Revisiting the historical scenario of a disease dissemination using genetic data and Approximate Bayesian Computation methodology: The case of Pseudocercospora fijiensis invasion in Africa.

The reconstruction of geographic and demographic scenarios of dissemination for invasive pathogens of crops is a key step toward improving the management of emerging infectious diseases. Nowadays, the reconstruction of biological invasions typically uses the information of both genetic and historical information to test for different hypotheses of colonization. The Approximate Bayesian Computation framework and its recent Random Forest development (ABC-RF) have been successfully used in evolutionary biology to decipher multiple histories of biological invasions. Yet, for some organisms, typically plant pathogens, historical data may not be reliable notably because of the difficulty to identify the organism and the delay between the introduction and the first mention. We investigated the history of the invasion of Africa by the fungal pathogen of banana Pseudocercospora fijiensis, by testing the historical hypothesis against other plausible hypotheses. We analyzed the genetic structure of eight populations from six eastern and western African countries, using 20 microsatellite markers and tested competing scenarios of population foundation using the ABC-RF methodology. We do find evidence for an invasion front consistent with the historical hypothesis, but also for the existence of another front never mentioned in historical records. We question the historical introduction point of the disease on the continent. Crucially, our results illustrate that even if ABC-RF inferences may sometimes fail to infer a single, well-supported scenario of invasion, they can be helpful in rejecting unlikely scenarios, which can prove much useful to shed light on disease dissemination routes.

Fungal adaptation to contemporary fungicide applications: the case of Botrytis cinerea populations from Champagne vineyards (France).

In addition to being one of the most acute problems impeding chemical control of fungal diseases, the evolution of fungicide resistance is an emblematic case of local adaptation to spatially heterogeneous and temporally variable selection pressures. Here we dissected the adaptation of Botrytis cinerea (the causal agent of grey mould) populations on grapes to several fungicides. We carried out a 2-year survey (four collection dates) on three treated/untreated pairs of plots from vineyards in Champagne (France) and monitored the frequency of four resistant phenotypes that are unambiguously associated with four distinct genotypes. For two loci under selection by currently used fungicides (MDR1 and MDR2), the frequencies of resistant mutations at vintage were greater in treated plots compared to untreated plots, showing that the effect of selection is detectable even at the plot scale. This effect was not detectable for two other loci under selection by previously used fungicides (BenR1 and ImiR1). We also found that treatment with currently used fungicides reduced B. cinerea effective population size, leading to a significant decrease in genic diversity and allelic richness in treated vs. untreated plots. We further highlight that even under ample drift and migration, fungal populations can present an efficient response to selection. Finally, for the four studied loci, the costs of fungicide resistance were estimated by modelling the decrease in the frequency of resistant mutations in the absence of treatment. We discuss the importance of these estimates for defining strategies for limiting or counteracting the local adaptation of pests to fungicides.

Adaptation of genetically monomorphic bacteria: evolution of copper resistance through multiple horizontal gene transfers of complex and versatile mobile genetic elements.

Copper-based antimicrobial compounds are widely used to control plant bacterial pathogens. Pathogens have adapted in response to this selective pressure. Xanthomonas citri pv. citri, a major citrus pathogen causing Asiatic citrus canker, was first reported to carry plasmid-encoded copper resistance in Argentina. This phenotype was conferred by the copLAB gene system. The emergence of resistant strains has since been reported in Réunion and Martinique. Using microsatellite-based genotyping and copLAB PCR, we demonstrated that the genetic structure of the copper-resistant strains from these three regions was made up of two distant clusters and varied for the detection of copLAB amplicons. In order to investigate this pattern more closely, we sequenced six copper-resistant X. citri pv. citri strains from Argentina, Martinique and Réunion, together with reference copper-resistant Xanthomonas and Stenotrophomonas strains using long-read sequencing technology. Genes involved in copper resistance were found to be strain dependent with the novel identification in X. citri pv. citri of copABCD and a cus heavy metal efflux resistance-nodulation-division system. The genes providing the adaptive trait were part of a mobile genetic element similar to Tn3-like transposons and included in a conjugative plasmid. This indicates the system's great versatility. The mining of all available bacterial genomes suggested that, within the bacterial community, the spread of copper resistance associated with mobile elements and their plasmid environments was primarily restricted to the Xanthomonadaceae family.

Bridgehead invasion of a monomorphic plant pathogenic bacterium: Xanthomonas citri pv. citri, an emerging citrus pathogen in Mali and Burkina Faso.

