Carbonarius Strains Research Articles

Unlocking the Transcriptional Reprogramming Repertoire between Variety-Dependent Responses of Grapevine Berries to Infection by Aspergillus carbonarius.

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.

Inhibitory Effect of GRAS Essential Oils and Plant Extracts on the Growth of Aspergillus westerdijkiae and Aspergillus carbonarius Strains.

The effect of essential oils (obtained using hydrodistillation) and plant extracts (ethanolic, aqueous, and hexanic extractions) of 10 different plants cultivated in Brazil were tested using the diffusion agar method, with the objective of evaluating the inhibitory effect of the oils and extracts on the mycelial growth of Aspergillus westerdijkiae NRRL 3174 and A. carbonarius RC 2054 (UNRC). Of the 40 essential oils and plant extracts analyzed, oregano essential oil and plant extract, rosemary essential oil, and the clove ethanolic extract were the best choice to obtain the growth parameters (radial growth rates (mm day−1) and lag phase (h)) due the good results presented and the volume of oil/extract obtained. Comparing all the essential oils and plant extracts that were tested for growth parameters, the best results were obtained for the clove ethanolic extract for both strains assayed. These results demonstrated an outstanding potential use of some of these products in prevention of fungal contamination in food. However, further studies need to be conducted to determine the ability of these oils and extracts to inhibit or reduce ochratoxin A production.

Ochratoxigenic fungi in post-fermented tea and inhibitory activities of Bacillus spp. from post-fermented tea on ochratoxigenic fungi

The contamination of ochratoxigenic fungi in the process of pile-fermentation of post-fermented tea is objective, but the fact that ochratoxigenic fungi have almost no risk to the safe drinking of post-fermented tea products has been widely concerned. This study aims to identify the diversity of ochratoxigenic fungi in post-fermented tea and to reveal the inhibitory effect of Bacillus strains isolated from post-fermented tea on ochratoxigenic fungi. Results showed: 8.33% of A. niger strains, 30% of A. tubingensis strains, 100% of A. carbonarius strains, A. nidulans strains, P. verrucosum strains and A. ochraceus strains isolated from post-fermented tea have the ability to produce ochratoxin A (OTA), and there are significant differences in the OTA production ability between different strains. The six different species of Bacillus isolated from post-fermented tea coexisted with ochratoxigenic fungi in liquid media can not only inhibit the growth of ochratoxigenic fungi, but also inhibit the production of OTA. Bacillus strains cocultured with ochratoxigenic fungi on the tea matrix also have significant effects on the growth of OTA-producing fungi and the effects of inhibiting fungi are influenced by the order of inoculation, culture time and the difference of fungal species. Bacillus crude antifungal extracts have inhibitory effects on the growth of OTA-producing fungi and anti-fungal substances can be roughly divided into two categories: peptides with molecular weight ≤10 kDa, and anti-fungal proteins with molecular weight >10 kDa according to their molecular weight. The research results have important guiding value for the correct understanding of the hazards of fungal contamination in post-fermented tea and searching for new technologies of producing post-fermented tea aimed at reducing the safety risks of OTA.

Transcriptome analysis of non-ochratoxigenic Aspergillus carbonarius strains and interactions between some black aspergilli species

Aspergillus carbonarius consistently produces large amounts of ochratoxin A (OTA), a mycotoxin with nephrotoxic effects on animals and humans. In the present study, we analyzed the transcriptional changes associated to OTA production in three atypical non-ochratoxigenic strains of A. carbonarius. In addition, in vitro interactions between ochratoxigenic strains of A. carbonarius and A. niger and non-ochratoxigenic strains of A. carbonarius and A. tubingensis were studied in order to evaluate their potential for controlling OTA production. RNA-seq analysis revealed that there are 696 differentially expressed genes identified in the three non-OTA-producing strains, including 280 up-regulated and 333 down-regulated genes. A functional and gene ontology enrichment analysis revealed that the processes related to metabolic and oxidation processes, associated with functions such as oxidoreductase and hydrolase activity were down regulated. All the genes related with OTA biosynthesis in A. carbonarius were the most down-regulated genes in non-ochratoxigenic strains. We also showed that these strains possess a deleterious mutation in the AcOTApks gene required for OTA biosynthesis. Moreover, one of these strains gave the best control of OTA production resulting in an OTA reduction of 98–100% in co-inoculation with an ochratoxigenic strain of A. niger and an OTA reduction of 79–89% with an ochratoxigenic strain of A. carbonarius. Results of this study provided novel insights into the knowledge of the OTA biosynthetic pathway in these non-ochratoxigenic wild strains, and showed the biocontrol potential of these strains.

