Effect of preharvest fungicides and interacting fungi on Aspergillus carbonarius growth and ochratoxin A synthesis in dehydrating grapes

AIMS: To evaluate the effect of preharvest grape pesticides in Aspergillus section Nigri infection in dehydrating grapes and the final ochratoxin A (OTA) content. Additionally, the effect of coinoculation of moulds frequently isolated from grapes and raisins on Aspergillus section Nigri infection was studied. METHODS AND RESULTS: Fungicide-treated grapes were inoculated with Aspergillus carbonarius, Aspergillus niger aggregate, Eurotium amstelodami and Penicillium janthinellum in different combinations, then dehydrated by reducing a(w) for 20 days. The percentages of colonized grapes treated with fungicides were, in general, lower, but no differences were observed among fungicides. The untreated grapes always showed higher concentrations of OTA, regardless of the inoculum applied. In general, Chorus was the most effective antifungal treatment in reducing OTA accumulation in grapes during dehydration. Penicillium janthinellum reduced Aspergillus section Nigri colonization and OTA accumulation in grapes during dehydration. CONCLUSIONS: The four preharvest fungicides studied reduced the Aspergillus section Nigri growth and OTA production by A. carbonarius during dehydration of grapes. SIGNIFICANCE AND IMPACT OF THE STUDY: The success of these chemical treatments might depend on the mycobiota composition of grapes.     

Ochratoxin A-producing species in grapes and sun-dried grapes and their relation to ecophysiological factors

AIMS: To explain the dominance of OTA-producing fungal species in sun-dried grapes for special wine production through an ecophysiological approach. METHODS AND RESULTS: Grapes at different ripening stages, sun-dried grapes and raisins were analysed for fungal presence, and isolates identified. Aspergillus section Nigri incidence in grapes increased with grape maturation. In the ecophysiological study five isolates (Alternaria alternata, Cladosporium herbarum, Penicillium decumbens, Aspergillus carbonarius, A. niger aggregate and A. section Nigri uniseriate) were inoculated in SNM medium at four a(w) (0.82-0.97) and incubated at 20, 30 and 40 degrees C for 18 d. Isolates were also inoculated in pairs to evaluate fungal interactions recording their growth rates and indexes of dominance. Aspergillus section Nigri grew in a wider range of temperature and a(w), had higher growth rates than the others under most of the conditions tested and showed behaviour usually dominant. CONCLUSIONS: The presence of A. section Nigri is predominant in grapes during harvesting and sun-drying period likely because of a better adaptation to hot and humid environment. SIGNIFICANCE AND IMPACT OF THE STUDY: The duration of the drying period should be reduced as much as possible without compromising the quality of the final product, or drying the grapes in controlled chambers with dry hot air flow.     

Discrimination of Aspergillus niger and other Aspergillus species belonging to section Nigri by PCR assays

Aspergillus species included in section Nigri are common in plant products and processed food, such as grapes, cereals, coffee and derivatives, particularly in warm and tropical climates. Two of these species, A. carbonarius and A. niger, are known to produce ochratoxin A (OTA), a potent nephrotoxin and carcinogenic to human (group 2B). Recognition of the several species of this section is difficult and requires considerable expertise using conventional methods based on morphological features. In this work we describe rapid, sensitive and robust assays based on the PCR technique to discriminate the main species included in section Nigri: A. japonicus, A. heteromorphus, A. ellipticus and the two morphologically indistinguishable species of the A. niger aggregate: A. niger and A. tubingensis. The species-specific primers have been designed on the basis of ITS (internal transcribed spacers of rDNA units) sequence comparisons obtained from several Aspergillus strains and have been tested in a number of strains from different origins and hosts. These PCR assays, based on multi-copy sequences, are highly sensitive and specific and represent a good tool for an early detection of OTA-producing Aspergillus species in order to prevent OTA from entering the food chain.     

