Aspergillus spp., distribution, population composition and ochratoxin A production in wine producing vineyards in Greece

Vineyard surveys of 11 wine producing grape cultivars, were carried out in sixteen vineyards, in five winemaking regions in Greece, during 2002 and 2003. The occurrence of various Aspergillus spp. in bunches of berries at setting, veraison and ripening at harvest time was investigated. Aspergillus niger aggregate and A. carbonarius were predominantly isolated from sampled berries. Although the prevailing Aspergillus spp. isolates belonged to A. niger aggregate, isolates of A. carbonarius were the most efficient ochratoxin A (OTA) producers. Of 50 tested isolates of A. carbonarius 42% produced amounts of OTA, exceeding 25 ppb, while none of the 85 isolates of A. niger aggregate tested produced above 16 ppb.     

Australian research on ochratoxigenic fungi and ochratoxin A

The presence of the mycotoxin, ochratoxin A (OTA), has been reported in Australian grape products. Comprehensive surveys of Australian wines have determined that the frequency and level of OTA contamination are low. Aspergillus carbonarius is the primary OTA-producing species associated with grapes in Australia, and all isolates tested to date produce OTA. Aspergillus niger is isolated more frequently from vineyards, however, few strains produce OTA. A. carbonarius and A. niger exist as saprophytes in the top layer of soil beneath vines, from where they are thought to be blown onto bunches. The level of A. carbonarius in soil may be reduced by temperatures above or below the optimum temperature for survival (25 degrees C), by high soil moisture content, and by modifications to tillage and mulching practices. A. carbonarius is an opportunistic pathogen of damaged berries. In the absence of damage, spores may exist on berry surfaces without causing visible rots. Aspergillus rots are associated with black Aspergillus species, primarily A. niger, A. carbonarius and A. aculeatus. The potential for such rots is increased with berry damage, inoculum coverage and berry maturity. Susceptibility to berry splitting is related, in part, to bunch structure, and may be variety-dependent or influenced by rainfall, irrigation and canopy management. Black Aspergillus spp. are closely associated with berries near the main stem of the bunch. During winemaking, around 80% of the OTA initially present in grapes is removed, primarily with the skins and pulp during pressing. Additional reductions occur with the removal of precipitated grape and yeast solids. Bentonite in white wine and yeast hulls in red wine were the most effective non-carbonaceous fining agents for the removal of OTA.     

Ochratoxin A occurrence and formation in Portuguese wine grapes at various stages of maturation

A total of 93 Portuguese grape samples destined for wine production were examined for the presence of ochratoxin A (OTA) and the OTA producing fungi Aspergillus carbonarius and A. niger aggregate. Samples came from 11 vineyards from four winemaking regions in the North and South of the Portuguese mainland, during the harvest seasons of 2001, 2002 and 2003. Grapes were examined at 3 maturation stages, from setting to the harvesting period, to evaluate when contamination with OTA producing fungi and OTA synthesis occur. The detection of fungi in grape samples was made by plating methods with and without surface disinfection. OTA was formed by 14% of the 650 isolates tested. Most of the OTA producing strains (96%) were isolated at harvest time. At this stage, the percentage of grape samples with OTA producing strains detected without surface disinfection was 56%. With surface disinfection, A. carbonarius was isolated from 10% of the samples. OTA was detected in grapes at the 3 maturation stages. The average OTA concentrations in 60 samples at pea berry (28 samples), early veraison (22 samples) and ripe berry (20 samples) were 263, 149 and 35 ng/kg, respectively. Experiments with an A. carbonarius strain demonstrated that OTA production differs significantly with the composition of the berries at different maturation stages (P<0.001), with a mean value of OTA production at pea berry, early veraison and ripe berry of 3402, 1530 and 22 mug/kg, respectively. The production of OTA by A. carbonarius was correlated positively and negatively with the total acidity of grapes (r(s)=0.855, P<0.001) and reducing sugars content (r(s)=-0.835, P<0.001), respectively. Our data demonstrate that OTA synthesis in grapes occurs since early maturation stages.     

