Occurrence of ochratoxigenic fungi and ochratoxin A in grapes from a Tunisian vineyard

The occurrence of ochratoxin A (OTA) and the identification of the ochratoxigenic microbiota in Tunisian grapes were studied for the first time. Black aspergilli were the dominant genus among the filamentous fungi isolated from grapes and were the only potential OTA-producing fungi found. The most abundant species were member of Aspergillus niger aggregate (63%) and Aspergillus carbonarius (36%). Uniseriate aspergilli were rarely present (1%). Of the A. carbonarius isolates, 97% were OTA positive but only 3% of the A. niger aggregate isolates were OTA positive. During grape maturation, the frequency of black aspergilli increased due to increase of the numbers of A. carbonarius. Musts (n=24) obtained from grapes collected at the different sampling times were analyzed for their OTA content. Up to 37% of the musts contained OTA at levels varying between 0.59 and 2.57 microg/l. The amounts of OTA in musts increased as grapes matured. These results indicate that A. carbonarius is the main cause of OTA contamination of Tunisian grapes.     

Occurrence of ochratoxin A in Australian wine

A survey of 601 samples of Australian wine for the presence of ochratoxin A (OA) was undertaken. The study sampled predominantly bottled wines, approximately equal numbers of red and white wines, wines made from a range of grape varieties and wines from all the major producing areas and some of the minor ones. A validated HPLC assay that did not utilise expensive immunoaffinity columns was developed for quantifying OA in wine, with limits of detection and quantification of 0.02 and 0.05 μg/L respectively. OA was detected in 90 (15.0%) of the 601 samples, but at uniformly low levels, with 85% of positive samples containing less than 0.2 μg/L OA. Only one sample exceeded 0.5 μg/L OA and no sample exceeded 1.0 μg/L OA. No region of Australia appeared more or less likely to produce wines containing OA.     

Occurrence of ochratoxin A in wine and ochratoxigenic mycoflora in grapes and dried vine fruits in South America

Grape and wine production in South America represents about 6.6% and 10% respectively of the world grape and wine production. The available information on the ochratoxigenic mycoflora and ochratoxin A (OTA) presence in wine grapes, wines, grape juices and dried vine fruits is limited. Surveys have been carried out in Argentina and Brazil which showed that Aspergillus niger aggregate are predominant in the Argentinean varieties while from the Brazilian varieties the species A. niger, Aspergillus ochraceus and Aspergillus carbonarius were isolated. A mycobiota survey from wine grapes in Argentina showed that while Alternaria alternata was predominant, Aspergillus section Nigri species were isolated from 60% of samples. About 41% of black Aspergilli isolates produced OTA with levels ranging from 2 to 24.5 ng mL(-1). In another study, about 83% of A. carbonarius isolates from dried vine fruits produced OTA, with levels ranging from 2 to 5200 ng mL(-1). A survey of grape juices and wines of Brazilian, Argentinean and Chilean origin were found to contain very low levels of OTA. Studies are in progress in Latin America on the ecophysiology of ochratoxigenic fungi and OTA occurrence to reduce the impact of this toxin in the food chain.     

Mycobiota and ochratoxin A producing fungi from Spanish wine grapes

Grapes from three different regions with a long winemaking tradition in Spain were analysed at different growth stages in order to identify the ochratoxigenic mycobiota during three consecutive seasons. The correlation between meteorological parameters and ochratoxigenic fungi was studied and revealed a significant positive correlation between black aspergilli infection and temperatures in the month preceding each sampling date. No significant correlation was found with either relative humidity or rainfall. Biodiversity indexes were also calculated in this study. Black aspergilli species were the most abundant in grapes before harvest, and among them, Aspergillus carbonarius was the main ochratoxin A (OTA) producer species and represented 78-100% of the isolates tested. The results obtained support the key role of A. carbonarius as the main source of OTA contamination in grapes.     

