International Journal of Systematic and Evolutionary Microbiology (2014), 64, 3087–3103 DOI 10.1099/ijs.0.066712-0

Polyphasic taxonomic revision of the Ralstonia solanacearum species complex: proposal to emend the descriptions of Ralstonia solanacearum and Ralstonia syzygii and reclassify current R. syzygii strains as Ralstonia syzygii subsp. syzygii subsp. nov., 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 phylotype I and III strains as Ralstonia pseudosolanacearum sp. nov.

Irda Safni,13 Ilse Cleenwerck,2 Paul De Vos,2 Mark Fegan,14 Lindsay Sly1 and Ulrike Kappler1

Correspondence 1School of Chemistry and Molecular Biosciences, Faculty of Science, University of Queensland, Lindsay Sly St Lucia, Queensland 4072, Australia [email protected] 2BCCM/LMG Bacteria Collection, Laboratory of Microbiology, Ghent University, Ulrike Kappler K. L. Ledeganckstraat 35, B-9000 Ghent, Belgium [email protected]

The Ralstonia solanacearum species complex has long been recognized as a group of phenotypically diverse strains that can be subdivided into four phylotypes. Using a polyphasic taxonomic approach on an extensive set of strains, this study provides evidence for a taxonomic and nomenclatural revision of members of this complex. Data obtained from phylogenetic analysis of 16S-23S rRNA ITS gene sequences, 16S–23S rRNA intergenic spacer (ITS) region sequences and partial endoglucanase (egl) gene sequences and DNA–DNA hybridizations demonstrate that the R. solanacearum species complex comprises three genospecies. One of these includes the type strain of Ralstonia solanacearum and consists of strains of R. solanacearum phylotype II only. The second genospecies includes the type strain of Ralstonia syzygii and contains only phylotype IV strains. This genospecies is subdivided into three distinct groups, namely R. syzygii, the causal agent of Sumatra disease on clove trees in Indonesia, R. solanacearum phylotype IV strains isolated from different host plants mostly from Indonesia, and strains of the blood disease bacterium (BDB), the causal agent of the banana blood disease, a bacterial wilt disease in Indonesia that affects bananas and plantains. The last genospecies is composed of R. solanacearum strains that belong to phylotypes I and III. As these genospecies are also supported by phenotypic data that allow the differentiation of the three genospecies, the following taxonomic proposals are made: emendation of the descriptions of Ralstonia solanacearum and Ralstonia syzygii and descriptions of Ralstonia syzygii subsp. nov. (type strain

3Present address: Faculty of Agriculture, University of Sumatra Utara, Medan, 20155 North Sumatra, Indonesia. 4Present address: Department of Environment and Primary Industries, 5 Ring Rd, La Trobe University, Bundoora, Victoria 3083, Australia. Abbreviations: BDB, blood disease bacterium; ITS, intergenic spacer; MLSA, multilocus sequence analysis. The GenBank/EMBL/DDBJ accession numbers for the sequences determined in this study are KC757031–KC757076 and KC820937–KC820940 (16S- 23S rRNA ITS gene), KC756969–KC757029 (16S–23S rRNA gene ITS) and KC757078–KC757122 and KC820936 (egl gene), as detailed in Table S1. Nine supplementary tables and three supplementary figures are available with the online version of this paper.

066712 G 2014 IUMS Printed in Great Britain 3087 I. Safni and others

R 001T5LMG 10661T5DSM 7385T) for the current R. syzygii strains, Ralstonia syzygii subsp. indonesiensis subsp. nov. (type strain UQRS 464T5LMG 27703T5DSM 27478T) for the current R. solanacearum phylotype IV strains, Ralstonia syzygii subsp. celebesensis subsp. nov. (type strain UQRS 627T5LMG 27706T5DSM 27477T) for the BDB strains and Ralstonia pseudosolanacearum sp. nov. (type strain UQRS 461T5LMG 9673T5NCPPB 1029T) for the strains of R. solanacearum phylotypes I and III.

INTRODUCTION the causal agent of banana blood disease, which is one of the most destructive bacterial wilt diseases affecting bananas Ralstonia solanacearum is a soil-borne pathogen that causes (Musa acuminata) and plantains (Musa balbisiana6acumi- lethal vascular wilt diseases of over 200 plant species from nata) in Indonesia (Eden-Green, 1994). BDB was probably more than 50 families including solanaceous vegetable first isolated in the early 20th century and named crops, banana, ginger, custard apple, peanut, eucalyptus and ¨ many other crop plants (Hayward, 1994; Kelman, 1953). ‘Pseudomonas celebensis’(Gaumann, 1921). However, this Due to its lethality, persistence, wide host range and very name was not included in the Approved Lists of Bacterial broad geographical distribution, R. solanacearum is one of Names and, as type or reference cultures of ‘P. celebensis’no the most devastating bacterial plant pathogens known longer exist, the name has no standing in nomenclature. The (Elphinstone, 2005). bacterium is currently referred to as BDB and classified as a member of phylotype IV of the R. solanacearum species R. solanacearum is a heterogeneous species, as evidenced by complex. its large host range, pathogenic specialization and cultural and physiological properties, as well as its phylogeny Recently, Wicker et al. (2012) further subdivided the R. (Hayward, 2000). Following its discovery, R. solanacearum solanacearum species complex into eight clades based on was originally classified as a member of the genus ‘Bac- multilocus sequence analysis (MLSA). Phylotypes I and III terium’ (Smith, 1896). The application of DNA-based were each contained in a single clade (1 and 6, respec- methods eventually resulted in its transfer to the genus tively), while phylotype II consisted of four separate clades Burkholderia (Yabuuchi et al., 1992) and then to the genus (2–5). Phylotype IV was composed of two clades (7 and 8), Ralstonia (Yabuuchi et al., 1995). Despite being classified as with clade 7 including BDB and R. solanacearum phylotype a single species, it has been accepted since the 1980s that IV strains and clade 8 containing R. syzygii strains and R. different strains of R. solanacearum may show DNA–DNA solanacearum phylotype IV strains isolated from clove relatedness values below 70 % (De Vos, 1980; Palleroni & trees. Phylotype IV was reported to be the most divergent Doudoroff, 1971) and therefore might be members of and ancestral phylotype, although ongoing diversification different species. As a result, the term ‘species complex’ was observed within phylotypes I, II and III (Wicker et al., (Gillings & Fahy, 1994), referring to ‘a cluster of closely 2012). In addition, the MLSA study indicated that phylo- related bacteria whose individual members may represent types I and III are more closely related to each other than more than one species’ (Fegan & Prior, 2006), has been to phylotypes II and IV, and the dendrograms also clearly applied to R. solanacearum. documented the close relationship between the phylotype IV strains. Within the R. solanacearum species complex, four phylo- types are recognized (Prior & Fegan, 2005) that can be Whole-genome sequences of several representatives of the distinguished based on the sequences of their 16S–23S R. solanacearum species complex have become available in rRNA gene intergenic spacer (ITS) region and the hrpB and recent years, and these include three representatives of egl genes as well as comparative genomic hybridization phylotype I (GMI1000, Y45, FQY-4), six phylotype II (Fegan & Prior, 2005; Guidot et al., 2007). Phylotypes I, II strains (IIA: K60; CFBP 2957; IIB: IPO1609, UW 551, and III are composed of strains mainly from Asia, America Po82, Molk2) and one representative each of phylotype III and Africa, respectively, and surrounding islands, while (CMR15) and the three main phylotype IV groups (R. phylotype IV is primarily composed of strains from Indo- solanacearum, PSI 07; R. syzygii, R 24; BDB, R 229) (see nesia and some isolates from Japan, Australia and the Table S2, available in the online Supplementary Material). Philippines. Phylotype IV is the most diverse group, as it Additional genomes have been sequenced but are not pub- consists of strains assigned to R. solanacearum, Ralstonia licly available at present (https://www.genoscope.cns.fr/agc/ syzygii and the blood disease bacterium (BDB). R. syzygii is microscope/about/collabprojects.php). All complete genomes the causal agent of the Sumatra disease of clove trees in consist of two replicative units, a chromosome of approxi- Indonesia (Roberts et al., 1990) and its status as a separate mately 3.7 Mb and a megaplasmid of 1.6–2.3 Mb, which species in the genus Ralstonia hasbeenproven(Vaneechoutte together encode approximately 5000 proteins. Comparative et al., 2004). However, it is also clearly a member of the R. studies found that each genome contained between 400 and solanacearum species complex (Taghavi et al., 1996). BDB is 600 unique genes, regardless of whether or not the genomes

