Dr Tatiana Giraud

Dr Denis Faure

Pauline Blin

Kévin Robic

Slimane Khayi

Jérémy Cigna

Euphrasie Munier

Pauline Dewaegeneire

Angélique Laurent

Yan Jaszczyszyn

Kar-Wai Hong

Kok-Gan Chan

Amélie Beury

Sylvie Reverchon

Tatiana Giraud

Valérie Hélias

Denis Faure
email: denis.faure@cnrs.fr

Pattern and causes of the establishment of the invasive bacterial potato pathogen Dickeya solani and of the maintenance of the resident pathogen D. dianthicola

Keywords: competitive exclusion, Dickeya, invasion, plant pathogens, population genomics, quorumsensing

est destinée au dépôt

Biological invasions represent major threats to biodiversity as invasive species can replace native species. Evolutionary and ecological studies are of major importance for understanding the patterns and causes of biological invasions and plant and animal invasions have been extensively studied since the 19 th century [START_REF] Burns | Tests of alternative evolutionary models are needed to enhance our understanding of biological invasions[END_REF][START_REF] Cadotte | Preadaptation and naturalization of nonnative species: Darwin's two fundamental insights into species invasion[END_REF][START_REF] Macdougall | Plant invasions and the niche[END_REF]. Evolutionary and ecological approaches aim at elucidating the dynamics of invasions as well as the advantages of invaders that allow them to outcompete resident species (Facon et al., 2006;[START_REF] Gladieux | The population biology of fungal invasions[END_REF]. Although microbial invasions can represent important threats to ecosystems and human societies, they have been much less studied than invasive plants and animals [START_REF] Gladieux | The population biology of fungal invasions[END_REF][START_REF] Kinnunen | MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger dataset[END_REF][START_REF] Litchman | Invisible invaders: non-pathogenic invasive microbes in aquatic and terrestrial ecosystems[END_REF][START_REF] Mallon | Microbial invasions: the process, patterns, and mechanisms[END_REF][START_REF] Perkins | Invasion triangle: an organizational framework for species invasion[END_REF].

In this work, we used evolutionary and ecological approaches for understanding the patterns and causes of the establishment of the bacterial pathogen Dickeya solani in potato agrosystems and the maintenance of the resident, related competitor D. dianthicola. There are two major clades of potato bacterial pathogens in Europe, Pectobacterium and Dickeya, two sister genera among the order Enterobacterales (Gamma-proteobacteria). While some Pectobacterium species had settled since before the 1970s in potato agrosystems in Europe [START_REF] Pérombelon | Potato diseases caused by soft-rot erwinias: an overview of pathogenesis[END_REF], Dickeya species invaded later, in two successive waves. Mostly isolated from ornamental plants such as Dianthus, Dahlia and Begonia, D. dianthicola has been reported in Europe as early as the 1950s.

Isolation of D. dianthicola from Solanum tuberosum dates back to the 70's [START_REF] Toth | Dickeya species an emerging problem for potato production in Europe[END_REF][START_REF] Parkinson | Dickeya species relatedness and clade structure determined by comparison of recA sequences[END_REF]. Nowadays, D. dianthicola is considered an endemic pathogen of potato plants in Europe. It is present in all continents, including North-America, where it was associated with disease outbreaks in potato farms in 2015 [START_REF] Oulghazi | First report of Dickeya dianthicola causing blackleg disease on potato in Morocco[END_REF][START_REF] Patel | First report of Dickeya dianthicola causing blackleg and soft-rot on potato (Solanum tuberosum) in New Jersey[END_REF][START_REF] Sarfraz | First report of Dickeya dianthicola causing blackleg disease on potato plants in Pakistan[END_REF][START_REF] Toth | Dickeya species an emerging problem for potato production in Europe[END_REF]Wright et al., 2018).

Dickeya solani has emerged in potato production systems in Europe much more recently, in the early 2000s [START_REF] Toth | Dickeya species an emerging problem for potato production in Europe[END_REF]van der Wolf et al., 2014). Aside from potatoes, D. solani has been isolated from ornamental plants such as Hyacinthus, Iris and Muscari, but the primary host(s) of that species remain(s) uncertain [START_REF] Chen | First report of Dickeya solani causing soft-rot in imported bulbs of Hyacinthus orientalis in China[END_REF]Slawiak et al., 2009;van der Wolf et al., 2014). Population genomics revealed little genetic variation in D. solani, in agreement with a recent spread of this species with a bottleneck associated to the introduction and/or invasion [START_REF] Golanowska | Comparison of highly and weakly virulent Dickeya solani strains, with a view on the pangenome and panregulon of this species[END_REF][START_REF] Khayi | Population genomics reveals additive and replacing horizontal gene transfers in the emerging pathogen Dickeya solani[END_REF]. An analysis of 20 D. solani genomes identified a non-synonymous single nucleotide polymorphism (SNP) in each of the two fliC and fliN genes that are involved in bacterial motility [START_REF] Khayi | Population genomics reveals additive and replacing horizontal gene transfers in the emerging pathogen Dickeya solani[END_REF]. Horizontal gene transfer events from D. dianthicola to D. solani have been documented, adding or replacing genomic fragments [START_REF] Khayi | Population genomics reveals additive and replacing horizontal gene transfers in the emerging pathogen Dickeya solani[END_REF].

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The D. solani invader, as well as the D. dianthicola and Pectobacterium spp. residents, are necrotrophic pathogens causing similar symptoms, resulting in blackleg disease on stems and soft-rot disease of tubers (Charkowski 2018;[START_REF] Toth | Dickeya species an emerging problem for potato production in Europe[END_REF]. All these bacteria secrete plant cell-wall macerating enzymes (including the pectate lyases) and proliferate by assimilating the plant cell remains, including iron by producing the chrysobactin and achromobactin siderophores.

Pectate lyases, i.e., PelA, PelD and especially PelE, play a key role in the initiation of the plant cell wall maceration. Using pectin as a substrate, these enzymes release oligosaccharides that elicit expression of other plant-cell wall macerating enzymes in a positive feedback loop [START_REF] Duprey | Transcriptional start site turnover in the evolution of bacterial paralogous genes -the pelE-pelD virulence genes in Dickeya[END_REF]. Noticeably, the pelA gene is truncated, hence non-functional, in D. dianthicola [START_REF] Duprey | Transcriptional start site turnover in the evolution of bacterial paralogous genes -the pelE-pelD virulence genes in Dickeya[END_REF][START_REF] Des Essarts | Common and distinctive adaptive traits expressed in Dickeya dianthicola and Dickeya solani pathogens when exploiting potato plant host[END_REF]. The secretion of these virulence factors is a costly process that is tightly regulated, being induced by the perception of chemicals that inform the pathogen on plant host physiology (mainly plant sugars) and on the size of its own population (quorum-sensing signals) (Leonard, Hommais, Nasser, & Reverchon, 2017). Dickeya pathogens synthetize and sense two types of quorumsensing signals, the N-acylhomoserine lactones and Vfm compounds, which activate virulence gene expression when a threshold concentration of signals (reflecting the number of cells in the environment) is reached [START_REF] Crépin | Quorum sensing signaling molecules produced by reference and emerging soft-rot bacteria (Dickeya and Pectobacterium spp[END_REF][START_REF] Nasser | Vfm a new quorum sensing system controls the virulence of Dickeya dadantii[END_REF][START_REF] Potrykus | Regulators involved in Dickeya solani virulence, genetic conservation and functional variability[END_REF][START_REF] Potrykus | Interplay of classic Exp and specific Vfm quorum sensing systems on the phenotypic features of Dickeya solani strains exhibiting different virulence levels[END_REF]. By modulating the synthesis of virulence factors, the quorum-sensing signals participate in the transition from an oligotroph lifestyle in soils and surface waters, where Dickeya and Pectobacterium bacteria are rare, to a copiotroph lifestyle on a plant host, where they increase in number by several orders of magnitude [START_REF] Laurila | Characterization of Dickeya strains isolated from potato and river water samples in Finland[END_REF][START_REF] Potrykus | Biodiversity of Dickeya spp. isolated from potato plants and water sources in temperate climate[END_REF]. Functional differences such as metabolic capacities have been identified between D. solani and D.

dianthicola by comparative genomics, transcriptomics and biochemical approaches [START_REF] Bellieny-Rabelo | Transcriptome and comparative genomics analyses reveal new functional insights on key determinants of pathogenesis and interbacterial competition in Pectobacterium and Dickeya spp[END_REF][START_REF] Des Essarts | Common and distinctive adaptive traits expressed in Dickeya dianthicola and Dickeya solani pathogens when exploiting potato plant host[END_REF], but fitness differences within host plants remain only partially investigated [START_REF] Czajkowski | Virulence of 'Dickeya solani' and Dickeya dianthicola biovar-1 and -7 strains on potato (Solanum tuberosum)[END_REF][START_REF] Czajkowski | Systemic colonization of potato plants by a soilborne, green fluorescent protein-tagged strain of Dickeya sp. biovar 3[END_REF][START_REF] Des Essarts | Common and distinctive adaptive traits expressed in Dickeya dianthicola and Dickeya solani pathogens when exploiting potato plant host[END_REF][START_REF] Shyntum | The Impact of Type VI secretion system, bacteriocins and antibiotics on bacterial competition of Pectobacterium carotovorum subsp. brasiliense and the regulation of carbapenem biosynthesis by iron and the ferric-uptake regulator[END_REF]. Whether the potato host could be differentially exploited by D. dianthicola and D. solani and whether the two pathogens stably coexist by using different ecological niches or compete directly when exploiting this host remain unsolved questions, despite their importance for understanding the D. solani invasion.