Grape variety related trans-resveratrol induction affects Aspergillus carbonarius growth and ochratoxin A biosynthesis

The paper reports the results of a study performed to investigate the influence of the grape variety on the growth of Aspergillus carbonarius on grape berries and the correlation between the amount of ochratoxin A (OTA) and the content of trans-resveratrol produced after fungal contamination. Variations in the amount of OTA produced by the fungus are observed depending on both grape variety and on the induction of trans-resveratrol determined during the infection. The obtained data suggest that if an increase in trans-resveratrol production in grape berries occurs early after the fungal infection, the berry exploits this compound to control OTA synthesis. If the increase in trans-resveratrol concentration is delayed after fungal infection (40 h), a control of OTA accumulation can not be achieved. The possibility of exerting significant control of OTA biosynthesis by this phytoalexin seems to rely in the promptness of its production, as occurs also in other fungus plant interactions and, in turn, seems to be dependent also on grape cultivar. In this fungus-plant system, trans-resveratrol appears to represent a defence-related compound toward A. carbonarius and OTA contamination.     

Effect of carbendazim and physicochemical factors on the growth and ochratoxin A production of Aspergillus carbonarius isolated from grapes

Carbendazim is a systemic fungicide that is commonly used on several crops (tobacco, fruit, vegetables, cereals, etc.). This fungicide is used to control fungal infections in vineyards. It is indicated against Botrytis cinerea, Uncinula necator, Plasmopara viticola and other fungi and can be used either alone or coupled with other fungicides. However, there is a lack of in-depth studies to evaluate its effectiveness against growth of Aspergillus carbonarius isolated from grapes and OTA production. A medium based on red grape juice was used in this study. Preliminary studies were performed at 0.98 a(w) and 25 degrees C using carbendazim concentrations over a wide range (1-2000 ng/ml medium) to control both growth of a strain of A. carbonarius isolated from grape and its ability to produce OTA. As the lag phase increased considerably at levels > 1000 ng/ml of medium, detailed studies were carried out in the range 50-450 ng/ml of medium at 0.98-0.94 a(w) and 20-28 degrees C. Statistical analysis (multifactor ANOVA) of the data revealed that the three factors assayed and the interactions a(w)-carbendazim concentration and a(w)-temperature had significant effects on lag phase duration. The highest lag-times were observed at 0.94 a(w,) 20 degrees C, and with 450 ng carbendazim/ml. The three factors also had significant effects of the growth rate and there was an interaction between a(w) and temperature. The growth rate of A. carbonarius in these cultures is favoured by high water availability and relatively high temperatures. However, addition of carbendazim at the assayed levels did not significantly influenced fungal growth rate. Accumulation of OTA was studied as a function of four factors (the three previously considered, and time). All factors had significant effects on the accumulation of OTA. There were also two significant interactions (a(w)-temperature and temperature-time). On the basis of the results obtained, carbendazim does not increase the lag phase of A. carbonarius except at relatively low a(w) and temperatures. It does not substantially decrease fungal growth rate once growth is apparent but it appears to cause an increase in OTA accumulation in the medium at the doses assayed. Carbendazim, which is widely used against fungal infections in grape, can positively influence OTA production by A. carbonarius in field, which can increase OTA content in grape juices and wines.     

Impact of non-selective fungicides on the growth and production of ochratoxin A by Aspergillus ochraceus and A. carbonarius in barley-based medium

The aim of this study was to assess the influence of the non-selective fungicides mancozeb, copper oxychloride, and sulfur on the growth and capability for producing ochratoxin A (OTA) of ochratoxigenic isolates of Aspergillus carbonarius and A. ochraceus in barley-based medium. Lag phases and growth rates were determined for each fungicide at different doses, at 15°C and 25°C and at 0.97 a(w). Mancozeb at 40 mg l(-1 )inhibited fungal growth and provided lag phases >24 days at 10-20 mg l(-1) and 15°C. OTA was observed only at 25°C and doses <10 mg l(-1). At 15°C, copper oxychloride proved inhibitory at 800 mg l(-1), while at 25°C growth was not delayed and only high doses decreased OTA levels. Sulfur was inhibitory or provided large lag phases at 5-8 g l(-1) (at 15°C) while at 25°C growth took place even at 8 g l(-1), although OTA levels were low or undetectable. The antifungal activity decreased in the order mancozeb > copper oxychloride > sulfur, and was lower at 25°C than at 15°C. OTA accumulation was affected by the type of fungicide, dose, temperature and time. The efficacy of these fungicides on the growth of A. carbonarius and A. ochraceus and OTA production in barley-based medium is assessed for the first time.     

