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Grapevine (Vitis vinifera L.) is prone to fungal and oomycete diseases. Downy and powdery mildews and grey mold, are caused by Plasmopara viticola, Erisiphe necator and Botrytis cinerea, respectively. P. viticola and E. necator are obligatory biotrophs whereas B. cinerea is a necrotroph. In tolerant grapevine cultivars, plant-pathogen interaction induces defence responses, including metabolite and protein accumulation and hypersensitive reaction. Lipid and lipid-derived molecules may have a key role in the activation of defence mechanisms. Previous results suggest that V. vinifera cv Regent tolerance to P. viticola may be mediated in the first hours post inoculation by fatty acid (FA) associated signalling. In the present study we characterized FA modulation in V. vinifera cv Regent leaves upon inoculation with P. viticola, E. necator and B. cinerea and correlated FA modulation with the expression profiles of genes encoding the FA desaturases FAD6 and FAD8. In all the interactions, a progressive desaturation of stearic acid to α-linolenic acid, precursor of jasmonic acid, occurred, which was observed for a longer period against B. cinerea. Our results provide evidence of a distinct FA meditated signalling pattern in grapevine interaction with biotrophs and necrotrophs. While the interaction with the biotrophs may trigger a higher synthesis of polyunsaturated FA (PUFA) at early time-points with a tendency to return to basal levels, the interaction with B. cinerea may trigger a later and more durable induction of PUFA synthesis. In all interactions, membrane fluidity modulation occurred, which may be crucial to maintain cellular function during infection.

Grapevine is one of the most important fruit plants in the world, mainly due to its grapes and related products, with a highly economic and cultural importance. Every year, vineyards are affected by several pathogen outbreaks and the only way to control them is through preventive applications of agrochemicals every 12 to 15 days. This approach is not sustainable and not always effective. TheVitisgenus comprise different species that exhibit varying levels of resistance to pathogens, thus the understanding of the innate resistance/susceptibility mechanisms of these differentVitisspecies is crucial to cope with these threats. In this work, an untargeted metabolomics approach was followed, using Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR-MS), to analyse the metabolic chemical diversity of twoVitisspecies:Vitis rotundifolia(resistant to pathogens) andV. viniferacv. ‘Cabernet Sauvignon’ (susceptible to pathogens). Chemical formulas from bothVitiswere used to build Van Krevelen diagrams and compositional space plots, which do not require full metabolite identification and provide an easy comparison method. Based only on these visualization tools, it was shown that theV. rotundifoliametabolome is more complex than the metabolome ofV. viniferacv. ‘Cabernet Sauvignon’. Moreover, the regions that present a higher density are associated to lipids, polyketides and carbohydrates. Also,V. rotundifoliametabolome presented a higher ratio O/C compounds.

The domesticated species Vitis vinifera L. harbours many cultivars throughout the world that present distinctive phenology and grape quality. Not only have the grapes been used for human consumption, but the leaves are also used as a source of bioactive compounds and are present in the diets of several Mediterranean countries. We have selected seven different cultivars and performed elemental, fatty acid (FA) and pigment profiling. Total reflection X-ray fluorescence (TXRF) enabled the identification of 21 elements. Among them, Na, Ca and K were highly represented in all the cultivars and Zn was prevalent in V. vinifera cv. ‘Pinot noir’ and ‘Cabernet sauvignon’. Through gas chromatography, six FAs were identified, including omega-3 and omega-6 FA, the most abundant mainly in V. vinifera cv. ‘Tinta barroca’, ‘Pinot noir’ and ‘Cabernet sauvignon’. FA composition was used to determine nutritional quality parameters, namely atherogenic, thrombogenic, hypocholesterolemic/hypercholesterolemic and peroxidisability indexes as well as oxidability and oxidative susceptibility. Grapevine leaves were highlighted as a suitable source of health-promoting lipids. Given the popularity of “green” diets, we have also performed a leaf pigment analysis. Seventeen pigments including chlorophylls, trans-lutein, b-carotene and zeaxanthins were detected. ‘C19’ presented the highest content of most of the detected pigments.

Research insights arising from the sciences of Viticulture, Enology and Vitivinicultural Economy are crucial for responding to an everincreasing set of challenges - such as climate change, environmental issues and a global and more competitive market - faced by the players in the vitivinicultural sector. In this context, the scientific journals play a key role in spreading the knowledge acquired worldwide and contributing to the generation of new ideas and new research lines. The Ciência e Técnica Vitivinícola is one of the oldest and few scientific journals specifically devoted to these three branches of science. In 2020, twelve articles from several international research teams were published, covering a wide range of current topics, including: sustainability of viticulture and winemaking; modelling of climate impact on wine production; control of pests and diseases; management and adaptability of grapevine varieties; quality and differentiation of wine based on minority grapevine varieties, native yeasts from grapevine and winemaking technologies; wine authenticity and traceability; botrytized wines; human resource management in small wine companies.

The lack of graft success is a problem that causes economic loss to nursery growers and wine growers, especially because it is often expressed when the vineyard is already established. Grafting is a complex phenomenon, involving anatomical, physiological, biochemical and molecular interactions between the scion and the rootstock. For this reason, the lack of graft success is a complex problem to study. However, it is believed that through a joint analysis of the different mechanisms of graft success, one can advance more quickly in the knowledge of this phenomenon. Despite the scarce information available on the role of phenolic compounds in Vitis grafts, an analytical method was developed and validated for phenolic compounds quantification in vine graft tissues reviling some chemical markers for the cv. ‘Syrah’ with 110R and SO4 rootstocks. This work aims to determine, among phenolic acids and two flavanols, the chemical markers of graft compatibility between scions from two clones of cv. ‘Touriga Nacional’ (TN 21 and TN 112) and the rootstock 110R. For this purpose, extracts of grafting tissues were prepared at three different locations (above, below and at the grafting union), and were analyzed in three different stages of the grafting cycle. From all sections analyzed, the graft union tissue is the one to discriminate better the two combinations. Gallic acid, catechin and sinapic acid were found in higher amount at graft union in the less compatible combination later in graft development. Furthermore, the chlorogenic acid had a higher content in the more compatible combination at the early stage when compared to the less compatible graft.