Here, we combined epidemiological records in potato fields, experimental inoculations and

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This article is protected by copyright. All rights reserved diversity of strains and of ecological conditions, we then focused on D. solani and D. dianthicola to determine the ecological traits that could facilitate or constrain D. solani establishment, including competition or ecological niche differences between the two Dickeya species. Finally, we obtained the genomes of 56 D. solani isolates that we analyzed together with 20 genomes previously published [START_REF] Khayi | Population genomics reveals additive and replacing horizontal gene transfers in the emerging pathogen Dickeya solani[END_REF], revealing novel variations in D. solani population. We focused on the quorum-sensing gene vfmB, because it is known to be involved in virulence, to investigate whether its variation, which has not been studied so far, could be associated to invasion traits in D. solani.

Material and Methods

Pathogen sampling and identification in potato fields with blackleg disease symptoms

Pectinolytic bacteria were isolated from symptomatic plant tissues on crystal violet pectate medium [START_REF] Hélias | Two new effective semiselective crystal violet pectate media for isolation of Pectobacterium and Dickeya[END_REF]. Isolates were purified on agar plates and characterized at the genus (Dickeya and Pectobacterium) and species (D. solani and D. dianthicola) levels using the PCR primers listed in Table S1. Two sampling strategies were used.

From 2004 to 2015, a national inventory was conducted in French potato producing areas. Each year (with the exception of the year 2006), fields exhibiting blackleg symptoms were randomly sampled by collecting diseased plants from which isolates were recovered and characterized.

From 541 fields, around 1600 isolates were collected and taxonomically characterized. In parallel, from 2013 to 2016, 19 of the potato fields exhibiting more than 1% of blackleg incidence were deeply sampled. Around 30 plants with blackleg symptoms were collected along a transect in each field, and a single isolate was retained from each of the 548 plants. The resulting 548 isolates were characterized taxonomically.

Dickeya solani and D. dianthicola strains used in plant assays

Five D. dianthicola strains (RNS11-47-1-1A, CFBP1888, CFBP2982, CFBP2015, MIE34) and five D. solani strains (3337 = RNS08-23-3-1A, IPO2222, RNS05-1-2A, Ds0432.1, PPO9019) were used to produce plant inoculation assays. The strains were chosen to be representative of the diversity of the populations, i.e. from different geographical locations, isolation years or host of collection (potato plants vs ornamentals), and regarding phylogeny placement with neutral markers (Figure S1 and see characteristics and genome accession numbers in Table S2).

Phylogeny analyses were conducted using MEGA7 (Kumar, Stecher, & Tamura, 2016). Bacterial strains were stored at -80°C in glycerol (25% vol/vol). They were cultivated in TY medium (tryptone 5 g.L -1 ; yeast extract 3 g.L -1 ) with or without agar (15 g.L -1 ) at 28°C.

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This article is protected by copyright. All rights reserved In tuber assays, potato tubers (S. tuberosum var Bintje) were inoculated using a tip to inject 10 µL of calibrated pathogen suspensions. Based on previous results (Raoul des Essarts et al., 2019), we chose two different inoculum loads of strains and experimental populations (10 7 CFU and 10 5 CFU per tuber) to obtain a large range of symptom severity, to compare the capacity to initiate maceration at different population sizes, to investigate fitness of the two pathogens in two contrasting ecological conditions. After 5 days of incubation at 24°C, the tubers were cut in half

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This article is protected by copyright. All rights reserved and scored based on five aggressiveness classes according to the symptom severity in each tuber (from no symptom to the most severe symptoms; Figure 1c). For each strain and experimental population, at least 10 tubers were inoculated. A set of non-inoculated tubers was used as an asymptomatic control. In order to facilitate the presentation of the aggressiveness data in the figures, results are presented as normalized disease severity index (DSI) of which values, ranging from 0 to 100 (arbitrary unit), increase with symptom severity. DSI was calculated using the five aggressiveness classes assigned to 10 tubers, see formula in Supplementary Methods 1 (SM1).

Emerging lesions from potato stems and hyacinths and rotting tissue recovered from tubers at 5 dpi were collected and frozen at -80°C, and then used to quantify the abundance D. solani and D.

dianthicola pathogens within hosts by Taq Man qPCR as described in Supplementary Methods

(SM2

Dickeya solani population genomics

Total DNA was purified from each of 56 D. solani isolates (their characteristics in Figure S2) using MasterPure complete DNA and RNA purification kit (Epicentre, Illumina). After quantity and quality controls of extracted DNA using a NanoDrop (ND 1000) device and agarose gel electrophoresis at 1.0 % (w/v), paired-end libraries were constructed for each strain and then sequenced using High Output Sequencing kit with 75 × 2 cycles on and Illumina NextSeq500 sequencer (CNRS, Gif-sur-Yvette, France). For each strain, 2.4 to 18 million reads were obtained, corresponding to an average coverage ranging from 37× to 270×. Paired-end reads were trimmed (quality score threshold 0.05) and mapped against the D. solani reference genome, from the 3337 strain, at mild stringency threshold (80% identity on 50% read length) using CLC Genomics Workbench version 10.1.3 [START_REF] Khayi | Population genomics reveals additive and replacing horizontal gene transfers in the emerging pathogen Dickeya solani[END_REF]. The mapping was used for the detection of SNPs and InDels using the variant calling tool from CLC genomic workbench version 10.1.3.

Only SNPs and InDels (1 to 9 nucleotides) with a high occurrence (≥ 99 % of the reads) in the mapping step were retained. The representation of allelic variants in D. solani population was performed using PHYLOViZ [START_REF] Francisco | PHYLOViZ: Phylogenetic inference and data visualization for sequence based typing methods[END_REF].

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This article is protected by copyright. All rights reserved 2.5 Characterization of the fitness differences between Dickeya solani strains carrying the

VfmB Ser and VfmB Pro alleles

The structure of the VfmB proteins was modeled and represented using the Phyr2 and EzMol web portals [START_REF] Kelley | The Phyre2 web portal for protein modeling, prediction and analysis[END_REF][START_REF] Reynolds | EzMol: A Web Server Wizard for the Rapid Visualization and Image Production of Protein and Nucleic Acid Structures[END_REF]. Given the known role in virulence of the vfm gene cluster [START_REF] Nasser | Vfm a new quorum sensing system controls the virulence of Dickeya dadantii[END_REF] S3 andS4).

Plant inoculation assays (on potato tubers and stems) were performed following the same protocols as described above using pure strains and mixtures as inocula. In the figures, virulence was presented as the percentage of symptomatic plants (in potato stem infections assay) and aggressiveness by DSI values (in potato tuber infection assays).

To measure their relative fitness, co-inoculation assays were also performed with VfmB

Statistical analyses

The slopes of the dynamics of the percentages of Dickeya-, Pectobacterium-, D. solani-and D.

dianthicola-positive fields were analyzed using F-tests and ANCOVAs using GraphPad Prism Accepted Article version 8.4.2 for MacOS, GraphPad Software (www.graphpad.com). A Chi-squared method was used to test the non-random distribution of taxa in each field.

