Understanding how pathogenic fungal populations overcome plant resistance factors and interact with their environment to infect their hosts is the main objective of our research activity, which we address using both empirical and theoretical approaches. The team studies the epidemio-evolutionary dynamics of Venturia species, pathogens of Rosaceae plants, from the identification of genes under selection to the control of the pathogen at the landscape level, using both genetic and spatial diversification. Our research provides tools and methods to improve plant resistance durability.
Impact of apple domestication on evolutionary genetics and life traits history of its main pathogen Venturia inaequalis
Domestication is a process by which an organism adapts to new environments created by human. Recurrent reproduction and selection cycles of cultivated organisms lead to dramatic phenotypic and genotypic changes over evolutionary very short times (from hundreds to thousands generations). While domestication of many plants and animals is well studied, the impact on pathogens of such a rapid evolution from wild to cultivated hosts remains largely unknown.
The wild Central Asian apple Malus sieversii and the endemic European wild crabapple Malus sylvestris were identified as the main contributors to the genome of the cultivated apple Malus x domestica .In order to reveal the evolutionary history of V. inaequalis during apple domestication we sampled fungal populations on these two wild relatives as well as in cultivated orchards. Using clustering methods based on polymorphism of 10 SSR loci, we identified three distinct populations: (i) a large European population on domesticated and wild apples; (ii) a large Central Asian population on domesticated and wild apples in urban and agricultural areas; and (iii) a more geographically restricted population on M. sieversii in forests growing in the eastern mountains of Kazakhstan. We considered this last population as a relic of ancestral populations from which current populations found in human-managed habitats have diverged (ANR Emerfundis). Identification of this divergent population allowed us addressing both phenotypic and genetic modifications associated with apple domestication.
We first investigated changes in pathogenicity-related life history traits of V. inaequalis during apple domestication and subsequent host-range expansion on the wild European crabapple M. sylvestris. Based on pathogenicity tests, we showed that domestication of apple was associated with the acquisition of virulence and increased aggressiveness in the pathogen. Analyzing dispersion-related life history traits revealed that the shift from wild to domestic habitat has led to increased size and number of spores. Together, our results indicate that host domestication has strongly modified both genetic structure and important life traits of the pathogen.
We then carried out a population genomics study to elucidate the demo-genetic history of V. inaequalis (ANR GANDALF). We sequenced the genome of 40 Kazakh strains to study single nucleotide polymorphism (SNP). Twenty strains sampled in a non-agricultural mountain area (relic population) were compared to 20 strains from an anthropized area in plains. Using demographic inferences from site frequency spectra, we concluded that domesticated and wild populations diverged ca 5,000 years ago and have experienced a recent secondary contact (less than 100 years ago) with heterogeneous migration. These results revealed the occurrence of semi-pervasive barriers to gene flow (de Gracia thesis, Guitton thesis underway). Using coalescence analyses, we showed that it is possible to disentangle genetic barriers to gene flow caused by local adaptations and genetic incompatibilities revealed by secondary contacts. We first conducted genomic and geographic cline analyses at the 181 most discriminative non-synonymous SNPs using nine populations located along a gradient between wild and domestic habitats in Kazakhstan. These approaches based on 270 strains revealed i) a low level of introgression at these loci despite frequent contacts between the two populations; and ii) the direction of introgression is biased toward the wild population, indicating an introgression of genes of agricultural strains into the wild populations (INRA Escapade project).
Our studies revealed an original case of evolution for fungal pathogen populations: the existence of a secondary contact between divergent populations linked to the recent expansion of agriculture in this area (figure 1, unpublished). This provides a unique opportunity to identify endogenous and exogenous barriers responsible for reproductive isolation and genes involved in speciation. A cross between strains from wild and domestic habitat has been obtained in the lab to go further in identification of endogenous barriers and genetic architecture of different life traits of V. inaequalis. Analyses of aggressiveness and sporulation capacity of the 189 progenies are underway. Use of the 75K SNP genotyping Affymetrix chip developed in the lab will greatly facilitate localization of these life traits on the high density genetic map.
Emergence of Venturia inaequalis virulence in orchards from non-cultivated disease reservoir.