Phenological models applied to grapevines are valuable tools to assist in the decision of cultural practices related to winegrowers and winemakers. The two-parameter sigmoid phenological model was used to estimate the three main phenological stages of the grapevine development, i.e., budburst, flowering, and veraison. This model was calibrated and validated with phenology data for 51 grapevine varieties distributed in four wine regions in Portugal (Lisboa, Douro, D&atilde, o, and Vinhos Verdes). Meteorological data for the selected sites were also used. Hence, 153 model calibrations (51 varieties &times, 3 phenological stages) and corresponding parameter estimations were carried out based on an unprecedented comprehensive and systematized dataset of phenology in Portugal. For each phenological stage, the centroid of the estimated parameters was subsequently used, and three generalized sigmoid models (GSM) were constructed (budburst: d = &minus, 0.6, e = 8.6, flowering: d = &minus, 0.6, e = 13.7, veraison: d = &minus, 0.5, e = 13.2). Centroid parameters show high performance for approximately 90% of the varieties and can thereby be used instead of variety-specific parameters. Overall, the RMSE (root-mean-squared-error) is &lt, 7 days, while the EF (efficiency coefficient) is &gt, 0.5. Additionally, according to other studies, the predictive capacity of the models for budburst remains lower than for flowering or veraison. Furthermore, the F-forcing parameter (thermal accumulation) was evaluated for the Lisboa wine region, where the sample size is larger, and for the varieties with model efficiency equal to or greater than 0.5. A ranking and categorization of the varieties in early, intermediate, and late varieties was subsequently undertaken on the basis of F values. These results can be used to more accurately monitor and predict grapevine phenology during a given season, thus supporting decision making in the Portuguese wine sector.

Vitis vinifera, one of the most cultivated fruit crops, is susceptible to several diseases particularly caused by fungus and oomycete pathogens. In contrast, other Vitis species (American, Asian) display different degrees of tolerance/resistance to these pathogens, being widely used in breeding programs to introgress resistance traits in elite V. vinifera cultivars. Secondary metabolites are important players in plant defence responses. Therefore, the characterization of the metabolic profiles associated with disease resistance and susceptibility traits in grapevine is a promising approach to identify trait-related biomarkers. In this work, the leaf metabolic composition of eleven Vitis genotypes was analysed using an untargeted metabolomics approach. A total of 190 putative metabolites were found to discriminate resistant/partial resistant from susceptible genotypes. The biological relevance of discriminative compounds was assessed by pathway analysis. Several compounds were selected as promising biomarkers and the expression of genes coding for enzymes associated with their metabolic pathways was analysed. Reference genes for these grapevine genotypes were established for normalisation of candidate gene expression. The leucoanthocyanidin reductase 2 gene (LAR2) presented a significant increase of expression in susceptible genotypes, in accordance with catechin accumulation in this analysis group. Up to our knowledge this is the first time that metabolic constitutive biomarkers are proposed, opening new insights into plant selection on breeding programs.

The analysis of complex biological systems keeps challenging researchers. The main goal of systems biology is to decipher interactions within cells, by integrating datasets from large scale analytical approaches including transcriptomics, proteomics and metabolomics andmore specialized ‘OMICS’ such as epigenomics and lipidomics. Studying different cellular compartments allows a broader understanding of cell dynamics. Plant apoplast, the cellular compartment external to the plasma membrane including the cell wall, is particularly demanding to analyze. Despite our knowledge on apoplast involvement on several processes from cell growth to stress responses, its dynamics is still poorly known due to the lack of efficient extraction processes adequate to each plant system.Analyzing woody plants such as grapevine raises even more challenges. Grapevine is among the most important fruit crops worldwide and awider characterization of its apoplast is essential for a deeper understanding of its physiology and cellular mechanisms. Here, we describe, for the first time, a vacuum-infiltrationcentrifugationmethod that allows a simultaneous extraction of grapevine apoplastic proteins and metabolites from leaves on a single sample, compatible with high-throughput mass spectrometry analyses. The extracted apoplast from two grapevine cultivars, Vitis vinifera cv ‘Trincadeira’ and ‘Regent’, was directly used for proteomics and metabolomics analysis. The proteome was analyzed by nanoLC-MS/MS and more than 700 common proteinswere identified, with highly diverse biological functions. The metabolome profile through FT-ICR-MS allowed the identification of 514 unique putative compounds revealing a broad spectrum of molecular classes info:eu-repo/semantics/publishedVersion

Grapevine variety discrimination is important for growers to be able to certify plants, however, this is not simple to do using conventional ampelometric methods or DNA based methods. The present work proposes, to the best of the authors' knowledge, for the first time, to differentiate grapevine varieties using only spectroscopic stem information. A total of 1200 measurements were gathered non-destructively in the field. The spectroscopic data was processed with a combination of AdaBoost and random weight neural networks (RWNN) which is still rare in scientific literature. Ten grapevine varieties, 5 red and 5 white, were used: Cabernet Sauvignon (CSvar), one of the most widely planted varieties worldwide, and nine Portuguese autochthonous varieties, including Touriga Franca and Touriga Nacional (TNvar) that are very important in Port wine production. Portugal is known to be one of the countries with the largest number of autochthonous varieties in the world. In general, the overall correct classification percentages were significantly better than the 10% level of random classification. True positive rates for a classifier separating ten varieties varied between 41.7 and 70.8%, depending on the variety. The false positive rate (FPR) varied between 2.5 and 7.3%. TNvar and CSvar measurements were correctly classified in 70.8 and 70% of cases with FPR of 5.3 and 3.3%, respectively. The algorithms AdaBoost of extreme learning machines, random vector functional link with pseudoinverse and no regularization, support vector machines and random forest did not provide better results than the AdaBoost of RWNN.