European research on ochratoxin A in grapes and wine

European wine production represents about 70% of world production and thus is an important export commodity. Ochratoxin A (OTA) was first detected as a wine contaminant in 1996 and the role of Aspergillus section Nigri and A. carbonarius in OTA production discovered in Europe in 1999. Subsequently Europe-wide surveys have shown that A. carbonarius is predominantly responsible for OTA contamination of grapes, wine and vine fruits. Analyses of wine samples throughout Europe have shown that there is a gradient in OTA concentration with a decrease from red, to rose and to white wines. The latitude of production is an important factor in determining risk from OTA contamination. Some geographic regions in Southern Europe are more prone to contamination with the toxigenic species and OTA. Ochratoxin A has also been found in much higher concentrations (max. 53 mug/kg) in dried vine fruit than in wine suggesting that A. carbonarius can dominate the drying vine fruit ecosystem. There is a significant lack of knowledge in Europe on conducive climatic conditions preharvest and their relationship with levels of risk from OTA contamination in grapes and their fate in wine production. This needs to be integrated with cultivation system to maximise the prevention of OTA entering this food chain.     

Occurrence of toxigenic fungi in ochratoxin A contaminated liquorice root

Fungi associated with ochratoxin A (OTA)-contaminated liquorice root and their capabilities for OTA production were investigated. Medicinal materials of mouldy liquorice root were collected from herbal markets located in Jiangxi, Zhejiang, Henan provinces and Beijing, China, respectively. Sixteen fungal species belonging to Penicillium, Aspergillus, Eurotium, Fusarium, Mucor and Scopulariopsis were isolated; the fungal composition was different in each liquorice root sample. Penicillium polonicum was predominant, comprising 54% of the total isolates in the liquorice root sample from Jiangxi province, which was contaminated with OTA at the highest level. In other samples with lower OTA contents, species of Aspergillus and Eurotium were predominant. OTA production of representative strains on rice media was detected by LC-MS/MS; all Penicillium polonicum isolates and a P. chrysogenum were ochratoxigenic; OTA concentrations ranged from 6.94 to 217.37?ng?g(-1). This is the first study to report P. polonicum as an OTA-producing fungus. OTA contamination of mouldy liquorice root constitutes a major health hazard in consumption. This situation demands urgent and undivided attention.     

Occurrence of toxigenic fungi in ochratoxin A contaminated liquorice root

Fungi associated with ochratoxin A (OTA)-contaminated liquorice root and their capabilities for OTA production were investigated. Medicinal materials of mouldy liquorice root were collected from herbal markets located in Jiangxi, Zhejiang, Henan provinces and Beijing, China, respectively. Sixteen fungal species belonging to Penicillium, Aspergillus, Eurotium, Fusarium, Mucor and Scopulariopsis were isolated; the fungal composition was different in each liquorice root sample. Penicillium polonicum was predominant, comprising 54% of the total isolates in the liquorice root sample from Jiangxi province, which was contaminated with OTA at the highest level. In other samples with lower OTA contents, species of Aspergillus and Eurotium were predominant. OTA production of representative strains on rice media was detected by LC-MS/MS; all Penicillium polonicum isolates and a P. chrysogenum were ochratoxigenic; OTA concentrations ranged from 6.94 to 217.37?ng?g^?1. This is the first study to report P. polonicum as an OTA-producing fungus. OTA contamination of mouldy liquorice root constitutes a major health hazard in consumption. This situation demands urgent and undivided attention.     

Influence of nitrogen and carbon sources on the production of ochratoxin A by ochratoxigenic strains of Aspergillus spp. isolated from grapes

