**3.2 Black Aspergilli and OTA producers**

Black aspergilli appeared in all the tested samples, their incidence being higher at early veraison and ripening (Oliveri et al., 2006; Oliveri, 2006) (Fig.3 a-c)*.* According to macro- and micromorphological characteristics, they were identified and classified into two main groups, *A. niger* aggregate and *A. carbonarius* (Fig.3 g-n)*. A. ochraceus* has occasionally been detected in grape samples (Fig.3 d-f)*.* 

A subset of 66 strains was selected for further analysis. PCR assays supported the morphological identification. *A. niger*, *A. carbonarius* and *A. japonicus* were identified by target sequences for each species according to assays described by González-Salgado et al. (2005) and Patiño et al. (2005). In order to characterise the species in *A. niger* aggregate*,* i.e. *A. niger* and *A. tubingensis,* very difficult to differentiate by classical morphological criteria, the RFLP analysis with *Rsa*I was performed. This differentiation is very important to as to avoid overestimating toxicological contamination and related risks. A primer annealing site or restriction nuclease cleavage site was further confirmed by ITS sequencing which also confirmed the identity of the isolates (Oliveri et al., 2008).

The OTA production of isolates belonging to *A. carbonarius*, *A. niger*, *A. tubingensis* and *A. japonicus* was assessed by enzyme-linked immunosorbent assay (Oliveri et al., 2006b, 2008). 56% of strains were shown to produce OTA. *A. carbonarius* isolates were the strongest OTA producers with some of them producing high concentrations of OTA (>40 ppb).

### **3.3 Intraspecific variability**

A fAFLP protocol was used to assess specific and intraspecific variability (Oliveri et al., 2008). In agreement with other studies (Perrone et al., 2006a, 2006b), the AFLP technique generated enough polymorphism to differentiate between and within the species of black aspergilli. *A. niger, A. tubingensis*, *A. carbonarius* and *A. japonicus* strains were clearly differentiated, although *A. niger* strains clustered into two different groups. Intraspecific variability didn't correlate with the isolate origin. In fact isolates from different vineyards either of grape or the environment could also cluster in the same or in different clusters. Perrone et al. (2006a, 2006b) analyzed representative strains from the main wine producing

*Aspergillus niger* aggregate 125 94 128  *carbonarius* 43 49 38

 *ochraceus* 8 20 1 *Penicillium aurantiogriseum* 4 3 n.d.  *chrysogenum* 50 55 n.d.  *expansum* 63 22 15  *italicum* 29 19 14  *olsonii* n.d. 7 n.d.  *verrucosum* n.d. 1 n.d.

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

Black aspergilli appeared in all the tested samples, their incidence being higher at early veraison and ripening (Oliveri et al., 2006; Oliveri, 2006) (Fig.3 a-c)*.* According to macro- and micromorphological characteristics, they were identified and classified into two main groups, *A. niger* aggregate and *A. carbonarius* (Fig.3 g-n)*. A. ochraceus* has occasionally been

A subset of 66 strains was selected for further analysis. PCR assays supported the morphological identification. *A. niger*, *A. carbonarius* and *A. japonicus* were identified by target sequences for each species according to assays described by González-Salgado et al. (2005) and Patiño et al. (2005). In order to characterise the species in *A. niger* aggregate*,* i.e. *A. niger* and *A. tubingensis,* very difficult to differentiate by classical morphological criteria, the RFLP analysis with *Rsa*I was performed. This differentiation is very important to as to avoid overestimating toxicological contamination and related risks. A primer annealing site or restriction nuclease cleavage site was further confirmed by ITS sequencing which also

The OTA production of isolates belonging to *A. carbonarius*, *A. niger*, *A. tubingensis* and *A. japonicus* was assessed by enzyme-linked immunosorbent assay (Oliveri et al., 2006b, 2008). 56% of strains were shown to produce OTA. *A. carbonarius* isolates were the strongest OTA

A fAFLP protocol was used to assess specific and intraspecific variability (Oliveri et al., 2008). In agreement with other studies (Perrone et al., 2006a, 2006b), the AFLP technique generated enough polymorphism to differentiate between and within the species of black aspergilli. *A. niger, A. tubingensis*, *A. carbonarius* and *A. japonicus* strains were clearly differentiated, although *A. niger* strains clustered into two different groups. Intraspecific variability didn't correlate with the isolate origin. In fact isolates from different vineyards either of grape or the environment could also cluster in the same or in different clusters. Perrone et al. (2006a, 2006b) analyzed representative strains from the main wine producing

producers with some of them producing high concentrations of OTA (>40 ppb).

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

confirmed the identity of the isolates (Oliveri et al., 2008).

