoxigenic A. flavus strains are not in competition under natural conditions and may cohabit without inhibiting each other’s lifecycles unless their natural ratios are disturbed. Therefore, the decrease in aflatoxin B1 (AFB1) production may be indirectly due to the reduced population size of the aflatoxin-producing A. flavus strain [2]. Application of biocontrol agents during crop growth minimises the presence of toxigenic populations in the field. Products like Afla-Guard® and AflaSafe® use this principle [3].
While AFB1 is a secondary metabolite, its production is advantageous for the population’s survival as it acts as an antioxidant against accumulating reactive oxygen species (ROS) and oxylipins [4]. When grown at different densities, A. flavus undergoes an oxylipin-mediated developmental shift that regulates conidiospore, sclerotia, and aflatoxin production in corn kernels [5].
Competitive exclusion is not only caused by the occupation of the niche by the atoxigenic organism, but also by the release of small effector molecules, such as volatile organic compounds (VOCs) [6,7,8,9]. These small molecules can often affect the population size of the aflatoxigenic strain and, consequently, reduce mycotoxin biosynthesis. Without direct contact between the toxigenic and atoxigenic cultures, and only shared headspace, it was implied that A. flavus produced both inhibitory and stimulatory gases [5]. In opposition to this theory, thigmoregulation, the role of physical contact between the atoxigenic and toxigenic hyphae, was also suggested as part of the interaction. According to Rao et al. [10], the aflatoxin biosynthesis genes are downregulated in the aflatoxigenic strain when it is in contact with a non-aflatoxigenic strain compared to the control.
Essential questions like the ability to produce other mycotoxins, genetic stability, persistence in the field under climatic changes, and the mechanism(s) that allow control over aflatoxigenic strains [11,12] are always key to answering. However, several factors, like the nature of the interaction, must be considered to improve recent application techniques. Here, we isolated and characterised A. flavus strains to investigate fungal interactions for further applications.

2. Results

2.1. Identification and Characterisation of the Isolates

From different commodities, 22 Aspergillus flavus strains from 72 Aspergillus section Flavi isolates were identified by ITS1-ITS4 PCR (Table 1) and calmodulin (caM) gene sequencing.
The aflatoxin gene cluster was tested using multiplex PCR targeting norA, aflR, and omtA (Figure 1A, Table 1), and the strains were subsequently tested for AFB1 production (Table 1). Based on HPLC-MS/MS-determined secondary metabolites present on corn kernels with the different A. flavus cultures, an isolate without mycotoxin production (atoxigenic strain) and others that produce other secondary metabolites, such as speradine or aspergillin, were identified and used for further analysis (Figure 1B). Toxigenic strains that produced only AFB1 were also identified (Table 1).

2.2. Non-Aflatoxigenic Isolates Have High-Impact Mutations in Aflatoxin Gene Clusters

Various intron losses (partial or complete) affected the aflatoxin biosynthetic gene cluster in AMK 27, AMK 30, and AMK 37 strains. In contrast, only a single intron was affected by 3′ truncation in the case of the aflM gene of the aflatoxigenic AMK 9 strain. In all four strains, high- or moderate-impact mutations affected the aflatoxin gene clusters (Figure 2A).
While moderate-impact (predominantly missense) variants were common in all four strains, regardless of aflatoxin production capability, high-impact variants affected the genes aflL and/or aflLa in all tested strains. In contrast, in the non-aflatoxigenic strains, AMK 37, five different genes were affected, and in both AMK 27 and AMK 30, seven genes were affected with high-impact mutations (Figure 2A).
The usual amplified genes of the aflatoxin gene cluster are norA/aflE, verA/aflN and omtA/aflP (Figure 2). However, as shown in Figure 2A, all these genes, especially verA/aflN, are hotspots for moderate-impact mutations that can lead to false-negative PCR results. The norA/aflE encodes a norsolorinic acid reductase protein. The verA/aflN gene encodes an enzyme, aromatic alcohol reductase, which converts norsolorinic acid to averantin, an early intermediate in the aflatoxin biosynthetic pathway. This step is critical for the progression of aflatoxin biosynthesis. The omtA/aflP gene, as a part of the aflatoxin biosynthetic gene cluster, encodes the enzyme O-methyltransferase A. The enzyme catalyses the methylation of sterigmatocystin to form O-methyl sterigmatocystin, a critical step in the later stages of the aflatoxin biosynthesis. The activity of OmtA is essential in producing aflatoxins, particularly AFB1 and B2. Interestingly, AFB1 non-producing strains showed high-impact mutations that differed from those in the toxigenic strain AMK 9. Genes (alfF, aflU, aflaW, and aflY) were identified as highly impacted genes that were differentially affected in the toxigenic strain. Only the region affected by the same mutation intensity was in the verB/aflL region.

2.3. Aflatoxin B1 Production Is Negatively Correlated with Increasing Fungal Biomass

AFB1 production depended on the spore numbers used for inoculation in the MEA medium (Figure 3). In the case of the largest inoculated spore count, the size of the mould colonies was the smallest on MEA, but the coverage was total. As the number of spores used during inoculation decreased, the size of the fungal colonies increased, and the amount of hyphal coverage on the Petri dish decreased.
The amounts of AFB1 produced in these experimental settings were determined. The lowest inoculated spore volume produced the highest AFB1 amount on the MEA medium. However, in the case of higher quantities (103 spores/plate and 104 spores/plate, or 105 spores/plate and 106 spores/plate), there was no significant difference between the produced AFB1 concentrations (p < 0.05).

