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International audience; Cultivar mixtures slow polycyclic epidemics but may also affect the evolution of pathogen populations by diversifying the selection pressures exerted by their plant hosts at field scale. We compared the dynamics of natural populations of the fungal pathogen Zymoseptoria tritici in pure stands and in three binary mixtures of wheat cultivars (one susceptible cultivar and one cultivar carrying the recently broken-down Stb16q gene) over two annual field epidemics. We combined analyses of population "size" based on disease severity, and of population "composition" based on changes in the frequency of virulence against Stb16q in seedling assays with more than 3000 strains. Disease reductions were observed in mixtures late in the epidemic, at the whole-canopy scale and on both cultivars, suggesting the existence of a reciprocal protective effect. The three cultivar proportions in the mixtures (0.25, 0.5, and 0.75) modulated the decrease in (a) the size of the pathogen population relative to the two pure stands, (b) the size of the virulent subpopulation, and (c) the frequency of virulence relative to the pure stand of the cultivar carrying Stb16q. Our findings suggest that optimal proportions may differ slightly between the three indicators considered. We argue potential trade-offs that should be taken into account when deploying a resistance gene in cultivar mixtures: between the dual objectives "efficacy" and "durability," and between the "size" and "frequency" of the virulent subpopulation. Based on current knowledge, it remains unclear whether virulent subpopulation size or frequency has the largest influence on interepidemic virulence transmission.

Zymoseptoria triticiis the fungal pathogen responsible for Septoria tritici blotch on wheat. Disease outcome in this pathosystem is partly determined by isolate-specific resistance, where wheat resistance genes recognize specific fungal factors triggering an immune response. Despite the large number of known wheat resistance genes, fungal molecular determinants involved in such cultivar-specific resistance remain largely unknown. We identified the avirulence factorAvrStb9using association mapping and functional validation approaches. PathotypingAvrStb9transgenic strains onStb9cultivars, near isogenic lines and wheat mapping populations, showed thatAvrStb9interacts withStb9resistance gene, triggering an immune response.AvrStb9encodes an unusually large avirulence gene with a predicted secretion signal and a protease domain. It belongs to a S41 protease family conserved across different filamentous fungi in the Ascomycota class and may constitute a core effector.AvrStb9is also conserved among a globalZ. triticipopulation and carries multiple amino acid substitutions caused by strong positive diversifying selection. These results demonstrate the contribution of an ‘atypical’ conserved effector protein to fungal avirulence and the role of sequence diversification in the escape of host recognition, adding to our understanding of host-pathogen interactions and the evolutionary processes underlying pathogen adaptation.Author SummaryFungal avirulence (Avr) genes are involved in gene-for-gene relationships with host resistance genes.Avrgenes may at the same time target host defenses to allow infection and be recognized by a host resistance gene triggering a defense response. The fungusZymoseptoria triticicauses Septoria tritici blotch, a major disease of wheat worldwide.Z. triticipopulations rapidly adapt to selection pressures such as host resistance, leading to resistance breakdown. We report the identification of the avirulence geneAvrStb9based on genetic mapping, sequence polymorphisms and allele swapping.AvrStb9is involved in the interaction withStb9resistance gene following the gene-for-gene model, and its recognition hinders disease symptoms in hosts carrying the corresponding resistance gene. Unlike other knownZ. tritici Avreffectors,AvrStb9encodes for an unusually large Avr protein with a predicted protease S41 domain conserved among diverse ascomycete lineages. We also highlight several gene mutations likely involved in escapingStb9-mediated recognition.

Quantitative resistance is considered more durable than qualitative resistance as it does not involve major resistance genes that can be easily overcome by pathogen populations, but rather a combination of genes with a lower individual effect. This durability means that quantitative resistance could be an interesting tool for breeding crops that would not systematically require phytosanitary products. Quantitative resistance has yet to reveal all of its intricacies. Here, we delve into the case of the wheat/Septoria tritici blotch (STB) pathosystem. Using a population resulting from a cross between French cultivar Renan, generally resistant to STB, and Chinese Spring, a cultivar susceptible to the disease, we built an ultra-dense genetic map that carries 148,820 single nucleotide polymorphism (SNP) markers. Phenotyping the interaction was done with two different Zymoseptoria tritici strains with contrasted pathogenicities on Renan. A linkage analysis led to the detection of three quantitative trait loci (QTL) related to resistance in Renan. These QTL, on chromosomes 7B, 1D, and 5D, present with an interesting diversity as that on 7B was detected with both fungal strains, while those on 1D and 5D were strain-specific. The resistance on 7B was located in the region of Stb8 and the resistance on 1D colocalized with Stb19. However, the resistance on 5D was new, so further designated Stb20q. Several wall-associated kinases (WAK), nucleotide-binding and leucine-rich repeats (NB-LRR) type, and kinase domain carrying genes were present in the QTL regions, and some of them were expressed during the infection. These results advocate for a role of Stb genes in quantitative resistance and for resistance in the wheat/STB pathosystem being as a whole quantitative and polygenic.