3088 International Journal of Systematic and Evolutionary Microbiology 64 Taxonomic revision of the R. solanacearum species complex originated from the same phylotype (Remenant et al., 2010, and egl sequences included data for strains with published genome 2011). sequences as well as sequences deposited in GenBank as part of previous studies (Tables S1, S2 and S5). Recent work comparing eight complete genome sequences Strains were grown aerobically at 28 uC. For routine maintenance, from R. solanacearum (one from phylotype I, three from solid Casamino acid peptone glucose agar (CPG) medium, composed phylotype II, one from phylotype III and one each for the of (l21) 10 g peptone (Oxoid), 1 g casein hydrolysate (Bacto three subgroups of phylotype IV) also observed very close Casamino acids) and 5 g glucose, adjusted to pH 6.5–7.0 (Kelman, relationships between phylotype I and III strains, and the 1954), was used with incubation times of 2 days for strains of R. authors proposed a revision of the taxonomy of the R. solanacearum, 4 days for BDB and 6 days for strains of R. syzygii. For solanacearum species complex based on genome average fatty acid composition determination, Casamino acid (CA) medium nucleotide identity (ANI) values and genetic relatedness (Roberts et al., 1990) was used as the cultivation medium. DNA for DNA–DNA hybridization experiments and determination of the (Remenant et al., 2011). Phylotype II strains were to be G+C content was isolated from cells grown on tryptic soy agar maintained as R. solanacearum, while strains of phylotype (Oxoid CM131) or CA medium, except when growth on them IV of R. solanacearum, BDB and R. syzygii were proposed resulted in insufficient amounts of cell material. In that case, to be members of subspecies of a single novel species, cultivation was performed on charcoal medium [l21: 10 g yeast ‘Ralstonia haywardii’. Finally, strains of phylotypes I and III extract, 2 g activated charcoal (Norit SG), 10 g ACES buffer (Sigma), ] of R. solanacearum were proposed to form a single novel 17 g agar . The ACES buffer was dissolved in 500 ml distilled water at 50 uC and then mixed with a solution containing 40 ml 1 M KOH in species, ‘Ralstonia sequeirae’. 440 ml distilled water. This mixture was used to hydrate the other While the genome analyses were very informative, only ingredients. Cysteine hydrochloride (0.4 g) and Fe4(P2O7)3 (0.25 g) eight strains of the entire R. solanacearum species complex were dissolved either together (20 ml) or separately (10 ml each) in distilled water, filter-sterilized and then added to the medium after were analysed, rather than a set of strains representative of autoclaving. The final pH of the medium was 6.9. the diversity within the proposed novel taxa, and no genotypic or phenotypic characteristics suitable for their Molecular verification of strain identity. Multiplex PCR (Fegan & differentiation were reported (Remenant et al., 2011). Prior, 2005) and BDB-specific PCR (Tan, 2003) were used as Further, the proposal of the novel species ‘Ralstonia molecular diagnostic tools to verify the phylotype to which each strain haywardii’ for strains of phylotype IV of R. solanacearum, belonged and to determine whether the strain was a BDB, respectively. The primers are shown in Table S3. BDB and R. syzygii is not in accordance with the Rules of the International Code of Nomenclature of Prokaryotes Phenotypic characterization. Forty-seven classical phenotypic tests (De Vos & Tru¨per, 2000; Lapage et al., 1992), as the name (physiological and biochemical) were performed on the 68 strains of Ralstonia syzygii is validly published and must be retained. the R. solanacearum species complex (Table 1). These tests included the oxidase test (Kova´cs, 1956), catalase test (He et al., 1983), nitrate In this study, we report the results of a polyphasic taxo- reduction test by the method of Hayward (1964) with minor nomic study of 68 strains of the R. solanacearum species modification by using the medium of Van der Mooter et al. (1987), complex representing all four phylotypes, but with a hydrolysis of gelatin, starch and Tween 80 (Lelliot & Stead, 1987) and specific focus on strains belonging to the highly diverse tests for the utilization of carbohydrates (Hayward, 1995) with a minor phylotype IV (Table 1). Published data for a number of modification by using microtitre plates (French et al., 1995), arginine additional strains of phylotypes I, II, III and IV of R. dihydrolase, lysine decarboxylase, ornithine decarboxylase and the production of phenylalanine deaminases described by Møller (1955) in solanacearum were also used in our comparative analyses Collin et al. (1989), DNase (Collin et al., 1989), urease production (Anzai et al., 2000; Castillo & Greenberg, 2007; Fegan & (Lelliot & Stead, 1987), citrate utilization as described by Simmons Prior, 2006; Hayward, 1964; He et al., 1983; Horita & (1926) in Collin et al. (1989) and malonate utilization (Collin et al., Tsuchiya, 1999, 2000; Horita et al., 2005; Ivey et al., 2007; 1989). Bacterial motility was observed by growth in semi-solid motility Lebeau et al., 2011; Pastrik et al., 2002; Poussier et al., medium (SMM) as described by Kelman & Hruschka (1973). Growth 2000a, b; Prior & Steva, 1990; Roberts et al., 1990; was observed on MacConkey agar (Bridson, 1998; Nash & Krenz, 1991). All tests were conducted at 28 uC unless otherwise stated. Salanoubat et al., 2002; Taghavi et al., 1996; Villa et al., Tolerance of NaCl was observed on CPG (for R. solanacearum and BDB 2005; Wicker et al., 2007, 2012). The results obtained form strains) and CA agar medium (for R. syzygii strains) containing 0, 3 or the basis for a revision of the taxonomy of the R. 5 % NaCl. Additionally, phenotypic data for 21 strains of R. solanacearum species complex and also clearly identify solanacearum representing phylotypes I, II, III and IV (Hayward, genotypic and phenotypic characteristics that differentiate 1964; He et al., 1983; Horita & Tsuchiya, 1999; Horita et al., 2005; Prior the taxa contained in this species complex. & Steva, 1990; Roberts et al., 1990) were included in the comparisons carried out in this study (Table 2). For metabolic phenotypic fingerprinting, Biolog GN2 MicroPlate 96- METHODS well assays were performed according to the manufacturer’s instruc- tions. Results from phenotypic characterization were analysed with the Bacterial strains and growth conditions. The bacterial strains E-Workbench (InforBIO) program (Sugawara et al., 2003) using the used in this study are listed in Table 1. Additionally, published Dice, Euclid, simple matching and Jaccard coefficient algorithms. phenotypic data for 21 strains of R. solanacearum belonging to Dendrograms were constructed using UPGMA clustering (not shown). phylotypes I, II, III and IV (Hayward, 1964; He et al., 1983; Horita & Tsuchiya, 1999; Horita et al., 2005; Prior & Steva, 1990; Roberts et al., Determination of whole-cell fatty acid composition. The whole- 1990) were included and compared with the results obtained in our cell fatty acid composition was determined for 14 selected strains of study (Table 2). Phylogenetic analyses of 16S-23S rRNA ITS gene, ITS the R. solanacearum species complex and the type strain of Ralstonia http://ijs.sgmjournals.org 3089 I. Safni and others

Table 1. Bacterial strains investigated in this study

Taxa are named according to the names proposed in this study, with former names and phylotypes in brackets. 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; D.I.Y., Daerah Istimewa Yogyakarta, Indonesia; 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, Auckland, New Zealand; JT and JS, Laboratory of Phytopathology, CIRAD-FLHOR, La Re´union, 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 the Research Institute of Food Crops Biotechnology (RIFCB), Indonesia; R, Rothamsted Experimental Station, Institute of Arable Crops, Harpenden, UK; R-, Research Collection, Laboratory of Microbiology, Ghent University, Belgium; UQRS, University of Queensland Ralstonia solanacearum collection, University of Queensland, Brisbane, Australia; RUN, collection at CIRAD-FLHOR, La Re´union, France; T, Faculty of Agriculture, Gadjah Mada University, Indonesia.

