IJSEM Papers in Press. Published June 18, 2014 as doi:10.1099/ijs.0.066712-0

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Polyphasic taxonomic revision of the Ralstonia solanacearum species complex: proposal to emend the descriptions of R. solanacearum and R. syzygii and reclassify current R. syzygii strains as Ralstonia syzygii subsp. syzygii, R. solanacearum phylotype IV strains as Ralstonia syzygii subsp. indonesiensis subsp. nov., banana blood disease bacterium strains as Ralstonia syzygii subsp. celebesensis subsp. nov. and R. solanacearum phylotypes I and III strains as Ralstonia pseudosolanacearum sp. nov.

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Irda Safni1*, Ilse Cleenwerck2, Paul De Vos2, Mark Fegan1#, Lindsay Sly1, Ulrike Kappler1 1

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School of Chemistry and Molecular Biosciences, Faculty of Science, The University of Queensland, QLD 4072, Australia 2 BCCM/LMG Bacteria Collection, Laboratory of Microbiology, Ghent University, K.L. Ledeganckstraat 35, B-9000 Ghent, Belgium

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* Current address: Faculty of Agriculture, University of Sumatra Utara, Medan, 20155 North Sumatra, Indonesia

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# Current address: Department of Environment and 5 Ring Rd, La Trobe University, Bundoora, Victoria 3083, Australia

Primary

Industries

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Corresponding authors: 1.Lindsay Sly e-mail : [email protected] phone : +61 7 33741907

2. Ulrike Kappler e-mail : [email protected] phone : +61 7 33652978

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Running title : Taxonomic revision of the Ralstonia solanacearum species complex

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Subject category : New taxa, Proteobacteria

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Footnote : The Genbank accession numbers for the sequences of type strains of

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Ralstonia solanacearum, Ralstonia pseudosolanacearum sp. nov., Ralstonia syzygii

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subsp. indonesiensis subsp. nov. and Ralstonia syzygii subsp. celebesensis subsp.

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nov. are KC757031-KC757076, KC820937-KC820940 (16S rRNA gene),

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KC756969-KC757029 (16S-23S rRNA gene ITS), KC757078-KC757122 and 820936

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(egl gene)(Table S1)

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1

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ABSTRACT

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The Ralstonia solanacearum species complex has long been recognized as a group

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of phenotypically diverse strains that can be subdivided into four phylotypes. Using a

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polyphasic taxonomic approach on an extensive set of strains, this study provides

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evidence for a taxonomic and nomenclatural revision of members of this complex.

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Data obtained from phylogenetic analysis of 16S rRNA gene sequences, 16S-23S

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rRNA intergenic spacer (ITS) region sequences, partial endoglucanase (egl) gene

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sequences

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solanacearum species complex is comprised of three genospecies. One of these

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includes the type strain of R. solanacearum and consists of R. solanacearum

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phylotype II strains only. The second genospecies includes the type strain of R.

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syzygii and contains only phylotype IV strains. This genospecies is subdivided into

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three distinct groups, namely R. syzygii, the causal agent of Sumatra disease on

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clove trees in Indonesia, R. solanacearum phylotype IV strains isolated from different

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host plants mostly from Indonesia, and strains of the blood disease bacterium (BDB),

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the causal agent of the Banana Blood Disease, a bacterial wilt disease in Indonesia

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affecting bananas and plantains. The last genospecies is composed of R.

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solanacearum strains belonging to phylotypes I and III. As these genospecies are

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also supported by phenotypic data that allow the differentiation of the three

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genospecies, the following taxonomic proposals are made: emendation of the

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descriptions of R. solanacearum and R. syzygii, descriptions of Ralstonia syzygii

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subsp. syzygii (R 001T = LMG 10661T = DSM 7385T) for the current R. syzygii

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strains, Ralstonia syzygii subsp. indonesiensis subsp. nov. (UQRS 464T = LMG

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27703T = DSM 27478T) for the current R. solanacearum phylotype IV strains,

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Ralstonia syzygii subsp. celebesensis subsp. nov. (UQRS 627T = LMG 27706T =

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DSM 27477T) for the BDB strains and Ralstonia pseudosolanacearum sp. nov.

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(UQRS 461T = LMG 9673T = NCPPB 1029T) for the R. solanacearum phylotype I and

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III strains.

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2

and

DNA-DNA

hybridizations

demonstrate

that

the

Ralstonia

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INTRODUCTION

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Ralstonia solanacearum is a soil-borne pathogen that causes lethal vascular wilt

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diseases of over 200 plant species from more than 50 families including solanaceous

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vegetable crops, banana, ginger, custard apple, peanut, eucalyptus, and many other

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crop plants (Hayward, 1994; Kelman, 1953). Due to its lethality, persistence, wide

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host range and very broad geographical distribution, R. solanacearum is one of the

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most devastating known bacterial plant pathogens (Elphinstone, 2005).

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R. solanacearum is a heterogeneous species as evidenced by its large host range,

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pathogenic specialization, cultural and physiological properties, as well as its

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phylogeny (Hayward, 2000). Following its discovery, R. solanacearum was originally

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classified as a member of the genus Bacterium (Smith, 1896). The application of

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DNA based methods eventually resulted in its transfer to the genus Burkholderia

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(Yabuuchi et al., 1992) and then to the genus Ralstonia (Yabuuchi et al., 1995).

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Despite being classified as a single species, it has been accepted since the 1980s

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that different strains of R. solanacearum may have DNA homology values below

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70% (De Vos, 1980; Palleroni & Doudoroff, 1971) and therefore might be members

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of different species. As a result, the term ‘species complex’ (Gillings & Fahy, 1994),

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referring to ‘a cluster of closely related bacteria whose individual members may

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represent more than one species’ (Fegan & Prior, 2006) has been applied to R.

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solanacearum.

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Within the R. solanacearum species complex four phylotypes are recognized (Prior &

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Fegan, 2005) that can be distinguished based on the sequences of their 16S-23S

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rRNA gene ITS region, the hrpB and egl genes as well as comparative genomic

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hybridization (Fegan & Prior, 2005; Guidot et al., 2007). Phylotypes I, II and III are

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comprised of strains mainly from Asia, America and Africa and surrounding islands,

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respectively, while phylotype IV is primarily comprised of strains from Indonesia and

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some isolates from Japan, Australia and the Philippines. Phylotype IV is the most

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diverse group as it consists of strains assigned to R. solanacearum, Ralstonia

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syzygii and the blood disease bacterium (BDB). R. syzygii is the causal agent of the

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Sumatra disease of clove trees in Indonesia (Roberts et al., 1990) and its status as a

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separate species in the genus Ralstonia has been proven (Vaneechoutte et al., 3

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2004). However, it is also clearly a member of the R. solanacearum species complex

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(Taghavi et al., 1996). BDB is the causal agent of Banana Blood Disease, which is

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one of the most destructive bacterial wilt diseases affecting bananas (Musa

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acuminata) and plantains (Musa balbisiana x acuminata) in Indonesia (Eden-Green,

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1994). BDB was likely first isolated in the early 20 th century and named

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Pseudomonas celebensis (Gäumann, 1921). This name, however, was not included

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in the approved lists of bacterial names and as the type or reference cultures of P.

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celebensis no longer exist the name has no standing in nomenclature. The

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bacterium is currently referred to as BDB and classified as a member of the

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phylotype IV group of the R. solanacearum species complex.

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Recently, Wicker et al. (2012) further subdivided the R. solanacearum species

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complex into eight clades based on multilocus sequence analysis (MLSA).

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Phylotypes I and III were each contained in a single clade (1 and 6, respectively),

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while phylotype II consisted of 4 separate clades (2-5). Phylotype IV was composed

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of two clades (7-8), with clade 7 including BDB and R. solanacearum phylotype IV

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strains and clade 8 containing R. syzygii strains and R. solanacearum phylotype IV

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strains isolated from clove trees. Phylotype IV was reported to be the most divergent

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and ancestral phylotype, although ongoing diversification was observed within

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phylotypes I, II and III (Wicker et al., 2012). In addition, the MLSA study indicated

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that phylotypes I and III are more closely related to each other than to phylotypes II

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and IV, and the dendrograms also clearly documented the close relationship

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between the phylotype IV strains.

