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International audience; Objectives: On the last decade, ECC members has been defined by Hoffmann and Roggenkamp in 13 genetic clusters (I to XIII) based most exhaustively on hsp60 sequencing. More recently, Chavda et al. in 2016 proposed a new phylogenic classification by using both average nucleotide identity and a single-nucleotide polymorphism based phylogeny. The aim of this study is to improve the determination method based on hsp60.Methods: All the available ECC genomes (1.324) in the patric database v3.5.27 have been downloaded. The correct specie identification and the absence of contaminant specie were checked using the rMLST data base provided by PubMLST. The hsp60, rpoB and ampC genes were extract form all the genomes and clustered using cd-hit with an 99% identical cut off. A distance matrix between all the genomes based on the Kmer content was determined using Mash software and a neighbor joining tree (NJT) was constructed using the neighbor software available in the PHYLIP package. The tree and the molecular typing of all the strains were represented using itol.Results: Using Mash allowed to obtain a distance matrix of more than a thousand genomes in less than one hour. Clusters recovered in the NJT were in ad equation with the hsp60 clustering method for C11, C2, C5, C7. Nevertheless, genomes which belonged to the same clusters such as C9 or C4 appeared to be very divergent to each other. Furthermore, genomes which belonged to C13 seemed to be miss-classified C6. As the consequence, the hsp60 method could be not enough discriminant and precise to allow a correct clusterization and can lead to typing errors.The diversity of the rpoB gene sequences didn’t explained the phylogenetic clusters observed. Especially, it appeared that, C8 and C6 clusters shared the same rpoB sequences, as the consequence of what, rpoB sequences can’t be used as a clustering method. Interestingly, the diversity of the blaACT sequences improved the discrimination between the isolates and avoided mis-clusterization.Conclusion: The findings in this study provide an additional approach to identify in a more precise way and easily the cluster to which belongs the isolate of ECC.

International audience; Objectives: Members of the Enterobacter cloacae complex (ECC) are major opportunistic pathogens, especially among intensive care unit (ICU) patients. The aim of this study was to decipher the phenotypic and genomic evolution of an ECC clone that acquired a multidrug resistance phenotype in an ICU patient. Methods: During a four-month period, 8 MDR ECC strains were recovered from the same patient: 5 from clinical samples and 3 from rectal swabs. The patient benefited from an escalation of antibiotic treatment until he died. MIC values of 18 antibiotics were determined by the broth microdilution method. Whole-genome sequencing was performed for the 8 strains and a Single-Nucleotide Polymorphism (SNP) analysis was performed after alignment against the ECC reference genome (ATCC 13047). Results: In strains isolated from the 3rd month, a significant increase in MICs (at least 4 fold change) was observed for ceftazidime, cefepime, temocillin, ertapenem, tigecycline and chloramphenicol. All strains harbored the same antibiotic resistance genes against β-lactams, aminoglycosides, fluoroquinolones, sulphonamides, trimethoprime and chloramphenicol. The 8 MDR ECC belonged to the same cluster 6 (ECC6) and were genetically related. No genetic evolution was found in chromosomal genes usually involved in β-lactam resistance (i.e. ampD, ampR, nagZ, dacA, dacB, acrAB-tolC, ompK35, ompK36). The isolates differed by 5 SNPs located in metabolism genes and a deletion of 13 bases in romR, which encodes for TetR-type transcriptonal regulator. This latter is known to play a repressor role on the expression of porins such as ompK35, whose the downregulation might be involved in MIC increases observed here. Conclusion: Consequent and long-term antibiotic treatment in one patient indicated the selection of a unique MDR ECC6 population where antibiotic resistance evolved more by chromosomal modifications than acquired antibiotic resistance genes. The analysis of the different genetic features through all the 8 MDR ECC6 strains allowed us to highlight a candidate gene that could be implicated in carbapenem, chloramphenicol and tigecyclin resistance. Further phenotypic and molecular characterization by site-directed mutagenesis is in progress.

Ce numéro est constitué d’articles de synthèse des projets DEPHY EXPE publiés à l’occasion du Colloque National DEPHY EXPE, qui s’est déroulé le 28 mai 2019 à l'Assemblée Permanente des Chambres d'Agriculture (Paris). Nous vous invitons à découvrir le bilan et les enseignements de 19 projets conduits sur la période 2013-2018.; The agricultural specialization currently faces some limits. New synergies between crop and livestocksystems with mutual benefits seem necessary. These new crop-livestock systems offer a solution for amore sustainable agriculture on the scale of farms but also territories.The project aimed at promoting partnerships between crops and livestock specialized systems on aterritory scale, focusing on sociological, organizational and legal issues. The action was based on 7experimental groups in 3 different French regions (Centre-Val de Loire, Pays de la Loire, NouvelleAquitaine). The main objective was to analyse obstacles and catalysts to create complementarities at aterritory scale. The project identified key elements to develop complementarities (sociological analysis)and created new tools to support farms for emergence, stabilisation and formalisation of the exchangesand for securing them: a methodological guide, videos of successful experiences, testimonies andmethods records and a legal guide; Face aux limites de la spécialisation des systèmes d’exploitation de grandes cultures et d’élevages, unenouvelle complémentarité à bénéfices communs est nécessaire. Il s’agit de « recréer un système » pourune agriculture plus durable au niveau des territoires et non plus seulement au sein des exploitations.Le projet CER’EL s’est ainsi intéressé aux ressorts sociologiques, organisationnels et juridiques pourétablir une complémentarité territoriale innovante entre systèmes spécialisés de grandes cultures etd’élevages. En s’appuyant sur sept territoires pilotes représentant la diversité des situations rencontréesdans les trois régions partenaires (Centre – Val de Pays de la Loire et Nouvelle Aquitaine), le projetCER’EL a permis d’identifier des clefs de développement des complémentarités (analyse sociologique)et d’élaborer des outils concrets permettant aux conseillers d’accompagner les agriculteurs dansl’émergence, la stabilisation, la formalisation et la sécurisation des échanges : un guideméthodologique, des vidéos d’expériences réussies, des fiches méthodes et témoignages, ainsi qu’unguide juridique.

