Emmanuel Wicker

Genomes of three tomato pathogens within the Ralstonia solanacearum species complex reveal significant evolutionary divergence

BackgroundThe Ralstonia solanacearum species complex includes thousands of strains pathogenic to an unusually wide range of plant species. These globally dispersed and heterogeneous strains cause bacterial wilt diseases, which have major socio-economic impacts. Pathogenicity is an ancestral trait in R. solanacearum and strains with high genetic variation can be subdivided into four phylotypes, correlating to isolates from Asia (phylotype I), the Americas (phylotype IIA and IIB), Africa (phylotype III) and Indonesia (phylotype IV). Comparison of genome sequences strains representative of this phylogenetic diversity can help determine which traits allow this bacterium to be such a pathogen of so many different plant species and how the bacteria survive in many different habitats.ResultsThe genomes of three tomato bacterial wilt pathogens, CFBP2957 (phy. IIA), CMR15 (phy. III) and PSI07 (phy. IV) were sequenced and manually annotated. These genomes were compared with those of three previously sequenced R. solanacearum strains: GMI1000 (tomato, phy. I), IPO1609 (potato, phy. IIB), and Molk2 (banana, phy. IIB). The major genomic features (size, G+C content, number of genes) were conserved across all of the six sequenced strains. Despite relatively high genetic distances (calculated from average nucleotide identity) and many genomic rearrangements, more than 60% of the genes of the megaplasmid and 70% of those on the chromosome are syntenic. The three new genomic sequences revealed the presence of several previously unknown traits, probably acquired by horizontal transfers, within the genomes of R. solanacearum, including a type IV secretion system, a rhi-type anti-mitotic toxin and two small plasmids. Genes involved in virulence appear to be evolving at a faster rate than the genome as a whole.ConclusionsComparative analysis of genome sequences and gene content confirmed the differentiation of R. solanacearum species complex strains into four phylotypes. Genetic distances between strains, in conjunction with CGH analysis of a larger set of strains, revealed differences great enough to consider reclassification of the R. solanacearum species complex into three species. The data are still too fragmentary to link genomic classification and phenotypes, but these new genome sequences identify a pan-genome more representative of the diversity in the R. solanancearum species complex.

Ralstonia solanacearum strains from Martinique (French West Indies) exhibiting a new pathogenic potential

ABSTRACT We investigated a destructive pathogenic variant of the plant pathogen Ralstonia solanacearum that was consistently isolated in Martinique (French West Indies). Since the 1960s, bacterial wilt of solanaceous crops in Martinique has been caused primarily by strains of R. solanacearum that belong to either phylotype I or phylotype II. Since 1999, anthurium shade houses have been dramatically affected by uncharacterized phylotype II strains that also affected a wide range of species, such as Heliconia caribea, cucurbitaceous crops, and weeds. From 1989 to 2003, a total of 224 R. solanacearum isolates were collected and compared to 6 strains isolated in Martinique in the 1980s. The genetic diversity and phylogenetic position of selected strains from Martinique were assessed (multiplex PCRs, mutS and egl DNA sequence analysis) and compared to the genetic diversity and phylogenetic position of 32 reference strains covering the known diversity within the R. solanacearum species complex. Twenty-four representative isolates were tested for pathogenicity to Musa species (banana) and tomato, eggplant, and sweet pepper. Based upon both PCR and sequence analysis, 119 Martinique isolates from anthurium, members of the family Cucurbitaceae, Heliconia, and tomato, were determined to belong to a group termed phylotype II/sequevar 4 (II/4). While these strains cluster with the Moko disease-causing strains, they were not pathogenic to banana (NPB). The strains belonging to phylotype II/4NPB were highly pathogenic to tomato, eggplant, and pepper, were able to wilt the resistant tomato variety Hawaii7996, and may latently infect cooking banana. Phylotype II/4NPB constitutes a new pathogenic variant of R. solanacearum that has recently appeared in Martinique and may be latently prevalent throughout Caribbean and Central/South America.