葡萄中产赭曲霉毒素A炭黑曲霉的控制方法

炭黑曲霉(Aspergillus carbonarius)属曲霉属黑色曲霉菌,是葡萄中赭曲霉毒素A(ochratoxin A,OTA)主要产生菌,广泛存在于许多国家和地区的葡萄及其制品中,是造成葡萄酒中OTA污染的主要来源。控制这些霉菌的生长与产毒是从源头上减少葡萄原料及其制品OTA污染的关键环节,有关炭黑曲霉的污染规律及其控制方法方面的研究成为近年来的国际研究热点。根据已有研究,紫外线照射、杀菌剂、二氧化硫、纳他霉素、多酚、香精油、天然提取物和挥发性化合物等都能够在一定条件下对炭黑曲霉的生长与产生OTA的能力产生一定的抑制作用。最新研究发现,酵母、细菌和一些非产毒真菌及其代谢产物也对炭黑曲霉的生长和OTA产生具有良好抑制作用,而且具有无毒无害,安全性好等优点,在实际应用中展示出很好的应用潜力,越来越受到相关科研工作者和应用者的重视。本文主要围绕上述研究内容,对国内外近年来的相关研究进展进行综述。     

Development of a quantitative real-time PCR assay for the detection of Aspergillus carbonarius in grapes

Aspergillus carbonarius is the main species responsible for the production of ochratoxin A (OTA) in wine grapes. To monitor and quantify A. carbonarious in grapes, a quantitative real-time PCR assay was developed as a possible tool for predicting the potential ochratoxigenic risk. DNA extraction from grape berries was performed by using conventional extraction and clean up through EZNA Hi-bond spin columns. A TaqMan probe was used to quantify A. carbonarius genomic DNA in grape berries samples. An exogenous internal positive control was used to overcome DNA recovery losses due to matrix inhibition. The quantification of fungal genomic DNA in naturally contaminated grape was performed using the TaqMan signal versus spectrophotometrically measured DNA quantities (Log10) calibration curve with a linearity range from 50 to 5 x 10(-4) ng of DNA. A positive correlation (R2=0.92) was found between A. carbonarious DNA content and OTA concentration in naturally contaminated grape samples. This is the first application of TaqMan real-time PCR for identifying and quantifying A. carbonarius genomic DNA occurring in grapes. The rapid DNA extraction method for grapes, together with the commercial availability of reagents and instrumentation, allows to perform a remarkable number of reproducible assays (96-well format) in less than 4 h.     

Managing ochratoxin A risk in the grape-wine food chain

The main source of ochratoxin A (OTA) in the wine food chain is the infection of grapes by "black aspergilli" in the field. OTA-producing black aspergilli include principally Aspergillus carbonarius, followed by A. niger and possibly A. tubingensis. They are opportunistic fungi that develop particularly on damaged berries at ripening, although they may occur and form OTA on grapes from veraison to harvest. Climatic conditions (high humidity and temperature) and geographical location are important factors favouring OTA accumulation in grape berries. The severity of aspergillus rot is influenced by excessive irrigation and rainfall prior to harvest, which causes berry splitting. In addition, berry wounds caused by insect attack provide preferential entries for black aspergilli. High OTA levels occur in grapes severely damaged by the grape moth, Lobesia botrana, particularly in Mediterranean areas. Some grape varieties display greater susceptibility to aspergillus rot due to intrinsic genetic characteristics and bunch conformation (i.e. compact>sparse). Control measures for toxigenic mycoflora in the vineyards must consider these critical control points. Proper fungicidal and insecticidal treatments can reduce OTA contamination. Nevertheless, knowledge about the fate of OTA and its distribution in wine and winery by-products is important to manage OTA risk in contaminated stock. In our wine-making experiments, only 4% of the OTA present in grapes remained in the wine--the majority is retained in pressed grape pomaces. OTA concentration remained unchanged in wine after a 1-year aging as well as in all liquid fractions collected during vinification (i.e. must, free run wine, and wine after first and second decantation). Activated carbon can reduce OTA levels in wine but negatively affects wine quality.     