International audience; Introduction :Les entérobactéries sécrétrices de béta-lactamase à spectre étendu (BLSE) sont responsables d’une augmentation de la morbi-mortalité infectieuse. Cette étude s’est intéressée à la diversité génomique des souches d’E. coli sécrétrices de BLSE au de CHU de Caen sur une période de 5 ans.Matériels et méthodes :Cinquante-trois souches représentant la première indentification d’une BLSE chez des patients uniques, isolées entre 2012 et 2016, à partir d’écouvillonnages rectaux ont été séquencées en génome entier. Les caractéristiques des bactéries : Sequence-Type (ST), sérotype, gènes de résistance et gènes de virulence ont été déterminés in silico grâce aux bases de données.Résultats :Les prélèvements provenaient de 16 différents services, majoritairement les services de médecine, de chirurgie puis de réanimation (40%, 31%, 29%). Le sex ratio H/F était de 0,9. L’âge moyen des patients était de 64 ans ; 55% avaient été hospitalisés dans les 3 mois précédents ; 51% ont reçu une antibiothérapie au cours de leur hospitalisation et 14% avaient voyagé hors de France.Au total, 34 différents ST et 35 sérotypes ont été retrouvés. Les populations E. coli les plus fréquentes étaient les ST131 O25:H4 (n=6), ST10 O89:H9 (n=3) et ST162:O9:H9 (n=2). Onze différentes béta-lactamases ont été détectées, les plus fréquentes étaient : CTX-M-1 (n=18), CTX-M-14 (n=9), CTX-M-15 (n=7). Pour 27 patients (49%), la souche d’E. coli BLSE a été identifiée avant 48h d’hospitalisation, principalement CTX-M-14. Tandis que l’enzyme « nosocomiale » était volontiers CTX-M-15 (délai moyen d’acquisition 19 jours, min/max : 0/66 jours). Enfin, CTX-M-14 et SHV-12 étaient significativement plus retrouvées pour les souches d’E. coli circulantes au sein des services de réanimation (p=0,02, p=0,02) supposant une circulation diffuse de souches épidémiques.Conclusion :Cette étude d’épidémiologie génomique a permis de révéler une grande diversité de populations des souches d’E. coli sécrétrices de BLSE au CHU de Caen depuis 2012. Cela supporte que dans 50% des cas le portage de ces bactéries était probablement d’origine communautaire (détection dans les 48h après l’admission). D’autres souches hospitalières, communautaires et vétérinaires sont en cours de séquençage afin d’augmenter la puissance de l’étude sur leurs attributions et leurs circulations en Normandie qui semblent assez étendues.

International audience

Epidemiological surveillance of plant pathogens based on genotyping methods is mandatory to improve disease management strategies. In the Southwest Indian Ocean (SWIO) islands, bacterial wilt (BW) caused by the Ralstonia solanacearum species complex (RSSC) is hampering the production of many sustainable and cash crops. To thoroughly analyze the genetic diversity of the RSSC in the SWIO, we performed a wide sampling survey (in Comoros, Mauritius, Reunion, Rodrigues, and Seychelles) that yielded 1,704 isolates from 129 plots, mainly from solanaceous crops. Classification of the isolates to the four major RSSC phylogenetic groups, named phylotypes, showed that 87% were phylotype I, representing the most prevalent strain in each of the SWIO islands. Additionally, 9.7% were phylotype II, and 3.3% were phylotype III; however, these isolates were found only in Reunion. Phylotype IV (2 isolates), known to be restricted to Indonesia-Australia-Japan, was reported in Mauritius, representing the first report of this group in the SWIO. Partial endoglucanase (egl) sequencing, based on the selection of 145 isolates covering the geographic and host diversity in the SWIO (also including strains from Mayotte and Madagascar), revealed 14 sequevars with Reunion and Mauritius displaying the highest sequevar diversity. Through a multilocus sequence analysis (MLSA) scheme based on the partial sequencing of 6 housekeeping genes (gdhA, gyrB, rplB, leuS, adk, and mutS) and 1 virulence-associated gene (egl), we inferred the phylogenetic relationships between these 145 SWIO isolates and 90 worldwide RSSC reference strains. Phylotype I was the most recombinogenic, although recombination events were detected among all phylotypes. A multilocus sequence typing (MLST) scheme identified 29 sequence types (STs) with variable geographic distributions in the SWIO. The outstanding epidemiologic feature was STI-13 (sequevar I-31), which was overrepresented in the SWIO and obviously reflected a lineage strongly adapted to the SWIO environment. A goeBURST analysis identified eight clonal complexes (CCs) including SWIO isolates, four CCs being geographically restricted to the SWIO, and four CCs being widespread beyond the SWIO. This work, which highlights notable genetic links between African and SWIO strains, provides a basis for the epidemiological surveillance of RSSC and will contribute to BW management in the SWIO.

International audience; Nous présentons dans ce travail les études préliminaires dans le but de créer une nouvelle formation pratique en salle blanche sur la thématique de fabrication de substrats SOI (Silicium on Insulator). La méthode de fabrication est basée sur le procédé Smart-CutTM (Soitec/CEA-Leti). L’objectif de ces premiers travaux est de démontrer la faisabilité du procédé avec des wafers de taille réduite (2 pouces) dans un environnement loin des standards (en termes de propreté) normalement requis pour la réalisation du procédé. Le procédé comprend une première étape d’implantation d’ions H, puis une phase de collage moléculaire et d’activation de la fracture par recuit. Des taux de transfert de 80% à 90% ont été obtenus ouvrant la voie vers un enseignement sur la fabrication de SOI par le procédé Smart-CutTM.

