Fungal diversity and occurrence of mycotoxin producing fungi in tropical vineyards.

Grapevine cultivars are distributed worldwide, nevertheless the fermentation of its grape berries renders distinct wine products that are highly associated to the local fungal community. Despite the symbiotic association between wine and the fungal metabolism, impacting both the terroir and mycotoxin production, few studies have explored the vineyard ecosystem fungal community using both molecular marker sequencing and mycotoxin production assessment. In this study, we investigated the fungal community of three grapevine cultivars (Vitis vinifera L.) in two tropical vineyards. Illumina MiSeq sequencing was performed on two biocompartments: grape berries (GB) and grapevine soil (GS); yielding a total of 578,495 fungal internal transcribed spacer 1 reads, which were used for taxonomic classification. GB and GS fungal communities were mainly constituted by Ascomycota phylum. GS harbors a significant richer and more diverse fungal community than GB. Among GB samples, Syrah grape berries exclusively shared fungal community included wine-associated yeasts (e.g. Saccharomycopsis vini) that may play key roles in wine terroir. Mycotoxin production assessment revealed the high potential of Aspergillus section Flavi and Penicillium section Citrina isolates to produce aflatoxin B1-B2 and citrinin, respectively. This is the first study to employ next-generation sequencing to investigate vineyard associated fungal community in Brazil. Our findings provide valuable insights on the available tools for fungal ecology assessment applied to food products emphasizing the coexistence between classical and molecular tools.

Impact of grapevine red blotch disease on primary and secondary metabolites in 'Cabernet Sauvignon' grape tissues.

The grapevine red blotch disease (GRBD) was first noticed in 2008, impacting grape ripening. In general, GRBD reduces grape and wine quality resulting in significant economic losses. The purpose of the present study was to evaluate the effect of GRBD on agronomical parameters of 'Cabernet Sauvignon' vines at harvest. Using a metabolomics approach, the influence on primary and secondary metabolite profiling in skin + pulp/flesh and seeds were also determined. GRBD influenced °Brix and berry weight, as well as primary and secondary metabolites in both tissues. 1D 1H NMR was effective in quantifying the main primary and secondary metabolites affected by GRBD. RP-HPLC was similarly able to quantify the main phenolics affected. Multivariate analysis showed the influence of the virus on grape metabolites using both tools in two berry tissues. The effectiveness of both tools to describe sample variability was compared and the most affected metabolites in each tissue could be identified.

Trunk Girdling Increased Stomatal Conductance in Cabernet Sauvignon Grapevines, Reduced Glutamine, and Increased Malvidin-3-Glucoside and Quercetin-3-Glucoside Concentrations in Skins and Pulp at Harvest.

Girdling is a traditional horticultural practice applied at fruit set or other phenological stages, and is used mostly as a vine management. In grapevines, it is used primarily for table grapes to improve berry weight, sugar content, color, and to promote early harvest. The objective of this study was to evaluate the effect of trunk girdling applied at veraison, in 'Cabernet Sauvignon' wine grapes (Vitis vinifera L.), on agronomical and physiological parameters during vine development from the onset of ripening (veraison) to harvest, and additionally to quantify the effect of girdling on primary and secondary metabolism. Girdling was applied 146 days after pruning (dap) at veraison, when berry sampling for metabolomics and agronomical evaluations commenced, with a further three sampling dates until harvest, at 156 dap (30% maturation, 10 days after girdling-dag), 181 dap (70% maturation, 35 dag), and 223 dap (commercial harvest, 77 dag). Skin/pulp and seed tissues were extracted separately and metabolomics was performed using one-dimensional proton nuclear magnetic resonance (1D 1H NMR) spectroscopy and high performance liquid chromatography (HPLC-DAD). At harvest, girdling significantly increased stomatal conductance (gs) in vines, decreased glutamine concentrations, and increased anthocyanin and flavonol concentrations in the skin/pulp tissues of grape berries. Berry weight was reduced by 27% from 181 dap to harvest, and was significantly higher in grapes from girdled vines at 181 dap. Sugars, organic acids, and other amino acids in skin/pulp or seeds were not significantly different, possibly due to extra-fascicular phloem vessels transporting metabolites from leaves to the roots. Using a metabolomics approach, differences between skin/pulp and seeds tissues were meaningful, and a greater number of secondary metabolites in skin/pulp was affected by girdling than in seeds. Girdling is a simple technique that could easily be applied commercially on vine management to improve berry color and other phenolics in 'Cabernet Sauvignon' grapes.

