Touriga Nacional Research Articles

Preliminary sensory evaluation of grpe pomace flour sweet cookies

The sustainable use of resources and industrial waste is an important objective for environmental preservation and in the search for potential new foods and ingredients. Considering the size and importance of the wine industry, the use of winemaking waste in a sustainable manner is important for producers. Given the nutritional characteristics of this by-product, its use as an ingredient may be an interesting source of income, especially in Portugal, where around 183 thousand tons of this waste are generated annually. In addition to studies regarding the nutritional characteristics and active constituents, it is necessary to evaluate the acceptability of potential buyers to different food matrices resulting from the incorporation of grape pomace flour. This study was designed to evaluate the acceptability of sweet cookies with the incorporation of different percentages of grape pomace flour resulting from the vinification of Arinto and Touriga Nacional grape varieties. The sensory assessment of 149 individuals was evaluated using a questionnaire developed for this purpose. The results obtained are not conclusive, but there is a tendency for the preference for cookies with the incorporation of grape pomace flour at 15% versus incorporation of 20%, independent of the cast used.

Authentication of Douro DO monovarietal red wines based on anthocyanin profile: Comparison of partial least squares – discriminant analysis, decision trees and artificial neural networks

In this study, the feasibility of discriminating monovarietal red wines from Touriga Nacional (TN), Touriga Franca (TF), and Tinta Roriz (TR) from Douro Demarcated Region (DDR) using the anthocyanin profile was investigated in combination with partial least squares – discrimination analysis (PLS-DA), classification and regression trees (CART) and artificial neural networks (ANN). CART and ANN were more accurate in the classification of monovarietal wines according to their origin with a 100% classification rate in the test set. PLS-DA presented an accuracy of 91.4%. The variables most important for monovarietal wines authentication were petunidin-3-glucoside, malvidin-3-acetylglucoside, and cyanidin-3-glucoside for the PLS-DA, petunidin-3-glucoside, peonidin-3-acetylglucoside, and malvidin-3-acetylglucoside for CART analysis, and malvidin-3-acetylglucoside, petunidin-3-glucoside, malvidin-3-glucoside, peonidin-3-coumaroylglucoside and delphinidin-3-glucoside for ANN. The results demonstrate that the anthocyanin profile of Touriga Nacional, Touriga Franca, and Tinta Roriz Douro Denomination of Origin monovarietal red wines combined with multivariate analysis is an effective tool for verifying their authenticity.

Sustainable Replacement Strategies for Bentonite in Wine Using Alternative Protein Fining Agents

Protein stability is an important quality attribute in wines and protein haze will lead to consumer rejection. Traditionally, stability is achieved by bentonite addition; however, environmental concerns and disposal problems mean that alternatives are required to achieve the same goal. In this study, the use of Sacharomyces paradoxus, chitosan, polystyrene, carboxymethyl cellulose, and bentonite were evaluated. Trials in finished wines were agitated for 10 h overnight and analyzed for turbidity and color characteristics spectrophotometrically. Experiments were conducted with wines that are expected to develop protein instabilities, Muscat Canelli, White Zinfandel, Cabernet Sauvignon blanc de noir, Barbera rosé, and Touriga Nacional. Results indicate that S. paradoxus can help with the removal of proteins from wine. Wines with low protein instability can be stabilized with S. paradoxus as well as polystyrene and chitosan to a lesser degree. All fining agents except for bentonite show efficiency variability between white and red wines. With an average protein reduction around 50%, none of the alternative fining methods could reach the efficiency level of bentonite. Experiments in a model system confirm the findings and explain some of the mechanisms involved, for example the specificity of chitosan and challenges related to the use of yeast as a fining agent.