Interacting climate change factors (CO2 and temperature cycles) effects on growth, secondary metabolite gene expression and phenotypic ochratoxin A production by Aspergillus carbonarius strains on a grape-based matrix

Little is known on the impact that climate change (CC) may have on Aspergillus carbonarius and Ochratoxin A (OTA) contamination of grapes, especially in the Mediterranean region where in CC scenarios temperature are expected to increase by +2–5 °C and CO2 from 400 to 800/1200 ppm. This study examined the effect of (i) current and increased temperature in the alternating 11.5 h dark/12.5 h light cycle (15–28 °C vs 18–34 °C), representative of the North Apulia area, South Italy and (ii) existing and predicted CO2 concentrations (400 vs 1000 ppm), on growth, expression of biosynthetic genes (AcOTApks, AcOTAnrps, AcOTAhal, AcOTAp450, AcOTAbZIP) and regulatory genes of Velvet complex (laeA/veA/velB, “velvet complex”) involved in OTA biosynthesis and OTA phenotypic production by three strains of A. carbonarius. The experiments made on a grape-based matrix showed that elevated CO2 resulted in a general stimulation of growth and OTA production. These results were also supported by the up-regulation of both structural and regulatory genes involved in the OTA biosynthesis. Our work has shown for the first time that elevated CO2 concentration in the Mediterranean region may result in an increased risk of OTA contamination in the wine production chain.

Intraspecific variability of growth and ochratoxin A production by Aspergillus carbonarius from different foods and geographical areas

Ochratoxin A (OTA) is a nephrotoxic mycotoxin naturally found in a wide range of food commodities throughout the world. Aspergillus carbonarius is the most important source of OTA in food commodities such as wine, grapes and dried vine fruits and is also responsible for the formation of OTA in coffee.The aim of this study was to determine the simultaneous effect of three culture media (Czapek Yeast Extract Broth (CYB); Synthetic Grape Juice Medium (SGM) and White grape juice (WGJ)) at three water activity (aw) levels (0.90; 0.95 and 0.98–0.99), and three incubation temperatures (15 °C, 25 °C and 35 °C) on the growth and OTA production by 16 strains of A. carbonarius. The strains were mainly isolated from grapes from areas with a Mediterranean climate. All the strains were confirmed for identity by sequencing of the calmodulin gene. The assay was performed in microtiter plates, determining the absorbance at 530 nm and the concentration of OTA after 1, 2, 4 and 10 days of incubation.No significant differences were observed in absorbance values between the strains. The highest absorbance values were recorded in CYB at 0.99 aw and at 0.95 aw after 10 days of incubation at 25 °C and 35 °C. None of the strains were able to grow at 0.90 aw and 15 °C in any culture media after 10 days of incubation. OTA concentration was statistically higher at 15 °C than at 25 °C or 35 °C. The highest significant OTA values were obtained at 0.98–0.99 aw and the best culture media for OTA production was CYB, followed by WGJ and SGM. While strains isolated from Mediterranean climate foods had a similar behavior despite being isolated from different geographical areas, OTA concentration produced by one Robusta coffee strain from Thailand was statistically higher at 25 °C than at 15 °C. This would suggest that the type of food matrices and consequently the adaptation of A. carbonarius strains to different climatic conditions would have a greater influence on the ecophysiology of the strains than only their geographical origin.

A comparison of electronic nose and gas chromatography–mass spectrometry on discrimination and prediction of ochratoxin A content in Aspergillus carbonarius cultured grape-based medium

This study investigated discrimination and prediction of ochratoxin A (OTA) in three Aspergillus carbonarius strains cultured grape-based medium using E-nose technology and GC–MS analysis. Results showed that these strains cultured medium samples were divided into four groups regarding their log 10 OTA value using an equispaced normal distribution analysis. Partial least squares-discriminant analysis (PLS-DA) revealed that GC–MS PLS-DA model only separated the low OTA level medium samples from the rest OTA level samples, whereas all the OTA level samples were segregated from each other using E-nose PLS-DA model. Partial least squares regression (PLSR) analysis indicated that an excellent prediction performance was established on the accumulation of OTA in these medium samples using E-nose PLSR, whereas GC–MS PLSR model showed a screening performance on the OTA formation. These indicated that E-nose analysis could be a reliable method on discriminating and predicting OTA in A. carbonarius strains under grape-based medium.

