Grape colour phenotyping: development of a method based on the reflectance spectrum.

INTRODUCTION: The colour of fruit is an important quality factor for cultivar classification and phenotyping techniques. Besides the subjective visual evaluation, new instruments and techniques can be used. OBJECTIVES: This work aims at developping an objective, fast, easy and non-destructive method as a useful support for evaluating grapes' colour under different cultural and environmental conditions, as well as for breeding process and germplasm evaluation, supporting the plant characterization and the biodiversity preservation. MATERIALS AND METHODS: Colours of 120 grape varieties were studied using reflectance spectra. The classification was realized using cluster and discriminant analysis. Reflectance of the whole berries surface was also compared with absorption properties of single skin extracts. RESULTS: A phenotyping method based on the reflectance spectra was developed, producing reliable colour classifications. A cultivar-independent index for pigment content evaluation has also been obtained. CONCLUSIONS: This work allowed the classification of the berry colour using an objective method.

Phenotyping Key Fruit Quality Traits in Olive Using RGB Images and Back Propagation Neural Networks.

To predict oil and phenol concentrations in olive fruit, the combination of back propagation neural networks (BPNNs) and contact-less plant phenotyping techniques was employed to retrieve RGB image-based digital proxies of oil and phenol concentrations. Fruits of cultivars (×3) differing in ripening time were sampled (~10-day interval, ×2 years), pictured and analyzed for phenol and oil concentrations. Prior to this, fruit samples were pictured and images were segmented to extract the red (R), green (G), and blue (B) mean pixel values that were rearranged in 35 RGB-based colorimetric indexes. Three BPNNs were designed using as input variables (a) the original 35 RGB indexes, (b) the scores of principal components after a principal component analysis (PCA) pre-processing of those indexes, and (c) a reduced number (28) of the RGB indexes achieved after a sparse PCA. The results show that the predictions reached the highest mean R2 values ranging from 0.87 to 0.95 (oil) and from 0.81 to 0.90 (phenols) across the BPNNs. In addition to the R2, other performance metrics were calculated (root mean squared error and mean absolute error) and combined into a general performance indicator (GPI). The resulting rank of the GPI suggests that a BPNN with a specific topology might be designed for cultivars grouped according to their ripening period. The present study documented that an RGB-based image phenotyping can effectively predict key quality traits in olive fruit supporting the developing olive sector within a digital agriculture domain.

Microclimate of Grape Bunch and Sunburn of White Grape Berries: Effect on Wine Quality.

This research aimed to evaluate the composition of wines made with white grapes which are particularly susceptible to sunburn symptoms due to the absence of anthocyanin. Sunburn is a complex physiological dysfunction leading to browning or necrosis of berry tissues. In vintage 2021, the canopy of 'Verdeca' grapevines grown in Salento, South Italy, was differently managed by sun exposing or shading the bunches. Micrometeorological conditions were studied at different levels. Grapes were vinified, comparing the winemaking with and without skin maceration. The vegetative-productive balance of plants was not substantially modified. On the contrary, a significant effect was observed on the quality and quantity of grapes produced: smaller berries with sunburn symptoms were found on unshaded bunches. This influenced the percentage distribution among skin, pulp and seeds, causing a decrease in must yield of up to 30%. The pH was significantly higher in macerated wines made using shaded grapes, due to a lower titratable acidity and to significant impacts on the acid profile. Obviously, maceration produced a higher extraction of phenolics in wines, which reached their maximum in wines made with sunburned grapes. The absorbance at 420 nm, index of yellow color, was also significantly higher in sunburned grapes, indicating greater oxidation. Even though excessive grape sun-exposure could negatively affect the perception of white wines made without maceration (resulting in more oxidative character), the sensory quality of orange/amber wines was not significantly impacted by the presence of sunburned grapes. Thus, this winemaking technique could be particularly interesting to set up a production strategy adapted to viticultural regions strongly affected by climate change.

Metabolomic NMR analysis and organoleptic perceptions of pomegranate wines: Influence of cultivar and yeast on the product characteristics.