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Whole genome sequences of several representatives of the R. solanacearum

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species complex have become available in recent years and these include three

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representatives of phylotype I (GMI1000, Y45, FQY-4), six phylotype II strains (IIA:

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K60; CFBP 2957; IIB: IPO1609, UW 551, Po82, Molk2) and one representative each

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of phylotype III (CMR15) and the three main phylotype IV groups (R.solanacearum –

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PSI 07, R. syzygii – R 24, BDB – R 229) (Table S2). Additional genomes have been

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sequenced

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(https://www.genoscope.cns.fr/agc/microscope/about/collabprojects.php).

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complete genomes consist of two replicative units, a chromosome of approximately

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3.7 Mb and a megaplasmid of 1.6 - 2.3 Mb which together encode approximately 4

but

are

not

publically

available

at

present All

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5,000 proteins. Comparative studies found that each genome contained between

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400-600 unique genes, regardless of whether the genomes originated from the same

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phylotype or not (Remenant et al., 2011; Remenant et al., 2010).

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Recent work comparing eight complete R. solanacearum genome sequences (one

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from phylotype I, three from phylotype II, one from phylotype III and one each for the

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three subgroups of phylotype IV) also observed very close relationships between

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phylotype I and III strains, and the authors proposed a revision of the taxonomy of

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the R. solanacearum species complex based on genome ANI values and genetic

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relatedness (Remenant et al., 2011). Phylotype II strains were to be maintained as

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R. solanacearum while phylotype IV R. solanacearum, BDB and R. syzygii strains

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were proposed to be subspecies of a single new species, “Ralstonia haywardii”. And,

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finally, R. solanacearum phylotype I and phylotype III strains were proposed to form

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a single new species, “Ralstonia sequeirae”.

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While the genome analyses were very informative, only eight strains of the entire R.

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solanacearum species complex were analysed instead of a set of strains

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representative of the diversity within the proposed new taxa, and no genotypic and

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phenotypic characteristics suitable for their differentiation were reported (Remenant

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et al., 2011). Further, the proposal of the new species “Ralstonia haywardii" for the

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phylotype IV R. solanacearum, BDB and R. syzygii strains is not in accordance with

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the International Code of Nomenclature of Prokaryotes (De Vos & Trüper, 2000;

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Lapage et al., 1992) as the name R. syzygii is validly published and should be

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retained.

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In this study, we report the results of a polyphasic taxonomic study of 68 strains of

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the R. solanacearum species complex representing all four phylotypes, but with a

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specific focus on strains belonging to the highly diverse phylotype IV group (Table 1).

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Published data for a number of additional R. solanacearum phylotype I, II and III

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strains were also used in our comparative analyses (Anzai et al., 2000; Castillo &

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Greenberg, 2007; Fegan & Prior, 2006; Hayward, 1964; He et al., 1983; Horita &

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Tsuchiya, 1999; Horita & Tsuchiya, 2000; Horita et al., 2005; Ivey et al., 2007;

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Lebeau et al., 2011; Pastrik et al., 2002; Poussier et al., 2000a; Poussier et al.,

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2000b; Prior & Steva, 1990; Roberts et al., 1990; Salanoubat et al., 2002; Taghavi et 5

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al., 1996; Villa et al., 2005; Wicker et al., 2012; Wicker et al., 2007). The results

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obtained form the basis for a revision of the R. solanacearum species complex

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taxonomy and also clearly identify genotypic and phenotypic characteristics that

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differentiate the taxa contained in this species complex.

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METHODS

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Bacterial strains and growth conditions

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The bacterial strains used in this study are listed in Table1. Additionally, published

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phenotypic data for 21 strains of R. solanacearum belonging to phylotypes I, II, III

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and IV (Hayward, 1964; He et al., 1983; Horita & Tsuchiya, 1999; Horita et al., 2005;

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Prior & Steva, 1990; Roberts et al., 1990) were included and compared to the results

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obtained in our study (Table 2). Phylogenetic analyses of 16S, ITS and egl

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sequences included data for strains with published genome sequences as well as

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sequences deposited in GenBank as part of previous studies (Tables S1, S2, S5).

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Strains were grown aerobically at 28°C. For routine maintenance solid casamino

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acid peptone glucose agar medium (CPG) (g/l), composed of peptone (Oxoid) 10 g;

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casein hydrolysate (Bacto casamino acids) 1 g; glucose 5 g and the pH adjusted to

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6.5-7.0 (Kelman, 1954), was used with incubation times of two days for R.

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solanacearum, four days for BDB and six days for R. syzygii strains. For fatty acid

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composition determination, Casamino acid (CA) medium (Roberts et al., 1990) was

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used as cultivation medium. DNA for the DDH experiments and determination of the

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mol% G+C content was isolated from cells grown on Tryptic Soy agar (Oxoid

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CM131) or CA medium, except when growth on them resulted in insufficient amounts

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of cell material. In that case cultivation was performed on charcoal medium (g/l):

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yeast extract 10 g; activated charcoal (Norit SG) 2 g; ACES buffer (Sigma) 10 g; agar

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17 g. The ACES buffer was dissolved in 500 ml distilled water at 50 °C then mixed

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with a solution containing 40 ml of 1N KOH in 440 ml distilled water. The mixture was

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used to hydrate the other ingredients. Cysteine hydrochloride 0.4 g and Fe4(P2O7)3

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0.25 g, were dissolved either together (20 ml) or separately (10 ml each) in distilled

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water, filter sterilized and then added to the medium after autoclaving. The final pH of

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the medium was 6.9.

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6

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Molecular verification of strain identity

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Multiplex Polymerase Chain Reaction (Multiplex-PCR) (Fegan & Prior, 2005) and

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BDB-specific PCR (Tan, 2003) were used as a molecular diagnostic tool to verify the

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phylotype to which each strain belonged and to determine if the strain was a BDB,

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respectively. The primers are shown in Table S3.

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Phenotypic characterization

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Forty-seven classical phenotypic tests (physiological and biochemical) were

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performed on the 68 R. solanacearum species complex strains (Table 1). These

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tests included the oxidase test (Kovacs, 1956), catalase test (He et al., 1983), nitrate

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reduction test by the method of Hayward (1964) with minor modification by using the

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medium of Vandermooter (1987), gelatine hydrolysis (Lelliot & Stead, 1987), starch

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hydrolysis (Lelliot & Stead, 1987), Tween 80 hydroysis (Lelliot & Stead, 1987), and

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tests for the utilization of carbohydrates (Hayward, 1995) with a minor modification

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by using microtiter plates (French et al., 1995), arginine dihydrolase, lysine

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decarboxylase, ornithine decarboxylase and the production of phenylalanine

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deaminases described by Møller (1955) in Collin et al. (1989), DNase (Collin et al.,

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1989), urease production (Lelliot & Stead, 1987), citrate utilization as described by

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Simmons (1926) in Collins et al. (1989), and malonate utilization (Collin et al., 1989).

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Bacterial motility was observed by growth in semisolid motility medium (SMM) as

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described by Kelman and Hruschka (1973). Growth was observed on MacConkey

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agar (Bridson, 1998; Nash & Krenz, 1991). All tests were conducted at 28 °C unless

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otherwise stated. Tolerance to sodium chloride was observed on CPG (for R.

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solanacearum and BDB strains) and CA agar medium (for R. syzygii strains)

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containing 0, 3, and 5% NaCl. Additionally, phenotypic data for 21 strains of R.

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solanacearum representing phylotypes I, II, III and IV (Hayward, 1964; He et al.,

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1983; Horita & Tsuchiya, 1999; Horita et al., 2005; Prior & Steva, 1990; Roberts et

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al., 1990) were included in the comparisons carried out in this study (Table 2).

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For metabolic phenotypic fingerprinting, Biolog GN2 MicroPlate 96 well assays

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(Biolog, Hayward, CA, USA) were performed according to the manufacturer’s

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instructions. Results from phenotypic characterization were analyzed with the E7

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Workbench (InforBIO) program (Sugawara et al., 2003) using the Dice, Euclid,

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Simple matching, and Jaccard coefficient algorithms. Dendrograms were constructed

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using UPGMA clustering (data not shown).

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Determination of whole-cell fatty acid composition

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The whole-cell fatty acid composition was determined for fourteen selected strains of

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the R. solanacearum species complex and the type strain of R. mannitolilytica (Table

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1) using an Agilent Technologies 6890N gas chromatograph (Santa Clara, CA, USA).

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Cultivation of the strains, extraction and analysis of the fatty acid methyl esters were

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performed according to the recommendations of the Microbial Identification System,

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Sherlock version 3.10 (MIDI). Fatty acids were extracted from cells harvested from

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cultures grown for 48 h at 28 °C under aerobic conditions on CA medium (Roberts et

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al., 1990). The peaks of the profiles were identified using the TSBA50 identification

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library version 5.0.

