A MLSA-MLST scheme to investigate the real evolutionary dynamics within the Ralstonia solanacearum species complex : [P01-06]

The soilborne beta-proteobacterium #Ralstonia solanacearum# is the causing agent of major plant diseases (bacterial wilt on several species, potato brown rot, Moko disease of banana) within tropical and subtropical areas, affecting both cash and subsistence crops. This species complex is constituted of four phylotypes, correlated with the geographical origin of strains (I, Asian; II, American; III, African; IV, Indonesian). Each phylotype is further subdivided in sequevars, on the basis of sequence divergence of the endoglucanase gene (egl); some of these sequevars have been associated to specific ecological features (host range, virulence at cool temperatures). Genomic structure and phylogeny of this species complex is now well understood, thank to recent CGH studies (2) and sequencing of several complete genomes. The subdivision in four phylotypes was clearly demonstrated, but the existence of different clades within each phylotype still had to be validated. The high genotypic and phenotypic plasticity of this organism has been illustrated by emergence of new pathogenic variants (3). It was demonstrated that this bacterium, naturally competent, is potentially subjected to recombination and horizontal genetic transfer (HGT). However, the real degrees of recombination occurring in natural populations, the dominant reproductive mode of this bacterium, the selection pressures structuring these populations, are still largely unknown. To clarify both phylogeny and evolutionary dynamics of the #R. solanacearum# species complex, we developed a MLSA-MLST scheme on a collection of 88 #R.solanacearum# strains classified in the four phylotypes and 51 sequevars described to date, and one strain of each of the close species #R. syzygii#, #R. pickettii#, #R. mannitolylitica# and #R. insidiosa#. Genes were chosen following previous MLSA approach (1) and reference studies; all were (i) evenly distributed across the two replicons, and distant of at least 100 kb, (ii) present in one single copy in the genomes. They consisted in six housekeeping genes (ppsA, rplB, gdhA, leuS, adk, gyrB), the DNA mismatch repair gene mutS, and two virulence-associated genes (egl, fliC). Phylogenies reconstructed from individual genes, and with concatenated genes, were globally congruent with each other. They allowed identifying several phylogenetically differentiated subdivisions, named clades, within some of the phylotypes. Within the phylotype I, one single clade was found. Within the phylotype II, the subclusters IIA and IIB were validated and four clades were found: (i) "brown rot" IIB/sequevar 1, 2 and Moko sequevar 3; (ii) sequevar 4 (Moko and emergent strains); (iii) "South-Eastern USA biovar 1" strains, or sequevar 7; (iv) "Antillean biovar 1" strains, and Moko sequevar 6 strains. Within the phylotype III, two clades were identified: (i) Austral Africa and Indian Ocean strains, and (ii) Central and Western African strains. Within the phylotype IV, two clades were found: (i) blood disease bacterium and Indonesian #R. solanacearum# strains, (ii) #R. syzygii#. The population structure of #R. solanacearum# was assessed by the MLST approach, at the global scale and at the phylotype scale. The global population structure appeared to be clonal; however phylotypes clearly differed in structure: phylotypes I and III displayed a recombining population structure, whereas phylotype II was highly clonal. Analyses of the different evolutionary forces structuring the #R. solanacearum# species complex are ongoing, and will be presented and discussed. A collection of reference strains for each clade will be proposed. (Résumé d'auteur

Complete genome sequence of the potato pathogen Ralstonia solanacearum UY031

Ralstonia solanacearum is the causative agent of bacterial wilt of potato. Ralstonia solanacearum strain UY031 belongs to the American phylotype IIB, sequevar 1, also classified as race 3 biovar 2. Here we report the completely sequenced genome of this strain, the first complete genome for phylotype IIB, sequevar 1, and the fourth for the R. solanacearum species complex. In addition to standard genome annotation, we have carried out a curated annotation of type III effector genes, an important pathogenicity-related class of genes for this organism. We identified 60 effector genes, and observed that this effector repertoire is distinct when compared to those from other phylotype IIB strains. Eleven of the effectors appear to be nonfunctional due to disruptive mutations. We also report a methylome analysis of this genome, the first for a R. solanacearum strain. This analysis helped us note the presence of a toxin gene within a region of probable phage origin, raising the hypothesis that this gene may play a role in this strain's virulence

Moko disease-causing strains of Ralstonia solanacearum" from Brazil extend known diversity in paraphyletic phylotype II

The epidemic situation of Moko disease-causing strains in Latin America and Brazil is unclear. Thirty-seven Ralstonia solanacearum strains from Brazil that cause the Moko disease on banana and heliconia plants were sampled and phylogenetically typed using the endoglucanase (egl) and DNA repair (mutS) genes according to the phylotype and sequevar classification. All of the strains belonged to phylotype II and a portion of the strains was typed as the Moko disease-related sequevars IIA-6 and IIA-24. Nevertheless, two unsuspected sequevars also harbored the Moko disease-causing strains IIA-41 and IIB-25, and a new sequevar was described and named IIA-53. All of the strains were pathogenic to banana and some of the strains of sequevars IIA-6, IIA-24, and IIA-41 were also pathogenic to tomato. The Moko disease-causing strains from sequevar IIB-25 were pathogenic to potato but not to tomato. These results highlight the high diversity of strains of Moko in Brazil, reinforce the efficiency of the egl gene to reveal relationships among these strains, and contribute to a better understanding of the diversity of paraphyletic Moko disease-causing strains of the R. solanacearum species complex, where the following seven distinct genetic clusters have been described: IIA-6, IIA-24, IIA-41, IIA-53, IIB-3, IIB-4, and IIB-25. (Résumé d'auteur

Story of an array based technology designed to grasp and detect the complex plant pathogen Ralstonia solanacearum