Mycoflora and ochratoxin-producing strains of Aspergillus section Nigri in wine grapes in Argentina

AIMS: The aims of this work were to evaluate the mycoflora and to identify the species of Aspergillus with the potential to produce ochratoxin A (OA) from different wine grape varieties from Mendoza, Argentina. Likewise, the capacity to produce OA by Aspergillus section Nigri was studied. METHODS AND RESULTS: Fifty samples of wine grapes were obtained from a winery of Mendoza province, Argentina. The surface-disinfection method was used for mycoflora determination using the medium dichloran 18% glycerol agar (DG18). Alternaria, Aspergillus and Penicillium were identified at species level. OA production was tested in 63 strains belonging to section Nigri. Alternaria genus was the most frequent (80% of the samples) followed by Aspergillus (70%). Alternaria alternata was the only specie identified from the Alternaria genus, followed by A. niger var. niger, A. flavus among others. From Penicillium genus, P. crysogenum was the most frequent specie. From 63 strains of Aspergillus section Nigri, 41.3% were OA producers. The levels of produced toxin ranged from 2 to 24.5 ng ml-1 of culture medium. CONCLUSIONS: The presence of ochratoxigenic strains of Nigri section in this substrate suggests that they may be an important source of OA in grapes from tropical and subtropical zones. Therefore, the industry should work further to diminish the growth of these fungi and mycotoxins formation in grapes, with the aim to reduce OA content in wine products. SIGNIFICANCE AND IMPACT OF THE STUDY: The wine grape contamination with A. alternata and Aspergillus section Nigri was significant.     

Aspergillus ibericus: a new species of section Nigri isolated from grapes

As part of a study on the ochratoxin producing mycoflora of grapes, several Aspergillus strains were isolated and tested for their ochratoxin A (OTA) producing abilities. Aspergillus strains of the section Nigri, which did not produce detectable amounts of OTA but which had a similar morphology to A. carbonarius, were isolated from wine grapes and/or dried vine fruit in Portugal and Spain. These strains, however, have characters that allow morphological distinction from the other species in the section, particularly the conidia size (5-7 microm), which allows separation of the species from the two most common biseriate species in section Nigri: A. carbonarius (7-9 microm) and A. niger and its aggregate species (3-5 microm). The strains are described here as belonging to a new species, named A. ibericus. The validation of this new taxon is supported further by analysis of the ITS-5.8S rDNA and calmodulin gene sequences and by analysis of the amplified fragment length polymorphism (AFLP) patterns, which were consistent in separating these strains from other species in the section. A. ibericus strains do not produce OTA therefore they are interesting for biotechnological exploration because many metabolites with commercial value are produced by other species in the section.     

Aspergillus species producing ochratoxin A: isolation from vineyard soils and infection of Semillon bunches in Australia

AIMS: The incidence of toxigenicity among Australian isolates of Aspergillus niger and Aspergillus carbonarius was assessed. Aspergillus rot and concomitant production of ochratoxin A (OA) in bunches inoculated with A. carbonarius were also investigated. METHODS AND RESULTS: Aspergillus niger and A. carbonarius were isolated from vineyard soils. Aspergillus niger was more widespread than A. carbonarius, and two restriction fragment length polymorphism types of A. niger, N and T, were present. Three of 113 A. niger isolates and all 33 A. carbonarius isolates produced OA. Aspergillus carbonarius was inoculated onto Semillon bunches with and without damage in the month before harvest. Damaged berries at greater than 12.3 (o) Bx were particularly susceptible to Aspergillus rot and production of OA, which was concentrated in severely mouldy berries. CONCLUSIONS: OA in Australian grapes results mainly from infection of berries by A. carbonarius. It is concentrated in discoloured, shrivelled berries. The potential for Aspergillus rot and OA production appears to commence after veraison and increase with berry damage and ripeness. SIGNIFICANCE AND IMPACT OF THE STUDY: Minimizing damage to grapes between veraison and harvest significantly reduces Aspergillus rot and OA formation. Monitoring the extent of Aspergillus rot in bunches infected with toxigenic Aspergillus spp. may give some indication of OA contamination.     