This work studies the influence of nitrogen and carbon source on ochratoxin A production by three Aspergillus isolates A. ochraceus (Aso2), A. carbonarius (Ac25) and A. tubingensis (Bo66), all isolated from grapes. A basal medium (0.01 g/l FeSO4.7H2O, 0.5 g/l MgSO4.7H2O, 0.5 g/l Na2HPO4.2H2O, 1.0 g/l KCl) was prepared. This medium was supplemented with different nitrogen sources, both inorganic [(NH4)3PO(4), 0.3 g/l plus NH4NO3, 0.2 g/l] and organic (histidine, proline, arginine, phenylalanine, tryptophan or tyrosine) at two concentrations (0.05 g/l or 0.3 g/l), and different carbon sources (sucrose, glucose, maltose, arabinose or fructose) at three concentrations (10 g/l, 50 g/l or 150 g/l). A medium with sucrose (18 g/l) and glucose (1 g/l) was also tested. After a 10-day incubation period at 25 degrees C the highest levels of OTA (44.0 ng/ml, 13.5 ng/ml and 0.49 ng/ml for A. ochraceus, A. carbonarius and A. tubingensis, respectively) were obtained in the cultures containing 150 g/l of arabinose and 0.05 g/l of phenylalanine. Analysis of variance of the data showed that there were significant differences (p-value 0.05) among the OTA levels in the cultures with regard to carbon source and isolate. No significant differences were detected in OTA production regarding nitrogen source, although 0.05 g/l of phenylalanine generally favoured OTA production in the cultures of the three isolates. The dynamics of toxin production in the cultures of each isolate using the optimized basal medium supplemented with 0.05 g/l of phenylalanine and 150 g/l of arabinose for a period of 42 days at 25 degrees C was also studied. The maximum level of OTA was detected on the 3rd day of incubation in A. tubingensis cultures and on the 35th and 43(rd) days of incubation in A. ochraceus and A. carbonarius, respectively. This is the first study in which defined media have been used to assess the influence of carbon and nitrogen sources on OTA production by isolates of OTA-producing species isolated from grapes and to analyse the dynamics of toxin production in these species in a defined culture medium. This optimized medium for OTA production is being used in current studies aimed at elucidating its biosynthetic pathway.     

Effect of chemical treatments on ochratoxigenic fungi and common mycobiota of grapes (Vitis vinifera)

The effect of the application of two fungicides (cyprodinil alone and a mixture of cyprodinil and fludioxonil) on the mycoflora of grapes, especially ochratoxigenic fungi, was studied. Different doses and application times were analyzed. Grape mycobiota was isolated and identified, and the classification of black aspergilli was carried out. We found that 81.7% of the isolates belonged to Aspergillus niger aggregate and 18.3% to Aspergillus carbonarius. The ability to produce ochratoxin A (OTA) was studied on Czapek yeast extract agar (CYA) medium in 238 isolates. Most A. carbonarius (97.2%) produced detectable amounts of OTA, while only 2.9% of the A. niger aggregate were OTA producers. Most of the isolates (58%) produced less than 2.5 microg OTA per g of CYA. That, together with the highest levels of black aspergilli detected near harvest, proved the reported theory that they are primarily responsible for OTA in grapes. The fungicides studied had a significant effect on black aspergilli in three of the four vineyards sampled, as the natural increase of black aspergilli when approaching harvest was in general lower in all the fields treated with fungicides as compared to the control treatment. A mixture of cyprodinil (37.5%) and fludioxonil (25%) applied at veraison and 21 days before harvest was the most effective treatment to prevent black aspergilli in grapes, together with a single application of this mixture at veraison followed by an application of cyprodinil (50%) 21 days before harvest. No OTA was detected in musts (n=112) produced from either the control treatment or the treated grapes.     

Occurrence of ochratoxin A producing fungi in wine and table grapes in Israel

A 3-year survey was conducted to assay the number of Aspergillus Section Nigri isolates and in vitro ochratoxin A (OTA) production capacity in 10 vineyards in Israel. The survey included field sampling of two wine cultivars, 'Sauvignon Blanc' and 'Cabernet Sauvignon' as well as the table grape cultivar 'Superior'. A total of 2114 isolates were analyzed and of those 161 isolates were shown to produce OTA. The major finding was that Aspergillus carbonarius (336 tested strains) is the most consistent producer of OTA, with approximately 35% of the isolates identified as positive in vitro. In comparison, 3.1% of other isolates from the Aspergillus niger aggregate (of 1432 strains) produced OTA in vitro. In contrast, none of the 346 tested strains with a uniseriate head morphology produced OTA. The incidence of infected berries was very low before veraison, while at harvest, this frequency was twice as high. In general, the composition of black Aspergilli did not differ during berry development. Generally, more OTA-producing isolates were isolated from the surface of table grapes cv. 'Superior' compared to 'Sauvignon Blanc'. None of the samples collected at harvest contained traces of OTA in the juice. This study shows that grapes in Israel are contaminated with ochratoxigenic species which represent a risk of OTA contamination.     

