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Summary: Grapevine downy mildew, caused by the oomycete Plasmopara viticola (P. viticola, Berk. & M. A. Curtis; Berl. & De Toni), is a global threat to Eurasian wine grapes Vitis vinifera. Although resistant grapevine varieties are becoming more accessible, P. viticola populations are rapidly evolving to overcome these resistances. We aimed to uncover avirulence genes related to Rpv3.1‐mediated grapevine resistance.We sequenced the genomes and characterized the development of 136 P. viticola strains on resistant and sensitive grapevine cultivars. A genome‐wide association study was conducted to identify genomic variations associated with resistant‐breaking phenotypes.We identified a genomic region associated with the breakdown of Rpv3.1 grapevine resistance (avrRpv3.1 locus). A diploid‐aware reassembly of the P. viticola INRA‐Pv221 genome revealed structural variations in this locus, including a 30 kbp deletion. Virulent P. viticola strains displayed multiple deletions on both haplotypes at the avrRpv3.1 locus. These deletions involve two paralog genes coding for proteins with 800–900 amino acids and signal peptides. These proteins exhibited a structure featuring LWY‐fold structural modules, common among oomycete effectors. When transiently expressed, these proteins induced cell death in grapevines carrying Rpv3.1 resistance, confirming their avirulence nature.This discovery sheds light on the genetic mechanisms enabling P. viticola to adapt to grapevine resistance, laying a foundation for developing strategies to manage this destructive crop pathogen. [ABSTRACT FROM AUTHOR]

Aims: Cheap, rapid tools for measuring emissions of Plasmopara viticola sporangia directly in the field are required to protect grapevines efficiently and sustainably against downy mildew. To this end, we adapted an existing loop‐mediated isothermal amplification (LAMP) protocol based on ITS2 sequences, coupled with a rotating‐arm sampler and simple cell lysis, for the in‐field measurement of airborne sporangia of P. viticola. Methods and Results: We estimated the sensitivity and specificity of the molecular reaction with an unpurified DNA template in controlled conditions, using the droplet digital PCR (ddPCR) as a reference. We show that the LAMP lower limit of quantification is 3.3 sporangia.m−3 air sampled. Cell lysis in KOH solution was less efficient than CTAB for DNA extraction, but the repeatability of the method was good. We tested this protocol directly in a plot at Chateau Dillon (Blanquefort, France) in which we monitored P. viticola sporangia concentrations from March to October 2020 (88 samples which revealed concentrations ranging from 0 to 243 sporangia.m−3). There was a significant quantitative correlation (R2 = 0.52) between ddPCR and LAMP results. Conclusion: LAMP analysis of an unpurified DNA matrix is a simple and reliable method for in‐field estimations of the concentration of airborne P. viticola sporangia. Significance and Impact of the Study: This study constitutes a first step towards the development of a regional grapevine downy mildew monitoring network in the vineyards of Bordeaux. [ABSTRACT FROM AUTHOR]

Downy mildew is a highly destructive disease of grapevine. Currently, monitoring for its symptoms is time-consuming and requires specialist staff. Therefore, an automated non-destructive method to detect the pathogen before the visible symptoms appear would be beneficial for early targeted treatments. The aim of this study was to detect the disease early in a controlled environment, and to monitor the disease severity evolution in time and space. We used a hyperspectral image database following the development from 0 to 9 days post inoculation (dpi) of three strains of Plasmopara viticola inoculated on grapevine leaves and developed an automatic detection tool based on a Support Vector Machine (SVM) classifier. The SVM obtained promising validation average accuracy scores of 0.96, a test accuracy score of 0.99, and it did not output false positives on the control leaves and detected downy mildew at 2 dpi, 2 days before the clear onset of visual symptoms at 4 dpi. Moreover, the disease area detected over time was higher than that when visually assessed, providing a better evaluation of disease severity. To our knowledge, this is the first study using hyperspectral imaging to automatically detect and show the spatial distribution of downy mildew on grapevine leaves early over time. [ABSTRACT FROM AUTHOR]