Understanding how virulence emerges is essential to manage the deployment of plant resistance genes. The rapid emergence of new virulent isolates is often hypothesized to arise from de novo mutations within avirulent populations already present in the agrosystem, as a response to the introduction of new resistant hosts. However, conclusions from our studies on the breakdown of the apple Rvi6 resistance gene, which is the most used resistance gene in worldwide apple breeding programs, do not match this scenario. Analysis of the genetic variation in the population virulent on Rvi6 cultivars at the European scale revealed that this virulent population diverged from the one present in the non-cultivated habitat ca 24 000 years ago (De Gracia thesis, Lemaire et al., New Phytologist). Actually, it originated from populations infecting the ornamental Japanese crabapple Malus floribunda, which is also the progenitor of the Rvi6 resistance. We demonstrated that introduction of this population in orchards was not followed by free gene flow with local avirulent populations, even several decades after the first reported breakdown. Using pathogenicity tests, population genomic analyses and genetic mapping, we identified two types of barriers impeding free gene flow :exogenous barriers due to other resistance genes present in non-Rvi6 cultivars or reduced fitness of the virulent population and endogenous barriers due to Dobzansky-Müller genetic Incompatibilities (DMI) accumulated during the divergence time (Leroy thesis). However, in 2009, we identified an experimental orchard with gene flow between these two divergent populations, thus providing the opportunity to study putative modifications of pathogenicity-related life history traits occurring during a secondary contact. We showed that hybrids exhibited a higher aggressiveness variance and we observed an invasion of the virulent trait. Moreover, we detected a favored introgression from avirulent to virulent genetic backgrounds (Leroy et al., New Phytologist).
Very recently, cloning of the AvrRvi6 gene in the lab has allowed us to exploit the huge collection of V. inaequalis available in the lab to analyze molecular events responsible for resistance breakdowns in Europe (unpublished data).
To our knowledge, this study is the first evidence that standing genetic variation in non-agricultural reservoirs may be responsible for resistance breakdown. This mechanism is problematic because it is expected to happen faster than de novo mutations in the avirulent population. To conclude, our main finding is that breeding for resistance can favor the introduction of an alien population. Subsequent secondary contact between divergent non-agricultural and agricultural pathogen populations leads to important evolutionary and epidemiological changes in pathogens. It is well exemplified with the Rvi6 breakdown and may stand true for other resistance genes in apple and others crops (Collaborative Project with Massey University and Plant Food Research, New Zealand and La Trobe, Australia and FED San Michelle, Italia)
Adaptation of pathogens to resistance factors and management of plant resistance
We combined both experimental and theoretical approaches to understand the adaptive capacity of fungal pathogens and to assess the spatial and temporal management of resistance genes on their durability (Collab. with the ResPom team at IRHS).
Erosion of quantitative trait loci (QTLs) in the field and selective pressure exerted by QTLs on V. inaequalis
Theoretical approaches predicted that host quantitative resistance selects for pathogens with a high level of pathogenicity, leading to erosion of the resistance. We investigated the erosion of apple quantitative resistance over time in three orchards planted with susceptible and quantitatively resistant apple genotypes (INRA GAP-SPE INRA project). Our study revealed that the use of quantitative resistance resulted in the emergence of a generalist pathogen population that has extended its pathogenicity range by performing similarly on susceptible and partially resistant genotypes (). This emphasizes the need to develop quantitative resistances conducive to trade-offs within the pathogen populations (Aramis/Smach INRA project). To understand how pathogens evolve in response to pressures exerted by their host plants, we compared the impact of broad and narrow spectrum resistance QTLs on V. inaequalis strains frequencies under controlled conditions. Our main finding is that apple broad spectrum QTLs do not exert any differential selection pressure on V. inaequalis whereas narrow-spectrum QTLs do. This will help reasoning strategies coupling breeding and deployment of quantitatively resistant cultivars in space: planting genotypes that carry broad-spectrum factors or planting a mixture of genotypes that carry different narrow-spectrum resistances.
Identification of QTL in Venturia inaequalis
A progeny of 160 strains obtained between strains differing in their aggressiveness have been phenotyped and genotyped using the 75K SNP Affymetrix chip.(RFI Funmagazine Project). A fine mapping of many life traits of V. inaequalis is expected.