Genomic diversity in ochratoxigenic and non ochratoxigenic strains of Aspergillus carbonarius

Ochratoxin A (OTA) is a mycotoxin with nephrotoxic effects on animals and humans. Aspergillus carbonarius is the main responsible for OTA contamination of grapes and derived products. We present the genome resequencing of four A. carbonarius strains, one OTA producer and three atypical and unique non-OTA producing strains. These strains were sequenced using Illumina technology and compared with a reference genome of this species. We performed some specific bioinformatics analyses in genes involved in OTA biosynthesis. Data obtained in this study revealed the high genomic diversity within A. carbonarius strains. Although some gaps of more than 1,000 bp were identified in non-ochratoxigenic strains, no large deletions in functional genes related with OTA production were found. Moreover, the expression of five genes of the putative OTA biosynthetic cluster was down regulated under OTA-inducing conditions in the non-ochratoxigenic strains. Knowledge of the regulatory mechanisms involved in OTA biosynthesis will provide a deeper understanding of these non-ochratoxigenic strains.

Chemometric Analysis of the Volatile Compounds Generated by Aspergillus carbonarius Strains Isolated from Grapes and Dried Vine Fruits.

Ochratoxin A (OTA) contamination in grape production is an important problem worldwide. Microbial volatile organic compounds (MVOCs) have been demonstrated as useful tools to identify different toxigenic strains. In this study, Aspergillus carbonarius strains were classified into two groups, moderate toxigenic strains (MT) and high toxigenic strains (HT), according to OTA-forming ability. The MVOCs were analyzed by GC-MS and the data processing was based on untargeted profiling using XCMS Online software. Orthogonal projection to latent structures discriminant analysis (OPLS-DA) was performed using extract ion chromatogram GC-MS datasets. For contrast, quantitative analysis was also performed. Results demonstrated that the performance of the OPLS-DA model of untargeted profiling was better than the quantitative method. Potential markers were successfully discovered by variable importance on projection (VIP) and t-test. (E)-2-octen-1-ol, octanal, 1-octen-3-one, styrene, limonene, methyl-2-phenylacetate and 3 unknown compounds were selected as potential markers for the MT group. Cuparene, (Z)-thujopsene, methyl octanoate and 1 unknown compound were identified as potential markers for the HT groups. Finally, the selected markers were used to construct a supported vector machine classification (SVM-C) model to check classification ability. The models showed good performance with the accuracy of cross-validation and test prediction of 87.93% and 92.00%, respectively.

A study on accumulation of volatile organic compounds during ochratoxin a biosynthesis and characterization of the correlation in Aspergillus carbonarius isolated from grape and dried vine fruit.

Volatile organic compounds (VOCs) and ochratoxin A (OTA) were detected in Aspergillus carbonarius strains (AF, SD26 and SD27) during a 7-day growing period, using HS-SPME-GC-MS and HPLC-MS/MS, respectively. Results showed that 33 VOCs were detected in these strains. The AF strain showed the different sesquiterpene profile compared with the other strains. Principal component analysis revealed that the AF strain was segregated from the other two strains regarding the VOC profile, especially the sesquiterpene profile. Ester metabolites were produced more at the beginning of the strain growth period, whereas extension of the strain incubation resulted in an intense accumulation of hydrocarbon compounds. The prediction models on the correlation between OTA and VOCs were established using partial least squares regression analysis. This analysis indicated that C-8 alcohols, ketones, and trans-nerolidol showed a close relation with the OTA synthesis.

The global regulator LaeA controls production of citric acid and endoglucanases in Aspergillus carbonarius.