Influence of Maturation Stages in Different Varieties of Wine Grapes ( Vitis vinifera) on the Production of Ochratoxin A and Its Modified Forms by Aspergillus carbonarius and Aspergillus niger.

Ochratoxin A is the main contaminant mycotoxin of grapes produced mainly by Aspergillus niger and Aspergillus carbonarius. Besides, it is possible that the formation of modified mycotoxin occurs through the plant defense mechanism or also by fungus actions itself. The objective of this study was to evaluate the influence of grape variety and maturation stage on the formation of OTA and modified mycotoxin. The determination of OTA was performed by high-performance liquid chromatography, and a high-resolution mass spectrometry was used for the detection of modified ochratoxin. A positive correlation was observed between the following grapes physicochemical parameters: pH, total soluble solids, total glycosides in glucose, total anthocyanin, and OTA levels produced by A. niger and A. carbonarius. Therefore, the higher the concentrations of these parameters, the greater the production of mycotoxin in grapes. Among the elected targets, we identified the 14-decarboxy-ochratoxin A in Muscat Italia variety at veraison and 15 days after the beginning of veraison stages; and ethylamide-ochratoxin A as a biomarker in the Syrah variety at the ripeness stage.

Improved sample preparation for GC-MS-SIM analysis of ethyl carbamate in wine.

An improved sample preparation procedure for analysis of carcinogenic ethyl carbamate (EC) in wine by GC-MS-SIM is proposed. Differences over AOAC reference procedure were: (1) use of EC-d5 as internal standard instead of less similar propyl carbamate; (2) extraction by diethyl ether instead of more toxic dichloromethane, and (3) concentration by vacuum automated parallel evaporation instead of more time and work consuming rotary evaporation. Mean recovery was 104.4%, intraday precision was 6.7% (3.4 µg L(-)(1)) and 1.7% (88.5 µg L(-)(1)), regression coefficient was 0.999 in the linear working range of 3-89 µg L(-)(1), and limits of detection and quantification were 0.4 and 1.2 µg L(-)(1). Applicability was demonstrated by analysis (in triplicate) of 5 wine samples. EC concentration ranged from 5.2 ± 0.2 to 29.4 ± 1.5 µg L(-)(1). The analytical method is selective, accurate, repeatable, linear, and has similar method performance as the reference method along with the several mentioned advantages.

Saccharomyces cerevisiae and non-Saccharomyces yeasts in grape varieties of the São Francisco Valley.

The aims of this work was to characterise indigenous Saccharomyces cerevisiae strains in the naturally fermented juice of grape varieties Cabernet Sauvignon, Grenache, Tempranillo, Sauvignon Blanc and Verdejo used in the São Francisco River Valley, northeastern Brazil. In this study, 155 S. cerevisiae and 60 non-Saccharomyces yeasts were isolated and identified using physiological tests and sequencing of the D1/D2 domains of the large subunit of the rRNA gene. Among the non-Saccharomyces species, Rhodotorula mucilaginosa was the most common species, followed by Pichia kudriavzevii, Candida parapsilosis, Meyerozyma guilliermondii, Wickerhamomyces anomalus, Kloeckera apis, P. manshurica, C. orthopsilosis and C. zemplinina. The population counts of these yeasts ranged among 1.0 to 19 × 10(5) cfu/mL. A total of 155 isolates of S. cerevisiae were compared by mitochondrial DNA restriction analysis, and five molecular mitochondrial DNA restriction profiles were detected. Indigenous strains of S. cerevisiae isolated from grapes of the São Francisco Valley can be further tested as potential starters for wine production.

Saccharomyces cerevisiae and non-Saccharomyces yeasts in grape varieties of the São Francisco Valley

The aims of this work was to characterise indigenous Saccharomyces cerevisiae strains in the naturally fermented juice of grape varieties Cabernet Sauvignon, Grenache, Tempranillo, Sauvignon Blanc and Verdejo used in the São Francisco River Valley, northeastern Brazil. In this study, 155 S. cerevisiae and 60 non-Saccharomyces yeasts were isolated and identified using physiological tests and sequencing of the D1/D2 domains of the large subunit of the rRNA gene. Among the non-Saccharomyces species, Rhodotorula mucilaginosa was the most common species, followed by Pichia kudriavzevii, Candida parapsilosis, Meyerozyma guilliermondii, Wickerhamomyces anomalus, Kloeckera apis, P. manshurica, C. orthopsilosis and C. zemplinina. The population counts of these yeasts ranged among 1.0 to 19 x 10(5) cfu/mL. A total of 155 isolates of S. cerevisiae were compared by mitochondrial DNA restriction analysis, and five molecular mitochondrial DNA restriction profiles were detected. Indigenous strains of S. cerevisiae isolated from grapes of the São Francisco Valley can be further tested as potential starters for wine production.

