Genotyping-by-sequencing-based high-resolution mapping reveals a single candidate gene for the grapevine veraison locus Ver1.

Veraison marks the transition from berry growth to berry ripening and is a crucial phenological stage in grapevine (Vitis vinifera): the berries become soft and begin to accumulate sugars, aromatic substances, and, in red cultivars, anthocyanins for pigmentation, while the organic acid levels begin to decrease. These changes determine the potential quality of wine. However, rising global temperatures lead to earlier flowering and ripening, which strongly influence wine quality. Here, we combined genotyping-by-sequencing with a bioinformatics pipeline on ∼150 F1 genotypes derived from a cross between the early ripening variety 'Calardis Musqué' and the late-ripening variety 'Villard Blanc'. Starting from 20,410 haplotype-based markers, we generated a high-density genetic map and performed a quantitative trait locus analysis based on phenotypic datasets evaluated over 20 years. Through locus-specific-marker-enrichment and recombinant screening of ∼1000 additional genotypes, we refined the originally postulated 5 Mb veraison locus, Ver1, on chromosome 16 to only 112 kb, allowing us to pinpoint the ethylene response factor (ERF) VviERF027 (VCost.v3 gene ID: Vitvi16g00942, CRIBIv1 gene ID: VIT_16s0100g00400) as veraison candidate gene. Furthermore, the early veraison allele could be traced back to a clonal 'Pinot' variant first mentioned in the 17th century. 'Pinot Precoce Noir' passed this allele over 'Madeleine Royale' to the maternal grandparent 'Bacchus Weiss' and, ultimately, to the maternal parent 'Calardis Musqué'. Our findings are crucial for ripening time control, thereby improving wine quality, and for breeding grapevines adjusted to climate change scenarios that have a major impact on agro-ecosystems in altering crop plant phenology.

Phased grapevine genome sequence of an Rpv12 carrier for biotechnological exploration of resistance to Plasmopara viticola.

The downy mildew disease caused by the oomycete Plasmopara viticola is a serious threat for grapevine and can cause enormous yield losses in viticulture. The quantitative trait locus Rpv12, mediating resistance against P. viticola, was originally found in Asian Vitis amurensis. This locus and its genes were analyzed here in detail. A haplotype-separated genome sequence of the diploid Rpv12-carrier Gf.99-03 was created and annotated. The defense response against P. viticola was investigated in an infection time-course RNA-seq experiment, revealing approximately 600 upregulated Vitis genes during host-pathogen interaction. The Rpv12 regions of the resistance and the sensitivity encoding Gf.99-03 haplotype were structurally and functionally compared with each other. Two different clusters of resistance-related genes were identified within the Rpv12 locus. One cluster carries a set of four differentially expressed genes with three ACCELERATED CELL DEATH 6-like genes. The other cluster carries a set of six resistance gene analogs related to qualitative pathogen resistance. The Rpv12 locus and its candidate genes for P. viticola resistance provide a precious genetic resource for P. viticola resistance breeding. Newly developed co-segregating simple sequence repeat markers in close proximity to the R-genes enable its improved applicability in marker-assisted grapevine breeding.

Confirmation and Fine Mapping of the Resistance Locus Ren9 from the Grapevine Cultivar 'Regent'.

Grapevine (Vitis vinifera ssp. vinifera) is a major fruit crop with high economic importance. Due to its susceptibility towards fungal and oomycete pathogens such as Erysiphe necator and Plasmopara viticola, the causal agents of powdery and downy mildew (PM and DM, respectively), grapevine growers annually face a major challenge in coping with shortfalls of yield caused by these diseases. Here we report the confirmation of a genetic resource for grapevine resistance breeding against PM. During the delimitation process of Ren3 on chromosome 15 from the cultivar 'Regent', a second resistance-encoding region on chromosome 15 termed Ren9 was characterized. It mediates a trailing necrosis associated with the appressoria of E. necator and restricts pathogen growth. In this study, we confirm this QTL in a related mapping population of 'Regent' × 'Cabernet Sauvignon'. The data show that this locus is located at the upper arm of chromosome 15 between markers GF15-58 (0.15 Mb) and GF15-53 (4 Mb). The efficiency of the resistance against one of the prominent European PM isolates (EU-B) is demonstrated. Based on fine-mapping and literature knowledge we propose two possible regions of interest and supply molecular markers to follow both regions in marker-assisted selection.