Little is known about the impact of host immunity on sexual reproduction in fungal pathogens. In particular, it is unclear whether crossing requires both sexual partners to infect living plant tissues. We addressed this issue in a three-year experiment investigating different scenarios of Zymoseptoria tritici crosses on wheat according to the virulence (‘vir’) or avirulence (‘avr’) of the parents against a qualitative resistance gene. Co-inoculations (‘vir × vir’, ‘avr × vir’, ‘avr × avr’) and single inoculations were performed on a cultivar carrying the resistance gene (Cellule) and a susceptible cultivar (Apache), in the greenhouse. We assessed the intensity of asexual multiplication by scoring disease severity, and the intensity of sexual reproduction by counting the ascospores discharged from wheat residues. As expected, disease severity was more intense on Cellule for ‘vir × vir’ co-inoculations than for ‘avr × vir’ co-inoculations, with no disease for ‘avr × avr’. However, all types of co-inoculation yielded sexual offspring, whether or not the parental strains caused plant symptoms. Parenthood was confirmed by genotyping (SSR markers), and the occurrence of crosses between (co-)inoculated and exogenous strains (other strains from the experiment, or from far away) was determined. We found that symptomatic asexual infection was not required for a strain to participate in sexual reproduction, and that avirulent strains could be maintained asymptomatically “on” or “in” leaf tissues of plants carrying the corresponding resistant gene for long enough to reproduce sexually. In two of the three years, the intensity of sexual reproduction did not differ significantly between the three types of co-inoculation in Cellule, suggesting that crosses involving avirulent strains are not anecdotal. We discuss the possible mechanisms explaining the maintenance of avirulence in Z. tritici populations and supporting the potential efficacy of cultivar mixtures for limiting resistance gene breakdown.HighlightsAvirulent Zymoseptoria tritici strains can reproduce sexually in wheat plants carrying the corresponding resistant gene.Symptomatic infection of plant tissues is not essential for a strain to reproduce sexually.Avirulent strains can be maintained asymptomatically “on” or “in” leaf tissues of plants carrying the corresponding resistant gene for long enough to reproduce sexually.Crosses of virulent strains with virulent and avirulent strains in a plant host carrying the corresponding resistance gene can produce offspring with similar population sizes.Several possible scenarios for sexual crosses can explain the maintenance of avirulence in Zymoseptoria tritici populations evolving in a wheat canopy, particular in cultivar mixtures.

Cultivar mixtures slow polycyclic epidemics but may also affect the evolution of pathogen populations by diversifying the selection pressures exerted by their plant hosts at field scale. We compared the dynamics of natural populations of the fungal pathogen Zymoseptoria tritici in pure stands and in three binary mixtures of wheat cultivars (one susceptible cultivar and one cultivar carrying the recently broken-down Stb16q gene) over two annual field epidemics. We combined analyses of population ‘size’ based on disease severity, and of population ‘composition’ based on changes in the frequency of virulence against Stb16q in seedling assays with more than 3000 strains. Disease reductions were observed in mixtures late in the epidemic, at the whole canopy scale and on both cultivars, suggesting the existence of a reciprocal ‘protective’ effect. The three cultivar proportions in the mixtures (0.25, 0.5 and 0.75) modulated the decrease in (i) the size of the pathogen population relative to the two pure stands, (ii) the size of the virulent subpopulation, and (iii) the frequency of virulence relative to the pure stand of the cultivar carrying Stb16q. Our findings suggest that optimal proportions may differ slightly between the three indicators considered. We argued potential trade-offs that should be taken into account when deploying a resistance gene in cultivar mixtures: between the dual objectives ‘efficacy’ and ‘durability’, and between the ‘size’ and ‘frequency’ of the virulent subpopulation. Based on current knowledge, it remains unclear whether virulent subpopulation size or frequency has the largest influence on interepidemic virulence transmission.