Ochratoxigenic species from Spanish wine grapes

The ochratoxigenic mycobiota of grapes belonging to representative wine regions located along the Mediterranean coast of Spain at different developmental stages was identified. During the development of the berries, the occurrence of Aspergillus spp. increased while the percentage of berries contaminated by non-ochratoxin A (OTA) producing species such as Alternaria spp. and Cladosporium spp. decreased. Penicillium verrucosum, the only confirmed Penicillium spp. that is able to produce OTA, was not isolated. The contamination by OTA-producing species comes from the surface of the berries and not from the inner fruit. Black aspergilli were predominant among the different Aspergillus spp. isolated. All the Aspergillus carbonarius isolates were able to produce OTA at different concentrations. None of the isolates belonging to Aspergillus niger aggregate and to Aspergillus japonicus var. aculeatus were able to produce OTA. These results are a strong evidence of the contribution of A. carbonarius in the OTA contamination in wine grapes, mainly at the last developmental stages of the berries.     

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.     

Kinetics and spatial distribution of OTA in Aspergillus carbonarius cultures

The aim of this study was to characterize Ochratoxin A (OTA) production by Aspergillus carbonarius under different environmental conditions, and to elucidate the diffusion capacity of OTA throughout synthetic medium. One strain belonging to the species A. carbonarius isolated from vine dried fruit was single-point inoculated onto triplicate synthetic nutrient medium plates at two water activities (0.92 and 0.97) and two temperatures (20 and 30 degrees C). Daily radii were measured and OTA production was tested after 4, 7, 10, 14 and 18 days of incubation at four distances from the centre of colony (1-4 cm). OTA production was detected mainly at 0.97 a(w). Earlier production was detected at 30 degrees C (optimum for growth), whereas maximum OTA concentrations were found at 20 degrees C. OTA production was detected from mycelium that was only a few days old and attained its optimum when mycelium was 4-7 days old at 0.97 a(w). OTA diffusion was observed at 0.92 a(w) and 20 degrees C. Thus OTA production is discernable in young A. carbonarius mycelium and diffusion of the toxin has been shown to occur in a solid substrate.     

葡萄中碳黑曲霉的分离及其产生赭曲霉毒素A的研究

碳黑曲霉(Aspergillus carbonarius)是葡萄中产生赭曲霉毒素A(Ochratoxin A,OTA)的重要菌株.采集烟台赤霞珠(Cabernet sauvignon)葡萄,接种于孟加拉红培养基,从中分离到7株黑曲霉群(Aspergillus section black group)真菌,其中3株鉴定属于碳黑曲霉种,占黑色曲霉的43.8%.此3菌株分别接种在粮粒培养基上,静置培养,全部产生OTA,最高浓度达到1300μg/kg,而且,其中2株碳黑曲霉菌株在可可浆培养基上产生荧光,而从葡萄样品中未检出OTA.     

Effect of photoperiod and day-night temperatures simulating field conditions on growth and ochratoxin A production of Aspergillus carbonarius strains isolated from grapes

The effect of light and temperature regimes simulating day and night in the field in the months preceding grape harvest on Aspergillus carbonarius growth and ochratoxin A (OTA) production were investigated. Twelve-hour photoperiod affected positively A. carbonarius growth with no differences between incubating the mould at day temperature (28 degrees C) or alternating day/night temperatures (28 degrees C/20 degrees C). Slower growth, however, was observed with constant incubation at 20 degrees C. Under 12h-alternation periods of day and night temperatures, growth was faster at continuous darkness than under continuous light conditions. Light did not cause any morphological changes in the aspect of the colonies. No significant differences on OTA production were detected due to either fluctuating temperature or photoperiod. However, as photoperiod enhanced the growth of colonies, it also enhanced OTA accumulation. The ability of A. carbonarius to produce OTA reported in previous laboratory studies has been demonstrated to be stimulated in field conditions.     

Isolation of epiphytic yeasts with potential for biocontrol of Aspergillus carbonarius and A. niger on grape

Antagonistic yeasts were isolated from the epiphytic flora associated with grape berries cv. Negroamaro and identified at species level using molecular methods. A total of 144 yeast isolates were tested in a preliminary screening on agar to select isolates showing a killer activity against Aspergillus carbonarius and A. niger, the main species responsible for the accumulation of ochratoxin A in grape. Twenty-eight yeast isolates were selected for their inhibitory effects on the above fungal species and assayed by an in vitro nutritional competition test for their antagonistic capacity towards three selected ochratoxigenic strains. Six yeast isolates belonging to five species, namely 2 isolates of Issatchenkia orientalis and one each of Metschnikowia pulcherrima, Kluyveromyces thermotolerans, Issatchenkia terricola and Candida incommunis, were finally selected and screened on wounded grape berries for their ability to inhibit infection by ochratoxigenic moulds. With the exception of the K. thermotolerans isolate, when inoculated at 10(9) CFU/wound, the other five challenger yeasts reduced the A. carbonarius and A. niger colonization on grape berry (P<0.05). In particular, the best antagonistic activity was shown by the two I. orientalis isolates. Results suggest that antagonist yeasts with the potential to control A. carbonarius and A. niger on grape can be found among the microflora associated with the berries.     

