Portugal Spain within the berries at these early stages under field conditions can explain the low, or nonexistent levels of ochratoxin A identified in berries from these early growth stages.

Ecology of black Aspergilli

Fungi in Aspergillus Section Nigri can grow between 10 and 37°C, with 30-37°C considered optimal. Fungal growth and spore production are very limited at 10°C, and occur only when the available water (aw) is between 0.90 and 0.93. Aspergillus niger can produce 1-2*106 conidia/cm2 of culture surface area when growing in vitro on a synthetic medium with an aw of 0.93-0.97 (relative humidity = 93-97%) at 30-35°C (Parra and Magan, 2004). Aspergillus carbonarius produces no more than a third this number of conidia when cultured under similar conditions (Battilani et al., 2006d). The ability to produce large numbers of spores is common to all of the strains tested. Spore germination is rapid, and occurs within 24 hours with an aw of 0.90-0.99 and a temperature of 25-35°C, although germination can occur between 10 and 40°C. The optimal growth temperature for A. carbonarius is between 20 and 30°and between 30 and 35°C for members of the A. niger species aggregate.

Available water also is very important for black Aspergilli. The optimal aw for growth is 0.98, which is similar to the the aw in berries during ripening. Linear growth rate varies by species, with members of the A. niger species aggregate the fastest and A. carbonarius strains the slowest (Mitchell et al., 2003, 2004; Belli et al., 2004b). Aspergillus carbonarius grew best at an aw of 0.98-0.99, while growth by uniseriate strains or strains from the A. niger species aggregate were unchanged in growth across the aw interval of 0.90-0.995 (Mitchell et al., 2003; Belli et al., 2004c, 2005). Growth was never observed at an aw of 0.85 and only a few isolates could grow at an aw of 0.89.

The optimal conditions for growth and for ochratoxin A production were not the same. Aspergillus carbonarius could produce ochratoxin A at aws between 0.92 and 0.99 although the best toxin production occurred when the aw was between 0.95 and 0.99 (Belli et al., 2004b, 2005; Mitchell et al., 2004). Optimal ochratoxin A production occurred at 15-20°C and decreased significantly at 30-37°C (Mitchell et al., 2004; Belli et al., 2005).

Vineyard features and management

Meteorological conditions are important in determining if ochratoxin A will form in grapes, although proximity to the sea and the cropping system also are important. In Italy in 19992000, differences were detected in ochratoxin A content from three neighboring vineyards that were managed similarly with either Malvasia nera or Negroamaro grape varieties. In 1999, ochratoxin A levels were 0, low or high in the three vineyards, but the toxin was not detected in 2000 (Battilani et al., 2003a). Discriminant analysis based on a summation of degree-days and rain in late August-early September may be used to predict ochratoxin A presence in the vineyard (Battilani et al., 2006c).

The cropping system clearly has an effect, as do other factors, e.g., grape variety, trel-lising system and geographic location, whose effects may be confounded, and their individual effects difficult to resolve. Grape varieties are not universally susceptible to ochra-toxin A production either in vitro (Battilani et al., 2004a) or in field trials. In the field, the trellising system also may influence the incidence of black Aspergilli and the amount of ochratoxin A contamination. In particular, bunches that are closer to the soil appear more contaminated, even if a significant effect on the amount of ochratoxin A present has not yet been proven. The type of soil also can contribute significantly to the level of contamination with black Aspergilli, with clay soil being the most conducive.

Role of pests and diseases

Black Aspergilli are considered saprophytes, responsible for secondary rot, and wounds, of both mechanical and biological origin, are major entry sites. Data on the role of pests and diseases are limited, but good management of them in vineyards certainly results in a decrease in ochratoxin A content at harvest, as can be seen from a comparison of neighboring vineyards, managed with different approaches to crop protection (Kappes et al., 2006).

Lobesia botrana (Lepidoptera: Tortricidae) is the major grape berry moth in vineyards of Southern Europe, where it usually completes 3-4 generations a year, depending on the weather conditions in late summer. First generation larvae damage flowers, while the succeeding larval generations damage berries at different stages of maturity. Ochratoxin A content in berries and pest damage are correlated, probably due to wounds caused by pests and spore dissemination. Larvae can act as vectors by trapping conidia in the cuticle ornamentation, moving to healthy host plants, and facilitating colonization by tunneling berries (Cozzi et al., 2006).

Powdery mildew is the most conducive pathogen for black Aspergilli. Berries infected with powdery mildew often are misshapen, have rusty spots on the surface or split open during ripening when inoculum of black Aspergilli is readily available.

Control of Aspergillus carbonarius

Mycotoxin-producing fungi are not easy to control with fungicides because their reaction to the fungicide may be the opposite of that expected. Active ingredients reported as effective in reducing both fungal growth and ochratoxin A content in bunches include: mepani-pyrim, pyrimethanil, fluazinam, iprodione and a mixture cyprodinil and fludioxonil. Only the cyprodinil/fludioxonil combination was effective in field trials in France, Spain, Greece and Italy. The treatment was most effective 21 days before harvest (stage D). A second treatment at the earlier veraison stage (stage C) was recommended under high risk conditions. This combination of active ingredients was originally developed for the control of grey mold, caused by Botrytis cinerea, with the same application schedule (Kappes et al., 2006). Various biological control agents have been considered, including yeasts that occur naturally on grapes. So far, isolates of Cryptococcus laurentii and Aureobasidium pullulans are the most promising (Bleve et al, 2006).

Post harvest and wine making

Ochratoxin A is produced in vineyards, but its level may increase in bunches after harvest but before processing. The optimal conditions for minimizing ochratoxin A production during this time are not known. The temperature in the storage bin is important as is the length of drying time and the time that the grapes remain in the bins. Maintaining the bins at temperatures unfavorable for fungal growth and reducing the time before crushing both help prevent increases in ochratoxin A levels.

