Infection, Genetics and Evolution 8 (2008) 534540

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Infection, Genetics and Evolution

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Single nucleotide polymorphisms reveal multiple introductions into France of Plasmopara halstedii, the plant pathogen causing sunower downy mildew
Franc ois Delmotte a,*, Xavier Giresse a, Sylvie Richard-Cervera a, Jessica MBaya a, Felicity Vear b, Jeanne Tourvieille b, Pascal Walser b, Denis Tourvieille de Labrouhe b
a b

ge INRA, Institut des Sciences de la Vigne et du Vin, UMR 1065 Sante Ve tale (INRA-ENITAB), Centre de Recherche Bordeaux-Aquitaine, BP 81, 33883 Villenave dOrnon cedex, France INRA, UMR 1095 GEDEC (INRA-Universite Blaise Pascal), Centre de Recherches de Clermont-Ferrand, Domaine de Crouelle, 63100 Clermont-Ferrand, France

A R T I C L E I N F O

A B S T R A C T

Article history: Received 11 October 2007 Received in revised form 28 February 2008 Accepted 29 February 2008 Available online 18 March 2008 Keywords: Helianthus annuus Invasive parasite Oomycetes Pathotype Race SNP Virulence prole

Plasmopara halstedii, the causal agent of sunower downy mildew, displays a gene-for-gene interaction with its host plant, Helianthus annuus and other species of the genus. Monitoring of the evolution of virulent races in France over a 19-year period led to the identication of 14 different races (or pathotypes). Twelve expressed sequence tag (EST)-derived markers displaying SNPs and insertion deletion variations have recently been identied in P. halstedii. We used these markers to study the genetic structure and the evolution of sunower downy mildew races. Bayesian assignment analysis identied three genetically differentiated groups of isolates organized around the rst three races described in France. Strong genetic substructuring according to geographic origin of races was observed, conrming that these three groups corresponded to three separate introductions into France of isolates with different genetic and phenotypic backgrounds. Our results suggest that multiple introductions of P. halstedii isolates may have provided the raw material for more complex processes in the evolution of races, such as recombination between races or clonal evolution through mitotic instability. 2008 Elsevier B.V. All rights reserved.

1. Introduction Sunower downy mildew is caused by Plasmopara halstedii (Berlese & de Toni), an invasive species native to North America, where wild sunower (Helianthus annuus) is endemic. It was rst described in Indiana, Iowa and Minnesota in the early 1920s (Yong and Morris, 1927). This pathogen spreads into Europe, probably in the seeds of its host plant (Ioos et al., 2007), and was rst reported in Europe, in Russia, at the beginning of 1960s (Leppik, 1962; Novotelnova, 1966). Since 1992, P. halstedii has been subject to quarantine regulation in the European Union (directive 92/103/ CEE). P. halstedii is a homothallic oomycete, whose cycle is made up of a single sexual generation permitting overwintering and one or perhaps two asexual generations which occur during the growing season (Spring et al., 2006). Gene-for-gene interactions have been demonstrated between this pathogen and its host plant (Tourvieille de Labrouhe et al., 2000), and many physiological races (or pathotypes) have been characterized worldwide (Gulya, 2007; Gulya et al., 1991; Lafon et al., 1996; Moinard et al., 2006;

* Corresponding author. E-mail address: delmotte@bordeaux.inra.fr (F. Delmotte). 1567-1348/$ see front matter 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.meegid.2008.02.012

Molinero-Ruiz et al., 2002; Rashid, 1993; Spring et al., 1994; Zimmer, 1974). The introduction of genetic resistance into sunower varieties has been, and remains the most efcient method for controlling the disease. This resistance concerns a number of downy mildew races and is conferred by major genes, denoted Pl (Vear et al., 1997, 2003). In France, the monitoring of sunower downy mildew races has shown that, after a long period of stability, the effectiveness of the major resistance genes (Pl1, Pl2, Pl5 and Pl6) has recently been overcome. Fourteen different races have been described in France, nine of which emerged during the last 7 years. When sunower breeding programs were rst initiated in France, only one race of P. halstedii was known (race 100). This rst race of P. halstedii collected in Europe has a specic virulence prole different from that of American races. With changes in nomenclature, it has been called the European race (Sackston, 1981), race 1, and now race 100 (Tourvieille de Labrouhe, 1999). Until 1980s, race 100 was widely distributed throughout France and was the only race present in this country. Selection pressure on the pathogen was strong in 1980s, because all the hybrids on sale in France carried either Pl1 or Pl2, both of which confer resistance to race 100 (Sackston, 1981). At the end of this decade, two new virulent races (710 and 703) appeared in France: race 710 found in 1988 in the Indre (Tourvieille de Labrouhe, 1988)