The global regulatory protein LaeA is known for regulating the production of many kinds of secondary metabolites in Aspergillus species, as well as sexual and asexual reproduction, and morphology. In Aspergillus carbonarius, it has been shown that LaeA regulates production of ochratoxin. We have investigated the regulatory effect of LaeA on production of citric acid and cellulolytic enzymes in A. carbonarius. Two types of A. carbonarius strains, having laeA knocked out or overexpressed, were constructed and tested in fermentation. The knockout of laeA significantly decreased the production of citric acid and endoglucanases, but did not reduce the production of beta-glucosidases or xylanases. The citric acid accumulation was reduced with 74-96% compared to the wild type. The endoglucanase activity was reduced with 51-78%. Overexpression of LaeA seemed not to have an effect on citric acid production or on cellulose or xylanase activity.

RNA-Seq Reveals OTA-Related Gene Transcriptional Changes in Aspergillus carbonarius.

Ochratoxin A (OTA) is a mycotoxin harmful for animals and humans. Aspergillus carbonarius is the main responsible for OTA contamination of grapes and derived products. Gene transcriptional profiling of 4 A. carbonarius strains was carried out by RNA-Seq analysis to study transcriptome changes associated with OTA production. By comparing OTA inducing (OTAI) vs. non-inducing (OTAN) cultural conditions, a total of 3,705 differentially expressed genes (DEGs) (fold change > |2| and FDR ≤ 0.05) were identified. Several genes involved in primary metabolic processes, with particular regard to carbohydrate and amino acid metabolisms, secondary metabolic processes, transport, response to stress and sporulation were up-regulated by OTAI conditions at all the analysed sampling times (4, 6 and 8 DAI) or starting from 6 DAI. Highly up-regulated DEGs encoding enzymes involved in biosynthesis of secondary metabolites, oxidoreductases, transporters and transcription factors were examined for their potential involvement in OTA biosynthesis and related metabolic pathways. Differential expression of genes encoding polyketide synthases (pks), non-ribosomal peptide synthetases (nrps) and chloroperoxidase (cpo) was validated by RT-qPCR. Among clusters of co-regulated genes involved in SM biosynthesis, one putative OTA-gene cluster, including both pks and nrps genes, was detected in the A. carbonarius genome.

Real-Time PCR Assay Targeting the veA Gene for Quantification of Aspergillus carbonarius in Grapes

In this work, a SYBR Green I real-time PCR method has been developed for the detection and quantification of Aspergillus carbonarius in grapes by targeting the veA gene with a primer pair (veAF4/veAR4) that specifically amplifies a 91-bp PCR product. The quantification of the fungal DNA was performed by generation of standard curves for two A. carbonarius strains, using spectrophotometrically measured DNA quantities (Log) with a linearity range from 50 to 5 × 10(-4) ng of DNA. A high positive correlation (R(2) > 0.99) between exponential increases of DNA and real-time PCR threshold cycles showed a high amplification efficiency for the assay (E values 100.06 and 101.51%, respectively). Quantification of the fungal genomic DNA in grape samples artificially inoculated with A. carbonarius conidia was successfully performed with a minimum threshold of 10(4) conidia per g of grape berry. The assay developed would allow reliable, specific, and efficient detection and quantification of A. carbonarius in grapes.

Effect of interaction between Aspergillus carbonarius and non-ochratoxigenic grape-associated fungal isolates on growth and ochratoxin A production at different water activities and temperatures

The effect of water activity (0.90, 0.94, and 0.98 aw) and temperature (15, 20, and 25 °C) on the in vitro interactions between three ochratoxigenic strains of Aspergillus carbonarius (Ac-28, Ac-29, and Ac-33) and eleven non ochratoxigenic grape-associated fungal strains was assessed in this study. Fungal strains were allowed to grow in dual cultures on Synthetic Grape-juice Medium (SGM) for 15 days and fungal interactions were given a numerical score to obtain an Index of Dominance (ID) for each fungus. Results showed that in most cases A. carbonarius toxigenic strains were dominant against other fungal species. However, A. carbonarius presented mutual antagonism with A. section Nigri strains regardless of water activity (aw) and temperature. Moreover, interactions with Penicillium spinulosum and Cladosporium spp. at 15 °C, as well as Botrytis cinerea at 20 °C, showed that the antagonists were more competitive against A. carbonarius. In some cases, growth rates of A. carbonarius strains were either slightly stimulated or inhibited after interaction in dual cultures, depending on temperature, aw and competing species. Regarding OTA production, the presence of other species sometimes decreased the production or slightly enhanced it, depending on fungal competitor and environmental conditions. Overall, OTA production was higher at 15 °C/0.98 aw and 20 °C/0.98 aw for all target strains and at 20 °C/0.94 aw for Ac-33 strain only, but decreased at higher temperatures regardless of aw and interacting species.

