**3.1 Vineyard mycobiota**

Most studies address the biodiversity of grape mycoflora and above all of BA in countries and in wine producing areas. Recently we performed a two-year survey in a restricted geographical area to assess the population density and biodiversity of the mycoflora associated with the grapes, air and soil of the vineyards on the slopes of Mount Etna (eastern Sicily, Italy) where there is a long tradition of grape cultivation. This area is characterized by a temperate Mediterranean climate, with an average annual rainfall of 800 mm and high day/night temperature fluctuations. Moreover, the area is characterized by old and authoctonous wine-grape cultivars, i.e. Etna rosso DOC (Nerello mascalese, Nerello Cappuccio) and Etna Bianco (Carricante, Catarratto).

Special emphasis was made on toxigenic fungal species, e.g. *Aspergillus* Sect. *Nigri* spp. and *Penicillium* spp. (Oliveri, 2006; Oliveri et al., 2008). It is postulated that air movement deposits soil fungal spores onto grapes, because their incidence in air samples increases closer to the soil. So, healthy grape soil beneath the vines was ecologically monitored and its air was sampled at two different heights at the pea stage, early veraison and ripening using plating methods. Spore-producing filamentous fungi were detected, identified at the genus level, and then the *Aspergillus* and *Penicillium* strains were isolated and identified at the species level (Fig. 1).

Fig. 1**.** Scanning electron microscopy pictures of a) *A. carbonarius*, *A. niger* b) and c) *A. japonicus* spores, where spore ornamentation differences are clearly seen (bar = 1 µm) (Zeiss, DSM 940).

The most frequent genera isolated from air, soil and grapes by increasing order were *Aspergillus, Penicillium, Cladosporium* and *Rhizopus*.

the soil (Kazi et al., 2003a). Soil temperature could also affect the incidence of *A. carbonarius* in the soil; the optimal temperature for spore survival was around 25 °C, with counts

 Agronomic practices and biological and chemical treatments have been found to reduce BA colonization and OTA levels in grapes and grape-derived products (reviewed in Varga &

**3. Biodiversity in a restricted geographical area: A case study from the Mount** 

Most studies address the biodiversity of grape mycoflora and above all of BA in countries and in wine producing areas. Recently we performed a two-year survey in a restricted geographical area to assess the population density and biodiversity of the mycoflora associated with the grapes, air and soil of the vineyards on the slopes of Mount Etna (eastern Sicily, Italy) where there is a long tradition of grape cultivation. This area is characterized by a temperate Mediterranean climate, with an average annual rainfall of 800 mm and high day/night temperature fluctuations. Moreover, the area is characterized by old and authoctonous wine-grape cultivars, i.e. Etna rosso DOC (Nerello mascalese, Nerello

Special emphasis was made on toxigenic fungal species, e.g. *Aspergillus* Sect. *Nigri* spp. and *Penicillium* spp. (Oliveri, 2006; Oliveri et al., 2008). It is postulated that air movement deposits soil fungal spores onto grapes, because their incidence in air samples increases closer to the soil. So, healthy grape soil beneath the vines was ecologically monitored and its air was sampled at two different heights at the pea stage, early veraison and ripening using plating methods. Spore-producing filamentous fungi were detected, identified at the genus level, and then the *Aspergillus* and *Penicillium* strains were isolated and identified at the

a b c

**1μm 1μm 1μm** 

The most frequent genera isolated from air, soil and grapes by increasing order were

Fig. 1**.** Scanning electron microscopy pictures of a) *A. carbonarius*, *A. niger* b) and c) *A. japonicus* spores, where spore ornamentation differences are clearly seen (bar = 1 µm) (Zeiss,

decreasing at 15 °C and 35°C. Survival at 40 °C was poor (Kazi et al., 2003b, 2004).

Kozakiewicz, 2006).

**3.1 Vineyard mycobiota** 

species level (Fig. 1).

DSM 940).

Cappuccio) and Etna Bianco (Carricante, Catarratto).

*Aspergillus, Penicillium, Cladosporium* and *Rhizopus*.

**Etna Slopes** 

Fungi belonging to *Aspergillus* spp. were present in all the sampled matrices from the pea stage and they were predominant to *Penicillium* spp. from early veraison to ripening. Fungi from 6 genera were isolated on grapes at the ripening stage in eight different vineyards (Fig. 2).

Population densities of *Aspergillus* spp. in grape wash water ranged between 4.5 x 103 and 1.2 x 104 cfu ml-1.

The most frequent *Penicillium* species isolated from the vineyard eco-system were *P. chrysogenum*, *P. expansum* and *P.olsonii* (Tab. 2). *P. verrucosum* was isolated from only one soil sample. Among the *Aspergillus* species, the most frequent were from section *Nigri*; the level of contamination by *A. ochraceus* and *A. flavus* was low.

Fig. 2. Population composition (%) detected in 8 vineyards (V1-V8) from 20 grape samples, during the ripening stage and over a 2-year survey.

Mycoflora and Biodiversity of Black Aspergilli in Vineyard Eco-Systems 267

CYA MEA

*A.carbonarius*

CYA MEA

Fig. 3. Fungal colonies isolated from grape (a) , soil (b) and air (c) samples from vineyard. Colony morphologies and light microscopy pictures of conidiphores and conidia of

representative isolates belonging to *Aspergillus ochraceus* (d–f), *A. carbonarius* (g - i), *A. niger* (l - n).

*A. niger*

b

*A. ochraceus* d e <sup>f</sup>

g h i

MEA

l m n

a b c

a

CYA


n.d.= not detected

**<sup>a</sup>** in parenthesis the total number of analyzed samples for each source

Table 2. *Aspergillus* and *Penicillium* spp. isolated from air, soil and grape samples over a two year survey.