F. Delmotte et al. / Infection, Genetics and Evolution 8 (2008) 534540 Table 1 Name of race (pathotype), date of rst isolation in France and virulence proles for the 14 French reference isolates of P. halstedii used in this study Race namea Year of isolation 1966 1988 1989 1995 1995 2000 2001 2002 2002 2002 2004 2004 2004 2005 Virulence proles on differential hostsb D1 S S S S S S S S S S S S S S D2 R S S S S S S S S S S S S S D3 R S S R S R R R S S R S S S D4 R S R R R R S R R S S R S S D5 R R R R R R R R R R S R R S D6 R R R R R R R R R R R R R R D7 R R S R R R R S R R R S S R D8 R R S R R R R S R R R S S R D9 R R R R R S S S S S S S S R

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100 710 703 300 700 304 314 307 704 714 334 707 717 730
a b

According to Gulya et al. (1998). S: susceptible (compatible interaction); R: resistant (incompatible interaction).

and mostly restricted to the northern part of France, and race 703 which predominates in southern France, where it was rst described in 1989 (de Guenin, 1990). From 1989 to 1995, races 100, 703 and 710 were the only races found in French downy mildew populations (Meliala, 2001; Roeckel-Drevet et al., 1997). However, pathogen evolution has accelerated since 1995, due to the massive deployment of new resistance genes, such as Pl6 and Pl7, and as many as 11 new races of P. halstedii have been characterized (see Table 1 for a comprehensive chronology): 300, 700 (1995), 304 (2000), 314 (2001), 307, 704, 714 (2002), 334, 707, 717 (2004) and 730 (2005) (Moinard, 20022005; Tourvieille de Labrouhe et al., 2005). Today, the total population of French races can overcome the resistance genes in seven of the nine downy mildew differential lines of H. annuus, and the Pl2 and Pl6 genes have each been overcome by eight races (Moinard et al., 2006; Tourvieille de Labrouhe et al., 2000). Our understanding of the recurrent breakdown of sunower major resistance genes can be improved by new ndings concerning the key processes governing the evolution of P. halstedii populations. Knowledge of the evolutionary potential of plant pathogen species has improved the management of resistance genes and maximized their durability in space and time (McDonald and Linde, 2002). A population genetics approach can be used to evaluate the major forces driving evolutioni.e. selection, mutation, recombination, genetic drift and gene ow. New virulence proles in P. halstedii populations may evolve via several different mechanisms: (i) mutations in virulence genes in clonal lineages; (ii) recombination following crosses between different races; (iii) the introduction of races able to overcome the resistance of sunower varieties grown in one area. Sound knowledge of the population genetic structure of P. halstedii is required to distinguish between these possibilities. Previous genetic studies of P. halstedii in Europe have reported low levels of genetic and genotypic diversity in this species, with no clear genetic structure revealed with RAPD markers (Komjati et al., 2004; Roeckel-Drevet et al., 1997, 2003), ISSR (Intelmann and Spring, 2002) or ITS sequences (Spring et al., 2006; Thines et al., 2005). This precluded any reliable conclusions on the mode of reproduction, genetic structuring or the extent to which races are related in this species. It is possible that no genetic structure in P. halstedii populations was identied in previous studies because the molecular markers used were non-specic and insufciently polymorphic within this species. AFLP, ISSR and RAPD markers have been used in many studies of plant pathogens because they