2.4. Atoxigenic and Toxigenic Strains’ Interaction

The further studies were concentrated on the toxigenic and atoxigenic A. flavus strains as they were tested for interaction through presumable volatile and diffusible compounds on MEA. When separated in septated plates, the toxigenic (AMK 9) and atoxigenic A. flavus (AMK 27) strains did not show morphological changes (Figure 4). However, the cultures in the non-septated plates exhibited different colony morphology compared to those from the septated MEA plates. That was first explained by the different oxygen supplementation levels. Additionally, the toxigenic cultures on non-septated MEA plates exhibited differences when co-cultured with the atoxigenic strain. The toxigenic strain developed fewer aerial hyphae and produced more sclerotia than conidia; however, there was no difference in its sclerotium production when interacting with the atoxigenic strain. Interestingly, despite their ability to produce AFB1, the colonies were rarely in direct contact with each other. The presence of diffusible but, at the apparent temperature, non-volatile molecules could explain the phenomenon.
Analysis of whole plates from four biological replicates (n = 4) across three repeated experiments revealed clear differences in the metabolite profiles of the co-cultures. C-8 volatiles—notably 1-octen-3-ol, 3-octanone, 2-octenal,(E)-, and 2-octen-1-ol,(E)—were dominant components (Table 2). Compared with the atoxigenic colonies, toxigenic colonies produced significantly less 1-octen-3-ol but higher levels of 3-octanone (mean ± SD; see Table 2). Linoleic acid (18:2) concentrations did not differ significantly among co-cultures, suggesting that differences in the downstream metabolism of linoleic acid (e.g., altered oxylipin-processing enzyme expression or activity) rather than substrate availability underlie the observed volatile shifts (Figure 5). Importantly, toxinogen AMK 9–atoxinogen AMK 27 co-cultures exhibited an 82.5% reduction in total AFB1 (from 0.841 ± 0.04 µg/kg to 0.147 ± 0.02 µg/kg; p < 0.01), indicating that inter-strain metabolic interactions substantially suppress aflatoxin biosynthesis. Together, these results suggest that strain-dependent oxylipin processing may modulate volatile emission and aflatoxin production.