In all the plant assays, the non-inoculated plants and tubers remained asymptomatic, and this control condition was not considered in statistical analyses. The two symptom levels (with or without symptoms) of potato plants and hyacinths virulence assays, the five symptom classes in tuber aggressiveness assays, and gene expression levels were compared among treatments (the different pathogens) by Kruskal-Wallis tests (α = 0.05) because the data significantly deviated from normality. Then, post-hoc Tukey tests were run to assess what pairwise differences were significant. When the bacterial strains were inoculated separately on plants and tubers, we compared the D. solani and D. dianthicola species traits using each bacterial strain as a replicate of the considered species. When the bacterial strains were inoculated altogether to constitute an experimental population, we compared the D. solani and D. dianthicola species traits using at least two independent replicates of the experiments.

In competition assays, the hypothesis of balanced abundances of the two competitors was tested by comparing the calculated CI values to one using a Kruskal-Wallis test. Analyses were carried out with the software R (http://www.R-project.org) using the packages RcmdR and nparcomp.

Results

Little co-occurrence of D. dianthicola and D. solani in potato fields

A monitoring of the Pectobacterium and Dickeya populations was performed over one decade (2004)(2005)(2006)(2007)(2008)(2009)(2010)(2011)(2012)(2013)(2014)(2015) in France. Each year, we recorded the number of fields from which we isolated each taxon (Table S5). These data informed on the dynamics of the different pathogen populations.

Over the period, Dickeya was detected in 10% to 35% of the sampled fields (Figure 2a

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Aside from this national survey, we zoomed at the field level using a more extensive sampling strategy over a four-year period (2013)(2014)(2015)(2016). Along a transect in each of 19 sampled fields, we collected ca. 30 plants with blackleg symptoms and a single isolate was characterized from aerial symptoms of each plant, resulting in the sampling of 548 isolates (Figure 2c). The Pectobacterium populations were present in all the 19 sampled fields. In each of the 19 fields, the null hypothesis that Pectobacterium and Dickeya were randomly distributed was rejected (Chi- In a first assay, each strain was inoculated individually on 15 plants and the number of asymptomatic and symptomatic plants were counted at 70 days post inoculation (dpi). A Kruskal-Wallis test revealed differences between D. solani and D. dianthicola in terms of symptom incidence (k=7; DF=1; p=8 x 10 -3 ): D. dianthicola was found more virulent than D. solani (percentages of symptomatic plants of 61% ± 10 versus 15% ± 11; Figure 3a).

In a second plant assay, the five bacterial cultures (i.e., the five strains) of each species were assembled to constitute two experimental populations and each was inoculated on 15 plants. A mixture of the two species (10 strains) was also inoculated on 15 plants. The assay was duplicated (2 x 15 plants per treatment). We compared symptom incidence at 70 dpi. Kruskal-Wallis test revealed differences between the three treatments, i.e., D. solani strain mixture, D.

dianthicola strain mixture and species mixture (k=6.1; DF=2; p=0.04). Pairwise comparison (Post-

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hoc Tukey test) showed that D. solani was less virulent than D. dianthicola (F=-2.5; p= 0.03) and the species mixture (F=1.9; p= 0.10). However, symptom incidence caused by the species mixture did not differ from those of the D. dianthicola mixture (F=-0.5; p= 0.86). Post-hoc Tukey pvalues and mean value (± SE) of the percentage of symptomatic plants inoculated by the D.

dianthicola (53% ± 7), D. solani (23% ± 3) and mixed (47% ± 0) populations are presented in the figure 3b. To assess the dynamics of the appearance of symptomatic plants, we also drew a disease progress curve for each replicate (Figure S3a) and compared the areas under disease progress curve (Figure S3b). Pairwise comparisons (Post-hoc Tukey tests) confirmed that D.

solani was less virulent than D. dianthicola (F=-336; p<10 -11 ) and the species mixture (F=386; p<10 -11 ).

To evaluate which pathogen had emerged at the beginning of the infection process, hence to identify the most probable causative agent of the observed symptoms, we used qPCR to quantify pathogen loads. When the two species mixtures were inoculated separately, D. dianthicola and D. solani reached the mean values ± SE of 2 x 10 11 ± 1 x 10 4 cells and 3 x 10 9 ± 1 x 10 2 cells per gram of symptomatic stem tissues, respectively. Among the 14 co-infected plants exhibiting symptoms, eight lesions were randomly chosen for qPCR quantification of the pathogen abundance: this analysis revealed an excess of D. dianthicola with a CI median value of 10 -5

(Figure 3c). The eight CI values differed from one (Kruskal-Wallis test, k=9.5; DF=1; p= 2 x 10 -3 ), meaning that D. dianthicola outcompeted D. solani in blackleg tissues. Altogether, these data revealed a fitness advantage of D. dianthicola in terms of multiplication within lesions in the aerial parts of the host in either the absence or presence of the D. solani invader.

Dickeya dianthicola exhibited a fitness advantage over D. solani in potato plants with

wounded roots.

To evaluate whether plant wounding could benefit to the D. dianthicola resident or to the D. solani invader, pathogen mixtures were inoculated in pots containing plants with wounded roots.

Kruskal-Wallis test revealed differences between the three treatments, i.e., D. solani strain mixture, D. dianthicola strain mixture and species mixture (k=29.2; DF=2; p=4.6x10

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This article is protected by copyright. All rights reserved than a mixture of D. dianthicola and D. solani (F=386; p<10 -11 ) (Figure S3c-d). Because the virulence measured using symptom incidence at the final time point (70 dpi) and area under disease progress curve (from 1 to 70 dpi) gave similar results when comparing the two pathogens, only the final point of symptom incidence was retained for the analysis of the following plant assays, in order to facilitate the presentation of the data.

In the 27 emerging lesions collected from co-infected plants, qPCR quantification of the pathogens revealed an excess of D. dianthicola with a CI median value of 10 -5 . These CI values were different from one (Kruskal-Wallis test: k=19.5; DF=1; p=10 -5 ; Figure S3b).

Altogether, these experimental data revealed that the resident D. dianthicola was more effective at exploiting unwounded and wounded potato host plants than the D. solani invader, i.e. D.

dianthicola had a higher fitness regarding this component of its ecological niche.

3.4

Dickeya solani exhibited a fitness advantage over D. dianthicola in potato tubers.

We compared the D. dianthicola and D. solani ecological traits in potato tubers. Aggressiveness of the pathogens (i.e., disease severity) was compared using five symptom classes and a disease severity index (DSI) was calculated summarizing the observations of the symptom classes on 10 tubers.

In a first tuber rotting assay, each strain was inoculated separately at 10 7 or at 10 5 CFU on each set of 10 tubers. The two different inoculum loads allowed comparing the capacity of the pathogens to initiate maceration and exploit tubers in two contrasting ecological conditions. The assay was performed five times, so we collected symptom class information from 50 tubers for each bacterial strain and for each inoculation load. Kruskal-Wallis tests revealed differences between the two treatments, D. solani and D. dianthicola, both at high (k=3.2; DF=1; p=0.07) or at low (k=43.6; DF=1; p= 4 x 10 -11 ) inoculum dose. The DSI values were calculated for each set of 10 tubers and mean values ± SE across the five replicates reached 63 ± 13 for D. solani and 47 ± 4 for D. dianthicola with an inoculum of 10 7 CFU per tuber (Figure 4a) and 49 ± 8 for D. solani and 7 ± 4 for D. dianthicola with an inoculum of 10 5 CFU per tuber (Figure 4d).

In a second tuber assay, we used strain mixtures for each of the two species and an assembly of the two species to inoculate 10 tubers for each of 10 7 and 10 5 CFU per tuber. This assay was performed twice. Kruskal-Wallis tests revealed differences between the three treatments, i.e., D.

solani, D. dianthicola and the species mixture both at a high (k=51.3; DF=2; p=4.2x10 -11 ) and a low (k=22.4; DF=2; p=1.3x10 -5 ) pathogen load. At both bacterial loads, pairwise comparisons (Post-hoc Tukey tests) showed that aggressiveness was different between D. solani and D.

dianthicola (at a high load: F=2.7; p=0.04; at a low load: F=8.6; p< 10 -11 ). Aggressiveness also differed between D. solani and the species mixture (at a high load: F=2.9; p=0.01; at a low load:

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This article is protected by copyright. All rights reserved with an inoculum of 10 7 CFU per tuber (Figure 4b) and 69 ± 14 (D. solani), 16 ± 6 (D. dianthicola)

and 48 ± 10 (D. solani and D. dianthicola) with an inoculum of 10 5 CFU per tuber (Figure 4e).

Altogether, these tuber assays revealed a higher aggressiveness of D. solani compared to D.

dianthicola and to the species mixture.

