P.V. Martínez-Culebras

Mycobiota and mycotoxin producing fungi from cocoa beans

The present study reports on the natural mycobiota occurring in cocoa beans, paying special attention to the incidence of fungal species that are potential producers of mycotoxins. The results show that predominant fungi were different species of the genus Aspergillus belonging to section Flavi and Nigri. Of the 214 strains of Aspergillus section Flavi collected from cocoa beans, 120 were identified as A. flavus and 94 as A. tamarii. Of Aspergillus section Nigri 138 strains were isolated, with 132 belonging to A. niger aggregate and 6 to A. carbonarius species. Potential ability to produce aflatoxins (AFs) B1, B2, G1 and G2, cyclopiazonic acid (CPA) and ochratoxin A (OTA) was studied by isolate culture followed by HPLC analysis of these mycotoxins in the culture extracts. Results indicated that 64.1% and 34.2% of the A. flavus strains produced AFs and CPA, respectively. Most of the A. flavus strains presented moderate toxigenicity with mean levels of AFs ranging from 100 ng g(-1) to 1000 ng g(-1). All the CPA-producing strains of A. flavus were highly toxigenic producing >30 microg g(-1) of CPA. Furthermore, 98% of A. tamarii strains produced CPA and over 50% of them were highly CPA toxigenic. With respect to OTA-producing fungi, a high percentage of black aspergilli strains (49.2%) were able to produce OTA. Additionally, most of the OTA-producing isolates were of moderate toxigenicity, producing amounts of OTA from 10 microg g(-1) to 100 microg g(-1). These results indicate that there is a possible risk factor posed by AFs, CPA and OTA contamination of cocoa beans, and consequently, cocoa products.

Molecular characterization of the black Aspergillus isolates responsible for ochratoxin A contamination in grapes and wine in relation to taxonomy of Aspergillus section Nigri.

This work examines ochratoxigenic mycobiota in grapes by ap-PCR analysis sequence analysis of the ITS and IGS regions and ability to produce OTA. A comparison was also made with many reference strains of Aspergillus section Nigri. Based on ap-PCR profiles, derived from two microsatellite primers, three main groups were obtained by UPGMA cluster analysis corresponding to A. carbonarius, A. niger and A. tubingensis. The cophenetic correlation values corresponding to ap-PCR UPGMA analysis revealed a higher genetic variability in A. niger and A. tubingensis than in A. carbonarius. In addition, no genotypical differences could be established between OTA producers and nonproducers in all species analysed. Phylogenetic relationships inferred from ITS and IGS sequences are, mostly, congruent with earlier works. A. niger and A. tubingensis strains were closely related, but not identical, and they clustered into two distinct groups within the A. niger aggregate. Sequence analysis also showed genetic divergences between strains of A. foetidus and the rest of the Aspergillus section Nigri. Additionally, the phylogenetic analysis was consistent in separating a new group of ochratoxigenic strains, frequently isolated from grapes, named A. tubingensis-like. All strains of A. carbonarius analysed by sequence analysis had identical ITS and IGS sequences confirming the lack of significant genetic variability within this important ochratoxigenic species.

Development of a green fluorescent tagged strain of Aspergillus carbonarius to monitor fungal colonization in grapes.

An enhanced green fluorescent protein has been used to tag an OTA-producing strain of Aspergillus carbonarius (W04-40) isolated from naturally infected grape berries. Transformation of the fungus was mediated by Agrobacterium tumefaciens. The most efficient transformation occurred when the co-cultivation was done with 10(4) conidia due to higher frequency of resistance colonies (894 per 10(4) conidia) and lower background obtained. To confirm the presence of the hph gene in hygromycin resistant colonies, 20 putative transformants were screened by PCR analysis. The hph gene was identified in all the transformants. Variation on the expression levels of the eGFP was detected among the transformants and 50% of them appeared bright green fluorescent under the microscope. Microscopic analysis of all the bright fluorescent transformants revealed homogeneity of the fluorescent signal, which was clearly visible in the hyphae as well as in the conidia. eGFP expression in A. carbonarius was shown to be stable in all transformants. Confocal Laser scanning microscopy images of grape berries infected with the eGFP transformant demonstrated fungal penetration into the berry tissues. OTA production was importantly increased in the eGFP transformant in comparison with the wild type strain and pathogenicity on grape berries was slightly decreased after four days of inoculation. However, no differences in virulence were found after seven days of inoculation, thus allowing utilization of this eGFP mutant for in situ analysis of A. carbonarius infection of grape berries. To our knowledge, this is the first report describing the construction of a GFP-tagged strain belonging to Aspergillus section Nigri for monitoring Aspergillus rot on grape berries.