allow the rapid screening of many loci within a genome (Fry, 2001). However, these markers are dominant, anonymous and not specic to the target species. This lack of specicity is a major drawback in studies of obligate endoparasites, such as downy mildews. In contrast, single nucleotide polymorphisms (SNPs) are promising molecular markers for population genetics as they are widespread throughout the genome, codominant, specic and have a high resolving power. However, SNPs have been little used for analyses of the genetic structure of populations of fungi, and of plant pathogens in particular (Morin et al., 2004). One reason for this is that it has been difcult to characterize SNPs in non-model organisms, principally due to a lack of genomic data (Schlotterer, 2004). The development of new methods for screening for DNA polymorphism has rendered possible the extensive development of such makers for plant pathogen species (Brumeld et al., 2003; Schlotterer, 2004). We recently developed a set of 12 EST-derived markers displaying SNPs and insertiondeletion variations for genetic studies of sunower downy mildew populations (Giresse et al., 2007). The aim of this study was to investigate the evolutionary processes responsible for changes in the pathogenic features of P. halstedii. We used 12 EST-derived markers to perform a genetic analysis of races of P. halstedii characterized over a 19-year period of monitoring in France (19882006). We addressed four main questions, using a mixture of phenotypic characterization and molecular genotyping: how powerful are SNP and EST-derived markers for studies of the population structure of P. halstedii? What is the relative importance of outcrossing and clonal reproduction in the French P. halstedii population? How are the different races of P. halstedii related? Which evolutionary mechanisms are involved in the emergence of new races in this plant pathogenic species? 2. Materials and methods 2.1. Sample collection and DNA extraction We analyzed 24 isolates of P. halstedii collected in France between 1966 and 2006. Fourteen of these isolates are references
Table 2 Race (pathotype), collection site and date of isolation in France for the 24 isolates of P. halstedii of this study Race 100* 100 100 710* 710 710 710 710 703* 703 703 703 703 300* 700* 304* 314* 307* 704* 714* 334* 707* 717* 730* partement) Collection site (de Unknown Charente-Maritime Cher Indre Cher Unknown Maine-et-Loire ` Deux-Sevres Lot-et-Garonne Tarn Lot-et-Garonne Haute-Garonne Gers Aude Haute-Garonne Aude Manche Haute-Garonne ` Deux-Sevres Gers Charente Lot-et-Garonne Gers Tarn Year of collection 1966 1993 1993 1988 1993 2000 2004 2006 1989 1993 2001 2004 2006 1995 1995 2000 2001 2002 2002 2002 2004 2004 2004 2005

The asterisk (*) indicates isolates corresponding to the reference race (see Table 1).

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isolates, corresponding to the rst description of the race concerned in France (Table 1). The other 10 isolates (Table 2) were obtained from the French Plant Protection Service monitoring program (Moinard et al., 2006). Each sample resulted from the bulking of several infected sunower leaves collected on one or two plants in a eld. For samples corresponding to the rst identication of a race in France (Tables 1 and 2), these bulks were puried, i.e. a monozoosporangium isolation was achieve before multiplication of the isolate on a susceptible hostplant genotype (Sakr et al., 2007). 2.2. Race characterization Downy mildew races are dened on the basis of virulence proles on a set of differential hosts carrying different Pl resistance genes (Table 1). Resistance tests were performed as described by Cohen and Sackston (1974), with the modications proposed by Mouzeyar et al. (1993): 15 days after inoculation, plants were incubated for 48 h in a saturated atmosphere. Plants were scored as susceptible if sporulation was observed in cotyledons and leaves, and as resistant if no sporulation or only light sporulation was seen on cotyledons. Races were named according to the international nomenclature of P. halstedii races proposed by Gulya et al. (1998). 2.3. DNA extraction and molecular typing For each isolate, DNA was isolated from infected plant tissue, as previously described for P. viticola (Delmotte et al., 2006; Chen et al., 2007). We then used the 12 recently identied polymorphic EST-derived markers (Giresse et al., 2007) to genotype P. halstedii isolates. Pha6, Pha54, Pha56, Pha82, Pha99, Pha79 and Pha120 are seven SNPs transformed into cleaved amplied polymorphism sequence (CAPS) markers or directly screened by PCR-SSCP. Pha39, Pha42, Pha43, Pha106 and Pha47 correspond to ve insertion deletion (indel) polymorphisms automated on a Beckman Coulter Ceq8000 capillary sequencer or visualized directly by agarose gel electrophoresis (Giresse et al., 2007). 2.4. Statistical analysis The resolving power of EST-derived markers was evaluated with Multilocus 1.2 (Agapow and Burt, 2001), which calculates the percentage of discriminated multilocus genotypes commensurate to the number of combined loci after 1000 resamplings. Identical multilocus genotypes sharing the same alleles at all loci may result from distinct sexual reproduction events or clonal reproduction. We assessed the likelihood of copies of multilocus genotypes