Durum wheat (Triticum turgidumL. subsp.durum) landraces represent a prominent genetic resource for Mediterranean farming systems and breeding programs. Fourteen landraces sampled in Tunisia were genotyped with 9 microsatellite markers and characterized with 15 morphological descriptors, including resistance to the fungal diseaseSeptoria triticiblotch (STB). The genetic diversity, nearly was as important within landraces populations (45%) than between populations (54%). It was structured in seven genetic groups and was only partly explained by the variety name or the locality of origin. Populations were also greatly diversified phenotypically (Shannon-Weaver H’=0.54) with traits related to spike and awn colours being the most diversified. Resistance to STB was either qualitative in two populations or with varying degrees of quantitative resistance in the others. A Pst-Fstcomparison indicate a local adaptation of the populations. Overall, the genetic structure of Tunisian durum wheat landraces revealed a complex selection trajectory and seed exchanges between farmers.

Foliar plant pathogens require liquid or vapour water for at least part of their development, but their response and their adaptive tolerance to moisture conditions have been much less studied than other meteorological factors to date. We examined the impact on the wheat-Zymoseptoria tritici interaction of altering optimal moisture conditions conducive to infection. We assessed the responses in planta of 48 Z. tritici strains collected in two climatologically distinct locations (Ireland and Israel) to four high moisture regimes differing in the timing and the duration of uninterrupted exposure to saturated relative humidity (100% RH) during the first three days of infection. Individual- and population-level moisture reaction norms expressing how the sporulating area of a lesion change with the RH conditions were established based on visual assessments of lesion development at 14, 17 and 20 days post-inoculation (dpi). Our findings highlighted: (i) a critical time-dependent effect on lesion development of uninterrupted periods of exposure to 100% RH during these earliest infection stages; (ii) a marked interindividual variation in the sensitivity to RH conditions both in terms of strain average moisture response and plasticity; (iii) a higher tolerance – expressed at 14 dpi, not later – of the Israeli population to early interruption of optimal moisture conditions. By indicating that sensitivity to sub-optimal moisture conditions may vary greatly between Z. tritici individuals and populations, this study highlights the evidence of moisture adaptation signature in a plant pathogen. This suggests that understanding such variation will be critical to predict their response to changing climatic conditions.

International audience; This study provides empirical evidence for antagonistic density dependence mechanisms driving sexual reproduction in the wheat fungal pathogen Zymoseptoria tritici. Biparental crosses with 12 increasing inoculum concentrations, in controlled conditions, showed that sexual reproduction in Z. tritici was impacted by an Allee effect due to mate limitation and a competition with asexual multiplication for resource allocation. The highest number of ascospores discharged was reached at intermediate inoculum concentrations (from 5x10(4) conidiamL(-1) to 10(6) conidiamL(-1)). Consistent with these results for controlled co-inoculation, we found that the intensity of sexual reproduction varied with both cropping period and the vertical position of the host tissues in the field, with a maximum between 25 and 35cm above the ground. An optimal lesion density (disease severity of 30 to 45%) maximizing offspring (ascospores) number was established, and its eco-evolutionary consequences are considered here. Two ecological mechanisms may be involved: competition for resources between the two modes of reproduction (decrease in the host resources available for sexual reproduction due to their prior use in asexual multiplication), and competitive disequilibrium between the two parental isolates, due to differential interaction dynamics with the host, for example, leading to an imbalance between mating types. A conceptual model based on these results suggests that sexual reproduction plays a key role in the evolution of pathogenicity traits, including virulence and aggressiveness. Ecological knowledge about the determinants of sexual reproduction in Z. tritici may, therefore, open up new perspectives for the management of other fungal foliar pathogens with dual modes of reproduction.

