Corrigendum: Correlation between antioxidant and anticancer activity and phenolic profile of new Apulian table grape genotypes (V. Vinifera L.).

[This corrects the article DOI: 10.3389/fpls.2022.1064023.].

Use of Artificial Neural Networks and NIR Spectroscopy for Non-Destructive Grape Texture Prediction.

In this article, a combination of non-destructive NIR spectroscopy and machine learning techniques was applied to predict the texture parameters and the total soluble solids content (TSS) in intact berries. The multivariate models obtained by building artificial neural networks (ANNs) and applying partial least squares (PLS) regressions showed a better prediction ability after the elimination of uninformative spectral ranges. A very good prediction was obtained for TSS and springiness (R2 0.82 and 0.72). Qualitative models were obtained for hardness and chewiness (R2 0.50 and 0.53). No satisfactory calibration model could be established between the NIR spectra and cohesiveness. Textural parameters of grape are strictly related to the berry size. Before any grape textural measurement, a time-consuming berry-sorting step is compulsory. This is the first time a complete textural analysis of intact grape berries has been performed by NIR spectroscopy without any a priori knowledge of the berry density class.

Correlation between antioxidant and anticancer activity and phenolic profile of new Apulian table grape genotypes (V. Vinifera L.).

Grapes represent a significant source of phenolic compounds known for their health-promoting properties, such as antioxidant capacity on normal cells and prooxidant activity on tumor cells. The genotype highly affects the polyphenolic composition in grapes and, consequently, the nutritional quality of berries. This work aimed to characterize the phenolic composition, the antioxidant, and anticancer activity of grape skin extracts (GSEs) of nine new table grape genotypes selected from a breeding program to obtain new cultivars of seedless table grapes, well adapted to the climatic change and with higher nutraceutical properties. The grape polyphenolic profile was characterized by Ultra-High-Performance Liquid Chromatography/Quadrupole-Time of Flight mass spectrometry analysis. GSE antioxidant activity was determined by the ABTS, DPPH, and ORAC assays; GSE cell growth inhibition test was carried out in the Caco2 human cancer cell line. The nine GSEs showed different flavonoid and non-flavonoid profiles, and all possessed antioxidant activity, with the 'Aika N.', 'Turese N.', and 'Egnatia N.' the most active. As anticancer activity against the tested cancer cell line, 'Daunia N.' and 'Apenestae N.' showed the EC50 after 24 h of 35.60 µg/mL and 150.91 µg/mL, respectively. The relationship between polyphenolic profile and the antioxidant and anticancer activity of GSE was also investigated. Interestingly, among the different classes of polyphenolics, flavan-3-ols e proanthocyanidins showed the highest positive correlation with the anticancer activity of extracts. These findings can be helpful for the preparation of new extracts for the pharmaceutical and nutraceutical industry and geneticists working in vine breeding programs.

Native Vineyard Non-Saccharomyces Yeasts Used for Biological Control of Botrytis cinerea in Stored Table Grape.

Postharvest spoilage fungi, such as Botrytis cinerea, are considered the main cause of losses of fresh fruit quality and vegetables during storage, distribution, and consumption. The current control strategy is the use of SO2 generator pads whose application is now largely under observation. A high quantity of SO2 can be deleterious for fresh fruits and vegetables and it is not allowed in organic agriculture. For this reason, great attention has been recently focused on identifying Biological Control Agents (BCA) to implement biological approaches devoid of chemicals. In this direction, we carried out our study in isolating five different non-Saccharomyces yeast strains from local vineyards in the South of Italy as possible BCA. We performed both in vitro and in vivo assays in semi-commercial conditions on detached grape berries stored at 0 °C, simulating the temperature normally used during cold storage, and obtained relevant results. We isolated three M. pulcherrima strains and one L. thermotolerans strain able to largely antagonize the development of the B. cinerea, at both in vitro and in vivo conditions. In particular, we detected the ability of the three isolates of M. pulcherrima strains Ale4, N20/006, and Pr7 and the L. thermotolerans strain N10 to completely inhibit (100% in reduction) the mycelial growth of B. cinerea by producing fungistatic compounds. We found, using an extracellular lytic enzymes activity assay, that such activity could be related to lipid hydrolyzation, ß-1,3-glucanase and pectinase activity, and pectinase and protease activity, depending on the yeasts used. Results from our in vitro assays allowed us to hypothesize for M. pulcherrima strains Ale4 and N20/006 a possible combination of both the production of soluble metabolites and volatile organic compounds to antagonize against B. cinerea growth. Moreover, in semi-commercial conditions, the M. pulcherrima strain N20/006 and L. thermotolerans strain N10 showed relevant antagonistic effect also at low concentrations (with a significantly reduction of 'slip skin' incidence of 86.4% and 72.7%, respectively), thus highlighting a peculiar property to use in commercial development for organic agriculture and the handling process.