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Determination of DNA base composition and DNA-DNA hybridization

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High quality DNA was isolated using the method of Wilson (1987) with minor

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modifications (Cleenwerck et al., 2002). The DNA base composition was determined

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using High Performance Liquid Chromatography (HPLC) (Mesbah et al., 1989).

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DNA-DNA hybridizations were performed at 51 °C using a modified version

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(Cleenwerck et al., 2002; Goris et al., 1998) of the microplate method developed by

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Ezaki et al. (1989). Reciprocal reactions (A x B and B x A) were performed for each

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DNA pair from all strains and their variation was generally within the limits for this

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method (Goris et al., 1998).

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Phylogenetic analysis of 16S rRNA gene,16S-23S rRNA gene Intergenic Spacer

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(ITS) Region and partial endoglucanase (egl) gene sequences

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PCR amplification of 16S rRNA genes, 16S-23S rRNA ITS region and partial egl

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genes were performed as summarized in Table S4 using a PTC-100 programmable

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Thermal Controller (MJ Research, Inc., Waltham, MA, USA). PCR products were

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purified using a Qiaquick PCR Purification Kit (Qiagen). DNA sequencing was carried

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out at the Australian Genome Research Facility, The University of Queensland, St. 8

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Lucia, Australia.DNA sequences data were assembled using Chromas Pro version

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1.5 (Technelysium Pty Ltd) and aligned using CLUSTAL W (Larkin et al., 2007;

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Thompson et al., 1994).

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Phylogenetic trees were constructed based on the neighbour-joining (Saitou & Nei,

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1983), maximum likelihood, minimum evolution and unweighted average pair

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mathematical averaging (UPGMA) (Kidd & Sgaramella, 1971; Rzhetsky & Nei, 1993)

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methods as embedded in the MEGA (Molecular Evolutionary Genetic Analysis)

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software version 5.05 (Tamura et al., 2011). Bootstrap analysis was used with 1000

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replicates to test the statistical reliability of the phylogenetic trees. Trees generated

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using the maximum likelihood, minimum evolution and UPGMA algorithms (data not

278

shown) were similar to those generated using the Neighbour- joining algorithm.

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RESULTS & DISCUSSION

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The R. solanacearum species complex has long been known to be a heterogeneous

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collection of strains that share a high degree of 16S rRNA gene sequence similarity

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(98-100%) (Taghavi et al., 1996), but may exhibit DNA-DNA homologies of less than

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70% (De Vos, 1980; Palleroni & Doudoroff, 1971). In this study, we used a large set

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of strains collected in various parts of the world (Table 1) as well as published

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sequence data including genome data (Tables S1 & S5) with the aim to improve the

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taxonomy of the R. solanacearum species complex through a polyphasic taxonomic

288

approach.

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All strains investigated in this study (Table 1) were initially tested using a R.

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solanacearum phylotype specific multiplex PCR (data not shown) and a BDB specific

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PCR. These tests confirmed their previous classification (Fig. S1) and also validated

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the specificity of the PCR primers (121F/121R) (Table S3), that were developed for

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the identification of BDB (Tan, 2003).

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The phylogenetic relationships of strains of the R. solanacearum species complex

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(Table 1 & Table S5) based on their 16S rRNA gene, 16S-23S rRNA ITS region, and

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partial egl gene sequences, were determined. The egl gene encodes an 9

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endoglucanase that has been implicated in virulence of R. solanacearum strains

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(Fegan & Prior, 2005; Poussier et al., 2000a; Prior & Fegan, 2005; Saile et al., 1997;

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Villa et al., 2005).

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The overall average 16S rRNA gene sequence similarity of the investigated R.

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solanacearum species complex strains was 91.6% with minimum and maximum

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values of 81.0% and 100%. Within each of the four phylotypes the average 16S

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gene sequence similarity was higher than 90.9%. The sequence of the type strain of

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R. solanacearum, UQRS 426T, (phylotype II) exhibited 87.1, 93.8, 89.4% average

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sequence similarity to the members of phylotypes I, III and IV, respectively.

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Sequence similarities between R. solanacearum species complex strains and type

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strains of the other species of the genus Ralstonia such as the closely related R.

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mannitolilytica, were below 77.0%.

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The overall ITS sequence similarity among the strains of the R. solanacearum

313

complex was 82.0% with a range of 70-100%, while the average egl sequence

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similarity among the strains of the R. solanacearum species complex was 57.8%

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with a range of 18-100%.

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Neighbour-Joining evolutionary distance analyses of 16S rRNA gene sequences

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(1,342 bp) (Fig. S2) and 16S-23S rRNA ITS region sequences (520 nt) (Fig. 1) and

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partial egl gene sequences (703 nt) (Fig. S3) showed that members of the R.

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solanacearum species complex formed a coherent group within which the phylotype

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I, II, III and IV strains appeared as individual clusters. Additionally, within phylotype

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IV, BDB strains and R. syzygii strains each formed coherent, individual groups based

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on 16S-23S rRNA ITS region (Fig. 1) while the remaining phylotype IV strains formed

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two groups. The only exception to the described grouping was the sequence of R.

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syzygii strain R 24 that grouped with R. solanacearum PIV strains UQRS 533, 532

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and 518, all of which are isolated from clove trees. However, short branch lengths

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with low bootstrap support did not allow for any definite inference of phylogenetic

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relationships within phylotype IV to be made based on the ITS sequences. Phylotype

328

I and III strains appeared as closely related groups in the ITS based tree, which is

329

consistent with MLSA analyses of their phylogenetic relationships (Wicker et al.,

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2012) and comparative genome analyses (Remenant et al., 2010). 10

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In the egl based phylogenetic tree sequences of phylotype IV strains formed two

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separate clusters (Fig. S3). The first cluster contained all sequences from BDB

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strains and sequences of the R. solanacearum phylotype IV strains. The second

334

cluster was made up of the sequences of the remaining phylotype IV strains and

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contained three distinct clades, two of which were made up exclusively of sequences

336

from bacteria isolated from clove trees. Within this sequence cluster the majority of

337

R. syzygii strains formed a consistent cluster with only two strains (Strain R 24, for

338

which a genome sequence is available, and strain R 28 obtained from the NCBI

339

database), (Fig. S3) grouping with the three R. solanacearum phylotype IV strains

340

isolated from clove. Three Japanese strains of R. solanacearum phylotype IV

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isolated from potato were also part of this second sequence cluster, but formed a

342

distinct clade together with a single Indonesian strain of R. solanacearum phylotype

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IV isolated from tomato (Fig. S3). Phylotype I, II and III strains formed individual

344

clusters in the egl phylogenetic tree, with Phylotype II and III strains appearing as

345

closely related groups. Neither the geographical nor the host origin correlated with

346

distribution of strains in these clusters with the exception of the group of phylotype IV

347

strains isolated from clove trees (cluster 2, above).

348

The phylogenetic relationships within and between the four phylotypes (Fegan &

349

Prior, 2006; Prior & Fegan, 2005) have been investigated previously by various

350

researchers using analysis of the sequence similarities of different genes, including

351

16S rRNA, the 16S-23S ITS region, egl, hrpB, gdhA, adk, gyrB, gapA, ppsA and fliC

352

(Poussier et al., 2000a; Poussier et al., 2000b; Taghavi et al., 1996; Villa et al., 2005;

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Wicker et al., 2012). The 16S rRNA gene sequences of R. solanacearum and its

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close relatives have been shown to be almost indistinguishable (Fegan et al., 1998),

355

and that is also apparent in the data presented here. However, the use of more

356

specific genes, such as egl, hrpB, gdhA, adk, gyrB, gapA, ppsA and fliC and ITS

357

region showed the separation of the R. solanacearum species complex into several

358

groups that correspond to the current phylotyping division. Our study confirmed that

359

the sequences of 16S rRNA, the ITS region and egl genes formed four separate

360

clusters corresponding to the four phylotypes (Fegan & Prior, 2005; Prior & Fegan,

361

2005) as shown in Figs 1, S2 and S3.

362

11

363

DNA-DNA hybridization experiments were performed with selected strains from the

364

R. solanacearum species complex, representing the different phylogenetic groups,

365

and with the type strain of the closest related species, R. mannitolilytica with which

366

DNA-DNA relatedness values below 37% were obtained (Table 3). Within the R.