International audience; no abstract

International audience; The Rssc encompasses strains that are highly destructive worldwide causing bacterial wilt (BW) on a wide plant host range. BW was alread y described in Madagascar (phylotype I and III) but potato production was hampered by severe outbreaks. Controlling this disease is crucial for Malagasy potato producers. Knowledge of the genetic diversity and population structure of the pathogen was inves tigated, in fine to evaluate resistance of different potato cultivars. We characterized a large collection of strains (n=1224; 74 sites) surveyed from potato areas in 2013, by using different molecular tools. Phylotypes I, II and III were identified and most potato outbreaks were shown to be associated with brown rot strains IIB - 1, whereas members of phylotype I and III caused few. Along with reference strains, epidemic IIB - 1 strains (n=290) were genotyped through a MLVA scheme ( RS2 - MLVA10), which showed a single clonal complex distributed in wide agro - ecological areas, and genetically related to worldwide genotypes. This suggests this IIB - 1 lineage is busting potato production in Madagascar after being introduced (not cryptic) and spread through release of infected potato - seeds. To assess the population structure of long time recognized phylotype III strains, 251 strains were analyzed through the newly developed and highly discriminatory typing scheme RS3 - MLVA16. Malagasy population of phylotype III strains showed a broad genetic basis made of 132 haplotypes in at least 7 clonal complexes; and furthermore distinct from African strains, suggesting its endemic character. At an agro - ecological scale, two major clonal complexes were unified by haplotypes shared between individual areas and the remainder made of haplotypes derived from a unique genotype. This large - scale epidemiological study and the generated sets of data are reference models to further studies of Rssc population in Madagascar and the SWIO* , including evaluation of pathogen aggressiveness and host resistance. (Résumé d'auteur)

Bacterial wilt (BW) caused by Ralstonia solanacearum (Rs) is one of the most harmful plant disease in the world1 • Special careful should be paid to phylotype I due to its large host range, its worldwide distribution, and its high evolutionary potentiai2. However there is a Jack of studies focusing on population genetics of Rs, whereas basic knowledge on the dynarnics of pathogen genetic adaptation is now needed to predict plant resistance durability3, especially at the field scale. Thanks to a new ML V A scheme (ML V A-8) specifically tuned for field-scale microevolution of Rs phylotype :c4, we addressed the temporal and spatial dynamics of Rs populations' genetic structure in a field submitted to eggplant selective pressure. We set up a three years experimental field containing 3 microplots of resistant (E6) and susceptible eggplant (E8), two crops cycle were implanted each year (Cl to C5). No bacterial wilting was observed on resistant eggplant (E6) and we only isolated strains from latent infections on E6 during Cl, but no trade-offfor colonization of resistant eggplant was observed. On E8, we monitored an increase of incidence between Cl and the other cycle but virulence assay did not show a gain of virulence ofstrains between Cl and C2. Across eggplant cycles, the disease seemed to spread by host-to-host dissemination as well as by hot-spots of disease which finally merged. A clear genetic diversity reduction was observed in response to repetitive crop cycle of eggplant associated with the increased of frequency of two main haplotypes. However we failed to identify an effect of the host variety, the host's genetic background or the habitat of origin (soil, rhizosphere and stem) on the genetic structure of Rs populations. Our study helps to decipher the forces goveming of evolution of a field Rs population in response to host selective pressure, and the main outputs will be further discussed in the context of durable control ofBW in the field.

Ralstonia solanacearum is a species complex (Rssc), distributed worldwide and economically destructive. This soilborne bacterial plant pathogen encompasses an unusually wide hast range. The pathogen invades the roots and colonizes the xylem vessels causing bacterial wilt (BW) diseases. Strains distributed into four phylogenetic groups, based on ITS sequences called phylotypes that relates to geographical origin: phylotype 1 (Asia), phylotype Il (America), phylotype Ill (Africa), and phylotype IV (lndonesia-Japan). ln the South-West lndian Ocean (SWIO) islands, although R. solanacearum is regularly reported, no recent data is available about its genetic diversity and structure. Our main objectives are then to ge a significant collection of R. solanacearum strains to thoroughly analyze this genetic diversity; characterize the evolutionary forces that shape populations; and evaluate genetic resources for resistance to BW. 2971 R. solanacearum strains have been collected mainly from Solanaceae: Madagascar* (n=1224), Reunion (n=789), Mauritius (n=785), Mayotte (n=166), Seychelles (n=92), Rodrigues (n=Sl) and Comoros (n=S, to be completed). A preliminary broad molecular typing was done using the multiplex PCR (Fegan and Prier, 2005) showing 49.5% of phylotype I, 40 of phylotype Il, 10.4% of phylotype Ill, and 2 strains of phylotype IV. A selection of 126 strains that covered the geographic and host diversity was organized to assess phylogenetic relationships. Maximum Likelihood tree (PhyML) was performed on th partial endoglucanase (egl) amplification, along with a set of 100 international reference strains. Further characterizations are ongoing through a MLSA/MLST** scheme based on partial amplification of 7 genes; gdhA, mutS, adK, leuS, rplB, gyrB and egl. This is the first report of a large scale survey in the SWIO. lnterestingly we reported here the predominance of one sequevar (1-31) among the SWIO strains whereas the phylotype I is known to encompass a broad genetic basis (16 sequevars) Also sequevar IV-10 was described in Mauritius for the first time.

The Ralstonia solanacearum species complex (Rssc) is a highly diverse cluster of strains, many of which proved destructive worldwide causing bacterial wilt (BW) on a wide plant host range. Potato BW has been described for nearly 30 years in Madagascar highlands (phylotype I and Ill) without major impact on production. Recently, potato production was dramatically devastated by severe outbreaks. Controlling this disease is crucial for Malagasy potato producers and a first important step to achieve this goal is the identification of the strains involved and of their putative origin. Consequently, the genetic diversity and population structure of the Rssc was investigated in the major potato production areas in the highlands. By using different molecular tools we characterized a large collection of strains (n=1224; 75 sites) collected from potato production areas in 2013, April (n=763; 41 sites) and December (n=461; 33 sites). Present for a long time in Madagascar, Phylotypes I and Ill strains were little associated with potato BW outbreaks, while surprisingly, most outbreaks were associated with brown rot strains IIB-1. This constitutes the first report of phylotype IIB-1 strains in Madagascar. Along with reference strains, epidemic IIB-1 strains (n=285) were genotyped through the MLVA RS2-MLVA9 implemented from existing schemes (1, 2]. Malagasy phylotype Il strains separated into two clonal complexes (CC) including one major CCl grouping also most of the worldwide genotypes that distributed in wide agro-ecological areas. Fine scale genetic investigation of the outbreak suggested that the clonal expansion of this IIB- lineage affected potato production in Madagascar after being introduced and massively spread through propagation of latently infected potato-seeds. The survey also allowed the first study about the population structure of phylotype Ill strains. Two hundreds and seventeen phylotype Ill strains were genotyped with the highly discriminatory MLVA scheme RS3-MLVA16 that we newly developed [3]. MLVA distinguished a high genetic diversity in Malagasy population of phylotype Ill strains producing 117 haplotypes grouped in 11 clonal complexes. Malagasy strains were distinct from continental African strains, suggesting little recent immigration from the continent. Many CC a long with the large genetic diversity supported the endemic character of the phylotype Ill population in Madagascar. At an agro-ecological scale, phylotype Ill mostly structured in small clonal complexes associated with a single geographic origin each. But two major CCs grouped some haplotypes shared between individuals from different highland areas, and the remainder made of haplotypes derived from a unique origin supporting long distance dispersal most probably through human activities. Although the most popular Malagasy potato cultivars developed by CIP and FIFAMANOR were apparently developing resistance to phylotype Ill strains in the field, we reported no bacterial wilt resistance properties under artificial environment (growth chambers). As expected, the same potato cultivars were highly susceptible to expanding phylotype IIB-1 lineage.