Downy mildew, caused by the Oomycete Plasmopara viticola, is one of the most important diseases affecting the Eurasian grapevine, Vitis vinifera. The pathogen originated in Northern America and its presence was signalled for the first time into Europe in 1878. In this study, the genetic variability and structure of Italian P. viticola population was investigated by genotyping 106 P. viticola strains belonging to 12 different regions, at 31 microsatellite markers. As a result of the high percentage of missing data, 96 strains and 19 loci were retained for the data analysis. The overall Italian population presents absence of clones, evidence of sexual and asexual reproduction and a low genetic diversity, as expected for an introduced pathogen, but a slightly higher genetic diversity than that found in other European populations, based on allelic diversity at the investigated microsatellite loci. Out of 19 loci, half shows deviation from Hardy–Weinberg equilibrium and, indeed, structure analysis indicates the presence of two separate genetic clusters, with little but significantly different distribution according to geography (west–east gradient) and climatic conditions. Overall, the analysis of the P. viticola population, 140 years after its appearance in Italy, provides indication on the pathogen adaptability. This should be taken into consideration in the future for preserving the durability of disease resistant varieties in open field. In this view, all the disease control methods available should be integrated in order to reduce the selection of pathogen strains able to overcome plant resistance. [ABSTRACT FROM AUTHOR]

Downy mildews are obligate biotrophic oomycete pathogens that cause devastating plant diseases on economically important crops. Plasmopara viticola is the causal agent of grapevine downy mildew, a major disease in vineyards worldwide. We sequenced the genome of Pl. viticola with PacBio long reads and obtained a new 92.94 Mb assembly with high contiguity (359 scaffolds for a N50 of 706.5 kb) due to a better resolution of repeat regions. This assembly presented a high level of gene completeness, recovering 1,592 genes encoding secreted proteins involved in plant–pathogen interactions. Plasmopara viticola had a two-speed genome architecture, with secreted protein-encoding genes preferentially located in gene-sparse, repeat-rich regions and evolving rapidly, as indicated by pairwise d N /d S values. We also used short reads to assemble the genome of Plasmopara muralis, a closely related species infecting grape ivy (Parthenocissus tricuspidata). The lineage-specific proteins identified by comparative genomics analysis included a large proportion of RxLR cytoplasmic effectors and, more generally, genes with high d N /d S values. We identified 270 candidate genes under positive selection, including several genes encoding transporters and components of the RNA machinery potentially involved in host specialization. Finally, the Pl. viticola genome assembly generated here will allow the development of robust population genomics approaches for investigating the mechanisms involved in adaptation to biotic and abiotic selective pressures in this species. [ABSTRACT FROM AUTHOR]

Background: Grapevine phylloxera, an insect related to true aphids, is a major historic pest of viticulture only controlled through the selection of resistant rootstocks or through quarantine regulations where grapevine is cultivated own-rooted. Transcriptomic data could help understand the bases of its original life-traits, including a striking case of polyphenism, with forms feeding on roots and forms feeding in leaf-galls. Comparisons with true aphids (for which complete genomes have been sequenced) should also allow to link differences in life-traits of the two groups with changes in gene repertoires or shifts in patterns of expression. Results: We sequenced transcriptomes of the grapevine phylloxera (Illumina technology), choosing three life-stages (adults on roots or on leaf galls, and eggs) to cover a large catalogue of transcripts, and performed a de novo assembly. This resulted in 105,697 contigs, which were annotated: most contigs had a best blastx hit to the pea aphid (phylogenetically closest complete genome), while very few bacterial hits were recorded (except for Probionibacterium acnes). Coding sequences were predicted from this data set (17,372 sequences), revealing an extremely high AT-bias (at the third codon position). Differential expression (DE) analysis among root-feeding and gall-feeding showed that i) the root-feeding form displayed a much larger number of differentially expressed transcripts ii) root-feeding biased genes were enriched in some categories, for example cuticular proteins and genes associated with cell-cell signaling iii) leaf-galling-biased genes were enriched in genes associated with the nucleus and DNA-replication, suggesting a metabolism more oriented towards fast and active multiplication. We also identified a gene family with a very high expression level (copies totaling nearly 10 % of the reads) in the grapevine phylloxera (both in root and leaf galling forms), but usually expressed at very low levels in true aphids (except in sexual oviparous females). These transcripts thus appear to be associated with oviparity. Conclusions: Our study illustrated major intraspecific changes in transcriptome profiles, related with different life-styles (and the feeding on roots versus in leaf-galls). At a different scale, we could also illustrate one major shift in expression levels associated with changes in life-traits that occurred along evolution and that respectively characterize (strictly oviparous) grapevine phylloxera and (mostly viviparous) true aphids. [ABSTRACT FROM AUTHOR]