Simultaneous detection of the main black aspergilli responsible for ochratoxin A (OTA) contamination in grapes by multiplex real-time polymerase chain reaction

This paper reports a duplex real-time polymerase chain reaction (PCR) assay for the simultaneous detection of members of the Aspergillus niger aggregate and A. carbonarius, which are the main responsible species for ochratoxin A (OTA) contamination in grapes. This single tube reaction targets the β-ketosynthase and the acyl transferase domains of the poliketide synthase of A. carbonarius and the A. niger aggregate, respectively. Melting curve analysis using a SYBR Green I real-time PCR approach showed characteristic T m-values demonstrating the specific, efficient and balanced amplification of the two PCR fragments. Subsequently, a TaqMan real-time PCR approach was settled, using 6-carboxy-fluorescein group (FAM) and VIC®-labelled specific probes for automated detection. Results indicated no differences in sensitivity when using either the two sets of primers and probes in separate or in the same reaction. However, when both targets are in very different amounts, there is a preferential amplification of the target which is in more concentration. CT-values obtained in the presence of grape DNA were very similar to those observed when only fungal purified DNA was present, indicating that the grape DNA does not interfere in the real-time PCR reaction. This procedure provides a fast and accurate tool to monitor, in a single reaction, the presence of OTA-producing species in grapes which, to some extent, will facilitate OTA contamination surveys to guarantee food safety in the wine industry.

Genes differentially expressed by Aspergillus carbonarius strains under ochratoxin A producing conditions

Aspergillus carbonarius is an important ochratoxin A (OTA)-producing fungus that is responsible for toxin contamination of grapes and wine, coffee and cocoa. A suppression subtractive hybridization (SSH) approach was performed with two strains of A. carbonarius, antagonistic in their OTA-production ability, to identify genes whose expression is linked with the ability to produce OTA. BlastX analysis identified 109 differentially-expressed sequences putatively involved in the production of OTA, with significant similarities (E(value)<10(-5)) to sequences deposited in the NCBI non-redundant protein database. Of the 109 ESTs, 26% were involved in regulation processes, 15% corresponded to hypothetical proteins, 12% were involved in stress response and detoxification, 9% corresponded to transport and secretion processes, 7% corresponded to amino acid metabolism, 7% were involved in hydrolysis of energy reserves and 5% involved in secondary metabolism. Other unisequences showed homology to genes involved in protein synthesis and general metabolism. According to their sequence similarities to genes in the NCBI database, the possible functional roles they might play in the production and regulation of OTA are discussed. Worth noting is the high percentage of genes involved in regulation, including specific and global regulators. It is also important to note the high percentage of genes involved in the response to stress and detoxification.

Mycobiota and toxigenic Penicillium species on two Spanish dry-cured ham manufacturing plants

The present study reports the natural mycobiota occurring in dry-cured hams, and in particular on the incidence of mycotoxin-producing fungi. A total of 338 fungal colonies were isolated from three stages of production, these being the post-salting, ripening and aging stages in two manufacturing plants. The results show that fungi were more frequently isolated from the aging stage and that the predominant filamentous fungal genus isolated was Penicillium. Seventy-four of the 338 fungal strains were selected for identification at the species level by using morphological criteria and internal transcribed spacers sequencing. Of the 74 fungal strains, 59 were Penicillium strains. Sixteen Penicillium species were identified, with P. commune (24 strains) and P. chrysogenum (13 strains) being the most abundant. The potential ability to produce cyclopiazonic acid (CPA) and ochratoxin A (OTA) was studied by isolating the culture followed by HPLC analysis of these mycotoxins in the culture extracts. The results indicated that 25 (33.7%) of the 74 fungal strains produced CPA. Worth noting is the high percentage of CPA-producing strains of P. commune (66.6%) of which some strains were highly toxigenic. P. polonicum strains were also highly toxigenic. With respect to OTA-producing fungi, a low percentage of fungal strains (9.5%) were able to produce OTA at moderate levels. OTA-producing fungi belonged to different Penicillium species including P. chrysogenum, P. commune, P. polonicum and P. verrucosum. These results indicate that there is a possible risk factor posed by CPA and OTA contamination of dry-cured hams.

