Biodiversity and ITS-RFLP Characterisation of Aspergillus Section Nigri Isolates in Grapes from Four Traditional Grape-Producing Areas in Greece

Abstract

A study on the occurrence of Aspergillus section Nigri species on grapes from four traditional grape-producing areas in Greece during the 2011/2012 vintage, and their capability to produce OTA was conducted. One hundred and twenty-eight black aspergilli isolates were characterised at the species level initially by the use of morphological criteria in accordance with appropriate keys, followed by molecular characterisation performed with Polymerase Chain Reaction–Restriction Fragment Length Polymorphism (PCR-RFLP) of the 5.8 ribosomal RNA gene Internal Transcribed Spacer region (5.8 rRNA ITS). Restriction enzyme digestion of the ITS amplicons using the HhaI, HinfI and RsaI, endonucleases distinguished eleven different patterns of restriction fragment length polymorphism (RFLP), four for each of the HhaI and RsaI digests and three for HinfI. From a total number of 128 individual isolates, 124 were classified into four Aspergillus species corresponding to A. carbonarius, A. tubingensis, A. japonicus and A. ibericus, and the remaining 4 were classified as members of the A. niger aggregate. A. carbonarius and A. tubingensis being the main representative species were equally counted, with higher geographical representation of the former in southern and the latter in northern regions, respectively. All isolates were tested for their ochratoxigenic potential by use of High Performance Liquid Chromatography (HPLC) and Enzyme Linked Immuno Sorbent Assay (ELISA), resulting in significant interspecies differences in OTA production.

Screening and Identification of Novel Ochratoxin A-Producing Fungi from Grapes

“…Then, the reverse of CCA or CA with colonies was exposed to long wavelength UV light (365 nm) in the dark environment [8,46,47]. OTA-producing isolates would take on characteristic green fluorescence.…”

Quantification of Aspergillus carbonarius in grapes using a real time PCR assay

“…carbonarius is the major OTA producer among black Aspergilli and the principle contaminant of grapes for this mycotoxin (Spadaro et al, 2012). In a previous work, the representation of black Aspergilli in table and wine grapes in 4 main grape producing regions in Greece has been reported, showing that nearly 100% of all identified A. carbonarius isolates were OTA producers (Kizis et al, 2014). The Greek wine-grape variety Savatiano is the most widely cultivated variety in Greece and has an important economic share in the wine producing sector.…”

“…In order to test for fungal DNA presence in our samples, an initial end point PCR was performed using the ITS1/4 primers which are commonly used in various PCR-RFLP analyses (Borman et al, 2008;Kizis et al, 2014;Levin, 2012). As illustrated in Fig.…”

“…Previous studies have shown that the majority of A. carbonarius strains are able to produce OTA with different capacity (Kizis et al, 2014;Spadaro et al, 2012) and only few strains have been characterized as non-ochratoxigenic (Cabañes et al, 2013). Furthermore, OTA production seems to depend on the environmental factors which affect growth (Selouane et al, 2009).…”

Modelling the influence of temperature, water activity and sodium metabisulphite on the growth and OTA production of Aspergillus carbonarius isolated from Greek wine grapes

“…For the lag phase duration, the model presented underestimation (B f > 1) with higher average deviation between predictions and observations (42%). This variation in external validation could be attributed to intraspecific variability of fungal strains, since different strains of A. carbonarius present diverse mycotoxigenic potential (Kizis et al, 2014) and hence some authors have proposed the use of mixed inocula of different isolates to obtain an average representation within a fungal species (Garcia et al, 2014), probably resulting in better model building. In a previous work, Tassou et al (2007) working on SGM with A. carbonarius reported values of 1.31 and 1.59 for B f and A f , respectively, after performing external validation for the Rosso model, with independently derived data from the literature which is in line with the respective values reported in this work.…”