Ochratoxin A production during wine making has not been demonstrated. There is, however, a dynamic of ochratoxin A content in must and wine that enables toxin levels to vary with processing (Fig. 3). When bunches are crushed, the first step in wine making, most of the ochratoxin A is released into the must. In the maceration step, additional ochratoxin A is released, followed by an increase in toxin content for at least five days. Subsequent operations reduce ochratoxin A content, with the size of the decrease dependent upon several factors. During red wine making, ochratoxin A content is reduced by fermentation both in vitro and in commercial scale experiments. The efficacy of both alcoholic and ma-lolactic fermentations in reducing ochratoxin A depends upon the yeasts or bacterial strains involved and the level of contamination (Grazioli et al., 2006). The effect of clarification on ochratoxin A level depends solely upon the method or product used, with carbon based adjuvants usually the most effective (Silva et al., 2003). Finally, the ochratoxin A content is influenced by the individuals controlling the process.

In white wine-making, ochratoxin A content always decreases with the extent of the decrease dependent on choices made for the yeasts doing the alcoholic fermentation and the adjuvants added for clarification. Possible corrective actions during wine-making could be based on the use of carbon-based adjuvants for clarification because of their ability to absorb the toxin. Although these adjuvants are a good corrective tool in white wine making, more care must be exercised with red wines. A dosage of 10 g/l/hr is useful for reducing ochratoxin A while maintaining quality, with higher dosages having a negative effect on polyphenols and color. The use of yeasts or bacterial strains that can reduce ochratoxin A during alcoholic or malolactic fermentations require further evaluation.

Critical control points in grape production and processing

Critical control points can be defined both during grape production and processing. Early veraison is a crucial stage at which the berry status needs to be controlled, by checking for mechanical or insect damage and for visible black mold. Closer to ripening these visual checks should be repeated and accompanied by ochratoxin A analysis, particularly if the vineyard is located in a high risk area, if it is a high risk year, or if black mold is visible. Knowing the level of ochratoxin A present near ripening enables better post-harvest management. Ochratoxin A is not evenly distributed in vineyards, so the sampling protocol used is crucial and must result in a representative sample that neither oversamples nor misses potential hot spots (Battilani et al., 2006b). During wine making, control points could occur at all operational units, but if the must has low ochratoxin A content, then the wine can be considered safe. An additional control after the alcoholic or malolactic fermentation can be added if the ochratoxin A level in the must is above the legal limit.

Figure 3. Simulated dynamics of ochratoxin A content (ig/l) during wine making: A - White wine, B - Red wine. The dotted lines represent optimal cases, when all unit operations are managed to minimize ochratoxin A content. Worst case scenarios are represented by the solid lines. M = must; MA = maceration; C = clarification; AF = alcoholic fermentation; MLF = malolactic fermentation [after Grazioli et al. (2006) and Silva et al. (2003, 2005)].

Good agriculture (GAP) and manufacturing (GMP) practices

Following GAP and GMP protocols can minimize the amount of ochratoxin A present in the final product. Good management of the vineyard that follows correct fertilizing, irrigation and trellising practices is essential for minimizing ochratoxin A contamination. Controlling pests and diseases that can damage the berries is of particular importance. Fungicide selection should favor those with ingredients active against black Aspergilli, especially if high risk conditions occur. Harvesting must be at ripening and overripening must be avoided, especially if damaged berries with visible black mold are present. The interval between harvesting and processing should be minimized to prevent further fungal growth and ochratoxin A biosynthesis by A. carbonarius in the detached bunches. The temperature during this time should be between 15 and 30°C.

Elimination of bunches with visible black mold is strongly advised, not only for high quality production, but also to reduce ochratoxin A contamination. The addition of carbon-based adjuvants may be necessary if the ochratoxin A level in the must is high. For red wine, the must should be treated and for white wines the adjuvant(s) should be added during clarification. Ochratoxin A contamination can be reduced further by adding yeast or lactic acid bacteria that can either adsorb or degrade ochratoxin A during fermentation. Finally, normal procedures for sanitizing materials and machines must be followed.


Ochratoxin A is produced in vineyards, primarily by A. carbonarius. Meteorological conditions play the most important role in determining risk areas and years, and the prediction of risk levels can be used to design meaningful practices that reduce the risk of ochratoxin A

contamination. Farmers should follow GAP vineyard management practices and pay special attention to pest and disease control in high risk areas and years. Collaboration between farmers and wine makers to minimize the time that the grapes stand in the bins before crushing also is important. The last stage for prevention of ochratoxin A contamination occurs when the grape bunches are crushed, as ochratoxin A is not synthesized during the later stages of wine making. Adjuvants can be used, as necessary, to reduce the amount of ochratoxin A present in the final product.

Although ochratoxin A has been identified only recently in grape-derived products, there are many possible ways that the toxin content can be managed and reduced, if not eliminated. Further study to clarify the role of grape variety, trellising systems, the length of time between harvest and crushing, and the drying conditions, are needed to develop a Decision Support System and increase the safety of products for consumers.


Data reported were collected through the WINE-OCHRA RISK project (EC, Vth FP, QLK1-2001-01761). I thank J. Cabanes, Z. Kozakiewicz, A. Lebrihi, A. Lichter, N. Magan, G. Mulè, V. Sanchis, E. Tjamos, A. Venâncio and the members of their research teams for data collection, and C. Barbano, P. Giorni, S. Formenti, A. Pietri and A. Silva for their support of the activities managed through my laboratory.


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