Proteome analysis of the fungus Aspergillus carbonarius under ochratoxin A producing conditions.

Aspergillus carbonarius is an important ochratoxin A producing fungus that is responsible for mycotoxin contamination of grapes and wine. In this study, the proteomes of highly (W04-40) and weakly (W04-46) OTA-producing A. carbonarius strains were compared to identify proteins that may be involved in OTA biosynthesis. Protein samples were extracted from two biological replicates and subjected to two dimensional gel electrophoresis analysis and mass spectrometry. Expression profile comparison (PDQuest software), revealed 21 differential spots that were statistically significant and showed a two-fold change in expression, or greater. Among these, nine protein spots were identified by MALDI-MS/MS and MASCOT database and twelve remain unidentified. Of the identified proteins, seven showed a higher expression in strain W04-40 (high OTA producer) and two in strain W04-46 (low OTA producer). Some of the identified amino acid sequences shared homology with proteins involved in regulation, amino acid metabolism, oxidative stress and sporulation. It is worth noting the presence of a protein with 126.5 fold higher abundance in strain W04-40 showing homology with protein CipC, a protein with unknown function related with pathogenesis and mycotoxin production by some authors. Variations in protein expression were also further investigated at the mRNA level by real-time PCR analysis. The mRNA expression levels from three identified proteins including CipC showed correlation with protein expression levels. This study represents the first proteomic analysis for a comparison of two A. carbonarius strains with different OTA production and will contribute to a better understanding of the molecular events involved in OTA biosynthesis.

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.

Multiplex Detection of Aspergillus Species

Multiplex real-time polymerase chain reaction (PCR) provides a fast and accurate DNA-based tool for the simultaneous amplification of more than one target sequence in a single reaction. Here a duplex real-time PCR assay is described for the simultaneous detection of Aspergillus carbonarius and members of the Aspergillus niger aggregate, which are the main responsible species for ochratoxin A (OTA) contamination in grapes. This single tube reaction targets the beta-ketosynthase and the acyl transferase domains of the polyketide synthase of A. carbonarius and the A. niger aggregate, respectively.Besides, a rapid and efficient fungi DNA extraction procedure is described suitable to be applied in wine grapes. It includes a pulsifier equipment to remove conidia from grapes which prevents releasing of PCR inhibitors.

Effect of oxidant stressors and phenolic antioxidants on the ochratoxigenic fungus Aspergillus carbonarius

BACKGROUND There are few studies dealing with the relationship between oxidative stress and ochratoxin A (OTA) biosynthesis. In this work, we analyzed the effect of the oxidant stressor menadione and the antioxidants 3,5-di-tert-butyl-4-hydroxytoluene (BHT), catechin, resveratrol and a polyphenolic extract on growth, generation of reactive oxygen species (ROS), OTA production and gene expression of antioxidant enzymes of Aspergillus carbonarius. RESULTS Exposure to menadione concentrations higher than 20 µmol L(-1) led to increases in ROS and OTA levels and a decrease in growth rate. Exposure to 2.5-10 mmol L(-1) BHT also led to higher ROS and OTA levels, although growth rate was only affected above 5 mmol L(-1). Naturally occurring concentrations of catechin, resveratrol and polyphenolic extract barely affected growth rate, but they produced widely different effects on OTA production level depending on the antioxidant concentration used. In general, gene expression of antioxidant enzymes superoxide dismutase (SOD) and peroxiredoxin (PRX) was downregulated after exposure to oxidant and antioxidant concentrations that enhanced OTA production level. CONCLUSION Aspergillus carbonarius responds to oxidative stress, increasing OTA production. Nevertheless, the use of naturally occurring concentrations of antioxidant phenolic compounds to reduce oxidative stress is not a valid approach by itself for OTA contamination control in grapes.