Phenological models for predicting the grapevine flowering were tested using phenological data of 15 grape varieties collected between 1990 and 2014 in Vinhos Verdes and Lisbon Portuguese wine regions. Three models were tested: Spring Warming (Growing Degree Days – GDD model), Spring Warming modified using a triangular function – GDD triangular and UniFORC model, which considers an exponential response curve to temperature. Model estimation was performed using data on two grape varieties (Loureiro and Fernão Pires), present in both regions. Three dates were tested for the beginning of heat unit accumulation (t0 date): budburst, 1 January and 1 September. The best overall date was budburst. Furthermore, for each model parameter, an intermediate range of values common for the studied regions was estimated and further optimized to obtain one model that could be used for a diverse range of grape varieties in both wine regions. External validation was performed using an independent data set from 13 grape varieties (seven red and six white), different from the two used in the estimation step. The results showed a high coefficient of determination (R2: 0.59–0.89), low Root Mean Square Error (RMSE: 3–7 days) and Mean Absolute Deviation (MAD: 2–6 days) between predicted and observed values. The UniFORC model overall performed slightly better than the two GDD models, presenting higher R2 (0.75) and lower RMSE (4.55) and MAD (3.60). The developed phenological models presented good accuracy when applied to several varieties in different regions and can be used as a predictor tool of flowering date in Portugal.

Vitis vinifera can be divided into two subspecies, V. vinifera subsp. vinifera, one of the most important agricultural crops in the world, and its wild ancestor, V. vinifera subsp. sylvestris. Three flower types can be observed: hermaphrodite and female (on some varieties) in vinifera, and male or female flowers in sylvestris. It is assumed that the different flower types in the wild ancestor arose through specific floral patterns of organ abortion. A considerable amount of data about the diversity of sexual systems in grapevines has been collected over the past century. Several grapevine breeding studies led to the hypothesis that dioecy in vinifera is derived from a hermaphrodite ancestor and could be controlled by either, one or two linked genetic determinants following Mendelian inherence. More recently, experiments using molecular approaches suggested that these loci were located in a specific region of the chromosome 2 of vinifera. Based on the works published so far, its seems evident that a putative sex locus is present in chromosome 2. However, it is still not fully elucidated whether flower types are regulated by two linked loci or by one locus with three alleles. Nevertheless, several genes could contribute to sex determination in grapevine. This review presents the results from early studies, combined with the recent molecular approaches, which may contribute to the design of new experiments towards a better understanding of the sex inheritance in grapevine., This work was supported by the funded project PTDC/AGR-GPL/119298/2010 from Fundacao para a Ciencia e Tecnologia (FCT, Portugal), by UID/AGR/04129/2013 centre grant from FCT, Portugal (to LEAF) and by PEst-OE/BIA/UI4046/2014; UID/MULTI/04046/2013 centre grant from FCT, Portugal (to BioISI) and are supported by FCT fellowships JL Coito, MJN Ramos, H Silva, M Rocheta, respectively, SFRH/BD/85824/2012, SFRH/BD/110274/2015, SFRH/BD/111529/2015, SFRH/BPD/64905/2009. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Background Grafting is an intensive commercial practice required to protect the European grapevine against the Phylloxera pest. Rootstocks resistant to this pest are hybrids of American vine species with different levels of compatibility with European Vitis vinifera varieties. Aiming to understand what drives grafting compatibility in grapevine, a transcriptomic approach was used to search for master regulators of graft success. Two scion/rootstock combinations, with different levels of compatibility, were compared in a nursery-grafting context at two stages, at 21 and 80 days after grafting. Results In the most compatible combination, an earlier and higher expression of genes signaling the metabolic and hormonal pathways as well as a reduced expression of genes of the phenolic metabolism and of the oxidative stress response was observed. At 80 days after grafting a higher expression of transcription factors regulating vascular maintenance, differentiation and proliferation was obtained in the most compatible combination. Moreover, lower expression levels of microRNAs potentially targeting important transcription factors related to plant development was observed in the more compatible combination when compared to the less compatible one. Conclusion In this context, a set of regulators was selected as potential expression markers for early prediction of a compatible grafting.

The assessment of wine authenticity is of utmost importance in the current context of a growing market globalization. The strontium isotopic ratio 87Sr/86Sr is a well-established tool for dating and tracing the origin of rocks and minerals with special interest for wine traceability. A study was developed to examine the variation of 87Sr/86Sr in wines from Douro Valley taking into account the effects of vineyard location and grape variety. The 87Sr/86Sr of soils and wines from six vineyards were determined by using an ICP-MS based analytical procedure. A total of twenty-two monovarietal wines, obtained at micro vinification scale, from relevant white and red grapevine varieties for Douro region, were analysed. The range of 87Sr/86Sr values observed in soils and wines was of 0.708–0.725 and 0.711–0.717, respectively. The present study updates the scarce knowledge available on strontium isotopic ratios in soils and wines from Douro Valley, and its results will enlarge global databank on wine composition and support comparison with other world regions.

The role of phenolic compounds in graft compatibility has been widely studied in various fruit species. However, little information is available for Vitis spp. This work aims at investigating the differences in phenolic acid and flavanol content in various scion/rootstock systems and understanding if they can be used as chemical markers of graft compatibility, using combinations of the Syrah cultivar (clones FR 470 and FR 383) with two rootstocks (110R and SO4). For this purpose, extracts of grafting tissues collected above graft union, at the graft union and below graft union from each combination at crucial stages of the grafting cycle (callusing, rooting and end of cycle) were prepared and analysed by HPLC. The results obtained revealed that the contents in gallic, ferulic and sinapic acids are closely related to the level of compatibility of the studied grafting combinations. The less compatible combinations (Syrah383/110R and Syrah383/SO4) exhibit higher content of gallic acid and lower contents of ferulic and sinapic acids than the more compatible ones (Syrah470/110R and Syrah470/SO4). In addition, these phenolic acids accumulated in the graft union and in the adjacent sections as a response to grafting. A significant difference in their levels was detected between the above and below graft union sections, especially at the rooting stage. Therefore, they can be seen as chemical markers to be used in an early detection of graft compatibility, preferably performed at the rooting stage and targeting the sections above and below the graft union.