Incidence of toxigenic fungi and ochratoxin A in dried fruits sold in Brazil

A total of 117 dried fruit samples (black sultanas, white sultanas, dates, dried plums, dried figs and apricots) from different origins were analysed both for toxigenic fungi and for the presence of ochratoxin A. Amongst the fungi found, Aspergillus niger was predominant, with 406 isolates, of which 15% were ochratoxin A producers. They were followed by A. ochraceus, with 15 isolates and 87% ochratoxigenics, and A. carbonarius, with only five isolates of which 60% were ochratoxin A producers. The average infection rates for A. niger in black sultanas, plums, figs, dates and white sultanas were 22.0, 8.0, 4.0, 1.5 and 0.5%, respectively. The apricot samples were not contaminated by any fungi or ochratoxin A. Black sultana and dried figs contained the highest contamination with ochratoxin A, with 33 and 26.3% of the samples containing more than 5 µg kg^-1 respectively, while all the white sultanas, dates and plums had no sample that exceeded this limit.     

Aflatoxins and ochratoxin A in stored barley grain in Spain and impact of PCR-based strategies to assess the occurrence of aflatoxigenic and ochratoxigenic Aspergillus spp

Contamination of barley by moulds and mycotoxins results in quality and nutritional losses and represents a significant hazard to the food chain. The presence of aflatoxin B1 (AFB1), B2 (AFB2), G1 (AFG1) and G2 (AFG2) and ochratoxin A (OTA) in stored barley in Spain has been studied. Species-specific PCR assays were used for detection of Aspergillus flavus, A. parasiticus, A. ochraceus, A. steynii, A. westerdijkiae, A. carbonarius and A. niger aggregate in mycotoxin-positive barley samples at different incubation times (0, 1 and 2 days). Classical enumeration techniques (CFU/g) in different culture media for evaluation of Aspergillus in sections Flavi, Circumdati and Nigri were also used. One hundred and five barley kernel samples were collected in Spanish grain stores from 2008 to 2010, and analyzed using a previously optimized method involving accelerated solvent extraction, cleanup by immunoaffinity column, liquid chromatographic separation, post-column derivatization with iodine and fluorescence detection. Twenty-nine samples were contaminated with at least one of the studied mycotoxins. AFB1, AFB2, AFG1, AFG2, and OTA were detected in 12.4%, 2.9%, 4.8%, 2.9%, and 20% of the samples, respectively. Aflatoxins and OTA co-occurred in 4.8% of the samples. Maximum mycotoxin levels (ng/g) were 0.61 (AFB1), 0.06 (AFB2), 0.26 (AFG1), 0.05 (AFG2), and 2.0 (OTA). The results of PCR assays indicated the presence of all the studied species, except A. westerdijkiae. The PCR assays showed high levels of natural contamination of barley with the studied species of Aspergillus which do not correspond to the expected number of CFU/g in the cultures. These results suggest that a high number of non-viable spores or hyphae may exist in the samples. This is the first study carried out on the levels of aflatoxins and OTA in barley grain in Spain. Likewise, this is the first report on the presence of aflatoxigenic and ochratoxigenic Aspergillus spp. in barley grain naturally contaminated with those mycotoxins using a species-specific PCR approach.     

Occurrence of mycotoxins (ochratoxin A, deoxynivalenol) and toxigenic fungi in Moroccan wheat grains: impact of ecological factors on the growth and ochratoxin A production

The aim of the present work was to evaluate the contamination of some samples, taken from Moroccan wheat grains, by ochratoxin A (OTA), deoxynivalenol (DON) and the associated toxigenic fungi. Moreover, we focused on the influence of environmental factors on both the growth and OTA production by three strains of Aspergillus. The results showed that only few samples were contaminated by the two mycotoxins (2 samples for OTA and 7 for DON). The main isolated fungi belong to the Aspergillus, Penicillium and Fusarium genus; 74 Aspergillus and 28 Penicillium isolates were tested for their ability to produce OTA. Only 2 A. alliaceus and 14 A. niger were able to synthesize OTA. However, none of Penicillium isolates can produce this toxin under the conditions mentioned. In respect of the effects of the temperature and water activity (aw), the optimal conditions for the growth and OTA production were different. While the optimal conditions of growth for A. alliaceus and A. terreus are 30 degrees C and 0.98 aw, A. niger preferred 0.93-0.95 aw at 25 degrees C, whereas the optimal production of OTA was observed at 30 degrees C for both A. alliaceus and A. niger at 0.93 and 0.99 aw, respectively.     