Strain Other strain name(s) Area of origin Host/source

Ralstonia mannitolilytica LMG 6866T*d NCIMB 10805T London, UK Human blood Ralstonia solanacearum species complex Ralstonia solanacearum [R. solanacearum (phylotype II)] UQRS 426T LMG 2299T*d; K60T; NCPPB 325T USA Tomato UQRS 648 NCPPB 1331 India Potato UQRS 647 Molk2§ Philippines Banana UQRS 652d IPO1609§ The Netherlands Potato Ralstonia pseudosolanacearum sp. nov. [R. solanacearum (phylotype I)] GMI1000§ UQRS 442*d; JS 753 French Guyana Tomato NCPPB 1123 UQRS 649 Papua New Guinea Potato NCPPB 253 UQRS 650*D; LMG 2297 Mauritius Casuarina equisetifolia Ralstonia pseudosolanacearum sp. nov. [R. solanacearum (phylotype III)] UQRS 84 NCPPB 342 Zimbabwe Tobacco UQRS 460d NCPPB 216 Zimbabwe Potato NCPPB 1018 2 Angola UQRS 461T*D NCPPB 1029T; LMG 9673T Re´union, France Pelargonium capitatum UQRS 651d CMR15*§; RUN 133; CFBP 6941 Cameroon Tomato Ralstonia syzygii subsp. syzygii subsp. nov. [R. syzygii (phylotype IV)] LMG 10661T*Dd R 001T; DSM 7385T; ATCC 49543T West Sumatra, Indonesia Clove R 002 R 168; T 330; UQRS 530; NCPPB 3445 Indonesia Clove R 106 R 167; T 247; UQRS 523 Indonesia Clove R 165 T 325; UQRS 529 Indonesia Clove R 166 2 Indonesia Clove Ralstonia syzygii subsp. indonesiensis subsp. nov. [R. solanacearum (phylotype IV)] UQRS 85 MAFF 301558; JS 934 Japan Potato UQRS 92 T 6 West Java, Indonesia Tomato UQRS 95 T 9; R-46895 West Java, Indonesia Tomato UQRS 96 T 10 West Java, Indonesia Tomato UQRS 99 T 13 West Java, Indonesia Tomato UQRS 262 R 792 Indonesia Chilli pepper UQRS 264 T 19 West Java, Indonesia Tomato UQRS 265 T 20 West Java, Indonesia Tomato UQRS 267 T 22 West Java, Indonesia Tomato UQRS 268 T 23 West Java, Indonesia Tomato UQRS 271*Dd T 26; R-46896 West Java, Indonesia Tomato UQRS 272 T 27 West Java, Indonesia Tomato

3090 International Journal of Systematic and Evolutionary Microbiology 64 Taxonomic revision of the R. solanacearum species complex

Table 1. cont.

Strain Other strain name(s) Area of origin Host/source

UQRS 274 T 29 West Java, Indonesia Tomato UQRS 280 T 35 West Java, Indonesia Tomato UQRS 281 T 36 West Java, Indonesia Tomato UQRS 290 T 45 West Java, Indonesia Tomato UQRS 291 T 46; R-46897 West Java, Indonesia Tomato UQRS 463 PSI 36; R-46899 Indonesia Tomato UQRS 464T*Dd PSI 07T§; R-46900T; LMG 27703T; DSM Indonesia Tomato 27478T UQRS 518 NCPPB 3219; ICMP 9915 Indonesia Clove UQRS 524 R 142; R-46901 Indonesia Clove UQRS 532 R 220; R 045 West Sumatra, Indonesia Clove UQRS 533*Dd R 221; R 456; R 768; R-46902; LMG 27704; West Sumatra, Indonesia Clove DSM 27479 UQRS 548 R 780; R-46903 West Java, Indonesia Clove UQRS 549*Dd R 784; R-46904; LMG 27705; DSM 27480 West Java, Indonesia Potato UQRS 550 R 792 West Java, Indonesia Chilli pepper Ralstonia syzygii subsp. celebesensis subsp. nov. [BDB (phylotype IV)] UQRS 465 R 230; JT 657 West Java, Indonesia Banana UQRS 479 SSBD1 Central Java, Indonesia Banana UQRS 480*Dd R-46906; SSBD2 Bali, Indonesia Banana UQRS 481 SSB3; Y1 Indonesia Banana UQRS 519 ICMP 10000; R 230 Indonesia Banana UQRS 520 R 229§ ICMP 10001; T 389; LMG 27886* Indonesia Banana UQRS 534 R 223; T 439 North Sulawesi, Indonesia Banana UQRS 535 R 224; T 386 South Sulawesi, Indonesia Banana UQRS 536*Dd R-46907; R 225; T 380; T 412 South Sulawesi, Indonesia Banana UQRS 538 R 227; T 394; T 383 South Sulawesi, Indonesia Banana UQRS 539 R 228; T 381 South Sulawesi, Indonesia Banana UQRS 542 R 231; T 336 West Java, Indonesia Banana UQRS 543 R 233; T 379 South Sulawesi, Indonesia Banana UQRS 544 R 234; T 391 Indonesia Banana UQRS 546 R 506; T 340 West Java, Indonesia Banana UQRS 621 2 D.I.Y, Indonesia Banana UQRS 622 3A D.I.Y, Indonesia Banana UQRS 624 6 Central Java, Indonesia Banana UQRS 625 11A D.I.Y, Indonesia Banana UQRS 627T*Dd R-46908T; LMG 27706T; DSM 27477T;17T Central Java, Indonesia Banana UQRS 631 22A South Sulawesi, Indonesia Banana UQRS 633 2 North Sulawesi, Indonesia Banana UQRS 635 29A West Sumatra, Indonesia Banana UQRS 637*Dd R-46909; LMG 27707; DSM 27481; 31A West Sumatra, Indonesia Banana BB 2 Bali, Indonesia Banana

*Strain investigated in this study through DNA–DNA hybridization. DStrain for which the DNA G+C content was determined in this study. dStrain for which the fatty acid composition was determined in this study. §Strain for which a whole-genome sequence was determined by Remenant et al. (2010, 2011).

mannitolilytica (Table 1) using an Agilent Technologies 6890N gas profiles were identified using the TSBA50 identification library chromatograph. Cultivation of the strains and extraction and version 5.0. analysis of the fatty acid methyl esters were performed according to the recommendations of the Microbial Identification System, Determination of DNA base composition and DNA–DNA Sherlock version 3.10 (MIDI). Fatty acids were extracted from cells hybridization. High-quality DNA was isolated using the method of harvested from cultures grown for 48 h at 28 uC under aerobic Wilson (1987) with minor modifications (Cleenwerck et al., 2002). conditions on CA medium (Roberts et al., 1990). The peaks of the The DNA base composition was determined using HPLC (Mesbah http://ijs.sgmjournals.org 3091 I. Safni and others

Table 2. Strains for which previously published phenotypic data were included in our analyses

Strain Other strain name(s) Area of origin Host/source Reference

R. pseudosolanacearum [R. solanacearum (phylotype I)] NCPPB 253 UQRS 650; LMG 2297 Mauritius Casuarina equisetifolia Hayward (1964) NCPPB 1052 2 Malaysia Ginger Hayward (1964) GMI1000 UQRS 442; JS 753 French Guyana Tomato Prior & Steva (1990) S236 2 Nambour, Australia Tomato Prior & Steva (1990) M4 2 Guangdong, China Mulberry He et al. (1983) MAFF 211266 2 Hiroshima, Japan Tomato Horita & Tsuchiya (1999) R. solanacearum [R. solanacearum (phylotype II)] NCPPB 1331 UQRS 648 India Potato Hayward (1964) NCPPB 1487 2 Sri Lanka Potato Hayward (1964) K60T UQRS 426T; LMG 2299T USA Tomato Prior & Steva (1990) K136 2 Trinidad Tomato Prior & Steva (1990) K105 2 Florida, USA Tobacco Prior & Steva (1990) S225 2 Peru Tomato Prior & Steva (1990) S247 2 Colombia Tobacco Prior & Steva (1990) R. pseudosolanacearum [R. solanacearum (phylotype III)] NCPPB 216 UQRS 460 Zimbabwe Potato Hayward (1964) NCPPB 1029T UQS 461T; LMG 9673T Re´union, France Pelargonium capitatum Hayward (1964) NCPPB 1486 2 Uganda Groundnut Hayward (1964) R. syzygii subsp. syzygii [R. syzygii (phylotype IV)] R24 2 Indonesia Clove Roberts et al. (1990) R. syzygii subsp. indonesiensis [R. solanacearum (phylotype IV)] MAFF 211271 2 Shizuoka, Japan Potato Horita et al. (2005) MAFF 301559 2 Nagasaki, Japan Potato Horita et al. (2005) WP20 2 Luzon, Philippines Potato Horita et al. (2005) 28MF 2 Mindanao, Potato Horita et al. (2005) Philippines

