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The quantitative and qualitative impacts of anthocyanins on proanthocyanidin adsorption to grape-derived cell wall material were investigated in fifteen unique systems of varying temperatures, ethanol concentrations, and proanthocyanidin concentrations. Proanthocyanidin solutions were exposed to cell wall material and monitored for changes in concentration over 24 h. Increases in both temperature and ethanol resulted in a larger retention of proanthocyanidins in solution and typically faster adsorption kinetics. Analysis of the solution after exposure to cell wall revealed a significant reduction in the molecular weight of proanthocyanidins present in solution, suggesting that anthocyanins do not alter a previously described mechanism of preferentially binding large molecular weight molecules. Additionally, a reduction in polymeric pigment abundance was noted in most conditions, suggesting rapid formation of polymeric pigment in the model solution and preferential adsorption of the polymeric pigment to cell wall material. Compared to a previous study of proanthocyanidin adsorption in the absence of anthocyanins, a significantly larger percentage of proanthocyanidin material was lost via adsorption-up to 70% of available material. In a winemaking context, this may suggest a preferential loss of polymeric pigment via adsorption to cap cell wall material compared to non-pigmented proanthocyanidins and free anthocyanins.

BACKGROUND: Grafting is widely used in horticulture and rootstocks are known to modify scion growth and adaptation to soil conditions. However, the role of scion genotype in regulating rootstock development and functioning has remained largely unexplored. In this study, reciprocal grafts of two grapevine genotypes were produced as well as the corresponding homo-graft controls. These plants were subjected to a low phosphate (LP) treatment and transcriptome profiling by RNA sequencing was done on root samples collected 27 h after the onset of the LP treatment. RESULTS: A set of transcripts responsive to the LP treatment in all scion/rootstock combinations was identified. Gene expression patterns associated with genetic variation in response to LP were identified by comparing the response of the two homo-grafts. In addition, the scion was shown to modify root transcriptome responses to LP in a rootstock dependent manner. A weighted gene co-expression network analysis identified modules of correlated genes; the analysis of the association of these modules with the phosphate treatment, and the scion and rootstock genotype identified potential hub genes. CONCLUSIONS: This study provides insights into the response of grafted grapevine to phosphate supply and identifies potential shoot-to-root signals that could vary between different grapevine genotypes.

BACKGROUND: VviERF6Ls are an uncharacterized gene clade in Vitis with only distant Arabidopsis orthologs. Preliminary data indicated these transcription factors may play a role in berry development and extreme abiotic stress responses. To better understand this highly duplicated, conserved clade, additional members of the clade were identified in four Vitis genotypes. A meta-data analysis was performed on publicly available microarray and RNA-Seq data (confirmed and expanded with RT-qPCR), and Vitis VviERF6L1 overexpression lines were established and characterized with phenotyping and RNA-Seq. RESULTS: A total of 18 PN40024 VviERF6Ls were identified; additional VviERF6Ls were identified in Cabernet Sauvignon, Chardonnay, and Carménère. The amino acid sequences of VviERF6Ls were found to be highly conserved. VviERF6L transcripts were detected in numerous plant organs and were differentially expressed in response to numerous abiotic stresses including water deficit, salinity, and cold as well as biotic stresses such as red blotch virus, N. parvum, and E. necator. VviERF6Ls were differentially expressed across stages of berry development, peaking in the pre-veraison/veraison stage and retaining conserved expression patterns across different vineyards, years, and Vitis cultivars. Co-expression network analysis identified a scarecrow-like transcription factor and a calmodulin-like gene with highly similar expression profiles to the VviERF6L clade. Overexpression of VviERF6L1 in a Seyval Blanc background did not result in detectable morphological phenotypes. Genes differentially expressed in response to VviERF6L1 overexpression were associated with abiotic and biotic stress responses. CONCLUSIONS: VviERF6Ls represent a large and distinct clade of ERF transcription factors in grapevine. The high conservation of protein sequence between these 18 transcription factors may indicate these genes originate from a duplication event in Vitis. Despite high sequence similarity and similar expression patterns, VviERF6Ls demonstrate unique levels of expression supported by similar but heterogeneous promoter sequences. VviERF6L gene expression differed between Vitis species, cultivars and organs including roots, leaves and berries. These genes respond to berry development and abiotic and biotic stresses. VviERF6L1 overexpression in Vitis vinifera results in differential expression of genes related to phytohormone and immune system signaling. Further investigation of this interesting gene family is warranted.

Grapevine red blotch disease (GRBD) is a recently identified viral disease that affects grapevines. GRBD has been shown to impact grapevine physiology and grape composition by altering specific ripening events. However, no studies have been reported on the impact of GRBD on wine composition and its sensory attributes. This study evaluated the impact of GRBD on wine primary and secondary metabolites, in addition to its sensory properties, when making wines from Cabernet Sauvignon and Merlot grapes during two seasons. Wines made with GRBD-impacted fruit were lower in ethanol content when compared to wines made with grapes from healthy grapevines. This was attributed to the lower total soluble sugar (TSS) levels of diseased grapes due to delayed ripening at harvest. GRBD impacted wine phenolic composition by decreasing anthocyanin concentrations and increasing flavonol concentrations in some instances. Additionally, proanthocyanidin concentrations were also consistently higher in GRBD wines compared to wines made from healthy fruit. Descriptive analysis demonstrated that GRBD can impact wine style by altering aroma, flavor, and mouthfeel attributes. However, the extent of GRBD impact on wine composition and sensory properties were site and season dependent.

Bud necrosis (BN) is a common disorder that affects Vitis vinifera L. and reduces its potential yield. To minimize the losses caused by BN, the double pruning management was applied in Brazilian Southeast vineyards. In this management strategy plants are pruned at the winter to promote a vegetative cycle and then, at summer, to promote the reproductive cycle at optimal environmental conditions. To investigate the relationship of BN and the double pruning management RNA-seq libraries were sequenced from healthy and necrotic tissues at four different stages of the year. The comparison of differentially expressed genes in necrotic and non-necrotic tissues showed an enhanced expression of genes related to cell death possibly induced by endophytic microorganisms in the necrotic tissues. The de novo assembly, characterization and quantification of transcripts within the RNA-seq libraries showed that genes from the endophytic fungus Alternaria alternata, responsible for the production of toxic compounds were highly expressed under BN. Here we propose a model in which unfavorable conditions and reduced carbohydrate levels in buds can promote the switch from a biotrophic lifestyle to a necrotrophic lifestyle in the endophytic fungi, which seems to be involved in the development of BN.