Background and Aims Grapevine cultivation has a long tradition in Portugal. Presently, 343 cultivars are legally accepted for wine production. Two hundred and eighty eight accessions from the Portuguese National Ampelographic Collection (including 27 wild grapevines) were genotyped using single-nucleotide polymorphisms (SNP) and compared with the Instituto de Ciencias de la Vid y del Vino-SNP database to reveal identities, synonymies and homonymies. Methods and Results A set of 48 SNPs was used to profile the 288 accessions and 263 different genotypes were detected. A comparison with the Instituto de Ciencias de la Vid y del Vino-SNP database unveiled 14 new synonymies among Portuguese, Spanish and other international cultivars and confirmed known synonyms and homonyms. Most of the genotyped cultivars are not cultivated in Spain. Clustering using hierarchical and non-hierarchical methods did not reflect the Portuguese geographical viticulture regions. Conclusions New synonyms and homonyms were identified by SNP profiling of the Portuguese grapevine germplasm. Most of the genotyped cultivars are circumscribed to Portugal. Some cultivars are important in the Iberian context. Significance of the Study This study confirmed the uniqueness of the Portuguese grapevine genetic pool and disclosed many new synonyms within the cultivars authorised for wine production in Portugal. This study confirmed the suitability of the 48 SNP set for grapevine profiling. Results will help to manage the Portuguese germplasm and to adjust the Portuguese legal framework on the denomination of the cultivars authorised for wine production.

Budbreak date in grapevine is strictly dependent on temperature, and the correct simulation of its occurrence is of great interest since it may have major consequences on the final yield and quality. In this study, we evaluated the reliability for budbreak simulation of two modeling approaches, the chilling-forcing (CF), which describes the entire dormancy period (endo- and eco-dormancy) and the forcing approach (F), which only describes the eco-dormancy. For this, we selected six phenological models that apply CF and F in different ways, which were tested on budbreak simulation of eight grapevine varieties cultivated at different latitudes in Europe. Although none of the compared models showed a clear supremacy over the others, models based on CF showed a generally higher estimation accuracy than F where fixed starting dates were adopted. In the latter models, the accurate simulation of budbreak was dependent on the selection of the starting date for forcing accumulation that changes according to the latitude, whereas CF models were independent. Indeed, distinct thermal requirements were found for the grapevine varieties cultivated in Northern and Southern Europe. This implies the need to improve modeling of the dormancy period to avoid under- or over-estimations of budbreak date under different environmental conditions. This research was funded by the European Union’s Horizon 2020 Research and Innovation Programme, under the Clim4Vitis project: “Climate change impact mitigation for European viticulture: knowledge transfer for an integrated approach”, grant agreement no. 810176. It was also supported by FCT-Portuguese Foundation for Science and Technology, under the project UIDB/04033/2020 and the French National Research Agency (ANR) in the frame of the Investments for the Future Program, within the cluster of excellence COTE (ANR-10-LABX-45).

The diversity found among the Vitis vinifera L. species allows the production of wines with very different characteristics. The development of platforms suitable for food composition analysis is currently an emerging area. Among these, DNA biosensors have been developed for a wide variety of applications, ranging from food safety to authenticity. The main aim of this work was to study the detection capacity of the DNA-based optical biosensor using different V. vinifera matrices (leaf, must and wine). Genomic DNA was extracted from leaf, must and wine of three V. vinifera varieties and was tested on the long-period grating (LPG) DNA-based biosensor developed within our group. The biosensor was able to distinguish the varieties even using DNA extracted from complex matrices, revealing its potential to be applied in wine authenticity.

SUMMARYPhenological models are considered key tools for the short-term planning of viticultural activities and long-term impact assessment of climate change. In the present study, statistical phenological models were developed for budburst (BUD), flowering (FLO) and veraison (VER) of 16 grapevine varieties (autochthonous and international) from the Portuguese wine-making regions of Douro, Lisbon and Vinhos Verdes. For model calibration, monthly averages of daily minimum (Tmin), maximum (Tmax) and mean (Tmean) temperatures were selected as potential regressors by a stepwise methodology. Significant predictors included Tmin in January–February–March for BUD, Tmax in March–April for FLO, and Tmin, Tmax and Tmean in March–July for VER. Developed models showed a high degree of accuracy after validation, representing 0·71 of total variance for BUD, 0·83 for FLO and 0·78 for VER. Model errors were in most cases < 5 days, outperforming classic growing degree-day models, including models based on optimized temperature thresholds for each variety. Applied to the future scenarios RCP4·5/8·5, projections indicate earlier phenophase onset and shorter interphases for all varieties. These changes may bring significant challenges to the Portuguese wine-making sector, highlighting the need for suitable adaptation/mitigation strategies, to ensure its future sustainability.

Viticulture plays a key role in Portuguese economic growth. The possibility of obtaining early predictions of viticultural parameters, such as phenology, water status, yield, and potential wine characteristics, are of great value for growers and will influence the viticultural chain. The present study is a first approach to adapt the Simulateur mulTIdisciplinaire pour les Cultures Standard (STICS), a dynamic crop model, to Portuguese winegrowing conditions and grapevine varieties. Several site-specific parameters for climate, soil, and management practices were assessed. Varietal-specific parameters were measured from vineyards in the Douro and Lisboa winegrowing regions of Portugal. Three of the most important varieties in the country (Aragonez, Touriga-Franca, and Touriga-Nacional) were targeted for model calibration. Overall, the STICS crop model was successful in simulating yield, phenological stages, and water stress, but not alcohol content. The relatively high model accuracy will improve the performance of vineyard operations and winemaking practices, such as organizing harvests, vineyard intervention scheduling, irrigation procedures, and strategic planning of wineries. STICS may be feasible as a decision support tool for short- and long-term strategic planning in Portuguese viticulture, particularly taking into account the projected impacts of climate change. The efficiency gains obtained from the use of this crop model by wine industry stakeholders is expected to increase the competitiveness and sustainability of the wine sector in Portugal.

