International Journal of Systematic and Evolutionary Microbiology (2001), 51, 89–103 Printed in Great Britain A revision of Rhizobium Frank 1889, with an emended description of the genus, and the inclusion of all species of Agrobacterium Conn 1942 and Allorhizobium undicola de Lajudie et al. 1998 as new combinations: Rhizobium radiobacter, R. rhizogenes, R. rubi, R. undicola and R. vitis J. M. Young,1 L. D. Kuykendall,2 E. Martı!nez-Romero,3 A. Kerr4 and H. Sawada5 Author for correspondence: L. D. Kuykendall. Tel: j1 301 504 7072. Fax: j1 301 504 5449. e-mail: dkuykend!asrr.arsusda.gov 1 Landcare Research, Private Rhizobium, Agrobacterium and Allorhizobium are genera within the bacterial Bag 92170, Auckland, family Rhizobiaceae, together with Sinorhizobium. The species of New Zealand Agrobacterium, Agrobacterium tumefaciens (syn. Agrobacterium radiobacter), 2 Plant Sciences Institute, Agrobacterium rhizogenes, Agrobacterium rubi and Agrobacterium vitis, Beltsville Agricultural Research Center, USDA- together with Allorhizobium undicola, form a monophyletic group with all ARS, 10300 Baltimore Ave, Rhizobium species, based on comparative 16S rDNA analyses. Agrobacterium is Beltsville, MD 20705, USA an artificial genus comprising plant-pathogenic species. The monophyletic 3 Centro de Investigacio! n nature of Agrobacterium, Allorhizobium and Rhizobium and their common sobre Fijacio! nde phenotypic generic circumscription support their amalgamation into a single Nitro! geno, UNAM. AP 565-A, Cuernavaca, genus, Rhizobium. Agrobacterium tumefaciens was conserved as the type Morelos, Mexico species of Agrobacterium, but the epithet radiobacter would take precedence 4 419 Carrington Street, as Rhizobium radiobacter in the revised genus. The proposed new Adelaide, South Australia combinations are Rhizobium radiobacter, Rhizobium rhizogenes, Rhizobium 5000, Australia rubi, Rhizobium undicola and Rhizobium vitis. 5 National Institute of Agro- Environmental Sciences, 3-1-1 Kannondai, Tsukuba, Ibaraki 305-8604, Keywords: Rhizobiaceae, phenetic, phylogenetic, polyphasic, taxonomy Japan INTRODUCTION revision (Kersters & De Ley, 1984; Willems & Collins, 1993; Sawada et al., 1993b; de Lajudie et al., 1998b). Nitrogen-fixing bacteria that form symbiotic associ- In this paper, the relevant literature is reviewed and a ations with members of the Leguminosae, and related nomenclature is proposed that aims to reflect, as pathogenic bacteria, have been ascribed to the genera closely as possible, the natural polyphasic and phenetic Agrobacterium Conn 1942, Allorhizobium de Lajudie relationships of these taxa. et al. 1998b, Azorhizobium Dreyfus et al. 1988, Brady- rhizobium Jordan 1982, Mesorhizobium Jarvis et al. 1997, Phyllobacterium Kno$ sel 1984, Rhizobium Frank The original species nomenclature 1889 and Sinorhizobium Chen et al. 1988. For some The nomenclature of Rhizobium species was originally time, it has been clear that the nomenclature of shaped by the belief that a natural classification could Rhizobium and related genera does not accurately be based on the specificity of symbiotic plant range of describe their natural classification and is in need of bacterial strains and species. The recognition that ................................................................................................................................................. nodulation and specificity were characters of strains Abbreviations: ITS, internal transcribed spacer; LBP, local bootstrap carrying particular Sym plasmids, reviewed by probability; ME, minimum-evolution; ML, maximum-likelihood; MP, Martı!nez-Romero & Palacios (1990), and therefore maximum-parsimony; NJ, neighbour-joining. were taxonomically unreliable, has led to the aban- 01471 # 2001 IUMS 89 J. M. Young and others donment of this concept. In Rhizobium species, non- bacterium rubiacearum) causing hypertrophies in symbiotic strains have been reported for different plants as pathogenic (or oncogenic; meaning ‘causing species (Jarvis et al., 1989; Laguerre et al., 1993; tumours’) strains were also included in the family Segovia et al., 1991; Sobero! n-Cha! vez & Na! jera, 1989), Rhizobiaceae. Since then, revisions have been made indicating that symbiotic plasmids can be lost in nature at both the generic and specific levels. Based on a and are not essential for bacterial survival. Further- summary of clustering analyses of phenotypic char- more, functional plasmids can be transferred between acters, DNA–DNA reassociation data and other data, members of Rhizobium and Agrobacterium species Jordan (1982) revised the symbiotic nitrogen-fixing (Abe et al., 1998; Hooykaas et al., 1977; Martı!nez et bacteria into two genera, in which fast-growing, acid- al., 1987). Overviews of the relationships of bacterial