It remains a major challenge to identify the genes and mutations that lead to plant sexual differentiation. Here, we study the structure and evolution of the sex-determining region (SDR) in Vitis species. We report an improved, chromosome-scale Cabernet Sauvignon genome sequence and the phased assembly of nine wild and cultivated grape genomes. By resolving twenty Vitis SDR haplotypes, we compare male, female, and hermaphrodite haplotype structures and identify sex-linked regions. Coupled with gene expression data, we identify a candidate male-sterility mutation in the VviINP1 gene and potential female-sterility function associated with the transcription factor VviYABBY3. Our data suggest that dioecy has been lost during domestication through a rare recombination event between male and female haplotypes. This work significantly advances the understanding of the genetic basis of sex determination in Vitis and provides the information necessary to rapidly identify sex types in grape breeding programs.

Grapevine leafroll-associated virus 3 (GLRaV-3) is one of the most important viruses of grapevine but, despite this, there remain several gaps in our understanding of its biology. Because of its narrow host range - limited to Vitis species - and because the virus is restricted to the phloem, most GLRaV-3 research has concentrated on epidemiology and the development of detection assays. The recent discovery that GLRaV-3 can infect Nicotiana benthamiana, a plant model organism, makes new opportunities available for research in this field. We used RNA-seq to compare both V. vinifera and P1/HC-Pro N. benthamiana host responses to GLRaV-3 infection. Our analysis revealed that the majority of DEGs observed between the two hosts were unique although responses between the two hosts also showed several shared gene expression results. When comparing gene expression patterns that were shared between the two hosts, we observed the downregulation of genes associated with stress chaperones, and the induction of gene families involved in primary plant physiological processes. This is the first analysis of gene expression profiles beyond Vitis to mealybug-transmitted GLRaV-3 and demonstrates that N. benthamiana could serve as a useful tool for future studies of GLRaV-3-host interactions.

BACKGROUND: Grapevine is an economically important crop for which yield and berry quality is strongly affected by climate change. Large variations in drought tolerance exist across Vitis species. Some of these species are used as rootstock to enhance abiotic and biotic stress tolerance. In this study, we investigated the physiological and transcriptomic responses to water deficit of four different genotypes that differ in drought tolerance: Ramsey (Vitis champinii), Riparia Gloire (Vitis riparia), Cabernet Sauvignon (Vitis vinifera), and SC2 (Vitis vinifera x Vitis girdiana). RESULTS: Ramsey was particularly more drought tolerant than the other three genotypes. Ramsey maintained a higher stomatal conductance and photosynthesis at equivalent levels of moderate water deficit. We identified specific and common transcriptomic responses shared among the four different Vitis species using RNA sequencing analysis. A weighted gene co-expression analysis identified a water deficit core gene set with the ABA biosynthesis and signaling genes, NCED3, RD29B and ABI1 as potential hub genes. The transcript abundance of many abscisic acid metabolism and signaling genes was strongly increased by water deficit along with genes associated with lipid metabolism, galactinol synthases and MIP family proteins. This response occurred at smaller water deficits in Ramsey and with higher transcript abundance than the other genotypes. A number of aquaporin genes displayed differential and unique responses to water deficit in Ramsey leaves. Genes involved in cysteine biosynthesis and metabolism were constitutively higher in the roots of Ramsey; thus, linking the gene expression of a known factor that influences ABA biosynthesis to this genotypes increased NCED3 transcript abundance. CONCLUSION: The drought tolerant Ramsey maintained higher photosynthesis at equivalent water deficit than the three other grapevine genotypes. Ramsey was more responsive to water deficit; its transcriptome responded at smaller water deficits, whereas the other genotypes did not respond until more severe water deficits were reached. There was a common core gene network responding to water deficit for all genotypes that included ABA metabolism and signaling. The gene clusters and sub-networks identified in this work represent interesting gene lists to explore and to better understand drought tolerance molecular mechanisms.

BACKGROUND: Grape berry ripening is influenced by climate, the main component of the terroir of a place. Light and temperature are major factors in the vineyard that affect berry development and fruit metabolite composition. RESULTS: To better understand the effect of place on transcript abundance during the late stages of berry ripening, Cabernet Sauvignon berries grown in Bordeaux and Reno were compared at similar sugar levels (19 to 26 °Brix (total soluble solids)). Day temperatures were warmer and night temperatures were cooler in Reno. °Brix was lower in Bordeaux berries compared to Reno at maturity levels considered optimum for harvest. RNA-Seq analysis identified 5528 differentially expressed genes between Bordeaux and Reno grape skins at 22°Brix. Weighted Gene Coexpression Network Analysis for all expressed transcripts for all four °Brix levels measured indicated that the majority (75%) of transcript expression differed significantly between the two locations. Top gene ontology categories for the common transcript sets were translation, photosynthesis, DNA metabolism and catabolism. Top gene ontology categories for the differentially expressed genes at 22°Brix involved response to stimulus, biosynthesis and response to stress. Some differentially expressed genes encoded terpene synthases, cell wall enzymes, kinases, transporters, transcription factors and photoreceptors. Most circadian clock genes had higher transcript abundance in Bordeaux. Bordeaux berries had higher transcript abundance with differentially expressed genes associated with seed dormancy, light, auxin, ethylene signaling, powdery mildew infection, phenylpropanoid, carotenoid and terpenoid metabolism, whereas Reno berries were enriched with differentially expressed genes involved in water deprivation, cold response, ABA signaling and iron homeostasis. CONCLUSIONS: Transcript abundance profiles in the berry skins at maturity were highly dynamic. RNA-Seq analysis identified a smaller (25% of total) common core set of ripening genes that appear not to depend on rootstock, vineyard management, plant age, soil and climatic conditions. Much of the gene expression differed between the two locations and could be associated with multiple differences in environmental conditions that may have affected the berries in the two locations; some of these genes may be potentially controlled in different ways by the vinegrower to adjust final berry composition and reach a desired result.