International audience; The wheat pathogen Zymoseptoria tritici is a relevant fungal model organism for investigations of the epidemiological determinants of sexual reproduction. The objective of this experimental study was to determine which intrinsic factors, including parental fitness and timing conditions of infection, affect the numbers of ascospores produced. We first performed 28 crosses on adult wheat plants in semi controlled conditions, with 10 isolates characterized for their fitness traits. We validated the efficiency of the crossing method, opening up new perspectives for epidemiological studies. We found that the ability to reproduce sexually was determined, at least partly, by the parental genotypes. We also found that the number of ascospores released was correlated with the mean size of the sporulating lesions of the parental isolates on the one hand, and the absolute difference in the latent periods of these isolates on the other. No functional trade-off between the two modes of reproduction in Z. tritici was revealed: there was no adaptive compromise between pathogenicity (asexual multiplication on leaves) and transmission (intensity of sexual reproduction on wheat debris). Moreover, a few days' difference in the latent periods of the two parental isolates, such that one progressed more rapidly in the host tissue than the other, seemed to be slightly beneficial to ascosporogenesis. This may be because the first parental isolate breaks down host defenses, thereby facilitating infection for the other parental isolate. However, a larger difference (a few weeks), generated by leaving two to three weeks between the inoculations of the plant with the parental isolates, was clearly detrimental to ascosporogenesis. In this case, the host tissues were likely colonized by the first isolate, leaving less host resources available for the second, consistent with a competition effect during the asexual stage. (C) 2016 Elsevier Inc. All rights reserved.

This study provides empirical evidence for antagonistic density-dependence mechanisms driving sexual reproduction in the wheat fungal pathogen Zymoseptoria tritici. Biparental crosses with 12 increasing inoculum concentrations, in controlled conditions, showed that sexual reproduction in Z. tritici was impacted by an Allee effect due to mate limitation and a competition with asexual multiplication for resource allocation. We found that asexual multiplication was itself affected by competition for host resources between the two parental isolates, as illustrated by the decrease in asexual fruiting body density and sporulation intensity observed with increasing lesion density. Consistent with these results for controlled co-inoculation, we found that the intensity of sexual reproduction varied with both cropping period and the vertical position of the host tissues in the field. An optimal lesion density maximizing offspring (ascospores) number was established, and its eco-evolutionary consequences are considered here. Two ecological mechanisms may be involved: competition for resources between the two modes of reproduction (decrease in the host resources available for sexual reproduction due to their prior use in asexual multiplication), and competitive disequilibrium between the two parental isolates, due to differential interaction dynamics with the host, for example, leading to an imbalance between mating types. Conceptual models based on these results suggest that sexual reproduction plays a key role in the evolution of pathogenicity traits, including virulence and aggressiveness. Ecological knowledge about the determinants of sexual reproduction in Z. tritici may, therefore, open up new perspectives for the management of other fungal foliar pathogens with dual modes of reproduction.

This preprint has been reviewed and recommended by Peer Community In Evolutionary Biology (http://dx.doi.org/10.24072/pci.evolbiol.100039). The efficiency of plant resistance to fungal pathogen populations is expected to decrease over time, due to its evolution with an increase in the frequency of virulent or highly aggressive strains. This dynamics may differ depending on the scale investigated (annual or pluriannual), particularly for annual crop pathogens with both sexual and asexual reproduction cycles. We assessed this time-scale effect, by comparing aggressiveness changes in a localZymoseptoria triticipopulation over an eight-month cropping season and a six-year period of wheat monoculture. We collected two pairs of subpopulations to represent the annual and pluriannual scales: from leaf lesions at the beginning and end of a single annual epidemic, and from crop debris at the beginning and end of a six-year period. We assessed two aggressiveness traits – latent period and lesion size – on sympatric and allopatric host varieties. A trend toward decreased latent period concomitant with a significant loss of variability was established during the course of the annual epidemic, but not over the six-year period. Furthermore, a significant cultivar effect (sympatric vs. allopatric) on the average aggressiveness of the isolates revealed host adaptation, arguing that the observed patterns could result from selection. We thus provide an experimental body of evidence of an epidemiological trade-off between the intra- and inter-annual scales in the evolution of aggressiveness in a local plant pathogen population. More aggressive isolates were collected from upper leaves, on which disease severity is usually lower than on the lower part of the plants left in the field as crop debris after harvest. We suggest that these isolates play little role in sexual reproduction, due to an Allee effect (difficulty finding mates at low pathogen densities), particularly as the upper parts of the plant are removed from the field, explaining the lack of transmission of increases in aggressiveness between epidemics.

