Resistance breeding offers invaluable perspectives for environment-friendly crop protection, but its success may be limited by the breakdown of plant resistance by pathogen strains. With the breeding and use of varieties carrying multiple genetic resistances, grapevine (Vitis spp.) represents a distinctive model for perennials to investigate the agreement that pyramiding broadens and enhances the efficacy and durability of resistance. To this end, grapevine progenies segregating for four major resistance loci against Plasmopara viticola (Rpvs) were used to evaluate single and pyramided loci when confronted with naive and Rpv-breaking pathogen strains. In pyramiding, undefeated and defeated Rpvs provided either beneficial, neutral, or detrimental quantitative effects, depending on the loci combination and pathogen strain. In particular, the fact that defeated loci may compromise resistance highlighted important implications for the breeding of perennials. Thorough phenotypic investigations of pyramiding schemes emerge as a critical step for the effective and durable management of genetic resistances.

Black rot, caused by Phyllosticta ampelicida, is a notable example of a recent reemerging disease of grapevine (Vitis vinifera). The pathogen infects young, actively growing plant parts and may have a large effect on yield, even at low incidence. A strategy to control grapevine black rot is to develop resistant varieties through breeding programs aimed at introducing the resistance into the susceptible V. vinifera. However, a complete picture of the existing sources for resistance in the Vitis species is missing. In this work, we evaluated a collection of Vitis species for resistance to black rot in the vineyard in natural conditions of infection over a 3-year period and validated the results using a bioassay for the evaluation of resistance in semi-controlled conditions. Our results confirmed the resistance to black rot previously reported for several species and identified new sources of resistance. The majority of the species identified as resistant come from America, whereas almost all species of Asian origin are susceptible. Because they displayed strong resistance, under field conditions and after artificial inoculation, the new sources of resistance reported here have a high potential to be used in future breeding programs.

Grape varieties carrying “Resistance to Plasmopara viticola” (Rpv) loci represent an effective solution to mitigate the environmental impact of fungicide application in viticulture. However, P. viticola strains able to overcome major Rpv have become a main threat to their cultivation. Pyramiding resistance loci in the same variety enhances plant resistance, but interactions involving stacked and defeated Rpv and different P. viticola strains are poorly documented. Investigation of these interactions may uncover information for the efficient deployment of Rpv and durable resistances. In the present study, a grapevine offspring carrying single and pyramided Rpv1, Rpv3.1 and Rpv10 was phenotyped for the resistance to P. viticola using a naive strain and a strain virulent towards Rpv10. By using high-resolution phenotyping applied to grapevine phytoalexins and P. viticola metabolic biomarkers, we demonstrated that the efficacy of Rpv combinations and aggressiveness of P. viticola strains are differentiated in the early phase of infection. Quantitative early and late infection outcomes based on P. viticola lipid biomarkers were closely related and provided complementary information. In particular, they evidenced the residual effect of a defeated Rpv and its importance in pyramiding, thereby providing keys to streamline resistance research and utilization in grapevine. In contrast, the accumulation of grapevine stilbenoids depended on infection severity, Rpv combination and genotype, highlighting the need for caution in their use as resistance biomarkers. The implementation of pathogen metabolic biomarkers-based phenotyping in resistance breeding represents an innovation that can be adapted to other plant pathosystems.

Esca is the most destructive and predominant grapevine trunk disease. The chronic infections and vine mortality caused by esca syndrome lead to huge economic losses and threaten the sustainability of vineyards worldwide. Esca is caused by numerous wood-decay and wood-decay-associated fungi, but its full etiology remains unclear due to the grapevine trunk disease complex, making effective control methods challenging. As differences in esca susceptibility have already been observed among grapevine varieties, we investigated the presence of genetic factors that can explain these variations using a Riesling × Gewurztraminer progeny. Thanks to the destructive phenotyping of a 16-year-old vineyard plot, we discovered that the Gewurztraminer variety carries on chromosome 1 a locus linked to variations in trunk necrosis associated with esca, which we have named Esca Necrosis Susceptibility 1 (ENS1). Our study also suggests that there is a partial link between trunk vigor and necrosis due to esca. To our best knowledge, ENS1 is the first instance of the genetic factor identified as involved in the limitation of necrosis associated with grapevine esca. While the identification of ENS1 alone may not provide a complete resolution of the issue, this discovery nonetheless represents a first step toward a genetic solution and paves the way for broader genetic investigations in the future.

