Search Results (27)

Ochratoxin A (OTA) is a major mycotoxin contaminant in grapes and their products, and Aspergillus carbonarius is its main producer. Controlling the growth of A. carbonarius is therefore critical for mitigating OTA contamination. Plant-derived perillaldehyde, with good antifungal activity and safety, has garnered growing attention. However, current understanding of how perillaldehyde affects A. carbonarius growth and OTA production remains poorly characterized. In this study, we systematically investigated the antifungal and antimycotoxigenic effects of perillaldehyde against A. carbonarius and explored the underlying mechanisms. The results showed that perillaldehyde could alter the mycelial morphology and damage the cell integrity of A. carbonarius. Additionally, perillaldehyde could diminish the total antioxidant capacity and impair the energy metabolism of A. carbonarius. Transcriptome analysis showed that the expressions of all the known conserved OTA biosynthetic genes and two OTA transport-related genes were significantly down-regulated, indicating that perillaldehyde could directly inhibit their expression. In conclusion, perillaldehyde can significantly inhibit OTA production by directly disrupting OTA biosynthesis and transport and inhibiting the growth of A. carbonarius. Thus, perillaldehyde has the potential to be used as a natural fungicide or alternative food preservative in grapes and their products, owing to its strong antifungal and antimycotoxigenic effects on A. carbonarius. Full article

Histone post-translational modifications (HPTMs) can affect gene expression by rearranging chromatin structure. Between these, histone methylation is one of the most studied in filamentous fungi, and different conserved domains coding for methyltransferase were found in Aspergillus spp. genomes. In this work, the role of the histone methyltransferases AcDot1 and AcRmtA in the mycotoxigenic fungus Aspergillus carbonarius was investigated, obtaining knockout or overexpression mutants through Agrobacterium tumefaciens-mediated transformation (ATMT). A. carbonarius is responsible for grape-bunch rot, representing the major source of ochratoxin A (OTA) contamination on grapes. In vivo conditions, the deletion of Acdot1 or AcrmtA resulted in upregulation of growth when the isolates were cultivated on a minimal medium. The influence of Acdot1 on the OTA biosynthesis was differently affected by culture conditions. On rich media, an increase in OTA accumulation was observed, while on minimal medium, lower OTA concentrations were reported. The deletion of AcrmtA always resulted in lower OTA accumulation. However, the expression of OTA biosynthesis genes was regulated by both histone methyltransferases. Of the six analyzed OTA genes, three of them showed altered expression in the knockout mutants, and otaB and otaR1 were common between both mutants. Furthermore, both AcDot1 and AcRmtA play a role in oxidative stress response, induced by 1 mM hydrogen peroxide, by modulating growth, conidiation and OTA biosynthesis. Neither the deletion nor the overexpression of the Acdot1 or AcrmtA affected virulence, while both the sporulation and OTA production were negatively affected in vivo by the deletion of AcrmtA. Full article

Aflatoxin B1 (AFB1) and Ochratoxin A (OTA) are considered the most important mycotoxins in terms of food safety. The aim of this study was to evaluate the hepatotoxicity of AFB1 and OTA exposure in Wistar rats and to assess the beneficial effect of fermented whey (FW) and pumpkin (P) as functional ingredients through a proteomic approach. For the experimental procedures, rats were fed AFB1 and OTA individually or in combination, with the addition of FW or a FW-P mixture during 28 days. For proteomics analysis, peptides were separated using a LC-MS/MS-QTOF system and differentially expressed proteins (DEPs) were statistically filtered (p < 0.05) distinguishing males from females. Gene ontology visualization allowed the identification of proteins involved in important biological processes such as the response to xenobiotic stimuli and liver development. Likewise, KEGG pathway analysis reported the metabolic routes as the most affected, followed by carbon metabolism and biosynthesis of amino acids. Overall, the results highlighted a strong downregulation of DEPs in the presence of AFB1 and OTA individually but not with the mixture of both, suggesting a synergistic effect. However, FW and P have helped in the mitigation of processes triggered by mycotoxins. Full article

