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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.

Xanthomonas arboricola is a complex bacterial species which mainly attacks fruit trees and is responsible for emerging diseases in Europe. It comprises seven pathovars ( X. arboricola pv. pruni, X. arboricola pv. corylina, X. arboricola pv. juglandis, X. arboricola pv. populi, X. arboricola pv. poinsettiicola, X. arboricola pv. celebensis, and X. arboricola pv. fragariae), each exhibiting characteristic disease symptoms and distinct host specificities. To better understand the factors underlying this ecological trait, we first assessed the phylogenetic relationships among a worldwide collection of X. arboricola strains by sequencing the housekeeping gene rpoD . This analysis revealed that strains of X. arboricola pathovar populi are divergent from the main X. arboricola cluster formed by all other strains. Then, we investigated the distribution of 53 type III effector (T3E) genes in a collection of 57 X. arboricola strains that are representative of the main X. arboricola cluster. Our results showed that T3E repertoires vary greatly between X. arboricola pathovars in terms of size. Indeed, X. arboricola pathovars pruni, corylina, and juglandis, which are responsible for economically important stone fruit and nut diseases in Europe, harbored the largest T3E repertoires, whereas pathovars poinsettiicola, celebensis, and fragariae harbored the smallest. We also identified several differences in T3E gene content between X. arboricola pathovars pruni, corylina, and juglandis which may account for their differing host specificities. Further, we examined the allelic diversity of eight T3E genes from X. arboricola pathovars. This analysis revealed very limited allelic variations at the different loci. Altogether, the data presented here provide new insights into the evolution of pathogenicity and host range of X. arboricola and are discussed in terms of emergence of new diseases within this bacterial species.

Fluorescent amplified fragment length polymorphism revealed that strains of Xanthomonas axonopodis pv. phaseoli and Xanthomonas fuscans subsp. fuscans are genetically distinct and can be grouped into four genetic lineages. Four suppression subtractive hybridizations were then performed to isolate DNA fragments present in these bean pathogens and absent from closely related xanthomonads. Virulence gene candidates were identified such as homologs of hemagglutinins, TonB-dependent receptors, zinc-dependent metalloproteases, type III effectors, and type IV secretion system components. Unexpectedly, homologs of the type III secretion apparatus components (SPI-1 family), usually reported in animal pathogens and insect symbionts, were also detected.

IS Xax1 is a novel insertion sequence belonging to the IS 256 and Mutator families. Dot blot, Southern blot, and PCR analyses revealed that IS Xax1 is restricted to Xanthomonas axonopodis pv. phaseoli (variants fuscans and non-fuscans) and X. axonopodis pv. vesicatoria strains. Directed AFLP also showed that a high degree of polymorphism is associated with IS Xax1 insertion in these strains.

Xanthomonas campestris pv. campestris is the causal agent of black rot on Brassicaceae . The draft genome sequences of strains CFBP 1869 and CFBP 5817 have been determined and are the first ones corresponding to race 1 and race 4 strains, which have a predominant agronomic and economic impact on cabbage cultures worldwide.

Ralstonia (formerly Pseudomonas) solanacearum (E. F. Smith) Yabuuchi et al. (47) is the causal agent of bacterial wilt, a severe and devastating plant disease in most tropical and subtropical and some warm temperate areas (22). Moreover, it can also occur in cool temperate areas (9, 33). Many economically important food crops such as potatoes, tomatoes, and bananas are affected. The disease was recorded on several hundred plant species distributed in more than 50 families (23). The species R. solanacearum is a complex taxonomic unit in which strains display an important diversity at different levels (physiological, serological, genetic characteristics, and host range). In order to describe this intraspecific variability, several systems of classification have been proposed. Thus, the species was subdivided into five races according to its host range (7, 25, 35) and into six biovars based on the utilization of three disaccharides and three hexose alcohols (21, 24, 25). Fatty acid analysis (26, 42) and protein profiling (15) were also performed but did not further clarify the relationships among R. solanacearum strains. Restriction fragment length polymorphism (RFLP) analysis (involving nine probes, seven of which encode information required for virulence and the hypersensitive response) (12–14) has provided a new classification scheme dividing the species into 46 groups in relation to geographic origin of strains and sometimes host range. The species was then separated into two major groups, the “Asiaticum” and the “Americanum” divisions, which regrouped strains from Asia and America, respectively. Further investigations comparing sequences of 16S rRNA (30, 40, 43) or using PCR amplification with tRNA consensus primers (39) supported the separation according to geographic origin. Only a few strains originating from Africa, and only one from Reunion Island (21), were included in these previous studies. However, strains related to the three major biovars (1, 2, and 3) were isolated from various crops in Reunion (17). The aim of our study was to assess the genetic diversity within the local populations of R. solanacearum. Since we also wanted to develop molecular tools for the identification and detection of R. solanacearum biovars, we used the PCR-RFLP procedure to analyze the diversity. Recently, several authors have successfully performed PCR-RFLP analysis to assess genetic diversity among bacterial species (27, 28, 31, 44, 45). The hrp (hypersensitive reaction and pathogenicity) gene region, which is required by many phytopathogenic bacteria to produce symptoms on susceptible hosts and a hypersensitive reaction on resistant hosts or on nonhosts (1, 3, 4, 6, 18, 29), was explored for studying the variability within a collection of 120 strains isolated from different hosts over the five continents and belonging to biovars 1, 2, 3, and 4.