Molecular epidemiology studies further our understanding of migrations of phytopathogenic bacteria, the major determining factor in their emergence. Asiatic citrus canker, caused by Xanthomonas citri pv. citri, was recently reported in Mali and Burkina Faso, a region remote from other contaminated areas. To identify the origin and pathways of these emergences, we used two sets of markers, minisatellites and microsatellites, for investigating different evolutionary scales. Minisatellite typing suggested the introduction of two groups of strains in Mali (DAPC 1 and DAPC 2), consistent with microsatellite typing. DAPC 2 was restricted to Bamako district, whereas DAPC 1 strains were found much more invasive. The latter strains formed a major clonal complex based on microsatellite data with the primary and secondary founders detected in commercial citrus nurseries and orchards. This suggests that human activities played a major role in the spread of DAPC 1 strains via the movement of contaminated propagative material, further supported by the frequent lack of differentiation between populations from geographically distant nurseries and orchards. Approximate Bayesian Computation analyses supported the hypothesis that strains from Burkina Faso resulted from a bridgehead invasion from Mali. Multi-locus variable number of tandem repeat analysis and Approximate Bayesian Computation are useful for understanding invasion routes and pathways of monomorphic bacterial pathogens.

Investigating the genetic load of an emblematic invasive species: the case of the invasive harlequin ladybird Harmonia axyridis.

Introduction events can lead to admixture between genetically differentiated populations and bottlenecks in population size. These processes can alter the adaptive potential of invasive species by shaping genetic variation, but more importantly, they can also directly affect mean population fitness either increasing it or decreasing it. Which outcome is observed depends on the structure of the genetic load of the species. The ladybird Harmonia axyridis is a good example of invasive species where introduced populations have gone through admixture and bottleneck events. We used laboratory experiments to manipulate the relatedness among H. axyridis parental individuals to assess the possibility for heterosis or outbreeding depression in F1 generation offspring for two traits related to fitness (lifetime performance and generation time). We found that inter-populations crosses had no major impact on the lifetime performance of the offspring produced by individuals from either native or invasive populations. Significant outbreeding depression was observed only for crosses between native populations for generation time. The absence of observed heterosis is indicative of a low occurrence of fixed deleterious mutations within both the native and invasive populations of H. axyridis. The observed deterioration of fitness in native inter-population crosses most likely results from genetic incompatibilities between native genomic backgrounds. We discuss the implications of these results for the structure of genetic load in H. axyridis in the light of the available information regarding the introduction history of this species.

Using neutral cline decay to estimate contemporary dispersal: a generic tool and its application to a major crop pathogen.

Dispersal is a key parameter of adaptation, invasion and persistence. Yet standard population genetics inference methods hardly distinguish it from drift and many species cannot be studied by direct mark-recapture methods. Here, we introduce a method using rates of change in cline shapes for neutral markers to estimate contemporary dispersal. We apply it to the devastating banana pest Mycosphaerella fijiensis, a wind-dispersed fungus for which a secondary contact zone had previously been detected using landscape genetics tools. By tracking the spatio-temporal frequency change of 15 microsatellite markers, we find that σ, the standard deviation of parent-offspring dispersal distances, is 1.2 km/generation(1/2) . The analysis is further shown robust to a large range of dispersal kernels. We conclude that combining landscape genetics approaches to detect breaks in allelic frequencies with analyses of changes in neutral genetic clines offers a powerful way to obtain ecologically relevant estimates of dispersal in many species.

Recent range expansion and agricultural landscape heterogeneity have only minimal effect on the spatial genetic structure of the plant pathogenic fungus Mycosphaerella fijiensis.

Understanding how geographical and environmental features affect genetic variation at both the population and individual levels is crucial in biology, especially in the case of pathogens. However, distinguishing between these factors and the effects of historical range expansion on spatial genetic structure remains challenging. In the present study, we investigated the case of Mycosphaerella fijiensis-a plant pathogenic fungus that has recently colonized an agricultural landscape characterized by the presence of potential barriers to gene flow, including several commercial plantations in which disease control practises such as the use of fungicides are applied frequently, and low host density areas. We first genotyped 300 isolates sampled at a global scale on untreated plants in two dimensions over a 50 × 80-km area. Using two different clustering algorithms, no genetic structure was detected in the studied area, suggesting expansion of large populations and/or no influence of potential barriers. Second, we investigated the potential effect of disease control practises on M. fijiensis diversity by comparing populations sampled in commercial vs food-crop plantations. At this local scale, we detected significantly higher allelic richness inside commercial plantations compared with the surrounding food-crop plantation populations. Analysis of molecular variance indicated that 99% of the total genetic variance occurred within populations. We discuss the suggestion that high population size and/or high migration rate between populations might be responsible for the absence of any effect of disease control practises on genetic diversity and differentiation.

