Ochratoxigenic Black Species of Aspergilli in Grape Fruits of Northern Italy Identified by an Improved PCR-RFLP Procedure

Abstract

A collection of 356 isolates of Aspergillus spp. collected during 2006 and 2007 from grapevines in northern Italy were identified through Internal Transcribed Spacer based Restriction Fragment Length Polymorphism (ITS-RFLP) and tested for ochratoxin A (OTA) production. Restriction endonuclease digestion of the ITS products using the endonucleases HhaI, HinfI and RsaI, distinguished five different RFLPs. From each pattern, three samples were sequenced and the nucleotide sequences showed different species corresponding to Aspergillus niger, A. carbonarius, A. tubingensis, A. japonicus and A. aculeatus. By comparing the sequences of the ITS regions, also the uniseriate species A. japonicus and A. aculeatus could be differentiated by HinfI digestion of the ITS products. Among the aspergilli, A. niger was the major species associated with grapes during 2006 (57.4%), while A. carbonarius was the major species during 2007 (46.6%). All the strains of Aspergillus were tested for their ability to produce OTA on Yeast extract sucrose medium (YES), as it was tested as an optimal substrate for the evaluation of OTA production by black aspergilli. Out of 356 isolates, 63 (17.7%) isolates produced OTA ranging from 0.05 to 3.0 µg mL−1. Most of the ochratoxigenic isolates were A. carbonarius (46) in both years, but also some strains of A. tubingensis (11) and A. japonicus (6) produced lower amounts of OTA.

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

“…These results are in accordance with results presented by other authors, supporting the use of the Hha I, Hinf I and Rsa I restriction endonucleases for accurate characterization of A. carbonarius , A. tubingensis and, A. niger species [33], [39], [59], [60]. Two other digestion pattern combinations have resulted using the Hha I and Hinf I enzymes.…”

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).…”

“…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).…”

“…Subsequently, many studies have focused on the characterization of the grape fungal flora, determining Aspergillus, Botrytis and Penicillium genera as the main spoilers (Battilani and Pietri, 2002;Somma et al, 2012). With respect to OTA contamination, Aspergillus carbonarius is considered the major cause among OTA producers in grapes (Battilani et al, 2006;Cabañes et al, 2002;Spadaro et al, 2012). The premature detection of this species has been a case study by many researchers in order to control industrial quality by quantifying and reducing the levels of OTA producing fungal species in grapes, grapederivatives and wines (Battilani et al, 2006;Gashgari et al, 2011;Gonz alez-Salgado et al, 2009;Perrone et al, 2006).…”

Population Dynamics of Aspergillus Section Nigri Species on Vineyard Samples of Grapes and Raisins

“…Several PCR methods have been developed to detect ochratoxigenic Aspergillus species (1,9,19,28,32). Our laboratory recently designed PCR primer sets to distinguish among A. niger, A. welwitschiae, A. carbonarius, and A. tubingensis based on calmodulin gene sequence differences (20).…”