**2.1 Role in Ocratoxin A contamination**

Several surveys conducted in the Mediterranean, South America, and Australia reported fungal species belonging to *Aspergillus* section *Nigri* (also known as Black Aspergilli-BA) as

Mycoflora and Biodiversity of Black Aspergilli in Vineyard Eco-Systems 261

*A. japonicus/A. aculeatus* Uniseriate 4–5 Negative *A. niger* aggregate Biseriate 3–5 Positive (low %) *A. sclerotioniger* Biseriate 5–6 Positive *A. carbonarius* Biseriate 7–9 Positive (high %) *A. ibericus* Biseriate 5–7 Negative

Table 1. Some characteristics of the main black *Aspergillus* species (from Serra et al., 2006)

PCR primers for detecting BA target the generic sequences for identifying or characterizing the fungus taxonomy at the intraspecific level or the sequence of genes involved in mycotoxin biosynthesis which are not necessarily able to distinguish fungal species (Bau et al., 2005; Dao et al., 2005; Perrone et al., 2007; Sartori et al., 2006; Schmidt et al.. 2003, 2004;

Species-specific primers based on ITS sequence differences were developed for *A. ellipticus, A. heteromorphus, A. japonicus*, *A. niger* (Gonzales-Salgado et al., 2005), *A. carbonarius* and *A. ochraceus* (Patiño et al., 2005). Some others were developed for *A. carbonarius* identification based on SCAR primers (Pollastro et al., 2003; Pelegrinelli-Fungaro et al. 2004), whereas PCR-RFLP analysis is necessary to distinguish the strains of *Aspergillus niger* aggregate into two groups: *A. niger* and *A. tubingensis* (N and T type) (Accensi et al., 2001, Gonzalez-

More primers have been developed for the genes encoding the polyketide synthases (PKSs) involved in OTA biosynthesis in both *Aspergillus* and *Penicillium* (Ayoub et al., 2010; Atoui

Recent studies have reported the use of degenerate primers targeting the ketosynthase domain (KS) which identified a new pks gene from *A. carbonarius* (AC*pks*) (Atoui et al., 2006; Gallo et al., 2009). By screening *Aspergillus* isolates with AC*pks* specific primers, AC*pks* homologues appeared to be present in *A. sclerotioniger* and *A. ibericus* which are closely

A duplex real-time PCR assay for simultaneously detecting members of the *Aspergillus niger* aggregate and *A. carbonarius* was developed by López-Mendoza et al. (2009) and Selma et al. (2009). They targeted the beta -ketosynthase and acyl transferase domains of the poliketide synthase of *A. carbonarius* and the *A. niger* aggregate, the assay allowing preferential amplification at greater concentrations providing a fast and accurate tool to monitor, OTAproducing species in grapes in a single reaction. These approaches gave rise to molecular diagnostic assays based on expression profiling and which determine the molecular triggers

More recently real-time/quantitative PCR (qPCR) protocols have detected and quantified ochratoxigenic fungi, developed using constitutive genes (González-Salgado et al., 2009; Morello et al., 2007; Mulè et al., 2006) or genes involved in toxin biosynthesis (Atoui et al.,

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.

(µm) OTA production

Species Conidiophore Conidial size

Serra et al., 2005).

Salgado et al., 2005).

related to *A. carbonarius*.

et al., 2006; Dao et al., 2005; O'Callaghan et al., 2003).

controlling OTA biosynthesis in *Aspergillus* spp.

2007; Schmidt et al., 2004; Selma et al., 2009).

**2.3 European biodiversity monitoring** 

the major responsible for OTA contamination in grape (reviewed in Perrone et al., 2007). Several BA have been isolated from grape or from vineyard soil/air such as *A. niger* aggregate (namely *A. niger* sensu stricto, *A. tubingensis, A. foetidus,* and *A. brasiliensis)*, *A*. *carbonarius* and the uniseriate species *A. aculeatus, A. japonicus,* and *A. uvarum* (Medina et al., 2005; Perrone et al., 2008). Only a few species produce OTA among the *A. niger* aggregate, *A. carbonarius*, and *A. japonicus* (Battilani et al., 2003a). *A. ochraceus* (belonging to section *Circumdati),* although able to produce OTA, have only occasionally been isolated from grape. The most frequently occurring species are *A. niger* aggregate and *A. carbonarius*, respectively, although the highest percentage of OTA-producing strains has been detected in the latter species (Serra et al., 2005).