et al., 1989). DNA–DNA hybridizations were performed at 51 uC using the maximum-likelihood, minimum-evolution and UPGMA using a modified version (Cleenwerck et al., 2002; Goris et al., 1998) algorithms (not shown) were similar to those generated using the of the microplate method developed by Ezaki et al. (1989). Reciprocal neighbour-joining algorithm. reactions (A6B and B6A) were performed for each DNA pair from all strains and the variation was generally within the limits for this method (Goris et al., 1998). RESULTS AND DISCUSSION Phylogenetic analysis of 16S-23S rRNA ITS gene, 16S–23S The R. solanacearum species complex has long been known to rRNA gene ITS region and partial endoglucanase (egl) gene be a heterogeneous collection of strains that share a high degree sequences. PCR amplification of 16S-23S rRNA ITS genes, the of 16S-23S rRNA ITS gene sequence similarity (98–100 %) 16S–23S rRNA ITS region and partial egl genes was performed as (Taghavi et al., 1996), but may exhibit DNA–DNA relatedness summarized in Table S4 using a PTC-100 programmable Thermal Controller (MJ Research). PCR products were purified using a of less than 70 % (De Vos, 1980; Palleroni & Doudoroff, 1971). Qiaquick PCR Purification kit (Qiagen). DNA sequencing was In this study, we used a large set of strains collected in various carried out at the Australian Genome Research Facility, University of parts of the world (Table 1), as well as published sequence Queensland, St Lucia, Australia. DNA sequence data were assembled data including genome data (Tables S1 and S5), with the aim using Chromas Pro version 1.5 (Technelysium Pty Ltd) and aligned of improving the taxonomy of the R. solanacearum species using CLUSTAL W (Larkin et al., 2007; Thompson et al., 1994). complex through a polyphasic taxonomic approach. Phylogenetic trees were reconstructed based on the neighbour-joining All strains investigated in this study (Table 1) were initially (Saitou & Nei, 1987), maximum-likelihood, minimum-evolution and UPGMA (Kidd & Sgaramella-Zonta, 1971; Rzhetsky & Nei, 1993) tested using an R. solanacearum phylotype-specific multi- methods as embedded in the MEGA software version 5.05 (Tamura plex PCR (data not shown) and a BDB-specific PCR. These et al., 2011). Bootstrap analysis was used with 1000 replicates to test tests confirmed their previous classification (Fig. S1) and the statistical reliability of the phylogenetic trees. Trees generated also validated the specificity of the PCR primers (121F/

3092 International Journal of Systematic and Evolutionary Microbiology 64 Taxonomic revision of the R. solanacearum species complex

121R) (Table S3) that were developed for the identification distinct clades, two of which were made up exclusively of of BDB (Tan, 2003). sequences from bacteria isolated from clove trees. Within this sequence cluster, the majority of strains of R. syzygii The phylogenetic relationships of strains of the R. solana- formed a consistent cluster, with only two strains (strain R cearum species complex (Tables 1 and S5) were determined 24, for which a genome sequence is available, and strain R based on their 16S-23S rRNA ITS gene, 16S–23S rRNA ITS 28, obtained from the NCBI database) grouping with the region and partial egl gene sequences. The egl gene encodes three R. solanacearum phylotype IV strains isolated from an endoglucanase that has been implicated in virulence of clove (Fig. S3). Three Japanese strains of R. solanacearum strains of R. solanacearum (Fegan & Prior, 2005; Poussier phylotype IV isolated from potato were also part of this et al., 2000a; Prior & Fegan, 2005; Saile et al., 1997; Villa second sequence cluster, but formed a distinct clade together et al., 2005). with a single Indonesian strain of R. solanacearum phylotype The overall mean 16S-23S rRNA ITS gene sequence similarity IV isolated from tomato (Fig. S3). Phylotype I, II and III of the investigated R. solanacearum species complex strains strains formed individual clusters in the egl phylogenetic was 91.6 %, with minimum and maximum values of 81.0 and tree, with phylotype II and III strains appearing as closely 100 %. Within each of the four phylotypes, the mean 16S-23S related groups. Neither geographical nor host origin rRNA ITS gene sequence similarity was higher than 90.9 %. correlated with distribution of strains in these clusters with The sequence of the type strain of R. solanacearum, UQRS the exception of the group of phylotype IV strains isolated 426T (phylotype II), exhibited means of 87.1, 93.8 and 89.4 % from clove trees (cluster 2, above). sequence similarity to sequences of members of phylotypes I, The phylogenetic relationships within and between the four III and IV, respectively. Sequence similarities between R. phylotypes (Fegan & Prior, 2006; Prior & Fegan, 2005) solanacearum species complex strains and type strains of the have been investigated previously by various researchers other species of the genus Ralstonia, such as the closely using analysis of the sequence similarities of different related R. mannitolilytica, were below 77.0 %. genes, including 16S-23S rRNA ITS, egl, hrpB, gdhA, adk, The overall ITS sequence similarity among the strains of gyrB, gapA, ppsA and fliC, and the 16S–23S ITS region the R. solanacearum complex was 82.0 %, with a range of (Poussier et al., 2000a, b; Taghavi et al., 1996; Villa et al., 70–100 %, while the mean egl sequence similarity among 2005; Wicker et al., 2012). The 16S-23S rRNA ITS gene strains of the R. solanacearum species complex was 57.8 %, sequences of R. solanacearum and its close relatives have with a range of 18–100 %. been shown to be almost indistinguishable (Fegan et al., 1998), and that is also apparent in the data presented here. Neighbour-joining evolutionary distance analyses of 16S- However, the use of more specific genes, such as egl, hrpB, 23S rRNA ITS gene sequences (1342 bp) (Fig. S2), 16S–23S gdhA, adk, gyrB, gapA, ppsA and fliC, and the ITS region rRNA ITS region sequences (520 nt) (Fig. 1) and partial egl showed the separation of the R. solanacearum species gene sequences (703 nt) (Fig. S3) showed that members of complex into several groups that correspond to the current the R. solanacearum species complex form a coherent phylotyping division. Our study confirmed that the group, within which phylotypes I, II, III and IV appear as sequences of the 16S-23S rRNA ITS gene, the ITS region individual clusters. Additionally, within phylotype IV, and the egl gene formed four separate clusters correspond- strains of BDB and R. syzygii each formed coherent, ing to the four phylotypes (Fegan & Prior, 2005; Prior & individual groups based on the sequence of the 16S–23S Fegan, 2005), as shown in Figs 1, S2 and S3. rRNA ITS region (Fig. 1), while the remaining phylotype IV strains formed two groups. The only exception to the DNA–DNA hybridization experiments were performed described grouping was the sequence of R. syzygii strain R with selected strains from the R. solanacearum species 24, which grouped with R. solanacearum phylotype IV complex, representing the different phylogenetic groups, strains UQRS 533, UQRS 532 and UQRS 518, all of which and with the type strain of the closest related species, R. were isolated from clove trees. However, short branch mannitolilytica, with which DNA–DNA relatedness values lengths with low bootstrap support did not allow for any below 37 % were obtained (Table 3). Within the R. solana- definite inference of phylogenetic relationships within cearum species complex, the strains belonging to phylo- phylotype IV based on the ITS sequences. Phylotype I types I, III and IV showed DNA–DNA relatedness values ranging from 51 to 60 % with the type strain of R. and III strains appeared as closely related groups in the T ITS-based tree, which is consistent with MLSA analyses of solanacearum (LMG 2299 ), which indicates that these strains should not be classified as R. solanacearum (Table their phylogenetic relationships (Wicker et al., 2012) and 3). Among the phylotype IV strains, DNA–DNA related- comparative genome analyses (Remenant et al., 2010). ness values ranging from 67 to 100 % were found, indi- In the egl-based phylogenetic tree, sequences of phylotype cating that they should be classified within a single IV strains formed two separate clusters (Fig. S3). The first genomic species (Table 3). High DNA–DNA relatedness cluster contained all sequences from BDB strains and values were found between the BDB strains (88–100 %), sequences of the strains of R. solanacearum phylotype IV. and also between the phylotype IV R. solanacearum strains The second cluster was made up of the sequences of (88–100 %). The DNA–DNA relatedness between BDB and the remaining phylotype IV strains and contained three R. solanacearum phylotype IV strains ranged from 70 to http://ijs.sgmjournals.org 3093 I. Safni and others