Differential expression of transcription factor- and further growth-related genes correlates with contrasting cluster architecture in Vitis vinifera 'Pinot Noir' and Vitis spp. genotypes.

Grapevine (Vitis vinifera L.) is an economically important crop that needs to comply with high quality standards for fruit, juice and wine production. Intense plant protection is required to avoid fungal damage. Grapevine cultivars with loose cluster architecture enable reducing protective treatments due to their enhanced resilience against fungal infections, such as Botrytis cinerea-induced gray mold. A recent study identified transcription factor gene VvGRF4 as determinant of pedicel length, an important component of cluster architecture, in samples of two loose and two compact quasi-isogenic 'Pinot Noir' clones. Here, we extended the analysis to 12 differently clustered 'Pinot Noir' clones from five diverse clonal selection programs. Differential gene expression of these clones was studied in three different locations over three seasons. Two phenotypically opposite clones were grown at all three locations and served for standardization. Data were correlated with the phenotypic variation of cluster architecture sub-traits. A set of 14 genes with consistent expression differences between loosely and compactly clustered clones-independent from season and location-was newly identified. These genes have annotations related to cellular growth, cell division and auxin metabolism and include two more transcription factor genes, PRE6 and SEP1-like. The differential expression of VvGRF4 in relation to loose clusters was exclusively found in 'Pinot Noir' clones. Gene expression studies were further broadened to phenotypically contrasting F1 individuals of an interspecific cross and OIV reference varieties of loose cluster architecture. This investigation confirmed PRE6 and six growth-related genes to show differential expression related to cluster architecture over genetically divergent backgrounds.

Genetic Diversity, Population Structure, and Parentage Analysis of Croatian Grapevine Germplasm.

Croatian viticulture was most extensive at the beginning of the 20th century, when about 400 varieties were in use. Autochthonous varieties are the result of spontaneous hybridization from the pre-phylloxera era and are still cultivated today on about 35 % of vineyard area, while some exist only in repositories. We present what is the most comprehensive genetic analysis of all major Croatian national repositories, with a large number of microsatellite, or simple sequence repeat (SSR) markers, and it is also the first study to apply single nucleotide polymorphism (SNP) markers. After 212 accessions were fingerprinted, 95 were classified as unique to Croatian germplasm. Genetic diversity of Croatian germplasm is rather high considering its size. SNP markers proved useful for fingerprinting but less informative and practical than SSRs. Analysis of the genetic structure showed that Croatian germplasm is predominantly part of the Balkan grape gene pool. A high number of admixed varieties and synonyms is a consequence of complex pedigrees and migrations. Parentage analysis confirmed 24 full parentages, as well as 113 half-kinships. Unexpectedly, several key genitors could not be detected within the present Croatian germplasm. The low number of reconstructed parentages (19%) points to severe genetic erosion and stresses the importance of germplasm repositories.

Mutations in the miR396 binding site of the growth-regulating factor gene VvGRF4 modulate inflorescence architecture in grapevine.

Bunch rot caused by Botrytis cinerea infections is a notorious problem in grapevine cultivation. To produce high quality fruits, grapevine plants are treated with fungicides, which is cost intensive and harmful to the environment. Conversely, loose cluster bunches show a considerably enhanced physical resilience to bunch diseases. With the aim to identify genetic determinants that modulate the development of bunch architecture, we have compared loose and compact 'Pinot noir' clones. Loose cluster architecture was found to be correlated with increased berry size, elongated rachis and elongated pedicels. Using transcriptome analysis in combination with whole genome sequencing, we have identified a growth-regulating factor gene, VvGRF4, upregulated and harbours heterozygous mutations in the loose cluster clones. At late stages of inflorescence development, the mRNA pools of loose cluster clones contain predominantly mRNAs derived from the mutated alleles, which are resistant to miR396 degradation. Expression of the VvGRF4 gene and its mutated variants in Arabidopsis demonstrates that it promotes pedicel elongation. Taken together, VvGRF4 modulates bunch architecture in grapevine 'Pinot noir' clones. This trait can be introduced into other cultivars using marker-assisted breeding or CRISPR-Cas9 technology. Related growth-regulating factors or other genes of the same pathway may have similar functions.

Colonization of Different Grapevine Tissues by Plasmopara viticola-A Histological Study.