### Competing Interest Statement The authors have declared no competing interest.

Resistance breeding offers invaluable perspectives for environment-friendly crop protection, but its success may be limited by the breakdown of plant resistance by pathogen strains. This threat is particularly acute for perennial crops, which may be cultivated for several decades. With the increasing use of new varieties carrying multiple major resistance loci, grapevine ( Vitis spp.) represents a distinctive model to investigate the broad agreement that combining several resistance genes (pyramiding) enhances both resistance efficacy and durability. To this end, grapevine progenies segregating for four resistance loci against Plasmopara viticola ( Rpvs ) were used to evaluate the efficiency of single and pyramided major loci when confronted to naive and Rpv -breaking pathogen strains. In the context of polygenic resistance, both undefeated and defeated Rpvs provided significant quantitative effects. However, interactions between pyramided Rpvs were either beneficial, neutral or detrimental to the level of resistance, depending on the loci combination and pathogen strain. In particular, the fact that the presence of defeated resistance loci may compromise the resistance provided by functional major loci has important implications for crops resistance breeding. Thorough phenotypic investigations of pyramiding breeding schemes emerge as a critical step for the effective and durable management of genetic resistances and plant diseases. ### Competing Interest Statement The authors have declared no competing interest. Agence Nationale de la RechercheAgence Nationale de la Recherche, https://ror.org/00rbzpz17, ANR-22-CE92-0005 Deutsche ForschungsgemeinschaftDeutsche Forschungsgemeinschaft, , 504993256

European viticulture faces two critical challenges currently: climate change and its direct and indirect impacts in the one hand and the need to dramatically reduce pesticide use in the other hand. In response, breeding programs, such as the INRAE-ResDur program, have prioritized the development of grapevine varieties with disease resistance and improved adaptability to climate change. We took the opportunity of the important plant material produced during the INRAE-ResDur program over twenty years to dissect the genetic basis of key agronomic traits using a comprehensive approach. We conducted genome-wide association studies (GWAS) modeling both additive and non-additive genetic effects, alongside classical QTL mapping and a meta-GWAS that integrates data across multiple environments and pleiotropic effects. These analyses identified numerous loci associated with important traits related to yield, phenology, and stress tolerance. Notably, several loci showed significant genotype-by-environment (GxE) interactions, while others showed pleiotropic effects influencing multiple traits. Overall, 39 genomic regions were detected in QTL analysis in biparental populations, GWAS with additive and non-additive effects, and meta-GWAs analysis, accounting for the 13 agronomic traits. These results provide valuable molecular markers that can be applied in marker-assisted selection or knowledge-assisted genomic selection. This study highlights a complex polygenic architecture underlying agronomic traits in grapevine, involving multiple loci with both additive and interaction effects and underscores the need for integrative breeding strategies to achieve sustainable improvements in future viticulture. ### Competing Interest Statement The authors have declared no competing interest. INRAE BAP division, IB2022_SelGen-ResDur, IVD4 ResGrape CASDAR FranceAgriMer, ViRéVATE, INNOVRES OIV research grant, PhD thesis of Clémentine Borrelli CTNSP

The integration of genetic values in ecophysiological models for phenological stages allows us to predict the effect of loci in future conditions. Modern grapevine (Vitis vinifera L.) breeding programs aim to create new varieties resistant to biotic and abiotic stresses, simultaneously. Developmental stages may affect many physiological processes in grapevine, especially berry composition. The shifts of phenological stages observed in the context of climate change challenge the selection of new varieties. In this paper, we evaluate how genotypes derived from a breeding program aimed at developing disease-resistant varieties may adapt to future climatic conditions. Specifically, we examine the genetic variability of three key phenological periods (February 15 to budbreak, budbreak to flowering, and flowering to véraison) using an ecophysiological model based on thermal requirements. Using high-density genetic information, we identified more than 18 quantitative trait loci for three phenological periods. By combining relevant allelic effects, we virtually constructed both an early and late composite genotype and evaluated their potential adaptation to future climatic conditions, using the greenhouse gas IPCC emissions scenario RCP 8.5 and simulated meteorological data at a local scale. While the early composite genotype may not outperform V. vinifera cv. Chardonnay under these projected conditions, the late composite genotype appears to remain suitably adapted through at least 2060. Our approach enables the prediction of allele-specific advantages on phenological stages across a range of future climate scenarios.