3. Discussion

The gene cluster for AF biosynthesis is approximately 75 kb in A. flavus and A. parasiticus [1,19]. The norA/aflE, verA/aflN, omtA/aflP, and aflR genes, which are essential for aflatoxin production, are typically tested to identify the presence of the AFB1 production cluster. The presence of a 75 kb gene cluster responsible for aflatoxin production was investigated simultaneously by detecting the three gene segments using multiplex PCR. This experiment was conducted based on the work of Geisen [20], who found that primer pairs designed for these genes can distinguish between A. flavus and A. parasiticus strains, as well as other fungi. Rashid et al. [21] also investigated the presence of these three genes, as well as the ver1 gene and its ability to produce aflatoxins, in 157 A. flavus and 36 A. parasiticus isolates. The simultaneous presence of all four genes was characteristic of only one toxin-producing isolate of A. parasiticus. According to Lee et al. [22], amplification of the aflR gene can detect aflatoxigenic strains of A. flavus. Varga et al. [23] also amplified the aflR, norA/aflE, and omtA/aflP genes to demonstrate the aflatoxin-producing ability of Aspergillus spp. isolates. However, based on our tests, a toxigenic and a non-aflatoxin-producing (atoxigenic) A. flavus isolate cannot be distinguished using the norA/aflE and omtA/aflP genes because of moderate-impact mutations in these genes.
Aflatoxin biosynthesis and export are mediated by vesicles and endosomes. Within the enzymatic cascade involved in AF synthesis, the protein NorA/AflE (AFLA_139310) was identified as having succinylated lysine residues [24]. Lysine succinylation of NorA/AflE influences the development of sclerotia and the production of AFs in A. flavus. In a multiple PCR, norA/aflE was not amplified in the atoxigenic AMK 27 strain. Moderate-impact mutations in norA/aflE were detected in the genomes of the non-aflatoxigenic and atoxigenic strains. Still, not in the toxigenic strain, which could explain the low sclerotial production and AFB1 levels. A. flavus has two distinct morphotypes, namely the “L-type” with large sclerotia (average diameter > 400 μm) and the “S-type”, which produces small sclerotia (average diameter < 400 μm) [18,25,26]. Aflatoxin production and sclerotia development are closely related [27,28,29] and co-regulated through the VeA regulator protein [28,29]. Here, the toxigenic AMK 9 strain produced S-type sclerotia, whereas the atoxigenic AMK 27 strain did not form sclerotia on the same medium; it produced only conidiospores.
Unfunctional verA/aflN also leads to the failure of aflatoxin biosynthesis [30]. Interestingly, active AflN was found to play a role in conidiation, as poorly developed conidiophores and decreased expression of the BrlA transcription factor (involved in conidiophore development) were observed in ΔaflN [31]. However, poor conidiophore production was not observed in the atoxigenic AMK 27 strain. Nevertheless, omtA/aflP and ver-1/aflM upregulation have also been observed in the ΔaflN strain [30].
In A. flavus, high-impact variants were detected in nadA/aflYa, omtB/aflO, hypB/aflLa, verB/aflL, and cypA/aflU genes by Furukawa et al. [32]. Presented analysis revealed “hot spots” for high-impact mutations and, besides the cluster starting genes (norB/aflF and cypA/aflU) and the last three genes (moxY/aflW, ordB/aflX, and hypA/aflY) of the cluster, two more mutation-sensitive genes (ordA/alfQ and hypB/aflLa) were found with an increased possibility of harbouring high-impact mutations in non-aflatoxigenic isolates. Meanwhile, the toxigenic strain harboured only two high-impact mutation sites: hypB/aflLa and verB/aflL.
Odamtten et al. [33] showed that the production of AFB1 increased 3 to 12-fold when the amount of A. flavus decreased, and Etcheverry et al. [34] found that the highest levels of AF were achieved with the lowest amount of inoculum applied at non-optimal aw. Yan et al. [35] found that the initial high spore concentration in the glucose-containing medium increased AFB1 production. In contrast, the peptone-containing medium had the opposite effect.
The artificial increase of propagules with atoxigenic strains is an applied technique to maintain competitive exclusion, as AFB1 production negatively correlates with increased fungal biomass. The initial high spore concentration led to lower AFB1 levels and greater mycelial growth. Metabolic studies revealed that this was due to inhibition of fatty acid biosynthesis, reduced intermediates of the tricarboxylic acid cycle, and increased activity of the pentose phosphate pathway. When grown at different densities, A. flavus undergoes an oxylipin-mediated developmental shift that regulates spore, sclerotial, and aflatoxin production [5]. These results suggest that Aspergilli, sensing the available nutrient sources, influence the intensity of mycelium growth or aflatoxin synthesis. This gives them a selection advantage, as they can adapt even with a lower nutrient source.
The production of specific effectors by non-aflatoxigenic A. flavus strains can inhibit the growth of aflatoxin-producing strains. Biocontrol strains can inhibit colony propagation [36] and subsequent AFB1 contamination. That affects the population size of the aflatoxigenic strain and, consequently, reduces mycotoxin production. Therefore, the decrease in AFB1 production may indirectly result from the suppressed population size of the aflatoxin-producing A. flavus strain [2]. Our findings supported this theory as increasing the atoxigenic population correlated negatively with the found AFB1 production. The decrease in AF production, together with the population depression of the aflatoxigenic fungus [37], supports the theory of competitive exclusion through robust propagation and fast colonisation by the non-aflatoxigenic fungus [2]. The role of physical contact (thygmoregulation) between hyphae was also suggested in the biocontrol process [10], but we were unable to demonstrate it.
Elmassry et al. [38] identified seventeen VOC molecules (e.g., 1-octen-3-ol, 3-octanone, and 2-pentylfuran) that are conserved in fungi. Guo et al. [39] confirmed 1-octen-3-ol as a common fungal emission. The fungus-specific compounds identified in our study were all C8 volatiles (VOCs) that cause a characteristic “mushroom smell” and exhibit close metabolic relationships among fungi [40].
From a biological perspective, 1-octen-3-ol is a key “fungal smell” and a signalling molecule that can inhibit spore formation [41] or, controversially, induce conidiation [42]. 3-octanone, also a volatile component, attracts or repels insects and, in fungi, inhibits spore germination [42]. 2-octenal (E) and 2-octen-1-ol (E): more pungent, slightly irritating odours, may participate in different interactions (antimicrobial, insect repellent). 2-Octenal (E) increased patulin biosynthesis in Penicillium expansum [43]. Recently, exposure to 1-octen-3-ol gradients in toxigenic A. flavus cultures was positively correlated with mycotoxin production, including AFB1 and its precursors. Additionally, negative correlations were found between the 1-octen-3-ol treatments and the relative abundance of oxylipin compounds [18].
A. flavus oxylipins are quorum-sensing molecules (QSM), which allow them to detect the density of fungal biomass. In A. flavus, which contains, in addition to dioxygenase genes ppoA, ppoB and ppoC, and another gene termed ppoD, studies have shown that these genes are necessary for density-dependent phenomena and regulate AF production as well as seed colonisation [44]. Deletion of the ppo genes reduced the pathogenicity of A. flavus [17]. In VOC-treated toxigenic A. flavus, a higher relative abundance of polyketide mycotoxins was detected, coupled with reduced growth rates [18]. By combining information on linoleic acid, 1-octen-3-ol concentration, and morphological characteristics, in which the intensities of conidiation and sclerotia production differed between the two investigated strains, we concluded that LOX and HPO activities should be differentially expressed; however, this hypothesis requires further investigation. With low LOX activity, low extracellular oxylipin concentrations, increased sclerotia production, decreased conidiation [16], and a concomitant increase in AFB1 biosynthesis, toxigenic strains [5,16,17] exhibit similar patterns to our AMK 9 toxigenic isolate. Atoxigenic culture (AMK 27) morphology and VOC pattern suggested significantly higher enzyme activities, resulting in higher 1-octen-3-ol levels, while morphology suggested increased oxylipin production. A presumably high level of oxylipin could improve aerial hyphal growth and conidia development in the AMK 27 atoxigenic strain. In co-culturing, besides mutation in the aflatoxin gene cluster from the atoxigenic strain’s part, high enzyme activities in oxylipin biodegradation could lower the AFB1 synthesis rate.
As a conclusion, for a competitive exclusion, besides a stable mutation(s) in the aflatoxin gene cluster, differences in oxylipin metabolism also contribute to the antagonist effect between the two competing strains. At the same time, thigmoregulation and gas-phase molecules were not proven to participate in the interaction.