The qPCR analyses of 10 symptomatic tubers revealed that the abundance of the D. solani and D. dianthicola strain mixtures ranged from 2 x 10 9 ± 3 x 10 9 to 9 x 10 9 ± 9 x 10 9 cells per gram of rotted tissues (mean values ± SE). These data indicated no substantial differences in the capacity to exploit tubers in terms of population yield. In co-infection assays, qPCR quantification of the pathogens in 10 symptomatic tubers revealed an advantage in favor of D. solani regardless of the initial load (CI median value = 5.7; Figure 4c and4f). The CI values were different from one (Kruskal-Wallis tests: k=16.3; DF=1; p-value of 5 x 10 -5 with a high load; k=7.8; DF=1; p= 0.05 with a low load).

Overall, tubers constituted potato plant tissues in which D. solani initiated symptoms more efficiently (even with a low pathogen load), caused more damage (higher disease severity index)

and was more competitive than D. dianthicola (outcompeting D. dianthicola). Remarkably, the disease severity caused by D. solani was decreased in the presence of D. dianthicola.

Dickeya solani exhibited a fitness advantage over D. dianthicola in hyacinths.

The symptom incidence and competition were further evaluated in hyacinths, that represent important hosts as they constitute a reservoir or/and primary or intermediate host of D. solani. We In co-inoculation assays with species mixtures (Figure S5b), the qPCR quantification of the pathogens in five symptomatic tissues showed that the calculated CI were different from one

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Pectate lyases gene expression is higher in Dickeya solani than in D. dianthicola

The capacity of D. solani to initiate rotting with a low bacterial load suggested a differential expression of pectate lyase genes between the two species. To test this hypothesis, the expression levels of the pelA, pelD and pelE genes were quantified by RT-qPCR in the five D. solani and five D. dianthicola strains grown separately in a rich medium (in the absence of pectin) and in emerging lesions of potato stems and tubers (see a detailed protocol in SM2). The expression of two other virulence genes, cbsE and acsD, which are not involved in the plant cell wall degradation, but in iron-uptake, were also compared. Four RT-qPCR measurements were performed per condition and gene. For each gene, Kruskal-Wallis tests revealed differences between the six treatments (rich medium, stems and tubers for the two species, considering each strain as a replicate) for the different genes: pelA (k=17.1; DF=5 ; p=4x10 -3 ), pelD (k=25.1; DF=5 ; p=1x10 -4 ), pelE (k=24.9; DF=5; p=1x10 -4 ), cbsE (k=20.3; DF=5 ; p=1x10 -3 ) and acsD (k=21.3; DF=5; p=7x10 -4 ).

Pairwise post-hoc Tukey tests were used to compare gene expression between D. solani and D.

dianthicola. In the absence of pectin (Figure 5), the pelA (F=864.3; p< 10 -11 ), pelD (F=864.3; p< 10 -11 ) and pelE (F=2.9; p= 4 x 10 -3 ) genes were expressed at a higher level in D. solani than in D.

dianthicola. The expression levels of the cbsE (F=0.377; p=0.99) and acsD (F=-1.0; p=0.99) genes were not significantly different between species (Figure S6). In the absence of pectin, a higher expression of pectate lyase genes, especially pelE, could be considered as an advantage for initiating a rapid degradation of the plant cell wall polymers.

In symptomatic potato tubers and stems, some remarkable gene expression patterns could be noticed (Figures 5 andS6). The expression levels of pelD and pelE were higher in D. solani than in D. dianthicola (pelD: F=864.3; p< 10 -11 in tubers and F=4.5; p= 10 -4 in stems; pelE: F=4.5; p= 1.1 x 10 -4 in tubers and F=8.1; p < 10 -11 in stems). The pelA (F=864.3; p< 10 -11 ) and cbsE (F=3.4; p= 0.01) genes were expressed at higher levels in D. solani than in D. dianthicola stem tissues, but not in tubers (pelA: F=-0.09; p=1 and cbsE: F=0.67; p=0.99). The acsD expression level did not differ between the two species in diseased tubers (F=-1.5; p=0.9) and stems (F=0.5 p=0.9).

Overall, in plant symptoms, D. solani maintained a higher expression of the virulence genes pelD and pelE compared to D. dianthicola.

Dickeya solani genomics revealed two vfmB alleles at balanced frequencies

We used a population genomic approach to investigate additional ecological traits of the D. solani invading population. We studied SNPs and InDels in 76 D. solani genomes (Figure S2), 56 of which were assembled after Illumina sequencing in this study, while the 20 others had been

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This article is protected by copyright. All rights reserved previously acquired using the same sequencing approach [START_REF] Khayi | Population genomics reveals additive and replacing horizontal gene transfers in the emerging pathogen Dickeya solani[END_REF]. Out of the 76 genomes, 66 exhibited from 2 to 11 SNPs and InDels when compared to the D. solani 3337 reference genome (Figure S2). In the nine others, more than 100 SNPs were observed: some of these SNPs were clustered in regions of the chromosome (RNS13-30-1A, PPO9134, PPO9019, RNS13-31-1A, RNS13-48-1A, RNS15-102-1A, RNS07-7-3B) while others were distributed along the genome (RNS05-1-2A and RNS10-105-1A).

Focusing on the 66 genomes exhibiting less than 12 SNPs compared to the strain 3337, 45 nonsynonymous variations were identified in whole genomes (Table S6). Two-third of them (30 out of 45) were present in one genome only, while the 15 others were found in at least two genomes.

Among these 15, three SNPs were identical in four strains, two others in five strains and one was present in 19 genomes (Table S6 and Figure 6a). The most balanced frequencies consisted in a thymine (T) in 19 genomes and a cytosine (C) residue in the other 48 genomes at the position 2,930,940 in the reference D. solani 3337 genome (Figure 6a). The C to T modification caused a proline (VfmB Pro allele) to serine (VfmB Ser allele) change at the position 55 in the VfmB protein.

Such SNPs affecting an amino-acid in the vfmB gene retained our attention because the Vfm quorum-sensing system is involved in the regulation of some virulence traits in Dickeya [START_REF] Nasser | Vfm a new quorum sensing system controls the virulence of Dickeya dadantii[END_REF][START_REF] Potrykus | Interplay of classic Exp and specific Vfm quorum sensing systems on the phenotypic features of Dickeya solani strains exhibiting different virulence levels[END_REF].

A SNP-based tree of these 67 strains (Figure 6b We also investigated the presence of the VfmB Pro and VfmB Ser alleles in other Dickeya species.

The alignments of VfmB proteins retrieved from genomic databases revealed the occurrence of the VfmB Pro allele in D. dadantii, D. chrysanthemi and D. dianthicola genomes, while the VfmB Ser allele was found in D. zeae genomes (Figure S7). However, the low number of genomes available let open the possibility of the existence of additional alleles in each of these Dickeya species.

VfmB Ser strains were more aggressive than VfmB Pro strains in soft-rot assays

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The modeling of the D. solani VfmB Pro and VfmB Ser protein structures predicted a conformational difference in the beta-sheet structure of the virulence regulator VfmB (Figure 7a). This change is explained by the exceptional conformational rigidity of proline that strongly affects secondary structures such as alpha helices and beta sheets. The modeling data prompted us to test whether VfmB Pro -VfmB Ser variation could be associated with a change in aggressiveness. To compare the aggressiveness of D. solani VfmB Ser and VfmB Pro isolates, we used genomic data to identify a set of 8 isolates carrying either VfmB Pro (IPO2222, MIE35, AM3a and 3337) or VfmB Ser (Ds0432.1, RNS10-27-2A, Sp1a and M21a), in which the other variations were at different positions (Tables S3 andS4). Soft-rot assays using 10 inoculated potato tubers per strain revealed that the set of the VfmB Ser strains was more aggressive (Kruskal-Wallis test; k=9.5; DF=1; p= 2 x 10 -3 ) than that of the VfmB Pro strains (Figure 7b).

To test the existence of differential expression of virulence genes in tuber tissues, we compared transcriptomes of D. solani IPO2222 (VfmB Pro ) and Ds0432.1 (VfmB Ser ) (Figure 7c; Table S7).