Detection of ochratoxin A in tropical wine and grape juice from Brazil.

BACKGROUND: Ochratoxin A (OTA) is the main mycotoxin found in grapes, wines and grape juices and is considered one of the most harmful contaminants to human health. In this study, samples of tropical wines and grape juices from different grape varieties grown in Brazil were analysed for their OTA content by high-performance liquid chromatography. RESULTS: The detection and quantification limits for OTA were 0.01 and 0.03 µg L(-1) respectively. OTA was detected in 13 (38.24%) of the samples analysed, with concentrations ranging from < 0.03 to 0.62 µg L(-1). OTA was not detected in any of the grape juice samples. Most of the red wine samples proved to be contaminated with OTA (75%), while only one white wine sample was contaminated. However, the OTA levels detected in all samples were well below the maximum tolerable limit (2 µg L(-1)) in wine and grape juice established by the European Community and Brazilian legislature. CONCLUSION: The results of this study indicate a low risk of exposure to OTA by consumption of tropical wines and grape juices from Brazil.

Determination of metabolite profiles in tropical wines by 1H NMR spectroscopy and chemometrics.

Traditionally, wines are produced in temperate climate zones, with one harvest per year. Tropical wines are a new concept of vitiviniculture that is being developed, principally in Brazil. The new Brazilian frontier is located in the northeast region (São Francisco River Valley) in Pernambuco State, close to the equator, between 8 and 9 degrees S. Compared with other Brazilian and worldwide vineyards, the grapes of this region possess peculiar characteristics. The aim of this work is a preliminary study of commercial São Francisco River Valley wines, analyzing their metabolite profiles by (1)H NMR and chemometric methods.

A liquid chromatographic method optimization for the assessment of low and high molar mass carbonyl compounds in wines.

Carbonyl compounds (CC) play an important role in beverage aroma since they may affect flavor of wines, brandies, and beers, among others. For this reason, it is necessary to identify and quantify CC through adequate analytical techniques. This study is a proposal of both developing and optimization of a new analytical methodology that allows investigate C(1)-C(8 )CC in wines simultaneously by quantifying even those ones that are predominantly present in the adduct form hydroxylalkylsulfonic acids (HASA). The HASA dissociation is undertaken by specific alkaline media (pH 11). The developed methodology employed the LC with UV/VIS detection (lambda = 365 nm) technique under gradient elution in the way to reach both free-CC and bound-CC quantification. Results showed that binary gradient system using eluent A (MeOH/ACN/H(2)O 74.5:0.5:25% v/v/v) and eluent B (MeOH) reached the best separation condition of both lower and higher molecular mass CC. This proposed method allowed simultaneous quantification of formaldehyde, acetaldehyde, propanone, furfuraldehyde, butyraldehyde, benzaldehyde, hexanaldehyde, 2-ethyl-hexanaldehyde, E-pent-2-en-1-al, and cyclohexanone--all of them were found in white wine (Moscato Canelli) and red wine (Shiraz) produced in the São Francisco Valley, in the Northeastern Region of Brazil--although this optimized method may probably be suitable for quantification of propionaldehyde, isobutyraldehyde, heptanaldehyde, octanaldehyde, benzaldehyde, and E-hex-2-en-1-al as well. We could not prove if this method is also able to determine the latter CC group since we have not found these substances present in detectable levels in our real samples considered in this study.

1H NMR and chemometrics to characterize mature grape berries in four wine-growing areas in Bordeaux, France.

The biochemical composition of grape berries depends on the cultivar genome and is influenced by environmental conditions and growing practices, which vary according to origin and "terroir" (French word accounting for the factors of climate, soil, and cultural practices on grape and wine quality). The components currently measured to determine the potential quality of grapes for wine-making at harvest are sugars, acidity, pH, and total phenolics, referred to as "classic analysis". The aim of this work was to establish metabolic profiles using both conventional physicochemical analyses and 1H NMR spectrometry of the skin and pulp of mature berry extracts in order in four appellations situated in different locations in southern-western France (Bordeaux). Principal component analysis was applied to the physiochemical and 1H NMR data to investigate the variability of the grape composition and to characterize groups of samples. A significant clustering of the metabolic profile of pulps or skins in relation to their terroir was observed. Physicochemical analyses were more discriminant than 1H NMR data, but NMR spectroscopy allowed metabolic finger-printings using identified metabolites and some still nonattributed resonances.