We describe a standard method for characterizing the virulence profile of Plasmopara viticola, the causal agent of grapevine downy mildew. We used 33 European strains to inoculate six grapevine varieties carrying the principal factors for resistance to downy mildew (Rpv1, Rpv3.1, Rpv3.2, Rpv5, Rpv6, Rpv10, and Rpv12) and the susceptible Vitis vinifera 'Chardonnay'. For each interaction, we characterized the level of sporulation by image analysis and the intensity of the grapevine hypersensitive response by visual score. We propose a definition for the breakdown of grapevine quantitative resistances combining these two traits. Among the 33 strains analyzed, 28 are virulent on at least one resistance factor. We identified five different pathotypes across the 33 strains analyzed: two pathotypes overcoming a single resistance factor (vir3.1 and vir3.2) and three complex pathotypes overcoming multiple resistance factors (vir3.1,3.2; vir3.2,12; vir3.1,3.2,10). Our findings confirm the widespread occurrence of P. viticola strains overcoming the Rpv3 haplotypes (28 strains). We also detected the first breakdown of resistance to the Rpv 10 by a strain from Germany and the breakdown of Rpv 12 factors by a strain from Hungary. The pathotyping method proposed here and the associated differential host range lay the groundwork for the early detection of resistance breakdown in grapevines. This approach will also facilitate the monitoring of the evolution of P. viticola populations at large spatial scales. This is an essential step forward to promoting durable management of the resistant grapevine varieties currently available. [ABSTRACT FROM AUTHOR]

European grapevine is a complex holobiont composed of two plant genomes, that of the scion (Vitis vinifera L.) and the rootstock (Vitis spp.), and a multitude of microbial genomes that collectively form the microbiome. The grapevine microbiome has been extensively described over the last decade, primarily using metabarcoding approaches. Unfortunately, metabarcoding data alone provide little information on microbial functions and outcomes of plant-microbe interactions. Here we review knowledge about the microorganisms that have a demonstrated influence, positive or negative, on the performance of the grapevine holobiont. Our review encompasses bacteria, filamentous fungi, yeasts, oomycetes and viruses. It covers aboveground and belowground microorganisms, including arbuscular mycorrhizal and ectomycorrhizal fungi. We focus on taxa and functions that protect the plant against pathogens and pests, promote growth, increase tolerance to abiotic stresses and highlight those involved in disease and decline. As the outcomes of plant-microbe interactions are labile, we examine the dynamics and functions of grapevine-microbiome interactions over both the plant lifetime and the plant evolutionary history, beginning with plant domestication. Based on the knowledge and gaps we identify, we suggest field sampling designs, culture-based experiments, molecular tools and theoretical analysis methods, including shotgun metagenomics and network models, that could be used in future research to uncover and leverage the full functional potential of the grapevine microbiome. [ABSTRACT FROM AUTHOR]

The putative center of origin of Plasmopara viticola, the causal agent of grape downy mildew, is eastern North America, where it has been described on several members of the family Vitaceae (e.g., Vitis spp., Parthenocissus spp., and Ampélopsis spp.). We have completed the first large-scale sampling of P. viticola isolates across a range of wild and cultivated host species distributed throughout the above region. Sequencing results of four partial genes indicated the presence of a new P. viticola species on Vitis vulpina in Virginia, adding to the four cryptic species of P. viticola recently recorded. The phylogenetic analysis also indicated that the P. viticola species found on Parthenocissus quinquefolia in North America is identical to Plasmopara muralis in Europe. The geographicdistribution and host range of five pathogen species was determined through analysis of the internal transcribed spacer polymorphism of 896 isolates of P. viticola. Among three P. viticola species found on cultivated grape, one was restricted to Vitis interspecific hybrids within the northern part of eastern North America. A second species was recovered from V. vinifera and V. labrusca. and was distributed across most of the sampled region. A third species, although less abundant, was distributed across a larger geographical range, including the southern part of eastern North America. P. viticola clade aestivalis predominated (83% of isolates) in vineyards of the European winegrape V. vinifera within the sampled area, indicating that a single pathogen species may represent the primary threat to the European host species within eastern North America. [ABSTRACT FROM AUTHOR]

