The taxonomy of this group of fungi depends on the shape of the conidial heads with the distinction between uni- and biseriate conidial heads of particular importance (Raper and Fennell, 1965). Among the uniseriates, Aspergillus aculeatus and Aspergillus japonicus have been isolated from grapes. Identification of these species based on morphology is not easy, but is not particularly relevant since their ability to produce ochratoxin A has never been confirmed. Among the biseriates, Aspergillus carbonarius is easily distinguished by its large conidia. A high percentage of these strains (Teren et al., 1996; Heenan et al., 1998), or all biseriate strains according to other authors (Cabanes et al., 2002; Sage et al., 2002), are ochratoxin A producers. Aspergillus carbonarius strains that do not produce ochratoxin A are now regarded by some as a new species, Aspergillus ibericus (Serra et al., 2006b). Aspergillus carbonarius is now confirmed as the primary producer of ochratoxin A in grapes in Europe and Israel (Battilani et al., 2006c; Bejaoui et al., 2006; Belli et al., 2006; Guzev et al., 2006; Serra et al., 2006b; Tjamos et al., 2006).
Several other species also are in the biseriate group, but there is no consensus regarding their identification based on morphology. Molecular analyses distinguished two groups - niger and tubingensis - but did not clearly distinguish two species (Accensi et al., 1999; Perrone et al., 2006b). The niger group includes a low percentage of ochratoxin A producers (5-10%; Bau et al., 2006), but all of the members of the tubingensis group produced the toxin (Perrone et al., 2006a). Collectively these groups/species are termed the Aspergillus niger species aggregate or species complex.
Black Aspergilli usually overwinter in soil (Kazi et al., 2003), but they are present on berries from setting and their incidence increases as the fruit grows and matures, with their numbers peaking at ripening. By using a common protocol in several European countries and Israel (www.ochra-wine.com), the presence of black Aspergilli in grape berries was confirmed beginning at setting. The incidence of strains from Aspergillus Section Nigri was low, ~2% at setting increasing to ~5% as the berry increases to pea-size (Battilani et al., 2006a). From early veraison to ripening the frequency of Aspergillus Section Nigri strains generally increased, except in Italy where the highest level of infected berries was reached at early veraison. The incidence of berries infected by black Aspergilli at early veraison
was < 25% in most countries, but in Italy and Israel it was > 30%. At harvest, the incidence of black Aspergilli was particularly high in Greece, France and Israel, where fungi were isolated from > 50% of the berries, while < 20% of the berries were infected in Spain.
Among black Aspergilli isolated from berries, uniseriate isolates were the least common. They were never detected in Greece, were found only sporadically in Portugal, were found on ~ 1% of the berries in Spain and on about 10% of the berries in the other participating countries at harvest. Members of A. niger species aggregate were the principal group in all growth stages, including ripening. In all countries and years isolates from the A. niger species aggregate were more common than isolates of either uniseriate Aspergillus sp. or A. carbonarius. The incidence of the A. niger species aggregate varied widely with the highest incidence detected in France and Israel with ~ 60% of the berries infected.
Aspergillus carbonarius was isolated in all the countries and years evaluated, but its incidence was < 10%, with high variation among vineyards from Italy, Spain and Portugal. The highest incidence of A. carbonarius occurred at ripening in France. In experimental studies of A. carbonarius in the field in Australia, the conduciveness of the ripening period to infection by this fungus was confirmed under natural conditions. Mean A. carbonarius counts decreased between pre-bunch closure and veraison, and then increased between veraison and pre-harvest, with fungal incidence dependent on the risk of berry splitting (Leong et al., 2006).
The incidence of berries infected by black Aspergilli at harvesting is significantly related to latitude and longitude, with positive West ^ East and North ^ South gradients. Predictive maps of infected berry incidence had the same trend in different years, but the infection incidence was highest in the hottest and driest year. Thus, the meteorological con ditions are a part of the explanation of the spatial distribution of black Aspergilli in the Mediterranean basin (Battilani et al., 2006a).
The incidence of A. carbonarius was evaluated more critically in the WINE-OCHRA RISK project (Battilani et al., 2006a; Table 2) because of its known ability as an ochratoxin A producer. The incidence of A. carbonarius varied by country and by year. Portugal and Spain had the lowest incidence, ~10% in 2001 and 2002 and slightly higher in Spain in 2003. In Italy and Israel the incidence was ~20%. In Greece in 2003 and in France the incidence of A. carbonarius was > 40%. These data could be entered into ArcView (2002), a geostatistic data analysis program, to generate a map predictive of the incidence of A. carbonarius (Fig. 2). The incidence of A. carbonarius increased from West ^ East and from North ^ South within Europe. All of the samples from 2003 positive for ochratoxin A at harvest (ochratoxin A > 0.3 ng/g) were collected from areas with an incidence of A. carbonarius that was > 30% (Battilani et al., 2006c; Bejaoui et al., 2006; Guzev et al., 2006; Serra et al., 2006b).
All of the studies identified A. carbonarius as the main ochratoxin A producer, because of the high percentage of A. carbonarius strains that could produce toxin and the relatively high levels of toxin produced relative to other strains of black Aspergilli. In the WINE-OCHRA RISK project, 70-100% of the A. carbonarius strains could produce ochratoxin A when grown in vitro and evaluated with high performance liquid chromatography (HPLC), while only 2-20% of the strains from the A. niger species aggregate produced ochratoxin A (Battilani et al., 2006c, Bejaoui et al., 2006; Belli et al., 2006; Serra et al., 2006b). Most of the ochratoxin A producing strains were recovered at harvest or between veraison and ripening.
Ochratoxin A is produced while the grapes are in the vineyards and is not normally detected before early veraison (Battilani et al., 2004a). Bunches without visible symptoms may contain ochratoxin A, but berries with visible black molds were more contaminated. The presence of ochratoxin A in berries only during ripening is not due to inadequacy of the substrate at earlier stages. Aspergillus carbonarius produced ochratoxin A when the fungus was inoculated on homogenized wine grapes at different growth stages and belonging to different varieties. Ochratoxin A production was positively correlated with total acidity and negatively correlated with sugar content, which suggests that ochratoxin A biosynthesis is potentially higher in earlier growth stages (Serra et al., 2006b). I think that the fungus has problems penetrating the berry at the earlier stages and that the lack of fungal growth
Table 2. Percent incidence of Aspergillus carbonarius and black Aspergilli isolated from grapes at harvest in different European countries and Israel (after Battilani et al., 2006a).
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