**2.3 European biodiversity monitoring**

DNA-based fingerprinting techniques such as AFLP, RFLP. RAPD, ap-PCR, and the sequencing of subgenomic DNA fragments have drastically improved the understanding of the occurrence and biodiversity of *Aspergillus* spp. in grapes and vineyards worldwide.

Mycoflora and Biodiversity of Black Aspergilli in Vineyard Eco-Systems 263

both on fungal colonisation and the OTA content in bunches. BA were present in bunches from setting, colonising most berries at early veraison (Battilani et al., 2006). The detection of isolates belonging to the *A. niger* aggregate*,* A*. carbonarius* and uniseriate varied with growth stage. At setting and berry pea-sized stages, more than 50% of isolates belonged to uniseriate; starting from early veraison, the *A. niger* aggregate became dominant (about 50%) whereas A*. carbonarius* was around 20% from pea-size to harvesting. The region with the highest percentage of grapes berries colonised by the *A. niger* aggregate was Veneto, while the lowest was in central Emilia Romagna. The highest incidence of *A. carbonarius* was detected in Puglia and the lowest in Emilia Romagna and Veneto. The number of OTAproducing strains among BA, isolated in each vineyard at different growth stages, was

Molecular techniques to investigate strain variation in toxigenic and non-toxigenic black *Aspergillus* spp. showed that isolates of A*. carbonarius* and *A. niger* clustered into species groups, however, within species, strains displaying similar degrees of toxigenicity did not cluster together when characterized by RAPD techniques (Ilic et al., 2001, 2004). The profile of the *Aspergillus terreus* species isolated from dried grapes, analysed by RAPD, indicated great genomic diversity (Narasimhan & Asokan, 2010). Martínez-Culebras et al. (2009) recently carried out a study on ochratoxigenic mycobiota in grapes by ap-PCR sequence analysis of the ITS and IGS regions and their ability to produce OTA. Based on ap-PCR profiles, derived from two microsatellite primers, three main groups were obtained by UPGMA cluster analysis corresponding to *A. carbonarius*, *A. niger* and *A. tubingensis*. The cophenetic correlation values corresponding to ap-PCR UPGMA analysis showed higher genetic variability in *A. niger* and *A. tubingensis* than in *A. carbonarius*. In addition, no genotypical differences could be established between OTA producers and non-producers in all the species analysed. Regarding uniseriate black aspergilli, low divergence was found between *A. aculetus* and *A. uvarum*. OTA-production seems to be strain related since it was

found in different clusters, with either ap-PCR or IGS-ITS phylogenetic analysis.

disease and OTA accumulation in grapes (Cozzi et al., 2006).

Black aspergilli are affected by several factors in the vine environment, i.e., grape status , the number of damaged grape berries, meteorological conditions, vineyard location, the cropping system as well as chemical treatments (Battilani et al., 2003b, 2006; Belli et al., 2005; 2007a; Blesa et al*.,* 2006; Clouvel et al., 2008; Hocking et al., 2007; Leong et al., 2006). Generally fungi have been detected in vineyards and on grapes from setting. However, grape aspergilli increase gradually, reaching their maximum values at the beginning of veraison and ripening (Battilani et al., 2002). As *Aspergillus* species are not considered primary pathogens, various grape damage, such as attack by other fungi or mechanical injury, dramatically increases the risk of fungal infection by these species and OTA contamination (Serra et al., 2006; Belli et al., 2007b). However, grape damage due to insects, birds or other fungal infections, is the primary factor affecting the development of the

Some Australian studies have demonstrated that vineyard soil at a depth of 0–5 cm beneath the vines is the primary reservoir of black aspergilli (Clarke et al., 2003; Kazi et al. 2004; Leong et al. 2006). Concentrations were also higher in the soil directly beneath the vines compared to the inter-row area. It is postulated that air movement deposits spores from the soil onto the grapes berry surfaces, because BA spores in air samples were higher closer to

**2.4 Epidemiology in vineyard** 

generally very limited (an encouraging result) (Battilani et al., 2006).