The Rpv2 locus for total resistance to grapevine downy mildew is mapped to a 250 kb genomic region containing two NLR-type genes specific to V. rotundifolia. Downy mildew caused by the oomycete Plasmopara viticola is one of the most important diseases affecting grapevine. Resistant varieties are an environmentally friendly tool to control grapevine downy mildew. Efficient breeding for durable resistance requires knowledge of the underlying mechanisms. Here we aimed at identifying the molecular basis of Rpv2, a gene for total resistance to downy mildew derived from Vitis rotundifolia, and at characterizing its effect on pathogen development. Individuals from two populations segregating for Rpv2 were evaluated for resistance to downy mildew and genotyped. Following genetic mapping, markers flanking Rpv2 were used to screen new populations and identify recombinant individuals. Sequencing of recombinants and in silico chromosome painting was used to reduce the interval containing Rpv2. Comparative genomics inside the Vitaceae, involving de novo assembly of the V. rotundifolia Regale genome, allowed narrowing down the list of candidate genes. We restrict Rpv2 to a 250 kb genomic region that contains two resistance genes of the NLR type. Comparative genomics analyses could not find orthologs of both NLRs in the other Vitis species studied. We also show that Rpv2-mediated resistance leads to pathogen arrest early in the infection cycle. Our results show that Rpv2 belongs to the NLR family of resistance genes, contributing thus to understand the potential and risks of its use in breeding programmes and suggesting that combining NLR-type genes may lead to durable resistance

Downy mildew caused by the oomycete Plasmopara viticola is one of the most important diseases affecting grapevine. Resistant varieties are an environmentally-friendly tool to control grapevine downy mildew. Efficient breeding for durable resistance requires knowledge of the underlying mechanisms. Here we aimed at identifying the molecular basis of Rpv2 , a gene for extreme resistance to downy mildew derived from Vitis rotundifolia , and at characterizing its effect on pathogen development. Individuals from two populations segregating for Rpv2 were evaluated for resistance to downy mildew and genotyped. Following genetic mapping, markers flanking Rpv2 were used to screen new populations and identify recombinant individuals. Sequencing of recombinants and in silico chromosome painting was used to reduce the interval containing Rpv2 . Comparative genomics inside the Vitaceae , involving de novo assembly of the V. rotundifolia Regale genome, allowed narrowing-down the list of candidate genes. We restrict Rpv2 to a 250 kb genomic region that contains two resistance genes of the NLR type. Comparative genomics analyses could not find orthologs of both NLRs in the other Vitis species studied. We also show that Rpv2 -mediated resistance leads to pathogen arrest early in the infection cycle. Our results show that Rpv2 belongs to the NLR family of resistance genes, contributing thus to understand the potential and risks of its use in breeding programs and suggesting that combining NLR-type genes may lead to durable resistance The Rpv2 locus for extreme resistance to grapevine downy mildew is mapped to a 250 kb genomic region containing two NLR-type genes specific to V. rotundifolia .

Accelerating grapevine breeding for disease resistance and climate adaptation remains a major challenge due to long generation cycles. We conducted a comprehensive benchmarking of genomic (SNP), phenomic (FT-NIR), and metabolomic (untargeted LC-MS) prediction models across multiple traits in a biparental grapevine population phenotyped over three years. Seven statistical frameworks and four tissue x timepoint strategies (wood; vineyard leaves at budbreak and flowering; greenhouse leaves at flowering) were assessed for predictive performance. Cross-year and cross-population scenarios, involving two additional biparental populations, evaluated model robustness and transferability. Genomic prediction consistently yielded the highest accuracies (up to r = 0.80), with rrBLUP outperforming other models. Metabolomic models achieved intermediate accuracies and, in some cases matched genomic predictions, while phenomic models, though less accurate, provided a cost-effective option for early selection. Heritability analyses showed that metabolomic features were more genetically determined than NIR traits, which were highly sensitive to environmental variation. Integrating omic layers led to limited improvement, except in a few traits where genomic-metabolomic models provided modest gains. Prediction accuracy declined in across-population transfers, underscoring the importance of genetic relatedness. We propose a tiered selection strategy combining low-cost phenomic or metabolomic pre-screening under controlled conditions with genomic selection in advanced lines to accelerate the development of resilient grapevine cultivars. ### Competing Interest Statement The authors have declared no competing interest. INRAE BAP division, IB\_2023\_MetabOptimum, PhD thesis grant OIV research grant, PhD thesis of Clémentine Borrelli

