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.

Fig. 1

Agarose gel showing fingerprints of the hrp region (primer set pglA-F2 and hrcV-R2) after restriction with (a) HinfI and (b) RsaI. Lane M is Kb+ molecular size marker, lane 1 is strain 1609; lane 2 is strain KZR-5

Fig. 2

a Agarose gel of uncut genomic DNA of R. solancearum strains, showing the two circular replicons. Lane M: H. wingei chromosomal marker, lane 1: GMI1000 (biovar 3), lane 2: 1609, lane 3: 715, lane 4: KZR-5, lane 5: PA2, lane 6: PA5. Run conditions were: 0.8% chromosomal-grade agarose (1× TAE), switch time of 500 s, 3 V/cm, 14°C for 48 h. b Agarose gel showing pulsed field gel electrophoresis profiles of XbaI digested genomic DNA of R. solanacearum strains. Lane M: lambda marker, lane 1: 715, lane 2: 1609, lane 3: KZR-1, lane 4: KZR-2, lane 5: KZR-3, lane 6: KZR-5, lane 7: PA1, lane 8: PA2, lane 9: PA4, lane 10: PA5, lane 11: WA19, lane 12: WC76, lane 13: WC78. Arrows: polymorphic bands. Run conditions were: 1% pulsed field certified agarose (0.5× TBE), switch time 1–80 s, 6 V/cm, 14°C for 22 h

Fig. 3

Southern blot analysis of R. solanacearum genomic DNA after restriction with PstI and hybridisation with an ISRso3-AvaI/RsaI (610 bp) fragment as DNA probe. Lane M: Kb+ molecular size marker, lane 1: 1609, lane 2: KZR-2, lane 3: KZR-5, lane 4: KZR-1, lane 5: PA1, lane 6: PA2, lane 7: RA18, lane 8: SA31, lane 9: SB63, lane 10: WB48, lane 11: WC76

Fig. 4

Dendrogram obtained by cluster analysis with UPGA and Euclidean distance, using the PFGE and ISRso3 patterns of all R. solanacearum strains used in this study. Bands were scored as either present or absent. Genomotype defined by pulsotype (A–D) and ISRso3 group (1 or 2)

Fig. 5

Doubling times in 0.1× TSBS of selected R. solanacearum strains in hours (h) at a 28°C and b 16°C. Statistical classes (a, ab, b and x) are indicated. P < 0.05