Mycelial growth and ochratoxin A production by Aspergillus section Nigri on simulated grape medium in modified atmospheres

Aims:  To evaluate the impact of modified atmosphere packaging on in vitro growth of Aspergillus carbonarius and Aspergillus niger , and possible effects on ochratoxin A (OTA) biosynthesis.
Methods and Results:  Ochratoxigenic isolates belonging to the species A. carbonarius and A. niger were grown on a synthetic grapejuice medium (SNM) and packaged in combinations of controlled O₂ (1% and 5%) and CO₂ levels (0% and 15%), and in air as a control. Colony diameters were recorded every 3 days up to 21 days, and OTA was analysed after 7, 14 and 21 days. The greatest reductions in mycelial growth rate were observed at 1% O₂ followed by 1% O₂/15% CO₂, whereas 5% O₂ stimulated the growth of all isolates. OTA production by A. carbonarius and A. niger isolates was minimized at 1% O₂/15% CO₂ and 1% O₂, respectively, after 7 days of incubation. Maximal OTA accumulation after 7 days was observed for all isolates in the control pack and at 5% O₂.
Conclusions:  Of the atmospheres tested, only 1% O₂ combined with 15% CO₂ consistently reduced fungal growth and OTA synthesis by A. carbonarius and A. niger .
Significance and Impact of the Study:  Storage under modified atmospheres is unlikely to be suitable as the sole method for OTA minimization and grape preservation; other inhibitory factors are necessary.     

Fungi and ochratoxin A detected in healthy grapes for wine production

AIMS: The mycoflora of healthy grapes (i.e. without visible symptoms of rot) for wine production in Portuguese wine-making regions was assessed and its potential for ochratoxin A (OTA) production evaluated. The OTA content of grapes was also determined. METHODS AND RESULTS: A total of 386 fungal strains were isolated by plating methods. The most frequent genera found in grapes were non-ochratoxigenic species: Cladosporium (28%), Penicillium (24%), Botrytis (13%) and Aspergillus (9%). Two OTA-producing strains were isolated, belonging to the species Aspergillus carbonarius and Aspergillus ochraceus. OTA was detected in three of four grape samples, up to 116 ng l(-1). CONCLUSIONS: OTA is being produced in healthy berries by Aspergillus species, namely A. carbonarius, at levels below the maximum recommended limit of 2,000 ng l(-1) in wine. SIGNIFICANCE AND IMPACT OF THE STUDY: The OTA concentration detected in healthy Portuguese grapes does not represent a risk to wine regarding the legal limit established.     

Inhibition of species of the Aspergillus section Nigri and ochratoxin a production in grapes by fusapyrone

Fusapyrone (FP), an antifungal natural compound, was tested against the three main ochratoxigenic species of the Aspergillus section Nigri. The MICs at 24 h were 6.0, 11.6, and 9.9 mug/ml for Aspergillus carbonarius, Aspergillus tubingensis, and Aspergillus niger, respectively. Strong inhibition of growth and morphological changes were still observed at half the MIC after 7 days. The application of a 100 mug/ml FP solution in a laboratory assay on artificially inoculated grapes resulted in a significant reduction (up to 6 orders of magnitude) of A. carbonarius CFU counts. Dramatic reductions of the ochratoxin A (OTA) content, compared to the content of the positive control (average amount of OTA, 112.5 ng/g of grape; three experiments), were obtained with the application of either 100 or 50 mug/ml of FP (0.6 or 5.1 ng/g of grape, respectively).     