Resveratrol is a phytoalexin, stilbenoid compound with antioxidant properties attributable to its bioactive trans-resveratrol content. This study characterized the effects of over-the-counter (OTC) resveratrol nutritional supplements and a HPLC-purified resveratrol formulation, in human transmitochondrial age-related macular degeneration (AMD) retinal pigment epithelial (RPE) patient cell lines. These cell lines, which were created by fusing blood platelets obtained from dry and wet AMD patients with mitochondria-deficient (Rho0) ARPE-19 cells, had identical nuclei (derived from ARPE-19 cells) but different mitochondria that were derived from AMD patients. After resveratrol treatment, the levels of cell viability and reactive oxygen species production were measured. Results demonstrated that treatment with different resveratrol formulations improved cell viability and decreased reactive oxygen species generation in each AMD patient cell line. Although further studies are required to establish the cytoprotective potential of resveratrol under different physiological conditions, this novel study established the positive effects of OTC resveratrol supplements in macular degeneration patient cybrid cell lines in vitro.

Grapevine viruses are found throughout the viticultural world and have detrimental effects on vine productivity and grape and wine quality. This report provides a comprehensive and up-to-date review on grapevine viruses in Australia with a focus on "Shiraz Disease" (SD) and its two major associated viruses, grapevine virus A (GVA) and grapevine leafroll-associated virus 3 (GLRaV-3). Sensitive grapevine cultivars like Shiraz infected with GVA alone or with a co-infection of a leafroll virus, primarily GLRaV-3, show symptoms of SD leading to significant yield and quality reductions in Australia and in South Africa. Symptom descriptors for SD will be outlined and a phylogenetic tree will be presented indicating the SD-associated isolates of GVA in both countries belong to the same clade. Virus transmission, which occurs through infected propagation material, grafting, and naturally vectored by mealybugs and scale insects, will be discussed. Laboratory and field-based indexing will also be discussed along with management strategies including rogueing and replanting certified stock that decrease the incidence and spread of SD. Finally, we present several cases of SD incidence in South Australian vineyards and their effects on vine productivity. We conclude by offering strategies for virus detection and management that can be adopted by viticulturists. Novel technologies such as high throughput sequencing and remote sensing for virus detection will be outlined.

The genus Vitivirus in the family Betaflexiviridae includes eleven viruses known to infect grapevine: grapevine vitiviruses A, B, D, E, F, G, H, I, J, L and M (GVA-GVM). Three of these viruses, GVA, GVB and GVD, have been associated with the etiology of rugose wood disease in grapevine and cause agronomically significant losses. The other vitiviruses were more recently discovered and their effects on grapevine are undetermined. To certify grape material for propagation as virus tested, an updated reverse transcription PCR (RT-PCR) assay to detect all known vitiviruses is desirable. To accomplish this, multiple grapevine vitivirus sequences were aligned at the amino acid level to search for conserved motifs. Two highly conserved motifs were found at an ideal distance for RT-PCR detection in the RNA-dependent RNA polymerase region of the replicase protein. The amino acid motifs were back translated to create degenerate primers and used to successfully amplify all eleven grapevine vitiviruses. The RT-PCR primers were used to test a panel of vitivirus-infected vines for inclusivity as well as vines infected with closely related viruses in the Betaflexiviridae family (i.e. grapevine pinot gris virus and grapevine rupestris stem pitting-associated virus) for exclusivity. Broader use of these primers to detect vitiviruses in other plant hosts was investigated. In summary, an end-point RT-PCR assay that detects all the known grapevine vitiviruses and potentially other members of the genus Vitivirus has been developed. The universal assay represents an alternative to individual assays to reduce the work associated with the diagnosis of vitiviruses, including for regulatory purposes.

Grapevine leafroll disease is one of the most important virus diseases of grapevines and occurs in every major grape-growing region of the world. The vector-transmission mechanisms of the causative agent, Grapevine leafroll-associated virus 3 (GLRaV-3), remain poorly understood. We show that the vine mealybug, Planococcus ficus, feeds through a membrane feeding system on GLRaV-3 viral purifications from both V. vinifera and N. benthamiana and transmits the virus to test plants from plants from both species. Building on this strategy, we used an immunofluorescence approach to localize virions to two retention sites in P. ficus mouthparts. Assays testing molecules capable of blocking virus transmission demonstrated that GLRaV-3-transmission by P. ficus could be disrupted. Our results indicate that our membrane feeding system and transmission-blocking assays are a valid approach and can be used to screen other candidate blocking molecules.

The compositional authentication of wine is of great interest, as the geographic origin of the grapes is often associated with quality, uniqueness, and authenticity. Previous elemental fingerprinting studies mainly discriminated wines from different countries or regions within a country. Here, we report the use of element profiles to distinguish commercial Pinot noir wines from five sub-regional appellations or neighborhoods within one American viticultural area (AVA). Fifty-three single cultivar wines were collected over two harvests and analyzed using microwave plasma-atomic emission spectroscopy (MP-AES) and inductively coupled plasma-mass spectrometry (ICP-MS). Of 62 monitored elements that were quantified with fully validated methods, 24 and 32 elements differed significantly across the neighborhoods and vintages, respectively (p < 0.05). Targeted canonical variate analysis (CVA) explained 85%-90% of the variance ratio across the two vintages, indicating persistent and stable elemental fingerprints of wines at a sub-regional level. A sixth, newly founded neighborhood was correctly grouped separately from the others using a Soft Independent Modeling of Class Analogy (SIMCA), indicating the potential of elemental fingerprints for wine authenticity.

BACKGROUND: Vegetatively propagated clones accumulate somatic mutations. The purpose of this study was to better appreciate clone diversity and involved defining the nature of somatic mutations throughout the genome. Fifteen Zinfandel winegrape clone genomes were sequenced and compared to one another using a highly contiguous genome reference produced from one of the clones, Zinfandel 03. RESULTS: Though most heterozygous variants were shared, somatic mutations accumulated in individual and subsets of clones. Overall, heterozygous mutations were most frequent in intergenic space and more frequent in introns than exons. A significantly larger percentage of CpG, CHG, and CHH sites in repetitive intergenic space experienced transition mutations than in genic and non-repetitive intergenic spaces, likely because of higher levels of methylation in the region and because methylated cytosines often spontaneously deaminate. Of the minority of mutations that occurred in exons, larger proportions of these were putatively deleterious when they occurred in relatively few clones. CONCLUSIONS: These data support three major conclusions. First, repetitive intergenic space is a major driver of clone genome diversification. Second, clones accumulate putatively deleterious mutations. Third, the data suggest selection against deleterious variants in coding regions or some mechanism by which mutations are less frequent in coding than noncoding regions of the genome.