Societal Impact Statement Widespread adoption of disease‐resistant grapevine varieties presents a significant opportunity to revolutionize viticulture and address pressing environmental concerns. By reducing reliance on chemical pesticides, these varieties align with the European Commission's Green Deal objectives, striving for sustainable agricultural practices. Despite initial hesitancy among winegrowers and consumers, shifting attitudes towards sustainability and environmental consciousness are fostering increased acceptance of these varieties. As the market evolves, coupled with supportive policy measures for adoption, disease‐resistant vines are poised to significantly expand their presence in European vineyards. This transition not only mitigates environmental impact but also ensures the long‐term viability and resilience of viticulture. Summary Sustainable agriculture and most prominently the reduction of pesticides is one of the main goals of the European Unions (EU) agricultural policy. As viticulture uses around 70% of the fungicides in the EU, there is substantial pressure on winemakers to reduce their pesticide input. On top of the political goal, winegrowers face increased pressure from the public demanding a more sustainable production of wine. Since the introduction of downy mildew, powdery mildew, and other pests and pathogens from North America to Europe in the 19th century, substantial breeding efforts were undertaken to develop disease‐resistant varieties and reduce the amount of plant protection products needed for harvesting healthy grapes. Today's winegrowers in Europe can choose from many new varieties, allowing them to reduce fungicide input by 50–80% and produce high‐quality wines on par with the valued traditional cultivars. This review will provide an overview on the current situation of disease‐resistant varieties in European viticulture. Selected breeding institutions and private breeders will be introduced, and their most important grapevine varieties presented. Many resistant varieties are available for winegrowers today, however, even the most robust new varieties will not lead to a more sustainable viticulture if the market does not accept them. The adoption and acceptance of new varieties by winegrowers and consumers is still rather low, but the interest in these varieties is rapidly growing. The production of grafted vines of disease‐resistant grapevine varieties allows the estimation that the area of disease‐resistant varieties will significantly grow in the next years.

A major goal of modern grapevine (Vitis vinifera L.) breeding programs is the introgression of resistance genes along with desirable traits for better adaptation to climate change. Developmental stages have an impact on yield components and berry composition and are expected to shift towards earlier dates in the future. We investigated the genetic determinism of phenological stages in the progeny of a cross between two grapevine hybrids, each carrying several quantitative trait loci (QTL) for downy mildew and powdery mildew resistance. The dates of three phenological stages, budbreak, flowering and veraison, were recorded during three consecutive seasons for 209 genotypes in the vineyard. The phenotypic data analysed were the duration of three periods expressed in thermal time (degree-days): 15 February to budbreak, budbreak to flowering and flowering to veraison. High density parental and consensus genetic maps were constructed and used for QTL detection. Several QTL were detected for each period and the corresponding allelic effects were quantified and expressed in degree.days. Two virtual early and late genotypes were created by combining the relevant alleles. Using a previously validated ecophysiological model with simulated climate data for the RCP8.5 IPCC scenario, budbreak, flowering and veraison dates were predicted for the parents, Chardonnay, and the two virtual genotypes for each year up to 2100. Mean temperatures during the ripening period were calculated. The interest of the virtual genotypes in compensating for the expected shift in veraison dates will be discussed.