The expression of four genes found differentially expressed based on transcriptomics (pelE, impC, cytA and budA) was further investigated by RT-qPCR in the same samples used for transcriptomics: they found to be also upregulated (Figure S8). Overall, 170 differentially expressed genes (adjusted p-value < 0.05 and absolute log2 fold change > 2) were identified by comparative transcriptomics. Most of them (150 genes) were upregulated in Ds0432.1 carrying the VfmB Ser allele (Table S8). We studied more specifically 35 well-characterized D. solani virulence genes (Raoul des [START_REF] Des Essarts | Common and distinctive adaptive traits expressed in Dickeya dianthicola and Dickeya solani pathogens when exploiting potato plant host[END_REF]. We observed an enrichment of these virulence genes among the upregulated genes in D. solani Ds0432.1, while none of them was found among the downregulated genes (Table S9). These upregulated virulence genes in D. solani Ds0432.1 included the pectate lyase genes pelB, pelC, pelD, pelE and pelL, the protease genes ptrA, prtB and prtC, and the T6SS-related toxin/antitoxin genes hcp (Table S9). These upregulated genes encompassed virulence determinants that are already known to be regulated by the Vfm quorumsensing in D. dadantii [START_REF] Nasser | Vfm a new quorum sensing system controls the virulence of Dickeya dadantii[END_REF].

Fitness costs and gains associated with VfmB alleles in potato stems and tubers

We used the same eight isolates carrying either VfmB Pro (IPO2222, MIE35, AM3a and 3337) or

VfmB Ser (Ds0432.1, RNS10-27-2A, Sp1a and M21a) to constitute VfmB Pro and VfmB Ser experimental populations for comparing their fitness in plant assays.

In soft-rot assays (Figure 8a-b), each of the two experimental populations and their mixture was inoculated on 10 tubers and a symptom class was assigned to each tuber. Two inoculation loads were used (10 7 CFU or 10 5 CFU per tuber). This assay was performed in triplicate (3 x 10 tubers per treatment). Kruskal-Wallis tests revealed differences between the three treatments, i.e.,

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This article is protected by copyright. All rights reserved inoculation by D. solani VfmB Pro , D. solani VfmB Ser and their mixture, and the differences were significant at both high (k=17.6; DF=2; p=1.5x10 -4 ) and low (k=7. Altogether, the plant assays indicated that the VfmB Ser allele was associated to a higher aggressiveness in the rotted tubers compared to the VfmB Pro allele. This suggested that the VfmB Ser allele could more efficiently initiate maceration of tubers in the field and storage conditions. In contrast, the VfmB Ser allele seemed to confer a lower competitive fitness than the VfmB Pro allele when D. solani proliferates in rotted tubers and, particularly, in stem symptoms.

Such opposite gains across different conditions could contribute to the maintenance of balanced frequencies of the two alleles in D. solani populations.

Discussion

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This article is protected by copyright. All rights reserved By comparing ecological traits of D. solani and D. dianthicola, using multiple strains representative of the population diversity, we indeed found contrasted behaviors in these species with respect to aerial parts versus tubers of S. tuberosum. The resident D. dianthicola more efficiently exploited host stems after an inoculation of the wounded and unwounded roots than did the invader D. solani, i.e., causing higher symptom incidence, and exhibiting greater proliferation and relative fitness in competition assays. In contrast, D. solani more efficiently initiated the rotting process (especially at a low bacterial inoculum) and outcompeted D. dianthicola in tubers, with potential consequences on tubers in the fields and during storage. Hence, our assays suggest that infected tubers could facilitate the entry and establishment of the D. solani invader into the potato agrosystems. A contrasting defense response of potato foliage and tuber against Phytophtora infestans has been reported [START_REF] Gao | Contrasting potato foliage and tuber defense mechanisms against the late blight pathogen Phytophthora infestans[END_REF]. In addition to the intrinsic characteristics of D. solani and D. dianthicola, an organ-specific response of the plant defense system could similarly also contribute to their difference in behavior.

The capacity of D. solani to cause rotting at a lower bacterial load than D. dianthicola is likely at least partly due to a higher expression of the pel enzymes, especially PelE that acts as an initiator of the plant cell wall degradation [START_REF] Duprey | Transcriptional start site turnover in the evolution of bacterial paralogous genes -the pelE-pelD virulence genes in Dickeya[END_REF]. By comparing the expression of some pel genes in emerging lesions in potato stems and tubers, we observed a higher expression of the virulence genes pelD and pelE in D. solani compared to D. dianthicola. Remarkably, by comparing pel genes in several Dickeya species, [START_REF] Duprey | Transcriptional start site turnover in the evolution of bacterial paralogous genes -the pelE-pelD virulence genes in Dickeya[END_REF] showed that D. solani evolved specific regulatory sequences that contributes to a different expression level of the pelD and pelE genes compared to D. dianthicola. The pelA gene is truncated in D. dianthicola [START_REF] Duprey | Transcriptional start site turnover in the evolution of bacterial paralogous genes -the pelE-pelD virulence genes in Dickeya[END_REF][START_REF] Des Essarts | Common and distinctive adaptive traits expressed in Dickeya dianthicola and Dickeya solani pathogens when exploiting potato plant host[END_REF], but full-length and expressed in D. solani (as shown in this study), reinforcing their virulence arsenal. Other metabolic traits could also contribute to the fitness advantage of D. solani for exploiting tubers: comparative transcriptomics revealed a higher expression of the glyoxylate shunt in D. solani than in D. dianthicola, a pathway that contributes to exploit alternative carbon sources when sugar availability is low (Raoul des [START_REF] Des Essarts | Common and distinctive adaptive traits expressed in Dickeya dianthicola and Dickeya solani pathogens when exploiting potato plant host[END_REF].

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Beside the tripartite interaction between potato plant host, D. solani and D. dianthicola, additional environmental (soil and climate) and biological factors (microbiota including Pectobacterium species, other hosts and non-host plants) may facilitate or limit D. solani establishment [START_REF] Charkowsky | The changing face of bacterial soft-rot diseases[END_REF][START_REF] Shyntum | The Impact of Type VI secretion system, bacteriocins and antibiotics on bacterial competition of Pectobacterium carotovorum subsp. brasiliense and the regulation of carbapenem biosynthesis by iron and the ferric-uptake regulator[END_REF][START_REF] Toth | Dickeya species an emerging problem for potato production in Europe[END_REF]. We showed that D. solani efficiently outcompeted D. dianthicola in hyacinths, in line with the epidemiologic data supporting the bulb plants as potential intermediate hosts with an important role in the recurrent invasion of potato agrosystems [START_REF] Chen | First report of Dickeya solani causing soft-rot in imported bulbs of Hyacinthus orientalis in China[END_REF]Slawiak et al., 2009;van der Wolf et al., 2014).

Prophylactics may allow circumventing the propagation of D. solani from an agrosystem to another. Competition and facilitation processes have been well studied in pathogenic fungi [START_REF] Al-Naimi | Competition, facilitation, and niche differentiation in two foliar pathogens[END_REF][START_REF] Abdullah | Host-multi-pathogen warfare: pathogen interactions in co-infected plants[END_REF][START_REF] Gladieux | The population biology of fungal invasions[END_REF]Zhan & McDonald, 2013). Another factor that could reduce competition between the two Dickeya species is their natural low abundance in soils and surface waters, as well as in seed tubers that are subjected to prophylactic diagnosis. A small population size belonging to a single species could proliferate in a plant individual without any interactions with another Dickeya or Pectobacterium pathogen species. These effects linked to population size and dispersal are expected to delay invasion. In line with this hypothesis, the observed slow increase of the percentage of Dickeya-positive fields over one decade (2004)(2005)(2006)(2007)(2008)(2009)(2010)(2011)(2012)(2013)(2014)(2015) suggested that the Dickeya invasion was still ongoing. Among the emerging literature on microbial invasion, the D. solani pathogen appears as a good example illustrating how different ecological components (here, the plant host and a resident pathogen) should be considered to understand the biological invasion by a bacterial pathogen [START_REF] Cadotte | Preadaptation and naturalization of nonnative species: Darwin's two fundamental insights into species invasion[END_REF][START_REF] Germain | The 'filtering' metaphor revisited: competition and environment jointly structure invasibility and coexistence[END_REF].

The genome sequencing data indicating low diversity in D. solani suggest a bottleneck during introduction in Europe. The dispersal modes (either horizontally via soil, surface water, insects and some agricultural practices or vertically by asymptomatically contaminated seed tubers) could contribute to further bottlenecks in D. solani [START_REF] Charkowsky | The changing face of bacterial soft-rot diseases[END_REF][START_REF] Toth | Dickeya species an emerging problem for potato production in Europe[END_REF].