Europe is the historical cradle of viticulture, but grapevines (Vitis vinifera) have been increasingly threatened by pathogens of American origin. The invasive oomycete Plasmopara viticola causes downy mildew, one of the most devastating grapevine diseases worldwide. Despite major economic consequences, its invasion history remains poorly understood. We analyzed a comprehensive dataset of ∼2,000 samples, collected from the most important wine-producing countries, using nuclear and mitochondrial gene sequences and microsatellite markers. Population genetic analyses revealed very low genetic diversity in invasive downy mildew populations worldwide and little evidence of admixture. All the invasive populations originated from only one of the five native North American lineages, the one parasitizing wild summer grape (V. aestivalis). An approximate Bayesian computation-random forest approach allowed inferring the worldwide invasion scenario of P. viticola. After an initial introduction into Europe, invasive European populations served as a secondary source of introduction into vineyards worldwide, including China, South Africa, and twice independently, Australia. Only the invasion of Argentina probably represents a tertiary introduction, from Australia. Our findings provide a striking example of a global pathogen invasion resulting from secondary dispersal of a successful invasive population. Our study will also help designing quarantine regulations and efficient breeding for resistance against grapevine downy mildew. [Display omitted] • Population genetics approaches infer the origin and routes of P. viticola invasion • All invasive populations belong to only one of the five North American lineages • A first introduction occurred into Europe in the 1870s with wild grape import • European populations served as source for introductions into vineyards worldwide Downy mildew is a devastating grapevine disease. Fontaine et al. show that invasive populations worldwide originate from the North American lineage from wild Vitis aestivalis grapes. A first introduction of the pathogen into Europe occurred in the 1870s, from where further invasions occurred into other wine-producing areas worldwide. [ABSTRACT FROM AUTHOR]

Mating types are self-incompatibility systems that promote outcrossing in plants, fungi, and oomycetes. Mating-type genes have been widely studied in plants and fungi but have yet to be identified in oomycetes, eukaryotic organisms closely related to brown algae that cause many destructive animal and plant diseases. We identified the mating-type locus of Plasmopara viticola , the oomycete responsible for grapevine downy mildew, one of the most damaging grapevine diseases worldwide. Using a genome-wide association approach, we identified a 570-kb repeat-rich non-recombining region controlling mating types, with two highly divergent alleles. We showed that one mating type was homozygous, whereas the other was heterozygous at this locus. The mating-type locus encompassed 40 genes, including one encoding a putative hormone receptor. Functional studies will, however, be required to validate the function of these genes and find the actual determinants of mating type. Our findings have fundamental implications for our understanding of the evolution of mating types, as they reveal a unique determinism involving an asymmetry of heterozygosity, as in sex chromosomes and unlike other mating-type systems. This identification of the mating-type locus in such an economically important crop pathogen also has applied implications, as outcrossing facilitates rapid evolution and resistance to harsh environmental conditions. • Identification of the first oomycete mating-type locus using a GWAS approach • This 570-kb repeat-rich non-recombining region showed two highly divergent alleles • One mating type was heterozygous and the other homozygous, as in XY sex chromosomes Mating-type genes have been widely studied in plants and fungi but have yet to be identified in oomycetes. Dussert et al. utilize a genome-wide association approach to identify the mating-type locus of the oomycete plant pathogen Plasmopara viticola. The mating-type locus region displays similarity to heteromorphic sex chromosome systems. [ABSTRACT FROM AUTHOR]