National audience; Diversity in the South-West Indian Ocean. Surveys, phylotype analysis and phylogenetic tree by egl sequencing.

Ralstonia solanocearum is a species complex (Rssc), distributed worldwide and economically destructive. This soilborne bacterial plant pathogen encompasses an unusually wide host range. The pathogen invades the roots and coloniz.es the xylem vessels causing bacterial wilt (BW) diseases. Four phylogenetic groups called phylotypes structured the Rssc that relates to their geographic origin of evolution: phylotype I (Asia), phylotype II (America), phylotype III (Africa), and phylotype IV (Indonesia-Japan). In the South-West Indian Ocean (SWIO) islands, although R solanocearum is regularly reported, no recent data is available about its genetic diversity and structure. Our main objectives are then to get a significant collection of R.solanacearum strains to thoroughly analyze the genetic diversity of the strains; characterize the evolutionary forces that shape populations; and evaluate genetic resources for resistance to BW. Presently, 2971 R.solanocearum strains have been collected mainly from Solanaceae: Madagscar* (n=1224), Reunion (n=F789), Mauritius (n=693), Mayotte (n=166), Seychelles (n=92), and Comoros (n=5, to be completed). A preliminary broad molecular typing was done using the multiplex PCR (Fegan and Prior 2005)**, showing 46.6% of phylotype I, 42.3% of phylotype II, 11% of phylotype III, and 0.1% of phylotype IV. A selection of 126 strains that covered the geographic and host diversity was organized to assess phylogenetic relationships. Maximum Likelihood tree (PhyML) was performed on the partial endoglucanase (egl) amplification, along with a set of 1 OO international reference strains. Further characterizations are ongoing through a MLSA scheme based on partial amplification of 6 genes; gdhA, mutS, adK, leuS, rplB and gyrB. This is the first report of a large scale survey in the SWIO. Surprisingly we showed that phylotype I-31 is mainly represented and found in every prospected territory in the SWIO. Also Phylotype IV was first described in Mauritius.

International audience; MultiLocus Variable number of tandem repeat Analysis (MLVA) has been extensively used to examine epidemiological and evolutionary issues on monomorphic human pathogenic bacteria. MLVA is gaining popularity on agriculturally important bacterial plant pathogens as a tool to improve our understanding of their epidemiology. Xanthomonas citri pv. citri, the causal agent of Asiatic citrus canker, is a quarantine organism in several countries and a major threat for the citrus industry worldwide. We screened the genomes of X. citri pv. citri strain IAPAR 306 and of phylogenetically related strains for tandem repeats. Two MLVA schemes targeting minisatellites and microsatellites, respectively, were developed to assess the diversity of this monomorphic bacterium at various spatio-temporal scales. Microsatellites are useful for outbreak investigation. In contrast, minisatellites were used to decipher the global diversity of X. citri pv. citri and revealed four pathotype-specific lineages based on Discriminant Analysis of Principal Components (DAPC). A single lineage (DAPC1) comprised strains that were implicated in the major geographical expansion of X. citri pv. citri during the 20th century. When applied to the analysis of the emergence of in Africa, minisatellites revealed the presence of two different genetic lineages in addition to DAPC1 on this continent, consistent with SNP data derived from draft genome sequences. Using bacterial populations collected in two neighboring Western African countries, Mali and Burkina Faso, we applied these two genotyping schemes to investigate the origin and pathways of these emergences. Minisatellite and SNP data suggested the introduction of two groups of strains in Mali (DAPC1 and DAPC2). DAPC2 was restricted to Bamako district, whereas DAPC1 strains were found much more invasive. The latter strains formed a major clonal complex based on microsatellite data. This suggests that human activities played a major role in the spread of DAPC1 strains via the movement of contaminated propagative material, further supported by the low differentiation between populations from geographically distant nurseries and orchards and the fact that many nursery strains were assigned to the primary or secondary founder haplotypes identified in the minimum spanning tree. Approximate Bayesian Computation analyses supported the hypothesis that strains from Burkina Faso resulted from a bridgehead invasion from Mali. The new minisatellite scheme represents an opportunity for international X. citri pv. citri genotyping and data sharing. The data generated in this study was deposited in the Xanthomonas citri genotyping database (http://www.biopred.net/MLVA/). (Texte intégral)

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

[Departement_IRSTEA]Eaux [TR1_IRSTEA]RIVAGE; National audience; Degradation phenomena of materials and structures are a major industrial preoccupation. Indeed, they condition strategies on inspection, maintenance, repair and extending the service life of facilities. We introduce here the Theme 4 of the JFMS 2014 which was devoted to the probabilistic modelling of degradations caused by the mechanical behaviour and the natural environment of materials and structures. This document presents briefly the 6 papers related to the Theme 4 JFMS 2014 and it summarizes the contribution of these papers to the probabilistic modelling of degradations.; Les phénomènes de dégradation des matériaux et des structures constituent une préoccupation industrielle majeure. Ils conditionnent les stratégies d’inspection, maintenance, réparation et prolongation de la durée de vie des installations. Nous introduisons ici le Thème 4 des JFMS 2014 qui a été consacré à la modélisation probabiliste des dégradations dues au fonctionnement mécanique et à l’environnement naturel des matériaux et des structures. Ce document présente les 6 communications associées au Thème 4 des JFMS 2014 et synthétise l’apport de ces communications à la modélisation probabiliste des dégradations.