Occurrence of ochratoxin A-producing fungi in raw Brazilian coffee

Ochratoxin A (OA)-producing fungi were identified in coffee at different stages of maturation. The toxin was quantified in coffee during terrace drying and in coffee stored in barns. By direct plating, a high level of contamination (100%) was found in the coffee beans studied, with the genus Aspergillus representing 33.2%, of which Aspergillus ochraceus and Aspergillus niger represented 10.3 and 22.9%, respectively, of the strains isolated from the coffee beans. The capacity to produce ochratoxin was determined in 155 strains of A. ochraceus and A. niger using both the agar plug method and extraction with chloroform, giving positive results for 88.1% of the A. ochraceus strains and 11.5% of the A. niger strains. Analysis for OA in the terrace and barn coffee samples showed that, independent of cultivar, year harvested, or production region, all except one of the samples analyzed showed mycotoxin levels below the limit suggested by the European Common Market (8 microg/kg), thus indicating that the problem is restricted and due to severe faults in harvesting and storage practices.     

Advances in the molecular diagnosis of ochratoxin A-producing fungi

Ochratoxin A (OTA) is detected worldwide in various food and feed sources. The compound is produced by Penicillium nordicum and P. verrucosum, as well as by various species within the sections Nigri and Circumdati of the genus Aspergillus, with A. ochraceus and A. carbonarius known to be the predominant producers. Recently, various pairs of PCR primers based on AFLP, RFLP, RAPD and the calmodulin gene were developed to set up novel diagnostic approaches for OTA producers in the Aspergillus and Penicillium genera. Real-time PCR assays based on well-characterized genomic sequences in A. ochraceus and P. nordicum have also been set up. Since the application of such assays to the analysis of contaminated sample material was demonstrated in only a few cases, future studies should be focused on applying such methods in rapid, robust and user-friendly applications, and implementing them in HACCP concepts. The recent detection and characterization of OTA biosynthetic pathway genes in the Penicillium genus is an important step towards understanding what mechanisms influence production of the toxin in order to redesign production processes in the food and feed industry and to keep de-novo synthesis to a minimum.     

An easy screening method for fungi producing ochratoxin A in pure culture.

A simple screening method has been developed for detecting ochratoxin production by fungi, based on high-performance liquid chromatographic determinations on extracts obtained from agar plugs cut from pure Petri dish cultures. Two culture media. Yeast Extract Sucrose agar and Czapek Yeast Extract agar, and three extraction solvents (methanol, methylene chloride/formic acid, and methanol/formic acid) were compared. All of the isolates tested produced ochratoxin A in one or both culture media after 7 or 14 days of incubation. Based on the results obtained, the use of both culture media is recommended. As extraction solvent, either methanol or methanol-formic acid could be used. This method also provides quantitative information on the level of ochratoxin produced by the cultures. The simplicity of the method makes it very useful when many fungal isolates need to be screened.     

Influence of light on food relevant fungi with emphasis on ochratoxin producing species

