Ochratoxin A production by Aspergillus carbonarius on some grape varieties grown in Italy

Fungi responsible for the presence of Ochratoxin A (OTA) in grapes have been identified as belonging to the black aspergilli, Aspergillus section Nigri, among which Aspergillus carbonarius is the main producer. Despite the widespread occurrence of OTA in various types of wine, there is no specific information on the ability of black aspergilli to infect berries and to produce OTA in different grape varieties. The aim of this study was to investigate the susceptibility to A carbonarius of twelve grape varieties representative of a wide range of characteristics, including geographical distribution. In all the conditions of berry status (intact and damaged) and temperature (20 and 25 °C) tested, the incidence of colonised berries and their OTA contents were always low in ‘Bianco d'Alessano’, ‘Pampanuto’ and ‘Uva di Troia’. In contrast, ‘Cabernet Sauvignon’, ‘Trebbiano’ and ‘Verdeca’ showed high fungal incidence and OTA content. The information gathered is relevant for defining risk factors for OTA presence in grapes, even if these data need to be confirmed by observations in vineyards. Copyright © 2004 Society of Chemical Industry     

Effect of Lobesia botrana damages on black aspergilli rot and ochratoxin A content in grapes

Sixty-nine wine grape samples of two varieties (Bombino Nero and Uva di Troia) were collected from four vineyards in a high ochratoxin A (OTA) incidence grape-growing area in Apulia, southern Italy, during the 2001 and 2002 crop harvests. The levels of toxin, measured in the two year harvests, ranged from 0.02 to 681 ng/g of fresh berries. In both surveys, higher levels of contamination by black aspergilli and OTA were found in both intact and rotten berries originating from bunches damaged by Lobesia botrana larvae as compared to bunches without L. botrana attacks. All berry samples with an OTA level >1 ng/g were contaminated by black aspergilli with a CFU>10(6), and all but one of these samples belonged to the Lobesia damaged berries group. This is the first evidence of an interaction between L. botrana damaged berries and OTA contamination, in field.     

Fungi and mycotoxins in vineyards and grape products

Many fungi may occur on grapes during growth in the vineyard, but the main concern from the viewpoint of mycotoxin contamination is the black Aspergilli, Aspergillus carbonarius and A. niger. These fungi are capable of producing ochratoxin A (OA) which may contaminate grapes and grape products such as wine, grape juice and dried vine fruit. Understanding the ecology and physiology of the black Aspergilli can provide tools for management of OA at all stages of grape production and processing. In the vineyard, careful management of cultivation, irrigation and pruning can assist in minimising the levels of black Aspergilli in the soil, which in turn, can minimise contamination of grapes by these fungi. Minimising damage to grapes on the vine by the use of open vine canopies, grape varieties with resistance to rain damage and by the management of insect pests and fungal diseases (e.g., mildew, Botrytis bunch rot) can reduce the incidence of Aspergillus rot in mature berries. The risk of OA in table grapes can be minimised by careful visual inspection to avoid damaged and discoloured berries. In wine, harvesting grapes with minimal damage, rapid processing and good sanitation practices in the winery assist in minimising OA. During vinification, pressing of grapes, and clarification steps which remove grape solids, grape proteins and spent yeast can also remove a significant proportion of OA. For dried vine fruit production, avoiding berry damage, rapid drying, and final cleaning and sorting to remove dark berries can reduce overall OA levels in finished products.     

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.     

Wine contamination by ochratoxin A in relation to vine environment

Since 2006, a new EC regulation has imposed 2.0 microg/l as the upper limit for OTA concentration in wines (CR 1881/2006). OTA production results from a harmful combination of skin lesions and favourable conditions for A. carbonarius growth, namely the air temperature and humidity, and the berry sugar content. The aim of our research was to highlight in vivo interaction between the number of damaged berries and the conditions for fungus development in relation to OTA concentration in wine. A survey carried out on the 2005 production from 156 vineyards bordering on the French Mediterranean enabled a link to be made between information on crop management, vineyard characteristics, meteorological data and the OTA concentration in wine. With humid air conditions during the susceptible berry period of 20 days before harvest, combined with a large number of damaged berries and a temperature conducive to both fungus growth and OTA production, critical levels of OTA concentration in wine were observed. Within the range of average daily air temperatures investigated, from 17.2 to 22.8 degrees C, during the susceptible berry period, 21 degrees C appeared to be the lower limit below which fungus growth and OTA production were not sufficient to result in critical levels of OTA concentration in wine. In our experiment, an average daily air temperature under 21 degrees C corresponded to an average minimum daily air temperature under 15 degrees C, below which fungus growth stopped under in vitro conditions. The importance of the berry sugar content before harvest was also demonstrated and our results suggest that the risk of wine contamination by OTA might be predicted from the number of damaged berries and the berry sugar content, along with climate monitoring.     