4. Materials and Methods

4.1. Materials

All culture media used were purchased from VWR International Ltd. (Debrecen, Hungary) or Scharlab Hungary Ltd. (Debrecen, Hungary). All chemicals and reagents used were analytical or chromatographic grade and were purchased from VWR International (Debrecen, Hungary) and Merck Life Science Ltd. (Budapest, Hungary). Ultra-pure water was obtained from a Millipore Elix purification system (Merck Millipore, Darmstadt, Germany).

4.2. Isolation, Identification and Cultivation of Aspergillus spp.

Chloramphenicol-yeast extract-glucose agar was inoculated with randomly sampled ground grains (corn and wheat) at decimal dilutions and incubated at 30 °C for 5 days. The fungal colonies, identified microscopically as Aspergillus spp., were collected and streaked onto malt extract agar (MEA) medium (20 g glucose, 10 g malt extract, 5 g yeast extract, and 15 g agar/L).

4.3. Identification of the Aspergillus spp.

For genomic DNA preparation, the fungal cells were disrupted with MagNALyser Green (Roche Diagnostics GmbH, Mannheim, Germany). DNA isolation was performed using the Nucleospin Plant II kit (Macherey-Nagel GmbH & Co. KG, Düren, Germany) according to the manufacturer’s instructions.
Multiplex PCR for the norA, aflR, and omtA genes of the aflatoxin gene cluster, as well as ITS1-ITS4 [45] and calmodulin (CaM) [46], was performed as described by Kovács and Pusztahelyi [47]. For the purification of amplified gene sequences, the NucleoSpin Gel kit and PCR Clean-up kit (Macherey-Nagel GmbH & Co. KG, Düren, Germany) were used, and the resulting DNA samples were sequenced (BIOMI Ltd., Gödöllő, Hungary). The data obtained were analysed using MEGA 11 (version 11.0.13) and compared with the NCBI GenBank database [48].

4.4. Co-Cultivation of the Atoxigenic and Toxigenic Strains

Co-cultivation of the atoxigenic and toxigenic strains was performed in septated and non-septated Petri dishes containing MEA medium, with 5 µL of 107 spores/mL of A. flavus inoculated at a single point and incubated for 5 days at 30 °C. Spore concentration was determined in a hemocytometer (Bürker (Paul Marienfeld GmbH and Co KG, Lauda-Königshofen, Germany)).

4.5. Detection of Fungal Secondary Metabolites with HPLC-MS Technique

Cultures of ground maize kernels were collected in sterile Stomacher homogeniser bags (BagLight 400, Interscience, Saint Nom, France) and homogenised with chloroform in a Stomacher homogeniser (Masticator, IUL Instruments, Cl Ciutat, D’Asuncion, Barcelona, Spain) for 2 min, which was repeated twice. First, the homogenised samples were filtered through filter paper (Grade 292, Ahlstrom-Munksjo Munktell, Jönköping, Sweden) into spherical flasks and evaporated in Rotavapor R114 (Büchi Labortechnik AG, Flawill, Switzerland). Then, after adding 2 mL of mobile phase (methanol: water, 45:55), the solute was filtered through a Millex-GV 0.22 μm filter (Merck KGaA, Darmstadt, Germany).
The HPLC-MS device was set with a Waters Separations module (2695) and a Waters 2996 Photodiode Array detector (Waters, Milford, MA, USA). The metabolites were separated on an Agilent Zorbax SB-C18, 4.6 × 75 mm, 3.5 μm column (Agilent Technologies, Santa Clara, CA, USA). The flow rate was 0.5 mL/min, the elution time was 60 min, and the temperature was 40 °C. UV detection was performed at 225 nm. The methanol: water gradient was as follows (Table 3). The mass spectrum was captured by a Bruker microTOF-Q mass spectrometer (Bruker Daltonics GmbH, Bremen, Germany) equipped with an Atmospheric Pressure Chemical Ionization (APCI) ion source. The ion source temperature was 390 °C. Bruker Daltonics Data Analysis software (version 3.4) was used to evaluate the results. During the analysis, several secondary metabolites were targeted [49].

