The project will be conducted over a three-year period and it is structured in six workpackages (WP1-WP6) which are further divided into tasks. The completed and currently in progress deliverables are mentioned below:
Deliverable 1.1: List of fungal isolates from wine and table grapes from different vineyards in the country.+
Abstract: A field research was conducted at eight prefectures of the country in order to take samples from table and wine grapes for the isolation and characterization of the indigenous fungal flora. The sampling sites included the prefectures of Heraklion Crete, Attica, Corinth, Arcadia, Imathia, Florina, Pella and Thessaloniki. Special concern was given to the sampling from the vineyards in order to be as representative as possible by taking samples from different vine stumps following specific sampling protocol. A total of 33 samples were collected and included wine varieties such Sabbatiano, Agiorgitiko, Moshofilero, Xinomavro, Rhoditis, Muscat, Mandilaria, Vilana, Kotsifali, Syrah, Edessis, as well as varieties for table use such as Sultanina, Calmerian and Thompson seedless. From these samples fungal isolations by the method of direct plating in petri dishes was undertaken. Overall, 5251 fungal isolates were recorded, from which the vast majority (39 %) belonged to the genus Aspergillus. Other fungi with significant presence in grapes were Alternaria (12%), Fusarium (15%) and Cladosporium (11%). Special attention was given to the isolation of black Aspergilli (Aspergillus section Nigri) including ochratoxigenic strains like Aspergillus carbonarius. From this class 120 strains of fungi were isolated, which are going to be submitted for identification and determination of the toxigenic potential in further tasks.
Deliverable 2.1: List of fungal genera characterized from wine and table grapes in Greek vineyards .+
Abstract: The work undertaken includes the identification of black Aspergilli species isolated from wine and table grape varieties from four different geographical areas of the country. In total, 117 strains of Aspergillus section Nigri group were isolated and characterized by phenotypic and molecular techniques. The phenotypic tests included the growth of fungal isolates at three different culture media (Malt Extract Agar, Czapek Yeast-extract Agar and Czapek Yeast-extract 20% Sucrose agar). Moreover microscopically observation of fruiting bodies after incubation at 25° C for 7 days, was conducted. At the same time, molecular characterization of fungi with PCR-RFLP technique of 5.8S-ITS DNA region was undertaken. Results of strain characterization with microscopic observations showed that 60 strains (51.28 %) were identified as Aspergillus carbonarius, 55 strains (47.01 %) as Aspergillus niger / A. tubingensis and 1 strain (0.85 %) as Aspergillus japonicus / A. aculeatus. Molecular methods for strain identification confirmed the results of microscopic observations with a concordance rate of 98.3 %, since only two isolates were misclassified between the two methods.
Deliverable 2.2: Report on distribution and frequency of fungal isolates.+
Abstract: The distribution and frequency of isolated fungi from different geographical regions of the country and different grape varieties was investigated. Special emphasis was given at black Aspergilli group (Aspergillus section Nigri) which are characterised as OTA producers. According to identification results, the most frequent occurrence of black Aspergilli was recoreded in Crete (83%), followed by the Peloponnese (50%), Attica (38%) and Macedonia (15%), confirming that the most frequent isolation and higher percentage of A. section Nigri group and Aspergillus carbonarius were recorded for the vineyards located in the southern part of the Greek territory. In terms of the varieties investigated, Calmerian and Thomson Seedless from table grape varieties, and Sabbatiano and Agiorgitiko from wine grape varieties presented the highest incidence and distribution for black Aspergilli group and especially of A. carbonarius. The grape type of vineyards (table or wine grapes) did not appear to have a significant effect on fungi incidence of isolation since numbers recorded were similar.
Deliverable 2.3: Correlation of fungal isolates with geo-climatic characteristics of various sampling regions.+
Abstract: The different species of fungi isolated from grapes were associated with the geographical areas of vineyards, in order to investigate any correlation between fungal isolates and regions of grape sampling. For the correlation of the data, multivariate statistical method analysis was applied and particularly Principal Component Analysis (PCA) and Partial Least Squares Discriminant Analysis (PLS-DA). Based on the results, it was made possible to correlate the presence of black Aspergilli group (Aspergillus section Nigri) with the samples originating from Attica. Moreover, the genus Alternaria was associated with samples originating from Macedonia. In contrast, the genera Rhizopus and Botrytis have not presented any particular correlation with any geographical area as they were related with samples originating from Peloponnese, as well as from Macedonia and Attica. These observations are confirmed by the fungal ecophysiology, due to the fact that genera originating from samples of high altitude areas (Macedonia, Peloponnese) are more psychotropic (Alternaria, Rhizopus, Penicillium), while genera that thrive on warmer areas (Aspergillus, Fusarium) are characteristic of Attica (lowland) and Crete (insular area).
Deliverable 3.1: Ochratoxigenic potential of isolated fungal species based on qualitative and quantitative methods.+
Abstract: The evaluation of rapid methods of OTA assessment from the isolated fungi of Aspergillus section Nigri group was investigated. Specifically, a qualitative method for the assessment of OTA presence produced by these fungi was applied, using Coconut Cream Agar medium (CCA) for the evaluation of the fluorescence intensity produced by the toxin under ultraviolet light. The second is the official reference method for OTA quantification with reverse-phase HPLC with FLD, and the third is the semi-quantitative immunochemical method ELISA, with application of a specific quantitative assay of OTA (kit) which is commercially available for rapid determination of the toxin in food samples. The results of the qualitative method (CCA) showed that generally there was an agreement with the results obtained by the reference method (HPLC). However, four strains of A. carbonarius gave absence of fluorescence (no OTA production) on CCA medium, but showed OTA production when HPLC analysis was performed. Also, eight strains of the same fungal species gave positive results at CCA but without confirmation by the HPLC method. Results of the official reference method for OTA determination (HPLC), confirmed that the vast majority of A. carbonarius strains are strong producers of OTA, while members of group A. niger aggregate (A. niger, A. tubigensis) are mainly non-ochratoxigenic, although there were a few exceptions with low OTA potential. Finally, the immunochemical method (ELISA) gave satisfactory results, even if it wasn’t as accurate as the HPLC determination, showing an underestimation of OTA capacity, especially at high concentrations.
"This work has been supported by the FungalPrognosis project 242 of the ARISTEIA-I call co-funded by EC European Social Fund and the Greek General Secretariat of Research and Technology".