Ralstonia syzygii subsp. celebesensis UQRS 631 (KC757026) Ralstonia syzygii subsp. celebesensis UQRS 633 (KC757027) Ralstonia syzygii subsp. celebesensis UQRS 627T (KC757025) Ralstonia syzygii subsp. celebesensis UQRS 544 (KC757019) subsp. UQRS 536 (KC757014) 65 Ralstonia syzygii celebesensis Ralstonia syzygii subsp. celebesensis UQRS 535 (KC757013) Ralstonia syzygii subsp. celebesensis UQRS 520; R229** (KC757011) 0.005 Ralstonia syzygii subsp. celebesensis UQRS 480 (KC757008) Ralstonia syzygii subsp. celebesensis UQRS 479 (KC757007) Ralstonia syzygii subsp. celebesensis UQRS 465 (KC757006) 63 Ralstonia syzygii subsp. celebesensis UQRS 637 (KC757029) Ralstonia syzygii subsp. indonesiensis UQRS 463 (KC756999) Ralstonia syzygii subsp. indonesiensis UQRS 92 (KC756983) Ralstonia syzygii subsp. indonesiensis UQRS 265 (KC756989) Ralstonia syzygii subsp. indonesiensis UQRS 271 (KC756992) Ralstonia syzygii subsp. indonesiensis UQRS 280 (KC756995) Phylotype IV Ralstonia syzygii subsp. indonesiensis UQRS 290 (KC756997) subsp. UQRS 464T; PSI 07T** (KC757000) 99 Ralstonia syzygii indonesiensis Ralstonia syzygii subsp. indonesiensis UQRS 524; R142 (MF) Ralstonia syzygii subsp. indonesiensis UQRS 549 (KC757004) Ralstonia syzygii subsp. indonesiensis UQRS 550 (KC757103) subsp. R 165 (KC756980) 42 Ralstonia syzygii syzygii Ralstonia syzygii subsp. syzygii R 166 (KC956981) 63 Ralstonia syzygii subsp. syzygii R 002 (KC756978) Ralstonia syzygii subsp. syzygii R 001T; LMG 10661T (KC756977) 82 Ralstonia syzygii subsp. syzygii R 106 (KC756979) 55 Ralstonia syzygii subsp. indonesiensis UQRS 85; MAFF 301558 (KC756982) Ralstonia syzygii subsp. indonesiensis UQRS 95 (KC756984) 80 subsp. UQRS 518 (KC757001) 26 Ralstonia syzygii indonesiensis Ralstonia syzygii subsp. indonesiensis UQRS 532 (KC757002) 64 Ralstonia syzygii subsp. indonesiensis UQRS 533; R221 (MF) Ralstonia syzygii subsp. syzygii R24** (FR854086.1) Ralstonia pseudosolanacearum NCPPB505 (MF) Ralstonia pseudosolanacearum NCPPB 283 (MF) Ralstonia pseudosolanacearum NCPPB 332 (MF) Ralstonia pseudosolanacearum NCPPB 342 (KC756971) Phylotype III NCPPB 1018 (MF) 99 Ralstonia pseudosolanacearum Ralstonia pseudosolanacearum NCPPB 1029T; UQRS 461T (KC756973) Ralstonia pseudosolanacearum UQRS 651; CMR15** (KC756976) CIP 365 (MF) 51 94 Ralstonia pseudosolanacearum Ralstonia pseudosolanacearum ACH 092 (MF) 98 Ralstonia pseudosolanacearum UQRS 649 (KC756974) Ralstonia pseudosolanacearum FQY-4** (AGH84716.1) 73 Phylotype I Ralstonia pseudosolanacearum UQRS 442; GMI1000** (AL646052) Ralstonia pseudosolanacearum UQRS 650 (KC756975) Ralstonia pseudosolanacearum R 288 (AJ277775) Ralstonia pseudosolanacearum Y45** (AFWL00000000) Ralstonia solanacearum CFBP 2972 (MF) 89 Ralstonia solanacearum CFBP 715 (MF) Ralstonia solanacearum CFBP 2957** (FP885897.1) Ralstonia solanacearum Po82** (CP002819/20) Ralstonia solanacearum R133 (AJ277848) 99 37 Ralstonia solanacearum LMG 2299T**(KC756967) Ralstonia solanacearum UQRS 647; Molk2** (KC756968) R 283; NCPPB 4025 (AJ277777) Ralstonia solanacearum Phylotype II R 651 (AJ277768) 55 Ralstonia solanacearum Ralstonia solanacearum UQRS 648 (KC756969) Ralstonia solanacearum UQRS 652; IPO1609** (KC756970) 72 Ralstonia solanacearum UQRS 619; UW 551** (AKL01000061) Ralstonia solanacearum CIP 335 (AJ277767) Ralstonia solanacearum CIP 238 (AJ277854) Ralstonia solanacearum CIP 10 (MF) Ralstonia solanacearum NCPPB 2505 (AJ277853) Ralstonia pickettii ATCC 27511T (L28163)

3094 International Journal of Systematic and Evolutionary Microbiology 64 Taxonomic revision of the R. solanacearum species complex

Fig. 1. Neighbour-joining tree of 16S–23S rRNA ITS region sequences reflecting the phylogenetic relationships of members of the R. solanacearum species complex. Ralstonia pickettii ATCC 27511T was used as an outgroup. Proposed type strains are highlighted in bold. Bar, 0.5 % sequence dissimilarity. Numbers at branching points indicate bootstrap percentages derived from 1000 samples. **, Genome-sequenced strain.

92 %, whereas values around 70 % were obtained for each Using a selected range of phenotypic tests based on data of these groups compared to the type strain of R. syzygii. from this study (Tables S6–S8) and from six published Strains of phylotypes I and III showed DNA–DNA relat- papers (Hayward, 1964; He et al., 1983; Horita & Tsuchiya, edness below 70 % with the type strains of R. syzygii (53– 1999; Horita et al., 2005; Prior & Steva, 1990; Roberts et al., 58 %), R. solanacearum (phylotype II) (52–58 %) and R. 1990) (Table S9), however, some phenotypic features were mannitolilytica (31–32 %), and exhibited values between 72 identified that are useful in distinguishing the different taxa and 90 % among each other, indicating that they represent a within the R. solanacearum species complex (Table 4). single genospecies within the genus Ralstonia (Table 3). Strains of phylotypes I and III can be differentiated from all These findings match the data from a recent MLSA study phylotype II strains and the strains of phylotype IV, R. syzygii (Wicker et al., 2012) and are also consistent with the ANI and BDB by their ability to utilize trehalose (Table 4). The values reported for comparisons between the single sequenced strains of phylotype IV of R. solanacearum were able to genomes of the phylotype IV subgroups (Remenant et al., utilize acetic acid, D-glucosaminic acid, D-glucuronic acid, 2011). The high DNA–DNA relatedness between phylotype I p-hydroxyphenylacetic acid, propionic acid, D-saccharic and III strains is also in agreement with an earlier DNA–DNA acid and c-aminobutyric acid, while strains of both BDB hybridization study by Palleroni & Doudoroff (1971) in and R. syzygii were negative for these traits. Similarly, acid which strains belonging to biotypes 3 and 4 (currently was produced from lactose and maltose by strains of assigned to phylotype I) showed 79–100 % DNA–DNA phylotype IV of R. solanacearum, while strains of phylotypes relatedness with some strains belonging to former biotype 1 I and III, as well as BDB and R. syzygii, were negative for that are currently assigned to phylotype III. these features. The inability to utilize Tween 40, DL-lactic acid and L-histidine and the inability to grow on CPG Biolog GN2 metabolic fingerprinting assays (Table S6) and medium clearly differentiated R. syzygii from strains of both more than 40 classical phenotypic tests (Table S7) were R. solanacearum and BDB, which were able to utilize these carried out on the strains listed in Table 1. Overall, strains substrates and grew well on CPG medium. An interesting of R. solanacearum assigned to any of the four phylotypes observation in terms of diagnostic typing of phylotype IV were more metabolically versatile in the utilization of strains was that BDB can be distinguished from the other carbon substrates than strains of BDB or R. syzygii (Table phylotype IV strains by their ability to hydrolyse starch, for S6). Within phylotype IV, strains of R. solanacearum were which strains of both R. solanacearum and R. syzygii showed able to utilize 50 substrates on average, followed by BDB negative results. (mean number of 28 substrates) and R. syzygii strains (mean number of 10 substrates) (Table S6). Mean numbers The cellular fatty acid compositions of strains of the R. of 49, 47 and 53 substrates were utilized by strains of solanacearum species complex and R. mannitolilytica were also analysed, and the results are shown in Table 5. All phylotypes I, II and III, respectively. For each phylotype, a T number of core substrates could be identified that could strains, including R. mannitolilytica LMG 6866 , contained be utilized by all or the majority of strains tested. For the fatty acids C14 : 0,C16 : 0,C18 : 1v7c,C18 : 1 2-OH and phylotypes I, II and III, strains could utilize core sets of 34, C17 : 0 cyclo, as well as summed features 2 (iso-C16 : 1 I/C14 : 0 31 and 36 substrates, respectively (Table S8). Further, 3-OH) and 3 (C16 : 1v7c/iso-C15 : 0 2-OH) (Table 5). C16 : 0, within phylotype IV, strains of R. solanacearum were able C18 : 1v7c and summed features 2 and 3 were the most to utilize a core set of 34 substrates (21 of these used by all predominant fatty acid components detected in all strains strains, 13 used by at least 90 % of the strains), whereas (Table 5). The fatty acid profiles of strains of phylotypes I, BDB strains utilized 18 core substrates (11 used by all II, III and IV of R. solanacearum were similar overall, strains, seven used by at least 90 % of all the strains) and R. although a few differences were observed in the relative syzygii strains were able to utilize only six core substrates concentrations of individual components, some of which (all of these were used by at least 80 % of the strains tested) may be of diagnostic value (Table 5). (Table S8). These data clearly document the varying Based on a combination of genotypic and phenotypic data abilities of strains of R. solanacearum to utilize carbon of a large set of strains of the R. solanacearum species sources of varying types. In classical phenotypic tests, complex obtained in this study and on additional analyses high similarity was found among the strains of the R. of genome sequences, comparative genome hybridization solanacearum species complex (Table S7), a well-known arrays and MLSA data (Cao et al., 2013; Gabriel et al., 2006; feature of these bacteria and the origin of the difficulties Guidot et al., 2009; Li et al., 2011; Remenant et al., 2010, in classifying the various representatives of this species 2011; Salanoubat et al., 2002; Wicker et al., 2012; Xu et al., complex (Fegan & Prior, 2005; Harris, 1971; Palleroni & 2011), the following taxonomic proposals are made. The Doudoroff, 1971). species Ralstonia solanacearum is limited to strains of http://ijs.sgmjournals.org 3095 3096 others and Safni I.