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.     

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.     

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.     

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.     

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 and toxigenic potential of fungal isolates from Spanish grapes

This paper reports the results from an extensive survey of filamentous fungi isolated from wine‐producing grapes and their capacity to produce ochratoxin A (OTA) on Czapek Yeast Autolysate agar (CYA), in order to assess their potential for producing this toxin in grapes. Grapes were sampled from four Spanish wine‐producing regions during 2001. The fungal infection in berries increased with time, reaching 100% at harvest. A total of 386 isolates of Aspergillus section Nigri and 10 of Aspergillus section Circumdati were isolated and tested for their ability to produce OTA in CYA. 21 strains produced OTA (18 Aspergillus section Nigri and 3 Aspergillus section Circumdati). Aspergillus section Circumdati isolates produced higher amounts of OTA than Aspergillus section Nigri ones, with means of 10.76 µg g^?1 CYA and 1.42 µg g^?1 CYA, respectively. Despite this, black aspergilli are believed to be highly responsible for the OTA levels found in musts and wines, as it is more widespread in grapes. Musts (n = 40) produced from the grapes collected were analysed. 15% were found to contain OTA, concentrations ranging from 0.091 to 0.813 ng ml^?1 (detection limit: 0.07 ng ml^?1), but no correlation was found with the ochratoxigenic moulds isolated from grapes. Copyright © 2004 Society of Chemical Industry     

Yeasts isolated from three varieties of grapes cultivated in different locations of the Dolenjska vine-growing region, Slovenia

The number and diversity of yeasts on grape berry surfaces are influenced by several factors, such as grape variety, degree of grape maturity at harvest, climatological conditions, geographic location, physical damage of grapes, the intensity of pest management etc. Cvicek is a typical Slovene wine, which has obtained a special protection under the Slovene Wine Law for its geographical origin. This blended red wine is produced from different grape varieties (Vitis vinifera L.), mostly from red grapes of Zametovka and Modra frankinja and from white grapes of Kraljevina. The aim of this study was to evaluate the impact of geographical locations in the Dolenjska vine-growing region and to obtain precise information about the influence of different grape varieties on the composition of yeast community on grape berries. The restriction fragment length polymorphism of PCR-amplified fragments from the rDNA gene cluster (PCR RFLP of rDNA) has been used for the differentiation of yeast species. The standard identification procedure has been performed on representative strains that shared identical RFLP profiles. The number of yeasts and yeast species isolated varied according to different grape varieties, Zametovka, Modra frankinja and Kraljevina (V. vinifera L.) and according to different sampling location. On the surface of grape berries 13 different yeast species have been identified. Saccharomyces cerevisiae has not been found.     

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.     

冰酒：酒中贵妇

冰酒是一种由藤蔓上冰冻的葡萄酿制而成的甜酒。葡萄内的糖分和其他溶化的固体不会被冰冻．但是水会被冰冻．所以会产生非常甜的葡萄酒。冰冻发生在发酵之前,而不会在之后。不像用来酿制其他酒（如法国产的苏特恩白葡萄酒．托卡依葡萄酒,或者贵腐酒）的葡萄．制造冰酒用的葡萄不会被灰霉病和贵腐病影响。只有健康的葡萄会一直保持美好的外型,直到采摘的最佳时机。在北半球极特殊的情况下,采摘很有可能是在新年之后。这使得冰酒中有特色的沁凉甜度和高酸度达到平衡。当葡萄远离霉菌时,     

Ochratoxin A-producing fungi from grapes intended for liqueur wine production

The ochratoxigenic mycobiota of grapes intended for liqueur wines from four Spanish vineyards were studied. The specific wine-making technology of these wines requires overripening of the grapes on the vine or extended post-harvest exposure of the grapes in the sun. In every vineyard, samples were taken at three different developmental stages: veraison, harvesting time and after over-ripening. With the maturation of the berries there was a clear increase of Aspergillus spp. In the last sampling time studied, they were isolated from the 90.3% of the plated berries. Black aspergilli (mainly A. niger aggregate and A. carbonarius) were predominant among the different Aspergillus spp. isolated and constituted 98.5% of the total Aspergillus strains isolated. At harvesting time and after over-ripening, the percentage of colonized berries with A. carbonarius exceeded that of Aspergillus niger aggregate. Due to their low frequency of isolation, Penicillium spp. and Aspergillus spp. outside black aspergilli are not an important source of ochratoxin A in grapes for liqueur wine production. On the contrary, 98.5% of the A. carbonarius isolates screened were able to produce ochratoxin A. Although the possible participation of different ochratoxin A-producing species may occur, our results confirm that A. carbonarius is the most important source of ochratoxin A in liqueur wines, increasing its occurrence along the ripening of grapes.     