Survey of mycoflora and ochratoxin A in dried vine fruits from Argentina markets

AIMS: The aims of this work were to identify the mycoflora and to evaluate the natural occurrence of OA in dried vine fruits. Likewise, the capacity to produce OA by Aspergillus section Nigri was studied. MATERIALS AND METHODS: Fifty samples of dried vine fruits were obtained from Mendoza and San Juan provinces. The surface disinfection method was used for mycoflora determination using the medium dichloran 18% glycerol agar (DG18) and dichloran Rose Bengal chloramphenicol agar (DRBC). RESULTS: Statistical analysis demonstrated that the species A. niger var. niger and Aspergillus niger var. awamori were isolated in higher frequency from black dried vine fruits from DRBC and DG18 media (P < 0.01). OA was found in 74% of the dried vine fruits samples. Sixty-two strains (28%) of Aspergillus section Nigri, were OA producers. In the species A. carbonarius the highest percentages of ochratoxigenic strains were detected (82.6%). CONCLUSIONS: The presence of ochratoxigenic strains of Nigri section in dried vine fruits suggests that they may be an important source of OA in this substrate. Dried vine fruits can also be an important source of OA people who consume large amounts. SIGNIFICANCE AND IMPACT OF THE STUDY: The dried vine fruits contamination with Aspergillus section Nigri and OA was significant.     

Partitioning of ochratoxin A in mycelium and conidia of Aspergillus carbonarius and the impact on toxin contamination of grapes and wine

AIMS: Aspergillus carbonarius is an important ochratoxin A (OTA)-producing fungus which is responsible for toxin contamination of grapes and wine. The objectives of this study were to examine the partitioning of OTA in mycelium and conidia of a range of A. carbonarius strains on artificial grape juice and defined media, to determine the excretion patterns of OTA from these spores, and the effect of organic acids used in wine production on OTA excretion from conidia. METHODS AND RESULTS: The results showed that 60-70% of the OTA was accumulated in the conidia of a number of different isolates of A. carbonarius. Calculations showed that on different defined media, an amount of 0.011- to 0.1-pg OTA was present per conidium. The OTA in spores was found to be rapidly excreted into the medium during the initial few hours after conidial germination leading to an increase of OTA in must during maceration for wine production. The presence of tartaric acid inhibited OTA production, but malic acid enhanced this production during mycelial growth. These acids were also shown to affect the time course of germination and the rate of OTA excretion from conidia during germination. CONCLUSIONS: This study is the first to examine and show the partitioning of OTA into spores of strains of A. carbonarius and that rapid excretion of OTA from spores could be a reason for OTA accumulation in musts during wine production. SIGNIFICANCE AND IMPACT OF THE STUDY: Conidia of A. carbonarius could be a major source of OTA contamination of grapes used in wine production. This information could help in the development of effective prevention strategies to minimize wine contamination with this important mycotoxin.     

Study of Spanish grape mycobiota and ochratoxin A production by Isolates of Aspergillus tubingensis and other members of Aspergillus section Nigri

The native mycobiota of five grape varieties grown in Spain has been studied. Four (Bobal, Tempranillo, Garnacha, and Monastrell) were red varieties and one (Moscatel) was white. The main fungal genera isolated were Alternaria, Cladosporium, and Aspergillus. The isolation frequency of Aspergillus spp. section Nigri in contaminated samples was 82%. Ochratoxin A (OTA) production was assessed using yeast extract-sucrose broth supplemented with 5% bee pollen. Cultures of 205 isolates from this section showed that 74.2% of Aspergillus carbonarius and 14.3% of Aspergillus tubingensis isolates produced OTA at levels ranging from 1.2 to 3,530 ng/ml and from 46.4 to 111.5 ng/ml, respectively. No Aspergillus niger isolate had the ability to produce this toxin under the conditions assayed. Identification of the A. niger aggregate isolates was based on PCR amplification of 5.8S rRNA genes and its two intergenic spacers, internal transcribed spacer 1 (ITS1) and ITS2, followed by digestion with restriction endonuclease RsaI of the PCR products. The restriction patterns were compared with those from strains of A. niger CECT 2807 and A. tubingensis CECT 20393, held at the Spanish Collection of Type Cultures. DNA sequencing of the ITS1-5.8S rRNA gene-ITS2 region of the OTA-producing isolates of A. tubingensis matched 99 to 100% with the nucleotide sequence of strain A. tubingensis CBS 643.92. OTA determination was accomplished by liquid chromatography with fluorescence detection. OTA confirmation was carried out by liquid chromatography coupled to ion trap mass spectrometry. The results showed that there are significant differences with regard to the isolation frequency of ochratoxinogenic fungi in the different grape varieties. These differences were uncorrelated to berry color. The ability of A. tubingensis to produce OTA and the influence of grape variety on the occurrence of OTA-producing fungi in grapes are described in this report for the first time.     