Wine authenticity methods are in increasing demand mainly in Denomination of Origin designations. The DNA-based methodologies are a reliable means of tracking food/wine varietal composition. The main aim of this work was the study of High Resolution Melting (HRM) application as a screening method for must and wine authenticity. Three sample types (leaf, must and wine) were used to validate the three developed HRM assays ( Vv1 –705 bp; Vv2 –375 bp; and Vv3 –119 bp). The Vv1 HRM assay was only successful when applied to leaf and must samples. The Vv2 HRM assay successfully amplified all sample types, allowing genotype discrimination based on melting temperature values. The smallest amplicon, Vv3 , produced a coincident melting curve shape in all sample types (leaf and wine) with corresponding genotypes. This study presents sensitive, rapid and efficient HRM assays applied for the first time to wine samples suitable for wine authenticity purposes.

Introduction Graft incompatibility of Vitis spp is an unresolved worldwide problem with important economic consequences. Grafting comprises a complex set of morphological and physiological alterations, in which the phenolic compounds seem to be strongly involved. Therefore, a detailed analysis and recognition of structural phenolic compounds diversity in the two partners of a Vitis graft is of great importance to evaluate their role as markers of graft establishment. Objective To optimise a sample extraction method, and to develop and validate a high-performance liquid chromatography (HPLC) method for the simultaneous determination of phenolic acids and flavonols in the graft union so as to understand their behaviour in the metabolism of the scion–rootstock system, using compatible and incompatible combinations of a Syrah cultivar and two rootstocks (R110 and SO4). Methods Sixty extracts of Vitis grafting tissues were prepared and analysed by HPLC for the qualitative and quantitative determination of their phenolic profile. Results Among the phenolic compounds identified in the samples, one benzoic acid (gallic acid), three cinnamic acids (caffeic acid, ferulic acid and sinapic acid) and two flavonols (catechin and epicatechin) are potentially suitable as markers of graft incompatibility. Conclusion The method developed presents good performance and lends itself readily for application in routine analysis of the phenolic composition of Vitis grafting tissues to distinguish compatible and incompatible combinations in the graft callusing stage. Copyright © 2014 John Wiley & Sons, Ltd.

Monitoring the main grapevine phenological stages is a key procedure for optimizing vineyard activities and improving yield and quality attributes. Remote sensing may be an effective and practical monitoring tool, as data from on-board satellite sensors can measure vegetative growth. In the current study, a 12-year time series of four main phenophases (budburst, flowering, veraison and harvest) were obtained from an experimental vineyard located in Lisbon (Portugal). LANDSAT surface reflectances were used to calculate the Enhanced Vegetation Index (EVI) and derived metrics. Both time series were then analysed. Results show statistically significant relationships between vegetation metrics and phenological timings and intervals, such as the linkage between the peak greenness and flowering/veraison. The current study highlights the applicability of remote sensing to monitor grapevine phenology in both retrospective and real-time, bringing an added-value to the winemaking sector.

Plant phenolics have been for many years a theme of major scientific and applied interest. Grape berry phenolics contribute to organoleptic properties, color and protection against environmental challenges. Climate change has already caused significant warming in most grape-growing areas of the world, and the climatic conditions determine, to a large degree, the grape varieties that can be cultivated as well as wine quality. In particular, heat, drought and light/UV intensity severely affect phenolic metabolism and, thus, grape composition and development. In the variety Chardonnay, water stress increases the content of flavonols and decreases the expression of genes involved in biosynthesis of stilbene precursors. Also, polyphenolic profile is greatly dependent on genotype and environmental interactions. This review deals with the diversity and biosynthesis of phenolic compounds in the grape berry, from a general overview to a more detailed level, where the influence of environmental challenges on key phenolic metabolism pathways is approached. The full understanding of how and when specific phenolic compounds accumulate in the berry, and how the varietal grape berry metabolism responds to the environment is of utmost importance to adjust agricultural practices and thus, modify wine profile., This work was supported by European Union Funds (FEDER/COMPETE-Operational Competitiveness Programme) and by national funds (FCT-Portuguese Foundation for Science and Technology) under the project FCOMP-01-0124-FEDER-022692 and the research project PTDC/AGR-ALI/100636/2008. This work was also supported by the European project INNOVINE (ref. 311775), and from the networking activities within the European funded COST ACTION FA1106 “QualityFruit” and COST Action FA1003 “East-West Collaboration for Grapevine Diversity Exploration and Mobilization of Adaptive Traits for Breeding”., info:eu-repo/semantics/publishedVersion

Portugal has a long tradition in viticulture and a great number of grapevine cultivars. To analyze the genetic relations among wild vines from Portuguese populations and old Portuguese grapevine cultivars we use morphological traits and chloroplastidial microsatellites from 53 accessions of four distinct populations of Vitis vinifera L. ssp. sylvestris (Gmelin) and 57 accessions of Vitis vinifera L. ssp. vinifera from the Portuguese National Ampelographic Collection. Principal coordinate analyses with the scores obtain from the descriptors of both the accessions of sylvestris and vinifera vines revealed two groups. One group is formed by the wild vine population of Alcacer do Sal and three vinifera accessions Rufete, Seara Nova and Trincadeira das Pratas and a second group includes all the other wild vines and grapevine cultivars. A total of four different chlorotypes (A, B, C and D) are present in the vinifera accessions and two in the sylvestris accessions (A, B). Chlorotype A is the most frequent in all the plants analyzed and correspond to 75.4% of the grapevine cultivars and 66% of the wild vines. The mixed distribution of chlorotypes in the Portuguese cultivars and the predominance of chlorotype A both in its wild populations and cultivars reinforced the hypothesis that West Europe was a domestication center for Vitis vinifera L. ssp. vinifera.