Pomegranate (Punica granatum L.) fruits are a historical agricultural product of the Mediterranean basin that became increasingly popular in the latest years for being rich in antioxidants and other micronutrients, and are extensively commercialized as fruits, juice, jams and, in some Eastern countries, as a fermented alcoholic beverage. In this work, four different pomegranate wines specifically designed using combinations of two cultivars (Jolly Red and Smith) and two yeast starters with markedly different characteristics (Saccharomyces cerevisiae Clos and Saccharomyces cerevisiae ex-bayanus EC1118) were analyzed. The chemical characterization of the wines together with the originating unfermented juices was performed by 1H NMR spectroscopy metabolomic analysis. The full spectra were used for unsupervised and supervised statistical multivariate analysis (MVA), namely Principal Component Analysis (PCA), Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA), and sparse PCA (SPCA). The MVA of the wines showed a clear discrimination between the cultivars, and a smaller, yet significant, discrimination between the yeasts used. In particular, a higher content of citrate and gallate was observed for the Smith cv. and, on the contrary, a statistically significant higher content of fructose, malate, glycerol, 2,3 butanediol, trigonelline, aromatic amino acids and 4-hydrophenylacetate was observed in Jolly Red pomegranate wines samples. Significant interaction among the pomegranate cultivar and the fermenting yeast was also observed. Sensorial analysis was performed by a panel of testing experts. MVA of tasting data showed that the cultivar significantly affected the organoleptic parameters considered, while the yeast had a minor impact. Correlation analysis between NMR-detected metabolites and organoleptic descriptors identified several potential sensorially-active molecules as those significantly impacting the characteristics of the pomegranate wines.

Influence of freezing and heating conditions on grape seed flavan-3-ol extractability, oxidation, and galloylation pattern.

In cool-climate viticulture, the short growing season can influence grape seed maturation by reducing the apparent oxidation of flavan-3-ol monomers and associated increase in seed browning. A reduction in seed maturation increases the potential extraction of flavan-3-ol monomers into wine during maceration operations, heightening bitterness. Here, we carried out a 2 × 2 factorial experiment to test the ability of freezing and heating treatments to advance maturation (decrease flavan-3-ol, improve browning) of (Vitis vinifera L.) Pinot noir and Cabernet Sauvignon seeds over a 24-h incubation period. Only freezing significantly increased seed browning in both cultivars. Subsequent correlations with seed flavan-3-ol monomer concentrations suggest that freezing enhanced the oxidation of these compounds. Interestingly, natural ripening and freezing reduced galloylated flavan-3-ol monomers to a greater extent than non-galloylated ones. This study provides new information regarding the susceptibility of flavan-3-ol monomers to freezing and heating, and also suggests that freezing can advance the maturation the seeds of under-ripe red vinifera grapes.

Climate Change Impacts on Plant Phenology: Grapevine (Vitis vinifera) Bud Break in Wintertime in Southern Italy.

The effects of global warming on plants are not limited to the exacerbation of summer stresses; they could also induce dormancy dysfunctions. In January 2020, a bud break was observed in an old poly-varietal vineyard. Meteorological data elaboration of the 1951-2020 period confirmed the general climatic warming of the area and highlighted the particular high temperatures of the last winter. Phenological records appeared to be significantly correlated to wood hydration and starch reserve consumption, demonstrating a systemic response of the plant to the warm conditions. The eight cultivars, identified by single-nucleotide polymorphism (SNP) profiles and ampelographic description, grown in this vineyard showed different behaviors. Among them, the neglected Sprino, Baresana, Bianco Palmento, and Uva Gerusalemme, as well as the interspecific hybrid Seyve Villard 12.375, appeared to be the most interesting. Among the adaptation strategies to climate changes, the cultivar selection should be considered a priority, as it reduces the inputs required for the plant management over the entire life cycle of the vineyard. Hot Mediterranean areas, such as Salento, are a battlefront against the climate change impacts, and, thus, they represent a precious source of biodiversity for viticulture.