Impact of oak (<i>Q. pyrenaica</i> and <i>Q. pubescens</i>) and cherry (<i>P. avium</i>) wood chip contact on phenolic composition and sensory profile evolution of red wines during bottle storage

The majority of published work has studied the impact of wood chips on red wine composition by conducting analyses during wood chip contact or immediately after the removal of chips from wine. Less attention has been directed at the potential influence of prior chip-wine contact on the further phenolic and sensory evolution of red wines during bottle storage. Therefore, this work focuses on the evolution over a period of 18 months of several phenolic parameters and sensory characteristics of bottled Touriga Nacional red wines that had previously been in contact with toasted wood chips from cherry (Prunus avium) and two oak species (Quercus pyrenaica and Quercus pubescens) during 30 days of pre-bottling storage. Various global phenolic parameters, colour properties, individual anthocyanin content and sensory profile of the wines were studied at 6, 12 and 18 months of bottle storage. The results showed less decrease in the phenolic composition and red colour of wines which had prior contact with oak chips, as well as a less developed brown colour during bottle storage, compared to the wine previously in contact with cherry chips and the control wine. In addition, wine previously in contact with cherry chips always showed an evolution similar to the control wine. From a sensory point of view, the wines previously in contact with oak wood chips showed a tendency for higher aroma scores for “vanilla” and “coconut” descriptors and lower scores for “brown colour” during bottle storage than wines previously in contact with cherry chips and the control wine. The outcomes of this research could be of practical interest to winemakers since they could improve the knowledge of the impact of prior contact with wood chips in the future evolution of the red wines during bottle storage.

Response of Mycorrhizal 'Touriga Nacional' Variety Grapevines to High Temperatures Measured by Calorespirometry and Near-Infrared Spectroscopy.

Heat stress negatively affects several physiological and biochemical processes in grapevine plants. In this work, two new methods, calorespirometry, which has been used to determine temperature adaptation in plants, and near-infrared (NIR) spectroscopy, which has been used to determine several grapevine-related traits and to discriminate among varieties, were tested to evaluate grapevine response to high temperatures. ‘Touriga Nacional’ variety grapevines, inoculated or not with Rhizoglomus irregulare or Funneliformis mosseae, were used in this study. Calorespirometric parameters and NIR spectra, as well as other parameters commonly used to assess heat injury in plants, were measured before and after high temperature exposure. Growth rate and substrate carbon conversion efficiency, calculated from calorespirometric measurements, and stomatal conductance, were the most sensitive parameters for discriminating among high temperature responses of control and inoculated grapevines. The results revealed that, although this vine variety can adapt its physiology to temperatures up to 40 °C, inoculation with R. irregulare could additionally help to sustain its growth, especially after heat shocks. Therefore, the combination of calorespirometry together with gas exchange measurements is a promising strategy for screening grapevine heat tolerance under controlled conditions and has high potential to be implemented in initial phases of plant breeding programs.

Survival and metabolism of hydroxycinnamic acids by Dekkera bruxellensis in monovarietal wines

Volatile phenols in wines are responsible for unpleasant aromas, which negatively affect the quality of the wine. These compounds are produced from the metabolism of hydroxycinnamic acids, mainly by the yeasts Brettanomyces/Dekkera. Relevant data, potentially useful to support decisions on how to manage the risk of contamination of wines by Brettanomyces/Dekkera, according to the grape varieties used in the vinification, is important to the wine industry. Therefore, the aim of this work was to evaluate the survival and the metabolism of hydroxycinnamic acids by Dekkera bruxellensis in monovarietal wines. Yeast growth and survival were monitored in fifteen wines, five from each of the grape varieties Touriga Nacional, Cabernet Sauvignon and Syrah, inoculated with a strain of D. bruxellensis. Yeast culturable populations of 107 CFU mL−1 were reduced to undetectable numbers in 24 h in all wines. Plate counts of 104–106 CFU mL−1 were, however, detected after 48 h in most of Touriga Nacional and Cabernet Sauvignon wines and later in Syrah. Viability measurement by flow cytometry showed that a significant part of the populations was in a viable but non-culturable state (VBNC). The time required for the recovery of the culturable state was dependent on the wine, being longer on Syrah wines. Besides the production of ethylphenols, the metabolism of hydroxycinnamic acids by VBNC cells led to the accumulation of vinylphenols at relatively high levels, independently of the grape variety. The flow cytometry methodology showed a higher survival capacity of D. bruxellensis in Touriga Nacional wines, which corroborates with the higher amounts of volatile phenols found on this variety.

