Search Results (69)

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

Grapes are frequently contaminated by Aspergillus section Nigri fungi and ochratoxin A (OTA), with A. niger also capable of producing substantial fumonisin B₂ (FB₂) levels. Emerging evidence suggests that aflatoxigenic fungi may eventually replace ochratoxigenic fungi in certain regions due to better adaptation to changes in climatic conditions. However, research on the toxigenic fungal community and mycotoxins in grapes from organic vineyards remains limited. Research on Spanish conventional grapes is also deficient, with most of the available literature being outdated. The present study investigates the diversity of toxigenic fungi and the presence of mycotoxins in organically cultivated grape berries in Spain, which are renowned for their significant oenological tradition. This study employed species-specific PCR protocols for fungal characterization and optimized methods for the analysis of OTA, FB₂, and aflatoxin B₁ (AFB₁) by UPLC–ESI–MS/MS. The most prevalent species present were Aspergillus flavus, A. niger, A. parasiticus, A. steynii, A. carbonarius, and A. westerdijkiae (67.1%, 43.5%, 20.0%, 14.1%, 14.1%, and 11.8% of the samples, respectively). OTA was detected only in 16 samples (19%), averaging 0.48 ng/g and peaking at 0.7 ng/g, which were lower than previously reported for conventional grapes. There was no FB₂ or AFB₁ detected. This study is pioneering in its exploration of the occurrence of toxigenic mycobiota, beyond Nigri fungi, and subsequent potential for other serious mycotoxins to contaminate Spain’s organic grapes. Full article

Aspergillus carbonarius is one of the main pathogens responsible for postharvest diseases in fruits and is also one of the main ochratoxin A-producing strains. It not only causes significant economic losses but also poses a risk to human health. We found an inhibitory effect of dimethyl fumarate fumigation on the growth of A. carbonarius. To further explore its antifungal mechanism, this study elucidated the functions of key pathway-related genes through a transcriptomics analysis. A total of 1402 differentially expressed genes (DEGs) were identified, including 987 up-regulated and 415 down-regulated genes. Dimethyl fumarate was found to significantly inhibit the growth of A. carbonarius by disrupting cell integrity and obstructing mycelium growth and secondary metabolism. These findings provide a basis for the potential application of dimethyl fumarate in the food industry to inhibit A. carbonarius and subsequent ochratoxin A contamination. 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

Aspergillus carbonarius is the main producer of Ochratoxin A (OTA) in coffee. In the last few years, there has been an increasing interest in using yeast isolates as Biocontrol Agents to prevent OTA production in coffee cherries during the primary postharvest processing. Little is known about how climate change abiotic conditions of increased temperature (+2–4 °C), elevated CO₂ (existing levels of 400 vs. 1000 ppm), and increased drought stress will impact biocontrol resilience. This study examined the effect of a three-way interaction between temperature (27, 30, and 33 °C) x water activity (a_w) (0.90 and 0.95 a_w) x CO₂ level (400 vs. 1000 ppm) on the growth and OTA production of A. carbonarius and the resilience of three yeast strains’ biocontrol capacity on fresh coffee cherries. High a_w (0.95), CO₂, and temperature levels increased the production of OTA by A. carbonarius. All the yeast biocontrol strains significantly reduced A. carbonarius growth by at least 20% and OTA production by up to 85%. From the three strains used, the Meyerozyma caribbica strain (Y4) showed the best resilience to climate change, since it reduced both growth (50%) and OTA production (70%) under future scenarios of CO₂ and a_w at all temperatures tested, and should be the one selected for pilot scale experiments in Ivory Coast. Full article