Contrasting recombination patterns and demographic histories of the plant pathogen Ralstonia solanacearum inferred from MLSA

We used multilocus sequence analysis (MLSA) on a worldwide collection of the plant pathogenic Ralstonia solanacearum (Betaproteobacteria) to retrace its complex evolutionary history. Using genetic imprints left during R. solanacearum evolution, we were able to delineate distinct evolutionary complex displaying contrasting dynamics. Among the phylotypes already described (I, IIA, IIB, III, IV), eight groups of strains with distinct evolutionary patterns, named clades, were identified. From our recombination analysis, we identified 21 recombination events that occurred within and across these lineages. Although appearing the most divergent and ancestral phylotype, phylotype IV was inferred as a gene donor for the majority of the recombination events that we detected. Whereas this phylotype apparently fuelled the species diversity, ongoing diversification was mainly detected within phylotype I, IIA and III. These three groups presented a recent expanding population structure, a high level of homologous recombination and evidences of long-distance migrations. Factors such as adaptation to a specific host or intense trading of infected crops may have promoted this diversification. Whether R. solanacearum lineages will eventually evolve in distinct species remains an open question. The intensification of cropping and increase of geographical dispersion may favour situations of phylotype sympatry and promote higher exchange of key factors for host adaptation from their common genetic pool.

Broad Diversity of Ralstonia solanacearum Strains in Cameroon

In 2005, an extensive survey of bacterial wilt in Cameroon collected 110 strains of Ralstonia solanacearum from wilting tomato, potato, pepper, huckleberry (Solanum scabrum), sesame, and amaranth. The genetic diversity and phylogeny of selected strains from Cameroon were assessed by multiplex-polymerase chain reaction (PCR), race 3/biovar 2-specific PCR, and sequence analyses of the mutS and egl genes. These data were compared with those from 33 reference strains covering the known diversity within the R. solanacearum species complex. Strains isolated in Cameroon clustered into three of the four known phylotypes: I (Asian), II (American), and III (African). Lowland tomato strains belonged to phylotype I and were quite homogeneous. The strains belonging to phylotype II were genetically diverse, and partitioned into subclusters IIA and IIB (sequevar 1, race 3/biovar 2). Cameroon strains in the African phylotype III were distinct from reference strains from Zimbabwe or the Indian Ocean, highlighting the genetic diversity present within this phylotype. Strains from potatoes growing in the highlands of West Cameroon fell into both phylotypes II (race 3/biovar 2) and III. These phylotype II and III highland strains attacked both potato and tomato and could therefore pose an economic threat to potato and tomato crops throughout Central Africa. This is the first comprehensive report on the genetic diversity of R. solanacearum strains in Cameroon.

Specific behaviour of French Aphanomyces euteiches Drechs. populations for virulence and aggressiveness on pea, related to isolates from Europe, America and New Zealand

The pathogenic variability of Aphanomyces euteiches on pea was investigated using a collection of 88 pea-infecting isolates from France and 21 isolates from Denmark, Sweden, Norway, USA, Canada and New Zealand. Aggressiveness and virulence were assessed by scoring the root symptoms on a differential set of six pea genotypes. Eleven virulence types were characterised. The virulence type I, previously described as virulent on the whole set, was predominant and included the most aggressive isolates of all geographical origins. The other types were much less prevalent, existing as one to five isolates. Three virulence types (III, IV and V) contained no French isolates. The type III, avirulent on MN313, was composed of American isolates only, and resembled the ‘major group’ recently described in the USA. A wide range of aggressiveness was found within the virulence type I, and the French isolates appeared globally more aggressive than the foreign isolates. These findings indicate that isolates from the virulence type I should be used as references in breeding programs, and that pea lines PI180693 and 552 may be the most interesting resistance sources to date, despite their only partial resistance.

Effect of Allium fistulosum Extract on Ralstonia solanacearum Populations and Tomato Bacterial Wilt

To control bacterial wilt (Ralstonia solanacearum, phylotype IIB/4NPB), the antimicrobial effect of Allium fistulosum aqueous extract was assessed as a preplant soil treatment. Three concentrations of extract (100, 50, and 25%, 1:1 [wt/vol]) were evaluated by in vitro inhibition assay and in vivo experiments in a growth chamber. In vitro, A. fistulosum (100 and 50%) suppressed growth of R. solanacearum. Preplant treatment of the soil with A. fistulosum extract significantly reduced the R. solanacearum populations. No pathogen was detected in the soil after treatment with 100% concentrated extract from the third day after application until the end of the experiment. A. fistulosum also significantly reduced the incidence of tomato bacterial wilt. In the untreated control, the disease affected 61% of the plants whereas, with 100 and 50% extracts, only 6 and 14% of the plants, respectively, were affected. These results suggest that A. fistulosum extracts could be used in biocontrol-based management strategies for bacterial wilt of tomato.