The influence of light of varying wavelength on growth and ochratoxin A biosynthesis of Aspergillus carbonarius, A. niger, A. steynii and on Penicillium nordicum and P. verrucosum was analysed. For comparison the influence of light on various other food relevant fungi, including citrinin producers, was also tested. Generally the Aspergilli seem to be more resistant to light treatment than the Penicillia. Interestingly wavelengths from both sides of the spectrum, e. g. red (long wavelength, 627 nm) and blue (short wavelength 470-455 nm) had the strongest inhibitory effects on growth and ochratoxin A biosynthesis. Blue light generally had a stronger effect. Light of moderate wavelength, 590 to 530 nm, (yellow to green) had more a positive than a negative influence on growth or ochratoxin A biosynthesis compared to the control (dark incubation). The light effect on growth and ochratoxin A biosynthesis was dependent on the growth medium. In contrast to malt extract medium (MEA), YES medium, as an especially nutrient rich medium, had an attenuating effect on the reactivity against light. However the tendency of the response in both media was the same. Moreover, the light intensity strongly influences how the fungus reacts. Depending on the intensity and the resistance of the species a complete cessation of growth and/or inhibition of ochratoxin A biosynthesis could be achieved. Light irradiation has the opposite effect on ochratoxin A than citrinin, two mycotoxins which can be produced simultaneously in P. verrucosum. Citrinin was produced essentially under light conditions which inhibited ochratoxin A biosynthesis. The same was true for a derivative of ochratoxin, in particular a derivative of ochratoxin β in A. carbonarius. A. carbonarius produced high amounts of the ochratoxin β derivative under blue light when the production of ochratoxin A was ceased at the most inhibiting conditions used (MEA, royal blue light, 455 nm, 1700 lx). Light has a growth stalling but not inactivating effect on aerial mycelia. If a non-growing colony under light is shifted to the dark it immediately grows normally. However on spores blue light has a deactivating effect. After incubation of spores of P. verrucosum for 24 h under blue light up to 97% of the spores were no longer able to germinate. Again the spores of the Aspergilli were much more resistant.     

Occurrence of ochratoxin A-producing fungi in grapes grown in Italy

A study was carried out to investigate fungi present on grapes grown in Italy. Aspergillus and Penicillium spp. isolates were identified and studied in vitro, and their ability to produce ochratoxin A (OA) was investigated. The survey involved nine vineyards, three located in northern Italy and six located in southern Italy. In 1999 and 2000, bunches of grapes at different growth stages were collected from all nine vineyards, and berry samples were placed in moist chambers and incubated. The resultant fungal colonies were then transferred to petri dishes containing Czapek yeast agar and incubated at 25 degrees C for 7 days; the fungal isolates were identified and then cultivated in liquid Czapek yeast medium and evaluated for their ability to produce OA. During the survey, 508 isolates were collected, with 477 belonging to Aspergillus spp. and 31 belonging to Penicillium spp. Among the aspergilli, species of the Fumigati, Circumdati, and Nigri sections were identified, with species of the Nigri section (464 isolates) largely predominating; for species of the Nigri section, 108 isolates were uniseriate, 270 were biseriate, and 86 were identified as Aspergillus carbonarius. Black aspergilli isolated over the 2 years of the study showed a very similar pattern. On average, the biseriates represented about 60% of the isolates collected in both years and were followed by uniseriates (21%) and A. carbonarius (19%). The most toxigenic strains proved to be those of A. carbonarius; about 60% of these isolates were OA producers and produced the highest levels of OA. A. carbonarius was more frequent in the south, but in both areas the percentages of OA-producing isolates remained the same.     

Filamentous fungi producing ochratoxin a during cocoa processing in Cameroon

Ochratoxin A (OTA) is the main mycotoxin occurring in cocoa. A study was conducted in Cameroon to assess how filamentous fungi and toxigenesis were affected by the type of cocoa post-harvest treatment (boxes or heaps). The filamentous fungi isolated were almost identical when fermentation was carried out in boxes or heaps, with the presence of abundant black Aspergillus filamentous fungi: A. niger and A. carbonarius. Filamentous fungi were more abundant at the end of the harvesting season. Factors affecting bean integrity (poor handling, deferred processing) resulted in a qualitative and quantitative increase in contamination, when the total number of filamentous fungi could reach a maximum value of 5.5+/-1.4x10(7) CFU g(-1) and black Aspergilli a maximum value of 1.42+/-2.2x10(7) CFU g(-1). A toxigenesis study showed that Aspergillus carbonarius was the main OTA-producing strain isolated. Its maximum production could reach 2.77 microg g(-1) on rice medium. Aspergillus niger strains did not always produce OTA and their toxigenesis was much lower. Fermented dried cocoa from poor quality pods was the most contaminated by OTA: up to 48 ng g(-1).