BackgroundAll eukaryotes share a conserved network of processes regulated by the proteasome and fundamental to growth, development, or perception of the environment, leading to complex but often predictable responses to stress. As a specialized component of the ubiquitin-proteasome system (UPS), the RING finger domain mediates protein-protein interactions and displays considerable versatility in regulating many physiological processes in plants. Many pathogenic organisms co-opt the UPS through RING-type E3 ligases, but little is known about how insects modify these integral networks to generate novel plant phenotypes.ResultsUsing a combination of transcriptome sequencing and genome annotation of a grapevine galling species, Daktulosphaira vitifoliae, we identified 138 putatively secretory protein RING-type (SPRINGs) E3 ligases that showed structure and evolutionary signatures of genes under rapid evolution. Moreover, the majority of the SPRINGs were more expressed in the feeding stage than the non-feeding egg stage, in contrast to the non-secretory RING genes. Phylogenetic analyses indicated that the SPRINGs formed clusters, likely resulting from species-specific gene duplication and conforming to features of arthropod host-manipulating (effector) genes. To test the hypothesis that these SPRINGs evolved to manipulate cellular processes within the plant host, we examined SPRING interactions with grapevine proteins using the yeast two-hybrid assay. An insect SPRING interacted with two plant proteins, a cellulose synthase, CSLD5, and a ribosomal protein, RPS4B suggesting secretion reprograms host immune signaling, cell division, and stress response in favor of the insect. Plant UPS gene expression during gall development linked numerous processes to novel organogenesis.ConclusionsTaken together, D. vitifoliae SPRINGs represent a novel gene expansion that evolved to interact with Vitis hosts. Thus, a pattern is emerging for gall forming insects to manipulate plant development through UPS targeting.

Desorption of proanthocyanidins (PA) from grape cell wall material (CWM) was investigated in solutions of varying ethanol concentrations and increasing temperature. The results reveal the reversibility of PA-CWM interactions and the role that temperature and ethanol concentration play in the extent of PA desorption. Sequentially raising temperature from 15 to 35 °C resulted in desorption of up to 48% of the initial adsorbed PA. A comparison to a phenolic extraction model showed significant differences between the predicted and actual amount of PA that desorbed from the CWM. This suggests that the initial conditions of temperature and ethanol concentration must be considered when estimating PA extraction in red wine production. Under typical winemaking conditions, a significant amount of PA may be irreversibly adsorbed if exposed to CWM at low temperature (i.e., cold soak). A compositional analysis suggests the selective desorption of large molecular weight PA from CWM under all experimental conditions. Additionally, a preferential desorption of skin-derived PA over seed-derived PA was noted in the absence of ethanol.

Agricultural bioinoculants containing arbuscular mycorrhizal fungi represent a potential opportunity to reduce the dependence of grapevines (Vitis) on agrochemicals. This field study assessed the ability of four commercial bioinoculants to colonize grapevine roots and their effects on petiole nutrient concentration, berry composition, and root morphology of ‘Pinot noir’ (Vitis vinifera) grafted onto rootstock ‘Couderc 3309’ (Vitis riparia × Vitis rupestris) and ‘Riesling’ (V. vinifera) grafted onto ‘Couderc 3309’ and Selection Oppenheim four (Vitis berlandieri × V. riparia). Three bioinoculants increased root mycorrhizal colonization; however, regardless of the treatment, mycorrhizal fungal structures were enhanced. Grapevine petiole nutrient concentration was improved by bioinoculants. Root diameter, root length density, and specific root length increased with greater mycorrhizal root colonization. Using bioinoculants to reduce chemical fertilizers may be a good strategy to improve grapevine productivity and health in cool climates; however, the impact of mycorrhizal bioinoculants in the vineyard may differ among scion–rootstocks, edaphoclimatic conditions, and vineyard soil microbiomes.

Grapevine virus diseases are widely spread in all viticultural areas of the world and they are considered detrimental, their presence is the most limiting factor of plants performances and vineyard life lasting. A large number of viruses have been identified in grapevine. From them, reported for the first time in 2012, Grapevine Pinot gris virus (GPGV) is spread all over the world. This study brings new knowledge about the virus distribution in plants throughout one year, the organ and period of sampling, for a reliable diagnostic. Additionally, the influence of high temperature on the physiological processes was investigated. Plants belonging to the 'Negru mare' genotype, natural infected with GPGV and Grapevine fleck virus (GFkV), maintained in a greenhouse, constituted the biological material. ELISA analyses of different grapevine organs and tissues (apex, leaf blade, petiole, young shoot, tendril, inflorescence, cane) located along the length of the shoot, carried out on different phenophases (before flowering, after flowering, veraison, dormancy) revealed that the conductive tissue was the most appropriate for diagnosis in the active growth period when the air temperature did not exceed 30°C. The measurement of the leaf temperature in two moments of the day (morning and afternoon), when the air temperature records increasing values revealed that at high temperatures (above 37°C), as a result of the intensification of the transpiration processes, the temperature at the leaf level does not increase progressively with the air temperature. Measurements of stomatal opening in two periods of the day in infected and virus-free grapevines showed that GPGV+GFkV complex infection caused sometimes a significant increase in stomatal opening both in the morning and in the afternoon, which could be a deficiency in the management of water and gas exchanges in plants.