Confirmation of Xanthomonas axonopodis pv. manihotis Causing Cassava Bacterial Blight in Ivory Coast

D. Kone, Université Félix Houphouët-Boigny, UFR Biosciences, Laboratoire de Physiologie Végétale, 22 BP582 Abidjan, Côte d’Ivoire; S. Dao, C. Tekete, I. Doumbia, and O. Koita, Université des Sciences Techniques et Technologiques, Faculté des Sciences et Techniques, LBMA, Bamako, Mali; K. Abo, Institut National Polytechnique Félix Houphouët-Boigny (INPHB), ESA, Département ARA, Laboratoire de Phytopathologie, Yamoussoukro, Côte d’Ivoire; E. Wicker, CIRAD, UMR Peuplements Végétaux et Bioagresseurs en Milieu Tropical (PVBMT) Pôle de Protection des Plantes 7, chemin de l’IRAT F-97410 Saint Pierre La Réunion, France; and V. Verdier, IRD, Cirad, Univ. Montpellier, Interactions Plantes Microorganismes Environnement (IPME), 34394 Montpellier, France.

New Multilocus Variable-Number Tandem-Repeat Analysis (MLVA) Scheme for Fine-Scale Monitoring and Microevolution-Related Study of Ralstonia pseudosolanacearum Phylotype I Populations

ABSTRACT Bacterial wilt caused by the Ralstonia solanacearum species complex (RSSC) is considered one of the most harmful plant diseases in the world. Special attention should be paid to R. pseudosolanacearum phylotype I due to its large host range, its worldwide distribution, and its high evolutionary potential. So far, the molecular epidemiology and population genetics of this bacterium are poorly understood. Until now, the genetic structure of the RSSC has been analyzed on the worldwide and regional scales. Emerging questions regarding evolutionary forces in RSSC adaptation to hosts now require genetic markers that are able to monitor RSSC field populations. In this study, we aimed to evaluate the multilocus variable-number tandem-repeat analysis (MLVA) approach for its ability to discriminate genetically close phylotype I strains and for population genetics studies. We developed a new MLVA scheme (MLVA-7) allowing us to genotype 580 R. pseudosolanacearum phylotype I strains extracted from susceptible and resistant hosts and from different habitats (stem, soil, and rhizosphere). Based on specificity, polymorphism, and the amplification success rate, we selected seven fast-evolving variable-number tandem-repeat (VNTR) markers. The newly developed MLVA-7 scheme showed higher discriminatory power than the previously published MLVA-13 scheme when applied to collections sampled from the same location on different dates and to collections from different locations on very small scales. Our study provides a valuable tool for fine-scale monitoring and microevolution-related study of R. pseudosolanacearum phylotype I populations. IMPORTANCE Understanding the evolutionary dynamics of adaptation of plant pathogens to new hosts or ecological niches has become a key point for the development of innovative disease management strategies, including durable resistance. Whereas the molecular mechanisms underlying virulence or pathogenicity changes have been studied thoroughly, the population genetics of plant pathogen adaptation remains an open, unexplored field, especially for plant-pathogenic bacteria. MLVA has become increasingly popular for epidemiosurveillance and molecular epidemiology studies of plant pathogens. However, this method has been used mostly for genotyping and identification on a regional or global scale. In this study, we developed a new MLVA scheme, targeting phylotype I of the soilborne Ralstonia solanacearum species complex (RSSC), specifically to address the bacterial population genetics on the field scale. Such a MLVA scheme, based on fast-evolving loci, may be a tool of choice for field experimental evolution and spatial genetics studies.

Draft Genome Sequences of Nine Strains of Ralstonia solanacearum Differing in Virulence to Eggplant (Solanum melongena)

ABSTRACT Ralstonia solanacearum displays variability in its virulence to solanaceous crops. We report here the draft genome sequences of eight phylotype I strains and one phylotype III strain differing in virulence to the resistant eggplant genotype AG91-25. These data will allow the identification of virulence- and avirulence-related genes.