Table 3. DNA–DNA relatedness (%) among strains of the R. solanacearum species complex

Data were obtained in this study unless indicated otherwise. Values in parentheses give the difference between reciprocal hybridisations. ND, Not determined.

Strain DNA G+C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 content (mol%)

R. pseudosolanacearum [R. solanacearum (phylotype I)] 1. UQRS 650 66.5 100 2. UQRS 442 ND 90 (2) 100 R. pseudosolanacearum [R. solanacearum (phylotype III)] 3. UQRS 461T 66.1 73 (1) 72 (5) 100 4. CMR15 ND 76 (0) 75 (5) 78 (2) 100 R. syzygii subsp. indonesiensis [R. solanacearum (phylotype IV)] nentoa ora fSseai n vltoayMicrobiology Evolutionary and Systematic of Journal International 5. UQRS 271 65.2 ND ND ND ND 100 T 6. UQRS 464 65.5 61 (15) 62 (24) 60 (12) 60 (10) ND 100 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 R. syzygii subsp. celebesensis [BDB (phylotype IV)] 9. UQRS 480 66.0 ND ND ND ND 83 (.30) ND 83 (0) 78 (13) 100 10. UQRS 536 66.0 ND ND ND ND 92 (24) ND ND ND 88 (17) 100 T 11. UQRS 627 65.8 59 (9) 57 (5) 55 (1) 52 (2) 88 (8) ND ND ND 100 (14) 96 (20) 100 12. LMG 27886 63 (9) 64 (17) 59 (8) 56 (3) ND ND ND ND ND ND 100 (2) 100 13. UQRS 637 65.9 ND ND ND ND 73 (.30) ND 70 (30) 77 (25) 97 (17) 89 (28) ND ND 100 R. syzygii subsp. syzygii [R. syzygii (phylotype IV)] T 14. LMG 10661 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 R. solanacearum [R. solanacearum (phylotype II)] T a 15. LMG 2299 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 T b 16. R. mannitolilytica LMG 6866 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) 100

*Data taken from: a, Yabuuchi et al. (1995); b, De Baere et al. (2001). 64 http://ijs.sgmjournals.org

Table 4. Phenotypic characteristics that differentiate the taxa within the R. solanacearum species complex

Taxa are named according to the proposals of the current study, with their former names given below. Numbers of strains examined are given as number of strains examined in the present study (Table 1) + number of strains for which results were obtained from earlier publications (Table 2). +, 51–100 % of strains reacted positive; 2, 10–50 % of strains reacted negative. Shaded symbols show distinguishing characteristics for a particular taxon. Data that were obtained in this study and in other publications are given in brackets; other data were obtained in this study only.

Characteristic R. solanacearum R. pseudosolanacearum R. syzygii subsp. R. syzygii subsp. R. syzygii subsp. syzygii indonesiensis celebesensis

R. solanacearum R. solanacearum R. solanacearum R. syzygii R. solanacearum BDB (phylotype IV) (phylotype II) (phylotype I) (phylotype III) (phylotype IV) (phylotype IV) (n525) (n54+7) (n53+6) (n55+3) (n55+1) (n526+4)

Growth on CPG medium +++2 ++ Starch hydrolysis 2 [2] 2 [2] 2 + Acid production from: Lactose [+][2][2][2] + 2 Maltose [+][2][2][2] + 2 Fructose [+][+][+] 2 + 2 Utilization of Biolog GN2 aooi eiino the of revision Taxonomic MicroPlate substrates Tween 40 +++2 ++ DTrehalose 2 ++ 2 + 2 Acetic acid +++2 + 2 D-Glucosaminic acid 2222 + 2 D-Glucuronic acid +++2 + 2 p-Hydroxyphenylacetic acid 2 + 22 + 2 DL-Lactic acid +++2 ++

Propionic acid + 2 + 2 + 2 solanacearum R. D-Saccharic acid +++2 + 2 L-Histidine +++2 ++ c-Aminobutyric acid +++2 + 2 pce complex species 3097 3098 others and Safni I.

Table 5. Cellular fatty acid compositions of representative strains of taxa within the R. solanacearum species complex

Taxa are named according to the proposals of the current study, with their former names given below. Values are percentages of total fatty acids. For phylotypes of which more than one strain was analysed, values are means (SD). ND, Not detected or ,2%.

Fatty acid R. solanacearum R. pseudosolanacearum R. syzygii subsp. R. syzygii subsp. R. syzygii subsp. R. mannitolilytica syzygii indonesiensis celebesensis LMG 6866T

R. solanacearum R. solanacearum R. solanacearum R. syzygii R. solanacearum BDB (phylotype IV) (phylotype II) (phylotype I) (phylotype III) (phylotype IV) (phylotype IV)

LMG 2299T, UQRS 442 UQRS 460, LMG 10661T UQRS 271, UQRS 464T, UQRS 480, UQRS 536, UQRS 652 UQRS 651 UQRS 533, UQRS 549 UQRS 627T, UQRS 637

C14 : 0 4.8 (0.1) 4.6 5.5 (1.9) 2.7 4.4 (0.2) 4.7 (0.2) 4.9 nentoa ora fSseai n vltoayMicrobiology Evolutionary and Systematic of Journal International C16 : 0 19.6 (1.5) 19.7 17 (0.5) 18 21.5 (1.1) 21.4 (1.0) 22.8 C18 : 1 v7c 18.8 (0.2) 19.8 19.3 (1.5) 28.7 19.7 (1.7) 19.6 (4.8) 15.8 C16 : 0 2-OH ND ND ND ND ND ND 4.2 C16 : 1 2-OH 6.3 (2.6) 6.9 9.8 (3.1) ND 3.2 (0.5) 4.3 (1.9) 5.4 C18 : 1 2-OH 6.8 (0.2) 7.3 6.2 (1.1) 5.1 6.6 (0.7) 4.6 (0.5) 4.3 C17 : 0 cyclo 8.7 (2.3) 4.7 4.0 (2.6) 5.7 8.1 (2.9) 3.5 (0.8) 6.1 Summed feature 2* 10 (1.9) 9.7 10 (0.4) 11.9 9.5 (1.6) 15 (4.6) 10.4 Summed feature 3* 21.1 (0.5) 23.6 24.0 (8.4) 23.9 22.4 (4.1) 24.0 (2.1) 19.6