Utilization of AFLP markers for PCR-based identification of Aspergillus carbonarius and indication of its presence in green coffee samples

AIMS: The objective of this work was to test whether ochratoxin A (OTA) production of Aspergillus niger and A. carbonarius is linked to a certain genotype and to identify marker sequences with diagnostic value aiding identification of A. carbonarius, a fungus of major concern regarding OTA production in food and food raw materials. METHODS AND RESULTS: Aspergillus niger and A. carbonarius were isolated mainly from Brazilian coffee sources. The ability of isolates to produce OTA was tested by thin layer chromatography (TLC). Strains were genetically characterized by AFLP fingerprinting and compared with each other and with reference strains. Cluster analysis of fingerprints showed clear separation of A. niger from A. carbonarius strains. To obtain marker sequences, AFLP fragments were isolated from silver stained polyacrylamide gels, cloned and sequenced. Sequences obtained were used to develop species- specific PCR primers for the identification of A. carbonarius in pure culture and in artificially and naturally infected samples of green coffee. CONCLUSIONS: No clear correlation between genetic similarity of the strains studied and their potential to produce OTA was found. The PCR assays designed are a useful and specific tool for identification and highly sensitive detection of A. carbonarius. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed PCR assays allow specific and sensitive detection and identification of A. carbonarius, a fungus considered to be one of the major causative agents for OTA in coffee and grape-derived products. Assays may provide powerful tools to improve quality control and consumer safety in the food processing industry.     

Aspergillus carbonarius growth and ochratoxin A production on a synthetic grape medium in relation to environmental factors

AIMS: The effects of water activity (0.90-0.99 a(w)), temperature (15-37 degrees C), and their interaction on growth and ochratoxin A (OTA) production by eight isolates of Aspergillus carbonarius were investigated on synthetic nutrient medium (SNM) with composition similar to grapes. METHODS AND RESULTS: Growth data were modelled by an multiple linear regression and response surface models were obtained. Aspergillus carbonarius grew much faster at 30 degrees C than at the other temperature levels tested and its growth rate increased with increasing a(w), maximum growth rate being between 0.95 and 0.99 a(w). In general, isolates grew faster at 35-37 degrees C than at 20 degrees C, although no significant differences were found between these temperatures. OTA accumulation was also favoured by high a(w) levels, and although it was observed in the whole range of temperatures, maximum amounts were detected at 20 degrees C. No OTA was found at the most unfavourable growth conditions. CONCLUSIONS: Optimum a(w) level for growth seems to correspond with optimum for OTA production, meanwhile the most propitious temperature for the toxin production was below the best one for growth. SIGNIFICANCE AND IMPACT OF THE STUDY: Prediction of A. carbonarius growth would allow estimating their presence and therefore, the OTA production, as it was found that conditions for the toxin production were more limited than those permitting growth.     

Taxonomy and significance of black aspergilli

Members of Aspergillus section Nigri (formerly A. niger group) are distributed worldwide and are regarded as common food spoilage fungi. Some of them are widely used and studied for industrial purposes. They are common sources of extracellular enzymes and organic acids to be used in food processing and are also used in the production of traditional foods, especially in the Orient. Products produced by strains of Aspergillus niger hold the GRAS (Generally Recognised As Safe) status from the FDA. However some species in Aspergillus section Nigri can produce ochratoxin A, a nephrotoxic mycotoxin. In spite of their industrial importance, the taxonomy of black aspergilli ( Aspergillus section Nigri ) is not clear and many attempts have been made in order to find suitable taxonomic criteria. The aim of this paper is to provide an overview of the significance of black aspergilli focusing on all the approaches made in the taxonomy of this group of fungi. Some species, such as A. carbonarius and uniseriate species can be easily recognised. In the A. niger aggregate, although speciation at molecular level has been proposed, no morphological differences can be observed and species identification will therefore remain problematic. Phylogenetic analyses of ITS and 5.8S rDNA gene region of representative black Aspergillus species and a simple key to the most common species that can be easily distinguished by morphological criteria are also included.     