resulting from sexual reproduction, by calculating the probability Pgen(f), taking into account departure from HardyWeinberg equilibrium, as estimated for the sample set with GenClone 1.0 (Arnaud-Haond and Belkhir, 2007). Genepop version 3.2a was used to calculate expected and observed heterozygosities, unbiased estimates of FIS and FST (Weir and Cockerham, 1984). We investigated the genetic structure of P. halstedii races, using a Bayesian approach to genetic mixture analysis based on STRUCTURE v2.2 (Pritchard et al., 2000; Falush et al., 2003). This method can be used to estimate parameters separately from the posterior probability distribution of allele frequencies. Parameter estimation assumes panmixia, and that each locus is at Hardy Weinberg equilibrium (HWE) and independent of the others. Nonetheless, this Bayesian approach is quite robust even when some deviations from these assumptions occur. Using the admixture model, we estimated the number of genetic clusters, K, to which the pathogens should be assigned. Using the full data set, 310 parallel Markov chains were run for all models of K, with a burn-in of 10,000 iterations and a run length of 105 iterations following the burn-in. For each run, the ln likelihood of each model was calculated. The genetic relationships between French races was assessed by correspondence analysis (Benzecri, 1982) performed using the AFC procedure available in genetix 4.05 (Belkhir et al., 2001) on the matrix of multilocus genotypes. Allele shared distance (DAS, Jin and Chakraborty, 1993) were computed using Populations 1.2.28 software (Langella, 1999). A Mantel test was performed to assess the correlation between pairwise geographic and genetic distance (DAS) matrices for each isolate, using Genepop software. 3. Results 3.1. Genotypic structure Based on combinations of the 12 genomic markers, we identied 11 different multilocus genotypes among the 24 isolates analyzed. Three multilocus genotypes (MLG1, MLG2, MLG9) were found to have multiple isolates: ve isolates for MLG1, six for MLG2, ve for MLG9 (Table 3). A combination of eleven EST markers was sufcient to discriminate all the multilocus genotypes in the dataset, demonstrating the high resolving power of the markers (Fig. 1). Three races were represented by more than one isolate (race 100 represented by three isolates, races 703 and 710 each represented by ve isolates; Table 2). Isolates of the same race

Table 3 Multilocus genotypes (MLG) characterized using 12 EST-derived genomic markers on the 24 French isolates of P. halstedii MLG N Races Clustera EST-derived markers Pha6 MLG1 MLG2 MLG3 MLG4 MLG5 MLG6 MLG7 MLG8 MLG9 MLG10 MLG11 5 6 1 1 1 1 1 1 5 1 1 100, 300, 304 703, 307 700 707 730 314 334 704 710 714 717 1 2 2 2 2 3 3 3 3 3 3 2/2 1/1 1/1 1/1 1/1 2/2 1/1 2/2 1/1 2/2 1/1 Pha39 2/2 1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/1 Pha42 1/1 1/1 1/1 1/1 1/1 2/2 2/2 1/1 2/2 2/2 1/2 Pha43 1/1 1/1 1/2 1/1 2/2 2/2 1/1 2/2 2/2 2/2 1/2 Pha54 1/1 1/1 1/1 1/1 1/1 1/1 2/2 1/1 1/1 1/1 1/1 Pha56 1/1 1/1 1/1 1/1 1/1 2/2 1/1 2/2 2/2 2/2 2/2 Pha74 1/1 1/1 1/1 1/1 1/1 2/2 2/2 2/2 2/2 1/1 2/2 Pha79 3/3 2/2 1/2 2/2 1/1 1/1 1/1 1/1 1/1 1/1 1/1 Pha82 2/2 1/1 1/1 2/2 1/1 1/1 1/1 2/2 1/1 1/1 1/1 Pha99 2/2 1/1 1/1 1/1 1/1 1/1 1/1 2/2 1/1 1/1 2/2 Pha106 1/1 2/2 2/2 1/1 2/2 2/1 2/2 2/2 2/2 2/2 2/2 Pha120 2/2 1/1 1/1 1/1 1/1 2/2 1/1 1/1 1/1 2/2 1/1

For each MLG, we indicate the number of isolates (N), race and cluster, and for each locus, the two alleles found. a Cluster number was obtained from the Bayesian assignation analysis performed with STRUCTURE software.