The Interplay between Atmospheric Conditions and Grape Berry Quality Parameters in Portugal

The atmospheric conditions are a strong modulator of grape berry composition, but further research is required to better understand this relationship, which is particularly pertinent under the context of climate change. The present study assesses the relationship between interannual variability in atmospheric conditions (mean, maximum and minimum air temperatures and precipitation totals) on grape berry quality attributes in three main Portuguese wine regions—Douro, Dão and Alentejo—and targets two major varieties growing in Portugal (cv. Touriga Nacional and cv. Aragonez/Tempranillo). Berry weight, titratable acidity (TA), pH, potential alcohol (PA), anthocyanins and total phenols index (TPI) data, collected two to three weeks after the end of the veraison until technological maturity, since 1999 in Douro, 2004 in Alentejo and 2008 in Dão, were selected. Meteorological data were obtained from both automatic weather stations and a climatic database defined at a very-high-resolution grid (<1 km) (PTHRES). The influence of daily mean, maximum and minimum air temperatures (November–October) and precipitation totals (April to June and July to September) on the above-mentioned berry quality parameters were first explored to identify the months/periods more influential to grape berry composition. Different statistical approaches were subsequently carried out to explore in greater detail these relationships. At technological maturity, temperature was negatively correlated to berry weight, titratable acidity, anthocyanins and TPI, but was positively correlated to pH and potential alcohol. Moreover, lowest levels of berry weight and TA (and highest levels of pH) were more frequent in warmer regions, while the opposite was seen in the cooler regions. PA, TPI and anthocyanins at maturity did not show a clear trend across regions. In addition, the maturation parameters of each site were grouped into two clusters—years where the maturation parameter is higher (cluster 1) and years where it is lower (cluster 2)—and significant differences in monthly mean temperatures between clusters were found. Overall, temperatures at veraison and maturation periods (June–August) were more influential in determining grape berry composition at harvest. The influence of precipitation was dependent on location and variety. The results also suggested that berry composition in Alentejo is more sensitive to atmospheric variability, while Aragonez seems more resilient than Touriga Nacional. These outcomes are based on a systematized and unprecedentedly large grape berry quality database in Portugal and provided the grounds for the development of grape quality forecast models, either to be used operationally in each vintage or for assessing potential modifications in berry composition in response to changing climates.

Evaluation of the Extraction Temperature Influence on Polyphenolic Profiles of Vine-Canes (Vitis vinifera) Subcritical Water Extracts.

This work focused on evaluating the possibility of using vineyard pruning wastes from two Portuguese Vitis vinifera varieties; Touriga Nacional (TN) and Tinta Roriz (TR), as new potential ingredients for the nutraceutical industry. An environmentally friendly extraction technique; namely subcritical-water extraction (SWE), was employed. The overall results indicate that phenolic acids were the major class of compounds quantified; being gallic acid the principal one. The highest value for total phenolic content (TPC) was obtained for the TR extract at 250 °C (181 ± 12 mg GAE/g dw). In terms of antioxidant activity; the DPPH values for the extracts obtained at 250 °C were approximately 4-fold higher than the ones obtained at 125 °C; with TR extract presenting the highest value (203 ± 22 mg TE/g dw). Thus, the TR extract obtained through SWE at 250 °C was selected to evaluate the scavenging activity and the in vitro effects on cells due to the best results achieved in the previous assays. This extract presented the ability to scavenge reactive oxygen species (O2●-, HOCl and ROO●). No adverse effects were observed in HFF-1 viability after exposure to extract concentrations below 100 μg/mL. This work demonstrated that vine-canes extracts could be a potential ingredient to nutraceutical industry

Vine-Canes as a Source of Value-Added Compounds for Cosmetic Formulations.