International audience; Bacterial wilt, caused by Ralstonia solancearum (Rs) (Smith) Yabuuchi et al., is one of the most important diseases of potato crops worldwide, including Madagascar. From the literature, strains of Rs phylolypes III or 1 were pathogenic to Solanaceae in the highlands potato areas (1,2). From years these strains were managed through the deployment of resistant or tolerant potato cultivars thanks to joint 30-yeaTS of collaborative research undertaken by Fifamanor (3) and CIP (4). however, since the last five years severe epidemics of bacterial will were observed in the Vakinankaratra highlands. A quest ion arises about the durability of resistant or tolerant potato varieties. It was hypothesized that (i) highly aggressive strains may have emerged from the Rs populations or ( ii ) exotic strains could have been introduced. A large survey was organized to collect strains of Rs to investigate how diverse the actual Rs population was. A first approach focused on phylotype profiling (multiplex-PCR) and phylogenetic assignation (egl sequcncing) of a limited number of strains (n=32) isolated from highly diseased potato plots. Surprisingly, this first set of Rs strains were computed phylotype IIB - I based on egl-treeing including worldwide reference sequences that covered the known diversity. This is the first report of brown rot causing strains in Madagascar. From the survey, 763 strains have been collected from symptomatic plants in main representative potato growing areas of Vakinankaratra: 132 strains were phylotype III and 631 phylotype II B-I. Phylotype III strains were associated to sites with low bacterial will incidence. In contrast., phylotype IIB-I strains were always associated with severe outbreaks and found to be distributed over almost the entire area surveyed. Population structure and evolutionary dynamics o f lineages IIB-I and III are currently investigated by improving available MLVA (multilocus VNTR analysis) schemes (5.6) and by developing a specific MLVA scheme, respectively. (Résumé d' auteur)

International audience; Le flétrissement bactérien causé par Ralstonia solanacearum (Rs), une bétaproteobactérie vasculaire d'origine tellurique, est considéré comme j'une des phytobactérioses majeures au niveau mondial. Rs est considérée comme un complexe d'espèces subdivisé en 4 phylotypes dont le phylotype 1 a le plus fort potentiel évolutif et une prévalence mondialea. La résistance variétale, stratégie de lutte la plus prometteuse, reste cependant difficile du fait de l'extrême plasticité génomique et phénotypique de la bactérie. Les plus hauts ni veaux de résistance ont été identifiés chez l'aubergine mais la question de leur durabilité reste ouverte. Ainsi nous proposons d'étudier la durabilité de la résistance de l'aubergine AG9 125, portant le gène majeur Ers/ u, à travers le suivi évolutif de la structure génétique d'une population parcellaire de Rs phylotype 1 soumise à des cultures répétées de cette accession. Dans cette parcelle, ont été implantés un cycle de culture de tomate sensible suivi de trois cycles de culture d'accessions d'aubergine résistante (AG9 1-25) et sensible (MM738). L'incidence de la maladie a été suivi puis des bactéries ont été isolées et analysées avec un premier schéma MLVAc (MultiLocus VNTR Analysis) mais celui-ci s'est révélé trop peu résolutif. Nous avons alors développé un nouveau schéma MLVA après criblage in silico de trois génomes (deux de phylotype 1 et un de phylotype III ). Ce schéma à 8 loci minisatellites, spécifiques au phylotype l, est plus discriminant que le schéma précédent sur des populations parcellaires ou régionales. Cet outil a révélé une faible diversité génétique (O,215

International audience; Le genre des Citrus est sujet à un grand nombre de maladies économiquement impactantes. C'est le cas du Chancre Asiatique des Agrumes, causé par la bactérie Xathomonas citri pv. citri (Xcc). Organisme de quarantaine dans le bassin méditerranéen, ce pathogène est émergent dans plusieurs pays d'Afrique, dont l'Ethiopie en 2006 (1). Très peu d'informations fiables sont disponibles pour reconstruire l'histoire de l'émergence de la maladie dans ce pays. A cette fin , nous avons entrepris des analyses de génétique des populations sur 8 vergers échantillonnés dans un même paysage agrumicole, à l'aide de 14 marqueurs microsatellites. Des analyses descriptives classiques confirment le statut épidémique de la population Ethiopienne et suggèrent que les plantations auraient été infectées à partie d'une même source non échantillonnée. Ce scénario semble être par ailleurs plausible compte tenu de l'organisation locale des plantations qui partagent une même pépinière. Dans un second temps, nous avons voulu mieux comprendre le fonctionnement de cette épidémie en inférant les valeurs de paramètres démographiques et évolutifs c lés (tailles efficaces des populations, temps de divergence entre populations. sévérité d'éventuels goulets d'étranglement associés à la fondation des populations, paramètres mutationnels ...). Les méthodes d'inférence existantes ne pouvant s'appliquer au cas de populations clonales typées par des microsatellites, nous introduisons ici une méthode récente d'estimation de paramètres selon un scénario complexe; le calcul bayésien approché (ABC,2). Cette approche nous permettra une meilleure compréhension de l'émergence. et nous donnera ainsi des informations sur (i) la biologie de Xcc (nombre d'individus nécessaire pour créer une invasion, temps de génération,...) et ( ii) pour la reconstruction des routes d'invasions afin d'obtenir des informations épidémiologique nécessaires à la gestion de la maladie (éviter de nouvelles introductions, limiter la dissémination). (Résumé d'auteur)