The production of high-quality wines is strictly related to the correct management of the vineyard, which guarantees good yields and grapes with the right characteristics required for subsequent vinification. Winegrowers face a variety of challenges during the grapevine cultivation cycle: the most notorious are fungal and oomycete diseases such as downy mildew, powdery mildew, and gray mold. If not properly addressed, these diseases can irremediably compromise the harvest, with disastrous consequences for the production and wine economy. Conventional defense methods used in the past involved chemical pesticides. However, such approaches are in conflict with the growing attention to environmental sustainability and shifts from the uncontrolled use of chemicals to the use of integrated approaches for crop protection. Improvements in genetic knowledge and the availability of novel biotechnologies have created new scenarios for possibly producing grapes with a reduced, if not almost zero, impact. Here, the main approaches used to protect grapevines from fungal and oomycete diseases are reviewed, starting from conventional breeding, which allowed the establishment of new resistant varieties, followed by biotechnological methods, such as transgenesis, cisgenesis, intragenesis, and genome editing, and ending with more recent perspectives concerning the application of new products based on RNAi technology. Evidence of their effectiveness, as well as potential risks and limitations based on the current legislative situation, are critically discussed. [ABSTRACT FROM AUTHOR]

East Asian wild grapes show a high level of species diversity and have been widely recognized as important germplasm resources of wine, table grapes, and resistance to biotic and abiotic stresses for the grape industry. However, the deeper phylogenetic relationships and drivers of diversification of East Asian Vitis remain poorly understood. Hybridization and introgression events of East Asian Vitis are not well investigated, particularly at the genome‐wide scale. The phylogenetic relationships of East Asian Vitis are herein explored using nuclear and plastid genome data based on target enrichment (Hyb‐Seq). Seven major clades are recognized for East Asian Vitis based on the nuclear phylogenetic trees, but there is topological incongruence between concatenated and coalescent analyses. Furthermore, significant cytonuclear discordance is observed within East Asian Vitis. Species network analyses identified several hybridization events within East Asian Vitis. These interspecific hybridization events may have caused the topological discordances and relatively low support detected in our analyses. Ecological niche modeling shows that most of the diversification of East Asian Vitis species is driven by temperature and precipitation environmental variables. Sympatric parallel diversifications of major clades also may have facilitated the rich diversity in East Asian Vitis. [ABSTRACT FROM AUTHOR]

Black rot (BR), caused by Guignardia bidwellii, is an emergent fungal disease threatening viticulture and affecting several mildew-tolerant varieties. However, its genetic bases are not fully dissected yet. For this purpose, a segregating population derived from the cross 'Merzling' (hybrid, resistant) × 'Teroldego' (V. vinifera, susceptible) was evaluated for BR resistance at the shoot and bunch level. The progeny was genotyped with the GrapeReSeq Illumina 20K SNPchip, and 7175 SNPs were combined with 194 SSRs to generate a high-density linkage map of 1677 cM. The QTL analysis based on shoot trials confirmed the previously identified Resistance to Guignardia bidwellii (Rgb)1 locus on chromosome 14, which explained up to 29.2% of the phenotypic variance, reducing the genomic interval from 2.4 to 0.7 Mb. Upstream of Rgb1, this study revealed a new QTL explaining up to 79.9% of the variance for bunch resistance, designated Rgb3. The physical region encompassing the two QTLs does not underlie annotated resistance (R)-genes. The Rgb1 locus resulted enriched in genes belonging to phloem dynamics and mitochondrial proton transfer, while Rgb3 presented a cluster of pathogenesis-related Germin-like protein genes, promoters of the programmed cell death. These outcomes suggest a strong involvement of mitochondrial oxidative burst and phloem occlusion in BR resistance mechanisms and provide new molecular tools for grapevine marker-assisted breeding. [ABSTRACT FROM AUTHOR]

Grape (Vitis spp.) is one of the most important horticultural crops, cultivated worldwide on more than 7.3 million hectares for various purposes such as winemaking, fresh fruit consumption, rootstock, and ornamental plants. Based on the inter- and intraspecific morphological variability, several descriptor lists, manuals and ampelographic studies are available for identification. Among the organs, leaves have the most traits, while the young shoot, bunch and berry are also important in the characterization of the genotypes. Vitis species and cultivars are described by leaf morphological characterization developed in many ways for the identification of genotypes, to clarify synonymies and distinct clones or evaluate the diversity of wild Vitis taxa. Morphometric—also known as ampelometric—evaluation has an extensive background in the literature. However, for some reasons, only a part of the literature is cited, despite its significant scientific value. In this paper, we summarize the efforts of metric characterization of the grapevine leaf with the introduction of the scientific objectives and reviewing the studies showing the innovations in phenotyping during the past 120 years. [ABSTRACT FROM AUTHOR]

Knowledge about the growth vigor of grapevine rootstocks is required for scion-based rootstock selection and rootstock breeding. We performed this trial aiming to evaluate the multiplication and growth vigor of several rootstocks. Thirty-one rootstock genotypes were compared on their multiplication characteristics, phenological periods, and growth indicators across three consecutive seasons. The results suggested that the cuttings of most rootstocks had callus-forming indices (CFIs) over 0.5 except for '188-08' (0.28). The rooting rate of '420A' was 5%, while that of the rest of the rootstocks was greater than 48%. The internode lengths of the one-year-old vines were positively correlated with those (as well as cane lengths and pruning weights) of the adult vines. These rootstocks were grouped into three clusters based on the growth measurements across three seasons. Eight combinations of genetic backgrounds showed various effects on the growth indicators. The high-vigor cluster includes '1103P', '5BB', '225Ru', etc.; the medium-vigor cluster includes 'Dogridge', '101-14M', 'Fercal', etc.; and the low-vigor cluster includes 'Gloire', '3309C', 'Ganzin1', etc. The Vitis berlandieri parentage showed a higher vigor heredity, while the V. riparia showed a lower vigor heredity. These findings would contribute to rootstock nursery construction and provide references for vigor-based rootstock selection for grafts and parent selection for rootstock breeding. [ABSTRACT FROM AUTHOR]

The work purpose was to apply the analytical hierarchical process (AHP) method for ranking and selecting grapevine varieties, to assess the market demands for developing a program of setting up new plantations in a near future in Huşi Vineyard, one of the oldest vineyards of the Moldavian Hills Viticultural Region. Seven grapevine varieties of white wine (Aligoté, Băbească gri, Donaris, Ozana, Pinot gris, Riesling italian, Fetească regală) and fourteen criteria (quantitative and qualitative)were used in the AHP exercise. The analyses were carried out using the Expert Choice Desktop software. Ozana, Băbească gris and Aligoté genotypeswere selected as having the greatest potential for Huşi vineyardfrom the group of white wine genotypes taken into the study. The work can contribute to the maintaining of a durable viticulture, to quality white wines production and a more reliable future of the viticultural area. [ABSTRACT FROM AUTHOR]