Dynamics of the Fermentation Process and Chemical Profiling of Pomegranate (Punica granatum L.) Wines Obtained by Different Cultivar×Yeast Combinations.

Pomegranate (Punica granatum L.) is one of the historical tree crops in the Mediterranean region and is nowadays commercialized for its beneficial properties in the form of fruits, juice, jams and, in some East countries, as fermented juice (pomegranate wine). However, pomegranate wines are not established as a common beverage in Western countries. In this work, we produced pomegranate wines using two cultivars and two yeasts (Saccharomyces cerevisiae strain Clos and S. cerevisiae ex-bayanus strain EC1118) with contrasting characteristics. A comprehensive chemical profile of the wines was obtained. Notable differences were observed in the function of the cultivars and the yeasts. Different cultivar×yeast combinations provided wines with clearly different chemical profiles and specific features in the patterns of organic acids, phenolics, and volatile compounds. This highlights the opportunity to obtain tailored pomegranate wines with desired chemical profiles and, consequently, sensory properties, through management optimization of pomegranate winemaking. In this view, pomegranate wines have the potential to become an established beverage in Western countries.

Georgian Grapevine Cultivars: Ancient Biodiversity for Future Viticulture.

Grapevine (Vitis vinifera) is one of the most widely cultivated plant species of agricultural interest, and is extensively appreciated for its fruits and the wines made from its fruits. Considering the high socio-economic impact of the wine sector all over the world, in recent years, there has been an increase in work aiming to investigate the biodiversity of grapevine germplasm available for breeding programs. Various studies have shed light on the genetic diversity characterizing the germplasm from the cradle of V. vinifera domestication in Georgia (South Caucasus). Georgian germplasm is placed in a distinct cluster from the European one and possesses a rich diversity for many different traits, including eno-carpological and phenological traits; resistance to pathogens, such as oomycetes and phytoplasmas; resistance to abiotic stresses, such as sunburn. The aim of this review is to assess the potential of Georgian cultivars as a source of useful traits for breeding programs. The unique genetic and phenotypic aspects of Georgian germplasm were unraveled, to better understand the diversity and quality of the genetic resources available to viticulturists, as valuable resources for the coming climate change scenario.

Increase in seed tannin extractability and oxidation using a freeze-thaw treatment in cool-climate grown red (Vitis vinifera L.) cultivars.

Grape seed maturation involves the gradual oxidation of tannins, decreasing excessive bitterness and astringency in wine. In cool climates, this process is limited by the short growing season, affecting wine quality. A "freeze-thaw" treatment on seeds of red vinifera cultivars at veraison and harvest was used to evaluate the effect of oxidation and extractability on seed phenolic fractions. Freezing increased the extraction of total phenolics and o-diphenols quantified from fractionation (fraction 1, vacuolar tannins; fraction 2, hydrogen bonded tannins; fraction 3, covalently bonded tannins), especially at harvest. Despite this, colorimetry, microscopy, oxidation reactivity index (ORI), and correlations between the color index and fractions indicated that freezing disrupted vacuole integrity, enhancing oxidation in the seed coat. In conclusion, vacuolar tannins (which are the main seed phenolics extracted during fermentation) were highly correlated with seed color change, potentially providing information for winemaking in cool climate regions.

Oxidations in white grape (Vitis vinifera L.) skins: Comparison between ripening process and photooxidative sunburn symptoms.

