Black spot disease, caused by the fungus Diplocarpon rosae, is a major threat to garden roses, leading to significant defoliation and reduced plant vigour. Several isolates originating from the United States and Europe have been characterised as races with different pathogenicity using a differential set of 10 plant hosts. However, no isolates from France have been tested on this set, and the races present in this country remain unknown. This study investigated the pathogenicity of seven monoconidial D. rosae strains isolated in France on 17 rose genotypes, including the previously published differential host set. Our results revealed that four of these strains have the virulence pattern of race 8, the prevalent race in North America. However, the commercial rose cultivars: Surrey (cultivar: KORlanum), High Voltage (cultivar: BAIage) and Ramblin' Red (cultivar: RADramblin) showed intermediate reactions to three strains, suggesting additional infection mechanisms. The four resistance genes available in the host set (Rdr3, Rdr4, Rdr5, Rdr6) remained effective against all tested strains. This study provides the first information on the races of D. rosae present in France and will be useful in breeding programmes for black spot-resistant roses.

Venturia inaequalis is an Ascomycota fungus responsible for apple scab, the main disease in apple orchards. During infection, the pathogen colonizes the subcuticular space and secretes hundreds of effectors to promote virulence. To mitigate disease impact, resistance genes such as Rvi6, which encodes a membrane-localized receptor-like protein, have been introduced into apple cultivars. However, over the past three decades, Rvi6-mediated resistance has been circumvented in orchards, with the emergence of virulent V. inaequalis strains. Through comparative genomics analyses and functional validation by complementation, we identified AvrRvi6 as the fungal determinant that activates Rvi6-mediated immunity. Screening 122 V. inaequalis strains worldwide we identified 20 distinct AvrRvi6 alleles. Evolution analysis demonstrated that the emergence of virulent alleles predates the domestication of apple thus revealing that wild Malus species constitute a reservoir of virulence. Using AlphaFold structural modeling, we showed that AvrRvi6 belongs to an expanded family that adopts a MAX effector fold, originally described for cytoplasmic effectors of the blast fungus Magnaporthe oryzae. Transient expression in Nicotiana benthamiana demonstrated that AvrRvi6 triggers allele-specific apoplastic immunity and validated three different molecular mechanisms (mutation, partial deletion and transposon insertion in the promoter) to circumvent Rvi6 recognition. This study delivers the first characterization of a V. inaequalis avirulence factor, uncovers an unexpected apoplastic role for a MAX effector, and shows how ancient virulence strategies compromise resistance durability in apple. ### Competing Interest Statement The authors have declared no competing interest. Agence Nationale de la Recherche, https://ror.org/00rbzpz17, ANR-22-CPJ1-0038-01 RFI Objectif Végétal de la Région Pays de la Loire

Apple scab caused by the fungus Venturia inaequalis, is the most important disease of apples in all temperate countries. The main commercial apple cultivars are susceptible to this disease and the phytosanitary treatments applied in the orchard to get rid of it represent a considerable economic and environmental cost ( up to 30 fungicide applications year-1 against this disease). To combat apple scab, we are developing a totally new biocontrol strategy combining two inventions patented by INRAE. The first invention makes the fungus non-virulent. We target the sexual phase of V. inaequalis by forcing it to reproduce with non-pathogenic strains belonging to the forma specialis pyracanthae of V. inaequalis (PYR strains), which results in the generation of non-virulent progeny the following spring. The second invention consists of applying the PYR strains in the spring to trigger immunity in the apple tree, thus protecting it against a subsequent scab attack. This breakthrough technical itinerary should cause a drastic collapse in the size of the pathogenic population in the orchard and should thus make it possible to considerably reduce the use of fungicides. This project, co-constructed with phytopathologists, geneticists, orchards experimenters and economists of INRAE, professionals of apple sector (CTIFL, IFPC) aims to prove the concept of this innovative strategy and its sustainability in the orchard (4 experimental sites in France) and to evaluate the conditions of acceptance and appropriation of the inventions by the apple sector.