The majority of works about vine-canes are focused on the evaluation of their chemical composition and antioxidant potential. To the best of our knowledge, the possible applications of produced extracts in cosmetic formulations have never been explored. The aim of the present study was to evaluate the antioxidant properties of vine-canes subcritical water extracts for use as active ingredients in the cosmetic industry. For that, the phenolic content and antioxidant activity of six vine-cane varieties, namely Alvarinho and Loureiro from the Minho region and Touriga Nacional and Tinta Roriz (TR) from both the Douro and Dão regions, were evaluated through spectrophotometric and chromatographic methods. All extracts presented similar antioxidant activity and the highest phenolic content was reported for TR variety from the Douro region (33.7 ± 1.9 mg GAE/g dw). The capacity of vine-cane extracts to capture reactive oxygen species superoxide (O2∙−) was also studied, with the highest IC50 value being obtained for Loureiro variety (56.68 ± 2.60 µg/mL). Furthermore, no adverse effects on HaCaT and HFF-1 dermal cell lines in concentrations below 100 and 1000 μg/mL, respectively, were determined. Finally, Loureiro vine-cane extract was incorporated into a topical formulation, and physical and microbiological properties were within expected values, demonstrating that vine-canes extracts can be successfully incorporated in cosmetic products.

Vine-Canes Valorisation: Ultrasound-Assisted Extraction from Lab to Pilot Scale.

Wine production generates large amounts of vine-canes, a devalued by-product that could be used for the recovery of bioactive compounds. In this work, two vine-canes varieties, namely Touriga Nacional (TN) and Tinta Roriz (TR), were submitted to different ultrasound-assisted extraction (UAE) conditions. The highest phenolic and flavonoid content was observed for TR extract obtained at lab-scale without an ice bath and pilot-scale after 60 min of extraction (32.6 ± 2.1 and 26.0 ± 1.5 mg gallic acid equivalent/g dry weight (dw) and 9.5 ± 0.6 and 8.3 ± 0.8 mg epicatechin equivalents/g dw, respectively). Further, all extracts demonstrated a high antioxidant activity to scavenge DPPH free radicals with the best value reached by TR at the lab-scale without an ice bath after 30 min and pilot-scale extraction after 60 min (34.2 ± 2.4 and 33.4 ± 2.1 mg trolox equivalents/g dw, respectively). Extracts phenolic composition were also evaluated by HPLC, demonstrating that resveratrol, myricetin and catechin were the main compounds. According to our knowledge, this is the first time that a pilot scale of UAE of phenolic compounds from vine-canes was performed. This paper represents an important step to the use of UAE as an industrial process to recover bioactive compounds.

Silicates of Potassium and Aluminium (Kaolin); Comparative Foliar Mitigation Treatments and Biochemical Insight on Grape Berry Quality in Vitis vinifera L. (cv. Touriga National and Touriga Franca).

Grapevine physiology is influenced by several environmental factors, such as temperature, precipitation, potential evapotranspiration, and sunshine hours. Due to climatic changes, effects in grapevine physiology and consequently on the grape berry composition and quality have been observed. This work aims to make a comparative study of the effect of foliar mitigation treatment with kaolin (5%) and potassium silicates (0.1% and 0.05%) on the grape berry quality; namely on berry weight, pH, probable alcohol, total phenolics, tannins, total anthocyanins, monomeric anthocyanins, calcium, potassium, and magnesium composition from Portuguese grapevines (Vitis vinifera L. cv. Touriga Nacional and Touriga Franca). The results suggested that the phenolic composition and anthocyanin content differs between treatments while other parameters showed distinct behavior among the different applications. Qualitative parameters observed in the present study suggested non-significant changes upon both the applications.

Preliminary sensory evaluation of salty crackers with grape pomace flour

Grape pomace, a by-product of winemaking, contains numerous chemical compounds with high nutritional value and potential benefits for human health, which enable its use for product enrichment in different industries from cosmetics to food. The aim of this study was to incorporate the grape pomace flour of the Touriga Nacional and Arinto grapes varietals from the Alentejo region of Portugal in a recipe for salty crackers, which were submitted to sensory evaluation by applying a hedonic test to 53 individuals. The results of the evaluation show a clear tendency of preference for the crackers with 10% of grape pomace flour, both in the Touriga Nacional and Arinto samples, which presented the highest result averages.