International audience; MultiLocus Variable number of tandem repeats Analysis (MLVA) has been extensively used for addressing epidemiological and evolutionary questions 0 11 bacteria pathogenic to humans (Ciammaruconi el al., 2008). This typing scheme was shown especially useful for the so-called monomorphic pathogens, which can be efficiently genotyped with a high throughput by MLVA. In contrast, it has been scarcely used for bacterial plant pathogens despite the tremendous impact of some of them. We developed an optimized MLVA scheme targeting long tandem repeats (2: 10 bp) for assessing the global epidemiology of the c itrus pathogen Xanthomonas citri pv. citri (Xcc). Xcc is a quarantine organism in several countries and a significant threat for the citrus industry worldwide. We screened the genome of Xcc strain IAI'AR 306 and of phylogenetically related xanthomonads for tandem repeals. The resolution at 31 polymorphie loci (MLVA3 1) was assessed using 129 strains of Xcc representative of the currently known genetic and pathological diversity of the pathogen worldwide. Based on Discriminant Analys is of Principal Components (DAPC), seven mostly pathotypespecifie genetic clusters were defined. DAPC c1uster 1 had a much wider geographical distribution than others. This single DAPC c1uster has been primarily implicated in the major geographical extension of Xcc during the XXth century. Because of the suboptimal inter-laboratory reproducibility of AFLP and rep-PCR, MLVA-31 represents an opportunity for international genotyping data sharing. (Texte intégral)

International audience; Xanthomonads are plant-associated bacteria that establish neutral, commensal or pathogenic relationships with plants. The list of common characteristics shared by all members of the genus Xanthomonas is now well established based on the entire genome sequences that are currently available and that represent various species, numerous pathovars of X. axonopodis (sensu Vauterin et al., 2000), X. oryzae and X. campestris, and many strains within some pathovars. These ?-proteobacteria are motile by a single polar flagellum. Motility is an important feature involved in biofilm formation, plant colonization and hence considered as a pathogenicity factor. X. axonopodis pv. phaseoli var. fuscans (Xapf) is one of the causal agents of common bacterial blight of bean and 4834-R is a highly aggressive strain of this pathogen that was isolated from a seed-borne epidemic in France in 1998. We obtained a high quality assembled sequence of the genome of this strain with 454-Solexa and 2X Sanger sequencing. Housekeeping functions are conserved in this genome that shares core characteristics with genomes of other xanthomonads: the six secretion systems which have been described so far in Gram negative bacteria are all present, as well as their ubiquitous substrates or effectors and a rather usual number of mobile elements. Elements devoted to the adaptation to the environment constitute an important part of the genome with a chemotaxis island and dispersed MCPs, numerous two-component systems, and numerous TonB dependent transporters. Furthermore, numerous multidrug efflux systems and functions dedicated to biofilm formation that confer resistance to stresses are also present. An intriguing feature revealed by genome analysis is a long deletion of 35 genes (33 kbp) involved in flagellar biosynthesis. This deletion is replaced by an insertion sequence called ISXapf2. Genes such as flgB to flgL and fliC to fleQ which are involved in the flagellar structure (rod, P- and L-ring, hook, cap and filament) are absent in the genome of strain 4834-R that is not motile. Primers were designed to detect this deletion by PCR in a collection of more than 300 strains representing different species and pathovars of Xanthomonas, and less than 5% of the tested xanthomonads strains were found nonmotile because of a deletion in the flagellum gene cluster. We observed that half of the Xapf strains isolated from the same epidemic than strain 4834-R was non-motile and that this ratio was conserved in the strains colonizing the next bean seed generation. Isolation of such variants in a natural epidemic reveals that either flagellar motility is not a key function for fitness or that some complementation occurs within the bacterial population. (Résumé d'auteur)

Xanthomonas citri pv. citri (Xcc), the causal agent of Asiatic Citrus Canker, is an internationally major bacterial pathogen currently not reported from countries in the Mediterranean basin, including the EU. Some pathological diversity among strains was reported, leading to the description in Xcc of pathotypes differing in host range and aggressiveness. Xcc is recognized as a monomorphic pathogen and the very low intrapathovar diversity at housekeeping genes precludes the use of the increasingly popular MLST (MultiLocus Sequence Typing) for the global molecular surveillance of Xcc. We developed a new MLVA (MultiLocus Variable number of tandem repeats Analysis) scheme. It targeted 51 minisatellite loci dispersed over the Xcc genome with tandem repeat size ranging from 10 to 217-bp, thus allowing the use of standard agarose gel electrophoresis in laboratories that are not equipped with a genotyper. Among these loci, 34 were found to be polymorphic within a strain collection composed of strains from all continents where Xcc has been recorded, including recent outbreaks in Africa where the bacterium is reemerging. This typing scheme displayed a maximal typeability and repeatability. The produced data support the usefulness of this new MLVA scheme for global surveillance of Xcc. (Texte integral)

International audience

Xanthomonads are plant-associated bacteria that establish neutral, commensal or pathogenic relationships with plants. The list of common characteristics shared by all members of the genus Xanthomonas is now well established based on the entire genome sequences that are currently available and that represent various species, numerous pathovars of X. axonopodis (sensu Vauterin et al., 2000), X. oryzae and X. campestris, and many strains within some pathovars. These ?-proteobacteria are motile by a single polar flagellum. Motility is an important feature involved in biofilm formation, plant colonization and hence considered as a pathogenicity factor. X. axonopodis pv. phaseoli var. fuscans (Xapf) is one of the causal agents of common bacterial blight of bean and 4834-R is a highly aggressive strain of this pathogen that was isolated from a seed-borne epidemic in France in 1998. We obtained a high quality assembled sequence of the genome of this strain with 454-Solexa and 2X Sanger sequencing. Housekeeping functions are conserved in this genome that shares core characteristics with genomes of other xanthomonads: the six secretion systems which have been described so far in Gram negative bacteria are all present, as well as their ubiquitous substrates or effectors and a rather usual number of mobile elements. Elements devoted to the adaptation to the environment constitute an important part of the genome with a chemotaxis island and dispersed MCPs, numerous two-component systems, and numerous TonB dependent transporters. Furthermore, numerous multidrug efflux systems and functions dedicated to biofilm formation that confer resistance to stresses are also present. An intriguing feature revealed by genome analysis is a long deletion of 35 genes (33 kbp) involved in flagellar biosynthesis. This deletion is replaced by an insertion sequence called ISXapf2. Genes such as flgB to flgL and fliC to fleQ which are involved in the flagellar structure (rod, P- and L-ring, hook, cap and filament) are absent in the genome of strain 4834-R that is not motile. Primers were designed to detect this deletion by PCR in a collection of more than 300 strains representing different species and pathovars of Xanthomonas, and less than 5% of the tested xanthomonads strains were found nonmotile because of a deletion in the flagellum gene cluster. We observed that half of the Xapf strains isolated from the same epidemic than strain 4834-R was non-motile and that this ratio was conserved in the strains colonizing the next bean seed generation. Isolation of such variants in a natural epidemic reveals that either flagellar motility is not a key function for fitness or that some complementation occurs within the bacterial population.