Sequencing techniques were primarily useful at the species identification level, whereas fingerprinting techniques were exploited at the intraspecific level (Ferracin et al., 2009; Dachoupakan et al., 2009; Perrone et al., 2007). Although all these studies contribute to analysing the species composition and genetic diversity of grape mycobiota, no genotypical differences could be established between OTA producers and non producers (Ilic et al., 2001, 2004; Martinez-Culebraz et al., 2009; Chiotta et al., 2011). Moreover, there was no correlation between genotype, the ability to produce OTA and geographical origin (Niessen et al., 2005). Surveys conducted in Europe during the four-year EU project 'Wine-Ochra Risk' (QLK1-CT 2001-01761) indicated a significant correlation between the incidence of grape infected by black aspergilli, as potentially OTA producer, at harvest and in climatic conditions and geography (latitude and longitude); there was increasing incidence from West to East and North to South (Battilani et al., 2006). Aspergilli in vineyards varied depending on years and geographic areas: France, Greece and Israel were the areas with the highest incidence, followed by South Italy, Spain and Portugal (Abarca et al., 2001; Battilani et al., 2006; Guzev et al., 2006; Logrieco et al., 2007; Otteneder & Majerus., 2000; Sage et al., 2002). In countries with colder temperate climates such as Germany, Northern Hungary, the Czech Republic as well as the northern parts of Portugal, France and Italy, BA has not often been isolated from grapes, although sometimes OTA has been detected in wines. The identification of OTA producing *Penicillium* species from grapes in Northen Italy and France suggests they could be responsible for contamination in these regions (Battilani et al., 2001; Rousseau, 2004).

Surveys in 107 vineyards in the Mediterranean basin have identified four main *Aspergillus*  populations: *A. carbonarius*, *A. tubingensis*, *A. niger*, and a group of *Aspergillus* 'uniseriate' isolates morphologically indistinguishable from *A. japonicus* and *A. aculeatus.* The latter could be clearly distinguished by molecular tools such as AFLP, RFLP and sequence analyses (Bau et al*.* 2006; Perrone et al. 2006a, 2006b). Highest genetic variability was observed in the *A. niger* group due to its complexity and the difficulty of identifying it at species level by both AFLP and the sequencing of calmodulin and β-tubulin subgenomic fragments (Perrone et al., 2007). In Australian vineyards, Leong et al. (2007) documented the dominance of *A. niger* over *A. carbonarius* and *A. aculeatus.* Polyphasic studies using macroand micromorphology, secondary metabolite profiles, partial sequences of β-tubulin, calmodulin and ITS genes, and AFLP analysis led to the description of a new *Aspergillus* species: *A. ibericus*, which is closely related to *A. carbonarius* but unable to produce OTA (Serra et al. 2006) and *A. brasiliensis* belonging to the *A. niger* aggregate (Varga et al., 2007), both isolated from grapes in the Iberian Peninsula; *A. uvarum,* morphologically very similar to *A. japonicus* and *A. aculeatus*, but clearly distinct by the molecular analysis of grapes samples in Portugal, Italy, France, Israel, Greece and Spain.

In Italy, field surveys studied the fungi associated with grapes and their ability to produce OTA in different grape-growing areas (as regards grape variety and farming methods) in the north and south of the country (Battilani et al., 2002, 2006). Analysis of these grape samples revealed that *A. niger* aggregate was the prevalent species and *A. carbonarius* was mostly found in Southern Italy and Sicily (Lucchetta et al., 2010; Oliveri, 2007). *A. carbonarius* was never dominant at different growth stages, or in different geographical areas and years, but it was confirmed as the key fungus because of the high percentage of strong OTA producing isolates in the population.

In sixteen vineyards located in 13 provinces (including Modena, Imola, Ravenna, Brindisi and the warmest places such as Trapani and Ragusa), the effect of geographic area on fungal flora was confirmed, even though a major role was played by meteorological conditions,