From grape to wine: Fate of ochratoxin A during red, rose, and white winemaking process and the presence of ochratoxin derivatives in the final products

The presence of ochratoxin A (OTA) in wine is mainly due to the contamination of grapes by Aspergillus carbonarius and A. niger, still in the vineyard or at stages prior to winemaking. Throughout winemaking process, although there is a reduction in OTA levels, modified mycotoxins may also be formed. In fact, modified mycotoxins are compounds that normally remain undetectable during the conventional analysis used for the parent toxin. In this context, the current study aimed to evaluate the effect of grape variety and winemaking steps on OTA fate as well as the formation of modified ochratoxins. White, rose and red wines were prepared from Muscat Italia, Syrah and Touriga Nacional varieties, respectively. OTA was determined during different steps of winemaking by UHPLC-ESI-MS/MS. Identification of ochratoxin derivatives was performed using tandem MS experiments. A reduction of 90.72, 92.44 and 88.15% in OTA levels was observed for white, rose and red wines, respectively. Among the sought targets, the following ochratoxin-derived candidates were identified: ochratoxin β, ochratoxin α methyl ester, ochratoxin B methyl ester, ochratoxin A methyl ester, ethylamide ochratoxin A, ochratoxin C and ochratoxin A glucose ester. These results indicate that the formation of ochratoxin derivatives leads to an underestimation of total mycotoxin levels in wine and, therefore, the inclusion of techniques for multi-mycotoxins detection should be considered.

Fertility of buds and pruning recommendation of different grapevine varieties grown in altitude regions of Santa Catarina State, Brazil

ABSTRACT Low bud fertility index is frequently observed in different grapevine varieties grown in high altitude of Santa Catarina Sate; in many cases less than one cluster per bud is observed. The objective of this study was to assess bud fertility of twenty six Vitis vinifera L. varieties and from this information, to recommend the method of pruning more suitable for these varieties in high altitude conditions. The grapevine canes and buds were collected from production vineyards located in Sao Joaquim, Santa Catarina State, in two consecutive vintages. Fifteen cane cuttings containing 10 nodes per variety were collected to determine bud fertility and placed in a growth chamber with controlled relative humidity, light, temperature. After budbreak, buds were classified as fertile or non-fertile according to the presence or absence of the inflorescence. The bud fertility varies according to the variety and its position in the cane. Merlot, Cabernet Franc, Cabernet Sauvignon, Tinta Roriz and Tinta Caiada, which present higher fertility in basal buds, are recommended for short or long pruning. The varieties Chardonnay, Manzoni Bianco, Sauvignon Blanc, Sangiovese, Pinot Gris, Tempranillo, Pinot Noir, Syrah, Montepulciano, and Touriga Nacional should be pruned preferentially in long pruning. While the varieties Viognier, Glera (Prosecco), Fiano, Garganega, Vermentino, Nebbiolo, Teroldego, Rebo, Canaiolo Nero and Touriga Francesa must be pruned exclusively with long pruning in order to ensure adequate cluster production.

Molecular and Functional Characterization of Grapevine NIPs through Heterologous Expression in aqy-Null Saccharomyces cerevisiae.

Plant Nodulin 26-like Intrinsic Proteins (NIPs) are multifunctional membrane channels of the Major Intrinsic Protein (MIP) family. Unlike other homologs, they have low intrinsic water permeability. NIPs possess diverse substrate selectivity, ranging from water to glycerol and to other small solutes, depending on the group-specific amino acid composition at aromatic/Arg (ar/R) constriction. We cloned three NIPs (NIP1;1, NIP5;1, and NIP6;1) from grapevine (cv. Touriga Nacional). Their expression in the membrane of aqy-null Saccharomyces cerevisiae enabled their functional characterization for water and glycerol transport through stopped-flow spectroscopy. VvTnNIP1;1 demonstrated high water as well as glycerol permeability, whereas VvTnNIP6;1 was impermeable to water but presented high glycerol permeability. Their transport activities were declined by cytosolic acidification, implying that internal-pH can regulate NIPs gating. Furthermore, an extension of C-terminal in VvTnNIP6;1M homolog, led to improved channel activity, suggesting that NIPs gating is putatively regulated by C-terminal. Yeast growth assays in the presence of diverse substrates suggest that the transmembrane flux of metalloids (As, B, and Se) and the heavy metal (Cd) are facilitated through grapevine NIPs. This is the first molecular and functional characterization of grapevine NIPs, providing crucial insights into understanding their role for uptake and translocation of small solutes, and extrusion of toxic compounds in grapevine.