Oxidations in grape berries are gaining major interest as they affect grape characteristics and quality. Considering berries, Reactive Oxygen Species are involved in the responses to both ripening process and stresses, including photooxidative sunburn. Redox metabolism involves a multitude of chemical and enzymatic reactions. In this study, four white grape cultivars were examined for natural ripening and photooxidative sunburn effects (obtained in artificial conditions) on berry pigmentation, chemical composition and enzymatic activity. The measured parameters included reflectance spectra, pigmentation (including berry browning), content of photosynthetic pigments, organic acid profiles, antioxidant activity, concentrations of antioxidants (total phenolics, ascorbic acid and reduced glutathione), enzymatic activities (guaiacol peroxidases, ascorbate peroxidase and catalase). The effects of the treatment (natural ripening and artificial photooxidative sunburn) on each considered parameter are described in the paper. Photooxidative sunburn strongly affected the contents of antioxidants and chlorophylls, increased the browning index and modulated the enzymatic activities investigated. Samples clearly clustered depending on the oxidation status. Furthermore, the PCA highlighted the similarities and differences in the responses to oxidative stress during ripening and photooxidative sunburn. PCA produced five functions with eigenvalues higher than 1, representing 87.03% of the total variability. In particular, the scores of the function 1 discriminated the samples based on the oxidation status, while the function 2 separated the samples based on the sampling date, representing the physiological responses characteristic of ripening. Our work sheds light on this topic, and will allow a more conscious vineyard management, thus supporting the agricultural adaptation to climate changes.

Utilization of a freeze-thaw treatment to enhance phenolic ripening and tannin oxidation of grape seeds in red (Vitis vinifera L.) cultivars.

Phenolic ripening represents a major interest for quality wine producers. Nevertheless, climatic or genotypical limitations can often prevent optimal maturation process. During winemaking seeds can be easily separated and technologically processed to improve their quality. Relying on the key role of oxidation for phenolic ripening, a freeze-thaw treatment was proposed to improve the fruit quality for potential use in challenging growing conditions. The experiment was carried on in two distinctive viticultural areas, Michigan and Italy. Five cultivars (Cabernet Franc, Cabernet Sauvignon, Merlot, Pinot noir and Chambourcin) and six cultivars (Cabernet Sauvignon, Sangiovese, Syrah, Croatina, Barbera and Nebbiolo) were used in Michigan and Italy, respectively. Samples were collected at different phenological stages, to describe the natural ripening process and grape seeds were characterized before and after a freeze-thaw treatment. Colorimetric and spectrophotometric data highlighted similarities among natural and artificial seed ripening promising future applications for the wine industries.

Multi-parameter characterization of water stress tolerance in Vitis hybrids for new rootstock selection.

Drought in grapevine could be faced using tolerant rootstocks. The present work aims at the evaluation of 25 new genotypes potentially tolerant to drought by using recent methods of phenotypical screening (thermography and on-solid reaction spectroscopy). Plants were grown in well-watered and stressed field conditions. Proxi for transpiration, wood hydrophobicity and starch content were used to characterize and classify the genotypes. The predominant role of the environment was highlighted, nevertheless genotype and genotype × environment interaction showed significant variations as well. Hybrids were classified based on their steady, susceptible or adaptable behavior. The 14 most promising genotypes were identified, 5 of them showing two tolerance mechanisms. In the future, results from this experiment will support viticulture in water limited areas releasing new drought-tolerant interspecific hybrids to be tested after grafting with different scions.

Oxidized Polymeric Phenolics: Could They Be Considered Photoprotectors?

Photooxidative sunburn is the consequence of photosystem overexcitations. It results in tissue color changes as a result of chlorophyll degradation and accumulation of oxidized polymeric phenolics (OPPs), resulting from scavenging of reactive oxygen species (ROS). From a productive point of view, OPPs should be considered as damages, decreasing the economical and esthetical values of plants and crops. However, from a physiological perspective, OPPs could be also play a screening role against excessive photosynthetically active radiation (PAR), because they follow the criteria proposed for the identification of photoprotectors, as follows: (i) As a result of the complex conjugated double bond systems, OPPs absorb and, thus, screen the visible PAR. (ii) The accumulation of brown OPPs is well-known to be stimulated by light exposure, resulting in sunburn symptoms. (iii) OPPs induce PAR resistance; for example, the sunburned brown skin allows the fruit ripening to proceed without further interferences. (iv) The screen provided by the accumulated OPPs in death cells protect underlying tissues, demonstrating an increased resistance to radiation when other physiological processes are not functioning.

Iron deficiency stimulates anthocyanin accumulation in grapevine apical leaves.

