Dataset Information

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.

SUBMITTER: Spadaro D

PROVIDER: S-EPMC3317106 | biostudies-literature | 2012 Feb

REPOSITORIES: biostudies-literature

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Ochratoxigenic black species of Aspergilli in grape fruits of northern Italy identified by an improved PCR-RFLP procedure.

Spadaro Davide D Patharajan Subban S Lorè Alessia A Garibaldi Angelo A Gullino Maria Lodovica ML

Toxins 20120130 2

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 ...[more]

PMID: 22474565

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Project description:Fungicide applications constitute a management practice that reduces the size of fungal populations and by acting as a genetic drift factor, may affect pathogen evolution. In a previous study, we showed that the farming system influenced the population structure of the Aspergillus section Nigri species in Greek vineyards. The current study aimed to test the hypothesis that the differences in the population structure may be associated with the selection of fungicide-resistant strains within the black aspergilli populations. To achieve this, we determined the sensitivity of 102, 151, 19, and 22 for the A. uvarum, A. tubingensis, A. niger, and A. carbonarious isolates, respectively, originating either from conventionally-treated or organic vineyards to the fungicides fluxapyroxad-SDHIs, pyraclostrobin-QoIs, tebuconazole-DMIs, and fludioxonil-phenylpyrroles. The results showed widespread resistance to all four fungicides tested in the A. uvarum isolates originating mostly from conventional vineyards. In contrast, all the A. tubingensis isolates tested were sensitive to pyraclostrobin, while moderate frequencies of only lowly resistant isolates were identified for tebuconazole, fludioxonil, and fluxapyroxad. Sequencing analysis of the corresponding fungicide target encoding genes revealed the presence of H270Y, H65Q/S66P, and G143A mutations in the sdhB, sdhD, and cytb genes of A. uvarum resistant isolates, respectively. No mutations in the Cyp51A and Cyp51B genes were detected in either the A. uvarum or A. tubingensis isolates exhibiting high or low resistance levels to DMIs, suggesting that other resistance mechanisms are responsible for the observed phenotype. Our results support the initial hypothesis for the contribution of fungicide resistance in the black aspergilli population structure in conventional and organic vineyards, while this is the first report of A. uvarum resistance to SDHIs and the first documentation of H270Y or H65Q/S66P mutations in sdhB, sdhD, and of the G143A mutation in the cytb gene of this fungal species.

Project description:Recent studies have highlighted the role of the grapevine microbiome in addressing a wide panel of features, ranging from the signature of field origin to wine quality. Although the influence of cultivar and vineyard environmental conditions in shaping the grape microbiome have already been ascertained, several aspects related to this topic, deserve to be further investigated. In this study, we selected three international diffused grapevine cultivars (Cabernet Sauvignon, Syrah, and Sauvignon Blanc) at three germplasm collections characterized by different climatic conditions [Northern Italy (NI), Italian Alps (AI), and Northern Spain (NS)]. The soil and grape microbiome was characterized by 16s rRNA High Throughput Sequencing (HTS), and the obtained results showed that all grape samples shared some bacterial taxa, regardless of sampling locality (e.g., Bacillus, Methylobacterium, Sphingomonas, and other genera belonging to Alphaproteobacteria, Gammaproteobacteria, and Actinobacteria). However, some Operational Taxonomic Units (OTUs) could act as geographical signatures and in some cases as cultivar fingerprint. Concerning the origin of the grape microbiome, our study confirms that vineyard soil represents a primary reservoir for grape associated bacteria with almost 60% of genera shared between the soil and grape. At each locality, grapevine cultivars shared a core of bacterial genera belonging to the vineyard soil, as well as from other local biodiversity elements such as arthropods inhabiting or foraging in the vineyard. Finally, a machine learning analysis showed that it was possible to predict the geographical origin and cultivar of grape starting from its microbiome composition with a high accuracy (9 cases out of 12 tested samples). Overall, these findings open new perspectives for the development of more comprehensive and integrated research activities to test which environmental variables have an effective role in shaping the microbiome composition and dynamics of cultivated species over time and space.

Dataset Information

Ochratoxigenic black species of Aspergilli in grape fruits of northern Italy identified by an improved PCR-RFLP procedure.

Publications

Ochratoxigenic black species of Aspergilli in grape fruits of northern Italy identified by an improved PCR-RFLP procedure.

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