Revealing the Usefulness of Aroma Networks to Explain Wine Aroma Properties: A Case Study of Portuguese Wines

Wine aroma is the result of complex interactions between volatile compounds and non-volatile ones and individual perception phenomenon. In this work, an aroma network approach, that links volatile composition (chromatographic data) with its corresponding aroma descriptors was used to explain the wine aroma properties. This concept was applied to six monovarietal wines from Bairrada Appellation (Portugal) and used as a case study. A comprehensive determination of the wines’ volatile composition was done (71 variables, i.e., volatile components), establishing a workflow that combines extraction techniques and gas chromatographic analysis. Then, a bipartite network-based approach consisting of two different nodes was built, one with 19 aroma descriptors, and the other with the corresponding volatile compound(s). To construct the aroma networks, the odor active values were calculated for each determined compound and combined with the bipartite network. Finally, the aroma network of each wine was compared with sensory descriptive analysis. The analysis of the specific aroma network of each wine revealed that Sauvignon Blanc and Arinto white wines present higher fruity (esters) and sweet notes (esters and C13 norisoprenoids) than Bical wine. Sauvignon Blanc also exhibits higher toasted aromas (thiols) while Arinto and Bical wines exhibit higher flowery (C13 norisoprenoids) and herbaceous notes (thiols), respectively. For red wines, sweet fruit aromas are the most abundant, especially for Touriga Nacional. Castelão and Touriga Nacional wines also present toasted aromas (thiols). Baga and Castelão wines also exhibit fusel/alcohol notes (alcohols). The proposed approach establishes a chemical aroma fingerprint (aroma ID) for each type of wine, which may be further used to estimate wine aroma characteristics by projection of the volatile composition on the aroma network.

Grapevine variety identification using “Big Data” collected with miniaturized spectrometer combined with support vector machines and convolutional neural networks

Several experiments have been previously reported suggesting that the application of spectroscopy and machine learning allows the identification of grapevine varieties, however, up to now, the maximum number of varieties separated was twenty and the total number of sample spectra used does not go beyond the few hundreds. The present work aim is to answer the question: Is it possible to separate one variety from an enlarged group of other varieties when the number of samples is also significantly increased? With this in mind, a total of 35,833 spectra from leaves of 626 plants from 64 varieties were gathered for the study. This is a non-trivial evolution from previous works because it originates an increase in the variability of spectra which brings in a higher risk that a significant percentage of spectra of different varieties are equal and cannot be separated. Simultaneously, it was studied if a miniaturized and easy to use spectrometer could deliver data whose quality was enough to allow varieties separation even with data being collected in the field, non-destructively, and under uncontrolled solar lighting. This data was used to build support vector machines and convolutional neural networks for separating Touriga Nacional from 63 other varieties (including Touriga Franca) or Touriga Franca from 63 varieties (including Touriga Nacional), and the classification efficiencies are analysed.

Grapevine Phenology of cv. Touriga Franca and Touriga Nacional in the Douro Wine Region: Modelling and Climate Change Projections

Projections of grapevine phenophases under future climate change scenarios are strategic decision support tools for viticulturists and wine producers. Several phenological models are tested for budburst, flowering, and veraison and for two main grapevine varieties (cv. Touriga Franca and Touriga Nacional) growing in the Douro Demarcated Region. Four forcing models (Growing degree-days, Richardson, Sigmoid, and Wang) and three dormancy models (Bidabe, Smoothed Utah and Chuine), with different parameterizations and combinations, are used. New datasets, combing phenology with weather station data, widespread over the Douro wine region, were used for this purpose. The eight best performing models and parameterizations were selected for each phenophase and variety, based on performance metrics. For both cultivars, results revealed moderate performances (0.4 < R2 < 0.7) for budburst, while high performances (R2 > 0.7) were found for flowering and veraison, particularly when Growing degree-days or Sigmoid models are used, respectively. Climate change projections were based on a two-member climate model ensemble from the EURO-CORDEX project under RCP4.5. Projections depicted an anticipation of phenophase timings by 6, 8 or 10–12 days until the end of the century for budburst, flowering, and veraison, respectively. The inter-model variability is of approximately 2–4 days for flowering and veraison and 4–6 days for budburst. These results establish grounds for the implementation of a decision support system for monitoring and short-term prediction of grapevine phenology, thus promoting a more efficient viticulture.