Ochratoxin A (OTA) is a mycotoxin, a common contaminant of grapes and their derivatives, such as wine, and classified as possible human carcinogen (group 2B) by the International Agency for Research on Cancer (IARC). Aspergillus carbonarius is the main producer of OTA in grapes. The stability of the molecule and the poor availability of detoxification systems makes the control of A. carbonarius in vineyards the main strategy used to reduce OTA contamination risk. Several molecular methods are available for A. carbonarius detection, but the correlation between the abundance of fungal population and OTA contamination needs to be improved. This study aimed at the development of innovative quantitative PCR (qPCR) and digital droplet PCR (ddPCR) tools to quantify the mycotoxigenic fractions of A. carbonarius strains on grapes, based on the key gene AcOTApks in the pathway of OTA biosynthesis. Different primers/probe sets were assessed, based on their specificity and sensitivity. This method allowed to quantify up to 100 fg∙µL⁻¹ [cycle of quantification (Cq) = 37] and 10 fg∙µL⁻¹ (0.38 copies∙µL⁻¹) of genomic DNA (gDNA) from A. carbonarius mycelium in qPCR and ddPCR, respectively. The sensitivity as to artificially contaminated must samples was up to 100 conidia (Cq = 38) and 1 conidium (0.13 copies∙µL⁻¹) with qPCR and ddPCR, respectively. Finally, the methods were validated on naturally infected must samples, and the quantification of the fungus was in both cases highly correlated (r = +0.8) with OTA concentrations in the samples. The results showed that both analytical methods can be suitable for improving the sustainable management of OTA contamination in grapes and their derivatives. Full article

Aspergillus carbonarius is considered one of the main fungi responsible for black and sour rot in grapes, as well as the production of the carcinogenic mycotoxin ochratoxin A. The global regulatory methyltransferase protein LaeA controls the production of various secondary metabolites in Aspergillus species, as well as influences sexual and asexual reproduction and morphology. The goal of this study was to investigate the role of the regulatory gene AclaeA in physiology, virulence, and ochratoxin A (OTA) production by deleting this gene from the genome of a wild-type A. carbonarius strain. The evaluation data on the morphological characteristics, virulence experiments in three different grape varieties, and OTA analysis of ΔAclaeA mutants showed that the growth and the OTA production by ΔAclaeA strains were significantly reduced. The mutant strains were also less virulent, producing 40–50% less conidia in three different cultivars of grape berries. Additionally, the gene AclaeA was considerably repressed after the application of three commercial biopesticides (Trianum-P^®, Vacciplant^®, and Serenade^® Max) and the essential oils (EOs) cinnamon, geranium, and thyme, which were also shown to inhibit OTA biosynthesis in A. carbonarius. The study of the regulatory gene AclaeA can contribute to a broader understanding of the role of secondary metabolites during A. carbonarius—grape interactions, as well as the discovery of the mode of action of biological plant protection products and EOs against this mycotoxigenic fungus. Full article

Ochratoxin A (OTA) is a naturally occurring mycotoxin mainly produced by certain species of Aspergillus and Penicillium and is a serious threat to human health and food safety. Previous studies showed that Brevundimonas naejangsanensis ML17 can completely degrade 1 μg/mL of OTA. The aim of this study was to investigate the degradation effect of ML17 at different concentrations of OTA, and specifically, to investigate the mechanism of OTA degradation by ML17. The growth of ML17 was not affected by exposure to 6 μg/mL OTA within 24 h. ML17 could almost completely degrade 12 μg/mL of OTA within 36 h, converting it into the non-toxic OTα and L-phenylalanine. Transcriptomic analysis showed that 275 genes were upregulated, whereas three genes were downregulated in ML17 under the stress of 1 μg/mL OTA. Functional enrichment analysis showed that exposure to OTA enhanced translation, amide and peptide biosynthesis and metabolism, promoted oxidative phosphorylation, and increased ATP production. Further analysis revealed that, when exposed to OTA, ML17 exerted a stress-protective effect by synthesizing large amounts of heat shock proteins, which contributed to the correct folding of proteins. Notably, genes related to antioxidant activity, such as peroxiredoxin, superoxide dismutase, and glutaredoxin 3, were significantly upregulated, indicating that ML17 can resist the toxic effects of OTA through adjusting its metabolic processes, and the enzyme-coding gene0095, having OTA degradation activity, was found to be upregulated. This suggests that ML17 can achieve OTA degradation by regulating its metabolism, upregulating its antioxidant system, and upregulating enzyme-encoding genes with OTA degradation activity. Our work provides a theoretical reference for clarifying the mechanism of OTA degradation by ML17. Full article