Resistance of grapevine to Plasmopara viticola is associated with the hypersensitive reaction, accumulation of stilbenoids, and formation of callose depositions. Spectral characterization of infected leaf tissue of cvs 'Regent' and 'Solaris' with resistance genes Rpv 3-1 and Rpv 10 and Rpv 3-3 , respectively, suggested that resistance is not dependent on large-scale necrotization of host tissue. Reactions of the resistant cultivars and a reference susceptible to P. viticola were studied using hyperspectral imaging (range 400–1000 nm) at the tissue level and microscopic techniques. Resistance of both cultivars was incomplete and allowed pathogen reproduction. Spectral vegetation indices characterized the host response to pathogen invasion; the vitality of infected and necrotic leaf tissue differed significantly. Resistance depended on local accumulation of polyphenols in response to haustorium formation and was more effective for cv. 'Solaris'. Although hypersensitive reaction of some cells prevented colonization of palisade parenchyma, resistance was not associated with extensive necrotization of tissue, and the biotrophic pathogen survived localized death of penetrated host cells. Hyperspectral imaging was suitable to characterize and differentiate the resistance reactions of grapevine cultivars by mapping of the cellular response to pathogen attack on the tissue level and yields useful information on host–pathogen interactions. [ABSTRACT FROM AUTHOR]

‘Niagara Steck’ and ‘Niagara Variegada’ are two grapevine varieties that arose spontaneously as somatic mutants of ‘Niagara Rosada’. Berries characterize ‘Niagara Steck’ at young stages exhibiting a remarkable brown reticulated structure on the epidermis which develops into a brown-bronze russet-like structure as the berries ripe. The mature berries have strongly acidic flesh. ‘Niagara Variegada’ shows notable variegation in leaves and berries. Meiotic studies were carried out to ascertain if the mutant phenotypes could be related to any change in chromosome number, structure or other irregularity. Floral buds of both varieties at different developmental stages were collected and fixed for cytological analyses. Chromosome counts at diakinesis/metaphase I showed n=19 bivalents for both varieties. Univalent, trivalent, tetravalent chromosomes or chain configuration were not recorded ruling out the possibility of any alteration in the chromosome number or structure in both mutant varieties. However, low percentages of meiotic irregularities were recorded such as chromosome stickiness, laggards, non-oriented chromosomes, precocious chromosome segregation, tetrads with one microcyte, microspore fusion, and tetrad degeneration. The total percentage of abnormalities was higher in ‘Niagara Variegada’ (19.68%) than in ‘Niagara Steck’ (14.40%) which may have contributed to a lower percentage of pollen fertility (79.80%) when compared to ‘Steck’ (90.74%). The aforementioned varieties can be propagated by hardwood cuttings and constitute an interesting option for ornamentation of home backyards, patios, and gardens with the advantage that ‘Niagara Variegada’ bearing sweet edible berries.

Neste trabaho descreve-se a cultivar de videira EPAGRI 401-Villenave.

The contribution of vine cultivation to human welfare as well as the stimulation of basic social and cultural features of civilization has been great. The wide temporal and regional distribution created a wide array of genetic variants that have been used as propagating material to promote cultivation. Information on the origin and relationships among cultivars is of great interest from a phylogenetics and biotechnology perspective. Fingerprinting and exploration of the complicated genetic background of varieties may contribute to future breeding programs. In this review, we present the most frequently used molecular markers, which have been used on Vitis germplasm. We discuss the scientific progress that led to the new strategies being implemented utilizing state-of-the-art next generation sequencing technologies. Additionally, we attempted to delimit the discussion on the algorithms used in phylogenetic analyses and differentiation of grape varieties. Lastly, the contribution of epigenetics is highlighted to tackle future roadmaps for breeding and exploitation of Vitis germplasm. The latter will remain in the top of the edge for future breeding and cultivation and the molecular tools presented herein, will serve as a reference point in the challenging years to come.

The effects of temperature and ethanol concentration on the kinetics of anthocyanin adsorption and desorption interactions with five cell wall materials (CWM) of different composition were investigated. Using temperatures of 15 °C and 30 °C and model wine with ethanol concentrations of 0% and 15% (v/v) over 120 min, the adsorption and desorption rates of five anthocyanin-glucosides were recorded in triplicate. Small-scale experiments were conducted using a benchtop incubator to mimic a single berry fermentation. Results indicate that more than 90% of the adsorption occurs within the first 60 min of the addition of anthocyanins to CWM. However, desorption appears to occur much faster, with maximum desorption being reached after 30 min. The extent of both adsorption and desorption was clearly dependent not only on temperature and ethanol concentration but also on the CWM composition.

The aim of this study was to determine the effects of dietary grape seed polyphenols (GSP) supplementation during the late gestation and lactation period on reproductive performance, antioxidative status in serum, nutrient composition, and Ig content in colostrum of multiparous sows. On day 80 of gestation, a total of 64 sows with similar body condition were allocated to a completely randomized block design with 4 dietary treatments (n = 16 sows per treatment): 1) basal diet (CON, control group); 2) basal diet supplemented with 200 IU/kg vitamin E (200VE, positive control group); 3) basal diet supplemented with 200 mg/kg GSP (200GSP); and 4) basal diet supplemented with 300 mg/kg GSP (300GSP). The trial lasted 56 d until the piglets were weaned on day 21 of lactation. Reproductive performance, parameters of antioxidative status, and levels of progesterone (P4) and estradiol (E2) in serum, nutrient composition, and Ig content in colostrum of sows were determined. The number of dead fetuses was reduced, and farrowing survival was significantly improved in the litters from 300GSP-fed (P < 0.05). Preweaning survivability significantly increased in the litters from sows fed 200GSP and 200VE (P < 0.05). The activity of superoxide dismutase and glutathione peroxidase (GSH-Px) in the serum was significantly increased in sows fed 200GSP and 300GSP (P < 0.05). The activity of GSH-Px in the serum also significantly increased in sows fed 200VE (P < 0.05). Sows fed 300GSP had the greatest levels of P4 and E2 in the serum, which was significantly greater than sows fed 200VE and CON (P < 0.05). No significant differences were found among treatments for the content of solids-not-fat, fat, protein, and lactose in colostrum (P > 0.05). However, sows fed GSP had greater IgM and IgG content in colostrum compared with sows fed 200VE and CON (P < 0.05). In conclusion, dietary GSP supplementation during late gestation and lactation improved the farrowing survival and preweaning survivability, enhanced the antioxidant status and hormone levels in serum, and increased the IgM and IgG content in colostrum of sows.