367

solanacearum species complex, the strains belonging to phylotypes I, III and IV

368

showed DNA-DNA relatedness values ranging from 51 to 60% with the type strain of

369

R. solanacearum (LMG 2299T), which indicates that these strains should not be

370

classified as R. solanacearum (Table 3). Among the phylotype IV strains, DNA-DNA

371

relatedness values ranging from 67 to 100% were found, indicating that they should

372

be classified as a single genomic species (Table 3). High DNA-DNA relatedness

373

values were found between the BDB strains (88-100%), and also between the

374

phylotype IV R. solanacearum strains (88-100%). The DNA-DNA relatedness

375

between BDB and R. solanacearum phylotype IV strains ranged from 70 to 92%,

376

whereas values around 70% were obtained for each of these groups compared to

377

the type strain of R. syzygii. Strains of phylotype I and III showed a DNA-DNA

378

relatedness below 70% with the type strains of R. syzygii (53-58%), R.

379

solanacearum (phylotype II) (52-58%) and R. mannitolilytica (31-32%), and exhibited

380

values between 72 and 90% among each other, indicating that they represent a

381

single genospecies within the genus Ralstonia (Table 3). These findings match the

382

data of a recent MLSA study (Wicker et al., 2012) and are also consistent with the

383

ANI values reported for comparisons between the single sequenced genomes of the

384

phylotype IV subgroups (Remenant et al., 2011). The high DNA-DNA relatedness

385

between phylotype I and III strains is also in agreement with an earlier DNA-DNA

386

hybridization study by Palleroni and Doudoroff (1971) in which strains belonging to

387

biotypes 3 and 4 (currently assigned to phylotype I) showed 79-100% DNA-DNA

388

homology with some strains belonging to former biotype1 strains that are currently

389

assigned to phylotype III.

390

Biolog GN2 metabolic fingerprinting assays (Table S6) and more than 40 classical

391

phenotypic tests (Table S7) were carried out on the strains listed in Table 1. Overall

392

R. solanacearum strains assigned to any of the four phylotypes were more

393

metabolically versatile in the utilization of carbon substrates than BDB or R. syzygii

394

strains (Table S6). Within phylotype IV, R. solanacearum strains were able to utilize

395

50 substrates on average, followed by BDB (average number of 28 substrates) and 12

396

R. syzygii strains (average number of 10 substrates) (Table S6). An average number

397

of 49, 47 and 53 of substrates were utilized by strains of phylotypes I, II and III

398

respectively. For each phylotype a number of core substrates could be identified that

399

could be utilized by all or the majority of strains tested. For phylotype I, II and III

400

strains could utilize a core set of 34, 31 and 36 substrates respectively (Table S8).

401

Further, within phylotype IV R. solanacearum strains were able to utilize a core set of

402

34 substrates (21 of these used by all strains, 13 used by at least 90% of strains),

403

whereas BDB strains utilized 18 core substrates (11 used by all strain, 7 used by at

404

least 90% of all strains) and R. syzygii strains were able to utilize only 6 core

405

substrates (all of these were used by at least 80% of strains tested) (Table S8).

406

These data clearly document the varying abilities of R. solanacearum strains to

407

utilize carbon sources of varying types. In classical phenotypic tests high similarity

408

was found among the strains of the R. solanacearum species complex (Table S7), a

409

well known feature of these bacteria and the origin of the difficulties in classifying the

410

various representatives of this species complex (Fegan & Prior, 2005; Harris, 1971;

411

Palleroni & Doudoroff, 1971).

412 413

However, using a selected range of phenotypic tests based on data from this study

414

(Tables S6, S7, S8) and from six published papers (Hayward, 1964; He et al., 1983;

415

Horita & Tsuchiya, 1999; Horita et al., 2005; Prior & Steva, 1990; Roberts et al.,

416

1990) (Table S9), some phenotypic features useful to distinguish the different taxa

417

within the R. solanacearum species complex were identified (Table 4). Phylotype I

418

and III strains can be differentiated from all phylotype II and the phylotype IV, R.

419

syzygii and BDB strains by their ability to utilize D-trehalose (Table 4). The phylotype

420

IV R. solanacearum strains were able to utilize acetic acid, D-glucosaminic acid, D-

421

glucuronic acid, p-hydroxy phenylacetic acid, propionic acid, D-saccharic acid and γ-

422

amino butyric acid, while both BDB and R. syzygii strains were negative for these

423

traits. Similarly, acid is produced from lactose and maltose by phylotype IV R.

424

solanacearum strains, while phylotype I and III strains, as well as BDB and R.

425

syzygii were negative for this feature. The inability to utilize Tween 40, D,L-lactic

426

acid, L-histidine, and the inability to grow on CPG medium clearly differentiated R.

427

syzygii from both R. solanacearum and BDB strains, which were able to utilize those

428

substrate and grew well on CPG medium. An interesting observation in terms of 13

429

diagnostic typing of phylotype IV strains was that BDB can be distinguished from the

430

other phylotype IV strains by their ability to hydrolyse starch for which both R.

431

solanacearum and R. syzygii strains showed negative results.

432 433

The cellular fatty acid composition of R. solanacearum species complex strains and

434

R. mannitolilytica, was also analysed and results are shown in Table 5. All strains,

435

including R. mannitolilytica, contained the fatty acids C14:0, C16:0, C18:1 ω7c, C18:1 2OH,

436

and C17:0 cyclo as well as the summed features 2 (C16:1 iso I /C14:0 3OH) and 3 (C16:1

437

ω7c /C15:0 iso 2OH) (Table 4). C16:0, C18:1 ω7c and the summed features 2 and 3 were

438

the most predominant fatty acid components detected in all strains (Table 5). The

439

fatty acid profiles of R. solanacearum phylotypes I, II, III and IV were similar overall,

440

although a few differences were observed in the relative concentrations of individual

441

components some of which may be of diagnostic value (Table 5).

442 443

Based on a combination of genotypic and phenotypic data of a large set of strains of

444

the R. solanacearum species complex obtained in this study and based on additional

445

analyses of genome sequences, CGH arrays and MLSA data (Cao et al., 2013;

446

Gabriel et al., 2006; Guidot et al., 2009; Li et al., 2011; Remenant et al., 2011;

447

Remenant et al., 2010; Salanoubat et al., 2002; Wicker et al., 2012; Xu et al., 2011),

448

the following taxonomic proposals are made: limit the species R. solanacearum to

449

phylotype II strains and reclassify BDB and phylotype IV “R. solanacearum” strains

450

as novel subspecies of R. syzygii for which respectively the following names are

451

proposed: Ralstonia syzygii subsp. celebesensis subsp. nov. (type strain: UQRS

452

627T = LMG 27706T= DSM 27477T= R-46908T) and Ralstonia syzygii subsp.

453

indonesiensis subsp. nov. (type strain: UQRS 464T = LMG 27703T = DSM 27478T =

454

PSI 07T). The type strain of R. syzygii subsp. syzygii (which is created automatically

455

as a consequence) is the current type strain of R. syzygii, R 001T. There is a genome

456

sequence available for strain R 24 that was selected by Remenant et al. (2011) as

457

reference strain for the current R. syzygii species. However, this strain has shown

458

some unusual genotypic properties in both our analyses and those of Wicker et al.

459

(2012) which makes its usefulness as a reference strain of R. syzygii subsp. syzygii

460

doubtful (Fegan & Prior, 2005; Prior & Fegan, 2005).

14

461

Ralstonia pseudosolanacearum sp. nov. (type strain: UQRS 461T = LMG 9673T =

462

NCPPB 1029T) is proposed to incorporate “R. solanacearum” strains belonging to

463

phylotypes I and III, which form a single species based on data from this study, the

464

comparative genome analyses carried out by Remenant et al. (2010), the MLSA data

465

of Wicker et al. (2012) and the early studies by Palleroni and Doudoroff (1971) and

466

De Vos (1980). The proposed type strain of R. pseudosolanacearum is one of the

467

strains included in the original DNA-DNA hybridization experiments on R.

468

solanacearum strains (De Vos, 1980; Palleroni & Doudoroff, 1971). Classification of

469

strains of phylotypes I and III as a single species is warranted based on their

470

phylogenetic as well as overall genomic relatedness (Remenant et al., 2010). We

471

recognize that both R. pseudosolanacearum as well as the true R. solanacearum

472

strains (phylotype II) encompass a significant number of strains, and future work may

473

well result in the designation of subspecies for either species. This could be

474

especially true for the R. pseudosolanacearum strains, where a clear geographical

475

division of strains belonging to phylotype I and phylotype III exists. Within the ‘true’

476

R. solanacearum strains (phylotype II) 8 separate groups of strains were identified

477

using MLSA (Wicker et al., 2012) which also indicates that multiple subspecies may

478

have to be proposed. However, additional data are needed for such proposals, which

479

is outside the scope of the current study.