Targeting a polyketide synthase gene for Aspergillus carbonarius quantification and ochratoxin A assessment in grapes using real-time PCR

Aspergillus carbonarius is an ochratoxin producing fungus that has been considered to be responsible of the ochratoxin A (OTA) contamination in grapes and wine. In order to monitor and quantify A. carbonarius, a specific primer pair Ac12RL_OTAF/Ac12RL_OTAR has been designed from the acyltransferase (AT) domain of the polyketide synthase sequence Ac12RL3 to amplify 141 bp PCR product. Among the mycotoxigenic fungi tested, only A. carbonarius gave a positive result. This specific primer pair was also successfully employed in real-time PCR conjugated with SYBR Green I dye for the direct quantification of this fungus in grape samples. A positive correlation (R(2)=0.81) was found between A. carbonarius DNA content and OTA concentration in 72 grape samples, allowing for the estimation of the potential risk from OTA contamination. Consequently, this work offers a quick alternative to conventional methods of OTA quantification and mycological detection and quantification of A. carbonarius in grapes.     

Black aspergilli and ochratoxin A in grapes in Italy

The objective of this research was to investigate the presence of black aspergilli in grapes grown in Italy and to study the effect of environmental and cultural factors able to influence fungal incidence and ochratoxin A (OTA) presence. In this 3-year study, black aspergilli were frequently associated with grape berries; they were present in bunches starting from setting, colonising most berries at early veraison. Aspergillus carbonarius was never dominant at the different growth stages, or in different geographic areas and years, but it was confirmed as the key fungus because of the high percentage of strong OTA producer isolates in the population. The number of OTA producer strains, isolated in each vineyard at the different growth stages, was generally very limited and they were never statistically correlated to OTA content in bunches. The effect of geographic area on fungal flora was confirmed by statistical analysis, even though a major role was played by meteorological conditions, both on fungal colonisation and OTA content in bunches. Discriminant analysis gave promising perspectives for predicting OTA presence in vineyards in the future, based on summation of degree-day and rain in the period between 21st of August and 10th of September.     

Study of the effect of water activity and temperature on ochratoxin A production by Aspergillus carbonarius

The effect of water activity (aw) (0.78-0.99) and temperature (15 and 30 degrees C) on growth and production of ochratoxin A (OTA) of six Aspergillus carbonarius strains was studied in two culture media: Czapek yeast autolysate (CYA) agar and yeast extract sucrose (YES) agar, during a period of 30 days. The strains were selected to include different sources and different reported abilities to produce OTA and were characterized by RAPD and ITS-5.8S rDNA sequencing. CYA showed to be better culture medium than YES for OTA production in the isolates tested. OTA concentration was higher at 15 degrees C than at 30 degrees C. At 30 degrees C, ranges for OTA production were more restrictive than those for growth. OTA was produced from 0.86, 0.90 or 0.94 aw depending on the strain. At 15 degrees C, growth and OTA production were detected only in the 0.94-0.99 aw range. The molecular study performed showed that five of the strains were conspecific and no correlation was found between molecular data and the OTA production level or origin. The remaining strain had never been able to produce OTA and will probably represent a new species in the Aspergillus section Nigri. Our results show that A. carbonarius is able to grow and produce OTA in a wide range of water activities at both high and low temperatures.     

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.     

What is the source of ochratoxin A in wine?

During a microvinification trial using natural mouldy grapes from a research experimental vineyard, ochratoxin A (OTA) contaminated white wine was obtained. Potential OTA-producing mycobiota of grape samples used in this microvinification process was assessed. Only Aspergillus carbonarius isolates were detected as producers of OTA. Our report is a strong evidence of the contribution of A. carbonarius in the OTA contamination in wine.