Hybridization and adaptation to new hosts are important mechanisms of fungal disease emergence. Evaluating the risk of emergence of hybrids with enhanced virulence is then key to develop sustainable crop disease management. We evaluated this risk in Venturia inaequalis, the fungus responsible for the common and serious scab disease on Rosaceae hosts, including apple, pyracantha, and loquat. Field isolates from these three hosts and progenies obtained from five crosses between formae speciales isolates collected from pyracantha (f. sp. pyracantha) and apple (f. sp. pomi) were tested for their pathogenicity on the three hosts. We confirmed a strict host specificity between isolates from apple and pyracantha and showed that most isolates were able to cause disease on loquat. None of the 251 progeny obtained from five crosses between V. inaequalis f. sp. pyracantha and V. inaequalis f. sp. pomi could infect apple. If confirmed on more crosses, the inability of the hybrids to infect apple could lead to a novel biocontrol strategy based on a sexual hijacking of V. inaequalis f. sp. pomi by a massive introduction of V. inaequalis f. sp. pyracantha in apple orchards. This strategy, analogous to the sterile insect approach, could lead to the collapse of the population size of V. inaequalis and dramatically reduce the use of chemicals in orchards.

Understanding the relationships between host range and pathogenicity for parasites, and between the efficiency and scope of immunity for hosts are essential to implement efficient disease control strategies.In the case of plant parasites, most studies have focused on describing qualitative interactions and a variety of genetic and evolutionary models has been proposed in this context.Although plant quantitative resistance benefits from advantages in terms of durability, we presently lack models that account for quantitative interactions between plants and their parasites and the evolution of these interactions.Nestedness and modularity are important features to unravel the overall structure of host-parasite interaction matrices.Here, we analysed these two features on 32 matrices of quantitative pathogenicity trait data gathered from 15 plant-parasite pathosystems consisting of either annual or perennial plants along with fungi or oomycetes, bacteria, nematodes, insects and viruses.The performance of several nestedness and modularity algorithms was evaluated through a simulation approach, which helped interpretation of the results.We observed significant modularity in only six of the 32 matrices, with two or three modules detected.For three of these matrices, modules could be related to resistance quantitative trait loci present in the host.In contrast, we found high and significant nestedness in 30 of the 32 matrices.Nestedness was linked to other properties of plant-parasite interactions.First, pathogenicity trait values were explained in majority by a parasite strain effect and a plant accession effect, with no or minor parasite-plant interaction term.Second, correlations between the efficiency and scope of the resistance of plant genotypes, and between the host range breadth and pathogenicity level of parasite strains were overall positive.This latter result questions the efficiency of strategies based on the deployment of several genetically-differentiated cultivars of a given crop species in the case of quantitative plant immunity.

Leaf blotch caused by Alternaria spp. is a common disease in apple-producing regions. The disease is usually associated with one phylogenetic species and one species complex, Alternaria alternata and the Alternaria arborescens species complex (A. arborescens SC), respectively. Both taxa may include the Alternaria apple pathotype, a quarantine or regulated pathogen in several countries. The apple pathotype is characterized by the production of a host-selective toxin (HST) which is involved in pathogenicity towards the apple. A cluster of genes located on conditionally dispensable chromosomes (CDCs) is involved in the production of this HST (namely AMT in the case of the apple pathotype). Since 2016, leaf blotch and premature tree defoliation attributed to Alternaria spp. have been observed in apple-producing regions of central and south-eastern France. Our study aimed to identify the Alternaria species involved in apple tree defoliation and assess the presence of the apple pathotype in French orchards. From 2016 to 2018, 166 isolates were collected and identified by multi-locus sequence typing (MLST). This analysis revealed that all these French isolates belonged to either the A. arborescens SC or A. alternata. Specific PCR detection targeting three genes located on the CDC did not indicate the presence of the apple pathotype in France. Pathogenicity was assessed under laboratory conditions on detached leaves of Golden Delicious and Gala apple cultivars for a representative subset of 28 Alternaria isolates. All the tested isolates were pathogenic on detached leaves of cultivars Golden Delicious and Gala, but no differences were observed between the pathogenicity levels of A. arborescens SC and A. alternata. However, the results of our pathogenicity test suggest that cultivar Golden Delicious is more susceptible than Gala to Alternaria leaf blotch. Implications in the detection of the Alternaria apple pathotype and the taxonomic assignment of Alternaria isolates involved in Alternaria leaf blotch are discussed.