Plasmopara viticola, the downy mildew pathogen, is one of the most important pathogens in European viticulture. This oomycete infects grapevine leaves via zoospores that encyst at stomata. A primary germ tube then enters the substomatal cavity and develops a tubular network of hyphae that proliferate intercellularly and parasitize the leaf mesophyll cells by haustoria. Leaf infections have thus been the primary object of multiple studies concerning the physiology of the pathogen and defense reactions of grapevines. Besides leaves, this oomycete pathogen is able to spread throughout the plant tissue. As shown here by microscopy, it colonizes leaf petioles, shoots, berries and seeds. Evidence is provided showing that this process is facilitated by formation of special fan-shaped hyphae that seem to be necessary to overcome physical barriers in plant tissues. Physical obstacles are mainly constituted by vascular tissue in leaf veins, leaf petioles and shoots. In grapevine shoots, the mycelium seems to extend along the cambial layer between xylem and phloem tissue. Infected young berries are completely colonized on the inside. Older infected "leather berries" show glossy appositions of the fan-shaped hyphae at the inner side of the berry skin. The seeds from that stage of infestation are devoid of endosperm and embryo and biologically dysfunctional. Furthermore, a classification system for P. viticola infection based on the degree of infections in petioles and shoot tips is presented. This study contributes to a better understanding of downy mildew pathogenesis in grapevine, a prerequisite for efficient control measures.

Identification of co-located QTLs and genomic regions affecting grapevine cluster architecture.

Loose cluster architecture is an important aim in grapevine breeding since it has high impact on the phytosanitary status of grapes. This investigation analyzed the contributions of individual cluster sub-traits to the overall trait of cluster architecture. Six sub-traits showed large impact on cluster architecture as major determinants. They explained 57% of the OIV204 descriptor for cluster compactness rating in a highly diverse cross-population of 149 genotypes. Genetic analysis revealed several genomic regions involved in the expression of this trait. Based on the linkage of phenotypic features to molecular markers, QTL calculations shed new light on the genetic determinants of cluster architecture. Eight QTL clusters harbor overlapping confidence intervals of up to four co-located QTLs. A physical projection of the QTL clusters by confidence interval-flanking markers onto the PN40024 reference genome sequence revealed genes enriched in these regions.

Quantitative trait loci affecting pathogen resistance and ripening of grapevines.

Grapevines (Vitis vinifera L.) form the basis of viticulture, and are susceptible to diseases such as downy mildew (Plasmopara viticola) and powdery mildew (Erysiphe necator). Therefore, successful viticulture programs require the use of pesticides. Breeding for resistance is the only eco-friendly solution. Marker-assisted selection is currently widely used for grapevine breeding. Consequently, traits of interest must be tagged with molecular markers linked to quantitative trait loci (QTL). We herein present our findings regarding genetic mapping and QTL analysis of resistance to downy and powdery mildew diseases in the progenies of the GF.GA-47-42 ('Bacchus' × 'Seyval') × 'Villard blanc' cross. Simple sequence repeats and single nucleotide polymorphisms of 151 individuals were analyzed. A map consisting of 543 loci was screened for QTL analyses based on phenotypic variations observed in plants grown in the field or under controlled conditions. A major QTL for downy mildew resistance was detected on chromosome 18. For powdery mildew resistance, a QTL was identified on chromosome 15. This QTL was replaced by a novel QTL on chromosome 18 in 2003 (abnormally high temperatures) and 2004. Subsequently, both QTLs functioned together. Additionally, variations in the timing of the onset of veraison, which is a crucial step during grape ripening, were studied to identify genomic regions affecting this trait. A major QTL was detected on linkage group 16, which was supplemented by a minor QTL on linkage group 18. This study provides useful information regarding novel QTL-linked markers relevant for the breeding of disease-resistant grapevines adapted to current climatic conditions.

Expression profiling of genes involved in drought stress and leaf senescence in juvenile barley.