Iron chlorosis is a diffuse disorder affecting Mediterranean vineyards. Beside the commonly described symptom of chlorophyll decrease, an apex reddening was recently observed. Secondary metabolites, such as anthocyanins, are often synthetized to cope with stresses in plants. The present work aimed to evaluate grapevine responses to iron deficiency, in terms of anthocyanin metabolism (reflectance spectrum, total anthocyanin content, HPLC profile and gene expression) in apical leaves of Cabernet sauvignon and Sangiovese grown in hydroponic conditions. Iron supply interruption produced after one month an increasing of anthocyanin content associated to a more stable profile in both cultivars. In Cabernet sauvignon, the higher red pigment accumulation was associated to a lower intensity of chlorotic symptoms, while in Sangiovese, despite the activation of the metabolism, the lower anthocyanin accumulation was associated to a stronger decrease in chlorophyll concentration. Gene expression data showed a significant increase of anthocyanin biosynthesis. The effects on the expression of structural and transcription factor genes of phenylpropanoid pathway were cultivar dependent. F3H, F3'H, F3'5'H and LDOX genes, in Cabernet sauvignon, and AOMT1 and AOMT genes, in Sangiovese, were positively affected by the treatment in response to iron deficiency. All data support the hypothesis of an anthocyanin biosynthesis stimulation rather than a decreased degradation of them due to iron chlorosis.

Characterization of iron deficiency symptoms in grapevine (Vitis spp.) leaves by reflectance spectroscopy.

The work aims at the description of the iron deficiency symptoms in grapevine leaves by reflectance spectroscopy at the plant and leaf levels. 5 genotypes of Vitis spp. were selected and grown in hydroponic conditions with and without iron supply. 450 spectra were collected among basal, young and apical leaves, as well as veins and interveinal areas. Iron deficiency produced significant and characteristic modifications in the pigment accumulation, proportion and distribution in plants. Basal leaves resulted to have higher concentrations of photosynthetic pigments in stressed plants with respect to the control, probably due to compensation effects. Iron deficient plants had lower chlorophyll concentrations in young and apical leaves. In the apical zone, also the relative composition of pigments appeared to be modified, explaining the reddish-yellowish apex appearance of iron deficient vines. Finally, the pigment distribution along the shoot characterized the symptoms, as well as the spectral variations among veins and interveinal areas. These results could support future applications in vineyard management (e.g.: symptom identification and detection; precision fertilization) as well as breeding programs for new rootstock selections (e.g.: fast screenings of seedlings).

Pink berry grape (Vitis vinifera L.) characterization: Reflectance spectroscopy, HPLC and molecular markers.

Color has a fundamental role for the qualitative evaluation and cultivar characterization of fruits. In grape, a normally functional pigment biosynthesis leads to the accumulation of a high quantity of anthocyanins. In this work, 28 Vitis vinifera L. cultivars accumulating low anthocyanins in berries were studied to characterize the biosynthetic dysfunctions in both a phenotypic and genotypic point of view. Reflectance spectroscopy, HPLC profiles and molecular markers related to VvMybA1 and VvMybA2 genes allowed a detailed description of the pigment-related characteristics of these cultivars. Data were consistent concerning the heterozygosity of the non-functional allele in both investigated genes, resulting in a low colored phenotype as described by reflectance. However, the variability in berry colour among our samples was not fully explained by MybA locus, probably due to specific interferences among the biosynthetic pathways, as suggested by the anthocyanin profile variations detected among our samples. The results presented in this work confirmed the importance of the genetic background: grapes accumulating high levels of cyanidin-3-O-glucosides (di-substituted anthocyanin) are generally originated by white cultivar retro-mutations and they seem to preserve the anomalies in the flavonoid hydroxylases enzymes which negatively affect the synthesis of tri-substituted anthocyanins.

Stem Xylem Characterization for Vitis Drought Tolerance.