480 481

Emended description of Ralstonia solanacearum (Smith 1896) Yabuuchi et al.

482

1995

483

The description of Yabuuchi et al. (1995) is emended to include only strains

484

belonging to phylotype II (Fegan & Prior, 2006; Prior & Fegan, 2005). The

485

descriptions are based on Yabuuchi et al. (1995) and this study. Cells are Gram-

486

negative, rod-shaped that may be motile or non motile (the type strain is motile).

487

Catalase and oxidase positive. Able to grow at 28 °C on CPG medium. Some strains

488

reduce nitrate to gas as well as reduce nitrate to nitrite. Most strains can utilize

489

citrate, hydrolyse Tween 80 and urease. Arginine dihydrolase, lysine and ornithine

490

decarboxylase activity are negative. Acid is produced oxidatively from glucose,

491

mannose, fructose, glycerol, galactose and sucrose. Some strains produce acid from

492

lactose, maltose, cellobiose, dulcitol, mannitol, sorbitol, inositol, rhamnose, D-

493

arabinose, ethanol, raffinose, trehalose, xylose and adonitol. In Biolog GN2 15

494

MicroPlate tests, this bacterium utilizes dextrin, glycogen, Tween 40, Tween 80, D-

495

fructose, α-D-glucose, m-inositol, sucrose, pyruvic acid methyl ester, succinic acid

496

mono-methyl-ester, acetic acid, cis-aconitic acid, citric acid, D-galacturonic acid, D-

497

gluconic acid, D-glucuronic acid, α-hydroxybutyric acid, β-hydroxybutyric acid, α-

498

ketoglutaric acid, D,L-lactic acid, propionic acid, quinic acid,

499

sebacic acid, succinic acid, bromosuccinic acid, glucuronamide acid, L-alaninamide,

500

D-alanine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-histidine, L-

501

ornithine, L-proline, L-pyroglutamic acid, D-serine, L-serine, L-threonine, γ-amino

502

butyric acid and glycerol. The major cellular fatty acids are summed feature 3 (C16:1

503

ω7c /C15:0 iso 2OH), C16:0, C18:1 ω7c, summed feature 2 (C16:1 iso I /C14:0 3OH), C17:0

504

cyclo, C18:1 2OH, C16:1 2OH, and C14:0. Strains contain a signature nucleotide

505

sequence, AAGTTATGGACGGTGGAAGTC (Fegan & Prior, 2006; Prior & Fegan,

506

2005).

507

The type strain is UQRS 426T (= LMG 2299T = NCPPB 325T = K60T). The DNA G+C

508

content is 66.6 Mol% (HPLC method). The GenBank accession number of the 16S

509

rRNA gene of the type strain is EF16361.

D-saccharic acid,

510 511

Emended description of Ralstonia syzygii (Roberts et al., 1990) Vaneechoutte

512

et al. 2004

513

This species includes all phylotype IV strains. The description is based on the data of

514

Roberts et al. (1990), Vaneechoutte et al. (2004) and this study. Cells are Gram-

515

negative, non-motile, non-sporulating, non-capsulated, non-motile, straight rods with

516

rounded ends, approximately 0.5-0.6 x 1.0-2.5 µm in size, occurring singly, in pairs

517

or occasionally in short chains. Aerobic growth. Catalase and oxidase positive.

518

Growth is observed on MacConkey agar without NaCl, but not on 5% NaCl. Strains

519

contain a signature nucleotide sequence, ATTGCCAAGACGAGAGAAGTA (Fegan &

520

Prior, 2006; Prior & Fegan, 2005).

521

The type strain is R 001T (=LMG 10661T = DSM 7385T). The GenBank accession

522

number of the 16S rRNA gene of the type strain is U28237.

523

The species consists of three subspecies which can be differentiated on the basis of

524

phenotypic and pathogenicity characteristics as described below.

525

16

526

Description of Ralstonia syzygii subsp. syzygii (Roberts et al., 1990)

527

Ralstonia syzygii subsp. syzygii (sy.zy’gi.i N.L. gen. n. Syzygium generic name of the

528

clove tree; N.L. gen n. Syzygii of the genus Syzygium).

529

Cells are Gram-negative straight rods, approximately 1x1.5 – 3 µm, occurring singly

530

or in pairs at 28 °C on CA medium after 5-7 days. Growth is observed on

531

MacConkey agar without added NaCl. Unable to grow on CPG medium. Most strains

532

(>85%) utilize D-glucose, succinic acid, L-aspargine, L-aspartic acid, L-glutamic acid

533

and L-proline. Unable to utilize Tween 40, D-trehalose, D,L-lactic acid and L-

534

histidine. Other characteristics are given in Tables 3, S6 and S7. Major cellular fatty

535

acids are C18:1 ω7c, summed feature 3 (C16:1 ω7c /C15:0 iso 2OH), C16:0, summed

536

feature 2 (C16:1 iso I /C14:0 3OH), C17:0 cyclo and C18:1 2OH.

537

The type strain is R 001T (= LMG 10661T = DSM 7385T), isolated as a plant pathogen

538

from xylem tissues of the clove tree (Syzygium aromaticum). Strains may also be

539

isolated from other Syzygium spp., and from insect vectors (Hindola spp.) in

540

Indonesia. The DNA G+C content is 65.2 mol% (HPLC method) and 66-67 mol%

541

(buoyant density method). The GenBank accession number of the 16S rRNA gene of

542

the type strain is U28237.

543 544 545

Description of Ralstonia syzygii subsp. indonesiensis subsp. nov.

546

Ralstonia syzygii subsp. indonesiensis subsp. nov. (in.do.ne.si.en'sis. N.L. fem. adj.

547

indonesiensis, of or belonging to Indonesia).

548

Cells are Gram-negative, asporogenous rod shaped that maybe motile or non motile

549

(the type strain is motile). Colonies vary from fluid form to butyrous consistency with

550

white colour and a diameter of approximately 0.5 mm after 2-3 days of incubation at

551

28 °C on CPG medium. The colonies are irregular and convex shaped. Aerobic.

552

Growth is observed on MacConkey agar without the addition of NaCl. Catalase and

553

oxidase positive. Most strains (>85%) can reduce nitrate to gas and reduce nitrate to

554

nitrite. Citrate is utilized but not malonate. Most strains hydrolyse Tween 80 and

555

show DNase activity. Acid is produced oxidatively from glucose, lactose, maltose,

556

fructose, glycerol, D-arabinose, galactose and sucrose, but not from dulcitol, 17

557

mannitol, sorbitol, adonitol, salicin, melezitose, melibiose and inulin. Arginine

558

dihydrolase, lysine decarboxylase, ornithine decarboxylase and phenylalanine

559

deaminase are negative. Strains do not hydrolyse esculin, gelatine or starch. In

560

Biolog GN2MicroPlate assays, Tween 40, Tween 80, D-trehalose, D-fructose, D-

561

galactose, α-D-glucose, pyruvic acid methyl ester, succinic acid mono-methyl-ester,

562

acetic acid, cis-aconitic acid, citric acid, D-galacturonic acid, D-gluconic acid, D-

563

glucoronic acid, β-hydroxybutyric acid, p-hydroxy phenylacetic acid, α-ketoglutaric

564

acid, D,L-lactic acid, propionic acid, quinic acid, D-saccharic acid, sebacic acid,

565

succinic acid, bromosuccinic acid, D-alanine, L-alanine, L-asparagine, L-aspartic

566

acid, L-glutamic acid, L-histidine, L-proline, L-pyroglutamic acid, L-serine, and γ-

567

amino butyric acid are utilized. Other characteristics are given in Tables 3, S6 and

568

S7. Major cellular fatty acids are summed feature 3 (C16:1 ω7c /C15:0 iso 2OH), C16:0,

569

C18:1 ω7c, summed feature 2 (C16:1 iso I /C14:0 3OH), C17:0 cyclo and C18:1 2OH.