Food safety and quality awareness have reached significant relevance as consumers are more interested in authentic foods and drinks with specific organoleptic values. Among foodstuffs, grape products can be contaminated by Ochratoxin A (OTA), a mycotoxin that can occur in red grape after infection with Aspergillus carbonarius. The high affinity of grape pomace with OTA makes its use advantageous as an adsorbing/decontaminating material whether the pomace is fresh, has undergone pressing, or has undergone a stabilizing process. The effects of different grape repassage treatments on wine metabolic profiles were studied by ¹H NMR spectroscopy coupled with metabolomics. The relative quantification of discriminating metabolites for activated-carbon-treated samples revealed higher levels of ethyl acetate and succinate than for the grape-pomace-repassed wine samples. On the contrary, the latter exhibited a relatively high content of glycerol, lactate, tartaric, isobutanol, isopentanol, and polyphenols. Although a specific decrease in aromatic compounds such as gallic acid, tyrosine, and tyrosol was also observed compared with the controls, for the pomace-based processes, the activated carbon treatment led to a marked general impoverishment of the metabolomic profiles, with a reduction in organic acids and glycerol. The repassage of wine over the grape pomace did not significantly affect the quality attributes of the wine, offering an alternative natural adsorbing/decontaminating material for the removal of OTA. 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 widely distributed mycotoxin and potent carcinogen produced by several fungal genera, but mainly by Aspergillus carbonarius. Grape contamination occurs in vineyards during the period between veraison and pre-harvest, and it is the main cause of OTA’s presence in wine. The aim of the current study was the evaluation of 6 chemical and 11 biological plant protection products (PPPs) and biocontrol agents in commercial vineyards of the two important Greek white wine varieties cv. Malagousia and cv. Savatiano. The PPPs were applied in a 4-year vineyard study as single treatments or/and in combinations as part of IPM systems. Subsequently, nine strains of Aspergillus carbonarius were investigated for their sensitivity against seven active compounds of synthetic fungicides. During the multi-year field trials, various novel management systems, including consortia of biocontrol agents, were revealed to be effective against Aspergillus sour rot and OTA production. However, expected variability was observed in the experimental results, indicating the dynamic character of biological systems and highlighting the possible inconsistency of PPPs’ efficacy in a changing environment. Furthermore, the IPM systems developed effectuated an optimized control of A. carbonarius, leading to 100% inhibition of OTA contamination, showing the importance of using both chemical and biological PPPs for disease management and prevention of fungal fungicide resistance. Finally, the majority of A. carbonarius tested strains were found to be sensitive against the pure active compounds used (fludioxonil, azoxystrobin, chlorothalonil, tebuconazole, cyprodinil, pyrimethanil and boscalid), with only a few exceptions of developed resistance towards boscalid. Full article

Fructooligosaccharides are prebiotic sugars that are widely used in the production of functional foods, which can be produced enzymatically by the transfructosylation reaction of sucrose. This work aimed to optimize the production of an invertase with high transfructosylation activity from Aspergillus carbonarius PC-4 using pineapple crown as the inducer substrate and evaluate its biochemical properties. The culture medium was optimized using a Plackett–Burman experimental design and a central composite rotatable design, resulting in a maximum transfructosylation activity of 65.33 U/mL at 72 h of cultivation. The cultivation parameters were Yp/s = 1070.75 U/g and P_P = 2771.48 U/h, which showed an increase of 5.2-fold in the enzyme produced. The optimum temperature (50 °C) and pH (5.0) for the enzymatic activity were obtained by a CCR design. The enzyme showed a half-life of 60 min at 40 °C. In conclusion, the invertase produced from A. carbonarius PC-4 using agro-industrial waste (pineapple crown) and an inorganic nitrogen source (ammonium nitrate) exhibits high transfructosylation activity that can be used as a potential source for the production of fructooligosaccharides. 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

Aspergillus species create major postharvest problems due to the food losses caused by their mere presence and the hazardous mycotoxins they produce, such as aflatoxin B1 (AFB1) and ochratoxin A (OTA). These mycotoxins are mainly produced by A. flavus and A. carbonarius, respectively. In this study, we developed a rapid detection method for the two aforementioned species based on loop-mediated isothermal amplification (LAMP). The primers were designed to target genes belonging to the mycotoxin clusters pks and aflT for A. carbonarius and A. flavus, respectively. Result visualization was carried out in real time via the detection of fluorescent signals. The method developed showed high sensitivity and specificity, with detection limits of 0.3 and 0.03 pg/reaction of purified DNA of A. carbonarius and A. flavus, respectively. The assays were further implemented on inoculated nuts, including pistachios and almonds, after one-step crude DNA extraction. These tests revealed a detection level of 0.5 spore/g that shows the effectiveness of LAMP as a rapid method for detecting potentially toxigenic Aspergillus spp. directly in food. The validation of the assays included tests on a larger scale that further confirmed their sensitivity and specificity, as well as enabling the production of ready-to-use LAMP prototype kits. These kits are easy to use and aim to simplify the screening of food samples in order to monitor the presence of specific Aspergillus contaminations. Full article

