Whole genome characterization of strains belonging to the Ralstonia solanacearum species complex and in silico analysis of TaqMan assays for detection in this heterogenous species complex

Abstract

Identification and classification of members of the Ralstonia solanacearum species complex (RSSC) is challenging due to the heterogeneity of this complex. Whole genome sequence data of 225 strains were used to classify strains based on average nucleotide identity (ANI) and multilocus sequence analysis (MLSA). Based on the ANI score (>95%), 191 out of 192(99.5%) RSSC strains could be grouped into the three species R. solanacearum, R. pseudosolanacearum, and R. syzygii, and into the four phylotypes within the RSSC (I,II, III, and IV). R. solanacearum phylotype II could be split in two groups (IIA and IIB), from which IIB clustered in three subgroups (IIBa, IIBb and IIBc). This division by ANI was in accordance with MLSA. The IIB subgroups found by ANI and MLSA also differed in the number of SNPs in the primer and probe sites of various assays. An in-silico analysis of eight TaqMan and 11 conventional PCR assays was performed using the whole genome sequences. Based on this analysis several cases of potential false positives or false negatives can be expected upon the use of these assays for their intended target organisms. Two TaqMan assays and two PCR assays targeting the 16S rDNA sequence should be able to detect all phylotypes of the RSSC. We conclude that the increasing availability of whole genome sequences is not only useful for classification of strains, but also shows potential for selection and evaluation of clade specific nucleic acid-based amplification methods within the RSSC.

Quantitative detection of the Ralstonia solanacearum species complex in soil by qPCR combined with a recombinant internal control strain

“…However, as the number of sequenced genomes increases, some published primers might be found to be less specific. A recent in silico analysis based on 192 RSSC genomes indicated that false positive or false negative results were possible in previous PCR assays because of a lack of sufficient sequencing information to guarantee primer specificity and coverage ( 49 ). Similarly, a study of Pseudomonas aeruginosa revealed that a previously developed method based on restricted numbers of sequences could only detect 82.1% of tested strains, and the novel assay designed using 1,000 genomes of P. aeruginosa and 1,017 genomes of other pathogens could detect all tested strains ( 50 ).…”