*Summed features are groups of two or three fatty acids that could not be separated with the MIDI System. Summed feature 2 contained iso-C16 : 1 I and/or C14 : 0 3-OH; summed feature 3 contained C16 : 1 v7c and/or iso-C15 : 0 2-OH. 64 Taxonomic revision of the R. solanacearum species complex phylotype II, and BDB and strains of phylotype IV of R. and lysine and ornithine decarboxylases are negative. Acid solanacearum are reclassified as members of novel subspecies is produced oxidatively from glucose, mannose, fructose, of R. syzygii, for which the following names are respectively glycerol, galactose and sucrose. Some strains produce acid proposed: Ralstonia syzygii subsp. celebesensis subsp. nov. from lactose, maltose, cellobiose, dulcitol, mannitol, sorbi- T T T (type strain UQRS 627 5LMG 27706 5DSM 27477 5 tol, inositol, rhamnose, D-arabinose, ethanol, raffinose, R-46908T) and Ralstonia syzygii subsp. indonesiensis subsp. trehalose, xylose and adonitol. In Biolog GN2 MicroPlate nov. (type strain UQRS 464T5LMG 27703T5DSM 27478T5 tests, utilizes dextrin, glycogen, Tweens 40 and 80, D- PSI 07T). The type strain of Ralstonia syzygii subsp. syzygii fructose, a-D-glucose, myo-inositol, sucrose, pyruvic acid (which is created automatically as a consequence) is the methyl ester, succinic acid monomethyl ester, acetic acid, current type strain of Ralstonia syzygii, R 001T. There is a cis-aconitic acid, citric acid, D-galacturonic acid, D-gluconic genome sequence available for strain R 24 that was selected acid, D-glucuronic acid, a-hydroxybutyric acid, b-hydro- by Remenant et al. (2011) as the reference strain for the xybutyric acid, a-ketoglutaric acid, DL-lactic acid, propionic current species R. syzygii. However, this strain has shown acid, quinic acid, D-saccharic acid, sebacic acid, succinic some unusual genotypic properties in both our analyses and acid, bromosuccinic acid, glucuronamide acid, L-alani- those of Wicker et al. (2012) that make its usefulness as a namide, D- and L-alanine, L-asparagine, L-aspartic acid, reference strain of R. syzygii subsp. syzygii doubtful (Fegan & L-glutamic acid, L-histidine, L-ornithine, L-proline, L-pyr- Prior, 2005; Prior & Fegan, 2005). oglutamic acid, D-andL-serine, L-threonine, c-aminobutyric acid and glycerol. The major cellular fatty acids are summed Ralstonia pseudosolanacearum sp. nov. (type strain UQRS feature 3 (C16 : 1v7c/iso-C15 : 0 2-OH), C16 : 0,C18 : 1v7c, 461T5LMG 9673T5NCPPB 1029T) is proposed to incorp- summed feature 2 (iso-C16 : 1 I/C14 : 0 3-OH), C17 : 0 cyclo, orate strains of R. solanacearum belonging to phylotypes I C18 : 1 2-OH, C16 : 1 2-OH and C14 : 0. Strains contain a signature and III, which form a single species based on data from nucleotide sequence, 59-AAGTTATGGACGGTGGAAGTC this study, the comparative genome analyses carried out by (Fegan & Prior, 2006; Prior & Fegan, 2005), in the 16S- Remenant et al. (2010), the MLSA data of Wicker et al. 23S rRNA ITS gene sequence. (2012) and the early studies by Palleroni & Doudoroff T T (1971) and De Vos (1980). The proposed type strain of R. The type strain is UQRS 426 (5LMG 2299 5NCPPB T T + pseudosolanacearum is one of the strains included in the 325 5K60 ). The DNA G C content of the type strain original DNA–DNA hybridization experiments on strains is 66.6 mol% (HPLC method). The GenBank accession of R. solanacearum (De Vos, 1980; Palleroni & Doudoroff, number of the 16S-23S rRNA ITS gene sequence of the 1971). Classification of strains of phylotypes I and III as a type strain is EF016361. single species is warranted based on their phylogenetic as well as overall genomic relatedness (Remenant et al., 2010). Emended description of Ralstonia syzygii We recognize that both R. pseudosolanacearum as well as the (Roberts et al. 1990) Vaneechoutte et al. 2004 true R. solanacearum (phylotype II) encompass a significant number of strains, and future work may well result in the This species includes all phylotype IV strains. The designation of subspecies for one or both species. This could description is based on the data of Roberts et al. (1990), be especially true for R. pseudosolanacearum, where a clear Vaneechoutte et al. (2004) and this study. Cells are Gram- geographical division of strains belonging to phylotypes I negative, non-motile, non-sporulating, non-capsulated, and III exists. Within the strains of the true R. solanacearum non-motile, straight rods with rounded ends, approximately (phylotype II), eight separate groups of strains were 0.5–0.661.0–2.5 mm, occurring singly, in pairs or occasion- identified using MLSA (Wicker et al., 2012), which also ally in short chains. Aerobic growth. Catalase- and oxidase- indicates that multiple subspecies may have to be proposed. positive. Growth is observed on MacConkey agar without However, additional data are needed for such proposals, NaCl, but not on 5 % NaCl. Strains contain a signature 9 which are outside the scope of the current study. nucleotide sequence, 5 -ATTGCCAAGACGAGAGAAGTA (Fegan & Prior, 2006; Prior & Fegan, 2005), in the 16S-23S rRNA ITS gene sequence. The species consists of three Emended description of Ralstonia solanacearum subspecies that can be differentiated on the basis of (Smith 1896) Yabuuchi et al. 1995 phenotypic and pathogenicity characteristics. The description of Yabuuchi et al. (1995) is emended to The type strain is R 001T (5LMG 10661T5DSM 7385T). include only strains belonging to phylotype II (Fegan & The GenBank accession number of the 16S-23S rRNA ITS Prior, 2006; Prior & Fegan, 2005). The description is based gene sequence of the type strain is U28237. on Yabuuchi et al. (1995) and this study. Cells are Gram- negative and rod-shaped and may be motile or non-motile Description of Ralstonia syzygii subsp. syzygii (the type strain is motile). Catalase- and oxidase-positive. (Roberts et al. 1990) subsp. nov. Able to grow at 28 uC on CPG medium. Some strains reduce nitrate to gas as well as reducing nitrate to nitrite. Ralstonia syzygii subsp. syzygii (sy.zy9gi.i. N.L. gen. n. Most strains can utilize citrate, hydrolyse Tween 80 and Syzygium generic name of the clove tree; N.L. gen. n. syzygii produce urease. Tests for activities of arginine dihydrolase of the genus Syzygium). http://ijs.sgmjournals.org 3099 I. Safni and others

Cells are Gram-negative straight rods, approximately The type strain is UQRS 464T (5LMG 27703T5DSM 161.5–3 mm, occurring singly or in pairs at 28 uConCA 27478T5PSI 07T), which was isolated from potato in medium after 5–7 days. Growth is observed on MacConkey Indonesia. Strains have been isolated from tomato, potato, agar without added NaCl. Unable to grow on CPG medium. chilli pepper and clove. The DNA G+C content of the type Most strains (.85 %) utilize D-glucose, succinic acid, L- strain is 65.5 mol% (HPLC method) and 66.3 mol% asparagine, L-aspartic acid, L-glutamic acid and L-proline. (whole genome sequence method; Remenant et al., 2011). Unable to utilize Tween 40, trehalose, DL-lactic acid and L- The GenBank accession number of the 16S-23S rRNA ITS histidine. Other characteristics are given in Tables 3, S6 and gene sequence of the type strain is KC757057. S7. Major cellular fatty acids are C18 : 1v7c, summed feature 3 (C v7c/iso-C 2-OH), C , summed feature 2 (iso- 16 : 1 15 : 0 16 : 0 Description of Ralstonia syzygii subsp. C I/C 3-OH), C cyclo and C 2-OH. 16 : 1 14 : 0 17 : 0 18 : 1 celebesensis subsp. nov. The type strain is R 001T (5LMG 10661T5DSM 7385T), Ralstonia syzygii subsp. celebesensis (ce.le.be.sen9sis. N.L. isolated as a plant pathogen from xylem tissues of the clove fem. adj. celebesensis of or belonging to Celebes). tree (Syzygium aromaticum). Strains have also been isolated from other Syzygium species and from insect vectors The description is based on the data of Eden-Green et al. (Hindola spp.) in Indonesia. The DNA G+C content of (1988) and this study. Cells are Gram-negative rods, the type strain is 65.2 mol% (HPLC method) and 66– 0.862.5 mm, and non-motile. Colonies are typically round, 67 mol% (buoyant density method). The GenBank acces- mucoid, non-fluid and small (0.5–2 mm) after incubation at sion number of the 16S-23S rRNA ITS gene sequence of the 28 uC for 4–5 days on CPG medium. Mucoid colonies (2– type strain is U28237. 3 mm in size) with smooth margins and dark-red centres are produced on tetrazolium chloride medium and non- fluorescent, small, white and mucoid colonies on peptone Description of Ralstonia syzygii subsp. or nutrient agar media. Catalase- and oxidase-positive. indonesiensis subsp. nov. Aerobic. All known strains are unable to reduce nitrate to Ralstonia syzygii subsp. indonesiensis (in.do.ne.si.en9sis. nitrogen gas, but 80 % of strains can reduce nitrate to nitrite. N.L. fem. adj. indonesiensis of or belonging to Indonesia). Eden-Green et al. (1988) reported previously that this pathogen did not hydrolyse starch, but our study showed Cells are Gram-negative, asporogenous rods that maybe that starch is hydrolysed, with the exception of strain UQRS motile or non-motile (the type strain is motile). Colonies 520 (5R 229), for which the genome has been sequenced by vary from fluid form to butyrous consistency with white Remenant et al. (2011), but not aesculin or gelatin. Citrate colour and a diameter of approximately 0.5 mm after 2– and malonate are not utilized. Acid is produced oxidatively 3 days of incubation at 28 uC on CPG medium. Colonies from galactose. Acid production from glucose was recorded are irregular and convex. Aerobic. Growth is observed on as positive by Eden-Green et al. (1988), but our study found MacConkey agar without the addition of NaCl. Catalase- and that acid production is variable (72 % of the strains positive) oxidase-positive. Most strains (.85 %) can reduce nitrate to for this sugar. Testing by means of Biolog GN2 MicroPlates gas and reduce nitrate to nitrite. Citrate is utilized but not indicates that .90 % of strains utilize Tweens 40 and 80, malonate. Most strains hydrolyse Tween 80 and show DNase pyruvic acid methyl ester, succinic acid monomethyl ester, activity. Acid is produced oxidatively from glucose, lactose, a-ketoglutaric acid, DL-lactic acid, quinic acid, succinic acid, maltose, fructose, glycerol, D-arabinose, galactose and bromosuccinic acid, succinamic acid, D- and L-alanine, L- sucrose, but not from dulcitol, mannitol, sorbitol, adonitol, asparagine, L-aspartic acid, L-glutamic acid, L-histidine, salicin, melezitose, melibiose or inulin. Tests for arginine L-proline and L-serine. Trehalose is not utilized. Other dihydrolase, lysine decarboxylase, ornithine decarboxylase characteristics are given in Tables 3, S6 and S7. Major and phenylalanine deaminase are negative. Strains do not cellular fatty acids are summed feature 3 (C v7c/iso C hydrolyse aesculin, gelatin or starch. In Biolog GN2 16 : 1 15 : 0 2-OH), C16 : 0,C18 : 1v7c, summed feature 2 (iso-C16 : 1 MicroPlate assays, Tweens 40 and 80, trehalose, D-fructose, I/C14 : 0 3-OH), C16 : 1 2-OH, C18 : 1 2-OH and C17 : 0 cyclo. D-galactose, a-D-glucose, pyruvic acid methyl ester, succinic acid monomethyl ester, acetic acid, cis-aconitic acid, citric The type strain is UQRS 627T (5LMG 27706T5DSM T T acid, D-galacturonic acid, D-gluconic acid, D-glucuronic acid, 27477 5R-46908 ), which was isolated from banana b-hydroxybutyric acid, p-hydroxyphenylacetic acid, a-keto- plants in central Java, Indonesia. The DNA G+C content glutaric acid, DL-lactic acid, propionic acid, quinic acid, D- is 65.8 mol% (HPLC method). The GenBank accession saccharic acid, sebacic acid, succinic acid, bromosuccinic number of the 16S-23S rRNA ITS gene sequence of the acid, D-andL-alanine, L-asparagine, L-aspartic acid, type strain is KC757073. L-glutamic acid, L-histidine, L-proline, L-pyroglutamic acid, L- serine and c-aminobutyric acid are utilized. Other character- Description of Ralstonia pseudosolanacearum istics are given in Tables 3, S6 and S7. Major cellular fatty sp. nov. acids are summed feature 3 (C16 : 1v7c/iso-C15 : 0 2-OH), C16 : 0,C18 : 1v7c, summed feature 2 (iso-C16 : 1 I/C14 : 0 3-OH), Ralstonia pseudosolanacearum (pseu.do.so.la.na.ce.a9rum. C17 : 0 cyclo and C18 : 1 2-OH. Gr. adj. pseudeˆs false; N.L. fem. pl. gen. n. solanacearum a