Water and temperature relations of growth and ochratoxin A production by Aspergillus carbonarius strains from grapes in Europe and Israel

AIMS: This study investigated the in vitro effects of water activity (a(w); 0.85-0.987) and temperature (10-40 degrees C) on growth and ochratoxin A (OTA) production by two strains of Aspergillus carbonarius isolated from wine grapes from three different European countries and Israel on a synthetic grape juice medium representative of mid-veraison (total of eight strains). METHODS AND RESULTS: The synthetic grape juice medium was modified with glycerol or glucose and experiments carried out for up to 56 days for growth and 25 days for OTA production. The lag phase prior to growth, growth rates and ochratoxin production were quantified. Statistical comparisons were made of all factors and multiple regression analysis used to obtain surface response curves of a(w) x temperature for the eight strains and optimum growth and OTA production by A. carbonarius. The lag phase increased from <1 day at 25-35 degrees C and 0.98 a(w) to >20 days at marginal temperatures and water availabilities. Generally, most A. carbonarius strains grew optimally at 30-35 degrees C, regardless of solute used to modify a(w), with no growth at <15 degrees C. The optimum a(w) for growth varied from 0.93 to 0.987 depending on the strain, with the widest a(w) tolerance at 25-30 degrees C. There was no direct relationship among growth, environmental factors and country of origin of individual strains. Optimum conditions for OTA production varied with strain. Some strains produced optimal OTA at 15-20 degrees C and 0.95-98 a(w). The maximum OTA produced after 10 days was about 0.6-0.7 microg g(-1), with a mean production over all eight strains of 0.2 microg g(-1) at optimum environmental conditions. CONCLUSIONS: This work demonstrates that optimum conditions for OTA production are very different from those for growth. While growth rates differed significantly between strains, integration of the OTA production data suggests possible benefits for use of the information on a regional basis. SIGNIFICANCE AND IMPACT OF THE STUDY: Very little detailed information has previously been available on the ecology of A. carbonarius. This knowledge is critical in the development and prediction of the risk models of contamination of grapes and grape products by this species under fluctuating and interacting environmental parameters.     

Ochratoxin A-producing Aspergilli in Vietnamese green coffee beans

AIMS: To determine the incidence and severity of infection by ochratoxin A (OA)-producing fungi in Vietnamese green coffee beans. METHODS AND RESULTS: Aspergillus carbonarius, A. niger and yellow Aspergilli (A. ochraceus and related species in section Circumdati) were isolated by direct plating of surface-disinfected Robusta (65 samples) and Arabica (11 samples) coffee beans from southern and central Vietnam. Significantly, more Robusta than Arabica beans were infected by fungi. Aspergillus niger infected 89% of Robusta beans, whereas A. carbonarius and yellow Aspergilli each infected 12-14% of beans. OA was not produced by A. niger (98 isolates) or A. ochraceus (77 isolates), but was detected in 110 of 113 isolates of A. carbonarius, 10 isolates of A. westerdijkiae and one isolate of A. steynii. The maximum OA observed in samples severely infected with toxigenic species was 1.8 microg kg(-1); however, no relationship between extent of infection and OA contamination was observed. CONCLUSIONS: Aspergillus niger is the dominant species infecting Vietnamese coffee beans, yet A. carbonarius is the likely source of OA contamination. SIGNIFICANCE AND IMPACT OF STUDY: Vietnamese green coffee beans were more severely infected with fungi than the levels reported for beans from other parts of the world, yet OA contamination appears to be infrequent.     

