Phylogenetic Assignment of Ralstonia pseudosolanacearum (Ralstonia solanacearum Phylotype I) Isolated from Rosa spp.

Abstract

During the last two years, greenhouse cultivation of rose (Rosa spp.) in the Netherlands has been challenged by an uncommon bacterial disease. Affected plants suffered from chlorosis, stunting, wilting, and necrosis. The bacterial isolates obtained from the different Rosa spp. cultivars were all identified as phylotype I, sequevar 33 from the 'Ralstonia solanacearum species complex' (RSSC), actually reclassified as Ralstonia pseudosolanacearum. The work in this paper considers the genetic diversity and the phylogenetic position of 129 R. pseudosolanacearum isolates from the outbreak. This was assessed by AFLP based on four different primer combinations and MLP using partial sequences of the egl, mutS, and fliC genes. The AFLP revealed identical profiles for all the isolates, irrespective of their association with Rosa sp. propagating material, Rosa spp. plants for cut flowers, or water used in the different greenhouse cultivations. These AFLP profiles were unique and diverged from profiles of all other reference isolates in the RSSC included. Furthermore, MLP on egl, fliC, and mutS gene sequences clearly demonstrated that all R. pseudosolanacearum isolates clustered in phylotype I, as a distinct monophyletic group. Interestingly, this monophyletic group also included phylotype I strain Rs-09-161 from eggplant (Solanum melongena), isolated in 2009 in India. AFLP and MLP were both efficient in revealing the genetic divergence from the RSSC isolates included. The phylogenetic tree constructed from the AFLP profiles was, in general, in agreement with the one obtained from MLP. Both phylogenetic trees displayed a similar clustering, supported by high posterior probabilities. Both methodologies clearly demonstrated that the R. pseudosolanacearum isolates from Rosa spp. grouped in a monophyletic group inside phylotype I, with a particular correspondence to a strain present in India, as revealed in MLP.

Genetic and Pathogenic Characterization of Bacterial Wilt Pathogen, Ralstonia pseudosolanacearum (Ralstonia solanacearum Phylotype I), on Roses in Korea

“…All strains clustered with PD7195 and PD7216, and Rs-09-161 which were isolated from roses in Netherlands and eggplants ( S. melongena ) in India ( Fig. 3A ) ( Bergsma-Vlami et al, 2018 ; Ramesh et al, 2014 ), respectively, and as such the sequevar was determined to be I-33. In addition, the tree based on only egl sequences was grouped all 17 R. pseudosolanacearum strains isolated from Korean and Dutch roses, as well as strains CFBP 8468 and CFBP 8469, which caused bacterial wilt on Rodrigues Island into sequevar I-33 ( Fig.…”

“…To evaluate the sequevars of the 17 R. pseudosolanacearum strains isolated from Korean roses, multi locus phylogeny was applied, using three individual genes: the endoglucanase gene egl , the DNA repair gene mutS , and the flagellin gene fliC . The analysis was also conducted using only the egl gene, as described by Bergsma-Vlami et al (2018) . The amplicons for egl , mutS , and fliC were produced with Endo-F/Endo-R, mutS-RsF1570/mutS-RsR1926, and Rsol_fli_for/Rsol_ fli_rev primers, respectively ( Supplementary Table 2 ) ( Poussier et al, 2000 ; Schönfeld et al, 2003 ; Wicker et al, 2007 ), and each sequence was trimmed using Vector NTI suit 8.0 (Thermo Scientific, Waltham, MA, USA), and aligned with ClustalW.…”

“…These results indicate that this pathogenic bacterium has optimum conditions under which symptoms appear on roses, and management techniques involving usage of resistant cultivars and methods to avoid scars produced during leaf detaching should be applied. In addition, hierarchical classifications of 129 R. pseudosolanacearum strains isolated from roses in the Netherlands were conducted based on the phylotype and sequevar, and results were compared with an isolate from India ( Bergsma-Vlami et al, 2018 ). Bergsma-Vlami et al (2018) concluded that all strains were grouped into phylotype I and sequevar 33, which were well-matched with the strain from India, and therefore all strains were found to belong to the same monophyletic group.…”

“…In addition, hierarchical classifications of 129 R. pseudosolanacearum strains isolated from roses in the Netherlands were conducted based on the phylotype and sequevar, and results were compared with an isolate from India ( Bergsma-Vlami et al, 2018 ). Bergsma-Vlami et al (2018) concluded that all strains were grouped into phylotype I and sequevar 33, which were well-matched with the strain from India, and therefore all strains were found to belong to the same monophyletic group. Studies aimed at evaluating the genetic classification and pathogenicity of R. pseudosolanacearum have aimed to develop to cope with this emerging pathogen in the Netherlands.…”

Detection of Ralstonia pseudosolanacearum in drain water based on concentration, enrichment and the use of a duplex TaqMan PCR test

“…Recently in Europe, outbreaks of bacterial wilt in ornamental rose crops grown in glasshouses were described (Tjou‐Tam‐Sin et al ., 2017; Bergsma‐Vlami et al ., 2018). Rosa spp.…”