The loss of the inducible Aspergillus carbonarius MFS transporter MfsA leads to ochratoxin A overproduction

Ochratoxin A (OTA), a nephrotoxic compound produced by certain Aspergillus and Penicillium species, is one of the most abundant mycotoxins in food commodities. Aspergillus carbonarius is the main source of OTA in wine, grape juice and dried vine fruits. Although many studies have focused on OTA production by A. carbonarius, little is known about the genes related to OTA production and transport. We have found a transporter that belongs to the major facilitator superfamily (MfsA) which is highly expressed with a 102-fold induction in an ochratoxigenic A. carbonarius strain compared to a low OTA producer strain. The encoding mfsA gene shows similarity to the multidrug efflux transporter flu1 from Candida albicans. A high number of putative transcription factor binding sites involved in the response to stress were identified within the promoter of mfsA. Phenotypical analysis of ΔmfsA deletion mutants revealed that the loss of mfsA leads to a slight growth reduction and increased OTA production. We therefore hypothesize that MfsA could be a stress response transporter whose disruption could cause an increase in oxidative stress together with a stimulation of mycotoxin production.

Effect of indigenous mycobiota on ochratoxin A production by Aspergillus carbonarius isolated from soil

This study determined the biotic interaction between 30 non-toxigenic indigenous strains of Aspergillus niger aggregate, Aspergillus flavus, Trichoderma spp., Mucor spp., Cladosporium spp., Ulocladium spp., Curvularia spp., Absidia spp., Geotrichum spp. and Acremonium spp., isolated from soil destined for maize crops, with respect to their ability to prevent ochratoxin A (OTA) production by A. carbonarius on "in vitro" assay, on liquid and solid medium. OTA production was completely inhibited when A. carbonarius was inoculated in a interactive mixed culture with all A. niger aggregate strains assayed, a 80% of Trichoderma spp. strains, a 40% of Cladosporium spp. strains, Acremonium spp and Geotrichum spp; only one strain of A. flavus tested was able to completely inhibit the mycotoxin accumulation. OTA production increased when A. carbonarius ACS 8 was growing on liquid interactive mixed culture with Mucor spp strains. These results demonstrated that OTA production by Aspergillua carbonarius strain was significantly influenced by the presence of different non-toxigenic fungal strains when growing together on paired cultures.

Characterization and disruption of the cipC gene in the ochratoxigenic fungus Aspergillus carbonarius

Aspergillus carbonarius is considered the most important ochratoxin A (OTA) producing fungi among those causing OTA contamination in grapes and grape-derived products. CipC is a small protein with unknown function that was previously found to be highly up-regulated in an OTA producer strain of A. carbonarius in comparison to a non OTA producer strain. In this study, cipC was characterized and disrupted via Agrobacterium tumefaciens-mediated transformation in an ochratoxigenic A. carbonarius strain in order to study whether this gene has a role in OTA production. Sequence analysis indicated that the promoter region of cipC contains putative binding sites for transcription factors that regulate the utilization of nutrients, the stress response and detoxification processes, all factors that can influence mycotoxin biosynthesis. Although the ∆cipC mutant grew similarly to the wild type strain, the null mutant showed a much higher OTA production. Moreover, when A. carbonarius was grown under the oxidative stress conditions imposed by the presence of hydrogen peroxide, cipC gene expression was up-regulated. These results indicate that cipC is not directly involved in OTA biosynthesis, but sequence analysis of the A. carbonarius cipC gene promoter and the phenotype of the ΔcipC disrupted mutant suggests that CipC could be a stress response protein that would be up-regulated concomitantly with OTA production.