Color Stabilization of Apulian Red Wines through the Sequential Inoculation of Starmerella bacillaris and Saccharomyces cerevisiae.

Mixed fermentation using Starmerella bacillaris and Saccharomyces cerevisiae has gained attention in recent years due to their ability to modulate the qualitative parameters of enological interest, such as the color intensity and stability of wine. In this study, three of the most important red Apulian varieties were fermented through two pure inoculations of Saccharomyces cerevisiae strains or the sequential inoculation of Saccharomyces cerevisiae after 48 h from Starmerella bacillaris. The evolution of anthocyanin profiles and chromatic characteristics were determined in the produced wines at draining off and after 18 months of bottle aging in order to assess the impact of the different fermentation protocols on the potential color stabilization and shelf-life. The chemical composition analysis showed titratable acidity and ethanol content exhibiting marked differences among wines after fermentation and aging. The 48 h inoculation delay produced wines with higher values of color intensity and color stability. This was ascribed to the increased presence of compounds, such as stable A-type vitisins and reddish/violet ethylidene-bridge flavonol-anthocyanin adducts, in the mixed fermentation. Our results proved that the sequential fermentation of Starmerella bacillaris and Saccharomyces cerevisiae could enhance the chromatic profile as well as the stability of the red wines, thus improving their organoleptic quality.

NIR Analysis of Intact Grape Berries: Chemical and Physical Properties Prediction Using Multivariate Analysis.

Texture characteristics are valuable parameters in the perceived quality and overall acceptability of fresh fruit. The characterization of grape texture attributes, such as firmness and crunchiness, is usually performed by sensory analysis or instrumental texture analysis. Both methodologies are destructive. Hence, it is not possible to test multiple times or perform any other analysis on the same sample. In this article, near-infrared (NIR) spectroscopy was applied to intact berries of table grape cv. Regal Seedless. NIR spectra were employed to predict both the physical parameter "hardness", which is correlated with the crunchiness of berry flesh and the sweetness, which is correlated with the total soluble solids content (TSS, as °Brix). The chemometric analysis was carried out exclusively based on an open-source software environment, producing results readily usable for any operator, besides the specific level of experience with NIR spectroscopy.

Unraveling the Deep Genetic Architecture for Seedlessness in Grapevine and the Development and Validation of a New Set of Markers for VviAGL11-Based Gene-Assisted Selection.