The quasi-universality of nestedness in the structure of quantitative plantparasite interactions Moury Benoît, Audergon Jean-Marc, Baudracco-Arnas Sylvie, Ben Krima Safa, Bertrand François, Boissot Nathalie, Buisson Mireille, Caffier Valérie, Cantet Mélissa, Chanéac Sylvia, Constant Carole, Delmotte François, Dogimont Catherine, Doumayrou Juliette, Fabre Frédéric, Fournet Sylvain, Grimault Valérie, Jaunet Thierry, Justafré Isabelle, Lefebvre Véronique, Losdat Denis, Marcel Thierry C., Montarry Josselin, Morris Cindy E., Omrani Mariem, Paineau Manon, Perrot Sophie, Pilet-Nayel Marie-Laure, Ruellan Youna(2020), bioRxiv, 2021.03.03.433745, ver. 4 peer-reviewed and recommended by Peer Community in Evolutionary Biologyhttps://doi.org/10.1101/2021.03.03.433745

Secondary contact between crops and their wild relatives poses a threat to wild species, not only through gene flow between plants, but also through the dispersal of crop pathogens and genetic exchanges involving these pathogens, particularly those that have become more virulent by indirect selection on resistant crops, a phenomenon known as "pestification." Joint analyses of wild and domesticated hosts and their pathogens are essential to address this issue, but such analyses remain rare. We used population genetics approaches, demographic inference and pathogenicity tests on host-pathogen pairs of wild or domesticated apple trees from Central Asia and their main fungal pathogen, Venturia inaequalis, which itself has differentiated agricultural and wild-type populations. We confirmed the occurrence of gene flow from cultivated (Malus domestica) to wild (Malus sieversii) apple trees in Asian forests, potentially threatening the persistence of Asian wild apple trees. Pathogenicity tests demonstrated the pestification of V. inaequalis, the agricultural-type population being more virulent on both wild and domesticated trees. Single nucleotide polymorphism (SNP) markers and the demographic modelling of pathogen populations revealed hybridization following secondary contact between agricultural and wild-type fungal populations, and dispersal of the agricultural-type pathogen population in wild forests, increasing the threat of disease in the wild apple species. We detected an SNP potentially involved in pathogen pestification, generating an early stop codon in a gene encoding a small secreted protein in the agricultural-type fungal population. Our findings, based on joint analyses of paired host and pathogen data sets, highlight the threat posed by cultivating a crop near its centre of origin, in terms of pestified pathogen invasions in wild plant populations and introgression in the wild-type pathogen population.

Massive gene flow between crops and their wild relatives may threaten the genetic integrity of wild species. Such threats are now well documented, but little is known about indirect consequences involving the spillover of crop pathogens to wild plants or introgression between crop and wild pathogens. To address these questions, we used population genetics approaches, demographic inference and pathogenicity tests on host-pathogen pairs composed of wild or domesticated apple trees of Central Asia and their fungal pathogen, Venturia inaequalis , itself showing differentiated agricultural-type and wild-type populations. We confirmed the occurrence of gene flow from cultivated to wild apple trees in Asian forests, threatening the Asian wild apple genetic integrity. SNP markers and demographic modeling revealed the occurrence of a secondary contact followed by hybridization between agricultural-type and wild-type fungal pathogen populations, and the dispersal of the agricultural-type pathogen in wild forests. We detected a SNP predicting the ability of the fungus to parasitize the different host populations, which induced an early stop codon in a gene coding for a small secreted protein in the agricultural-type fungal population, thus representing a putative avirulence gene which function loss would enable to parasitize cultivated apples. Pathogenicity tests in fact revealed the pestification of V. inaequalis , with higher virulence of the agricultural-type population on both wild and domesticated trees. Our findings highlight the threat posed by cultivating a crop near its center of origin, with the invasion of a pestified pathogen on wild plants and introgression in the wild-type pathogen.### Competing Interest StatementThe authors have declared no competing interest.