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Fig. 1. Proportion of P. halstedii genotypes discriminated (number of multilocus genotypes divided by the total number of isolates) as a function of the number of loci combined in the analysis.

presented an identical multilocus genotype, indicating that the three races may correspond to three clonal lineages (Table 3). Conversely, two multilocus genotypes included more than one race: the rst multilocus genotype (MLG1) comprised races 100, 300 and 304 and the second (MLG2) comprised races 307 and 703 (Table 3). We checked that isolates with the same multilocus genotype belonged to the same clonal lineage, by calculating the probability of identical multilocus genotypes resulting from sexual reproduction events (taking the selng rate into account), using GenClone v1. We were able to reject this hypothesis at the P < 105 level, conrming that identical multilocus genotypes probably resulted from asexual reproduction. As clonal reproduction leads to strong deviation from panmixia, further population genetic analysis were performed on a dataset comprising only one copy of each multilocus genotype (Sunnucks et al., 1997). 3.2. Population structure and genetic relations of races The expected heterozygosity level was consistent with high levels of genetic diversity across loci (0.349 < HE < 0.677). Observed heterozygosity levels were much lower, with a mean HO of 0.026. Only three of the 11 distinct multilocus genotypes were heterozygous: two at two loci (MLG3, MLG11) and one at one loci (MLG6) (Table 3). For the bayesian analysis, a single splitting solution was found for K = 3 for the full data set (Evanno et al., 2005). Probabilities of assignment of each race to each cluster are shown in Fig. 2. Assuming an arbitrary threshold of q = 0.9 for assignment to clusters, 12 over the 14 races (85.7%) were assigned to one of the three clusters, indicating high level of genetic differentiation between clusters (FST = 0.637 with a probability P < 105). Fig. 3 shows the scores of the races on the rst two components of the factorial analysis. For each race, the probability of assignment obtained from the Bayesian analysis was reported on the correspondence analysis. Axis 1 (representing 34.2% of total genetic variability) discriminates cluster 1 from clusters 2 and 3. Axis 2 (representing 25.2% of the total genetic variability) separated races belonging to cluster 2 from races belonging to cluster 3 (Fig. 3). The rst cluster corresponded to a single multilocus genotype, including three races: 100 (N = 3), 300 (N = 1) and 304 (N = 1). The second cluster included four multilocus genotypes and ve races: 703 (N = 5), 307 (N = 1), 700 (N = 1), 730 (N = 1), 707 (N = 1). The third cluster included six races with different multilocus genotypes: 710 (N = 5), 334 (N = 1), 314 (N = 1), 714 (N = 1), 717 (N = 1) and 704 (N = 1). Two races (707, 334) could be considered as hybrid genotypes since they were assigned to two different group with a

Fig. 2. Probabilities of assignment of each race to each of the clusters inferred with structure software (white, gray, black). The date of the rst description in France of each race is indicated in brackets under the race name.

threshold >20% (Fig. 2). For each of the three differentiated genetic clusters, we found one of the three races described before 1990 in France: cluster 1 included race 100 (1966), cluster 2 race 703 (1989), and cluster 3 race 710 (1988). Moreover, the genetic groups identied by the Bayesian analysis clustered races according to their geographical origin: cluster 1 corresponds to races 100 plus two races found in south-eastern France (Aude), cluster 2

Fig. 3. Factorial correspondence analysis (FCA) performed on the multilocus genotypes of P. halstedii races based on 12 EST-derived markers. Probabilities of assignment of each race to each cluster are reported on the FCA analysis using a colored pie-chart: white = cluster 1, gray = cluster 2, black = cluster 3.

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Fig. 4. Geographic origin of 21 isolates of P. halstedii collected in France from 1966 to 2005. French reference races are shown in bold. Using a threshold value of 60%, each race was assignment to a cluster inferred from the Bayesian analysis (white = cluster 1, gray = cluster 2, black = cluster 3). The matrix of pairwise genetic distance (DAS) estimates between isolates (including repeated genotypes) was signicantly correlated with the matrix of geographical distance (Mantel test, P = 0.0293).