4.6. Aflatoxin Gene Cluster Analysis

Library preparation was performed using tagmentation with the Nextera DNA Flex Library Prep kit (Illumina, San Diego, CA, USA) according to the manufacturer’s protocol. Sequencing was performed using 150 bp paired-end reads on an Illumina NextSeq 500 system (Illumina Inc., San Diego, CA, USA), resulting in approximately 140× coverage of the nuclear genome. The Illumina FASTQ sequencing files were trimmed and filtered using fastp 1.0 [50] for further analysis. The Illumina reads of the isolates were mapped to the ASM901741v1 (GenBank accession: GCA_009017415.1) reference genome using BWA 0.7.17. [51]. Sorted BAM files were obtained from these using Samtools 1.7. [52] and Picard-tools 2.23.8. was used to mark duplicated reads [53].
BEDTools 2.30.0 [54] was used to calculate the median coverage of chromosomes in 10,000-base windows sliding every 5000 bases, and the median coverage of the whole chromosomes and the mitochondria. The coverage of the aflatoxin biosynthetic gene cluster was compared to the median coverage of the chromosomes and local sliding windows. The gene cluster in the .bam files was then manually inspected using IGV 2.11 [55]. This allowed the identification of exon/intron losses, as well as partial losses.
A cohort calling strategy was applied for allele calling after mapping was performed on 283 previously sequenced A. flavus and A. oryzae genomes downloaded from NCBI SRA. Using BAM files, local realignment around indels and joint variant calling and filtering for the isolates were performed with GATK 4.1.9.0. [53,56]. In the first step, genomic VCF files were generated using the GATK 4.1.9.0. Haplotype Caller, and joint genotyping of the gVCF files was performed. Using this initial VCF, we recalibrated base quality scores with GATK and called the BAM files again in gVCF mode. After this joint calling, either SNPs or INDELS were selected for the resulting VCF files. SNPs were filtered according to the parameters [57]: QD < 5.0; QUAL < 30.0; SOR > 3.0; FS > 60.0; MQ < 40.0; MQRankSum < −12.5; ReadPosRankSum < −8.0. INDELS were filtered according to QD < 5.0; QUAL < 30.0; FS > 60.0; ReadPosRankSum < −20.0. INDELS were then left-aligned. For the final VCF files, INDELS and SNPs were merged and filtered, and the non-variant sites were removed. We identified mutations in genes of the aflatoxin cluster that potentially affect protein function using SNPEff 5.1 [58] based on the reference genome’s annotation. VCF files were annotated with SNPEff, and the high and medium-effect variant positions were further analysed using SNPEff’s output table. Aspergillus flavus NRRL 3367 (ASM901741v1; GenBank accession: GCA_009017415.1) was applied as the reference strain.

4.7. Effect of Biomass Density on Aflatoxin Formation

A decimal dilution series was prepared from the spore suspension of Aspergillus flavus AMK 9, and 100 μL of spore suspension was spread on the MEA medium. After incubation, morphological differences were observed. Then, the samples were prepared to determine AFB1 concentration in the HPLC-FLD measurement.

4.8. Fungal Colonisation on Corn Kernels

50 g of Mv 251 (FAO 280) maize hybrid (Martonvásár, Hungary) was washed with 5% sodium hypochlorite, sterile distilled water, and 70% ethanol, then again with sterile distilled water. Then, the surface-sterilised kernels were inoculated with 106 spores/mL and incubated in the dark at 30 °C for 7 days. After incubation, the kernels were dried at 60 °C for 48 h and then ground. The AFB1 content was measured by HPLC-FLD.
The production of other secondary metabolites in corn kernels was also investigated. For the test, 50 g of Mv 251 was treated with 70% ethanol and washed with distilled water. Then, it was placed in a sterile Erlenmeyer flask and inoculated with 106 spores. The incubation period lasted 7 days at 30 °C in the dark. The AFB1 production was determined using the HPLC-FLD method. Colonisation was evaluated as the percentage of mould cover.

4.9. Detection of Aflatoxin B1 with HPLC-FLD

Sample preparation was done according to Section 2.2. HPLC-FLD detection of mycotoxins was performed on a Dionex Ultimate 3000 (Dionex Softron GmbH, Germering, Germany) system. The measurement was conducted according to Adácsi et al. [59]. The AFB1 content of 20 μL samples was determined by HPLC-FLD using a Hibar 125-4 Lichrospher 100RP-18 (5 μm) column (Merck KGaA, Darmstadt, Germany) with a flow rate of 1 mL/min and detected by a fluorescence detector at ex 360 nm, em 440 nm after post-column UV derivatization (Romer Labs, Tulln, Austria). In addition, Biopure Aflatoxin B1 analytical standard solution (Romer Labs, Tulln, Austria) was applied to the column. The relative standard deviation, as the absolute value of the coefficient of variation, was calculated and found to be under 10% in all cases.

