Marc Leconte

Evidence of genetic recombination in wheat yellow rust populations of a Chinese oversummering area.

Wheat yellow rust (Puccinia striiformis f.sp. tritici) (PST) has been described as a strongly clonal species in both European and Australian populations, with very limited molecular diversity but rapidly evolving virulences. Contrastingly, marked genetic diversity has been reported in Chinese PST populations. To test whether such variability could originate from oversummering areas, we assessed the diversity of virulence and molecular markers (AFLP and SSR) using 412 PST isolates from the highlands of Tianshui county in Gansu province. Very marked phenotypic and genotypic diversity (38% and 89%, respectively) was found. No genetic structure dependent on the sites sampled (Fst=0.004) or altitude distribution (Fst=0.0098) was detected, indicating important gene flow at the county scale. This study also revealed genetic recombination between molecular markers and thus strongly suggests the existence of a sexual or parasexual cycle in PST in Tianshui county. The observations of higher rates of sexual spore production in genotypes originating from Tianshui are the very first elements suggestive of the existence of a sexual cycle in this species.

Genetic structure and local adaptation of European wheat yellow rust populations: the role of temperature-specific adaptation

Environmental heterogeneity influences coevolution and local adaptation in host–parasite systems. This also concerns applied issues, because the geographic range of parasites may depend on their capacity to adapt to abiotic conditions. We studied temperature‐specific adaptation in the wheat yellow/stripe rust pathogen, Puccinia striiformis f.sp. tritici (PST). Using laboratory experiments, PST isolates from northern and southern France were studied for their ability to germinate and to infect bread and durum wheat cultivars over a temperature gradient. Pathogen origin × temperature interactions for infectivity and germination rate suggest local adaptation to high‐ versus low‐temperature regimes in south and north. Competition experiments in southern and northern field sites showed a general competitive advantage of southern over northern isolates. This advantage was particularly pronounced in the southern ‘home’ site, consistent with a model integrating laboratory infectivity and field temperature variation. The stable PST population structure in France likely reflects adaptation to ecological and genetic factors: persistence of southern PST may be due to adaptation to the warmer Mediterranean climate; and persistence of northern PST can be explained by adaptation to commonly used cultivars, for which southern isolates are lacking the relevant virulence genes. Thus, understanding the role of temperature‐specific adaptations may help to improve forecast models or breeding programmes.

Tracking costs of virulence in natural populations of the wheat pathogen, Puccinia striiformis f.sp.tritici.

BackgroundCosts of adaptation play an important role in host-parasite coevolution. For parasites, evolving the ability to circumvent host resistance may trade off with subsequent growth or transmission. Such costs of virulence (sensu plant pathology) limit the spread of all-infectious genotypes and thus facilitate the maintenance of genetic polymorphism in both host and parasite. We investigated costs of three virulence factors in Puccinia striiformis f.sp.tritici, a fungal pathogen of wheat (Triticum aestivum).ResultsIn pairwise competition experiments, we compared the fitness of near-isogenic genotypes that differed by a single virulence factor. Two virulence factors (vir4, vir6) imposed substantial fitness costs in the absence of the corresponding resistance genes. In contrast, the vir9 virulence factor conferred a strong competitive advantage to several isolates, and this for different host cultivars and growing seasons. In part, the experimentally derived fitness costs and benefits are consistent with frequency changes of these virulence factors in the French pathogen population.ConclusionOur results illustrate the variation in the evolutionary trajectories of virulence mutations and the potential role of compensatory mutations. Anticipation of such variable evolutionary outcomes represents a major challenge for plant breeding strategies. More generally, we believe that agro-patho-systems can provide valuable insight in (co)evolutionary processes in host-parasite systems.

Reduction in the sex ability of worldwide clonal populations of Puccinia striiformis f.sp. tritici.

