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08 Apr 2016

Studies of the Taxonomy of Banana Blood Disease Bacterium and Related Bacteria

Irda Safni, 2013
The University of Queensland

Blood disease of banana, caused by blood disease bacterium (BDB), is one of the most destructive wilt diseases affecting bananas and plantains in Indonesia, and is a threat to Australia and surrounding countries. Phylogenetically, BDB is confirmed as a close relative of the Ralstonia solanacearum species complex and is included as a member in the phylotype IV within this species complex, which is the most diverse phylotype as it contains two other taxa, Ralstonia syzygii and R. solanacearum.

In the literatures, the members of phylotypes I, II, III and IV of the R. solanacearum species complex are phenotypically diverse, however, they are closely related on the basis of sequencing of 16S rRNA, 16S-23S intergenic transcribed spacer (ITS) region, endoglucanase (egl) and polygalacturonase genes. It had been previously suggested that high DNA homology values were also found among the R. solanacearum biovars 1, 2 and 3 strains (currently assigned as either phylotypes I, II and III). Thus, the exact taxonomic position of BDB and the other members of the R. solanacearum species complex remain unresolved.

This thesis focused on the determination of the taxonomic position of BDB within the other members of the R. solanacearum species complex and the clarification of its relationships within this species complex using a polyphasic taxonomic approach on an extensive set of strains. The works embodied by this thesis provide significant evidence for a taxonomic and nomenclature revision of members of this species complex. Data from the sequencing of 16S rRNA, 16S-23S rRNA ITS region and DNA-DNA hybridisation (DDH) demonstrate that the R. solanacearum species complex should be separated into three genomic species. Phenotypic data including classical phenotypic, Biolog GN2 MicroPlate metabolic fingerprinting and chemotaxonomic methods also support this new insight. The first genospecies contains only R. solanacearum phylotype II strains including the type strain. The second genospecies is the merger of the three taxa included in phylotype IV strains to be a single genomic species, Ralstonia syzygii. The last genospecies includes R. solanacearum strains belonging to phylotypes I and III.

Several phenotypic and chemotaxonomic characteristics are proven to distinguish the three taxa in the genospecies R. syzygii (phylotype IV), therefore, three subspecies are proposed to represent R. syzygii, BDB and R. solanacearum within the four phylotype IV. Phenotypic methods including physiological and biochemical analyses, Biolog GN2 MicroPlate metabolic fingerprinting test as well as chemotaxonomic markers successfully identified several differentiating characteristics of the four phylotypes. The sequencing of 16S rRNA and 16S-23S ITS spacer region support the separation of the R. solanacearum into three genomic species. The three novel species cluster together in the phylogenetic tree derived from the 16S rRNA sequence analysis. Whereas, in the ITS dendogram phylotypes I and III strains cluster separately but their branches are next to each other, but all phylotype II and IV strains formed a single cluster each. However, the sequence similarity of the members of the R. solanacearum species complex was more than 97%, which cannot resolve their taxonomic issues. Using sequencing of egl gene, BDB strains were clearly separable from R. syzygii strains but not from R. solanacearum phylotype IV strains.

Repetitive PCR (rep-PCR) and multilocus sequence typing (MLST) were selected for determining the genetic diversity of 25 and 4 BDB strains respectively. BDB strains showed high degree of internal diversity on the basis of their fingerprint patterns. However, using the MLST data of four housekeeping genes (adk, gapA, gdhA and ppsA) and one megaplasmid (hrpB), the four BDB strains showed their high level of similarity.

Based on the data presented in this thesis, following taxonomic proposals are made: emendation of the descriptions of R. solanacearum and R. syzygii, descriptions of Ralstonia syzygii subsp. syzygii comb. nov. (R 001T = LMG 10661T = NCPPB 3446T) for the current R. syzygii strains, Ralstonia syzygii subsp. indonesiensis subsp. nov. (R-46900T) for the current R. solanacearum phylotype IV strains, Ralstonia syzygii subsp. celebensis subsp. nov. (R-46908T) for the BDB strains and Ralstonia pseudosolanacearum sp. nov. (LMG 9673T = NCPPB 1029T) for the R. solanacearum phylotype I and III strains.

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