570

The type strain is UQRS 464T (= LMG 27703T = DSM 27478T = PSI 07T) which was

571

isolated from potato in Indonesia. Strains may be isolated from tomato, potato, chilli

572

pepper, and clove. The DNA G+C content is 65.5 mol% (HPLC method) and 66.3

573

mol% (whole genome sequence method (Remenant et al., 2011)). The GenBank

574

accession number of the 16S rRNA gene of the type strain is KC757057.

575 576

Description of Ralstonia syzygii subsp. celebesensis subsp. nov.

577

Ralstonia syzygii subsp. celebesensis subsp. nov. (ce.le.bes.en’sis. N.L. fem. adj.

578

celebesensis, of or belonging to Celebes).

579

The description is based on the data of Eden-Green et al. (1988) and this study.

580

Cells are Gram-negative rod shaped, 0.8 µm x 2.5 µm in size and non-motile.

581

Colonies are typical round, mucoid, non-fluidal, small (0.5 – 2 mm) at 28 °C

582

incubated after 4-5 days on CPG medium. Mucoid colonies (2-3 mm in size), with

583

smooth margins and dark red centres are produced on tetrazolium chloride medium

584

and non-fluorescent, small, white and mucoid colonies on peptone or nutrient agar

585

media. Catalase and oxidase positive. Aerobic. All strains are unable to reduce

586

nitrate to nitrogen gas but 80% of strains can reduce nitrate to nitrite. Eden-Green et

587

al.(1988) previously reported this pathogen did not hydrolyse starch but our study

588

showed that starch is hydrolysed with the exception of strain UQRS 520 (R 229), for 18

589

which the genome has been sequenced by Remenant et al. (2011), but not esculin

590

or gelatine. Citrate and malonate are not utilized. Acid is produced oxidatively from

591

galactose. Acid production from glucose was recorded as positive (Eden-Green et

592

al., 1988), but our study observed variable acid production (72% of the strains) for

593

this sugar. Testing by means of Biolog GN2MicroPlate indicates that >90% of strains

594

utilize Tween 40, Tween 80, pyruvic acid methyl ester, succinic acid mono-methyl-

595

ester, α-ketoglutaric acid, D,L-lactic acid, quinic acid, succinic acid, bromosuccinic

596

acid, succinamic acid, D-alanine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic

597

acid, L-histidine, L-proline and L-serine. D-trehalose is not utilised. Other

598

characteristics are given in Tables 3, S6 and S7. Major cellular fatty acids are

599

summed feature 3 (C16:1 ω7c /C15:0 iso 2OH), C16:0, C18:1 ω7c, summed feature 2 (C16:1

600

iso I /C14:0 3OH), C16:1 2OH, C18:1 2OH, C17:0 cyclo.

601

The type strain is UQRS 627T (= LMG 27706T = DSM 27477T = R-46908T) which was

602

isolated from banana plants in Central Java, Indonesia. The DNA G+C content is

603

65.8 mol% (HPLC method). The GenBank accession number of the 16S rRNA gene

604

of the type strain is KC757073.

605 606

Description of Ralstonia pseudosolanacearum sp. nov.

607

Ralstonia pseudosolanacearum (pseu.doso.la.na.ce.a'rum. Gr. adj. pseudês, false;

608

N.L. fem. pl. gen. n. solanacearum, a bacterial specific epithet; N.L. fem. pl. gen. n.

609

pseudosolanacearum, false solanacearum, referring to Ralstonia solanacearum)

610

This species is limited to strains belonging to phylotypes I and III. Cells are Gram-

611

negative, non-sporulating rods that may be motile or non-motile (the type strain is

612

non-motile). Catalase and oxidase positive. Aerobic. Colonies have diameters of less

613

than 1 mm on CPG medium after one to three days of incubation at 28 °C. Most

614

strains reduce nitrate to gas. Unable to hydrolyse starch, esculin or gelatine. Most

615

strains utilized citrate. Unable to utilize malonate. Phenylalanine deaminase, DNase,

616

argininine dihydrolase, lysine decarboxylase and ornithine decarboxylase activity is

617

negative. Acid is produced oxidatively from glucose, inositol, mannose, fructose,

618

glycerol, galactose, raffinose, and sucrose. Using Biolog GN2 MicroPlate, >90% of

619

the strains utilize Tween 40, Tween 80, D-fructose, α-D-glucose, m-inositol, D-

620

trehalose, pyruvic acid methyl ester, succinic acid mono-methyl-ester, cis-aconitic 19

621

acid, citric acid, D-galacturonic acid, D-gluconic acid, D-glucuronic acid, β-

622

hydroxybutyric acid, D,L-lactic acid, bromosuccinic acid, glucuronamide, L-

623

alaninamide, D-alanine, L-alanine, L-asparagine, L-aspartic acid, L-histidine, L-

624

proline, L-pyroglutamic acid and L-serine. All strains are unable to produce acid from

625

L-rhamnose and ethanol. Other characteristics are given in Tables 3, S6 and S7.

626

The following major cellular fatty acid components are present: summed feature 3

627

(C16:1 ω7c /C15:0 iso 2OH), C18:1 ω7c, C16:0, summed feature 2 (C16:1 iso I /C14:0 3OH),

628

C16:1 2OH, C18:1 2OH, C14:0, and C17:0 cyclo. Strains contain signature nucleotide

629

sequences

630

ATTACSAGAGCAATCGAAAGATT(Fegan & Prior, 2006; Prior & Fegan, 2005).

631

The type strain is UQRS 461T (= NCPPB 10229T = LMG 9673T) isolated from

632

Pelargonium capitatum in Reunion Island, France. The DNA G+C content is 66.1 mol

633

% (HPLC method). The GenBank accession number of the 16S rRNA gene of the

634

type strain is KC757037.

CGTTGATGAGGCGCGCAATTT

or

635 636

ACKNOWLEDGMENTS

637

This research was supported by the Australian Centre for International Agricultural

638

Research (ACIAR), the University of Queensland, Australia, and the BCCM/LMG

639

Bacteria

640

Ledeganckstraat 35, B-9000 Ghent, Belgium. Financial support is acknowledged

641

from AusAID and the Directorate General of Higher Education of Indonesia

642

(DGHE/DIKTI) for scholarships to Irda Safni. The BCCM/LMG Bacteria Collection is

643

supported by the Federal Public Planning Service – Science Policy, Belgium. We

644

thank K. Engelbeen and C. Snauwaert for their technical assistance.

645

20

Collection,

Laboratory

of

Microbiology,

Ghent

University,

K.L.

646

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647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694

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917 918 919 920 921 922

26

923 924

Table 1. List of bacterial strains investigated in this study. Taxon

Former name (+ phylotype)

Ralstonia mannitolilytica

Strain

Alternate Strain No.

Area of origin

Host/Source

LMG 6866T ¶,,§

NCIMB 10805T

London, UK

Human blood

UQRS 426T

LMG 2299T ¶,§; K60T; NCPPB 325T

USA

Tomato

UQRS 648 UQRS 647 UQRS 652 § GMI1000†

NCPPB 1331 Molk2† IPO1609† UQRS 442¶,§; JS 753

India Philippines The Netherlands French Guyana

Potato Banana Potato Tomato

NCPPB 1123 NCPPB 253

UQRS 649 UQRS 650 ¶,*;LMG 2297

Papua New Guinea Mauritius

Potato Casuarina equisetifolia

UQRS 84

NCPPB 342

Zimbabwe

Tobacco

UQRS 460 § NCPPB 1018 UQRS 461T ¶,* UQRS 651 §

NCPPB 216 NCPPB 1029T; LMG 9673T CMR15 ¶,†; RUN 133; CFBP 6941

Zimbabwe Angola Reunion Island, France Cameroon

Potato

LMG 10661T ¶,*,§

R 001T; DSM 7385T; ATCC 49543T

West Sumatra, Indonesia

Clove

R 002

R 168; T 330; UQRS 530; NCPPB 3445 R 167; T 247; UQRS 523

Indonesia

Clove

Indonesia

Clove

Ralstonia solanacearum species complex: Ralstonia solanacearum

Ralstonia pseudosolanacearum sp. nov.

Ralstonia solanacearum (phylotype II)

Ralstonia solanacearum (phylotype I)

Ralstonia solanacearum (phylotype III)

Ralstonia syzygii subsp. syzygii comb.nov.

Ralstonia syzygii (phylotype IV)

R 106

27

Pelargonium capitatum Tomato

R 165 R 166

T 325; UQRS 529 -

Indonesia Indonesia

Clove Clove

Taxon

Former name (+ phylotype)

Strain

Alternate Strain No.

