Molecular epidemiology of Ralstonia solanacearum strains from plants and environmental sources in Portugal

Several outbreaks of bacterial wilt disease caused by the quarantine bacterium Ralstonia solanacearum were identified in Portugal. Intensive surveys recognized the bacterium as endemic in the main irrigated agricultural ecosystems. Between 1999 and 2006 all isolates of R. solanacearum were characterized as belonging to biovar 2A. In 2007, biovar 1 strains were recorded in potato fields under a confined area. A panel of 101 Portuguese isolates obtained from biotic and environmental samples was studied. Following a polyphasic approach, these isolates were analysed by SDS-PAGE of the whole cell proteins, MSP-PCR (csM13), rep-PCR (BOXA1R and ERIC-2) and FAFLP (EcoRI + 0/MseI + C). A 750 bp sequence of endoglucanase (egl) gene was studied for 17 representative isolates and 95 accessions retrieved from the GeneBank. Numerical analysis of protein profiles correlated quite well with biovar subphenotype, producing a unique megacluster (r = 71.1%). MSP-PCR was more discriminative (r = 62%). Rep-PCR approaches displayed higher polymorphism levels with ERIC 2 primer producing high diversity indexes (D and J'). FAFLP was the most reproducible method (95%) displaying 229 polymorphic characters and the highest evenness (J'). For all the methods small clusters disclosed a clonal origin for isolates with a common geographical origin/matrix. FAFLP identified an adaptative microevolution phenomenon for surface water strains. Polyphasic approach congruence highlighted the inability of individual methods to explain the whole diversity. Mr. Bayes egl-based phylogenetic tree allocated the 17 Portuguese isolates into the sub-clusters of narrow (nhr) and broad host range (bhr) of Phylotype II unveiling the epidemiological story of R. solanacearum in Portugal and identified different populations coexisting in the same habitats. This is the first report of the presence of R. solanacearum Phylotype II, bhr strains in Western Europe.