VeA and LaeA transcriptional factors regulate ochratoxin A biosynthesis in Aspergillus carbonarius

Ochratoxin A (OTA) is a mycotoxin with nephrotoxic, teratogenic and immunotoxic properties which represents a serious risk for human and animal health. Aspergillus carbonarius is considered the main OTA-producing species in grapes and products such as raisins, wine or juices, although it has also been isolated from coffee, cocoa and cereals. Till now not much information is available about regulatory mechanisms of OTA production by A. carbonarius. A better understanding of how environmental factors influence OTA production and which genes are involved in its regulation could help us design new control strategies. In this study, we have evaluated the role of VeA and LaeA transcriptional factors, which have been shown to regulate secondary metabolism in response to light in A. carbonarius. To this aim, veA and laeA genes were deleted in an ochratoxigenic A. carbonarius strain by targeted gene replacement using Agrobacterium tumefaciens-mediated transformation. Loss of veA and laeA in A. carbonarius yields to an organism with slight differences in vegetative growth but a strong reduction in conidial production. A drastic decrease of OTA production that ranged from 68.5 to 99.4% in ΔveA and ΔlaeA null mutants was also observed, which was correlated with a downregulation of a nonribosomal peptide synthetase involved in OTA biosynthesis. These findings suggest that VeA and LaeA have an important role regulating conidiation and OTA biosynthesis in response to light in A. carbonarius in a similar way to other fungi where functions of VeA and LaeA have been previously described. This is the first report of a transcriptional factor governing the production of OTA by A. carbonarius.

Environmental factors affect the activity of biocontrol agents against ochratoxigenic Aspergillus carbonarius on wine grape

The influence of temperature and relative humidity (RH) on the activity of three biocontrol agents—the yeast Metschnikowia pulcherrima LS16 and two strains of the yeast-like fungus Aureobasidium pullulans LS30 and AU34-2—against infection by A. carbonarius and ochratoxin A (OTA) accumulation in wine grape berries was investigated in lab-scale experiments. The presence of wounds on grape skin dramatically favored infection of berries by A. carbonarius strain A1102, since unwounded berries showed very low levels of infection at all conditions of RH and temperature tested. Artificially wounded berries pre-treated with the biocontrol agents were inoculated with the ochratoxigenic A. carbonarius strain A1102 and were incubated for 5days at two levels of RH (60% and 100%) and three different temperatures (20, 25 and 30°C). The three biocontrol agents were able to prevent infections at 60% RH and 20°C. At 60% RH and 25°C only strain AU34-2 achieved some protection on day 5, whereas at 30°C a limited biocontrol efficacy was evident only up to day 2. At 100% RH, LS16, LS30 and AU34-2 showed effective protection of grape berries at 20°C until the 5th day of incubation. The three biocontrol agents achieved significant protection at higher temperatures only until the 2nd day after the beginning of the experiment: all three strains at 25°C, and only strain LS16 at 30°C. After 5days, the three biocontrol agents were able to significantly reduce the level of OTA in berries at all the conditions tested. This occurred even when protection from infection was not significant, except at 30°C and 100% of RH for all the three strains, and at 25°C and 100% of RH for strain LS16. The biocontrol agents displayed a higher rate of colonization on grape berries at 20 and 25°C than at 30°C. The higher value of RH (100%) appeared to increase the rate of colonization, in particular at 20 and 25°C. Taken together, our results emphasize the significant influence of environmental factors on the effectiveness of biocontrol against A. carbonarius as well as on OTA contamination in wine grape berries, and the need for biocontrol agents that can cope with the environmental conditions that are conducive to attack by A. carbonarius.

Growth and ochratoxin A production by Aspergillus carbonarius at different pHs and grape maturation stages

The objective of this work was to study the effect of some factors, linked to grape composition during ripeness process, on the growth and ochratoxin A (OTA) production of Aspergillus carbonarius isolated from grapes. Aspergillus carbonarius isolates were tested (i) in vitro, in Czapek yeast autolysate agar (CYA) at different pHs (2·5-4·5) and incubation times (2-6 days), and (ii) in situ, in fresh grapes collected at different maturation stages. Aspergillus carbonarius was able to grow with the same intensity at the different maturation stages and pH levels tested. In general, a similar trend of OTA production by A. carbonarius in response to acidity in media and in grape was observed. Low pH level seemed optimal for maximum OTA production. Aspergillus carbonarius strains can strongly grow and produce OTA on grape from the early stages of maturation. Extrinsic environmental conditions at the harvest period and skin thickness are, probably, the mains factors contributing to OTA contamination of grapes at the end of maturation. The results lead to a better understanding of the critical point during grape maturation for the growth of ochratoxigenic fungi and the toxin production.