BACKGROUND: Drought stress in juvenile stages of crop development and premature leaf senescence induced by drought stress have an impact on biomass production and yield formation of barley (Hordeum vulgare L.). Therefore, in order to get information of regulatory processes involved in the adaptation to drought stress and leaf senescence expression analyses of candidate genes were conducted on a set of 156 barley genotypes in early developmental stages, and expression quantitative trait loci (eQTL) were identified by a genome wide association study. RESULTS: Significant effects of genotype and treatment were detected for leaf colour measured at BBCH 25 as an indicator of leaf senescence and for the expression level of the genes analysed. Furthermore, significant correlations were detected within the group of genes involved in drought stress (r = 0.84) and those acting in leaf senescence (r = 0.64), as well as between leaf senescence genes and the leaf colour (r = 0.34). Based on these expression data and 3,212 polymorphic single nucleotide polymorphisms (SNP) with a minor allele frequency >5% derived from the Illumina 9 k iSelect SNP Chip, eight cis eQTL and seven trans eQTL were found. Out of these an eQTL located on chromosome 3H at 142.1 cM is of special interest harbouring two drought stress genes (GAD3 and P5CS2) and one leaf senescence gene (Contig7437), as well as an eQTL on chromosome 5H at 44.5 cM in which two genes (TRIUR3 and AVP1) were identified to be associated to drought stress tolerance in a previous study. CONCLUSION: With respect to the expression of genes involved in drought stress and early leaf senescence, genotypic differences exist in barley. Major eQTL for the expression of these genes are located on barley chromosome 3H and 5H. Respective markers may be used in future barley breeding programmes for improving tolerance to drought stress and leaf senescence.

The transcription factor VvWRKY33 is involved in the regulation of grapevine (Vitis vinifera) defense against the oomycete pathogen Plasmopara viticola.

Grapevine (Vitis vinifera ssp. vinifera) is one of the most important fruit species; however, it is highly susceptible to various pathogens, which can cause severe crop losses in viticulture. It has been shown that several WRKY class transcription factors (TFs) are part of the signal transduction cascade, which leads to the activation of plant defense reactions against various pathogens. In the present investigation, a full-length cDNA was isolated from V. vinifera leaf tissue encoding a predicted protein, designated VvWRKY33, which shows the characteristics of group I WRKY protein family. VvWRKY33 induction correlates with the expression of VvPR10.1 (pathogenesis-related 10.1) gene in the leaves of the resistant cultivar 'Regent' after infection with Plasmopara viticola, whereas in the susceptible cultivar 'Lemberger' VvWRKY33 and VvPR10.1 are not induced. Corresponding expression of the TF and VvPR10.1 was even obtained in uninfected ripening berries. In planta, analysis of VvWRKY33 has been performed by ectopic expression of VvWRKY33 in grapevine leaves of greenhouse plants mediated via Agrobacterium tumefaciens transformation. In consequence, VvWRKY33 strongly increases resistance to P. viticola in the susceptible cultivar 'Shiraz' and reduces pathogen sporulation of about 50-70%, indicating a functional role for resistance in grapevine. Complementation of the resistance-deficient Arabidopsis thaliana Columbia-0 (Col-0) mutant line wrky33-1 by constitutive expression of VvWRKY33 restores resistance against Botrytis cinerea to wild-type level and in some complemented mutant lines even exceeds the resistance level of the parental line Col-0. Our results support the involvement of VvWRKY33 in the defense reaction of grapevine against different pathogens.

QTL analysis of flowering time and ripening traits suggests an impact of a genomic region on linkage group 1 in Vitis.

In the recent past, genetic analyses of grapevine focused mainly on the identification of resistance loci for major diseases such as powdery and downy mildew. Currently, breeding programs make intensive use of these results by applying molecular markers linked to the resistance traits. However, modern genetics also allows to address additional agronomic traits that have considerable impact on the selection of grapevine cultivars. In this study, we have used linkage mapping for the identification and characterization of flowering time and ripening traits in a mapping population from a cross of V3125 ('Schiava Grossa' × 'Riesling') and the interspecific rootstock cultivar 'Börner' (Vitis riparia × Vitis cinerea). Comparison of the flowering time QTL mapping with data derived from a second independent segregating population identified several common QTLs. Especially a large region on linkage group 1 proved to be of special interest given the genetic divergence of the parents of the two populations. The proximity of the QTL region contains two CONSTANS-like genes. In accordance with data from other plants such as Arabidopsis thaliana and Oryza sativa, we hypothesize that these genes are major contributors to control the time of flowering in Vitis.

Genetic diversity and population structure assessed by SSR and SNP markers in a large germplasm collection of grape.