Puccinia striiformis f.sp. tritici (PST), has so far been considered to reproduce asexually with until very recently no known alternate host, has a clonal population structure in the USA, Australia and Europe. However, recently, high genotypic diversity in Eastern Asia and recombinant populations in China has been reported. Variations in the ability for sexual reproduction could provide an explanation for such a geographical gradient in genotypic diversity. In order to address this hypothesis, we tested for the existence of a relationship between the ability to produce telia, sex-specific structures that are obligatory for sexual cycle, and the genetic diversity of populations measured using neutral markers, in a set of 56 isolates representative of six worldwide geographical origins. Clustering methods assigned these isolates to five genetic groups corresponding to their geographical origin, with eight inter-group hybrid individuals. Isolates representing China, Nepal and Pakistan displayed the highest telial production, while clonal populations from France and the Mediterranean region displayed very low telial production. The geographic cline in telial production corresponded to the gradient of genotypic diversity described during previous studies, showing a clear difference in telial production between clonal vs. diverse/recombinant populations. The higher mean Qst value (0.822) for telial production than the Fst value (0.317) suggested that telial production has more probably evolved through direct or indirect selection rather than genetic drift alone. The existence of high telial production in genetically diverse populations and its reduction in clonal populations is discussed with regard to evolution of sex, PST centre of origin and distribution of its alternative host.

Geographic limits of a clonal population of wheat yellow rust in the Mediterranean region

Most plant pathogens present complex life cycles, in which the clonal reproduction may impede the delimitation of population entities. By studying the genetic structure of the wheat yellow rust caused by Puccinia striiformis f.sp. tritici (PST), we highlighted difficulties impeding population delimitation in highly clonal species. Despite the high dispersal potential of PST, southern France isolates were shown to be divergent from a northwestern European population. A 2‐year survey was performed in the Mediterranean region to assess the geographic distribution of southern isolates: 453 isolates collected in 11 countries were genotyped using 15 simple sequence repeat markers. A subsample was analysed for virulence against 23 resistance genes. The dominant strain in the western Mediterranean region was further studied with amplified fragment length polymorphism markers to test for a geographic substructure. Both ‘individual’‐ and ‘population’‐centred analyses of polymorphism markers revealed two south‐specific groups: a predominant group, with a broad variability and a wide distribution in both western and eastern Mediterranean countries, and a minor group in the western Mediterranean. The east–west gradient of genetic diversity suggested gene flow from the Middle East with subsequent founder effects and genetic divergence, and demonstrated the local survival of a western Mediterranean population. The high frequency of the resistance gene Yr8 observed in cultivars from Tunisia and Algeria may contribute to maintain the north/south structure observed in France. In addition to migration and local adaptation, the dynamics of clonal lineage diversification and replacement should be considered to define population entities in strongly clonal species.

Isolation of ten microsatellite loci in an EST library of the phytopathogenic fungus Puccinia striiformis f.sp. tritici

We report the characterization of ten microsatellite markers in the fungus Puccinia striiformis f.sp. tritici, responsible for yellow rust disease on wheat. A published EST library was scanned for microsatellite motives, and over 15 selected EST sequences, 13 were successfully amplified and ten exhibited polymorphism over an international collection of 43 isolates. These new microsatellites, added to the few previously published ones, provide a sufficient set of markers to perform population genetic studies.

A rapid genotyping method for an obligate fungal pathogen, Puccinia striiformis f.sp. tritici, based on DNA extraction from infected leaf and Multiplex PCR genotyping

BackgroundPuccinia striiformis f.sp. tritici (PST), an obligate fungal pathogen causing wheat yellow/stripe rust, a serious disease, has been used to understand the evolution of crop pathogen using molecular markers. However, numerous questions regarding its evolutionary history and recent migration routes still remains to be addressed, which need the genotyping of a large number of isolates, a process that is limited by both DNA extraction and genotyping methods. To address the two issues, we developed here a method for direct DNA extraction from infected leaves combined with optimized SSR multiplexing.FindingsWe report here an efficient protocol for direct fungal DNA extraction from infected leaves, avoiding the costly and time consuming step of spore multiplication. The genotyping strategy we propose, amplified a total of 20 SSRs in three Multiplex PCR reactions, which were highly polymorphic and were able to differentiate different PST populations with high efficiency and accuracy.ConclusionThese two developments enabled a genotyping strategy that could contribute to the development of molecular epidemiology of yellow rust disease, both at a regional or worldwide scale.

cloncase: Estimation of sex frequency and effective population size by clonemate resampling in partially clonal organisms.