Altogether, 39 cereal flour samples taken from a Serbian market were analyzed for mycobiota and mycotoxin content, among which were six Triticum aestivum specimens, five Triticum dicoccum specimens, four Hordeum vulgare specimens, five Fagopyrum esculentum specimens, three Secale cereale specimens, five Triticum spelta specimens, four Avena sativa specimens, two Oryza sativa specimens, two Zea mays specimens, and one specimen each of Panicum miliaceum, Triticum monococcum, and Triticum turgidum ssp. turanicum. To determine the mycobiota content using dilution techniques, the flour samples were transferred to a non-selective DG18 nutrient. The number of coloniforming units (CFU/g) varied from less than 100 (in the case of five samples, namely, two O. sativa, and one specimen each of S. cereale, H. vulgare, and T. aestivum) to as high as 5000 CFU/g (S. cereale), 6000 (A. sativa), 11,000 (T. aesticum), and 40,000 (Z. mays). The identification of fungal genera and species was performed on Czapex-Dox Agar and Potato dextrose Agar on the basis of the isolates’ colony characteristics and the morphology of the examined reproductive organs. The isolated fungi belonged to the following genera: Aspergillus, Penicillium, Alternaria, and Fusarium. Species from these genera are well-known mycotoxin-producing fungi. Among the identified species were A. candidus, A. flavus, A. carbonarius, A. ochraceus, A. oryzae, P. solitum, P. citrinum, P. griseofulvum, P. brevicompactum, A. alternata, F. avenaceum, and F. graminearum. The mycotoxin content was determined via the ELISA technique using Eurofins Technologies Hungary KFT kits for aflatoxin B1, deoxynivalenol, total aflatoxins, ochratoxin A, and zearalenone. In the case of eighteen samples, the total aflatoxin content was above the limit of detection, and seven of these samples were contaminated with aflatoxin B1, eight were contaminated with ochratoxin A, two were contaminated with dexynivalenol, and one was contaminated with zearalenon. Two samples of T. aestivum were contaminated with one or more toxins (33%), and the number of samples contaminated three for T. dicoccum (60%), one for H. vulgare (25%), four for F. esculentum (80%), one for S. cereale (33%), two for T. spelta (40%), three for A. sativa (75%), two for O. sativa (100%), two for Z. mays (100%), one for P. miliaceum (100%), one for T. monococcum (100%), and one for T. turgidum ssp. turanicum (100%). Full article

Aspergillus carbonarius is known to produce the carcinogenic ochratoxin A (OTA) in grapes. The metabolism process before OTA biosynthesis influences the content and composition of the volatile compounds in grapes. In this study, a self-established method based on QuEChERS coupled with high-performance liquid chromatography–fluorescence detection (HPLC-FLD) was used to determine the OTA levels during a seven-day contamination period. The results showed that OTA was detected on the second day after contamination with A. carbonarius. Thus, the first day was considered as the critical sampling timepoint for analyzing the volatiles in grapes before OTA biosynthesis. Additionally, the volatile compounds in grapes were analyzed using headspace solid-phase microextraction gas chromatography–mass spectrometry (HS-SPME-GC-MS) and dispersive liquid–liquid microextraction gas chromatography–mass spectrometry (DLLME-GC-MS). The corresponding data were evaluated via multivariate data analysis using projection methods, including PCA and OPLS-DA. The results indicated significant differences in the nine volatile compounds in grapes contaminated with A. carbonarius before OTA biosynthesis. The results of the Pearson correlation analysis showed positive correlations between ethyl acetate, styrene, 1-hexanol and OTA; (E)-2-hexenal and nerolic acid were negatively correlated with OTA. Overall, these findings provide a theoretical basis for the early prediction of OTA formation in grape and grape products using GC-MS technology. 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