Seedless inheritance has been considered a quasi-monogenic trait based on the VvAGL11 gene. An intragenic simple sequence repeat (SSR) marker, p3_VvAGL11, is currently used to opportunely discard seeded progeny, which represents up to 50% of seedlings to be established in the field. However, the rate of false positives remains significant, and this lack of accuracy might be due to a more complex genetic architecture, some intrinsic flaws of p3_VvAGL11, or potential recombination events between p3_VvAGL11 and the causal SNP located in the coding region. The purpose of this study was to update the genetic architecture of this trait in order to better understand its implications in breeding strategies. A total of 573 F1 individuals that segregate for seedlessness were genotyped with a 20K SNP chip and characterized phenotypically during four seasons for a fine QTL mapping analysis. Based on the molecular diversity of p3_VvAGL11 alleles, we redesigned this marker, and based on the causal SNP, we developed a qPCR-HRM marker for high-throughput and a Tetra-ARMS-PCR for simple predictive analyses. Up to 10 new QTLs were identified that describe the complex nature of seedlessness, corresponding to small but stable effects. The positive predictive value, based on VvAGL11 alone (0.647), was improved up to 0.814 when adding three small-effect QTLs in a multi-QTL additive model as a proof of concept. The new SSR, 5U_VviAGL11, is more informative and robust, and easier to analyze. However, we demonstrated that the association can be lost by intragenic recombination and that the e7_VviAGL11 SNP-based marker is thus more reliable and decreases the occurrence of false positives. This study highlights the bases of prediction failure based solely on a major gene and a reduced set of candidate genes, in addition to opportunities for molecular breeding following further and larger validation studies.

FT-NIR Analysis of Intact Table Grape Berries to Understand Consumer Preference Driving Factors.

Fourier-transform near infrared spectroscopy (FT-NIR) is a technique used in the compositional and sensory analysis of foodstuffs. In this work, we have measured the main maturity parameters for grape (sugars and acids) using hundreds of intact berry samples to build models for the prediction of these parameters from berries of two very different varieties: "Victoria" and "Autumn Royal". Together with the chemical composition in terms of sugar and acidic content, we have carried out a sensory analysis on single berries. Employing the models built for sugars and acids it was possible to learn the sweetness and acidity of each berry before the destructive sensory analysis. The direct correlation of sensory data with FT-NIR spectra is difficult; therefore, spectral data were exported from the spectrometer built-in software and analyzed with R software using a statistical analysis technique (Spearman correlation) which allowed the correlation of berry appreciation data with specific wavelengths that were then related to sugar and acidic content. In this article, we show how it is possible to carry out the analysis of single berries to obtain data on chemical composition parameters and consumer appreciation with a fast, simple, and non-destructive technique with a clear advantage for producers and consumers.

Inter-varietal structural variation in grapevine genomes.

Grapevine (Vitis vinifera L.) is one of the world's most important crop plants, which is of large economic value for fruit and wine production. There is much interest in identifying genomic variations and their functional effects on inter-varietal, phenotypic differences. Using an approach developed for the analysis of human and mammalian genomes, which combines high-throughput sequencing, array comparative genomic hybridization, fluorescent in situ hybridization and quantitative PCR, we created an inter-varietal atlas of structural variations and single nucleotide variants (SNVs) for the grapevine genome analyzing four economically and genetically relevant table grapevine varieties. We found 4.8 million SNVs and detected 8% of the grapevine genome to be affected by genomic variations. We identified more than 700 copy number variation (CNV) regions and more than 2000 genes subjected to CNV as potential candidates for phenotypic differences between varieties.

Ampelometric Leaf Trait and SSR Loci Selection for a Multivariate Statistical Approach in Vitis vinifera L. Biodiversity Management.

High estimated heritability values were recently revealed for mature leaf traits in grape (Vitis vinifera L.), thus redeeming ampelography in the era of molecular markers. The "Organisation Internationale de la Vigne et du Vin (OIV)" set a list of hundreds of descriptors for grapevine in order to standardize ampelographic and ampelometric scores. Therefore, the selection and reduction of the number of OIV codes can represent a major goal for leaner biodiversity assessment studies. The identification of ampelometric traits associated with grape diversity allows to construct Classification Trees with chi squared automatic interaction detection (CHAID) algorithm, a stepwise model-fitting method that produces a tree diagram in which at each step the sample pool is splitted based on the independent variables statistically different for the dependent variable. A collection of 100 table and wine grapevines (Vitis vinifera L.) was characterized and evaluated by means of six microsatellites and twenty-two ampelometric traits on mature leaves. Nine ampelometric traits were selected by principal component analysis and employed to build the classification trees based on CHAID algorithm. The strategy can represent an effective tool for grape biodiversity management, right allocations, and identification of new grape genotypes, implemented by a further microsatellite investigation only when unsolved cases occur, allowing faster and cheaper results.