BACKGROUND: The economic importance of grapevine has driven significant efforts in genomics to accelerate the exploitation of Vitis resources for development of new cultivars. However, although a large number of clonally propagated accessions are maintained in grape germplasm collections worldwide, their use for crop improvement is limited by the scarcity of information on genetic diversity, population structure and proper phenotypic assessment. The identification of representative and manageable subset of accessions would facilitate access to the diversity available in large collections. A genome-wide germplasm characterization using molecular markers can offer reliable tools for adjusting the quality and representativeness of such core samples. RESULTS: We investigated patterns of molecular diversity at 22 common microsatellite loci and 384 single nucleotide polymorphisms (SNPs) in 2273 accessions of domesticated grapevine V. vinifera ssp. sativa, its wild relative V. vinifera ssp. sylvestris, interspecific hybrid cultivars and rootstocks. Despite the large number of putative duplicates and extensive clonal relationships among the accessions, we observed high level of genetic variation. In the total germplasm collection the average genetic diversity, as quantified by the expected heterozygosity, was higher for SSR loci (0.81) than for SNPs (0.34). The analysis of the genetic structure in the grape germplasm collection revealed several levels of stratification. The primary division was between accessions of V. vinifera and non-vinifera, followed by the distinction between wild and domesticated grapevine. Intra-specific subgroups were detected within cultivated grapevine representing different eco-geographic groups. The comparison of a phenological core collection and genetic core collections showed that the latter retained more genetic diversity, while maintaining a similar phenotypic variability. CONCLUSIONS: The comprehensive molecular characterization of our grape germplasm collection contributes to the knowledge about levels and distribution of genetic diversity in the existing resources of Vitis and provides insights into genetic subdivision within the European germplasm. Genotypic and phenotypic information compared in this study may efficiently guide further exploration of this diversity for facilitating its practical use.

Alterations in grapevine leaf metabolism upon inoculation with Plasmopara viticola in different time-points.

Grapevines are easily infected by plant pathogens. It was found that resistant grapevines induce a wide range of phenolics upon the pathogen-infection. In this study in order to gain insight into these processes in different time-points the metabolic changes during the interaction of two grapevine cultivars, 'Regent' (resistant) and 'Trincadeira' (susceptible), with the downy mildew pathogen (Plasmopara viticola) were investigated. Nuclear magnetic resonance (NMR) spectroscopy on leaf extracts was used at several time points after experimental inoculation. A wide range of metabolites were identified using various two-dimensional (2D)-NMR techniques. Multivariate data analysis characterized both the resistant and the susceptible cultivars and their response against the pathogen. Metabolites responsible for their discrimination were identified as a fertaric acid, caftaric acid, quercetin-3-O-glucoside, linolenic acid, and alanine in the resistant cultivar 'Regent', while the susceptible 'Trincadeira' showed higher levels of glutamate, succinate, ascorbate and glucose. This study portrays the analytical capability of NMR spectroscopy and multivariate data analyses methods for the metabolic profiling of plant samples. The results obtained will underline the role of phenylpropanoids and flavonoids in resistance against biotic stresses which in turn provides a firm platform for the metabolic engineering of grapevine cultivars with higher resistance towards pathogens.

Cultivar-specific kinetics of gene induction during downy mildew early infection in grapevine.

The oomycete pathogen Plasmopara viticola (Berk. et Curt.) Berl. et de Toni is the causing agent of the destructive downy mildew disease in grapevine. Despite the advances towards elucidation of grapevine resistance mechanisms to downy mildew, increased knowledge of the biological and genetic components of the pathosystem is important to design suitable breeding strategies. Previously, a cDNA microarray approach was used to compare two Vitis vinifera genotypes Regent and Trincadeira (resistant and susceptible to downy mildew, respectively) in field conditions. The same cDNA microarray chip was used to confirm field-based results and to compare both genotypes under greenhouse conditions at 0, 6, and 12 h post-inoculation with P. viticola. Results show that when comparing both cultivars after pathogen inoculation, there is a preferential modulation of several defense, signaling, and metabolism associated transcripts in Regent. Early transcriptional changes are discussed in terms of genetic background and resistance mechanism. This study is the first to directly compare resistant and susceptible cultivars responses as early as 6 hpi with P. viticola, providing several candidate genes potentially related to the expression of resistance traits.

Rpv10: a new locus from the Asian Vitis gene pool for pyramiding downy mildew resistance loci in grapevine.

