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Genetic and phenotypic diversity of Ralstonia solanacearum biovar 2 strains obtained from Dutch waterways.

ABSTRACT: A novel set of Ralstonia solanacearum biovar 2 isolates was obtained, at several sampling occasions, from Dutch waterways, sediment and bittersweet plants and their genetic and phenotypic diversity was investigated. As reference strains, two previously-described strains obtained from diseased potato plants, denoted 1609 (The Netherlands) and 715 (Bangladesh), were included in the analyses. All novel isolates showed BOX and GTG5 PCR based genomic profiles similar to those of the reference strains. Also, PCR-restriction fragment length polymorphism analysis of the phcA and hrp genomic regions, as well as sequence analysis of six selected genomic loci, revealed great homogeneity across the strains. In contrast, pulsed field gel electrophoresis of restricted genomic DNA revealed the distribution of all strains across four groups, denoted pulsotypes A through D (pulsotypes C and D had one representative each). Moreover, pulsotype B, consisting of five strains, could be separated from the other pulsotypes by a divergent genomic fingerprint when hybridized to a probe specific for insertion element ISRso3. Representatives of pulsotypes A, B and C were selected for growth and metabolic studies. They showed similar growth rates when grown aerobically in liquid media. Assessment of their metabolic capacity using BIOLOG GN-2 revealed a reduced utilization of compounds as compared to the reference strains, with some variation between strains.

SUBMITTER: Stevens P

PROVIDER: S-EPMC2797627 | biostudies-literature | 2010 Feb

REPOSITORIES: biostudies-literature

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Genetic and phenotypic diversity of Ralstonia solanacearum biovar 2 strains obtained from Dutch waterways.

Stevens Patricia P van Elsas Jan Dirk JD

Antonie van Leeuwenhoek 20091204 2

A novel set of Ralstonia solanacearum biovar 2 isolates was obtained, at several sampling occasions, from Dutch waterways, sediment and bittersweet plants and their genetic and phenotypic diversity was investigated. As reference strains, two previously-described strains obtained from diseased potato plants, denoted 1609 (The Netherlands) and 715 (Bangladesh), were included in the analyses. All novel isolates showed BOX and GTG5 PCR based genomic profiles similar to those of the reference strains ...[more]

PMID: 19960250

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Project description: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.

Project description:BackgroundRalstonia solanacearum, the causal agent of bacterial wilt, is a genetically diverse bacterial plant pathogen present in tropical and subtropical regions of the world that infects more than 200 plant species, including economically important solanaceous crops. Most strains of R. solanacearum are only pathogenic at temperatures between 25 to 30°C with strains that can cause disease below 20°C considered a threat to agriculture in temperate areas. Identifying key molecular factors that distinguish strains virulent at cold temperatures from ones that are not is needed to develop effective management tools for this pathogen. We compared protein profiles of two strains virulent at low temperature and two strains not virulent at low temperature when incubated in the rhizosphere of tomato seedlings at 30 and 18°C using quantitative 2D DIGE gel methods. Spot intensities were quantified and compared, and differentially expressed proteins were sequenced and identified by mass spectrometry (MS/MS).ResultsFour hundred and eighteen (418) differentially expressed protein spots sequenced produced 101 unique proteins. The identified proteins were classified in the Gene Ontology biological processes categories of metabolism, cell processes, stress response, transport, secretion, motility, and virulence. Identified virulence factors included catalase (KatE), exoglucanase A (ChbA), drug efflux pump, and twitching motility porin (PilQ). Other proteins identified included two components of a putative type VI secretion system. We confirmed differential expression of 13 candidate genes using real time PCR techniques. Global regulators HrpB and HrpG also had temperature dependent expression when quantified by real time PCR.ConclusionsThe putative involvement of the identified proteins in virulence at low temperature is discussed. The discovery of a functional type VI secretion system provides a new potential virulence mechanism to explore. The global regulators HrpG and HrpB, and the protein expression profiles identified suggest that virulence at low temperatures can be partially explained by differences in regulation of virulence factors present in all the strains.

Dataset Information

Genetic and phenotypic diversity of Ralstonia solanacearum biovar 2 strains obtained from Dutch waterways.

Publications

Genetic and phenotypic diversity of Ralstonia solanacearum biovar 2 strains obtained from Dutch waterways.

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