Spatiotemporal Changes in Varietal Resistance to Wheat Yellow Rust in France Reveal an Increase in Field Resistance Level During the Period 1985-2018.

Monitoring spatiotemporal changes in varietal resistance and understanding its drivers seem essential to managing plant diseases but require having access to the genetic basis of disease resistance and to its deployment. In this study, we focused on yellow rust (Puccinia striiformis f. sp. tritici) for three decades in France, by using field adult plant resistance levels, Yr race-specific resistance genes of varieties, presence of Puccinia striiformis f. sp. tritici pathotypes and their virulence profiles, and systematic surveys of the acreages of bread wheat varieties available at a yearly survey time and at a district level. Based on these data, we studied spatiotemporal changes in varietal resistance over the period from 1985 to 2018 in 54 French administrative districts (hereafter "departments") by using a set of relevant indicators weighted by the relative acreage proportion of the varieties sown at the department level. Our analyses revealed an increase in varietal resistance over decades that would be due to the accumulation of both quantitative resistance and different race-specific resistance genes. We suggest that, beyond breeders, several actors, including examination offices, agricultural advisory services, and farmers, may have had a substantial influence on these spatiotemporal changes, promoting more resistant varieties and the rapid replacement of newly susceptible varieties by still resistant ones at the beginning of each epidemic.

Origin, migration routes and worldwide population genetic structure of the wheat yellow rust pathogen Puccinia striiformis f.sp. tritici.

Analyses of large-scale population structure of pathogens enable the identification of migration patterns, diversity reservoirs or longevity of populations, the understanding of current evolutionary trajectories and the anticipation of future ones. This is particularly important for long-distance migrating fungal pathogens such as Puccinia striiformis f.sp. tritici (PST), capable of rapid spread to new regions and crop varieties. Although a range of recent PST invasions at continental scales are well documented, the worldwide population structure and the center of origin of the pathogen were still unknown. In this study, we used multilocus microsatellite genotyping to infer worldwide population structure of PST and the origin of new invasions based on 409 isolates representative of distribution of the fungus on six continents. Bayesian and multivariate clustering methods partitioned the set of multilocus genotypes into six distinct genetic groups associated with their geographical origin. Analyses of linkage disequilibrium and genotypic diversity indicated a strong regional heterogeneity in levels of recombination, with clear signatures of recombination in the Himalayan (Nepal and Pakistan) and near-Himalayan regions (China) and a predominant clonal population structure in other regions. The higher genotypic diversity, recombinant population structure and high sexual reproduction ability in the Himalayan and neighboring regions suggests this area as the putative center of origin of PST. We used clustering methods and approximate Bayesian computation (ABC) to compare different competing scenarios describing ancestral relationship among ancestral populations and more recently founded populations. Our analyses confirmed the Middle East-East Africa as the most likely source of newly spreading, high-temperature-adapted strains; Europe as the source of South American, North American and Australian populations; and Mediterranean-Central Asian populations as the origin of South African populations. Although most geographic populations are not markedly affected by recent dispersal events, this study emphasizes the influence of human activities on recent long-distance spread of the pathogen.

Inferring the contribution of sexual reproduction, migration and off-season survival to the temporal maintenance of microbial populations: a case study on the wheat fungal pathogen Puccinia striiformis f.sp. tritici.

Understanding the mode of temporal maintenance of plant pathogens is an important domain of microbial ecology research. Due to the inconspicuous nature of microbes, their temporal maintenance cannot be studied directly through tracking individuals and their progeny. Here, we suggest a series of population genetic analyses on molecular marker variation in temporally spaced samples to infer about the relative contribution of sexual reproduction, off-season survival and migration to the temporal maintenance of pathogen populations. We used the proposed approach to investigate the temporal maintenance of wheat yellow rust pathogen, Puccinia striiformis f.sp. tritici (PST), in the Himalayan region of Pakistan. Multilocus microsatellite genotyping of PST isolates revealed high genotypic diversity and recombinant population structure across all locations, confirming the existence of sexual reproduction in this region. The genotypes were assigned to four genetic groups, revealing a clear differentiation between zones with and without Berberis spp., the alternate host of PST, with an additional subdivision within the Berberis zone. The lack of any differentiation between samples across two sampling years, and the very infrequent resampling of multilocus genotypes over years at a given location was consistent with limited over-year clonal survival, and a limited genetic drift. The off-season oversummering population in the Berberis zone, likely to be maintained locally, served as a source of migrants contributing to the temporal maintenance in the non-Berberis zone. Our study hence demonstrated the contribution of both sexual recombination and off-season oversummering survival to the temporal maintenance of the pathogen. These new insights into the population biology of PST highlight the general usefulness of the analytical approach proposed.