Integration of apple resistant cultivars in low-fungicide input systems
Evaluation of the sustainability of disease control strategies in the fields is poorly documented. In a 5-year study in apple orchards, we demonstrated the importance to integrate resistant cultivars in low-fungicide input systems. Removal of leaf litter in autumn to suppress fungal inoculum associated with spraying of fungicides in case of moderate or high risks of scab infection resulted in a sustainable control of scab on a partially resistant cultivar, using half the number of fungicide treatments that are usually applied on susceptible cultivars. Moreover, this low input system even delayed the breakdown of the major Rvi6 resistance gene of the Ariane cultivar. (Didelot et al., Agriculture, Ecosytems, and Environment)
Construction and spatial deployment of genotypes with QTLs and resistance genes.
Our theoretical studies allow gaining insights into the principles of durable management of plant resistance genes. They allow providing guidance for choosing appropriate associations of cultivars and optimizing diversification strategies. Moreover, our theoretical results will help breeders developing principles for sustainable deployment of QTLs.
Timing of pathogen adaptation to hosts carrying several resistance genes
The sustainable use of multi-resistant crops requires an understanding of how their use in agro-systems affects the adaptation speed of the pathogens (Modemave and INRA Modelling Projects). We built a stochastic model to estimate the emergence time of a mutant pathogen overcoming multiple resistance genes. Our results revealed the importance of stochastic mutation and migration processes in the estimation of the pathogen adaptation rate. In particular, accounting for the stochastic migration process alters the criteria for the critical proportion of multi-resistant host to be deployed in order to impede pathogen adaptation. We identified growth and migration rates that allow pathogens to adapt to a multi-resistant host, even when it is deployed on only small proportions. We also showed that deployment of the multi-resistant host only is the most effective strategy. However, if in the neighborhood there are hosts carrying some of the components of the multi-resistance, it is better to use a mixture of monogenic resistances instead of the multi-resistant host. From our results, it is evident that explicit modelling of stochastic processes underlying evolutionary dynamics can help elucidating the principles of the sustainable use of resistance genes in population-wide management strategies.
Timing of pathogen adaptation to quantitative resistance
The progressive adaptation of pathogens to quantitative resistance is poorly understood, which makes difficult to predict the durability of such resistance or to derive principles for its durable deployment (Modemave and INRA Modelling Projects). To study the dynamics of pathogen adaptation in response to quantitative plant resistance affecting pathogen reproduction rate and its colonizing capacity, we have developed a stochastic model for the continuous evolution of a pathogen population on a quantitatively resistant host. We assumed that pathogens adapt to a resistant host through the progressive restoration of their reproduction rate, colonizing capacity, or both. Our model suggested that a combination of QTLs that affects distinct pathogen traits was more durable if the evolution of repressed traits was antagonistic. Otherwise, quantitative resistance that reduced only pathogen reproduction was more durable. Thus, in order to alter the progressive adaptation of pathogens, QTLs that decrease the pathogen’s maximum capacity to colonize must be combined with QTLs that decrease the spore production per lesion or the infection efficiency or that increase the latent period.
Management of quantitative resistance: wheat rust and apple scab as case studies
To clarify principles for the successful use of quantitative plant resistance in disease management, we built a parsimonious model that describes the dynamics of competing pathogenic strains spreading through a mixture of cultivars that carry quantitative resistances. Using this model parameterized for the wheat–yellow rust system, we demonstrated that a more effective use of quantitative resistance in mixtures involves reinforcing the effect of the highly resistant cultivars rather than replacing them. We highlighted the fact that a judicious deployment of quantitative resistance in two- or three-component mixtures makes it possible to reduce disease severity using only small proportions of a highly resistant cultivar. Applying our model to the apple-scab pathosystem in an apple cider orchard planted according to derived planting strategies, we conclude that the optimization of spatial arrangement of cultivars in mixture can increase the mixture performance and enlarge the cultivar choice for mixture composition (unpublished data). (Vergers de demain, Casdar Project).
Available resources in the lab
5800 strains of Venturia spp stored in the lab collection
89 genomes sequences of Venturia spp (Illumina and Pac Bio) and RNA seq data
Many crosses between Venturia strains
480 strains génotyped with a 75K SNP Affymetrix chip