International audience; Expanding global trade and the domestication of ecosystems have greatly accelerated the rate of emerging infectious fungal diseases, and host-shift speciation appears to be a major route for disease emergence. There is therefore an increased interest in identifying the factors that drive the evolution of reproductive isolation between populations adapting to different hosts. Here, we used genetic markers and cross-inoculations to assess the level of gene flow and investigate barriers responsible for reproductive isolation between two sympatric populations of Venturia inaequalis, the fungal pathogen causing apple scab disease, one of the fungal populations causing a recent emerging disease on resistant varieties. Our results showed the maintenance over several years of strong and stable differentiation between the two populations in the same orchards, suggesting ongoing ecological divergence following a host shift. We identified strong selection against immigrants (i.e. host specificity) from different host varieties as the strongest and likely most efficient barrier to gene flow between local and emerging populations. Cross-variety disease transmission events were indeed rare in the field and cross-inoculation tests confirmed high host specificity. Because the fungus mates within its host after successful infection and because pathogenicity-related loci prevent infection of nonhost trees, adaptation to specific hosts may alone maintain both genetic differentiation between and adaptive allelic combinations within sympatric populations parasitizing different apple varieties, thus acting as a ‘magic trait’. Additional intrinsic and extrinsic postzygotic barriers might complete reproductive isolation and explain why the rare migrants and F1 hybrids detected do not lead to pervasive gene flow across years

Poster; Poster about Hydroids from Baa Atoll, Maldives Archipelago

International audience; An important branch of evolutionary biology strives to understand how divergent selection for an ecologically important trait can foster the emergence of new species specialized on different niches. Such ecological speciation is usually difficult to achieve because recombination between different subsets of a population that are adapting to different environments counteracts selection for locally adapted gene combinations. Traits pleiotropically controlling adaptation to different environments and reproductive isolation are therefore the most favourable for ecological speciation, and are thus called “magic traits”. We used genetic markers and cross-inoculations to show that pathogenicity-related loci are responsible for both host adaptation and reproductive isolation in emerging populations of Venturiainaequalis, the fungus causing apple scab disease. Because the fungus mates within its host and because the pathogenicity-related loci prevent infection of the non-host trees, host adaptation pleiotropically maintains genetic differentiation and adaptive allelic combinations between sympatric populations specific to different apple varieties. Such “magic traits” are likely frequent in fungal pathogens, and likely drive the emergence of new diseases.&nbsp

Correspondance: editorial.office@molecolres.com, UMR CBGP; International audience; This article documents the addition of 277 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Ascochyta rabiei, Cambarellus chapalanus, Chionodraco hamatus, Coptis omeiensis, Cynoscion nebulosus, Daphnia magna, Gerbillus nigeriae, Isurus oxyrinchus, Lates calcarifer, Metacarcinus magister, Oplegnathus fasciatus, Pachycondyla verenae, Phaethon lepturus, Pimelodus grosskopfii, Rotylenchulus reniformis, Scomberomorus niphonius, Sepia esculenta, Terapon jarbua, Teratosphaeria cryptica and Thunnus obesus. These loci were cross-tested on the following species: Austropotamobius italicus, Cambarellus montezumae, Cambarellus puer, Cambarellus shufeldtii, Cambarellus texanus, Chionodraco myersi, Chionodraco rastrospinosus, Coptis chinensis, Coptis chinensis var. brevisepala, Coptis deltoidea, Coptis teeta, Orconectes virilis, Pacifastacus leniusculus, Pimelodus bochii, Procambarus clarkii, Pseudopimelodus bufonius, Rhamdia quelen, Sepia andreana, Sepiella maindroni, Thunnus alalunga, Thunnus albacares, Thunnus maccoyii, Thunnus orientalis, Thunnus thynnus and Thunnus tonggol.

The genetic characterization of isolates and structure analysis of pathogenic bacterial populations are useful to investigate their epidemiology. Short-term epidemiological questions such as the identification of inoculum sources or the interconnection of different outbreaks could be approached by molecular tracing. #Xanthomonas citri# pv. #citri# (Xcc), the causal agent of Asiatic citrus canker, is a bacterium displaying little genetic diversity, based on genotyping tools such as MLST or rep-PCR. It presents low levels of recombination, which result in clonal populations. For pathogens such as Xcc, common genotyping tools are often not discriminant enough to address questions at a rather small time or spatial scale. Variable Number of Tandem Repeats targeting 14 loci were developed to detect variations and to discriminate between isolates within a strain collection from Vietnam, where Asiatic citrus canker is endemic with no current control, and from Brazil, where the pathogen is currently under containment in Saõ Paulo state. Four hundred and sixty four haplotypes out of 557 Vietnamese Xcc isolates from twelve provinces were identified. The Structure model-based clustering algorithm allows inference on genetic ancestry and identified two populations, V1 and V2, that have distinctive allelic frequencies and a Nei diversity index of 0.77 and 0.21, respectively. V1 was distributed in all provinces whereas V2 was limited in three northern provinces. A clonal complex analysis by eBurst produced 35 complexes, where all haplotypes are a single locus variant (SLV) of at least one other haplotype in the group, and 313 singletons. Nine complexes were distributed among different provinces. The largest clonal complex included 108 Xcc-V2 strains from three provinces with the founder located in a national citrus propagation center. Two hundred and twenty seven haplotypes were identified out of the 308 isolates in our Brazilian collection. Two ancestral populations were most significantly inferred by the Structure Bayesian approach. Population B1 was only found in the two southern states of Rio Grande do Sul (RS) and Santa Catarina (SC). Population B2 was inferred in the states of Parana (PR), Saõ Paulo (SP) and RS. Twenty-six clonal complexes, all within SP state, and 147 singletons were identified. Most clonal complexes were composed of strains from a single location and year of isolation, indicative of epidemic clonality. Allelic richness calculated by the rarefaction method and diversity index were a little higher for the group of strains from SC, RS and PR than for SP strains which are under eradication, with values of 6.22 / 0.67 and 5.25 / 0.54, respectively. At a larger level, allelic richness and diversity index were greater in Vietnam than in Brazil with values of 12.9 / 0.74 and 8.4 / 0.60, respectively. Molecular epidemiology and population's genetics may contribute to improve and refine our knowledge in the epidemiology of plant pathogenic bacteria even in the case of genetically monomorphic pathogens.