Background Downy mildew is a plant disease that affects all cultivated European grapevine varieties. The disease is caused by the oomycete Plasmopara viticola. The current strategy to control this threat relies on repeated applications of fungicides. The most eco-friendly and sustainable alternative solution would be to use bred-resistant varieties. During breeding programs, some wild Vitis species have been used as resistance sources to introduce resistance loci in Vitis vinifera varieties. To ensure the durability of resistance, resistant varieties are built on combinations of these loci, some of which are unfortunately already overcome by virulent pathogen strains. The development of a high-throughput machine learning phenotyping method is now essential for identifying new resistance loci. Results Images of grapevine leaf discs infected with P. viticola were annotated with OIV 452-1 values, a standard scale, traditionally used by experts to assess resistance visually. This descriptor takes two variables into account the complete phenotype of the symptom: sporulation and necrosis. This annotated dataset was used to train neural networks. Various encoders were used to incorporate prior knowledge of the scale's ordinality. The best results were obtained with the Swin transformer encoder which achieved an accuracy of 81.7%. Finally, from a biological point of view, the model described the studied trait and identified differences between genotypes in agreement with human observers, with an accuracy of 97% but at a high-throughput 650% faster than that of humans. Conclusion This work provides a fast, full pipeline for image processing, including machine learning, to describe the symptoms of grapevine leaf discs infected with P. viticola using the OIV 452-1, a two-symptom standard scale that considers sporulation and necrosis. If symptoms are frequently assessed by visual observation, which is time-consuming, low-throughput, tedious, and expert dependent, the method developed sweeps away all these constraints. This method could be extended to other pathosystems studied on leaf discs where disease symptoms are scored with ordinal scales.

Downy mildew of grapevine (Vitis vinifera), caused by the oomycete Plasmopara viticola, is an important disease that is present in cultivation areas worldwide, and using resistant varieties provides an environmentally friendly alternative to fungicides. DOWNY MILDEW RESISTANT 6 (DMR6) from Arabidopsis is a negative regulator of plant immunity and its loss of function confers resistance to downy mildew. In grapevine, DMR6 is present in two copies, named VvDMR6-1 and VvDMR6-2. Here, we describe the editing of VvDMR6-1 in embryogenic calli using CRISPR/Cas9 and the regeneration of the edited plants. All edited plants were found to be biallelic and chimeric, and whilst they all showed reduced growth compared with non-transformed control plants, they also had reduced susceptibility to P. viticola. Comparison between mock-inoculated genotypes showed that all edited lines presented higher levels of salicylic acid than controls, and lines subjected to transformation presented higher levels of cis-resveratrol than controls. Our results identify VvDMR6-1 as a promising target for breeding grapevine cultivars with improved resistance to downy mildew.

The strong societal demand to reduce pesticide use and adaptation to climate change challenges the capacities of phenotyping new varieties in the vineyard. High-throughput phenotyping is a way to obtain meaningful and reliable information on hundreds of genotypes in a limited period. We evaluated traits related to growth in 209 genotypes from an interspecific grapevine biparental cross, between IJ119, a local genitor, and Divona, both in summer and in winter, using several methods: fresh pruning wood weight, exposed leaf area calculated from digital images, leaf chlorophyll concentration, and LiDAR-derived apparent volumes. Using high-density genetic information obtained by the genotyping by sequencing technology (GBS), we detected 6 regions of the grapevine genome [quantitative trait loci (QTL)] associated with the variations of the traits in the progeny. The detection of statistically significant QTLs, as well as correlations (R2) with traditional methods above 0.46, shows that LiDAR technology is effective in characterizing the growth features of the grapevine. Heritabilities calculated with LiDAR-derived total canopy and pruning wood volumes were high, above 0.66, and stable between growing seasons. These variables provided genetic models explaining up to 47% of the phenotypic variance, which were better than models obtained with the exposed leaf area estimated from images and the destructive pruning weight measurements. Our results highlight the relevance of LiDAR-derived traits for characterizing genetically induced differences in grapevine growth and open new perspectives for high-throughput phenotyping of grapevines in the vineyard.

CREA Research Centre for Viticulture and Enology, viale XXVIII Aprile 26, 31015, Conegliano (TV), Italy INRAE, Université de Strasbourg, UMR 1131, 28 rue de Herrlisheim, 68000, Colmar, France Department of Agricultural, Food, Environmental and Animal Sciences, University of Udine, via delle Scienze 206, 33100, Udine (UD), Italy ______________________________________________________________________________