Selection on some genes may also have further reduced genetic diversity through selective sweeps across the whole genomes as these bacteria are clonal. Allelic changes related to quorum-sensing systems were already observed in different plant and animal pathogens along the host-colonization process [START_REF] Feltner | LasR variant cystic fibrosis isolates reveal an adaptable quorumsensing hierarchy in Pseudomonas aeruginosa[END_REF][START_REF] Guidot | Multihost experimental evolution of the pathogen Ralstonia solanacearum unveils genes involved in adaptation to plants[END_REF][START_REF] Tannières | Quorum-quenching limits quorum-sensing exploitation by signal-negative invaders[END_REF], indicating that balancing selection contributed to maintain their variability.

Remarkably, we observed balanced frequencies of two alleles in the VfmB protein involved in the Vfm-type quorum-sensing in Dickeya bacteria. The chemical structure of the Vfm signal is not known, but it is known to be involved in tuber rotting and upregulation of some virulence genes including pectate lyases (pel genes), proteases (prt genes) and cellulases (cel genes) [START_REF] Nasser | Vfm a new quorum sensing system controls the virulence of Dickeya dadantii[END_REF][START_REF] Potrykus | Interplay of classic Exp and specific Vfm quorum sensing systems on the phenotypic features of Dickeya solani strains exhibiting different virulence levels[END_REF]. Using tuber assays, we observed a slightly higher

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This article is protected by copyright. All rights reserved aggressiveness in VfmB Ser strains compared to VfmB Pro strains. Using transcriptomics of D. solani pathogens recovered from tuber symptoms, we confirmed an enrichment of upregulated genes, including pel and prt genes, in the VfmB Ser strain Ds0432.1 compared to VfmB Pro strain IPO2222.

Because of the role of the VfmB protein in Vfm signaling is not completely elucidated, it was premature to go deeper into the mechanistic characterization of the VfmB Ser and VfmB Pro alleles.

In a near future, the reconstruction of allelic mutants and comparison with the wild strain with otherwise the same genetic background should help characterizing the role of VfmB in Vfm quorum-sensing and ecology of D. solani.

In this work, we strongly suggest that the VfmB Ser strains are more aggressive than the VfmB Pro strains in potato tubers, but not more virulent in potato stem assays, and that the VfmB Ser strains were less competitive than the VfmB Pro strains in tuber and stem symptoms. There is a very low number of SNPs/InDels (<12) in the genomes beyond those in Vfm, however, functional genetics will be required to fully validate the causal role of the Vfm alleles.

In conclusion, this study using complementary approaches, multiple strains representative of the population diversity and multiple ecological conditions, brings novel insights allowing a better understanding of the pattern and causes of the D. solani invasion into potato production agrosystems, and the reasons why D. dianthicola nevertheless persisted. More broadly, this study contributes to our understanding the ecological determinants of pathogen invasion and of the conditions for the maintenance of endemic competitors. This article is protected by copyright. All rights reserved This article is protected by copyright. All rights reserved

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  Pattern and causes of the establishment of the invasive bacterial potato pathogen Dickeya solani and of the maintenance of the resident pathogen D. dianthicola Pauline Blin, Kévin Robic, Slimane Khayi, Jeremy Cigna, Euphrasie Munier, Pauline Dewaegeneire, Angélique Laurent, Yan Jaszczyszyn, Kar-wai Hong, Kok-gan Chan, et al. To cite this version: Pauline Blin, Kévin Robic, Slimane Khayi, Jeremy Cigna, Euphrasie Munier, et al.. Pattern and causes of the establishment of the invasive bacterial potato pathogen Dickeya solani and of the maintenance of the resident pathogen D. dianthicola. Molecular Ecology, 2020, 10.1111/mec.15751. hal-03028685 1 Introduction

  population genomics to contribute to our understanding of the patterns and ecological determinants of D. solani invasion. Epidemiological records over one decade in France allowed to compare the relative abundances of the D. solani invader, the D. dianthicola resident and Pectobacterium spp. Using experimental populations and plant assays in greenhouse, with a

2. 3

 3 Plant and tuber inoculations by D. solani and D. dianthicola strains and experimental populations In our plant and tuber assays, we use the term virulence as the capacity of the pathogen to parasitize a host (symptom incidence) and the term aggressiveness as the degree of damage caused by a pathogen on a host (symptom or disease severity). The terms used to describe pathogens behaviors (virulence, symptom incidence, aggressiveness, disease severity index, fitness and competitive index) are defined more extensively in Supplementary Methods 1 (SM1). We designed the whole plant experiments based on our knowledge of the pathosystem, to investigate contrasting conditions relevant for the disease expression in fields. Each bacterial strain was cultivated individually before being used for plant and tuber inoculations, either separately or assembled in populations. To constitute experimental populations of D. solani, D. dianthicola, or mixtures of the two species, cell suspensions of different strains were assembled just before plant inoculation. Potato plants (S. tuberosum var. Bintje) and hyacinths (Hyacinthus orientalis var. Delft blue) were cultivated in horticultural compost in individual pots (2 L) in a greenhouse at around 23°C (minimum 20°C maximum 28°C) with a 12-hour photoperiod. For mimicking plant infection by soil pathogens, potato plants (three weeks post-tuber plantation) and hyacinths (four weeks post-bulb plantation) were inoculated by watering the substrate with a pathogen cell suspension at 10 9 colony-forming units (CFU) per pot. This inoculation load was calibrated to obtain a large range (20 to 60%) of symptom incidence in the unwounded potato plant condition (Raoul des[START_REF] Des Essarts | Biocontrol of the potato blackleg and soft rot diseases caused by Dickeya dianthicola[END_REF]. In a wounded condition, the potato roots were wounded with a sterile knife before infection. The symptom monitoring was adapted from Raoul des[START_REF] Des Essarts | Biocontrol of the potato blackleg and soft rot diseases caused by Dickeya dianthicola[END_REF]. The number of symptomatic potato plants (blackleg disease) and hyacinths (soft-rot disease) was recorded twice a week (see examples of symptoms in Figure1ab). For each strain and experimental population and unwounded and wounded conditions, the number of symptomatic and asymptomatic plants (hence two symptom classes that were used in statistical comparisons) was counted at 70 days post infection (dpi) using 15 potato plants (up to 25 potato plants when indicated) and 8 hyacinths. A set of non-inoculated plants was used as an asymptomatic control. In order to facilitate visualization of the virulence data in the figures, they are presented as a percentage of symptomatic plants (symptom incidence).

  Ser and VfmB Pro experimental populations, and their relative abundance was quantified by shot gun sequencing of DNA extracted from inoculum and from lesions. From 12 to 28 million reads were obtained for each sample, corresponding to an average coverage of D. solani genomes ranging from 180× to 420×. Sequencing (75 × 2 cycles) was performed using an Illumina NextSeq500 at the I2BC platform (CNRS, Gif-sur-Yvette, France) and Illumina MiSeq platform (University of Malaya, Kuala Lumpur, Malaysia). The trimmed reads were mapped on D. solani vfmB gene for quantifying the relative abundance of the two VfmB Ser and VfmB Pro alleles in each sample using the CLC genomic workbench version 10.1.3. The relative abundances of the alleles permitted calculation of CI values of the VfmB Ser and VfmB Pro populations (see SM1). A CI value equal to one indicated an equal fitness between D. solani VfmB Pro and VfmB Ser populations, CI value greater than one indicated a fitness advantage to VfmB Pro , and CI value less than one indicated a fitness advantage to VfmB Ser . Finally, we used transcriptomics to identity the genes that were differentially expressed between IPO2222 (VfmB Pro ) and Ds0432.1 (VfmB Ser ) D. solani strains when they grew inside potato tubers (SM2). Transcriptomes were compared as described by Raoul des Essarts et al. (2019).