Area of origin

Host/Source

Ralstonia syzygii subsp. indonesiensis subsp. nov.

Ralstonia solanacearum

UQRS 85

MAFF 301558; JS 934

Japan

Potato

UQRS 92 UQRS 95 UQRS 96 UQRS 99 UQRS 262 UQRS 264 UQRS 265 UQRS 267 UQRS 268 UQRS 271 ¶,*,§ UQRS 272 UQRS 274 UQRS 280 UQRS 281 UQRS 290 UQRS 291 UQRS 463 UQRS 464T ¶,*,§

T6 T 9; R-46895 T 10 T 13 R 792 T 19 T 20 T 22 T 23 T 26; R-46896 T 27 T 29 T 35 T 36 T 45 T 46; R-46897 PSI 36; R-46899 PSI 07T†; R-46900T; LMG 27703T; DSM 27478T NCPPB 3219; ICMP 9915 R 142; R-46901 R 220; R 045

West Java, Indonesia West java, Indonesia West java, Indonesia West java, Indonesia Indonesia West java, Indonesia West Java, Indonesia West Java, Indonesia West Java, Indonesia West Java, Indonesia West Java, Indonesia West Java, Indonesia West Java, Indonesia West Java, Indonesia West Java, Indonesia West Java, Indonesia Indonesia Indonesia

Tomato Tomato Tomato Tomato Chilli pepper Tomato Tomato Tomato Tomato Tomato Tomato Tomato Tomato Tomato Tomato Tomato Tomato Tomato

(phylotype IV)

UQRS 518 UQRS 524 UQRS 532 UQRS 533 ¶,*,§ UQRS 548 UQRS 549 ¶,*,§

28

Indonesia Indonesia West Sumatra, Indonesia West Sumatra, Indonesia

R 221; R 456; R 768; R46902; LMG 27704; DSM 27479 R 780; R-46903 R 784; R-46904; LMG 27705;

West Java, Indonesia West Java, Indonesia

Clove Clove Clove Clove

Clove Potato

Ralstonia syzygii subsp. celebesensis subsp. nov.

BDB (phylotype IV)

Species name

Former name (+ phylotype)

Ralstonia syzygii subsp. celebesensis subsp. nov.

BDB (phylotype IV)

29

UQRS 550 UQRS 465

DSM 27480 R 792 R 230; JT 657

West Java, Indonesia West Java, Indonesia

Chilli pepper Banana

UQRS 479 UQRS 480 ¶,*,§ Strain

SSBD1 R-46906; SSBD2 Alternate Strain No.

Central Java, Indonesia Bali, Indonesia Area of origin

Banana Banana Host/Source

UQRS 481

SSB3; Y1

Indonesia

Banana

UQRS 519 UQRS 520

Indonesia Indonesia

Banana Banana

UQRS 534

ICMP 10000; R 230 R 229†, ICMP 10001; T 389; LMG 27886 ¶ R 223; T 439

Banana

UQRS 535

R 224; T 386

UQRS 536 ¶,*,§

R-46907; R 225; T 380; T 412

UQRS 538

R 227; T 394; T 383

UQRS 539

R 228; T 381

UQRS 542 UQRS 543

R 231; T 336 R 233; T 379

UQRS 544 UQRS 546 UQRS 621 UQRS 622 UQRS 624 UQRS 625 UQRS 627T ¶,*,§ UQRS 631

R 234; T 391 R 506; T 340 2 3A 6 11A R-46908T; LMG 27706T; DSM 27477T; 17T 22A

North Sulawesi, Indonesia South Sulawesi, Indonesia South Sulawesi, Indonesia South Sulawesi, Indonesia South Sulawesi, Indonesia West Java, Indonesia South Sulawesi, Indonesia Indonesia West Java, Indonesia D.I.Y, Indonesia D.I.Y, Indonesia Central Java, Indonesia D.I.Y, Indonesia Central Java, Indonesia

UQRS 633

-

South Sulawesi, Indonesia North Sulawesi,

Banana Banana Banana Banana Banana Banana Banana Banana Banana Banana Banana Banana Banana Banana Banana

UQRS 635

29A

UQRS 637 ¶,*,§

R-46909; LMG 27707; DSM 27481; 31A -

BB

Indonesia West Sumatra, Indonesia West Sumatra, Indonesia Bali, Indonesia

Banana Banana Banana

925 926 927 928 929 930 931 932 933 934 935 936 937 938 939

¶

Strain investigated through DNA-DNA hybridization in this study *Strain for which the DNA G+C content has been determined in this study § Strain for which the fatty acid composition has been determined in this study † Strain for which a whole genome sequences has been determined by Remenant et al. (2010, 2011) ATCC, American Type Culture Collection, Rockville, Md, USA; BB, SSBD and Y, isolates supplied by Dr. Siti Subandiyah, Gadjah Mada University, Indonesia; CFBP, French Collection of Plant associated bacteria, France; DSM, Deutsche Sammlung von Mikroorganismen und Zellkulturen Braunschweig, Germany; GMI, M. Arlat and P. Barberies, CNRS-INRA, Auzeville, Castanet-Tolosan, Cedex, France; ICMP, International Collection of Microorganisms from Plants, Landcare Research, Mt .Albert, Auckland, New Zealand; JT & JS, The Laboratory of Phytopathology, CIRAD-FLHOR, La-Reunion, France; LMG, Belgian Coordinated Collection of Microorganisms, Laboratory of Microbiology, Ghent University, Belgium; MAFF, Ministry of Agriculture Forestry and Fisheries, National Institute of Agrobiological Resources, Japan; NCIMB, National Collection of Industrial Bacteria, Aberdeen, UK; NCPPB, National Collection of Plant Pathogenic Bacteria, Harpenden, UK; PSI, isolates supplied by Research Institute of Food Crops Biotechnology (RIFCB), Indonesia; R, Rothamsted Experimental Station, Institute of Arable Crops, Hapenden, UK; R-, Research Collection, Laboratory of Microbiology, Ghent University, Belgium; UQRS, University of Queensland of Ralstonia solanacearum collection, The University of Queensland, Brisbane, Australia; RUN, collection at CIRAD-FLHOR, La-Reunion, France; T, Faculty of Agriculture, Gadjah Mada University, Indonesia

30

940

Table 2. List of strains for which previously published phenotypic data were included in our analyses Taxon

Former name (+ phylotype)

Strain

Alternate Strain No.

Area of origin

Host/Source

Source

Ralstonia pseudosolanacearum

Ralstonia solanacearum

NCPPB 253

UQRS 650; LMG 2297

Mauritius

Casuarina equisetifolia

(Hayward, 1964)

(Phylotype I)

NCPPB 1052

Malaysia

Ginger

(Hayward, 1964)

French Guyana

Tomato

(Prior & Steva, 1990)

GMI1000

Ralstonia solanacearum

Ralstonia solanacearum

UQRS 442; JS 753

S236

-

Nambour, Australia

Tomato

(Prior & Steva, 1990)

M4

-

Guangdong, China

Mulberry

(He et al., 1983)

MAFF 211266

-

Hiroshima, Japan

Tomato

(Horita & Tsuchiya, 1999)

India

Potato

(Hayward, 1964)

NCPPB 1331

UQRS 648;

(Phylotype II) -

Ceylon

Potato

(Hayward, 1964)

T

USA

Tomato

(Prior & Steva, 1990)

K136

-

Trinidad

Tomato

(Prior & Steva, 1990)

K105

-

Florida, USA

Tobacco

(Prior & Steva, 1990)

S225

-

Peru

Tomato

(Prior & Steva, 1990)

S247

-

Columbia

Tobacco

(Prior & Steva, 1990)

NCPPB 1487 K60

Ralstonia pseudosolanacearum

Ralstonia syzygii subsp. syzygii

31

Ralstonia solanacearum

T

UQRS 426 ; LMG 2299T

NCPPB 216

UQRS 460;

Zimbabwe

Potato

(Hayward, 1964)

NCPPB 1029T

UQS 461T; LMG 9673T

Reunion Island, France

Pelargonium capitatum

(Hayward, 1964)

(Phylotype III)

Ralstonia syzygii

NCPPB 1486

-

Uganda

Groundnut

(Hayward, 1964)

R 24

-

Indonesia

Clove

(Roberts et al., 1990)

(Phylotype IV)

Taxon

Former name (+ phylotype)

Strain

Ralstonia syzygii subsp. indonesiensis

Ralstonia solanacearum

MAFF 211271

32

Alternate Strain No.