A review of potassium nutrition in grapevines with special emphasis on berry accumulation

Potassium (K) is essential for vine growth and yield. Grape berries are a strong sink for K, particularly during ripening. Excess K levels in grape berries may have a negative impact on wine quality, mainly because it decreases free tartaric acid resulting in an increase in the pH of grape juice, must and wine. In Australia, high K status is common in most vineyards, which reflects the high K and high pH values of most Australian grape juice. This necessitates pH adjustment during the vinification process, and tartaric acid addition is a common practice in most Australian wineries. High K concentration may also lead to excessive loss of the additional tartaric acid by precipitation as potassium bitartrate and, as a consequence, pH adjustment becomes more difficult and expensive. Ensuring naturally low K levels in the berry will help reduce costs of input and waste management at the winery. Potential vineyard management options to manipulate berry K accumulation include selective use of rootstock/scion combination, canopy management and irrigation strategies. However, the impact of these practices on determining the optimum K concentration requires careful calibration of production parameters and the desirable grape juice and wine quality in relation to tissue K concentration. This paper reviews and discusses the possible functions of K in grape berries, translocation of K into the berry, and genetic and cultural factors that may affect the accumulation of K in the berry. This will help to identify the key research and management strategies needed for controlling K concentrations in grape berries.     

Black Aspergillus species as ochratoxin A producers in Portuguese wine grapes

To evaluate the incidence of fungi producing ochratoxin A (OA) in Portuguese wine grapes, a survey was conducted in 11 vineyards, from four winemaking regions each with distinct climatic conditions. From setting to the harvesting period, a total of 1,650 berries were sampled by plating methods. Out of 370 aspergilli and 301 Penicillium strains isolated, 14% of the aspergilli were OA-producing strains. None of the penicillia were OA-producing strains. The black aspergilli were predominant (90%). All Aspergillus strains were tested in vitro for OA production and all were preserved in the Micoteca da Universidade do Minho (MUM) culture collection. Most of the Aspergillus carbonarius (97%) and 4% of the Aspergillus niger aggregate strains were OA producers. Almost all ochratoxigenic strains were isolated at harvest time, mainly in the regions with a Mediterranean climate. In the vineyards sampled, the percentage of colonized berries with ochratoxigenic strains was up to 38%. The vineyards from the region with Atlantic influences, with high rainfall, exhibited the lowest occurrence of Aspergillus and ochratoxigenic strains, 0% to 10% and 0% to 2% colonized berries, respectively. Data obtained here supports the hypothesis that A. carbonarius and occasionally A. niger, are the main producers of OA in grapes. In this study, the highest incidence of these fungi occurred in vineyards with a Mediterranean climate.     

Black aspergilli and ochratoxin A production in French vineyards

A survey on the occurrence on grape of black Aspergillus species and their capability to produce ochratoxin A (OTA) was conducted in France over three years (2001-2003) in 10 vineyards from four winemaking regions with different geographical locations and climatic conditions. During 2001 and 2002, from setting to harvest, the total numbers of fungal isolates were respectively 721 and 711 increasing in 2003 to reach 1035. The Aspergillus genus was essentially represented by Section Nigri (99%) and it was predominant (80%+/-4.6) when compared to Penicillium (20%+/-4.6). Regardless of sampling year, 32.5% (+/-sigma=1.26) of the fungal isolates were OTA producers and 93% (+/-sigma=2.65) belonging to black aspergilli. The ochratoxigenic potential of the isolates and their occurrence on grapes revealed that Aspergillus carbonarius was the main OTA producer (up to 37.5 mug/g). At harvest time, the fungal population was maximal and this was the most critical period influencing OTA contamination. Grapes from Languedoc-Roussillon region were most infested with ochratoxigenic fungi and had the highest concentrations of OTA (up to 2.8 ng/g).     

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.