  ). A moderate increase of the percentage of Dickeya-positive fields was observed over time (F= 5.05; DFn= 1; DFd= 9; p= 0.05; R² = 0.36). Although decreasing in incidence, the resident Pectobacterium species remained the most widespread (67% in the most recent sampling year 2015). As expected, the slopes of the dynamics of the percentage of Dickeya-and Pectobacterium-positive fields diverged (F= 10.11; DFn= 1; DFd= 18; p= 5 x 10 -3 ). Among the Dickeya isolates, only two species were identified, D. dianthicola and D. solani (Figure2b). The slopes of D. dianthicola-positive (F= 1.35; DFn= 1; DFd= 9; p= 0.28) and D. solani-positive (F= 3.10; DFn=1; DFd= 9; p= 0.11) field percentages were not different from base line and were not different from each other (F= 0.26; DFn = 1; DFd = 18; p= 0.62). This suggests that the D. solani invasion did not occur at the expense of D. dianthicola in French potato agrosystems.

  squared test: DF= 1; p< 0.05), meaning that Pectobacterium and Dickeya co-occurred less often than expected under random distribution. The hypothesis that D. solani and D. dianthicola were randomly distributed along the transect was also rejected in each of the 16 Dickeya-positive fields (Chi-squared tests: DF= 1; p< 0.05) but one (field #13; DF= 1; p= 0.24). These field data showed little co-occurrence of taxa at the field levels, both for genera (Dickeya and Pectobacterium) and species (D. solani and D. dianthicola). This bias in the symptomatic plants can result from a nonrandom distribution of the taxa among populations in seed tubers or/and soils and surface waters before plant infection, or from competitive exclusion during the infection process of potato plants and tubers. In the following, we focused on the two species D. solani and D. dianthicola to compare experimentally their fitness in the course of plant and tuber infection, and in particular to test the hypothesis of competitive exclusion.3.2 Dickeya dianthicola exhibited a fitness advantage over D. solani in potato plants.Plant assays were performed in the greenhouse to compare the incidence of blackleg symptoms after inoculation by D. dianthicola or D. solani on non-wounded potato plants. Five D. dianthicola strains (RNS11-47-1-1A, CFBP1888, CFBP2982, CFBP2015, MIE34) and five D. solani strains (3337 = RNS08-23-3-1A, IPO2222, RNS05-1-2A, Ds0432.1, PPO9019) were used in these assays.

  -7 ). Pairwise comparisons (post-hoc Tukey tests) showed that the numbers of plants with symptoms differed between inoculations with D. solani and D. dianthicola (F=-4.5; p=1.9x10 -5 ) and with the mixture of D. dianthicola and D. solani (F=6.1; p=2.7x10 -9 ). Mean ± SE of the percentage of symptomatic plants reached 33% ± 0 for D. solani, 83% ± 10 for D. dianthicola and 93% ± 7 for a mixture of D. dianthicola and D. solani, indicating a stronger virulence of D. dianthicola as compared to D. solani (FigureS4a). Pairwise comparisons (post-hoc Tukey tests) of the areas under the disease curves also confirmed that D. solani was less virulent than D. dianthicola(F=-386; p<10 -11 ) and

F=2. 2

 2 ; p=0.07). A DSI value was calculated for each set of 10 tubers. Mean value ± SE of the DSI values reached 83 ± 0 (D. solani), 63 ± 3 (D. dianthicola) and63 ± 8 (D. solani and D. dianthicola)

  expected a higher symptom incidence and growth advantage of D. solani on hyacinth host as compared to D. dianthicola. The five D. dianthicola and five D. solani bacterial strains were inoculated separately on eight plants per strain and the number of symptomatic and asymptomatic plants were counted. At 70 dpi, a Kruskal-Wallis test revealed differences between D. solani and D. dianthicola in terms of symptom incidence (k=3.03; DF=1; p=0.08): D. solani was found more virulent than D. dianthicola; means ± SE of the percentage of symptomatic plants reached 43% ± 17 and 23% ± 9, respectively (FigureS5a).

(

  Kruskal-Wallis test: k=7.8; DF=1; p= 5 x 10 -3 ). The CI median value of 4 x 10 9 indicated a high competitive advantage of D. solani over D. dianthicola in rotted tissues of hyacinths. Bulb plants thus appeared promoting competitive exclusion of D. dianthicola by D. solani, which would lead to an enrichment in D. solani in the bulb plant agrosystems.

)

  revealed that the D. solani isolates carrying VfmB Ser were placed in two different sub-clusters, one with only two VfmB Ser D. solani strains, EU3296 and Ds0432.1, and the other mainly VfmB Ser strains. This observation suggests that the VfmB Ser allele could have appeared independently in two different D. solani lineages, thus representing convergence. The VfmB Ser allele was present in 25% (19 out of 76) of all analyzed D. solani genomes. A similar percentage (27%, 17 out of 63) was observed when only the French isolates were considered. Using the isolation year of these 63 D. solani isolates, we drew the dynamics of the percentage of the VfmB Ser alleles, which was found to fluctuate over the past decade (2005-2015) with a peak in 2013-2014 (Figure 6c).

  In blackleg symptom assays (Figure8c), the D. solani VfmB Pro and D. solani VfmB Ser experimental populations and their mixture were inoculated on non-wounded plants and the number of symptomatic and asymptomatic plants were counted. The assay was performed in triplicate: two assays with 25 plants and one assay with 15 plants per treatment. Kruskal-Wallis tests revealed no difference between the three treatments, i.e., the D. solani VfmB Pro strain mixture, the D. solani VfmB Ser strain mixture and their mixture (k=0.29; DF=2; p=0.86). Shotgun sequencing of bacteria recovered from symptoms allowed calculation of CI values in 16 coinfected plants (Figure8c). These CI values were different from one (Kruskal-Wallis test; k=6.6; DF=1; p= 9 10 -3 ) with a median CI of 14, showing a fitness advantage of the VfmB Pro allele in emerging lesions in the stems.

  Two successive invasions of Dickeya potato pathogens occurred in Europe: first D. dianthicola in the middle of the 20 th century, this pathogen being now considered as endemic, and second, D. solani at the beginning of the 21 st century. Dickeya solani and D. dianthicola cause similar symptoms on potato plants, so that they are expected to compete for the same resources. Over ten years, our epidemiologic surveys revealed a successful establishment of D. solani together with a maintenance of D. dianthicola. Our experiments contributed to explain such puzzling coexistence by contrasted advantages in different parts of the plants and therefore partially different ecological niches.

  Such an environment-dependent advantage of VfmB ser and VfmB Pro alleles in plant infection assays predicts balancing selection in natural populations. The VfmB ser allele could thus provide an advantage when competition is reduced at the beginning of the establishment in potato agrosystems and under a high dispersal condition. In contrast, VfmB ser could be outcompeted by VfmB Pro when D. solani is already established. Our epidemiologic data revealed up and down variation of VfmB ser relative abundance in field populations over the past decade, in agreement with an increase of competition between D. solani VfmB alleles in the more recent sampling year.

Figure 1

 1 Figure 1 Symptoms caused by Dickeya pathogens. Symptoms (white arrows) caused by Dickeya solani and Dickeya dianthicola on potato stems (a) and hyacinths (b). The five symptom classes (0 to 4) used to compare the aggressiveness of Dickeya solani and Dickeya dianthicola on potato tubers (c).

Figure 2

 2 Figure 2 Dickeya and Pectobacterium prevalence from potato fields exhibiting blackleg symptoms. Percentage of Pectobacterium-containing and Dickeya-containing fields (a) and that of D. solani-containing fields and D. dianthicola-containing fields (b) were calculated each year from 2004 to 2015 (with the exception of 2006). (c) Number and relative abundance of the Pectobacterium, D. dianthicola and D. solani isolates collected in 19 symptomatic fields sampled from 2013 to 2016. A hierarchical clustering paired group method delineated four pathogen population groups, i.e. Pectobacterium only (I: 3 fields), D. solani and Pectobacterium (II: 9 fields), D. dianthicola and Pectobacterium (III: 3 fields), and D. dianthicola, D. solani and Pectobacterium (IV: 4 fields).