Sequencing techniques were primarily useful at the species identification level, whereas fingerprinting techniques were exploited at the intraspecific level (Ferracin et al., 2009; Dachoupakan et al., 2009; Perrone et al., 2007). Although all these studies contribute to analysing the species composition and genetic diversity of grape mycobiota, no genotypical differences could be established between OTA producers and non producers (Ilic et al., 2001, 2004; Martinez-Culebraz et al., 2009; Chiotta et al., 2011). Moreover, there was no correlation between genotype, the ability to produce OTA and geographical origin (Niessen et al., 2005). Surveys conducted in Europe during the four-year EU project 'Wine-Ochra Risk' (QLK1-CT 2001-01761) indicated a significant correlation between the incidence of grape infected by black aspergilli, as potentially OTA producer, at harvest and in climatic conditions and geography (latitude and longitude); there was increasing incidence from West to East and North to South (Battilani et al., 2006). Aspergilli in vineyards varied depending on years and geographic areas: France, Greece and Israel were the areas with the highest incidence, followed by South Italy, Spain and Portugal (Abarca et al., 2001; Battilani et al., 2006; Guzev et al., 2006; Logrieco et al., 2007; Otteneder & Majerus., 2000; Sage et al., 2002). In countries with colder temperate climates such as Germany, Northern Hungary, the Czech Republic as well as the northern parts of Portugal, France and Italy, BA has not often been isolated from grapes, although sometimes OTA has been detected in wines. The identification of OTA producing *Penicillium* species from grapes in Northen Italy and France suggests they could be responsible for contamination in these regions (Battilani et al., 2001; Rousseau, 2004). Surveys in 107 vineyards in the Mediterranean basin have identified four main *Aspergillus*  populations: *A. carbonarius*, *A. tubingensis*, *A. niger*, and a group of *Aspergillus* 'uniseriate' isolates morphologically indistinguishable from *A. japonicus* and *A. aculeatus.* The latter could be clearly distinguished by molecular tools such as AFLP, RFLP and sequence analyses (Bau et al*.* 2006; Perrone et al. 2006a, 2006b). Highest genetic variability was observed in the *A. niger* group due to its complexity and the difficulty of identifying it at species level by both AFLP and the sequencing of calmodulin and β-tubulin subgenomic fragments (Perrone et al., 2007). In Australian vineyards, Leong et al. (2007) documented the dominance of *A. niger* over *A. carbonarius* and *A. aculeatus.* Polyphasic studies using macroand micromorphology, secondary metabolite profiles, partial sequences of β-tubulin, calmodulin and ITS genes, and AFLP analysis led to the description of a new *Aspergillus* species: *A. ibericus*, which is closely related to *A. carbonarius* but unable to produce OTA (Serra et al. 2006) and *A. brasiliensis* belonging to the *A. niger* aggregate (Varga et al., 2007), both isolated from grapes in the Iberian Peninsula; *A. uvarum,* morphologically very similar to *A. japonicus* and *A. aculeatus*, but clearly distinct by the molecular analysis of grapes

samples in Portugal, Italy, France, Israel, Greece and Spain.

producing isolates in the population.

In Italy, field surveys studied the fungi associated with grapes and their ability to produce OTA in different grape-growing areas (as regards grape variety and farming methods) in the north and south of the country (Battilani et al., 2002, 2006). Analysis of these grape samples revealed that *A. niger* aggregate was the prevalent species and *A. carbonarius* was mostly found in Southern Italy and Sicily (Lucchetta et al., 2010; Oliveri, 2007). *A. carbonarius* was never dominant at different growth stages, or in different geographical areas and years, but it was confirmed as the key fungus because of the high percentage of strong OTA

In sixteen vineyards located in 13 provinces (including Modena, Imola, Ravenna, Brindisi and the warmest places such as Trapani and Ragusa), the effect of geographic area on fungal flora was confirmed, even though a major role was played by meteorological conditions, both on fungal colonisation and the OTA content in bunches. BA were present in bunches from setting, colonising most berries at early veraison (Battilani et al., 2006). The detection of isolates belonging to the *A. niger* aggregate*,* A*. carbonarius* and uniseriate varied with growth stage. At setting and berry pea-sized stages, more than 50% of isolates belonged to uniseriate; starting from early veraison, the *A. niger* aggregate became dominant (about 50%) whereas A*. carbonarius* was around 20% from pea-size to harvesting. The region with the highest percentage of grapes berries colonised by the *A. niger* aggregate was Veneto, while the lowest was in central Emilia Romagna. The highest incidence of *A. carbonarius* was detected in Puglia and the lowest in Emilia Romagna and Veneto. The number of OTAproducing strains among BA, isolated in each vineyard at different growth stages, was generally very limited (an encouraging result) (Battilani et al., 2006).

Molecular techniques to investigate strain variation in toxigenic and non-toxigenic black *Aspergillus* spp. showed that isolates of A*. carbonarius* and *A. niger* clustered into species groups, however, within species, strains displaying similar degrees of toxigenicity did not cluster together when characterized by RAPD techniques (Ilic et al., 2001, 2004). The profile of the *Aspergillus terreus* species isolated from dried grapes, analysed by RAPD, indicated great genomic diversity (Narasimhan & Asokan, 2010). Martínez-Culebras et al. (2009) recently carried out a study on ochratoxigenic mycobiota in grapes by ap-PCR sequence analysis of the ITS and IGS regions and their ability to produce OTA. Based on ap-PCR profiles, derived from two microsatellite primers, three main groups were obtained by UPGMA cluster analysis corresponding to *A. carbonarius*, *A. niger* and *A. tubingensis*. The cophenetic correlation values corresponding to ap-PCR UPGMA analysis showed higher genetic variability in *A. niger* and *A. tubingensis* than in *A. carbonarius*. In addition, no genotypical differences could be established between OTA producers and non-producers in all the species analysed. Regarding uniseriate black aspergilli, low divergence was found between *A. aculetus* and *A. uvarum*. OTA-production seems to be strain related since it was found in different clusters, with either ap-PCR or IGS-ITS phylogenetic analysis.