OTA contamination of dried vine fruit was also found to be due to black aspergilli in Europe, including Spain, Hungary and other parts of the world such as Argentina and Australia (Varga & Kozakiewicz, 2006). In spite of the higher incidence of species belonging to the *A. niger* aggregate found in vineyards, only 5-10 % of *A. niger* OTA-producing strains were detected, whereas more than 50% and, in some studies up to 100%, in *A. carbonarius* (Battilani et al. 2006; Heenan et al,. 1998 Perrone et al., 2006a; Serra et al., 2005). Other *Aspergillus* species, such as *A. helicotrix*, *A. ellipticus* and A*. heteromorphus, A. ochraceus* are rare (Bau et al., 2005)

#### **2.2 Isolation and Identification tools**

Black aspergilli are isolated and identified at genus and species level by morphological criteria: colour, density and colony appearance (layer colour, wrinkled, umbilical, thick or flat) and microscope observation (conidial head, conidiophore and conidia characters) in accordance with appropriate keys (Klick, 2002; Klick and Pitt, 1988; Pitt & Hocking, 1999, 2009; Samson et al., 2004, 2007). The taxonomy of *Aspergillus* section *Nigri* is widely studied but although identification at section level is quite easy, at species level it is much more complex since morphologically taxa differences are very subtle requiring taxonomic expertise.

For macromorphological observations, Czapek yeast autolysate (CYA), malt extract autolysate (MEA), Czapek yeast autolysate with 5% NaCl (CYAS) agar, yeast extract-sucrose (YES) agar, oatmeal agar (OA) and Czapek agar (CZA) are used (Samson et al., 2004). Some differential growth media e.g. DYSG Agar, Coconut Cream Agar (Heenan et al., 1998) and MEA-B (Pollastro et al., 2006) may facilitate the recognition of ochratoxigenic black aspergilli (Samson et al., 2007). Useful physiological features are very good growth and sporulation at 37 °C as well as growth and acid production on CREA agar (Samson et al., 2004). Species can be identified by micromorphological analysis of the fungal structures by light microscopy. Scanning Electron Microscopy (SEM) is helpful for vesicle observation which is necessary for distinguishing between uniseriate (i.e. *A. aculeatus*, *A. japonicus*) and biseriate species (ie. *A. carbonarius*, *A. ibericus*, *A. niger*) and conidia ornamentation which can distinguish between *A. niger* aggregate, *A. carbonarius* and *A. ibericus* (Serra et al., 2006; Varga et al., 2000) (Table 1).

As for other fungal species studies based on molecular sequence analysis of ribosomal and ubiquitous genes (ITS, IGS, calmodulin, ß-tubulin, elongation factor) and polymorphisms by obtained Amplified Fragment Length Polymorphism (AFLP), Random Amplified Polymorphic DNA (RAPD) and microsatellites, have been performed for Aspergilli isolated from grapes. These studies have provided useful information on the taxonomy of BA and methods for their detection, identification and monitoring (reviewed in Abarca et al., 2004; Geiser et al., 2007; Niessen et al., 2005; Perrone et al., 2007, 2009; Samson et al., 2007).

the major responsible for OTA contamination in grape (reviewed in Perrone et al., 2007). Several BA have been isolated from grape or from vineyard soil/air such as *A. niger* aggregate (namely *A. niger* sensu stricto, *A. tubingensis, A. foetidus,* and *A. brasiliensis)*, *A*. *carbonarius* and the uniseriate species *A. aculeatus, A. japonicus,* and *A. uvarum* (Medina et al., 2005; Perrone et al., 2008). Only a few species produce OTA among the *A. niger* aggregate, *A. carbonarius*, and *A. japonicus* (Battilani et al., 2003a). *A. ochraceus* (belonging to section *Circumdati),* although able to produce OTA, have only occasionally been isolated from grape. The most frequently occurring species are *A. niger* aggregate and *A. carbonarius*, respectively, although the highest percentage of OTA-producing strains has been detected

OTA contamination of dried vine fruit was also found to be due to black aspergilli in Europe, including Spain, Hungary and other parts of the world such as Argentina and Australia (Varga & Kozakiewicz, 2006). In spite of the higher incidence of species belonging to the *A. niger* aggregate found in vineyards, only 5-10 % of *A. niger* OTA-producing strains were detected, whereas more than 50% and, in some studies up to 100%, in *A. carbonarius* (Battilani et al. 2006; Heenan et al,. 1998 Perrone et al., 2006a; Serra et al., 2005). Other *Aspergillus* species, such as *A. helicotrix*, *A. ellipticus* and A*. heteromorphus, A. ochraceus* are