Area of origin

Host/Source

Source

-

Shizuoka, Japan

Potato

(Horita et al., 2005)

MAFF 301559

-

Nagasaki, Japan

Potato

(Horita et al., 2005)

WP20

-

Luzon, Philippines

Potato

(Horita et al., 2005)

28MF

-

Mindanao, Philippines

Potato

(Horita et al., 2005)

(Phylotype IV)

941

Table 3. DNA-DNA relatedness among the Ralstonia solanacearum species complex strains

Taxon

Former name (+ phylotyp e)

Strain

G+C content (mol%)

1

Ralstoni a pseudo solanac earums p. nov

R. 1. UQRS solanacea 650 rum (phylotype I)

66.5

100

UQRS 442

ND

90 (2)

100

R. 3. UQRS solanacea 461T rum (phylotype III)

66.1

73 (1)

72 (5)

100

ND

76 (0)

75 (5)

78 (2)

65.2

ND

ND

ND

65.5

61 (15)

62 (24)

60 (12)

60 (10)

ND

100

2.

4. Ralstoni a syzygii subsp. indones iensis subsp. nov.

Ralstoni a syzygii subsp. celebes ensis subsp. nov.

CMR15

R. 5. UQRS solanacea 271 rum (phylotype IV)

6.

UQRS

2

3

4

5

6

7

8

9

10

11

12

100 100

T

464

7.

UQRS 549

66.0

ND

ND

ND

ND

100 (>30)

ND

100

8.

UQRS 533

65.5

ND

ND

ND

ND

ND

ND

88 (25)

100

BDB 9. UQRS (phylotype 480 IV)

66.0

ND

ND

ND

ND

83 (>30)

ND

83 (0)

78 (13)

100

10. UQRS

66.0

ND

ND

ND

ND

92 (24)

ND

ND

ND

88 (17)

100

65.8

59 (9)

57 (5)

55 (1)

52 (2)

88 (8)

ND

ND

ND

100 (14)

96 (20)

100

63 (9)

64 (17)

59 (8)

56 (3)

ND

ND

ND

ND

ND

ND

100 (2)

536

11. UQRS 627T

12. LMG 27886

33

100

13

14

15

16

13. UQRS

65.9

ND

ND

ND

ND

73 (>30)

ND

70 (30)

77 (25)

97 (17)

89 (28)

ND

ND

100

637 Ralstoni a syzygii subsp. syzygii

R. syzygii 14. LMG (phylotype 10661T IV)

65.2

58 (14)

56 (9)

54 (1)

53 (1)

75 (8)

73 (26)

70 (6)

72 (37)

71 (7)

ND

67 (5)

71 (1)

69 (5)

100

Ralstoni a solanac earum

R. 15. LMG solanacea 2299T rum (phylotype II)

66.6*

58 (5)

56 (11)

55 (0)

52 (11)

51 (26)

ND

57 (9)

52 (2)

53 (7)

ND

55 (5)

60 (6)

53 (11)

55 (1)

100

66.2¶

31 (1)

ND

32 (7)

ND

ND

ND

31 (14)

37 (2)

34 (14)

ND

ND

ND

35 (1)

31 (5)

29 (0)

Ralstoni a mannito lilytica

942 943 944 945

16. LMG T

6866

ND: not determined *

(Yabuuchi et al., 1995) (De Baere et al., 2001)

¶

34

100

946 947 948

Table 4. Phenotypic characteristics that differentiate the taxa within the R. solanacearum species complex +, 51-100% of strains reacted positive; –, 10-50% of strains reacted negative. The shaded symbol, the distinguished characteristics each traits for particular taxon. Taxon

Ralstonia solanacearum

Former name (+ phylotype)

R. solanacearum (phylotype II)

Ralstonia pseudosolanacearum sp. nov.

Ralstonia syzygii subsp. indonesiensis subsp. nov.

Ralstonia syzygii subsp. celebesensis subsp. nov.

(n=41+72)

R. solanacearum (phylotype I) (n=3+6)

R.. solanacearum (phylotype III) (n=5+3)

R. syzygii (phylotype IV) (n=5+1)

R. solanacearum (phylotype IV) (n=26+4)

BDB (phylotype IV) (n=25)

+3

+3

+3

-3

+3

+3

-3

-4

-3

-4

-3

+3

+4 +4

-4

-4 -4

+3

-4

-4 -4

+4

+4

+4

-3

+ +3

-3 -3

+3 -3 +3

+3 +3

+3 +3

-3

+3

+3 -3

3

+

3

3

3

-

-

-3 -3 -3

+3

3

-3 -3

+3

+3

+3

-3

+3

-3

-3

+3

-3

-3

+3

-3

+3

+3

+3

-3

+3

+3

+3 +3

-3

+3

3

3

-3 -3

+3 +3

-3 -3

Feature Growth on CPG medium Starch hydrolysis Acid production from: Lactose Maltose Fructose Utilization of Biolog GN2 MicroPlate substrates: Tween 40 D-Trehalose Acetic acid DGlucosaminic acid D-Glucuronic acid p-Hydroxy phenylactic acid D,L-Lactic acid Propionic Acid D-Saccharic

35

-

+

+

+

3

+

3

+

3

-3

Acid L-Histidine γ-Amino Butyric acid

949 950 951 952

1

+3 +3

, strains used in this current study , strains obtained from other publications 3 , Data was obtained only from this current study 4 , Data was obtained from this current study and from other publication 2

36

+3 +3

+3 +3

-3 -3

+3 +3

+3 -3

953

Table 5. Cellular fatty acid composition (%) of representative strains of taxa within the Ralstonia solanacearum species complex Taxon

Former name (+ phylotype)

Strains1

Ralstonia solanacearum

Ralstonia solanacearum (phylotype II)

Ralstonia pseudosolanacearum

14:0

1 6 : 0

18:1 ω7c

16: 0 2O H

16:1 2OH

18:1 2OH

17:0 cyclo

SF2

SF3

LMG 2299T, UQRS 652

4.8 (0.1)1

1 9. 6 (1 .5 )

18.8 (0.2)

ND

6.3 (2.6)

6.8 (0.2)

8.7 (2.3)

10 (1.9)

21.1 (0.5)

Ralstonia solanacearum (phylotype I)

UQRS 442

4.6

1 9. 7

19.8

ND

6.9

7.3

4.7

9.7

23.6

Ralstonia solanacearum (phylotype III)

UQRS 460, UQRS 651

5.5 (1.9)

1 7 (0 .5 )

19.3 (1.5)

ND

9.8 (3.1)

6.2 (1.1)

4.0 (2.6)

10 (0.4)

24.0 (8.4)

Ralstonia syzygii subsp. syzygii

Ralstonia syzygii (phylotype IV)

LMG 10661T

2.7

1 8

28.7

ND

ND

5.1

5.7

11.9

23.9

Ralstonia syzygii subsp. indonesiensis

Ralstonia solanacearum (phylotype IV)

UQRS 271, UQRS 464T, UQRS 533, UQRS 549

4.4 (0.2)

2 1. 5 (1 .1 )

19.7 (1.7)

ND

3.2 (0.5)

6.6 (0.7)

8.1 (2.9)

9.5 (1.6)

22.4 (4.1)

Ralstonia syzygii subsp. celebesensis

BDB (phylotype IV)

UQRS 480, UQRS 536, UQRS 627T, UQRS 637

4.7 (0.2)

2 1. 4 (1 .0 )

19.6 (4.8)

ND

4.3 (1.9)

4.6 (0.5)

3.5 (0.8)

15 (4.6)

24.0 (2.1)

LMG 6866T

4.9

2 2. 8

15.8

4.2

5.4

4.3

6.1

10.4

19.6

Ralstonia mannitolilytica

37

954

1

955

ND, not detected or < 2.00; SF2, summed feature 2 (16:1 iso I /14:0 3OH); SF3, summed feature 3 (16:1 ω7c /15:0iso 2OH)

, For phylotypes of which more than 1 strain was analysed, standard deviation values are given in brackets.

38

956 957 958 959 960

Fig.1. Neighbour-joining tree of 16S-23S rRNA ITS region sequences reflecting the phylogenetic relationships of members of the Ralstonia solanacearum species complex. The type strain of Ralstonia pickettii is used as an outgroup. Bar, 1% sequence dissimilarity. Numbers at branching points indicate bootstrap percentage derived from 1000 samples. ** genome sequenced-strain, Bold font: proposed type strain.

961

39