Xanthomonas citri pv. mangiferaeindicae (Xcm) est une bactérie pathogène de deux espèces de la famille des Anacardiacées : une espèce cultivée, le manguier et une peste végétale, le faux poivrier, fréquemment rencontrée en bordure de vergers. Des études d'inoculations croisées ont montré que leur pouvoir pathogène présente une spécialisation d'hôte : les souches originaires de manguier sont peu agressives sur faux poivrier et inversement. Une forte spécialisation pouvant parfois mener à une spéciation en sympatrie, quelles sont les conséquences de cette spécialisation sur les populations naturelles du pathogène ? Existe-t-il une structuration des populations de Xanthomonas présentes sur ces deux plantes ? En quoi la connaissance d'une éventuelle structure permet d'inférer l'histoire évolutive entre ces deux types de souches ? Pour répondre à ces questions, nous avons analysé la diversité génétique de souches isolées de manguier et faux poivrier dans trois localités. Nous avons choisi une approche MLVA (MultiLocus Vntr Analysis) pour son fort pouvoir discriminant et pour la pertinence du modèle évolutif sous-jacent. Douze séquences minisatellites identifiées à partir du génome de X. c. pv. citri ont été utilisées dans cette étude. La diversité génétique moyenne mesurée pour ces populations est plus élevée sur manguier que sur faux poivrier. Les indices de différenciation génétique et l'analyse des complexes clonaux montrent que la différenciation est plus forte entre populations issues d'hôtes différents qu'entre localités. Les distances génétiques entre souches sont cependant faibles, confirmant la proximité génétique des deux types de souches qui appartiennent au même pathovar. Les populations forment un continuum génétique : certaines souches isolées de manguier sont plus proches de souches de faux poivrier que d'autres souches de manguier. La spécialisation d'hôte de Xcm semble être un événement récent n'ayant pas abouti à un phénomène de spéciation.

Xanthomonas citri# pv. #mangiferaeindicae# (Xcm) is a bacterium attacking two plant species of the anacardiaceae family : mango (#Mangifera indica#) and pepper tree (#Schinus terebinthifolius#), which is a pest frequently found bordering mango orchards. Cross-inoculation indicated that there is a host specialization: strains isolated on mango are weakly pathogenic when artificially inoculated on pepper tree and strains from pepper tree are weakly pathogenic on mango. A strong host specialization can lead to a sympatric speciation, therefore we wondered what are the consequences of the observed specialization onto natural populations of the pathogen. What are the evolutionary and epidemiological relationships between these populations? What tools may help to describe evolution at a very small geographic and probably time scale? Can this example help to understand host shifting in xanthomonads? To address such questions we analysed the genetic diversity of populations of Xcm isolated on mango and pepper tree in three places. A MultiLocus Vntr Analysis (MLVA) approach was used because of its high discriminating power and because it allows to make precise evolutionary hypotheses. Twelve minisatellite sequences were identified based on the complete genomic sequence of #X. citri# pv. #citri#. The average genetic diversity is higher for populations isolated in mango orchards than on pepper tree. Genetic differentiation indices and clonal complexes indicate that differentiation is stronger between populations isolated on different hosts than between population isolated on the same host in different places. Genetic distances between strains are low, confirming the evolutionary relatedness of the two types of strains and their classification as a single pathovar. Populations are structured as a genetic continuum, with some strains isolated from mango more closely related to some strains isolated on pepper tree than from some of the mango strains. Host specialization of Xcm is probably a recent event which did not (yet) result in speciation.

Le chancre bactérien des agrumes causé par Xanthomonas citri pv. citri (Xcc) est largement répandu en Asie dont il est originaire. Sa présence au Vietnam date au moins du début du XXème siècle. L'absence de marqueurs génétiques polymorphes et indépendants a limité notre connaissance de la structure des populations de Xcc. L'objectif était d'analyser la structure des populations de Xcc à différentes échelles pour mieux comprendre l'épidémiologie de cette maladie. Le génotypage a été conduit en ciblant 14 loci VNTRs (Variable Number of Tandem Repeats) et 3 séquences insertionnelles par la méthode de Ligation-Mediated-PCR (IS-LM-PCR) sur une collection de 557 isolats de Xcc collectés dans 12 provinces du Vietnam en 2006. Le nombre d'haplotypes a été respectivement de 191 et 464 pour les marqueurs IS-LM-PCR et VNTRs. La recherche de discontinuités génétiques au sein de l'ensemble de la population associées ou non à la localisation géographique a été effectuée par des approches bayésiennes (GENELAND et STRUCTURE) pour les deux types de marqueurs. Les résultats congruents ont permis d'assigner les individus à deux populations différentes (Pl et P2). P1 est présente dans toutes les provinces alors que P2 est présente seulement dans trois provinces du nord avec un indice de diversité de Nei HT de 0,77 et 0,21, respectivement. L'analyse de complexes clonaux par une approche eBurst sur les données VNTRs a produit 35 complexes clonaux et 313 singletons à partir des 464 haplotypes. Neuf complexes clonaux seulement étaient distribués dans différentes provinces. Le plus gros contenait 108 souches de P2 originaires de trois provinces dont le fondateur primaire a été localisé dans un centre de propagation des agrumes. L'existence de déséquilibre de liaison significatifs entre tous les loci VNTRs indique une structure fortement clonale au sein de cette population vietnamienne de Xcc avec des évènements de recombinaison rares. Ces résultats suggèrent (1) que la population P2 est récemment introduite ou émergente au Vietnam et (2) le rôle important du matériel végétal dans la dissémination de la bactérie. Il reste à vérifier si la structure épidémique apparente de P2 peut être attribuée à une plus grande capacité adaptative ou épidémique de cette population ou si une rapide dispersion par l'homme en est majoritairement responsable. (Texte intégral)