**3.2 Black Aspergilli and OTA producers** 

detected in grape samples (Fig.3 d-f)*.* 

**3.3 Intraspecific variability** 

*flavus* 18 21 1

**n. samples a air Soil Grapes**  ( 224) (112) (160)

**Genus Species**

n.d.= not detected

year survey.

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).

Mycoflora and Biodiversity of Black Aspergilli in Vineyard Eco-Systems 269

from grape samples within the same vineyard proved that mixed populations of *A. niger* and *A. carbonarius* were present and most of them were OTA producers (Fig. 4). As for larger population studies, no correlation was found between genotypes and mycotoxin production

This chapter summarizes data on grape mycoflora, toxigenic fungi and mycotoxin contamination at the pre-harvesting, harvesting and processing stages. Grape rotting and spoilage can be caused by a variety of fungal species, including *Botrytis cinerea*, *Penicillium*, *Aspergillus*, *Alternaria* and *Cladosporium*. In recent years, black *Aspergillus* species (Section *Nigri*) and in particular *A. carbonarius* and *A. niger* aggregate have been described as the main source of grape contamination with the mycotoxin ochratoxin A. In this chapter, we highlighted how *Aspergillus* species distribution on European grapes may occur and vary in relation to meteorological conditions and geographical areas and several studies have shown an increase in the amount of OTA in warmer climates. The literature on various molecular methods used for species identification is reviewed and a critical evaluation of the usefulness of various techniques and genomic loci for the species identification of black aspergilli is presented. Reports of the occurrence of black aspergilli in vineyards and their potential toxigenicity must be reconsidered on the basis of the wide molecular biodiversity found within morphologically undistinguishable strains of this section. Mycotoxin production is a characteristic of the species, so by studying the species intraspecific biodiversity can predict potential mycotoxin hazards. Different isolates belonging to the black aspergilli species showed varying abilities to produce OTA so it becomes utmost importance to guarantee a quality control of the grapes and grape derived products,

Abarca, M.L., Accensi, F., Bragulat, M.R. & Cabañes, F.J. (2001). Current importance of

Abarca, M.L., Accens,i F., Cano, J., Cabañes, F.J. (2004). Taxonomy and significance of black aspergilli. *Antonie Van Leeuwenhoek,* Vol. 86, pp. 33–49, ISSN 0003-6072 Accensi, F., Cano, J., Figuera, L., Abarca, M.L. & Cabañes, F.J. (1999). New PCR method to

Atoui, A., Dao, P., Mathieu, F. & Lebrihi, A. (2006). Amplification and diversity analysis of

Atoui, A., Mathieu, F. & Lebrihi, A. (2007). Targeting a polyketide synthase gene for

ochratoxin A-producing *Aspergillus* spp. *Journal of Food Protection,* Vol. 64, pp. 903–

differentiate species in the *Aspergillus niger* aggregate. *FEMS Microbiology Letters*,

ketosynthase domains of putative polyketide synthase genes in *Aspergillus ochraceus* and *Aspergillus carbonarius. producers of ochratoxin A. Molecular Nutrition &* 

*Aspergillus carbonarius* quantification and ochratoxin A assessment in grapes using real-time PCR. *International Journal of Food Microbiology*, Vol. 115, pp. 313–318, ISNN

(Martinez-Culebraz et al., 2009).

through accurate contaminant mycoflora identification.

Vol. 180, No.2, pp. 191–196, ISSN 0378-1097

*Food Research*, Vol. 50, pp. 448-493, ISSN 1613-4133

906, ISSN 0362-028X

0168-1605

**4. Conclusion** 

**5. References** 

MPVCT: Micothèque of Institute of Plant Pathology, University of Catania, Italy; USA; MUCL: Micothèque de L'Universitè Catholique de Lovain, Belgium \* Reference strains

Fig. 4. UPGMA dendrogram obtained from fAFLP analysis with the selective primer pair E-AT (Cy5-labelled) and M-CT on 17 isolates (10 *A. niger*, 3 *A. tubingensis,* 4 *A. carbonarius*) and 2 reference strains isolated from grape samples in the same vineyard. Fragments between 50 and 600 bp were analysed with PHYLIP® v. 3.66 software

European countries (Italy, France, Spain, Portugal, Greece and Israel) and the four main groups were obtained by AFLP clustering analysis of the strains, three of them showing a well-defined homogeneous population/species with intraspecific homology higher than 48%: *A.carbonarius*, *A. tubingensis* and *Aspergillus* 'uniseriate'. The fourth cluster, called *A. niger* 'like', showed low homology with *A. niger* 'type strain' and high internal heterogeneity. The intra-population variability of *Aspergillus* Section *Nigri* strains isolated

from grape samples within the same vineyard proved that mixed populations of *A. niger* and *A. carbonarius* were present and most of them were OTA producers (Fig. 4). As for larger population studies, no correlation was found between genotypes and mycotoxin production (Martinez-Culebraz et al., 2009).