Together with stomatal conductance and root conductivity, the stem water reserve and transport systems could be regulatory mechanisms able to participate in the regulation of the plant water status. Lianas, such as Vitis spp., minimize the trunk support role, and stems have evolved to improve their ability in water transport. In this work, stems of 10 different Vitis species were studied in relation to their expected drought tolerance using reflectance spectroscopy. Spectra were measured before (T0) and after coloration with Sudan IV dye. The T0 spectral signature showed characteristic species features. The partial least squares (PLS) regression and the self-organizing map (SOM) neural network analysis were able to predict the expected drought tolerance score; thus, reflectance spectroscopy was demonstrated to be a useful technique for drought tolerance phenotyping. These methods could be applied for the preliminary selection of new rootstocks/cultivars. Wood composition variation appeared to be correlated with the water stress susceptibility. To clarify this relationship, the attention was focused on the wood hydrophobicity. Sudan IV is a microscopy dye traditionally used to underline suberin, waxes, and, in general, hydrophobic substances. Differences between rough and colored spectra evidenced the absorption band of Sudan IV with a maximum at 539 nm. The coloration intensity was used to develop a hydrophobicity index. The obtained values were correlated with the expected drought tolerance score. Therefore, hydrophobic compounds seem to play an important role in water use efficiency, and an hydrophobic barrier in the xylem tissue appears to be a protective mechanism against water stress.

Evaluation of tannins interactions in grape (Vitis vinifera L.) skins.

Tannins have a central role in grapevine berries both for their physiological and enological implications. In the skin tissue they can be in vacuolar solution, or associated to the cell walls through weak or strong physicochemical interactions. The present work aims to separate vacuolar, non-covalently and covalently bonded tannins fractions. A specific extraction procedure was developed. A first extraction in ethanol at low temperature allowed the quantification of vacuolar tannins. An urea treatment followed by an ethanol extraction at room temperature was able to separate non-covalently bonded compounds. Finally an acid catalysis was used to break down proanthocyanidin covalent bonds. The method was validated on ripe grape samples of three cultivars, on berries developed in two sun exposure conditions. The Ethephon treatment effect was also evaluated. Beside the method development, a preliminary evaluation of the cultivar, exposition and Ethephon treatment effects are discussed.

Characterization of Grape (Vitis vinifera L.) Berry Sunburn Symptoms by Reflectance.

Sunburn can affect grape quality both for chemical modifications and by visual impact of the browning. Optical properties of 17 white grape accessions were investigated in the visible region using a noninvasive instrument. Reflectance spectra were obtained using a Jaz System spectrometer. Browning was induced by exposing grape bunches to direct sunlight at 12:30 p.m. for 5 h. During the experiment, the global solar radiation ranged from 2.6 to 2.7 MJ m-2 h-1 and the air temperature from 24.3 to 29.2 °C; the exposed berries reached a temperature of 34.2 °C in comparison to the 30.4 °C of the shaded ones. Differences between the spectra of controlled and exposed berries mainly emphasized the loss in chlorophyll and the formation of brown compounds. A positive correlation between the chlorophyll concentration and berry browning was proposed. Developing rapid, noninvasive, and low-cost methods based on reflectance spectroscopy could support grapevine variety characterization with respect to sunburn susceptibility as well as study of the physiological processes involved in the symptoms' appearance.

Tuning color variation in grape anthocyanins at the molecular scale.

Anthocyanins are the main grape pigments. Due to their aromatic cyclic arrangements, they are able to absorb the radiation in the low energy range of the visible spectrum. In the fruit of Vitis vinifera L., the five main anthocyanidins (cyanidin, peonidin, delphinidin, petunidin and malvidin) are present as 3-O-glucosides, as well as their acetyl, p-coumaroyl and caffeoyl ester forms. Despite the huge number of experimental studies dedicated to the anthocyanin profile analysis of grapes and wines, the complete theoretical elucidation of the optical properties of grape anthocyanins is missing. The present work carried out this task through quantum chemistry calculations based on time-dependent density functional theory (TD-DFT), compared to experimental spectra. The differences in visible absorption spectra between the most common grape anthocyanins were rationalized according to B-ring substitution, glucosylation and esterification. A particular attention was given to the intra-molecular copigmentation effect, demonstrating the existence of an intra-molecular charge transfer excited state for the p-coumaroyl and caffeoyl ester forms.