In this genome report, we describe the sequencing and annotation of the genome of the wine grape Carménère (clone 02, VCR-702). Long considered extinct, this old French wine grape variety is now cultivated mostly in Chile where it was imported in the 1850s just before the European phylloxera epidemic. Genomic DNA was sequenced using Single Molecule Real Time technology and assembled with FALCON-Unzip, a diploid-aware assembly pipeline. To optimize the contiguity and completeness of the assembly, we tested about a thousand combinations of assembly parameters, sequencing coverage, error correction and repeat masking methods. The final scaffolds provide a complete and phased representation of the diploid genome of this wine grape. Comparison of the two haplotypes revealed numerous heterozygous variants, including loss-of-function ones, some of which in genes associated with polyphenol biosynthesis. Comparisons with other publicly available grape genomes and transcriptomes showed the impact of structural variation on gene content differences between Carménère and other wine grape cultivars. Among the putative cultivar-specific genes, we identified genes potentially involved in aroma production and stress responses. The genome assembly of Carménère expands the representation of the genomic variability in grapes and will enable studies that aim to understand its distinctive organoleptic and agronomical features and assess its still elusive extant genetic variability. A genome browser for Carménère, its annotation, and an associated blast tool are available at http://cantulab.github.io/data.

Phenolic extraction is a critical part of red wine making. Though empirical models of phenolic extraction kinetics exist, the current level of mechanistic understanding does not allow for accurate predictions. In this work, we propose a mechanistic model for the extraction of phenolics from grape skins and seeds as a function of temperature and ethanol. This model examines the release of phenolics, the adsorption of phenolics onto grape material, and the disappearance of anthocyanins from solution. Additionally, we performed epifluorescence microscopy to explore our finding that seed tannins' release rate appears independent of concentration, and found that the grape seed appears to ablate over fermentation. We also determined the activation energy of anthocyanin disappearance, in good agreement with similar systems. The proposed model results in an excellent fit, and increases the understanding of phenolic extraction and the ability to predict and optimize product outcome in red wine making.

Transcriptomics has been widely applied to study grape berry development. With few exceptions, transcriptomic studies in grape are performed using the available genome sequence, PN40024, as reference. However, differences in gene content among grape accessions, which contribute to phenotypic differences among cultivars, suggest that a single reference genome does not represent the species' entire gene space. Though whole genome assembly and annotation can reveal the relatively unique or "private" gene space of any particular cultivar, transcriptome reconstruction is a more rapid, less costly, and less computationally intensive strategy to accomplish the same goal. In this study, we used single molecule-real time sequencing (SMRT) to sequence full-length cDNA (Iso-Seq) and reconstruct the transcriptome of Cabernet Sauvignon berries during berry ripening. In addition, short reads from ripening berries were used to error-correct low-expression isoforms and to profile isoform expression. By comparing the annotated gene space of Cabernet Sauvignon to other grape cultivars, we demonstrate that the transcriptome reference built with Iso-Seq data represents most of the expressed genes in the grape berries and includes 1,501 cultivar-specific genes. Iso-Seq produced transcriptome profiles similar to those obtained after mapping on a complete genome reference. Together, these results justify the application of Iso-Seq to identify cultivar-specific genes and build a comprehensive reference for transcriptional profiling that circumvents the necessity of a genome reference with its associated costs and computational weight.

Two novel negative-stranded (ns)RNA viruses were identified by high throughput sequencing in grapevine. The genomes of both viruses, named grapevine Muscat rose virus (GMRV) and grapevine Garan dmak virus (GGDV), comprise three segments with each containing a unique gene. Based on sequence identity and presence of typical domains/motifs, the proteins encoded by the two viruses were predicted to be: RNA-dependent RNA polymerase (RdRp), nucleocapsid protein (NP), and putative movement protein (MP). These proteins showed the highest identities with orthologs in the recently discovered apple rubbery wood viruses 1 and 2, members of a tentative genus (Rubodvirus) within the family Phenuiviridae. The three segments of GMRV and GGDV share almost identical sequences at their 5' and 3' termini, which are also complementary to each other and may form a panhandle structure. Phylogenetics based on RdRp, NP and MP placed GMRV and GGDV in the same cluster with rubodviruses. Grapevine collections were screened for the presence of both novel viruses via RT-PCR, identifying infected plants. GMRV and GGDV were successfully graft-transmitted, thus, they are the first nsRNA viruses identified and transmitted in grapevine. Lastly, different evolutionary scenarios of nsRNA viruses are discussed.

Embryo rescue technology plays an important role in seedless grape breeding. However, the efficiency of embryo rescue, including the embryo formation, germination, and seedling rates, is closely related to the parental genotypes, degree of abortion, growth medium, and plant growth regulators. In this study, we investigated the effects of different concentrations of paclobutrazol (PAC), a plant growth regulator, and embryo collection times on the embryo formation, germination, and seedling rates for different hybrid combinations of grape breeding varieties used for their aroma and cold-resistance traits. The results showed that the different PAC concentrations had varying impacts on the development of ovules and embryos from the different grape varieties. The embryo formation rates of the ‘Sultanina Rose’בBeibinghong’ and ‘Kunxiang Seedless’בTaishan-2’ crosses were the highest under the 5.1 μmol L−1 PAC treatment. The 1.0 μmol L−1 PAC treatment was optimal for the germination and seedling development of the ‘Sultanina Rose’בBeibinghong’ embryos, whereas the 0.2 μmol L−1 PAC treatment induced the highest germination rate for the ‘Sultanina Rose’בKunxiang Seedless’ cross. The optimal sampling times for each cross varied as 39 d after pollination (DAP) for the ‘Flame Seedless’בMuscat Hamburg’ cross, 46 DAP for the ‘Kunxiang Seedless’בBeibinghong’ cross, and 41 DAP for the ‘Ruby Seedless’בBeibinghong’ and ‘Fantasy Seedless’בShuangyou’ crosses. Moreover, the medium modified with 0.5 g L−1 of indole-3-butyric acid allowed the malformed seedlings to develop into plantlets and achieve larger progenies. This study provides a useful basis for further studies into grape embryo rescue and could improve breeding efforts for new seedless grape varieties.