Figure 3 Figure 4

 34 Figure 3 Symptom incidence and fitness of Dickeya dianthicola and D. solani in potato plants. In a, mean value and standard error (SE) of the percentage (%) values of plants exhibiting blackleg symptoms, which were measured after inoculation of each of the five D. solani strains (3337, IPO2222, RNS05-1-2A, Ds0432.1, PPO9019) and five D. dianthicola strains (RNS11-47-1-1A, CFBP1888, CFBP2982, CFBP2015, MIE34) on 15 plants. The p-value of the Kruskal-Wallis test comparing of the symptomatic classes between the two species is indicated below the graph. In b, mean value and SE between two replicates of the percentages (%) of plants exhibiting blackleg symptoms which was measured on 15 plants inoculated by D. dianthicola and D. solani populations and their mixture. The pvalues of the pairwise comparisons (Post-hoc Tukey tests) of the symptomatic classes are indicated below the graph, when p ≤ 0.1. In c, competitive index (CI) values between D. solani and D. dianthicola populations were calculated in eight emerging lesions of co-infected plants and revealed a competitive advantage of D. dianthicola: the CI median (= 2.4 10 -5 ) is represented by a thick bar; p-values resulting from Kruskal-Wallis tests testing difference from one are indicated. Legend: * for 0.05<p≤0.1; ** for 0.01<p≤0.05 and *** for p ≤0.01.Accepted Article

Figure 5

 5 Figure 5 Expression of the pectate lyase genes pelA, pelD and pelE. The expression level of the pelA, pelD and pelE genes was evaluated in each of the five D. solani strains (3337, IPO2222, RNS05-1-2A, Ds0432.1, PPO9019) and five D. dianthicola strains (RNS11-47-1-1A, CFBP1888, CFBP2982, CFBP2015, MIE34), grown in three conditions: a rich culture medium in the absence of pectin (exponential growth phase) and symptomatic tubers (at 5 days post infection) and emerging lesions in stems. Relative expression was measured four times and normalized using the rpoB and yafS gene expression. In the graphs, the mean values and standard error (SE) of gene expression from all strains of a given species are indicated, as well as p-values of pairwise comparisons by Tukey tests. Legend: * for 0.05<p≤0.1; ** for 0.01<p≤0.05 and *** for p ≤0.01.

Figure 6

 6 Figure 6 Population genomics of Dickeya solani. In a, scan of 67 D. solani genomes revealed that the genome position 2,930,940, (according the D. solani 3337 genome), in the vfmB gene, was the most balanced non-synonymous variation, with alternated VfmB Pro (71%) and VfmB Ser (29%) alleles. In b, single nucleotide polymorphism (SNP)-based tree of D. solani strains using PHYLOViZ; the name of the French isolates is indicated in blue font; the VfmB Ser and VfmB Pro strains used in the plant assays are underlined. In c, dynamics of the VfmB Ser allele (%) among the D. solani isolates along the sampling period 2005-2015 in France.

Figure 7

 7 Figure 7 Characterization of the VfmB alleles. In a, using the Escherichia coli AidB protein (the PDB accession is 3DJL) as a model, the Phyre2-predicted structure of the VfmB protein showed conformational differences in the beta-sheet associated with the VfmB Pro and VfmB Ser alleles; the Pro55 and Ser55 positions are in red color in the VfmB representations obtained using the EzMol web server. In b, mean values and standard error (SE) of disease severity index (DSI) values, which were calculated by recording symptom classes on 10 tubers infected by 10 7 CFU of each of the D. solani strains carrying either VfmB Pro (IPO2222, MIE35, AM3a and 3337) or VfmB Ser (Ds0432.1, RNS10-27-2A, Sp1a and M21a); the p-value of the Kruskal-Wallis test comparing the four VfmB Pro and four VfmB Ser strains using symptom classes is indicated below the graph. In c, comparative transcriptome of D. solani 3337 (VfmB Pro ) and Ds0432.1 (VfmB Ser ) recovered from soft-rot lesions in potato tubers; upregulated virulence genes were enriched in Ds0432.1 (VfmB Ser ) as illustrated by the pelB, pelC, pelC, hcpA and prtA genes (closed circles).

Figure 8

 8 Figure 8 Aggressiveness, symptom incidence and fitness assays of two Dickeya solani experimental populations expressing the VfmB alleles. Soft-rot assays were conducted by inoculating each tuber by either 10 7 colony-forming units (CFU) (a) or 10 5 CFU (b) of the VfmB Pro and VfmB Ser D. solani populations and a mixture of the two. In a and b, each disease severity index (DSI) value (measuring aggressiveness) was calculated using symptomatic classes observed on 10 tubers; the assays were performed in triplicate. The p-values of the pairwise Tukey tests comparing the symptom classes are indicated, when p ≤ 0.1. In c, percentage (%) of plants exhibiting blackleg symptoms was measured on plants inoculated by the VfmB Pro and VfmB Ser D. solani populations and a mixture of the two. These assays measuring symptom incidence were performed in triplicate. Kruskal-Wallis test revealed no difference between the three treatments using symptom classes (p = 0.86). In a, b and c, competitive index (CI) values of VfmB Pro and VfmB Ser populations were calculated using allele counts based on shotgun sequencing of bacterial populations recovered from co-infected tissues. Median values of CI are represented as thick lines and reached 1.7 (8 repeats in a), 2.0 (3 repeats in b) and 14.0 (16 repeats in c) in the three treatments. Statistical differences between CI values and 1 (Kruskal-Wallis test) revealed that neither D. solani VfmB Pro or D. solani VfmB Ser had significant advantage in tubers inoculated with a high load (p = 0.3 in a), but D. solani VfmB Pro was more competitive in tubers inoculated with a low load (p = 0.04 in b) and in stems (p = 9 x 10 -3 in c). Legend: * for 0.05<p≤0.1; ** for 0.01<p≤0.05 and *** for p ≤0.01.

  Figure 1

  Figure 4

  Soft rot on tubers (DSI)This article is protected by copyright. All rights reservedAccepted Article

  2; DF=2; p=0.03) pathogen loads. Whatever the bacterial load, pairwise comparisons (Post-hoc Tukey tests) showed that aggressiveness (i.e. symptom severity) was different between D. solani VfmB Pro and D. solani In tubers, the VfmB Pro allele thus appeared neutral or to confer a moderate advantage as compared to the VfmB Ser allele.

	VfmB Ser (at a high load: F=4.6; p=9.5 x 10 -6 ; at a low load: F=0.8; p= 0.01). The D. solani VfmB Ser
	population was more aggressive than the D. solani VfmB Pro population (DSI mean value ± SE of
	92 ± 2 for VfmB Ser vs 73 ± 3 for VfmB Pro at a high load and 45 ± 2 for VfmB Ser vs 26 ± 9 for
	VfmB Pro at a low load; Figure 8a-b). VfmB Ser D. solani strains thus more efficiently damaged
	tubers than VfmB Pro D. solani strains.
	When the VfmB Ser and VfmB Pro D. solani experimental populations were co-infected in tubers,
	shotgun sequencing of the pathogens recovered from symptoms allowed to calculate CI values
	(Figure 8a-b). With a load of 10 7 CFU per tuber, the CI values (8 values; median = 1.3) were not
	different from one (Kruskal Wallis test; k=0.8; DF=1; p= 0.4). With a load of 10 5 CFU per tuber,
	the CI values (3 values; median = 2) differed from one (Kruskal Wallis test; k=4.4; DF=1; p=
	0.04).

ACKNOWLEDGEMENTS

PB was supported by a PhD grant from Université Paris-Sud (Université Paris-Saclay), KR was supported by a PhD grant from FN3PT/inov3PT with the support of ANRT (N°2016/0800). KGC thanks the financial support from the University of Malaya (FRGS grant FP022-2018A and HIR grant H-50001-A000027). This work has benefited from the facilities and expertise of the high throughput sequencing platform and the plant culture facilities at I2BC (Gif-sur-Yvette). This work was supported by CNRS (SE2016-2017-2018), French Federation of Seed Potato Growers

Accepted Article

This article is protected by copyright. All rights reserved van der Wolf, J. M., Nijhuis, E. H., Kowalewska, M. J., Saddler, G. S., Parkinson, N., Elphinstone, J. G., … Manulis, S. (2014). Dickeya solani sp. nov., a pectinolytic plant-pathogenic bacterium isolated from potato (Solanum tuberosum). International Journal of Systematic and Evolutionary Microbiology, 64, 768-774. Wright, D., Bwye, A., Banovic, M., Baulch, J., Wang, C., Hair, S., … Kehoe, M. (2018). First report of Dickeya dianthicola in potatoes in Australia. Plant Disease, 102, 2029. Zhan, J., & McDonald, B. A. (2013). Experimental measures of pathogen competition and relative fitness. Annual Reviews of Phytopathology, 51,

DATA ACCESSIBILITY

DNA sequences: Genbank accessions VYSC00000000; VZQE00000000; VZQF00000000; VZQG00000000; VZQH00000000; VZQI00000000; VZQK00000000; VZQJ00000000; VZQL00000000; VZQM00000000

Accepted Article

This article is protected by copyright. All rights reserved (FN3PT/inov3PT), Agence Nationale de la Recherche (ANR-15-CE21-0003) and LabEx Saclay Plant Sciences-SPS (ANR-10-LABX-0040-SPS).

AUTHOR

CONFLICT OF INTEREST

No potential conflict of interest was disclosed.

ORCID

Denis Faure orcid.org/0000-0002-5379-8867

Accepted Article