producing strains were retained in Rhizobium and nitrogen-fixing genera are given in Young (1994), slow-growing, alkali-producing strains were allocated Young & Haukka (1996) and Martı!nez-Romero & to Bradyrhizobium as Bradyrhizobium japonicum Caballero-Mellado (1996). Jordan 1982. Three species in Rhizobium, R. legumino- sarum (amalgamating the former species of R. When they first proposed the names Agrobacterium leguminosarum, R. phaseoli and R. trifolii), R. tumefaciens and Agrobacterium rhizogenes, Smith & meliloti and Rhizobium loti were listed in the first Townsend (1907) and Riker et al. (1930) followed the edition of Bergey’s Manual of Systematic Bac- custom of giving names that reported a distinctive teriology (Jordan, 1984). Since then, the genus Brady- character of the species, in this case their pathogenic rhizobium has been shown to be on a phylogenetic symptoms. Agrobacterium tumefaciens (Smith and branch distant from the Rhizobium species. Two Townsend 1907) Conn 1942 (type species) was the species, Bradyrhizobium elkanii Kuykendall et al. 1993 name given to strains of Agrobacterium capable of and Bradyrhizobium liaoningense Xu et al. 1995, and inducing tumorigenic reactions in a wide range of host other related slow-growing, symbiotic nitrogen-fixing plant species, although some of these tumorigenic strains have also been reported within this genus. The strains isolated from Vitis spp. appeared to be specific genus Azorhizobium, with a single species, Azo- to grape. Agrobacterium rhizogenes (Riker et al. 1930) rhizobium caulinodans, was proposed for stem-nodu- Conn 1942 comprised strains capable of inducing a lating strains from Sesbania rostrata (Dreyfus et al., hairy-root (rhizogenic) reaction in host plants. Agro- 1988). This taxon is also distantly related to other bacterium rubi (Hildebrand 1940) Starr and Weiss taxa with hypertrophying capabilities. 1943 referred to strains capable of inducing tumori- genic reactions in the canes of Rubus spp. and appeared Other significant revisions have been made to taxa to have a relatively limited host range. Recently, the closely related to, and including, Rhizobium species. strains specific to Vitis spp., referred to above, Chen et al. (1988) proposed a separate genus, Sino- were named Agrobacterium vitis Ophel and Kerr rhizobium, to include Rhizobium fredii Scholla and 1990. Agrobacterium radiobacter (Beijerinck and van Elkan 1984 and the new species Sinorhizobium xinjian- Delden 1902) Conn 1942 comprised non-pathogenic gense Chen et al. 1988. Subsequently, R. meliloti was Agrobacterium strains. Although Holmes & Roberts transferred to the genus (de Lajudie et al. 1994) and (1981) offered an alternative classification, the appli- Sinorhizobium sahelense de Lajudie et al. 1994, Sino- cation of the names Agrobacterium rhizogenes and rhizobium terangae de Lajudie et al. 1994, Sino- Agrobacterium tumefaciens on the basis of distinct rhizobium medicae Rome et al. 1996 and Sinorhizobium pathogenic characters was supported by Kersters & De arboris and Sinorhizobium kostiense (Nick et al., 1999) Ley (1984) and has since been used by many workers have been proposed as new species. R. loti and some (examples are Jarvis et al., 1986; Sawada et al., 1992, other recently described Rhizobium species are dis- 1993b; Weibgen et al., 1993; Bouzar et al., 1993). tinguished from Rhizobium and Sinorhizobium by comparative 16S rDNA sequence data, in having a Present classification of the family Rhizobiaceae slower growth rate (indicating underlying metabolic differences) and by a distinct fatty acid profile (Jarvis et The rhizobia. A number of revisions and additions to al., 1996). For the species, Jarvis et al. (1997) proposed the taxonomy of rhizobia (family Rhizobiaceae) have a new genus, Mesorhizobium, to include Mesorhizo- been made in the past 20 years. The Approved Lists of bium loti (type species), Mesorhizobium ciceri Nour Names of Bacteria (Skerman et al., 1980) recorded in et al. 1994, Mesorhizobium huakuii Chen et al. 1991, the genus Rhizobium all then-known bacteria capable Mesorhizobium mediterraneum Nour et al. 1995 and of nodulation and nitrogen fixation in symbiotic re- Mesorhizobium tianshanense Chen et al. 1995. Meso- lationships with plants in the family Leguminosae. rhizobium amorphae Wang et al. 1999 and Meso- Symbiotic species were Rhizobium leguminosarum (type rhizobium plurifarium de Lajudie et al. 1998a are new species), Rhizobium japonicum, Rhizobium lupini, species in the genus. Recently, a genus containing a Rhizobium meliloti, Rhizobium phaseoli and Rhizo- single species, Allorhizobium undicola de Lajudie et al. bium trifolii. The Agrobacterium species Agrobacterium 1998b, has been proposed for a population of nodu- tumefaciens, Agrobacterium rhizogenes