Wild populations of Vitis vinifera L.␣have been located in Portugal. Morphological characterization was carried out in three populations located in Alcacer do Sal, Castelo Branco, and Montemor-o-Novo, and then compared using multivariate discriminant analysis. These populations were from three different hydrological basins, therefore cross-pollination was not possible. It was verified that in each population all plants were different. The data suggest that the frequency of female and male plants is rather variable in wild populations. The morphology of the adult leaf, from the Alcacer do Sal population, had particular features when compared with Castelo Branco and Montemor-o-Novo populations, which were more homogeneous. The length of teeth compared with width at the end of the base, and the density of prostrate hairs between and on main veins (lower side) were the variables which allowed the best discrimination among populations.

The wine sector is a billion euro business and therefore subjected to multiple attempts of fraudulent practices. This requires the development of rapid and reliable methods to detect such situations. Several methodologies have been developed based on the chemical profiles of the wines, but they are limited due to the environmental conditions that cannot be controlled. The use of DNA-based detection systems are an emergent research field that have been extended to a wide variety of food prod- ucts and are still the most reliable methods for varietal identification. However these methods are not suitable for geographical determination. Soil related fingerprints have a primary role considering that there is a relationship between the elemental compo- sition of wine and the composition of the provenance soil. WineBioCode is a project aiming to define the best strategy for wine authenticity based on a multidisciplinary approach. Two DNA-based strategies have been developed based on Real-time PCR and a label free optical biosensor platform. Both platforms enabled successful identification of specific DNA-targets when applied to Vitis vinifera L., and can be applied throughout the grape-wine chain. The methods are complementary and can be used in dif- ferent situations, according to the requirements. The geographical evaluation has been assessed by the strontium 87 Sr/86 Sr isotope ratio determination involving soil evaluation in the vineyards followed by its assay in the wine samples. The results are being integrated in order to establish the best procedure to be undertaken for wine fingerprinting, including varietal composition and geographical origin, therefore fulfilling the requirements of the geographical denominations in wine certification.

Characteristics such as production yield, alcoholic level, acidity and anthocyanin levels vary among grape varieties. Since these characteristics are highly correlated with the final wine quality, it is important to be able to detect and correctly identify the grape varieties (cultivars) present in musts and wines. This is particularly relevant in controlling the quality and authenticity of monovarietal wines. In this study, molecular methods based on residual DNA analysis of Vitis vinifera L. cultivars were applied to musts and wines produced from five different cultivars, namely Touriga Franca, Fernao Pires, Tinta Barroca, Tinto Cao and Marselan. Initially, three DNA extraction methods were compared and optimised for the isolation of DNA from must and wine and then six nuclear and two chloroplast microsatellite markers were used to identify single-varietal and blends of two varieties of musts prepared in laboratory and single-varietal wines produced in microvinifications. Preliminary results on multivarietal musts indicated a possible relationship between the proportion of each variety in the mixture and the signal intensity of the alleles obtained in an automatic sequencer, suggesting that it could be possible to quantify the presence of each variety in the mixture. A method for DNA extraction from wine was developed and shown to be adequate for microsatellite amplification. Results have shown for the first time that markers targeting short DNA fragments at chloroplast genomes are useful tools to detect grapevine DNA in wines. This suggests that it may be possible to apply these techniques in controlling wine quality and origin certification, as well as in detecting wine falsification.

The characterization of the metabolites accumulated in the grapes of specific cultivars grown in different climates is of particular importance for viticulturists and enologists. In the present study, the metabolite profiling of grapes from the cultivars, Alvarinho, Arinto and Padeiro de Basto, of two Portuguese Controlled Denomination of Origin (DOC) regions (Vinho Verde and Lisboa) was investigated by gas chromatography-coupled time-of-flight mass spectrometry (GC-TOF-MS) and an amino acid analyzer. Primary metabolites, including sugars, organic acids and amino acids, and some secondary metabolites were identified. Tartaric and malic acids and free amino acids accumulated more in grapes from vines of the DOC region of Vinho Verde than DOC Lisboa, but a principal component analysis (PCA) plot showed that besides the DOC region, the grape cultivar also accounted for the variance in the relative abundance of metabolites. Grapes from the cultivar, Alvarinho, were particularly rich in malic acid and tartaric acids in both DOC regions, but sucrose accumulated more in the DOC region of Vinho Verde., This work was supported by European Union Funds (FEDER/COMPETE-Operational Competitiveness Programme) and by the Portuguese national funds (FCT—Portuguese Foundation for Science and Technology) under the project FCOMP-01-0124-FEDER-022692, the research project FCOMP-010124-FEDER-008760 (ref. FCT PTDC/AGR-ALI/100636/2008), and the PhD grants, no. SFRH/BD/ 64587/2009 to VM and SFRH/BD/74257/2010 to HN This work also benefited from the networking activities within the European project, INNOVINE (ref. 311775), and the European COST action (FA1106 “QualityFruit” and FA1003 “East-West Collaboration for Grapevine Diversity Exploration and Mobilization of Adaptive Traits for Breeding”)., info:eu-repo/semantics/publishedVersion

