In order to investigate the comparability of microsatellite profiles obtained in different laboratories, ten partners in seven countries analyzed 46 grape cultivars at six loci (VVMD5, VVMD7, VVMD27, VVS2, VrZAG62, and VrZAG79). No effort was made to standardize equipment or protocols. Although some partners obtained very similar results, in other cases different absolute allele sizes and, sometimes, different relative allele sizes were obtained. A strategy for data comparison by means of reference to the alleles detected in well-known cultivars was proposed. For each marker, each allele was designated by a code based on the name of the reference cultivar carrying that allele. Thirty-three cultivars, representing from 13 to 23 alleles per marker, were chosen as references. After the raw data obtained by the different partners were coded, more than 97% of the data were in agreement. Minor discrepancies were attributed to errors, suboptimal amplification and visualization, and misscoring of heterozygous versus homozygous allele pairs. We have shown that coded microsatellite data produced in different laboratories with different protocols and conditions can be compared, and that it is suitable for the identification and SSR allele characterization of cultivars. It is proposed that the six markers employed here, already widely used, be adopted as a minimal standard marker set for future grapevine cultivar analyses, and that additional cultivars be characterized by means of the coded reference alleles presented here. The complete database is available at http://www.genres.de/eccdb/vitis/ Cuttings of the 33 reference cultivars are available on request from the Institut National de la Recherche Agronomique Vassal collection (didier.vares@ensam.inra.fr).

In the Vitaceae, viniferins represent a relatively restricted group of trans-resveratrol oligomers with antifungal properties, thus enabling plants to cope with pathogen attack. The aim of this study was to perform isolation and structural characterization of the whole class of viniferins accumulating in the leaves of hybrid Vitis vinifera (Merzling × Teroldego) genotypes infected with Plasmopara viticola . Infected leaves of resistant plants were collected 6 days after infection, extracted with methanol, and prepurified by flash chromatography using ENV+ and Toyopearl HW 40S resins. Further fractionation using normal-phase preparative chromatography and then reversed-phase preparative chromatography allowed isolation of 14 peaks. The isolated compounds were identified using advanced mass spectrometry techniques and extensive one- and two-dimensional nuclear magnetic resonance measurements, UV, CD, optical properties, and molecular mechanic calculations. The results demonstrated the presence in infected leaves of seven dimers (six stilbenes and one stilbenoid), of which four were new in grapevine (ampelopsin D, quadrangularin A, E-ω-viniferin, and Z-ω-viniferin), four trimers (three stilbenes and one stilbenoid), of which two (Z-miyabenol C and E-cis-miyabenol C) were new in grapevine, three tetramer stilbenoids, all new in grapevine, isohopeaphenol, ampelopsin H, and a vaticanol C-like isomer. The isolation of a dimer deriving from the condensation of (+)-catechin with trans-caffeic acid also indicated that other preformed phenolics are structurally modified in tissues infected with P. viticola.

Downy mildew (Plasmopara viticola) is one of the most destructive diseases of the cultivated species Vitis vinifera. The use of resistant varieties, originally derived from backcrosses of North American Vitis spp., is a promising solution to reduce disease damage in the vineyards. To shed light on the type and the timing of pathogen-triggered resistance, this work aimed at discovering biomarkers for the defense response in the resistant variety Bianca, using leaf discs after inoculation with a suspension of P. viticola. We investigated primary and secondary metabolism at 12, 24, 48, and 96 h post-inoculation (hpi). We used methods of identification and quantification for lipids (LC-MS/MS), phenols (LC-MS/MS), primary compounds (GC-MS), and semi-quantification for volatile compounds (GC-MS). We were able to identify and quantify or semi-quantify 176 metabolites, among which 53 were modulated in response to pathogen infection. The earliest changes occurred in primary metabolism at 24–48 hpi and involved lipid compounds, specifically unsaturated fatty acid and ceramide; amino acids, in particular proline; and some acids and sugars. At 48 hpi, we also found changes in volatile compounds and accumulation of benzaldehyde, a promoter of salicylic acid-mediated defense. Secondary metabolism was strongly induced only at later stages. The classes of compounds that increased at 96 hpi included phenylpropanoids, flavonols, stilbenes, and stilbenoids. Among stilbenoids we found an accumulation of ampelopsin H + vaticanol C, pallidol, ampelopsin D + quadrangularin A, Z-miyabenol C, and α-viniferin in inoculated samples. Some of these compounds are known as phytoalexins, while others are novel biomarkers for the defense response in Bianca. This work highlighted some important aspects of the host response to P. viticola in a commercial variety under controlled conditions, providing biomarkers for a better understanding of the mechanism of plant defense and a potential application in field studies of resistant varieties.