Biodegradation of ochratoxin A by Aspergillus section Nigri species isolated from French grapes: a potential means of ochratoxin A decontamination in grape juices and musts

Ochratoxin A (OTA) is a very dangerous mycotoxin, the presence of which is often reported in different foods, as well as in beverages such as grapes, grape juices and wines. Detoxifying these products is therefore of prime importance in protecting consumer health, and biological approaches have been the most promising methods. In this report, 40 isolates representing the black apergilli species Aspergillus carbonarius, A. niger aggregate and A. japonicus, isolated on French grapes, were assessed for OTA degradation capacities in CZAPEK yeast extract broth (CYB) and in a synthetic grape juice medium (SGM) contaminated with OTA at 2 mg L(-1) (5 microM). It was clearly observed that in both media these fungi had the ability to degrade OTA to OTalpha (ochratoxinalpha). However, there were differences between the media used and species tested during OTA degradation. In SGM and CYB, 77% and 45% of the isolates, respectively were able to degrade more than 80% of the OTA. Despite a better growth on SGM, specific OTA degradation was higher on CYB for most of the isolates. Kinetic studies carried out on SGM with three black Aspergillus isolates all showed different OTA degradation rates. After 9 days of incubation, OTalpha had decreased, whereas an unknown compound appeared. A. niger could be the first interesting species for OTA detoxification processes, followed by A. japonicus.     

OTA-producing fungi isolated from stored cocoa beans

Aims:  The aim of this study was to identify fungal populations in unroasted cocoa beans stored in Spain in order to evaluate the ochratoxin A (OTA)-production ability of certain Aspergillus isolates.
Methods and Results:  Twenty batches of cocoa beans from different origins and with different OTA content were selected for this study. Three Aspergillus carbonarius and 13 Aspergillus niger aggregate strains isolated from these cocoa bean samples were selected to evaluate their OTA synthesis ability, being the only A. carbonarius isolates which are OTA producers [−1 culture medium; LOD = 6  μ g kg⁻¹ culture medium].
Conclusions:  No correspondence was found between the OTA levels in cocoa beans and the presence of OTA-producing fungi. Nonetheless, some samples contained A. carbonarius with a high OTA-producing ability and, consequently, specific fungal controls should be set up during storage to avoid this toxin.
Significance and Impact of the Study:  Toxigenic fungi in cocoa beans are not well understood. This study attempted to identify these fungi and evaluate their OTA-producing ability.     

Ochratoxin A production and amplified fragment length polymorphism analysis of Aspergillus carbonarius, Aspergillus tubingensis, and Aspergillus niger strains isolated from grapes in Italy

Ochratoxin A is a potent nephrotoxin and a possible human carcinogen that can contaminate various agricultural products, including grapes and wine. The capabilities of species other than Aspergillus carbonarius within Aspergillus section Nigri to produce ochratoxin A from grapes are uncertain, since strain identification is based primarily on morphological traits. We used amplified fragment length polymorphisms (AFLPs) and genomic DNA sequences (rRNA, calmodulin, and beta-tubulin genes) to identify 77 black aspergilli isolated from grape berries collected in a 2-year survey in 16 vineyards throughout Italy. Four main clusters were distinguished, and they shared an AFLP similarity of <25%. Twenty-two of 23 strains of A. carbonarius produced ochratoxin A (6 to 7,500 microg/liter), 5 of 20 strains of A. tubingensis produced ochratoxin A (4 to 130 microg/liter), 3 of 15 strains of A. niger produced ochratoxin A (250 to 360 microg/liter), and none of the 19 strains of Aspergillus "uniseriate" produced ochratoxin A above the level of detection (4 microg/liter). These findings indicate that A. tubingensis is able to produce ochratoxin and that, together with A. carbonarius and A. niger, it may be responsible for the ochratoxin contamination of wine in Italy.