INTRODUCTION – Common limits for phenotyping are costs in terms of equipment, specialized researchers, and time. Moreover, phenotyping requires specific methods in relation to the studied traits, and each method is generally designed to highlight very specific characteristics of the singular trait. Thus, the obtained results are not easily comparable between different institutions. However the data sharing is clearly the most powerful approach to limit the costs and efforts optimizing the obtained results in term of number of studied accessions. It should be stressed that funds are another great limit in research, especially in East countries, where the most interesting cultivar variability is preserved. 28 AIMS AND SCOPES – The main purpose of this work is the establishment of a phenotyping network able to produce comparable data among the Pan-European grapevine collections. MATERIALS AND METHODS – Two protocols were proposed: one for phenology and, the other, for enocarpological traits. Data were recorded in two consecutive vintages: 2012 and 2013. Concerning phenology, the extended BBCH scale (universal and flexible) was adapted to our objects by moving it into a chrono- phenological scale. Then, a cubic curve was selected for modelling the phenological courses. Thus, computing for each accession specific coefficients, it was possible to obtain the relative phenograms. Considering the different site climatic conditions, it was possible to spatialize the results, obtaining reference phenological maps. Concerning eno- carpological traits, the attention was focused on characteristics with a clear impact on the production quality, such as berry morphology (weight, diameters, number of seeds …) and composition (sugar, titratable acidity, anthocyanin and phenolic contents …). The protocol was organized to concatenate the data acquisition, with the objective to generate new derived variables. The methods were selected to be easily reproduced in all the involved collections, without the necessity of expensive facilities. RESULTS AND DISCUSSIONS – The first year of dataset was used to evaluate the methods. Two publications are already available confirming the protocols quality. Concerning phenology, both the variability between accessions grown into the same collection and the phenological shifts between the reference varieties cultivated in different areas were highlighted by the protocol. Matching climatic and phenological information of reference cultivars, phenophases maps were obtained. The performances obtained by the eno-carpological protocol were satisfactory both in a global point of view and in a singular collection evaluation. The project obtained an impressive participation, considering the volunteer contribution without any material funding to cover the work costs. Twenty-four institutions collaborate to the data collection. 1724 accessions were evaluated: 931 (583 in 2012 and 348 in 2013) for phenology and 793 (424 in 2012 and 369 in 2013) for eno-carpological descriptions. CONCLUSIONS AND POSSIBLE APPLICATIONS – The protocol sharing allows the data comparison among different grapevine collections. The possible future application for this dataset could be related to scientific studies of genetic associations. Moreover, the improved knowledge of minor and neglected cultivars could have a direct impact on the industrial production, giving information concerning new varieties for future introduction in viticultural areas.

Graft incompatibility of Vitis spp is an unresolved worldwide problem with important economic consequences. Grafting comprises a complex set of morphological and physiological alterations, in which the phenolic compounds seem to be strongly involved. Therefore, a detailed analysis and recognition of structural phenolic compounds diversity in the two partners of a Vitis graft is of great importance to evaluate their role as markers of graft establishment.To optimise a sample extraction method, and to develop and validate a high-performance liquid chromatography (HPLC) method for the simultaneous determination of phenolic acids and flavonols in the graft union so as to understand their behaviour in the metabolism of the scion-rootstock system, using compatible and incompatible combinations of a Syrah cultivar and two rootstocks (R110 and SO4).Sixty extracts of Vitis grafting tissues were prepared and analysed by HPLC for the qualitative and quantitative determination of their phenolic profile.Among the phenolic compounds identified in the samples, one benzoic acid (gallic acid), three cinnamic acids (caffeic acid, ferulic acid and sinapic acid) and two flavonols (catechin and epicatechin) are potentially suitable as markers of graft incompatibility.The method developed presents good performance and lends itself readily for application in routine analysis of the phenolic composition of Vitis grafting tissues to distinguish compatible and incompatible combinations in the graft callusing stage.

Testing for Grapevine leafroll-associated virus 1 (GLRaV-1) is mandatory in certification schemes of propagation material within the EU. Accurate and reliable diagnostic assays are necessary for implementation of this measure. During routine detection of GLRaV-1, using double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and reverse transcription (RT) followed by polymerase chain reaction (PCR), evidence was obtained that positive samples could be overlooked by either or both detection methods. With the aim of improving serological detection tools for GLRaV-1, a total of 20 isolates were analyzed and 83 new complete capsid protein (CP) gene sequences were obtained. This set, together with the CP sequences available at GenBank was used for a comprehensive in silico analysis. To obtain a specific antibody able to recognize all known CP variants, conserved regions with suitable antigenicity profile were identified along the deduced CP AA sequences and a 14 AA sequence was chosen for commercial peptide synthesis and immunization. Initially polyclonal antibodies were produced and tested, followed by purification of the respective monospecific antibody and conjugation with alkaline phosphatase or fluorescein isothiocyanate (FITC). These serological tools were tested successfully on all the available positive samples and proved adequate for in situ immunoassay (ISIA). Further testing showed that the monospecific antibody could also be used in tissue print immunoblotting (TPIB), a technique that allows rapid processing of large sample sets, which is highly suitable to implement protocols ensuring that, for each vine analyzed, enough random samples are taken and processed, before certification.

Grapevine (Vitis vinifera L.) is the most widely cultivated and economically important fruit crop in the world. In the different Portuguese agro-ecosystems, grapevine plays an impor‐ tant role either as a border culture or as an extensive crop. The surface area used by vine‐ yards amounts to 4.9 % of the arable land [1], representing 240,000 ha, being the 7th largest area in the world and the 4th in the European Union [2]. In 2011 Portugal produced 5.9 mil‐ lion hectoliters of which 2.9 million hectoliters were exported, making the country the 12th world wine producer [2]. There are fourteen wine regions with Protected Geographical Indi‐ cation (Figure 1) and 31 wine areas with Designation of Origin status including Porto, estab‐ lished since 1756, the oldest legally established wine production region in the world. Each one of the wine regions has a particular set of grapevine cultivars adapted to its specific ter‐ roirs. Officially there are 343 cultivars allowed to be use in wine production in Portugal [3].

