The Characterization of Pathotypes in Grapevine Downy Mildew Provides Insights into the Breakdown of Rpv3, Rpv10, and Rpv12 Factors in Grapevines.

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 Rpv10 by a strain from Germany and the breakdown of Rpv12 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.

Identification of the First Oomycete Mating-type Locus Sequence in the Grapevine Downy Mildew Pathogen, Plasmopara viticola.

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.

A High-Quality Grapevine Downy Mildew Genome Assembly Reveals Rapidly Evolving and Lineage-Specific Putative Host Adaptation Genes.

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 dN/dS 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 dN/dS 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.

Soft selective sweeps in fungicide resistance evolution: recurrent mutations without fitness costs in grapevine downy mildew.

Adaptation produces hard or soft selective sweeps depending on the supply of adaptive genetic polymorphism. The evolution of pesticide resistance in parasites is a striking example of rapid adaptation that can shed light on selection processes. Plasmopara viticola, which causes grapevine downy mildew, forms large populations, in which resistance has rapidly evolved due to excessive fungicide use. We investigated the pathways by which fungicide resistance has evolved in this plant pathogen, to determine whether hard or soft selective sweeps were involved. An analysis of nucleotide polymorphism in 108 field isolates from the Bordeaux region revealed recurrent mutations of cytb and CesA3 conferring resistance to quinone outside inhibiting (QoI) and carboxylic acid amide (CAA) fungicides, respectively. Higher levels of genetic differentiation were observed for nucleotide positions involved in resistance than for neutral microsatellites, consistent with local adaptation of the pathogen to fungicide treatments. No hitchhiking was found between selected sites and neighbouring polymorphisms in cytb and CesA3, confirming multiple origins of resistance alleles. We assessed resistance costs, by evaluating the fitness of the 108 isolates through measurements of multiple quantitative pathogenicity traits under controlled conditions. No significant differences were found between sensitive and resistant isolates, suggesting that fitness costs may be absent or negligible. Our results indicate that the rapid evolution of fungicide resistance in P. viticola has involved a soft sweep.

Draft Genome Sequence of Plasmopara viticola, the Grapevine Downy Mildew Pathogen.

Plasmopara viticola is a biotrophic pathogenic oomycete responsible for grapevine downy mildew. We present here the first draft of the P. viticola genome. Analysis of this sequence will help in understanding plant-pathogen interactions in oomycetes, especially pathogen host specialization and adaptation to host resistance.

Adaptation of a plant pathogen to partial host resistance: selection for greater aggressiveness in grapevine downy mildew.

An understanding of the evolution of pathogen quantitative traits in response to host selective pressures is essential for the development of durable management strategies for resistant crops. However, we still lack experimental data on the effects of partial host resistance on multiple phenotypic traits (aggressiveness) and evolutionary strategies in pathogens. We performed a cross-inoculation experiment with four grapevine hosts and 103 isolates of grapevine downy mildew (Plasmopara viticola) sampled from susceptible and partially resistant grapevine varieties. We analysed the neutral and adaptive genetic differentiation of five quantitative traits relating to pathogen transmission. Isolates from resistant hosts were more aggressive than isolates from susceptible hosts, as they had a shorter latency period and higher levels of spore production. This pattern of adaptation contrasted with the lack of neutral genetic differentiation, providing evidence for directional selection. No specificity for a particular host variety was detected. Adapted isolates had traits that were advantageous on all resistant varieties. There was no fitness cost associated with this genetic adaptation, but several trade-offs between pathogen traits were observed. These results should improve the accuracy of prediction of fitness trajectories for this biotrophic pathogen, an essential element for the modelling of durable deployment strategies for resistant varieties.

Simultaneous quantification of sporangia and zoospores in a biotrophic oomycete with an automatic particle analyzer: disentangling dispersal and infection potentials.

Quantitative pathogenicity traits drive the fitness and dynamics of pathogens in agricultural ecosystems and are key determinants of the correct management of crop production over time. However, traits relating to infection potential (i.e. zoospore production) have been less thoroughly investigated in oomycetes than traits relating to dispersal (i.e. sporangium production). We simultaneously quantified sporangium and zoospore production in a biotrophic oomycete, for the joint assessment of life-cycle traits relating to dispersal and infection potentials. We used an automatic particle analyzer to count and size the sporangia and/or zoospores produced at t = 0 min (no zoospore release) and t = 100 min (zoospore release) in 43 Plasmopara viticola isolates growing on the susceptible Vitis vinifera cv. Cabernet Sauvignon. We were able to differentiate and quantify three types of propagules from different stages of the pathogen life cycle: full sporangia, empty sporangia and zoospores. The method was validated by comparing the sporangium and zoospore counts obtained with an automatic particle analyzer and under a stereomicroscope (manual counting). Each isolate produced a mean of 5.8 ± 1.9 (SD) zoospores per sporangium. Significant relationships were found between sporangium production and sporangium size (negative) and between sporangium size and the number of zoospores produced per sporangium (positive). However, there was a significant positive correlation between total sporangium production and total zoospore production. This procedure can provide a valid quantification of the production of both sporangia and zoospores by oomycetes in large numbers of samples, facilitating joint estimation of the dispersal and infection potentials of plant pathogens in various agro-ecological contexts.