The Venturia genus comprises fungal species that are pathogens on Rosaceae host plants, including V. inaequalis and V. asperata on apple, V. aucupariae on sorbus and V. pirina on pear. Although the genetic structure of V. inaequalis populations has been investigated in detail, genomic features underlying these subdivisions remain poorly understood. Here, we report whole genome sequencing of 87 Venturia strains that represent each species and each population within V. inaequalis. We present a PacBio genome assembly for the V. inaequalis EU-B04 reference isolate. The size of selected genomes was determined by flow cytometry, and varied from 45 to 93 Mb. Genome assemblies of V. inaequalis and V. aucupariae contain a high content of transposable elements (TEs), most of which belong to the Gypsy or Copia LTR superfamilies and have been inactivated by Repeat-Induced Point mutations. The reference assembly of V. inaequalis presents a mosaic structure of GC-equilibrated regions that mainly contain predicted genes and AT-rich regions, mainly composed of TEs. Six pairs of strains were identified as clones. Single-Nucleotide Polymorphism (SNP) analysis between these clones revealed a high number of SNPs that are mostly located in AT-rich regions due to misalignments and allowed determining a false discovery rate. The availability of these genome sequences is expected to stimulate genetics and population genomics research of Venturia pathogens. Especially, it will help understanding the evolutionary history of Venturia species that are pathogenic on different hosts, a history that has probably been substantially influenced by TEs.

Genetic resistance is a useful strategy to control plant disease, but its effectiveness may be reduced over time due to the emergence of pathogens able to circumvent the defenses of the plant. However, the pyramiding of different resistance factors in the same plant can improve the effectiveness and durability of the resistance. To investigate the potential for this approach in apple to control scab disease we surveyed scab incidence in two experimental orchards located at a distance of more than 300 km planted with apple genotypes carrying quantitative resistance and major gene resistance alone or in combination. Our results showed that the effectiveness of pyramiding in controlling scab was dependent on the site and could not be completely explained by the effectiveness level of the resistances alone.

Quantitative resistance has gained interest in plant breeding for pathogen control in low-input cropping systems. Although quantitative resistance frequently has only a partial effect and is difficult to select, it is considered more durable than major resistance (R) genes. With the exponential development of molecular markers over the past 20 years, resistance QTL have been more accurately detected and better integrated into breeding strategies for resistant varieties with increased potential for durability. This review summarizes current knowledge on the genetic inheritance, molecular basis, and durability of quantitative resistance. Based on this knowledge, we discuss how strategies that combine major R genes and QTL in crops can maintain the effectiveness of plant resistance to pathogens. Combining resistance QTL with complementary modes of action appears to be an interesting strategy for breeding effective and potentially durable resistance. Combining quantitative resistance with major R genes has proven to be a valuable approach for extending the effectiveness of major genes. In the plant genomics era, improved tools and methods are becoming available to better integrate quantitative resistance into breeding strategies. Nevertheless, optimal combinations of resistance loci will still have to be identified to preserve resistance effectiveness over time for durable crop protection.