4.10. Headspace Solid Phase Micro Extraction Gas Chromatography Mass Spectrometry (HS-SPME-GC-MS) Analysis

Co-cultivation of the atoxigenic and toxigenic strains was performed in non-septated Petri dishes containing MEA medium, with 2 µL of 107 spores/mL A. flavus (AMK 9 and AMK 27) inoculated by point inoculation and incubated for 7 days at 30 °C. The initial spore concentration was determined in a hemocytometer (Bürker, Paul Marienfeld GmbH and Co KG, Lauda-Königshofen, Germany).
Agar plates were stored at −80 °C, homogenised, and kept frozen in liquid N2. One gram of the sample was weighed into 25 mL headspace vials. For sampling and analysis, an Agilent (Santa Clara, CA, USA) 7890B GC equipped with a Gerstel (Gerstel GmbH & Co. KG, Mülheim an der Ruhr, Germany) multipurpose autosampler (MPS) and CIS4 inlet coupled to an Agilent 5977B MS (Agilent Technologies, Inc., Santa Clara, CA, USA) was utilised. Helium 6.0 was used as a carrier gas. HS-SPME sampling was performed using a Gerstel MPS (Gerstel GmbH & Co. KG, Ruhr, Germany). Samples were incubated at 85 °C for 10 min, then extracted with an 80 µm-thick DVB/Carbon WR/PDMS phase SPME 1 cm fibre at 85 °C for 25 min, inserted into the septumless head of the GC CIS4 inlet kept at 270 °C, and thermally desorbed for 5 min. To increase throughput, the MPS was set to overlapping mode, allowing subsequent sample extractions to start during GC runtime.
The GC system was equipped with a J&W HP-5MS UI 30 m × 0.25 mm × 0.25 µm capillary column (Agilent Technologies, Inc., Santa Clara, CA, USA). The column flow was 1.15 mL/min, the septum purge flow was 3 mL/min, and a split ratio of 10 was applied at the start of analysis. After 3 min of desorption, a split flow of 57.5 mL/min was set. The oven temperature programme was as follows: 40 °C held for 2 min, then increased by 5 °C/min to 160 °C, then by 15 °C/min to 280 °C. As a post-run function, the column was flushed by heating it to 320 °C at a column flow of 1.5 mL/min for 3 min, then returned to initial conditions, resulting in a total GC runtime of 42 min, including cool-down and equilibration for the next injection.
For MS detection, EI ionisation (70 eV) was used, and the MS was tuned and calibrated with perfluorotributylamine, according to the manufacturer’s instructions. The auxiliary heater was set to 250 °C, the MS source to 230 °C, and the MS quad to 150 °C. Mass spectra were collected in the scan acquisition mode, with a cut time of 1 min. For identification, the scan event was configured to monitor the m/z range 35–500 at 9 scans/s with a 0.1 m/z step size. The reproducibility and linearity of the GC-MS method were verified. HS-SPME conditions were optimised for pepper leaf samples to detect the broadest possible range of components. Mass Hunter Workstation Qualitative Navigator B.08.00 and Quantitative Analysis B.09.00 software tools (Agilent Technologies, Inc., Santa Clara, CA, USA) were used for evaluation and quantitation. Identification of compounds was based on background-subtracted mass spectra that were identified with the help of the NIST/EPA/NIH Mass Spectral Library v17 (2017) and the Wiley Registry® of Mass Spectral Data (10th edition; 2014), and by utilising n-alkane retention indices with a C7-30 n-alkane mix (Sigma-Aldrich, St. Louis, MI, USA). The highest consistent library hit (exceeding 75% similarity with reverse search for mass spectra), and retention index score matches were accepted for the identification of volatile compounds. After subtracting the VOC from blank and untreated agar plates, the differentiating compounds were quantified based on the most intense 3–10 ions (EIC), yielding 80–90% of the total ion response for each compound to improve selectivity. To establish a matrix-matched calibration for 1-octen-3-ol and 3-octanone and n-alkane series of C7-30 (Merck/Millipore Sigma, Burlington, MA, USA), they were spiked in a mix by adding 2 µL ethanol containing 40–100–200–400 ng to untreated agar samples through the closed HS vials, for n-alkanes, 200 ng was spiked as a one-point calibration. The calibration obtained for 3-octanone was used to quantify 2-octenal (E) and 2-octen-1-ol (E) relative to 3-octanone. Heneicosane was used to calibrate linoleic and oleic acids; their LOQs were 2 ng/g. 20 ng/g LOQ was detected for 1-octen-3-ol, 3-octanol and 2-octen-1-ol, E-, while for 2-octenal (E)-, LOQ was 10 ng/g.

4.11. Statistics

The results were analysed using paired Student t-tests with a significance level of p < 0.05.

Author Contributions

Conceptualisation, T.P.; methodology, T.P., S.K., L.N. and K.Á.H.; validation, K.Á.H., T.P., L.N. and W.P.P.; investigation, B.B., S.K., W.P.P., T.P., L.N. and K.Á.H.; resources, T.P. and I.P.; writing—original draft preparation, S.K.; writing—review and editing, T.P.; visualisation, S.K.; supervision, I.P.; project administration, T.P. All authors have read and agreed to the published version of the manuscript.

Funding

This research is funded by the National Research, Development, and Innovation Fund of Hungary project no. 2018-1.2.1-NKP-2018-00002. Project no. STARTING 152400 has been implemented with the support provided by the Ministry of Culture and Innovation of Hungary from the National Research, Development and Innovation Fund, financed under the National Research Excellence Program STARTING_25 subprogram funding scheme. Project no. TKP2020-IKA-04 has been implemented with the support provided by the Ministry of Culture and Innovation of Hungary from the National Research, Development and Innovation Fund, financed under the Thematic Excellence Programme 2020 funding scheme.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The AMK 27 atoxigenic strain has been stored as a Patent Deposit at NCAIM culture collection.