Understanding the recent colonization history of a plant pathogenic fungus using population genetic tools and Approximate Bayesian Computation.

Understanding the processes by which new diseases are introduced in previously healthy areas is of major interest in elaborating prevention and management policies, as well as in understanding the dynamics of pathogen diversity at large spatial scale. In this study, we aimed to decipher the dispersal processes that have led to the emergence of the plant pathogenic fungus Microcyclus ulei, which is responsible for the South American Leaf Blight (SALB). This fungus has devastated rubber tree plantations across Latin America since the beginning of the twentieth century. As only imprecise historical information is available, the study of population evolutionary history based on population genetics appeared most appropriate. The distribution of genetic diversity in a continental sampling of four countries (Brazil, Ecuador, Guatemala and French Guiana) was studied using a set of 16 microsatellite markers developed specifically for this purpose. A very strong genetic structure was found (F(st)=0.70), demonstrating that there has been no regular gene flow between Latin American M. ulei populations. Strong bottlenecks probably occurred at the foundation of each population. The most likely scenario of colonization identified by the Approximate Bayesian Computation (ABC) method implemented in DIYABC suggested two independent sources from the Amazonian endemic area. The Brazilian, Ecuadorian and Guatemalan populations might stem from serial introductions through human-mediated movement of infected plant material from an unsampled source population, whereas the French Guiana population seems to have arisen from an independent colonization event through spore dispersal.

The chestnut blight fungus world tour: successive introduction events from diverse origins in an invasive plant fungal pathogen.

Clonal expansion has been observed in several invasive fungal plant pathogens colonizing new areas, raising the question of the origin of clonal lineages. Using microsatellite markers, we retraced the evolutionary history of introduction of the chestnut blight fungus, Cryphonectria parasitica, in North America and western Europe. Combining discriminant analysis of principal components and approximate Bayesian computation analysis, we showed that several introduction events from genetically differentiated source populations have occurred in both invaded areas. In addition, a low signal of genetic recombination among different source populations was suggested in North America. Finally, two genetic lineages were present in both invaded areas as well as in the native areas, suggesting the existence of genetic lineages with a high capacity to establish in diverse environments and host species. This study confirmed the importance of multiple introductions, but questioned the role of genetic admixture in the success of introduction of a fungal plant pathogen.

Contrasting introduction scenarios among continents in the worldwide invasion of the banana fungal pathogen Mycosphaerella fijiensis.

Reconstructing and characterizing introduction routes is a key step towards understanding the ecological and evolutionary factors underlying successful invasions and disease emergence. Here, we aimed to decipher scenarios of introduction and stochastic demographic events associated with the global spread of an emerging disease of bananas caused by the destructive fungal pathogen Mycosphaerella fijiensis. We analysed the worldwide population structure of this fungus using 21 microsatellites and 8 sequence-based markers on 735 individuals from 37 countries. Our analyses designated South-East Asia as the source of the global invasion and supported the location of the centre of origin of M. fijiensis within this area. We confirmed the occurrence of bottlenecks upon introduction into other continents followed by widespread founder events within continents. Furthermore, this study suggested contrasting introduction scenarios of the pathogen between the African and American continents. While potential signatures of admixture resulting from multiple introductions were detected in America, all the African samples examined seem to descend from a single successful founder event. In combination with historical information, our study reveals an original and unprecedented global scenario of invasion for this recently emerging disease caused by a wind-dispersed pathogen.

Inferences on pathogenic fungus population structures from microsatellite data: new insights from spatial genetics approaches.

Landscape genetics, which combines population genetics, landscape ecology and spatial statistics, has emerged recently as a new discipline that can be used to assess how landscape features or environmental variables can influence gene flow and spatial genetic variation. We applied this approach to the invasive plant pathogenic fungus Mycosphaerella fijiensis, which causes black leaf streak disease of banana. Around 880 isolates were sampled within a 50 × 50 km area located in a fragmented banana production zone in Cameroon that includes several potential physical barriers to gene flow. Two clustering algorithms and a new F(ST) -based procedure were applied to define the number of genetic entities and their spatial domain without a priori assumptions. Two populations were clearly delineated, and the genetic discontinuity appeared sharp but asymmetric. Interestingly, no landscape features matched this genetic discontinuity, and no isolation by distance (IBD) was found within populations. Our results suggest that the genetic structure observed in this production area reflects the recent history of M. fijiensis expansion in Cameroon rather than resulting from contemporary gene flow. Finally, we discuss the influence of the suspected high effective population size for such an organism on (i) the absence of an IBD signal, (ii) the characterization of contemporary gene-flow events through assignation methods of analysis and (iii) the evolution of the genetic discontinuity detected in this study.