Quantitative plant resistance is supposed to be more durable than qualitative resistance for the control of plant diseases. However, it has been experimentally shown that erosion of quantitative resistance can occur. Cumulation of quantitative resistance loci (QRLs) in the same cultivar is considered to improve the efficiency and durability of quantitative resistance, but the choice of QRLs to be combined is of crucial importance. This study investigated whether the combination of QRLs acting on different stages of pathogen development could improve the efficiency of resistance in the apple scab pathosystem. The efficiencies of three QRLs were evaluated against 10 isolates of Venturia inaequalis and the stages of pathogen development that were affected by the QRLs were defined microscopically. A gain in the efficiency of resistance was observed when QRLs were pyramided compared to when they acted alone. Thanks to the combined effects of the individual QRLs, the pyramiding of the three QRLs hindered fungal development at different stages: before the penetration of the plant cuticule, after the penetration with hypersensitivity reaction, and during the colonization and asexual reproduction. These effects were dependent on the V. inaequalis isolates. These results suggest that the gain in efficiency of resistance by pyramiding may derive from the combination of different and complementary molecular mechanisms underlying QRLs. Thus, the resistance achieved from pyramiding such a combination of QRLs should be durable.

Quantitative plant resistance affects the aggressiveness of pathogens and is usually considered more durable than qualitative resistance. However, the efficiency of a quantitative resistance based on an isolate-specific Quantitative Trait Locus (QTL) is expected to decrease over time due to the selection of isolates with a high level of aggressiveness on resistant plants. To test this hypothesis, we surveyed scab incidence over an eight-year period in an orchard planted with susceptible and quantitatively resistant apple genotypes. We sampled 79 Venturia inaequalis isolates from this orchard at three dates and we tested their level of aggressiveness under controlled conditions. Isolates sampled on resistant genotypes triggered higher lesion density and exhibited a higher sporulation rate on apple carrying the resistance allele of the QTL T1 compared to isolates sampled on susceptible genotypes. Due to this ability to select aggressive isolates, we expected the QTL T1 to be non-durable. However, our results showed that the quantitative resistance based on the QTL T1 remained efficient in orchard over an eight-year period, with only a slow decrease in efficiency and no detectable increase of the aggressiveness of fungal isolates over time. We conclude that knowledge on the specificity of a QTL is not sufficient to evaluate its durability. Deciphering molecular mechanisms associated with resistance QTLs, genetic determinants of aggressiveness and putative trade-offs within pathogen populations is needed to help in understanding the erosion processes.

In pathogens, introgressions through secondary contacts between divergent populations from agricultural and nonagricultural disease reservoirs are expected to have crucial evolutionary and epidemiological implications. Despite the importance of this question for disease management, experimental demonstrations of these implications remain scarce. Recently, we identified a virulent population of the apple scab pathogen Venturia inaequalis that migrated from nonagricultural hosts to European domestic apple orchards. Here, we investigated the occurrence of gene flow between agricultural and nonagricultural populations sampled in two orchards, and thereafter its consequences on the pathogenicity of hybrids. Population genetic structure and demographic inferences based on the genotypes of 104 strains revealed a high amount of gene flow between the two populations in one orchard. In this site, mating between populations was made possible by the presence of a common host. Our results revealed an invasion of the virulent trait in the agricultural population; a main direction of introgression in hybrids from the agricultural to nonagricultural genetic backgrounds; and a population of hybrids with transgressive traits. We demonstrate a secondary contact with gene flow between divergent populations of pathogens. Our findings highlight evolutionary and epidemiological changes in pathogens and have concrete implications for sustainable disease management.

As reproductive units, seeds are essential for the survival of plant species and for their role in stand establishment. Seeds are also a crucial input for agriculture, as they are the vector of genetic progress and of different plant protection treatments. Germination is the first step in plant development and is a complex process (Bewley and Black, 1994; Nonogaki et al., 2010; Rajjou et al., 2012). It is a critical phase in the plant life cycle during which quiescent seeds are reactivated to produce seedlings. Germination sensus stricto begins with uptake of water by the seed and terminates with the initiation of growth of the embryo axis and radicle protrusion (Come and Thevenot, 1982; Nonogaki et al., 2007). During this initial stage, seed water content increases in direct relation with the moisture content of the environment, allowing seed reserves to be hydrolyzed and metabolism to restart. Seed water content has to reach a threshold value to allow germination and to maintain this value until the radicle emerges (Bewley and Black, 1994; Kigel and Galili, 1995). Subsequent seedling growth depends