Acknowledgments

The authors acknowledge Erika Sőrés and Csaba Kiss for technical support.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
AFAflatoxin
AFB1Aflatoxin B1
VOCVolatile Organic Carbons
ROSReactive Oxygen Species
LOQLimit of Quantification
MEAMalt Extract Agar
DWDry Weight

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Microorganisms 14 00473 g001
Figure 1. Characterisation of the Aspergillus section Flavi isolates. (A) Multiplex PCR for three genes of the aflatoxin gene cluster: omtA, aflR and norA. (1.) 1 kb DNA ladder; (2.) Multiplex PCR result for AMK 9 isolate. (B) HPLC-UV chromatograms recorded at 225 nm of the selected fungal isolates inoculated on ground corn. (1.) Chromatogram of uninoculated ground corn at 225 nm; (2.) chromatogram of AMK 37 inoculated corn; (3.) chromatogram of AMK 9 inoculated corn. Compounds indicated were identified by APCI-MS/MS.
Figure 1. Characterisation of the Aspergillus section Flavi isolates. (A) Multiplex PCR for three genes of the aflatoxin gene cluster: omtA, aflR and norA. (1.) 1 kb DNA ladder; (2.) Multiplex PCR result for AMK 9 isolate. (B) HPLC-UV chromatograms recorded at 225 nm of the selected fungal isolates inoculated on ground corn. (1.) Chromatogram of uninoculated ground corn at 225 nm; (2.) chromatogram of AMK 37 inoculated corn; (3.) chromatogram of AMK 9 inoculated corn. Compounds indicated were identified by APCI-MS/MS.
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Figure 2. (A) Presentation of the occurrence of the high- and moderate-impact variations in genes of the aflatoxin biosynthetic gene cluster for two non-aflatoxigenic (AMK 30, AMK 37), an atoxigenic (AMK 27) and a toxigenic (AMK 9) Aspergillus flavus strain. The bars present the frequency of mutations in a specific gene. (B) Organisation of the aflatoxin gene cluster, including the old and new cluster gene nomenclatures. This figure was adapted from Caceres et al. [13]. Red dotted lines indicate the binding sites of the regulatory Cys6Zn2-type DNA-binding protein AflR in the above pathway cluster.
Figure 2. (A) Presentation of the occurrence of the high- and moderate-impact variations in genes of the aflatoxin biosynthetic gene cluster for two non-aflatoxigenic (AMK 30, AMK 37), an atoxigenic (AMK 27) and a toxigenic (AMK 9) Aspergillus flavus strain. The bars present the frequency of mutations in a specific gene. (B) Organisation of the aflatoxin gene cluster, including the old and new cluster gene nomenclatures. This figure was adapted from Caceres et al. [13]. Red dotted lines indicate the binding sites of the regulatory Cys6Zn2-type DNA-binding protein AflR in the above pathway cluster.
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Figure 3. Biomass effect on aflatoxin B1 production. Differences in the production of aflatoxin B1 as a function of the inoculation spore count of Aspergillus flavus AMK 9 on MEA medium (n = 3). The same letter designation indicates the non-significant difference at the p < 0.05 level.
Figure 3. Biomass effect on aflatoxin B1 production. Differences in the production of aflatoxin B1 as a function of the inoculation spore count of Aspergillus flavus AMK 9 on MEA medium (n = 3). The same letter designation indicates the non-significant difference at the p < 0.05 level.
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Figure 4. Point inoculations of toxigenic (Tox; AMK 9) and atoxigenic (Atox; AMK 27) Aspergillus flavus colonies on MEA in septated (upper row) and non-septated (lower row) Petri dishes.
Figure 4. Point inoculations of toxigenic (Tox; AMK 9) and atoxigenic (Atox; AMK 27) Aspergillus flavus colonies on MEA in septated (upper row) and non-septated (lower row) Petri dishes.
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Microorganisms 14 00473 g005
Figure 5. In fungi, the lipoxygenase enzymes (LOX) oxidise linoleic acid (C18:2). As a result, hydroperoxides [e.g., in fungi, (8E,10R,12Z)-10-hydroperoxyoctadeca-8,12-dienoic acid (10-HpODE)] are produced. Hydroperoxidase (HPO) cleaves these hydroperoxides into C8 aldehydes and alcohols. The main products are 1-octen-3-ol (“mushroom alcohol”), 3-octanone (a ketone), 2-octenal (E) (an unsaturated aldehyde), and 2-octen-1-ol (E) (an unsaturated alcohol). Since HpODE oxylipin is a quorum-sensing molecule (QSM), low oxylipin levels would be displayed with low mycelial growth and conidiospore development, while increasing the number of sclerotia. Low HpODE and high 1-octane-1-ol result in increased aflatoxin B1 (AFB1) production. Further transformations between the metabolites are possible: 1-octen-3-ol and 3-octanone can be interconverted by oxidation/reduction (catalysed by alcohol dehydrogenases and ketone reductases). 2-octenal (E) → 2-octen-1-ol (E) conversion by possible reduction (aldehyde → alcohol conversion by alcohol dehydrogenase) can also take place [5,16,17,18].