Black aspergilli are isolated and identified at genus and species level by morphological criteria: colour, density and colony appearance (layer colour, wrinkled, umbilical, thick or flat) and microscope observation (conidial head, conidiophore and conidia characters) in accordance with appropriate keys (Klick, 2002; Klick and Pitt, 1988; Pitt & Hocking, 1999, 2009; Samson et al., 2004, 2007). The taxonomy of *Aspergillus* section *Nigri* is widely studied but although identification at section level is quite easy, at species level it is much more complex since morphologically taxa differences are very subtle requiring taxonomic expertise. For macromorphological observations, Czapek yeast autolysate (CYA), malt extract autolysate (MEA), Czapek yeast autolysate with 5% NaCl (CYAS) agar, yeast extract-sucrose (YES) agar, oatmeal agar (OA) and Czapek agar (CZA) are used (Samson et al., 2004). Some differential growth media e.g. DYSG Agar, Coconut Cream Agar (Heenan et al., 1998) and MEA-B (Pollastro et al., 2006) may facilitate the recognition of ochratoxigenic black aspergilli (Samson et al., 2007). Useful physiological features are very good growth and sporulation at 37 °C as well as growth and acid production on CREA agar (Samson et al., 2004). Species can be identified by micromorphological analysis of the fungal structures by light microscopy. Scanning Electron Microscopy (SEM) is helpful for vesicle observation which is necessary for distinguishing between uniseriate (i.e. *A. aculeatus*, *A. japonicus*) and biseriate species (ie. *A. carbonarius*, *A. ibericus*, *A. niger*) and conidia ornamentation which can distinguish between *A. niger* aggregate, *A. carbonarius* and *A. ibericus* (Serra et al., 2006; Varga et al., 2000) (Table 1). As for other fungal species studies based on molecular sequence analysis of ribosomal and ubiquitous genes (ITS, IGS, calmodulin, ß-tubulin, elongation factor) and polymorphisms by obtained Amplified Fragment Length Polymorphism (AFLP), Random Amplified Polymorphic DNA (RAPD) and microsatellites, have been performed for Aspergilli isolated from grapes. These studies have provided useful information on the taxonomy of BA and methods for their detection, identification and monitoring (reviewed in Abarca et al., 2004;

Geiser et al., 2007; Niessen et al., 2005; Perrone et al., 2007, 2009; Samson et al., 2007).

in the latter species (Serra et al., 2005).

**2.2 Isolation and Identification tools** 

rare (Bau et al., 2005)


Table 1. Some characteristics of the main black *Aspergillus* species (from Serra et al., 2006)

PCR primers for detecting BA target the generic sequences for identifying or characterizing the fungus taxonomy at the intraspecific level or the sequence of genes involved in mycotoxin biosynthesis which are not necessarily able to distinguish fungal species (Bau et al., 2005; Dao et al., 2005; Perrone et al., 2007; Sartori et al., 2006; Schmidt et al.. 2003, 2004; Serra et al., 2005).

Species-specific primers based on ITS sequence differences were developed for *A. ellipticus, A. heteromorphus, A. japonicus*, *A. niger* (Gonzales-Salgado et al., 2005), *A. carbonarius* and *A. ochraceus* (Patiño et al., 2005). Some others were developed for *A. carbonarius* identification based on SCAR primers (Pollastro et al., 2003; Pelegrinelli-Fungaro et al. 2004), whereas PCR-RFLP analysis is necessary to distinguish the strains of *Aspergillus niger* aggregate into two groups: *A. niger* and *A. tubingensis* (N and T type) (Accensi et al., 2001, Gonzalez-Salgado et al., 2005).

More primers have been developed for the genes encoding the polyketide synthases (PKSs) involved in OTA biosynthesis in both *Aspergillus* and *Penicillium* (Ayoub et al., 2010; Atoui et al., 2006; Dao et al., 2005; O'Callaghan et al., 2003).

Recent studies have reported the use of degenerate primers targeting the ketosynthase domain (KS) which identified a new pks gene from *A. carbonarius* (AC*pks*) (Atoui et al., 2006; Gallo et al., 2009). By screening *Aspergillus* isolates with AC*pks* specific primers, AC*pks* homologues appeared to be present in *A. sclerotioniger* and *A. ibericus* which are closely related to *A. carbonarius*.

A duplex real-time PCR assay for simultaneously detecting members of the *Aspergillus niger* aggregate and *A. carbonarius* was developed by López-Mendoza et al. (2009) and Selma et al. (2009). They targeted the beta -ketosynthase and acyl transferase domains of the poliketide synthase of *A. carbonarius* and the *A. niger* aggregate, the assay allowing preferential amplification at greater concentrations providing a fast and accurate tool to monitor, OTAproducing species in grapes in a single reaction. These approaches gave rise to molecular diagnostic assays based on expression profiling and which determine the molecular triggers controlling OTA biosynthesis in *Aspergillus* spp.

More recently real-time/quantitative PCR (qPCR) protocols have detected and quantified ochratoxigenic fungi, developed using constitutive genes (González-Salgado et al., 2009; Morello et al., 2007; Mulè et al., 2006) or genes involved in toxin biosynthesis (Atoui et al., 2007; Schmidt et al., 2004; Selma et al., 2009).