Aspergillus carbonarius causes severe decays on berries in vineyards and is among the main fungal species responsible for grape contamination by ochratoxin A (OTA), which is the foremost mycotoxin produced by this fungus. The main goal of this study was to investigate at the transcriptome level the comparative profiles between two table grape varieties (Victoria and Fraoula, the white and red variety, respectively) after their inoculation with a virulent OTA-producing A. carbonarius strain. The two varieties revealed quite different transcriptomic signatures and the expression profiles of the differential expressed genes (DEGs) highlighted distinct and variety-specific responses during the infection period. The significant enrichment of pathways related to the modulation of transcriptional dynamics towards the activation of defence responses, the triggering of the metabolic shunt for the biosynthesis of secondary metabolites, mainly phenylpropanoids, and the upregulation of DEGs encoding phytoalexins, transcription factors, and genes involved in plant–pathogen interaction and immune signaling transduction was revealed in an early time point in Fraoula, whereas, in Victoria, any transcriptional reprogramming was observed after a delay. However, both varieties, to some extent, also showed common expression dynamics for specific DEG families, such as those encoding for laccases and stilbene synthases. Jasmonate (JA) may play a critical modulator role in the defence machinery as various JA-biosynthetic DEGs were upregulated. Along with the broader modulation of the transcriptome that was observed in white grape, expression profiles of specific A. carbonarius genes related to pathogenesis, fungal sporulation, and conidiation highlight the higher susceptibility of Victoria. Furthermore, the A. carbonarius transcriptional patterns directly associated with the regulation of the pathogen OTA-biosynthesis gene cluster were more highly induced in Victoria than in Fraoula. The latter was less contaminated by OTA and showed substantially lower sporulation. These findings contribute to uncovering the interplay beyond this plant–microbe interaction. Full article

Using degenerated primers (LC1–LC2c) and two novel primer pairs, namely (KSLB–LC6) for Aspergillus niger and (AFl1F–LC2) for Aspergillus tubingensis, created for the acyl transferase (AT) and the KS sequences of fungal PKSs genes, a 700 pb PCR-derived DNA fragment was isolated from Aspergillus carbonarius, Aspergillus niger, and Aspergillus tubingensis. Testing was performed on DNA from most of the black Aspergillus species currently known to exist. This article describes the identification and characterisation of a portion of a novel putative OTA-polyketide synthase gene in A. tubingensis “AT Pks,” A. niger “AN Pks,” and A. carbonarius “AC Pks”. Phylogenetic methods were used to align and evaluate the sequences. The study’s primers demonstrated broad application, and several Aspergillus species from the section Nigri, particularly A. niger and A. tubingensis, were amplified satisfactorily. Predicted amino acid sequences known as “AC Pks” showed 66–81% similarity to several polyketide synthase genes, whereas “AN Pks” and “AT Pks” showed 68–71% and 81–97% similarity, respectively. The AT and KS sequences were linked to PKSs engaged in various mycotoxin production routes, including ochratoxin A, and they seemed to be specific for a specific kind of fungal PKSs. The sequences that have been reported in this paper are particularly useful in finding new fungal PKS gene clusters. Full article