Background and Aims:In recent years, increasing summer temperature, coupled with reduced and erratic rainfall during the growing season, has induced accelerated fruit ripening in several regions, resulting in an undesirable increase in wine alcohol concentration. This study was designed to evaluate the impact of canopy and water management on grape sugar and flavonoid accumulation, with the goal of reducing wine alcohol concentration while conserving or enhancing the concentration of phenolic substances. Methods and Results: In 2011 and 2012, two irrigation treatments (I -irrigated and DI -deficit irrigated) and two canopy heights (HC -high canopy and SC -short canopy) were applied in a Merlot vineyard. No interactions between treatments were observed, and thus independent results were obtained; DI berries had significantly higher sugar concentration (+5%) than that of I in both years and higher wine alcohol concentration only in 2012. Short canopy berries had lower sugar concentration (−4%) and lower wine alcohol (−8%) (only in 2011) than that of HC. Anthocyanins and tannins in berry and wine were increased by water deficit and not affected by severe trimming. Conclusions: Deficit irrigation did not reduce berry sugar concentration and wine alcohol concentration but did enhance desirable wine attributes. Berry sugar concentration and alcohol concentration in wine were reduced by SC in one of the two seasons. Water deficit and severe trimming showed independent effects on berry composition. Significance of the Study: Severe canopy reduction at early stages of ripening can reduce sugars without affecting the accumulation of anthocyanins in Merlot. Conversely, DI applied before veraison, despite promoting anthocyanins accumulation, may also increase berry sugar concentration at harvest.

BackgroundDowny mildew, caused by the oomycete Plasmopara viticola, is a serious disease in Vitis vinifera, the most commonly cultivated grapevine species. Several wild Vitis species have instead been found to be resistant to this pathogen and have been used as a source to introgress resistance into a V. vinifera background. Stilbenoids represent the major phytoalexins in grapevine, and their toxicity is closely related to the specific compound. The aim of this study was to assess the resistance response to P. viticola of the Merzling × Teroldego cross by profiling the stilbenoid content of the leaves of an entire population and the transcriptome of resistant and susceptible individuals following infection.ResultsA three-year analysis of the population's response to artificial inoculation showed that individuals were distributed in nine classes ranging from total resistance to total susceptibility. In addition, quantitative metabolite profiling of stilbenoids in the population, carried out using HPLC-DAD-MS, identified three distinct groups differing according to the concentrations present and the complexity of their profiles. The high producers were characterized by the presence of trans-resveratrol, trans-piceid, trans-pterostilbene and up to thirteen different viniferins, nine of them new in grapevine.Accumulation of these compounds is consistent with a resistant phenotype and suggests that they may contribute to the resistance response.A preliminary transcriptional study using cDNA-AFLP selected a set of genes modulated by the oomycete in a resistant genotype. The expression of this set of genes in resistant and susceptible genotypes of the progeny population was then assessed by comparative microarray analysis.A group of 57 genes was found to be exclusively modulated in the resistant genotype suggesting that they are involved in the grapevine-P. viticola incompatible interaction. Functional annotation of these transcripts revealed that they belong to the categories defense response, photosynthesis, primary and secondary metabolism, signal transduction and transport.ConclusionsThis study reports the results of a combined metabolic and transcriptional profiling of a grapevine population segregating for resistance to P. viticola. Some resistant individuals were identified and further characterized at the molecular level. These results will be valuable to future grapevine breeding programs.

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The development of new resistant varieties to the oomycete Plasmopara viticola (Berk.& Curt) is a promising way to combat downy mildew (DM), one of the major diseases threatening the cultivated grapevine (Vitis vinifera L.). Taking advantage of a segregating population derived from “Merzling” (a mid-resistant hybrid) and “Teroldego” (a susceptible landrace), 136 F1 individuals were characterized by combining genetic, phenotypic, and gene expression data to elucidate the genetic basis of DM resistance and polyphenol biosynthesis upon P. viticola infection. An improved consensus linkage map was obtained by scoring 192 microsatellite markers. The progeny were screened for DM resistance and production of 42 polyphenols. QTL mapping showed that DM resistance is associated with the herein named Rpv3-3 specific haplotype and it identified 46 novel metabolic QTLs linked to 30 phenolics-related parameters. A list of the 95 most relevant candidate genes was generated by specifically exploring the stilbenoid-associated QTLs. Expression analysis of 11 genes in Rpv3-3+/− genotypes displaying disparity in DM resistance level and stilbenoid accumulation revealed significant new candidates for the genetic control of stilbenoid biosynthesis and oligomerization. These overall findings emphasized that DM resistance is likely mediated by the major Rpv3-3 haplotype and stilbenoid induction.

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