BACKGROUND:DNA metabarcoding, commonly used in exploratory microbial ecology studies, is a promising method for the simultaneous in planta-detection of multiple pathogens associated with disease complexes, such as the grapevine trunk diseases. Profiling of pathogen communities associated with grapevine trunk diseases is particularly challenging, due to the presence within an individual wood lesion of multiple co-infecting trunk pathogens and other wood-colonizing fungi, which span a broad range of taxa in the fungal kingdom. As such, we designed metabarcoding primers, using as template the ribosomal internal transcribed spacer of grapevine trunk-associated ascomycete fungi (GTAA) and compared them to two universal primer widely used in microbial ecology. RESULTS:We first performed in silico simulations and then tested the primers by high-throughput amplicon sequencing of (i) multiple combinations of mock communities, (ii) time-course experiments with controlled inoculations, and (iii) diseased field samples from vineyards under natural levels of infection. All analyses showed that GTAA had greater affinity and sensitivity, compared to those of the universal primers. Importantly, with GTAA, profiling of mock communities and comparisons with shotgun-sequencing metagenomics of field samples gave an accurate representation of genera of important trunk pathogens, namely Phaeomoniella, Phaeoacremonium, and Eutypa, the abundances of which were over- or under-estimated with universal primers. CONCLUSIONS:Overall, our findings not only demonstrate that DNA metabarcoding gives qualitatively and quantitatively accurate results when applied to grapevine trunk diseases, but also that primer customization and testing are crucial to ensure the validity of DNA metabarcoding results.

BACKGROUND:Arthropod-borne diseases remain a leading cause of human morbidity and mortality and exact an enormous toll on global agriculture. The practice of insecticide-based control is fraught with issues of excessive cost, human and environmental toxicity, unwanted impact on beneficial insects and selection of resistant insects. Efforts to modulate insects to eliminate pathogen transmission have gained some traction and remain future options for disease control. RESULTS:Here, we report a paratransgenic strategy that targets transmission of Xylella fastidiosa, a leading bacterial pathogen of agriculture, by the Glassy-Winged Sharpshooter (GWSS), Homalodisca vitripennis. Earlier, we identified Pantoea agglomerans, a bacterial symbiont of the GWSS as the paratransgenic control agent. We genetically engineered P. agglomerans to express two antimicrobial peptides (AMP)-melittin and scorpine-like molecule (SLM). Melittin and SLM were chosen as the effector molecules based on in vitro studies, which showed that both molecules have anti-Xylella activity at concentrations that did not kill P. agglomerans. Using these AMP-expressing strains of P. agglomerans, we demonstrated disruption of pathogen transmission from insects to grape plants below detectable levels. CONCLUSION:This is the first report of halting pathogen transmission from paratransgenically modified insects. It is also the first demonstration of paratransgenic control in an agriculturally important insect vector.

Feeding by the three-cornered alfalfa hopper, Spissistilus festinus (Say) (Hemiptera: Membracidae) results in girdling of grapevine petioles and shoots. Its significance as an economic pest of grape has increased since it was shown to transmit Grapevine red blotch virus (GRBV) in a greenhouse study. However, the status of grapevines as a reproductive host for S. festinus remained undetermined. Adult S. festinus were caged onto three regions of the grapevines: apical shoot, green shoot, and dormant cane. Their ability to reproduce was determined by weekly destructive sampling for 7 wk. Successful oviposition and nymphal emergence were observed on apical and green shoots, but not on dormant canes. However, insect development beyond the second nymphal instar did not occur. Knowledge of S. festinus reproduction on grapevines will be an important consideration in designing management guidelines to minimize the spread of GRBV in vineyards.

Proanthocyanidins are complex polymers of flavan-3-ol monomers and play a key sensory and health role in foods and beverages. We describe here a novel method for characterizing wine proanthocyanidins using a theoretical database comprised of the chemical formula and exact mass of 996 compounds. The database was constructed using the four primary grape and wine proanthocyanidin monomers: (epi)catechin, (epi)catechin-3-O-gallate, (epi)gallocatechin, and (epi)gallocatechin-3-O-gallate, each combined in all possible combinations up to a polymerization of 10. The database was queried against spectra collected using ultrahigh performance liquid chromatography (UHLPC) with a hydrophilic interaction liquid chromatography (HILIC) column and coupled to a high-resolution accurate mass quadrupole time-of-flight mass spectrometer (Q-TOF MS). Two wine samples produced with different post fermentation maceration were analyzed using the presented method to demonstrate application for analysis of diverse proanthocyanidins. The first sample was pressed immediately at the end of fermentation when all sugar had been utilized and the second received eight weeks of post fermentation maceration. The HILIC column combined with high resolution tandem mass spectrometry and database matching provided tentative identification of 89 compounds with excellent resolution and without the need for two-dimensional separations. The identified compounds were visualized with Kendrick mass analysis, a simple technique allowing for rapid visualization of which compounds are present in a given sample.

A genotyping technology has been developed based on nine microsatellite loci (VVS2, VVMD5, VVMD7, VVMD27, VVMD28, VVMD25, VVMD32, VrZAG62, and VrZAG79). It can be used for efficient, accurate, and fast identification of varieties and forms of Vitis grapes. The proposed approach includes multiplex PCR of all loci followed by electrophoretic analysis of DNA fragments in one capillary of a genetic analyzer. The application of an additional length standard, an allelic ladder, consisting of all possible DNA fragments of the analyzed microsatellite loci, is one of the key features of the technology, which ensures the accuracy and reproducibility of the results. The advantage of the proposed technology is the possibility of standardizing and automating the procedure using 96-well plates, which opens the possibility of conducting mass analyses. As a result of the study of the varieties and forms of the Vitis genus species, genetic passports were created, according to which a dendrogram was constructed, reflecting the genetic relationship of the studied samples. The developed technology makes it possible to distinguish varieties and wild-growing forms of grapes; it can be used for their identification and determination of the genetic distance between them, as well as for assessment of planting material and protection of breeders' rights. [ABSTRACT FROM AUTHOR]