Morphological variability in leaves and molecular characterization of novel table grape candidate cultivars (Vitis vinifera L.).

The present work report the characterization of twenty-one table grapes candidate cultivars plus five registered ones included as reference, by means of 47 ampelographic traits, 23 ampelometric measurements and six microsatellite loci. The final goal of the research was to analyse the possibility of reducing the number of morphological and molecular tools required for a precise and effective description of a grape genotype or cultivar. This would be of great help for future biodiversity description on a larger sample of more than 300 table grapes accessions today grown at the 'Consiglio per la Ricerca e la sperimentazione in Agricoltura (C.R.A.)-Unità di ricerca per l'uva da tavola e la vitivinicoltura in ambiente mediterraneo (Bari-Italy)'. OIV ampelographic traits showed a clear distinction among all twenty-six genotypes analysed, suggesting the relevant morphological variability investigated. Principal component analysis based on ampelometric traits revealed main veins ON(3), ON(4) and O(3)N(4); ratios between main veins; angles between main veins and of petiolar sinus, to be the most effective records in differentiating cultivars, for a total variation of 69.9 % described by the first three components. Molecular analysis based on six microsatellite loci was performed on all genotypes, providing a detailed molecular profile and a dendrogram of genetic similarity, in which all genotypes were clearly distinguishable. Finally, with the goal of using the minimum possible number of markers to differentiate genotypes, microsatellites VVMD5 and VVMD27 were selected to be sufficient to distinguish among all the candidate cultivars included in the analysis, representing a possible 'step by step' approach when a molecular characterization has to be undertaken on a large number of genotypes, by first testing few markers and increasing their number only if necessary.

Validation assay of p3_VvAGL11 marker in a wide range of genetic background for early selection of stenospermocarpy in Vitis vinifera L.

DNA markers technology, derived from research in molecular biology and genomics, offers great promise for plant breeding, allowing the "molecular breeding" via marker-assisted selection. Grapevine genomic resources allowed, in recent years, the characterization at molecular level of genes involved in interesting phenotypes such as stenospermocarpic seedlessness, a trait really appreciated by consumers. Recent studies in table grapes revealed that the VvAGL11 gene, member of the D-lineage MADS-box family, controls the ovule identity, and thus potentially playing an important role in stenospermocarpy. Intragenic markers of VvAGL11 have been found and tested for breeding purposes. In the present paper, we describe an in deep assay on a total of 475 genotypes derived by our own grape germplasm and seeded × seedless crosses F1 offspring, to evaluate and verify the "diagnostic" power of VvAGL11 in marker-assisted selection. We found only 8/475 that were seeded and carried the seedless-associated allele in the STS p3_VvAGL11. However, and most importantly, there were no seedless varieties without such allele. We validated the marker as a 100 % effective tool for early negative selection of stenospermocarpy in Vitis vinifera L. crosses.

Sangiovese and its offspring in southern Italy.

This paper demonstrates the importance of different approaches such as ampelography, historical researches, and molecular analysis to reveal direct parent-child relationship. The aim of this paper was to highlight the degree of relationship to five varieties spread in southern Italy, through ampelographic and molecular characterization: Sangiovese, Mantonico di Bianco, Gaglioppo di Cirò, Mantonicone, and Nerello Mascalese. Molecular characterization was carried out through 52 SSR molecular markers, showing that Sangiovese and Mantonico di Bianco are the parents of Gaglioppo di Cirò, Mantonicone, and Nerello Mascalese. Ampelographic description was performed using the method developed by the Organisation Internationale de la Vigne et du Vin. This analysis identifies three distinct groups: the first brings together Sangiovese and the two offspring Nerello Mascalese and Gaglioppo di Cirò, while Mantonico di Bianco and Mantonicone are positioned at a distance from the first and between them. Using molecular characterization, supported by the ampelographic one, we showed that Gaglioppo di Cirò, Mantonicone, and Nerello Mascalese, three varieties recovered in the southern regions of Italy, such as Calabria and Sicily, originated by the cross between a nationally spread grape variety as Sangiovese and a Calabria autochthonous vine as Mantonico di Bianco.