Aspergillus westerdijkiae, known as the major ochratoxin A (OTA) producer, usually occurs on agricultural crops, fruits, and dry-cured meats. Microorganisms produce OTA to adapt to the high osmotic pressure environment that is generated during food processing and storage. To investigate the relationship between OTA biosynthesis and the high osmolarity glycerol (HOG) pathway, the transcription factor AwHog1 gene in A. westerdijkiae was functionally characterised by means of a loss-of-function mutant. Our findings demonstrated that the growth and OTA production of a mutant lacking AwHog1 decreased significantly and was more sensitive to high osmotic media. The ΔAwHog1 mutant displayed a lower growth rate and a 73.16% reduction in OTA production in the wheat medium compared to the wild type. After three days of culture, the growth rate of the ΔAwHog1 mutant in medium with 60 g/L NaCl and 150 g/L glucose was slowed down 19.57% and 13.21%, respectively. Additionally, the expression of OTA biosynthesis genes was significantly reduced by the deletion of the AwHog1 gene. The infection ability of the ΔAwHog1 mutant was decreased, and the scab diameter of the pear was 6% smaller than that of the wild type. These data revealed that transcription factor AwHog1 plays a key role in the osmotic response, growth, OTA production, and pathogenicity in A. westerdijkiae. Full article

Ochratoxin A (OTA), as a common mycotoxin, has seriously harmful effects on agricultural products, livestock and humans. There are reports on the regulation of SakA in the MAPK pathway, which regulates the production of mycotoxins. However, the role of SakA in the regulation of Aspergillus westerdijkiae and OTA production is not clear. In this study, a SakA deletion mutant (ΔAwSakA) was constructed. The effects of different concentrations of D-sorbitol, NaCl, Congo red and H₂O₂ on the mycelia growth, conidia production and biosynthesis of OTA were investigated in A. westerdijkiae WT and ΔAwSakA. The results showed that 100 g/L NaCl and 3.6 M D-sorbitol significantly inhibited mycelium growth and that a concentration of 0.1% Congo red was sufficient to inhibit the mycelium growth. A reduction in mycelium development was observed in ΔAwSakA, especially in high concentrations of osmotic stress. A lack of AwSakA dramatically reduced OTA production by downregulating the expression of the biosynthetic genes otaA, otaY, otaB and otaD. However, otaC and the transcription factor otaR1 were slightly upregulated by 80 g/L NaCl and 2.4 M D-sorbitol, whereas they were downregulated by 0.1% Congo red and 2 mM H₂O₂. Furthermore, ΔAwSakA showed degenerative infection ability toward pears and grapes. These results suggest that AwSakA is involved in the regulation of fungal growth, OTA biosynthesis and the pathogenicity of A. westerdijkiae and could be influenced by specific environmental stresses. Full article

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. Full article

Aspergillus niger produces carcinogenic ochratoxin A (OTA), a serious food safety and human health concern. Here, the ability of A. niger CBS 513.88 to produce OTA using different carbon sources was investigated and the underlying regulatory mechanism was elucidated. The results indicated that 6% sucrose, glucose, and arabinose could trigger OTA biosynthesis and that 1586 differentially expressed genes (DEGs) overlapped compared to a non-inducing nutritional source, peptone. The genes that participated in OTA and its precursor phenylalanine biosynthesis, including pks, p450, nrps, hal, and bzip, were up-regulated, while the genes involved in oxidant detoxification, such as cat and pod, were down-regulated. Correspondingly, the activities of catalase and peroxidase were also decreased. Notably, the novel Gal4-like transcription factor An12g00840 (AnGal4), which is vital in regulating OTA biosynthesis, was identified. Deletion of AnGal4 elevated the OTA yields by 47.65%, 54.60%, and 309.23% using sucrose, glucose, and arabinose as carbon sources, respectively. Additionally, deletion of AnGal4 increased the superoxide anion and H₂O₂ contents, as well as the sensitivity to H₂O₂, using the three carbon sources. These results suggest that these three carbon sources repressed AnGal4, leading to the up-regulation of the OTA biosynthetic genes and alteration of cellular redox homeostasis, ultimately triggering OTA biosynthesis in A. niger. Full article