French population of P. halstedii. Previous genetic studies of P. halstedii suffered from a lack of variability (Komjati et al., 2004; Roeckel-Drevet et al., 1997, 2003; Spring et al., 2006; Thines et al., 2005; Intelmann and Spring, 2002), whereas the EST-derived markers used here displayed high levels of resolving power and intraspecic polymorphism. Unlike the ngerprints obtained with dominant markers (Intelmann and Spring, 2002; Roeckel-Drevet et al., 1997, 2003), these codominant markers accurately estimated intra-individual heterozygosity. Moreover, whereas previous studies on P. halstedii has not indicated any relationship between genetic prole and race, EST-derived markers made it possible to demonstrate the presence of relationships between races, and to assess those relationships using assignment methods. Finally, these markers are specic and could therefore be used for the highthroughput genotyping of isolates directly from sporulating lesions collected from host leaves, avoiding the need for laborintensive isolate subculture (Giresse et al., 2007). Because Oomycetes are diploid organisms, the use of codominant markers is much more powerful in population genetic studies. Microsatellite markers may have provided an interesting alternative to SNPs for population genetic studies of this species, as reported for Plasmopara viticola, a closely related species causing grapevine downy mildew (Delmotte et al., 2006; Chen et al., 2007). They are also reliable, codominant and specic, but may in some cases be very difcult to isolate (Dutech et al., 2007). We, therefore, believe that SNPs may be the most useful type of marker in diploid plant pathogenic species for which genomic nucleotide sequences are available. 4.2. Genetic structure and evolution of races

comprises races from the south-western region of France and cluster 3 brings together races found in western and northern France (with the exception of one 710 isolated in the Gers) (Table 2, Fig. 4). Conrming this result, the matrix of pairwise genetic distance (DAS) estimates between isolates was signicantly correlated with the matrix of geographical distance (Mantel test, P = 0.0293), indicating that the spatial distribution of races in France is not random (Fig. 4). Calculations of the genetic distance within each cluster indicated that cluster 3 was more heterogeneous than cluster 2 (Table 4). The clusters were well differentiated, with a particularly marked distinction between cluster 1 and clusters 2 and 3 (Table 4). This result was conrmed by the identication of two private alleles for cluster 1, at loci Pha39 and Pha79 (Table 3). 4. Discussion 4.1. The value of EST-derived markers for genetic studies of plant pathogens The use of EST-derived markers for this study resulted in the rst comprehensive description of the genetic structure of the
Table 4 Allele shared distance within and between the three clusters, as dened by the Bayesian analysis conducted with STRUCTURE software Clusters Intracluster C1 C2 C3 Intercluster C1C2 C1C3 C2C3 DAS 0 0.167 0.336

0.536 0.691 0.297

The nding that P. halstedii races cluster into three genetically differentiated groups, each including one of the rst races described in France (i.e. 100, 703, 710), sheds new light on sunower downy mildew evolution. Races 100, 710 and 703 correspond to three clonal lineages strongly differentiated from each other genetically and with different virulence proles (Table 1). These results reect multiple introductions of this pathogen in France: the rst corresponding to race 100 (before 1966), which is now widely distributed throughout France, the second to race 710 in northern France (before 1988) and the third to race 703 in south-western France (before 1989). These three introductions of P. halstedii may have provided the raw genetic materials for more complex evolutionary processes, such as recombination between races or the accumulation of mutations in clonal lineages (clonal evolution), in the emergence of new races. Oomycetes have already been reported to display a large range of processes generating phenotypic variability, including interspecic hybridization (Brasier et al., 1999), recombination resulting from sexual reproduction, and genome instability caused by mitotic recombination, gene conversion, transposable elements or dispensable chromosomes (Kamoun, 2003). Progress in genetic studies of Phytophthora is very rapid, but very little is known about the population genetics of downy mildews which are obligate parasite. Our study provides several important insights into the genetic basis of phenotypic evolution in P. halstedii. First, our data indicate that occasional recombination events between genetically differentiated races may have led to the emergence of new races: race 334 is intermediate between races 710 and 703 (Figs. 2 and 3), indicating that it may have resulted from crosses between isolates from clusters 2 and 3. In this case, the recombination of genetically differentiated races has generated a new phenotypic prole, characterized by the ability of races 334 to overcome the resistance gene of the differential host D5 (Table 1). Similarly, both genetic (Figs. 2 and 3) and phenotypic