Epicoccum layuense a potential biological control agent of esca-associated fungi in grapevine.

Epicoccum is a genus of ascomycetes often associated with the mycobiome of grapevines (Vitis vinifera). Epicoccum spp. are found in the soil, phyllosphere, as well as in the wood, where they interact both with the plant and with other endophytes and pathogens. Wood pathogens involved in the esca disease complex, a grapevine trunk disease, are particularly concerning in viticulture, as current control strategies have proven unsatisfactory. This study investigated the interaction among Epicoccum spp. and three esca-associated fungi, with the aim of establishing whether they are suitable candidates for biological control.A screening conducted in vitro, by means of dual culture, revealed that all tested Epicoccum spp. inhibited the growth of pathogens Phaeomoniella chlamydospora and Fomitiporia mediterranea, while only some of them inhibited Phaeoacremonium minimum. Epicoccum layuense E24, identified as the most efficient antagonist, was tested in rooted grapevine cuttings of cultivars Cabernet Sauvignon and Touriga Nacional, under greenhouse conditions, against P. chlamydospora and P. minimum. This study revealed that the inoculation of E. layuense E24 produced a successful colonization of the wood of grapevines; in addition it did not impair the growth of the plants or induce the appearance of symptoms in leaves or in wood. Moreover, grapevines colonized by E. layuense E24 showed a considerable decrease in the wood symptomatology caused by the inoculated pathogens (by 31–82%, depending on the pathogen/grapevine cultivar), as well as a reduction in their frequency of re-isolation (60–74%).Our findings suggest that E. layuense E24 is a promising candidate for its application in biological control, due to its antagonistic interaction with some esca-associated fungal pathogens.

Mycorrhizal Inoculation Differentially Affects Grapevine's Performance in Copper Contaminated and Non-contaminated Soils.

Plant inoculation with arbuscular mycorrhizal fungi (AMF) is increasingly employed to enhance productivity and sustainability in agricultural ecosystems. In the present study, the potential benefits of AMF inoculation on young grapevines replanted in pots containing vineyard soil with high Cu concentration were evaluated. For this purpose, one-year-old cv. Touriga Nacional grapevines grafted onto 1103P rootstocks were further inoculated with Rhizoglomus irregulare or Funneliformis mosseae, or left non-inoculated, and maintained in a sterilized substrate under greenhouse conditions for three months. After this time, half of the plants were transplanted to containers filled with an Arenosol from a vineyard which had been artificially contaminated or not with 300 mg kg−1 of Cu. At the end of the growing season, soil nutrient concentration, soil dehydrogenase activity and mycorrhizal colonization rate were analyzed. Grapevine performance was assessed by measuring several vegetative growth and physiological parameters as well as nutrient concentrations in leaves and roots. In the non-contaminated soil, R. irregulare- and F. mosseae-inoculated plants had significantly greater root biomass than the non-inoculated ones. However, the opposite effect was observed in the Cu-contaminated soil, where non-inoculated plants performed better regarding shoot and root development. Concerning nutrient levels, an increase in Cu, Mg and Mn concentrations was observed in the roots of plants growing in the contaminated soil, although only Mn was translocated to leaves. This led to a large increase in leaf Mn concentrations, which was significantly higher in non-inoculated and F. mosseae- inoculated plants than in the R. irregulare- inoculated ones. Copper contamination induced a general decrease in leaf N, P and Fe concentrations as well as chlorosis symptoms. The largest decrease in N and P was observed in F. mosseae- inoculated plants, with 73 and 31.2%, respectively. However, these plants were the ones with the least decrease in Fe concentration (10% vs. almost 30% in the other two inoculation treatments). In conclusion, this study indicates that soil Cu levels can modify the outcome of AMF inoculations in young grapevines, disclosing new AMF-plant associations potentially relevant in vineyards with a tradition of Cu-based fungicide application.