Ochratoxin A (OTA) is a well-known mycotoxin with wide distribution in food and feed. Fungal genome sequencing has great utility for identifying secondary metabolites gene clusters for known and novel compounds. A comparative analysis of the OTA-biosynthetic cluster in A. steynii, A. westerdijkiae, A. niger, A. carbonarius, and P. nordicum has revealed a high synteny in OTA cluster organization in five structural genes (otaA, otaB, ota, otaR1, and otaD). Moreover, a recent detailed comparative genome analysis of Aspergilli OTA producers led to the identification of a cyclase gene, otaY, located in the OTA cluster between the otaA and otaB genes, encoding for a predicted protein with high similarity to SnoaLs domain. These proteins have been shown to catalyze ring closure steps in the biosynthesis of polyketide antibiotics produced in Streptomyces. In the present study, we demonstrated an upregulation of the cyclase gene in A. carbonarius under OTA permissive conditions, consistent with the expression trends of the other OTA cluster genes and their role in OTA biosynthesis by complete gene deletion. Our results pointed out the involvement of a cyclase gene in OTA biosynthetic pathway for the first time. They represent a step forward in the understanding of the molecular basis of OTA biosynthesis in A. carbonarius. Full article

Ochratoxin A (OTA) usually contaminates agricultural products such as grapes, oatmeal, coffee and spices. Light was reported as an effective strategy to control spoilage fungi and mycotoxins. This research investigated the effects of light with different wavelengths on the growth and the production of OTA in Aspergillus ochraceus and Aspergillus carbonarius. The results showed that the growth of both fungi were extremely inhibited by UV-B. Short-wavelength (blue, violet) significantly inhibited the production of OTA in both fungi, while the inhibitory effect of white was only demonstrated on A. ochraceus. These results were supported by the expression profiles of OTA biosynthetic genes of A. ochraceus and A. carbonarius. To clarify, the decrease in OTA production is induced by inhibition or degradation; therefore, the degradation of OTA under different wavelengths of light was tested. Under UV-B, the degradation rate of 10 μg/mL OTA standard pure-solution samples could reach 96.50% in 15 days, and the degradation effect of blue light was relatively weak. Furthermore, infection experiments of pears showed that the pathogenicity of both fungi was significantly decreased under UV-B radiation. Thus, these results suggested that light could be used as a potential target for strategies in the prevention and control of ochratoxigenic fungi. Full article

Aspergillus carbonarius is the principal fungal species responsible for ochratoxin A (OTA) contamination of grapes and derived products in the main viticultural regions worldwide. In recent years, co-expressed genes representing a putative-OTA gene cluster were identified, and the deletion of a few of them allowed the partial elucidation of the biosynthetic pathway in the fungus. In the putative OTA-gene cluster is additionally present a bZIP transcription factor (AcOTAbZIP), and with this work, A. carbonarius ΔAcOTAbZIP strains were generated to study its functional role. According to phylogenetic analysis, the gene is conserved in the OTA-producing fungi. A Saccharomyces cerevisiae transcription factor binding motif (TFBM) homolog, associated with bZIP transcription factors was present in the A. carbonarius OTA-gene cluster no-coding regions. AcOTAbZIP deletion results in the loss of OTA and the intermediates OTB and OTβ. Additionally, in ΔAcOTAbZIP strains, a down-regulation of AcOTApks, AcOTAnrps, AcOTAp450, and AcOTAhal genes was observed compared to wild type (WT). These results provide evidence of the direct involvement of the AcOTAbZIP gene in the OTA biosynthetic pathway by regulating the involved genes. The loss of OTA biosynthesis ability does not affect fungal development as demonstrated by the comparison of ΔAcOTAbZIP strains and WT strains in terms of vegetative growth and asexual sporulation on three different media. Finally, no statistically significant differences in virulence were observed among ΔAcOTAbZIP strains and WT strains on artificially inoculated grape berries, demonstrating that OTA is not required by A. carbonarius for the pathogenicity process. Full article