Inferring reproductive and demographic parameters of populations is crucial to our understanding of species ecology and evolutionary potential but can be challenging, especially in partially clonal organisms. Here, we describe a new and accurate method, cloncase, for estimating both the rate of sexual vs. asexual reproduction and the effective population size, based on the frequency of clonemate resampling across generations. Simulations showed that our method provides reliable estimates of sex frequency and effective population size for a wide range of parameters. The cloncase method was applied to Puccinia striiformis f.sp. tritici, a fungal pathogen causing stripe/yellow rust, an important wheat disease. This fungus is highly clonal in Europe but has been suggested to recombine in Asia. Using two temporally spaced samples of P. striiformis f.sp. tritici in China, the estimated sex frequency was 75% (i.e. three‐quarter of individuals being sexually derived during the yearly sexual cycle), indicating strong contribution of sexual reproduction to the life cycle of the pathogen in this area. The inferred effective population size of this partially clonal organism (Nc = 998) was in good agreement with estimates obtained using methods based on temporal variations in allelic frequencies. The cloncase estimator presented herein is the first method allowing accurate inference of both sex frequency and effective population size from population data without knowledge of recombination or mutation rates. cloncase can be applied to population genetic data from any organism with cyclical parthenogenesis and should in particular be very useful for improving our understanding of pest and microbial population biology.

Effects of wheat varietal resistance level and rainfall characteristics on splash dispersal of Septoria tritici blotch

Septoria tritici blotch is an important splash-dispersed disease, causing high yield losses in Europe. Plant disease propagation results from spore dispersal and susceptibility of plant tissues. An experiment was performed in order to study differents aspects of the disease dispersal cycle. Three wheat varieties with contrasted resistance levels were grown in greenhouse conditions until flowering. Plant canopies of each variety received rains of two different raindrop diameter distributions generated by a rain simulator. A linear inoculum source consisting of an aqueous suspension of spores was placed in the middle of each canopy. Horizontal and vertical spore fluxes were measured using traps composed of microscope slides. Varietal resistance was assessed in parallel. After incubation, leaves sampled in canopies were collected and scanned. Spore traps slides were photographed using a microscope combined with a digital camera. Disease area measurement and automatic spore counting were achieved using an image analysis software. Both disease and spore fluxes decreased with the distance from the inoculum source and lower mean raindrop diameter. Disease levels depended on variety and leaf level. Vertical and horizontal gradients of spore fluxes and disease varied in function of rain type and variety. Combining all these results made it possible to disentangle components of splash-dispersed disease propagation for a single dispersal event.

Cultivar architecture modulates spore dispersal by rain splash: A new perspective to reduce disease progression in cultivar mixtures

Cultivar mixtures can be used to improve the sustainability of disease management within farming systems by growing cultivars that differ in their disease resistance level in the same field. The impact of canopy aerial architecture on rain-splash dispersal could amplify disease reduction within mixtures. We designed a controlled conditions experiment to study single splash-dispersal events and their consequences for disease. We quantified this impact through the spore interception capacities of the component cultivars of a mixture. Two wheat cultivars, differing in their aerial architecture (mainly leaf area density) and resistance to Septoria tritici blotch, were used to constitute pure stands and mixtures with 75% of resistant plants that accounted for 80% of the canopy leaf area. Canopies composed of 3 rows of plants were exposed to standardized spore fluxes produced by splashing calibrated rain drops on a linear source of inoculum. Disease propagation was measured through spore fluxes and several disease indicators. Leaf susceptibility was higher for upper than for lower leaves. Dense canopies intercepted more spores and mainly limited horizontal spore transfer to the first two rows. The presence of the resistant and dense cultivar made the mixed canopy denser than the susceptible pure stand. No disease symptoms were observed on susceptible plants of the second and third rows in the cultivar mixture, suggesting that the number of spores intercepted by these plants was too low to cause disease symptoms. Both lesion area and disease conditional severity were significantly reduced on susceptible plants within mixtures on the first row beside the inoculum source. Those reductions on one single-splash dispersal event, should be amplified after several cycle over the full epidemic season. Control of splash-dispersed diseases within mixtures could therefore be improved by a careful choice of cultivars taking into consideration both resistance and architecture.