Evidences for an alternative genealogy of 'Sangiovese'.

Two different hypothesis for the parentage of 'Sangiovese', the most important and widespread Italian winegrape, have been proposed by some previous studies. We screened our grapevine collection, mostly comprising south Italian cultivars collected to preserve biodiversity, to asses kinships. Surprisingly we found two previously unreported candidate parents for 'Sangiovese'. The first putative parent is 'Ciliegiolo' a well know variety already addressed as relative of 'Sangiovese'; the second putative parent is 'Negrodolce', an old local variety we recovered and was considered lost during the last century. In order to obtain a stronger statistical support for this new kinship, we tested seventy different microsatellite markers but only 57 were found reliable. The new proposed parentage stood well even with such a in depth molecular analysis whereas only one discrepancy was found in one of the 57 microsatellite marker analyzed. This discrepancy is certainly due to a null-allele and therefore it should not impair our hypothesis but it points out limits of the microsatellites profiling as a pedigree research method considering that this is the third different kinship proposed so far for 'Sangiovese'. Thus in this article, by means of detailed molecular fingerprinting, we provide a completely new strong evidence for a south Italian origin of 'Sangiovese' and we discuss our findings comparing our data with those previously reported by other authors.

HPLC-DAD-ESI-MS analysis of flavonoid compounds in 5 seedless table grapes grown in Apulian Region.

UNLABELLED: Flavonoids present in skin extracts of red seedless table grape varieties Summer Royal, Autumn Royal, and Crimson, and white seedless varieties Carati and Thompson were analyzed by HPLC-DAD-MS, in 3 y of study (2006 to 2008). The anthocyanins, delphinidin-3-O-glucoside, cyanidin-3-O-glucoside, petunidin-3-O-glucoside (with their corresponding p-coumaroyl derivatives), peonidin-3-O-glucoside, and malvidin-3-O-glucoside (with their corresponding acetyl, caffeoyl, and p-coumaroyl derivatives) were found. In addition the flavonols quercetin-3-O-glucuronide, quercetin-3-O-rutinoside, quercetin-3-O-glucoside, kaempferol-3-O-glucoside, kaempferol-3-O-galactoside, and the flavan-3-ols procyanidin B1, procyanidin B2, and catechin were also detected. Anthocyanins were the main flavonoids in red grapes ranging from 24 (Crimson) to 500 (Summer Royal) mg/kg fresh weight of grapes; consistent levels of flavonols and flavan-3-ols were also quantified in all varieties. To determine the effective climatic influence on flavonoids content in field conditions, viticultural practices have been developed, that could exclude the effects of direct solar radiation from confounding the assessment of those related to thermal conditions alone. A strong positive correlation was determined between flavonoids and temperature data that seem to be responsible for the difference of these metabolites along the years; furthermore, it has been possible to define a linear relationship (R(2) = 0.6871, P = 0.0057) between thermal amplitude and total flavonoids values in the red grapes. PRACTICAL APPLICATION: Grapes are economically the most important fruit species in the world and approximately 30% of its production is used as fresh fruit. Because of the very important role of flavonoids in food quality as well as their health-promoting properties, and considering that our experiments were performed along 3 consecutive years, gathered results in this research are quite promising to give a useful information on the flavonoid contents and their evolution in 5 seedless table grapes that are widespread in Mediterranean regions but also in California and South America, and are grown in a viticultural climate (Apulia, South Italy) very close to some regions of Spain, Turkey, Tunisia, and Israel.