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(Table 1) data have provided strong evidence to suggest that race 707 resulted from the recombination of races 703 and 304. These examples clearly illustrate the contribution of outcrossing events to the emergence of a new race in the French population of P. halstedii. Second, our results provide evidence to suggest that a clonal lineage (one multilocus genotype) can include several races (Table 3). This suggests that mutations in a clonal lineage may lead to the emergence of new races in the same genetic background: this is the cases for races 100, 300 and 304 and for races 703 and 307 (Figs. 2 and 3; Table 3). Two non-exclusive mechanisms may be responsible for this phenotypic variability in avirulence determinants. The rst of these mechanisms is recombination within the lineage via the homothallic fusion of gametangia. This hypothesis is certainly the most likely, given the high selng rate of sunower downy mildew inferred from our analysis. Alternatively, genetic recombination via parasexual events may have generated this variability as it has been shown recently that mitotic recombination between different isolates of P. halstedii may be involved in the generation of new phenotypes within a population (Spring and Zipper, 2006). In this case, mitotic recombination within a genetic lineage would be required, and no such phenomenon has ever been described. The importance of mutation events in race evolution has already been highlighted in several Oomycetes plant pathogens such as Bremia lactucae (Lebeda and Petrzelova, 2004; Lebeda and Zinkernagel, 2003) and Phytophtora infestans (Andrivon, 1994). Although surprising at rst sight, the greater importance of mutation than of recombination in populations able to outcross may be related to the large number of clonal cycles of fungal multiplication during epidemics. The combination of large populations, due to asexual reproduction of the pathogen, with strong selective pressures induced by resistance genes in the plant is likely to favor the emergence of new virulent races. Finally, the origin of ve of the reference races (314, 700, 704, 714, 717, 730) remains unexplained. First, they may have resulted from introduction via seed transportation, provided that the introduced isolates had new virulence proles in a genetic background similar to that of race 710 or 703 (Fig. 2). If this was the case, it would raise questions about identication of the source populations of this plant pathogen recently introduced into France. Also, there was a long delay between observation of races 703 and 710 and appearance of the new races (11 years, in spite of a concerted effort to detect new races), which except for race 700, have never been reported in the USA. Second, the new races may have arisen via the accumulation of mutations in established races (as discussed above). This hypothesis is probably the least likely, as it implies very high mutation rates for molecular markers to account for the genetic distances observed between races of the same cluster (difference of 10 alleles between the most distant races of cluster 3). Third, a two-step process, involving crosses between two races generating new phenotypic proles followed by several generations of selng resulting in the random xation of one parental allele at each locus may have occurred. This would generate races resembling either race 710 or race 703. It is difcult to determine with certainty which of these hypotheses is correct based solely on an analysis of the genetic structure of natural populations of the pathogen. Improvements in our understanding of the importance of these mechanisms would require investigations of race evolution under controlled experimental conditions. 5. Conclusion The EST-derived markers used in this study displayed high levels intraspecic polymorphism which made it possible to infer

the reproductive mode of P. halstedii and to assess the relationships among French races. The biology of P. halstedii is indicative of a pathogen species for which there is a high risk of the population adapting to plant resistance genes: the pathogen population is large and this pathogen produces oospores, which can survive in the soil in the absence of crops, and displays obligate sexual recombination, mainly by selng, and asexual multiplication. The potential of P. halstedii to evolve new races has also been enhanced by the introduction of several genetically differentiated genotypes: repeated introductions of P. halstedii isolates, combined with the selective pressure exerted by host resistance genes, may have greatly accelerated the evolution of new races in France (DesprezLoustau et al., 2007; Novak, 2007).

Acknowledgments This work was supported by the CETIOM (Centre Technique agineux Me tropolitains). We thank Cyril Interprofessionnel des Ole Dutech and Marie-Laure Desprez-Loustau for useful comments on an earlier version of the manuscript, and Julie Sappa for correcting the English. References
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