FungalPrognosis Work Packages
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. Specifically:
WP1: Isolation of filamentous fungi from wine and table grapes.+
Description: There is lack of information in the international literature referring to black Aspergilli’s field dynamics. The reason is that the black Aspergilli, or Aspergillus section Nigri group, were not believed to belong to the classical pathogens of grapes. Soon after the first reports of OTA presence in wine, researchers started isolating ochratoxigenic black Aspergillus spp. from berries of vineyards, believing that such species were the main source of contamination with the toxin. Although the possible participation of different OTA producing species has been observed in the earlier studies, there is a strong evidence of the significant contribution of A. carbonarius in the OTA contamination of grape products. This is supported by the high incidence of ochratoxin-producing isolates within A. carbonarius and the low incidence of the typical OTA producing species A. ochraceous and P. verrucosum. Based on recent studies, Greece is an area which has a higher isolation percentage of A. carbonarius when compared with other countries of the Mediterranean basin, as well as higher contamination in its vineyards by black Aspergilli at harvesting time, sometimes exceeding 50% of berries. These findings amplify the need of recording the mycoflora dynamics on the field, isolate and identify the responsible species for OTA production, and test their ochratoxigenic ability.
Task 1.1: Grape sampling
Task 1.2: Fungal isolation
WP2: Identification of fungal isolates using conventional and molecular techniques.+
Description: The most frequent fungal genera reported to be present on grapes include Alternaria, Aspergillus, Botrytis, Cladosporium, and Penicillium (Bau et al., 2005). However, the diversity of filamentous fungi on grapes has not been extensively investigated at species level. The present work package will describe the total mycoflora, with main emphasis on the Aspergillus section Nigri group and OTA producing species in grapes and will present their distribution and frequency of isolation. Moreover, effort will be make to correlate the above-mentioned recordings with the geo-climatic characteristics of the studied regions, the culturing techniques followed and compare them with similar studies from countries along the Mediterranean basin. It needs to be noted however, that culture-dependent methods with microscopic and macroscopic examination are traditionally used to identify filamentous fungi, but may fail to identify the complete diversity of fungi present. Moreover, morphological and physiological characteristics are influenced by culture conditions and consequently this approach can provide incomplete or ambiguous results, whereas these methods are also laborious and time consuming. Therefore, the trend today is to employ molecular methodologies for highly specific identification.
Task 2.1 Conventional identification of fungal isolates.
Task 2.2 Identification of fungal isolates with molecular methods.
WP3: Rapid qualitative and quantitative screening of isolated fungi for ochratoxin A production.+
Description: Numerous analytical methods have been described so far to detect toxigenic fungi based on toxin production in natural or synthetic substrates, followed by extraction, purification and detection by thin layer chromatography (TLC) or high performance liquid chromatography (HPLC). In general, these methods are time-consuming, laborious and are not suitable when many fungal isolates need to be screened. In the proposed project, the isolates will be tested for their ochratoxigenic ability according to the method of Bragulat et al. (2001), a rapid method for detecting OTA production, which quantifies the toxin in pure culture. The method has several advantages over other conventional methods which use large amounts of natural substrates or culture media, and consequently they are quite costly in terms of solvents and labour. The use of ELISA (Enzyme linked immune-sorbent assay) for OTA analysis will also be employed as an important and very rapid method, because it is easy to use and due to the large number of samples that can be processed at the same time (up to 90 samples for each ELISA kit in 45 minutes). In terms of qualitative screening, the method of coconut cream agar will be used, as it has been proved very effective for rapid screening of toxigenic fungi. The method is based on mycotoxin detection by fluorescence under long wave ultraviolet radiation.
Task 3.1 Qualitative rapid OTA screening method.
Task 3.2 Quantitative rapid OTA screening methods (HPLC-FD & ELISA method).
WP4: Ecophysiological studies.+
Description: Fungal growth is influenced by several environmental parameters, with temperature and water activity (aw) regarded as the principal controlling factors determining the potential for growth. The differences of temperature among the viticultural regions, the daily mean temperature, the reduction of water availability within the grapes due to sugar content increases and culturing techniques influence growth and OTA production. Nevertheless, there are more factors such as the use of fungicides or interactions with other fungi that can also affect growth and OTA production. Apart from aw and temperature additional ecological factors will be taken into account such as fungicides, essential oils and other inhibitors like natamycin. Special attention will be given to fungal species belonging to Aspergillus section Nigri that include the most relevant OTA producing species. In addition, interactions with other grape-associated fungi will be studied, alone and in combination with other environmental factors.
Task 4.1 Effect of different ecological factors on fungal responses (growth and toxin production).
Task 4.2 Predictive modelling.
WP5: Development of a Real Time PCR approach for detection of ochratoxigenic fungi.+
Description: The molecular work that has done so far for the discovery of the gene sequences of fungi species has enabled the gene clusters involved in the biosynthetic pathways for mycotoxin production. However, there is limited information about the genes involved in the OTA biosynthesis. Among the ochratoxigenic fungi, different pks (polyketide synthase) genes involved in OTA biosynthesis have been identified. Regardless of the extended work in this field, the researchers have concentrated on elucidating the role of the different genes in these clusters and their associated enzymes to understand the importance in terms of structural or regulatory function. It is critical to develop molecular approaches which could use the identified genes as target genes for identification and quantification of OTA producing fungi. Thus, the aim of this WP is to develop a Real Time PCR procedure for rapid, sensitive and specific detection and quantification of OTA producing strains.
Task 5.1 Detection of OTA producing fungi.
Task 5.2 OTA production and quantification.
WP6: Dissemination of the project outcome+
Description: It is anticipated that one seminar per year will be conducted for all interested student and Academic staff of AUA. In addition, one seminar will be organized with the collaboration of the Greek Food Industry Association (SEVT), for managers and technical personnel employed in relevant companies. Moreover, publications in scientific journals, national and international conferences will be made throughout the duration of the project. Finally, dissemination will be made possible to any interested party through the special web site developed for the project.
"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".