Figure 5. In fungi, the lipoxygenase enzymes (LOX) oxidise linoleic acid (C18:2). As a result, hydroperoxides [e.g., in fungi, (8E,10R,12Z)-10-hydroperoxyoctadeca-8,12-dienoic acid (10-HpODE)] are produced. Hydroperoxidase (HPO) cleaves these hydroperoxides into C8 aldehydes and alcohols. The main products are 1-octen-3-ol (“mushroom alcohol”), 3-octanone (a ketone), 2-octenal (E) (an unsaturated aldehyde), and 2-octen-1-ol (E) (an unsaturated alcohol). Since HpODE oxylipin is a quorum-sensing molecule (QSM), low oxylipin levels would be displayed with low mycelial growth and conidiospore development, while increasing the number of sclerotia. Low HpODE and high 1-octane-1-ol result in increased aflatoxin B1 (AFB1) production. Further transformations between the metabolites are possible: 1-octen-3-ol and 3-octanone can be interconverted by oxidation/reduction (catalysed by alcohol dehydrogenases and ketone reductases). 2-octenal (E) → 2-octen-1-ol (E) conversion by possible reduction (aldehyde → alcohol conversion by alcohol dehydrogenase) can also take place [5,16,17,18].
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Table 1. Characterisation of selected Aspergillus flavus strains by ITS homology and the closest homologues, result of the multiplex PCR of the aflatoxin gene cluster’s genes, secondary metabolite profile, and colonisation capabilities on corn. The AMK 27 strain was identified as a natural atoxigenic isolate.
Table 1. Characterisation of selected Aspergillus flavus strains by ITS homology and the closest homologues, result of the multiplex PCR of the aflatoxin gene cluster’s genes, secondary metabolite profile, and colonisation capabilities on corn. The AMK 27 strain was identified as a natural atoxigenic isolate.
Isolate CodeHighest Homology to
(NCBI)		PCR Detection of AF Gene Cluster GenesSecondary
Metabolite
(µg/mL)		Colonisation #
norAaflRomtA
AMK 9Aspergillus flavus GFRS30
ITS1-100%, ITS4-100%		+++AFB1: 0.643 ± 0.09****
AMK 27Aspergillus flavus GFRS30
ITS1-98%, ITS4-100%		−+−− $**
AMK 30Aspergillus flavus PHY35
ITS1-97%, ITS4-99%		+++3-OH-speradine A,
speradine I,
aspergilline D		**
AMK 37Aspergillus flavus GFRS30
ITS1-98%, ITS4-100%		+++speradine F**
$ Limit of Quantification (LOQ) was 0.02 ng/mL AFB1. # % mould coverage on Mv 251 corn kernels. ****—100%; **—50% coverage.
Table 2. Volatile metabolites from co-culturing point inoculations of toxigenic (Tox; AMK 9) and atoxigenic (Atox; AMK 27) Aspergillus flavus colonies on non-septated MEA dishes (n = 4) differed from MEA metabolites. Significant differences are shown with different letters (p < 0.05). Lowercase letters are used to compare results for the same component. Compounds marked in grey also contribute to plant distress [14,15].
Table 2. Volatile metabolites from co-culturing point inoculations of toxigenic (Tox; AMK 9) and atoxigenic (Atox; AMK 27) Aspergillus flavus colonies on non-septated MEA dishes (n = 4) differed from MEA metabolites. Significant differences are shown with different letters (p < 0.05). Lowercase letters are used to compare results for the same component. Compounds marked in grey also contribute to plant distress [14,15].
Co-InoculationLinoleic Acid
(ng/g DW)		1-octen-3-ol
(ng/g DW)		3-octanone
(ng/g DW)		2-octenal (E)-
(ng/g DW)		2-octen-1-ol, (E)-
(ng/g DW)		AFB1
(µg/kg)
Atox-Atox8.77 ± 15.15a1161 ± 117a26.2 ± 6.08c10.9 ± 1.92b33.7 ± 5.55b<0.01c
Tox-Tox4.85 ± 2.35a752 ± 64b **194 ± 37.9a **19.7 ± 4.43a *59.6 ± 15.3a **0.841 ± 0.04a
Atox-Tox10.14 ± 9.05a753 ± 86.9b **82.7 ± 30.8b *10.3 ± 1.92b38.9 ± 12.1b0.147 ± 0.02b **
MEA<2b<20c<20d5.20 ± 2.00c<20cN.D.
DW, dry weight; * p < 0.05; ** p < 0.01; N.D. not determined.
Table 3. Programmed gradient elution on HPLC-MS.
Table 3. Programmed gradient elution on HPLC-MS.
Time (min)MeOH (%)H2O (%)
01090
508020
548020
54.11090
601090
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Kovács, S.; Brendzsák, B.; Pfliegler, W.P.; Nagy, L.; Hamow, K.Á.; Pócsi, I.; Pusztahelyi, T. Mutations and Metabolic Factors Affecting Interaction of Toxigenic and Atoxigenic Aspergillus flavus. Microorganisms 2026, 14, 473. https://doi.org/10.3390/microorganisms14020473

AMA Style

Kovács S, Brendzsák B, Pfliegler WP, Nagy L, Hamow KÁ, Pócsi I, Pusztahelyi T. Mutations and Metabolic Factors Affecting Interaction of Toxigenic and Atoxigenic Aspergillus flavus. Microorganisms. 2026; 14(2):473. https://doi.org/10.3390/microorganisms14020473

Chicago/Turabian Style

Kovács, Szilvia, Barbara Brendzsák, Walter P. Pfliegler, Lajos Nagy, Kamirán Áron Hamow, István Pócsi, and Tünde Pusztahelyi. 2026. "Mutations and Metabolic Factors Affecting Interaction of Toxigenic and Atoxigenic Aspergillus flavus" Microorganisms 14, no. 2: 473. https://doi.org/10.3390/microorganisms14020473

APA Style

Kovács, S., Brendzsák, B., Pfliegler, W. P., Nagy, L., Hamow, K. Á., Pócsi, I., & Pusztahelyi, T. (2026). Mutations and Metabolic Factors Affecting Interaction of Toxigenic and Atoxigenic Aspergillus flavus. Microorganisms, 14(2), 473. https://doi.org/10.3390/microorganisms14020473

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