A comparison of 15 field isolates of grapevine leafroll-associated virus 5 (GLRaV-5) was conducted, based on the analysis of nucleotide sequences of two viral ORFs: the coat protein (CP) and the heat shock protein 90 homolog (HSP90h). After amplification and cloning, the population of viral sequences was analyzed for each isolate, revealing the within-isolate presence of sequence variants for both genes, with one or more major CP variants. Phylogenetic analysis showed the gene sequence variants detected to be exclusive for each isolate. These data, together with estimates of genetic diversity and positive selection, did not reveal evidence of vector-mediated transfer of GLRaV-5. Conversely, a strong effect of host vegetative propagation on divergence dynamics of GLRaV-5 variants was suggested by the isolates from this work The phylogeny of the CP gene further revealed clustering of GLRaV-5 isolates into eight lineages, four of which were detected in our study, revealing a higher diversity than previously described. The information gathered also contributes to firmly establishing GLRaV-5 as a cohesive taxonomic group within the ampeloviruses.

Wine quality and market value greatly depend on the grapevine varietal composition, which may be characteristic of specific regions. In order to defend the distinct regions, Denominations of Origin were defined to protect against fraudulent practices. In this study, we evaluated the efficiency of two microsatellite-based systems (microsatellite (SSR) and intermicrosatellite (ISSR)) for must varietal composition determination and their potential role in certification purposes. Eleven Vitis vinifera L. varieties from leaf and monovarietal must DNA samples were screened with six SSR and 14 ISSR primers to discriminate polymorphisms. Principal coordinates analysis was performed with DCENTER on the resultant data using unweighted pair group mathematical average and revealed that ISSRs markers were not suitable for certification procedures, whereas nuclear SSR markers presented a complete correspondence between leaf and must samples, demonstrating that they were adequate for traceability purposes.

The genetic variability and population structure of grapevine leafroll-associated virus 3 (GLRaV-3) variants were updated by examining the diversity within the viral coat protein (CP) gene among 174 isolates belonging to a collection of Vitis vinifera representing most of the Portuguese varieties. Phylogenetic analysis revealed the existence of five well-defined clusters. Three of these correspond to previously defined groups, another corresponds to variants from Chile for which only one sequence has been previously identified, and an additional new group includes only Portuguese variants. A typing tool based on asymmetric PCR-ELISA (APET) was developed within the frame of this population structure. This tool was used to assess the prevalence of each phylogenetic group among the infected grapevine varieties. Although most of the isolates harbour variants from groups 1 and 2, variants from the remaining three groups exist in a number of varieties, reinforcing the notion that they are genuine genomic variants and are not isolated, atypical cases.

As the ancestor of cultivated grape, Vitis vinifera subsp. sylvestris represents a unique, invaluable genetic resource for the improvement of cultivated grapevines. Recently, five populations of wild grapevines were identified in Portugal. Sixty vines were characterized with 11 nuclear SSR markers and further compared with 70 genotypes of Portuguese Vitis vinifera subsp. sativa. The obtained data demonstrate moderate genetic differentiation between wild grapevine populations and moderate to high genetic differentiation between wild and cultivated grapevines. However, the identification of high degrees of similarity between wild and cultivated grapes (up to 87%) and a putative parent–progeny relationship between wild and cultivated grapes with 17 additional SSR markers is indicative of gene flow between local wild grapevine populations and Portuguese domesticated vines. Also, the ancestry of some Azorean cultivars was ascertained. The obtained data further support the hypothesis of several domestication centres, with Portugal, Spain, and Italy playing a particular role after the last glaciation, giving rise to many of the Western European cultivars.

To assess the different origins of Portuguese grapevine varieties, we used six nuclear and four chloro- plastidal microsatellites as molecular markers, in order to compare the genetic structure of native wild-vines with native grapevine varieties. Both native subspecies have a great diversity, and a high interrelationship across the six nuclear microsatellites. Although identical numbers of alleles were found in each population, their distribu - tion was different in the vinifera and sylvestris subspecies. Portuguese wild-vines have only c hlorotypes A and B; A being the most frequent. The fifty-seven analysed Portuguese varieties have chlorotypes A, B, C, and D. The most frequent was the chlorotype A (75%), followed by D (21%). The results obtained reinforced the idea of Western Europe as having been one of the domestication centres for the grapevine, with contributions from the Eastern European gene pool. The observed genetic structure is a starting point from which to clarify the high number of native cultivars found in Portugal, and reinforces their probable origin in the Iberian Peninsula.

Aim: To investigate the characteristics, relationships and trends in the phenology of four winegrape varieties and associated temperature relationships in the Lisbon wine region (LWR), between 1990 and 2011.Methods and results: Budburst, flowering and véraison dates of red (Castelão and Aragonez, syn. Tempranillo) and white (Chasselas and Fernão Pires) varieties were taken from an experimental vineyard in the LWR. Harvest dates were determined based on a similar maturity level for all varieties. From these data, varietal characteristics, temporal trends as well as relationships between phenology and temperature were assessed through stepwise multivariate linear regressions. Flowering was the most sensitive to temperature in the preceding months (March-April). Differences/similarities between the phenological timing of the different varieties are presented. With few exceptions, no trends were found in phenophases over the 1990-2011 period, whereas several significant negative slopes were displayed for phenological intervals, suggesting a role for accumulated thermal effects in phenological timing. Strong correlations were observed between phenophases, especially between flowering and véraison.Conclusion: The study highlights the key role played by temperature on phenology, particularly during springtime. Furthermore, an increase in temperature during that period will cause an advance in the timing of the following phenological events. Given the significant trends found, phenological shifts may occur in the long term, emphasizing the need to assess varietal characteristics and responses to regional climate.Significance and impact of the study: The present work is the first attempt to systematically examine temporal trends in phenology and corresponding relationships with temperature in a Portuguese viticultural area, providing valuable information on the development and suitability of grapevine varieties, which determine viticultural practices and winegrower’s income.

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.