3100 International Journal of Systematic and Evolutionary Microbiology 64 Taxonomic revision of the R. solanacearum species complex bacterial specific epithet; N.L. fem. pl. gen. n. pseudosola- Bridson, E. Y. (1998). The Oxoid Manual, 8th edn. Basingstoke, UK: nacearum false solanacearum, referring to Ralstonia sola- Oxoid. nacearum). Cao, Y., Tian, B., Liu, Y., Cai, L., Wang, H., Lu, N., Wang, M., Shang, S., Luo, Z. & Shi, J. (2013). Genome sequencing of Ralstonia This species is limited to strains belonging to phylotypes I solanacearum FQY_4, isolated from a bacterial wilt nursery used for and III. Cells are Gram-negative, non-sporulating rods that breeding crop resistance. Genome Announc 1, e00125-13. may be motile or non-motile (the type strain is non- Castillo, J. A. & Greenberg, J. T. (2007). Evolutionary dynamics of motile). Catalase- and oxidase-positive. Aerobic. Colonies Ralstonia solanacearum. Appl Environ Microbiol 73, 1225–1238. have diameters of less than 1 mm on CPG medium after 1– Cleenwerck, I., Vandemeulebroecke, K., Janssens, D. & Swings, J. 3 days of incubation at 28 uC. Most strains reduce nitrate (2002). Re-examination of the genus Acetobacter, with descriptions of to gas. Unable to hydrolyse starch, aesculin or gelatin. Most Acetobacter cerevisiae sp. nov. and Acetobacter malorum sp. nov. Int J strains utilize citrate. Unable to utilize malonate. Tests for Syst Evol Microbiol 52, 1551–1558. phenylalanine deaminase, DNase, arginine dihydrolase, Collin, C. H., Lyne, P. M. & Grange, J. M. (1989). Microbiological lysine decarboxylase and ornithine decarboxylase activities Methods, 6th edn. London: Butterworth. are negative. Acid is produced oxidatively from glucose, De Baere, T., Steyaert, S., Wauters, G., Des Vos, P., Goris, J., inositol, mannose, fructose, glycerol, galactose, raffinose Coenye, T., Suyama, T., Verschraegen, G. & Vaneechoutte, M. and sucrose. Using Biolog GN2 MicroPlates, .90 % of the (2001). Classification of Ralstonia pickettii biovar 3/‘thomasii’ strains strains utilize Tweens 40 and 80, D-fructose, a-D-glucose, (Pickett 1994) and of new isolates related to nosocomial recurrent myo-inositol, trehalose, pyruvic acid methyl ester, succinic meningitis as Ralstonia mannitolytica sp. nov. Int J Syst Evol Microbiol 51, 547–558. acid monomethyl ester, cis-aconitic acid, citric acid, D- galacturonic acid, D-gluconic acid, D-glucuronic acid, b- De Vos, P. (1980). A new classification of the genus Pseudomonas based hydroxybutyric acid, DL-lactic acid, bromosuccinic acid, on DNA-rRNA hybridisation. PhD thesis, University of Ghent, Ghent, Belgium. glucuronamide, L-alaninamide, D- and L-alanine, L-aspar- agine, L-aspartic acid, L-histidine, L-proline, L-pyroglutamic De Vos, P. & Tru¨ per, H. G. (2000). Judicial Commission of the International Committee on Systematic Bacteriology. IXth Inter- acid and L-serine. All strains are unable to produce acid national (IUMS) Congress of Bacteriology and Applied Microbiology. from L-rhamnose and ethanol. Other characteristics are Minutes of the meetings, 14, 15 and 18 August 1999, Sydney, given in Tables 3, S6 and S7. The following major cellular Australia. Int J Syst Evol Microbiol 50, 2239–2244. fatty acid components are present: summed feature 3 Eden-Green, S. J. (1994). Diversity of Pseudomonas solanacearum and (C16 : 1v7c/iso-C15 : 0 2-OH), C18 : 1v7c,C16 : 0, summed related bacteria in South East Asia: new directions for moko disease. feature 2 (iso-C16 : 1 I/C14 : 0 3-OH), C16 : 1 2-OH, C18 : 1 2- In Bacterial Wilt: the Disease and its Causative Agent, Pseudomonas OH, C14 : 0 and C17 : 0 cyclo. Strains contain signature solanacearum, pp. 25–34. Edited by A. C. Hayward & G. L. Hartman. nucleotide sequences 59-CGTTGATGAGGCGCGCAATTT Wallingford, UK: CAB International. or 59-ATTACSAGAGCAATCGAAAGATT (Fegan & Prior, Eden-Green, S. 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Fluorometric type strain is KC757037. deoxyribonucleic acid-deoxyribonucleic acid hybridization in micro- dilution wells as an alternative to membrane-filter hybridization in which radioisotopes are used to determine genetic relatedness among ACKNOWLEDGEMENTS bacterial strains. Int J Syst Bacteriol 39, 224–229. Fegan, M. & Prior, P. (2005). How complex is the ‘‘Ralstonia This research was supported by the Australian Centre for solanacearum species complex’’. In Bacterial Wilt Disease and the International Agricultural Research (ACIAR), the University of Ralstonia solanacearum Species Complex, pp. 449–461. Edited by Queensland, Australia, and the BCCM/LMG Bacteria Collection. C. Allen, A. C. Hayward & P. Prior. St Paul, MN: American Financial support is acknowledged from AusAID and the Directorate Phytopathological Society. General of Higher Education of Indonesia (DGHE/DIKTI) for scholarships to I. S. The BCCM/LMG Bacteria Collection is supported Fegan, M. & Prior, P. (2006). 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