A population derived from a cross between grapevine breeding strain Gf.Ga-52-42 and cultivar 'Solaris' consisting of 265 F1-individuals was genetically mapped using SSR markers and screened for downy mildew resistance. Quantitative trait locus (QTL) analysis revealed two strong QTLs on linkage groups (LGs) 18 and 09. The locus on LG 18 was found to be identical with the previously described locus Rpv3 and is transmitted by Gf.Ga-52-42. 'Solaris' transmitted the resistance-related locus on LG 09 explaining up to 50% of the phenotypic variation in the population. This downy mildew resistance locus is named Rpv10 for resistance to Plasmopara viticola. Rpv10 was initially introgressed from Vitis amurensis, a wild species of the Asian Vitis gene pool. The one-LOD supported confidence interval of the QTL spans a section of 2.1 centi Morgan (cM) corresponding to 314 kb in the reference genome PN40024 (12x). Eight resistance gene analogues (RGAs) of the NBS-LRR type and additional resistance-linked genes are located in this region of PN40024. The F1 sub-population which contains the Rpv3 as well as the Rpv10 locus showed a significantly higher degree of resistance, indicating additive effects by pyramiding of resistance loci. Possibilities for using the resistance locus Rpv10 in a grapevine breeding programme are discussed. Furthermore, the marker data revealed 'Severnyi' × 'Muscat Ottonel' as the true parentage for the male parent of 'Solaris'.

NMR metabolic fingerprinting based identification of grapevine metabolites associated with downy mildew resistance.

Grapevine (Vitis vinifera ssp. vinifera L.) and grapes have been extensively studied due to their numerous nutritional benefits and health affecting activities. In this study, metabolite fingerprinting of crude leaf extracts, based on (1)H nuclear magnetic resonance (NMR) spectroscopy and multivariate data analyses, has been used for the metabolic characterization of six different grapevine cultivars including downy and powdery mildew resistant 'Regent' and susceptible 'Lemberger' among others. Several two-dimensional (2D)-NMR techniques were also employed leading to the identification of a number of different types of compounds. Principal component analysis (PCA), hierarchical cluster analysis (HCA), and partial least-squares-discriminant analysis (PLS-DA) of the processed (1)H NMR data revealed clear differences among the cultivars. Metabolites responsible for the discrimination in different grapevine cultivars belong to major classes, that is, organic acids, amino acids, carbohydrates, phenylpropanoids and flavonoids. A differentiation of the cultivars based on their resistance to downy mildew infection was also achieved, and metabolites associated with this trait, namely, quercetin-3-O-glucoside and a trans-feruloyl derivative, were identified. On the basis of these results, the distribution of different plant metabolites among the different grapevine cultivars is presented.

A framework map from grapevine V3125 (Vitis vinifera 'Schiava grossa' x 'Riesling') x rootstock cultivar 'Börner' (Vitis riparia x Vitis cinerea) to localize genetic determinants of phylloxera root resistance.

Grapevine rootstock cultivar 'Börner' is a hybrid of Vitis riparia and Vitis cinerea Arnold that shows high resistance to phylloxera (Daktulosphaira vitifoliae Fitch). To localize the determinants of phylloxera root resistance, the susceptible grapevine V3125 (Vitis vinifera 'Schiava grossa' x 'Riesling') was crossed to 'Börner'. Genetic framework maps were built from the progeny. 235 microsatellite markers were placed on the integrated parental map. They cover 1,155.98 cM on 19 linkage groups with an average marker distance of 4.8 cM. Phylloxera resistance was scored by counting nodosities after inoculation of the root system. Progeny plants were triplicated and experimentally infected in 2 years. A scan of the genetic maps indicated a quantitative trait locus on linkage group 13. This region was targeted by six microsatellite-type markers newly developed from the V. vinifera model genome sequence. Two of these appear closely linked to the trait, and can be useful for marker-assisted breeding.

Characterization of Plasmopara-resistance in grapevine using in vitro plants.

Although the exact mechanisms by which grapevine cells operate to reduce disease incidence caused by the downy mildew fungus Plasmopara viticola are not fully elucidated, our cytological results obtained from infected in vitro-plants confirm that enhanced disease resistance is associated with an expression of distinct reactions in a chronological order. An increased production of reactive oxygen species (superoxide radicals, 4-6 hours post infection, hpi) was followed by a hypersensitive response (6-8 hpi), an increased activity of peroxidase in cells flanking the infection area and in the vascular tissue (10-12 hpi) and an increased production, accumulation or conversion of phenolic compounds (12-15 hpi). These mechanisms seem also to be present in susceptible varieties as shown after an inoculation with non-host oomycetic pathogens on the basis of peroxidase activity, but they do not become activated after P. viticola infection. The investigation of the peroxidase activity in leaves at several time points after an infection with P. viticola indicated that there is a strong correlation between the POX activity in leaves of in vitro-plants and the resistance of grapevine plants to P. viticola in the field.