New Races of Puccinia striiformis Found in Europe Reveal Race Specificity of Long-Term Effective Adult Plant Resistance in Wheat.

Resistance to Puccinia striiformis was examined in nine wheat recombinant inbred lines (RILs) from a cross between 'Camp Rémy' (resistant parent) and 'Récital' (susceptible parent) using an isolate of a strain common to the northwestern European population before 2011 (old) and two additional isolates, one representing an aggressive and high-temperature-adapted strain (PstS2) and another representing a virulence phenotype new to Europe since 2011 (new). The RILs carried different combinations of quantitative trait loci (QTL) for resistance to P. striiformis. Under greenhouse conditions, the three isolates gave highly contrasting results for infection type, latent period, lesion length, and diseased leaf area. The PstS2 isolate revealed Yr genes and QTL which conferred complete resistance in adult plants. Six QTL had additive effects against the old isolate whereas the effects of these QTL were significantly lower for the new isolate. Furthermore, the new isolate revealed previously undetected resistance in the susceptible parent. Disease severity under field conditions agreed with greenhouse results, except for Camp Rémy being fully resistant to the new isolate and for two RILs being susceptible in the field. These results stress the need of maintaining high genetic diversity for disease resistance in wheat and of using pathogen isolates of diverse origin in studies of host resistance genetics.

Virulence Dynamics and Regional Structuring of Puccinia striiformis f. sp. tritici in France Between 1984 and 2009.

Understanding of long-term virulence dynamics of pathogen populations in response to host resistance gene deployment is of major importance for disease management and evolutionary biology. We monitored the virulence dynamics of Puccinia striiformis f. sp. tritici, the causal agent of wheat stripe rust, over 25 years in France. Virulence dynamics was explained by estimates of area associated with resistance genes carried by farmers' cultivars. The epidemics assessed through disease severity significantly correlated with the number of P. striiformis f. sp. tritici isolates collected each year, used to describe virulence dynamics. In the south, the dominance of the Mediterranean pathotype 6E16 and the cultivation of a susceptible cultivar were associated with an epidemic from 1997 to 1999. In the north, five epidemics occurred due to successive acquisition of virulence to the resistance genes Yr7, Yr6, Yr9, Yr17, and Yr32, either by acquisition of the virulence in the previous dominant pathotype or by incursion or selection of one or two new pathotypes. Frequency of pathotypes with Vr7 and Vr6 declined with the reduction in the cultivation of corresponding Yr gene cultivars, whereas the virulence Vr9 persisted longer than the cultivation of Yr9 cultivars. Although the first pathotypes carrying Vr9 decreased, this virulence persisted in other pathotypes even in the absence of Yr9 cultivars. At the regional level, Yr9 cultivars in the north caused a shift from high Vr6 frequency to high Vr9 frequency whereas, in the central region, where Yr9 cultivars were rare, Vr6 remained prevalent.

Stem rust on barberry species in Europe: Host specificities and genetic diversity.

The increased emergence of cereal stem rust in southern and western Europe, caused by the pathogen Puccinia graminis, and the prevalence of alternate (sexual) host, Berberis species, have regained attention as the sexual host may serve as source of novel pathogen variability that may pose a threat to cereal supply. The main objective of the present study was to investigate the functional role of Berberis species in the current epidemiological situation of cereal stem rust in Europe. Surveys in 11 European countries were carried out from 2018 to 2020, where aecial infections from five barberry species were collected. Phylogenetic analysis of 121 single aecial clusters of diverse origin using the elongation factor 1-α gene indicated the presence of different special forms (aka formae speciales) of P. graminis adapted to different cereal and grass species. Inoculation studies using aecial clusters from Spain, United Kingdom, and Switzerland resulted in 533 stem rust isolates sampled from wheat, barley, rye, and oat, which confirmed the presence of multiple special forms of P. graminis. Microsatellite marker analysis of a subset of 192 sexually-derived isolates recovered on wheat, barley and rye from the three populations confirmed the generation of novel genetic diversity revealed by the detection of 135 multilocus genotypes. Discriminant analysis of principal components resulted in four genetic clusters, which grouped at both local and country level. Here, we demonstrated that a variety of Berberis species may serve as functional alternate hosts for cereal stem rust fungi and highlights the increased risks that the sexual cycle may pose to cereal production in Europe, which calls for new initiatives within rust surveillance, epidemiological research and resistance breeding.

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.

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

BACKGROUND: Costs 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). RESULTS: In 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. CONCLUSION: Our 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.

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.

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.

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.

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.

BACKGROUND: Puccinia 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. FINDINGS: We 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. CONCLUSION: These 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.