The ancient soil borne plant vascular pathogen Ralstonia solanacearum evolved and adapted to cause severe damage on an unusually wide range of plants. Based on the phylotype classification, it was questioned how genetically and phenotypically diverse strains of Ralstonia solanacearum may be, in an attempt to produce adapted diagnostic tools. A pangenomic microarray was first used to better describe and understand the phylogeny of R. solanacearum, especially three particular ecotypes in the phylotype II: (i) Brown rot strains from sequevars IIB-1 and IIB-2, historically known as race 3 biovar 2 and clonal; (ii) new pathogenic variants from sequevar IIB-4NPB that lack pathogenicity to Cavendish banana but can infect many other plant species; and (iii) Moko disease-causing strains from sequevars IIB-3, IIB-4, and IIA-6, historically known as race 2, that cause wilt on banana, plantain, and Heliconia. Results revealed a phylogeographic structure within Brown rot strains, allowing European outbreak strains of Andean and African origins to be distinguished, and also showed a close but distinct relationship between Moko ecotype IIB-4 and the emerging IIB-4NPB strains. Based on those results, it is proposed to develop new DNA-based diagnostic tools, including array and routine PCR, to ensure better detection of this complex plant pathogen. (Résumé d'auteur

Deciphering phenotypic diversity of Ralstonia solanacearum strains pathogenic to potato : [P2-08]

Based on the phylotype classification scheme, we questioned how genetically how genetically and phenotypically diverse may strains of #Ralstonia solanacearum# pathogenic to potato are. We studied 129 representative European and Mediterranean strains of #R. solanacearum# along with 57 reference strains known to cover the genetic diversity in this species. Phylogeny analysis was done on partial endoglucanase (egl) gene sequences. Virulence and pathogenicity to potato, tomato, and eggplant was established at two ranges of temperature 24-30°C (tropical) and 15-24°C (temperate) and tests on banana were only conducted at 24-30°C. Results confirm the literature that the ability to cause wilt on Solanaceae and specially on potato was of polyphyletic nature and shared by strains in all phylotypes I, IIA, IIB, III, and IV, showing a wide genetic basis for pathogenicity on these hosts. We also noticed that strains that were known to cause wilt on Musaceae solely: phylotypes IIA sequevar 6 (IIA-6), IIB-3, and IIB-4, were also able to trigger wilt on Solanaceae, especially on susceptible potato, tomato and eggplant, and also on some resistant tomato for the phylotype IIB-4. In the same time, we isolated Brown rot potato strains belonging to phylotypes IIB-1 and IIB-2, but also strains belonging to phylotype IIB-27, from symptomless banana plants, also suggesting the polyphyletic nature of the Moko disease causing strains. That said, from an epidemiological point of view, no Moko disease causing strains have ever been isolated from a wilted tomato or potato in the field, although it is now apparent they were able to trigger wilt in these hosts. But from a phylogenetic point of view, these two well known ecotypes address the question of host adaptation mechanisms, which may have undergone a similar bottleneck evolution. On the contrary, cold-tolerance ability on Solanaceae is only shared among the clonal brown rot phylotype IIB-1, which gathered the great majority of European and Mediterranean (EuMr) strains. All other strains were not able to trigger wilt or were drastically less aggressive on these hosts. A total of 5 strains out of 106 originating or intercepted in EuMr countries were not distributed into the phylotype IIB-1 and showed that despite the great proportion of brown rot phylotypes IIB-1 strains on EuMr territory, other phylotypes may represent a potential threat to Solanaceae production. This study highlighted the strain LNPV24.25, which was isolated from a diseased tomato and belong to the phylotype IIB-4NPB that gather highly aggressive and virulent emerging strains. This indicates, from our knowledge, the first report of an emerging strain in France. These findings showed that pathogenicity traits of genetically identified strains still need to be understood, especially in the perspective of post-genomics comparative analysis to understand bacterial speciation in the #R. solanacearum# species complex. (Résumé d'auteur

Diagnóstico de Ralstonia solanacearum em tomateiro.

Pretende-se, com esta publicação, fornecer informações sobre os processos envolvidos na identificação da doença, coleta e envio de material para análise laboratorial, técnicas de identificação e armazenamento de cepas do patógeno.bitstream/item/84927/1/cot-92.pd

Evaluation de I'effet antibactérien d'extrait de bois d'inde (Pimento racemosa) contre I'agent pathogène Ralstonia solanacearum responsable du flétrissement bacterien de la tomate, en zone tropicale humide de la Martinique

Le flétrissement bactérien est une des pathologies végétales les plus problématiques pour les cultures maraichères, principalement dans les régions tropicales et subtropicales, Cl climat chaud et humide, et notamment Cl la Martinique. L'agent pathogène responsable de cette maladie, Ralstonia solanacearum est une bactérie particulièrement virulente qui s'attaque principalement aux solanacées. Dans le cadre de recherche de moyens de gestion du flétrissement bactérien, nous proposons ici de tester le pouvoir antibactérien d'un extrait végétal de bois d'inde (Pimenta racemosa). L'effet est teste sur la croissance in vitro de R. solanacearum par la technique de milieu amende; et in vivo, par suivi de populations de R. solanacearum dans un sol naturel amende en huile essentielle, et par suivi de l'incidence du flétrissement bactérien par observation de symptôme sur tomate. Les résultats montrent un effet antibactérien in vitro et in vivo de l'extrait végétal de bois d'inde sur R. solanacearum, et un effet suppresseur sur l''incidence de la maladie. Ces résultats offrent d'intéressantes perspectives de lutte biologique contre le flétrissement bactérien. Cette étude pourra être poursuivie par des essais en champs. (Résumé d'auteur