On the Way to the Technological Development of Newly Selected Non-Saccharomyces Yeasts Selected as Innovative Biocontrol Agents in Table Grapes.

Post-harvest decay of fresh table grapes causes considerable annual production losses. The main fungal agents of decay both in pre- and post-harvest are B. cinerea, Penicillium spp., Aspergillus spp., Alternaria spp., and Cladosporium spp. To date, the use of agrochemicals and SO2 are the main methods to control grape molds in pre- and postharvest, respectively. Significant improvements, however, have already been made in to apply innovative and more environmentally sustainable control strategies, such as Biological Control Agents (BCAs), which can reduce disease severity in both pre- and post-harvest. In this study, 31 new non-Saccharomyces yeast strains, isolated from berries of native Apulian table grape genotypes, were tested for their in vivo effectiveness against grey mold of table grapes, resulting in two St. bacillaris ('N22_I1' and 'S13_I3'), one S. diversa ('N22_I3'), one A. pullulans ('OLB_9.1_VL') and one H. uvarum ('OLB_9.1_BR') yeast strains that were marked as efficient and good BCAs. Their mechanisms of action were characterized through in vitro assays, and additional characteristics were evaluated to assess the economic feasibility and viability for future technological employment. Their effectiveness was tested by reducing the working concentration, their antagonistic effect on a wide range of fungal pathogens, their ability to survive in formulations with long shelf life, and their safety to human health.

Pesticide Residues and Berry Microbiome after Ozonated Water Washing in Table Grape Storage.

Nowadays, different systems for reducing pesticides in table grapes are being tested at different production stages either in the field or in postharvest. The present study tested ozonated water treatments at the beginning of the cold storage of the Princess® seedless table grape variety to reduce the residue contents of some pesticides and to evaluate their effect on gray mold and the berry microbiome. An ozone generator capable of producing an ozone concentration ranging from 18 to 65 Nm3 was utilized for obtaining three ozone concentration levels in water: 3, 5 and 10 mg/L. Ozonated water was placed in a 70 L plastic box where 500 g grape samples closed in perforated plastic clamshell containers were immersed utilizing two washing times (5 and 10 min). Overall, six ozonated water treatments were tested. After the ozonated water treatments, all samples were stored for 30 days at 2 °C and 95% relative humidity to simulate commercial practices. The pesticide residue contents were determined before the ozonated water treatments (T0) and 30 days after the cold storage (T1). The treatments with ozonated water washing reduced the pesticide residues up to 100%, while the SO2 control treatment reduced the pesticide residues ranging from 20.7 to 60.7%. Using 3 mg/L ozonated water to wash grapes for 5 min represented the optimal degradation conditions for all of the analyzed pesticides, except for fludioxonil, which degraded better with a washing time of 10 min. The ozone treatments did not significantly reduce the gray mold and the fungal and bacterial microbiome, while a relevant reduction was observed in the yeast population.

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.].

Preliminary Studies on the In Vitro Interactions Between the Secondary Metabolites Produced by Esca-Associated Fungi and Enological Saccharomyces cerevisiae Strains.

Esca-affected vines alter the carbohydrate metabolism, xylem transport of water and photosynthesis and show regular grapes (but berries do not reach maturity), and phenolic compounds are reduced in concentration, oxidate and polymerizate. Pullulan and a mixture of scytalone and isosclerone (9:1; w/w), secondary metabolites produced in vitro and in planta by Phaeoacremonium minimum (syn. P. aleophilum) and Phaeomoniella chlamydospora, were assayed against the strains Byosal HS1 and IOC 18-2007 in microvinifications with synthetic grape must. The presence of pullulan and pentaketides mix affects the growth and metabolism of the tested Saccharomyces cerevisiae strains. Assays at 100 and 1000 µg mL-1 inhibited the growth of both strains, while no effects were recorded when evaluated at 1 and 5 µg mL-1. In comparison with the controls, pullulan and the scytalone/isosclerone mixture at 10 µg mL-1 had a growth reduction, a lower alcohol yield, reduced the concentration of tartaric acid and malic acid; and slowed down the production of lactic acid, acetic acid and total polyphenol content of the tested S. cerevisiae strains. These metabolites could be applied as an alternative to the sulfite addition in the early stages of vinification to support the action of selected Saccharomyces. Appealing is the subtractive action of pullulan against tartaric acid. Further data are needed to confirm and validate the enological performance in freshly pressed grape juice.

Transcriptomic and methylation analysis of susceptible and tolerant grapevine genotypes following Plasmopara viticola infection.

Downy mildew, caused by the biotrophic oomycete Plasmopara viticola, is one of the most economically significant grapevine diseases worldwide. Current strategies to cope with this threat rely on the massive use of chemical compounds during each cultivation season. The economic costs and negative environmental impact associated with these applications increased the urge to search for sustainable strategies of disease control. Improved knowledge of plant mechanisms to counteract pathogen infection may allow the development of alternative strategies for plant protection. Epigenetic regulation, in particular DNA methylation, is emerging as a key factor in the context of plant-pathogen interactions associated with the expression modulation of defence genes. To improve our understanding of the genetic and epigenetic mechanisms underpinning grapevine response to P. viticola, we studied the modulation of both 5-mC methylation and gene expression at 6 and 24 h post-infection (hpi). Leaves of two table grape genotypes (Vitis vinifera), selected by breeding activities for their contrasting level of susceptibility to the pathogen, were analysed. Following pathogen infection, we found variations in the 5-mC methylation level and the gene expression profile. The results indicate a genotype-specific response to pathogen infection. The tolerant genotype (N23/018) at 6 hpi exhibits a lower methylation level compared to the susceptible one (N20/020), and it shows an early modulation (at 6 hpi) of defence and epigenetic-related genes during P. viticola infection. These data suggest that the timing of response is an important mechanism to efficiently counteract the pathogen attack.

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.