We investigated whether the origin of primary inoculum of the fungal wheat pathogen Zymoseptoria tritici can be inferred from evidence of adaptation to host cultivars. We compared the aggressiveness of two pathogen populations collected locally, the first considered as resident (collected from debris in a wheat cv. Soissons monoculture plot) and the second considered as immigrant (collected from leaf lesions in a 300 m apart wheat cv. Soissons plot containing no debris, exposed to an inoculum pool of distant origin), with the aggressiveness of a third population (collected from early leaf lesions in the same monoculture plot) the origin of which we wanted to determine (local vs. distant). The three populations were sampled twice, in 2009 and 2012, from a 6-year field trial. Latent period and sporulating area of 6 x 12 isolates were assessed in greenhouse on adult plants of cv. Soissons and of Apache, another cultivar commonly grown around the field for several years. Firstly, we detected a differential host adaptation: after several years of monoculture, the resident pathogen populations became less adapted to the other cultivar. Secondly, we showed that when the inoculum pressure was high (2009), the aggressiveness profile of the third pathogen population was more similar to that of the resident populations than of the immigrant. This indicates that early lesions in the monoculture plot were mostly caused by within-field (local) primary inoculum. The comparison of aggressiveness profiles when the inoculum pressure was lower (2012) was less conclusive, suggesting that the tested population could have a mixed origin.

Fusarium head blight (FHB) is one of the most damaging diseases of wheat. FHB is caused by a species complex that includes two genera of Ascomycetes: Microdochium and Fusarium. Fusarium graminearum , Fusarium culmorum , Fusarium poae , and Microdochium nivale are among the most common FHB species in Europe and were chosen for these experiments. Field studies and surveys show that two or more species often coexist within the same field or grain sample. In this study, we investigated the competitiveness of isolates of different species against isolates of F. graminearum at the scale of a single spike. By performing point inoculations of a single floret, we ensured that each species was able to establish independent infections and competed for spike colonization only. The fungal colonization was assessed in each spike by quantitative PCR. After establishing that the spike colonization was mainly downwards, we compared the relative colonization of each species in coinoculations. Classical analysis of variance suggested a competitive interaction but remained partly inconclusive because of a large between-spike variance. Further data exploration revealed a clear exclusion of one of the competing species and the complete absence of coexistence at the spike level.

Head blight (HB) is one of the most damaging diseases on wheat, inducing significant yield losses and toxin accumulation in grains. Fungal pathogens responsible for HB include the genus Microdochium , with two species, and the toxin producer genus Fusarium , with several species. Field studies and surveys show that two or more species can coexist within a same field and coinfect the same plant or the same spike. In the current study, we investigated how the concomitant presence of F. graminearum and another of the HB complex species influences the spike colonization and the toxin production by the fungi. To study these interactions, 17 well-characterized isolates representing five species were inoculated alone or in pairs on wheat spikes in greenhouse and field experiments. The fungal DNA in the grains was estimated by quantitative PCR and toxin contents (deoxynivalenol and nivalenol) by ultraperformance liquid chromatography-UV detection-tandem mass spectrometry. The responses of the different isolates to the presence of a competitor were variable and isolate specific more than species specific. The development of the most aggressive isolates was either unchanged or a slightly increased, while the development of the less aggressive isolates was reduced. The main outcome of the study was that no trend of increased toxin production was observed in coinoculations compared to single inoculations. On the contrary, the amount of toxin produced was often lower than expected in coinoculations. We thus conclude against the hypothesis that the co-occurrence of several HB-causing species in the same field might aggravate the risk linked to fusarium toxins in wheat production.

Fusarium head blight in wheat spikes is associated with production of mycotoxins by the fungi. Although flowering is recognized as the most favourable host stage for infection, a better understanding of infection timing on disease development and toxin accumulation is needed. This study monitored the development of eight characterized isolates of F. graminearum, F. culmorum and F. poae in a greenhouse experiment. The fungi were inoculated on winter wheat spikes before or at anther extrusion, or at 8, 18 and 28 days later. Disease levels were estimated by the AUDPC and thousand-kernel weight (TKW). The fungal biomass (estimated by qPCR) and toxin concentration (deoxynivalenol and nivalenol, estimated by UPLC-UV-MS/MS) were measured in each inoculated spike, providing a robust estimation of these variables and allowing correlations based on single-individual measurements to be established. The toxin content correlated well with fungal biomass in kernels, independently of inoculation date. The AUDPC was correlated with fungal DNA, but not for early and late infection dates. The highest disease and toxin levels were for inoculations around anthesis, but early or late infections led to detectable levels of fungus and toxin for the most aggressive isolates. Fungal development appeared higher in kernels than in the chaff for inoculations at anthesis, but the opposite was found for later inoculations. These results show that anthesis is the most susceptible stage for FHB, but also clearly shows that early and late infections can produce significant disease development and toxin accumulation with symptoms difficult to estimate visually.