gen. Akwhizobïum umdieda nov., sp. WOV., nitrogen-fixing bacteria that efsicieratiy nodulate Neptmía natans in Senegai Philippe de Lajudie,'i2 Etike Laurent-Fulele,' Anne will em^,*^^ Urbain Torck12 Renata Coopman,' Matthew D. col lin^,^ Kare1 KerstersI2 Bernard Dreyfuslt and Monique t Gillis2 Author for correspondence: Monique Gillis. Tel: +32 9 264 5117. Fax: $32 9 264 5092/5346. e-mail: [email protected] 1 Laboratoire de A group of nodule isolates from Neptunia natans, an indigenous stem- Microbiologie des Sols, nodulated tropical legume found in waterlogged areas of Senegal, was ORSTOM BP 1386, Dakar, studied. Polyphasic taxonomy was performed, including SDS-PAGE of total Senegal, West Africa / 1 proteins, auxanography using API galleries, host-plant specificity, PCR-RFLP of 2 Laboratorium voor Microbiologie, Universiteit the internal transcribed spacer region between the 16s and the 23s rRNA Gent, K.-L. coding genes, 165 rRNA gene sequencing and DNA-DNA hybridization. It was Ledeganckstraat, 35, B- demonstrated that this group is phenotypicallyand phylogenetically separate 9000 Ghent, Belgium from the known species of Rhizobium, Sinorhizobium, Mesorhizobium, Microbiology Department, Agrobacterium, Bradyrhizobium and Azorhizobium. Its closest phylogenetic Reading Laboratory, as Institute of Food Research, neighbour, deduced by 165 rRNA gene sequencing, is Agrobacterium vitis Earley Gate, Whiteknights (962O/O sequence homology). The name Allorhizobium undicola gen. nov., sp. Road, Reading RG6 6BZ, nov., is proposed for this group of bacteria, which are capable of efficient UK nitrogen-fixing symbiosis with Neptunia natans, and the type strain is ORS 99ZT (= LMG 118753. Keywords: Allarhizobium undicola, Neptunia natans, tropical rhizobia, polyphasic taxonomy, nitrogen fìxation INTRODUCTION Bacteria enter natural wounds caused by splitting of the epidermis and emergence of young lateral roots. Neptunia natans L.f. (Druce), previously Neptunia Bacteria spread first intercellularly, then through oleracea Lour., McVaugh 1987 (Subba Rao et al., intercellular infection threads towards the meriste- 1995), is an annual aquatic legume that is indigenous matic cells of the nodule (Schaede, 1940). The vascular to waterlogged areas of Senegal. N. natans produces bundles of the nodules are connected to the vasculature floating stems and roots containing white, spongy, of the adventitious roots and not to that of the stem, 11 internodal tissue and nodes with bright-red nodules indicating that they are root nodules rather than true and adventitious roots (Allen & Allen, 1981 ; Schaede, stemnodules (Schaede, 1940;James et al., 1992; Subba 1940). The mode of root infection of N.natans (Subba Rao et al., 1995). N.natans is being evaluated as green . r. Rao et al., 1995) is similar in many respects to that of manure for rice cultivation in India and is consumed in other tropical legumes, such as Aeschynomene ameri- South-East Asia (Subba Rao et al., 1995). N.natans cana (Napoli et al.? 1975), Neptuiiiapleria (James et al., nodule bacterial isolates have been reported to induce 1992) and Sesbania rostrata (Ndoye et al., 1994). small, white ineffective nodules on Medicago sativa and Ornithopus spp. (Subba Rao et al., 1995) but not on roots of Cicer arietinun?, Lupinus albus, Lupiizus angustifolius, Viciafaba, Trifolium subterraneunz, Glv- tPresent address: LSTM ORSTOMKIRAD-Foret, Baillarguet, BP 5035, cine max and Macroptiliuni atropurpureum. N. natans 34032 MontpellierCedex 1, France. was recently. reported to be nodulated by Mesorhi- Abbreviations: ITS, internal transcribed spacer; YMA, yeast mannitol agar: YEB, yeast extract peptone medium; PI, tryptone yeast extract zobiunz plurifariuni strains isolated from Acacia (de medium. Lajudie e$al., 1998). N. natms nodule isolates have The EMBL accession number Forthe 165 rRNA gene sequence of strain LMG been r-ported to be fast growers (Dreyfus et al., 1984) 11875 reported in this paper is Y17047. but have not yet been taxonomically characterized. P. de Lajudíe and others Phylogenetically, rhizobia belong in the alpha-:! sub- Following the proposition of Sawada et al. (1993) to class of the Proteobacteria (Stackcbrandi et al., 1988; name Agrobacterium bv. 1 strains and Agrobacterium Sawada et al., 1993; Willems & Collins, 1993; Yanagi bv. 2 strains Agrobacterium radiobacter and Agro- & Yamasato, 1993; Young, 1991), and several genera bacterium rhizogenes respectively, Bouzar (1994) re- have been recognized, i.e. Rhizobium, Bradyrhizobium, quested a Judicial Opinion to decide whether Agro- Azorhizobium, Sinorhizo bium and Mesorhizo bium (for bacterium radiobacter or Agrobíicterium ttiinefaciens a review see Young & Haukka, 1996). Polyphasic should be the type species of Agrobacteriunz. Because taxonomy has revealed that members of the genus this decision is still pending, we use here the no- Rhizobium are phylogenetically intertwined with mem- menclature proposed by Kersters & De Ley (1984). bers of the genus Agrobacterium, to which they are Here we study a new group of fast-growing rhizobia more closely related than to Azorhizobiaan and Brady- isolated from N. natans nodules collected in Dakar and , rhizobium (Sawada et al., 1993; Willems & Collins, in the Sine Saloum region of Senegal, where this plant 1993; Yanagi & Yamasato, 1993). In Agrobacteriurn, grows naturally. We performed whole-cell protein the original species (Agrobacterizan tumefaciens, Agro- analysis by SDS-PAGE, PCR-RFLP of the internal bacterium radiobacter and Agrobacterium rhizogenes) transcribed spacer (ITS) region between 16s and 23s were created on the basis of their phytopathogenic rRNA genes, 16s rRNA gene sequencing, DNA-DNA properties, which are mainly governed by plasmid- hybridizations, and auxanographic tests using API 50 borne genes and do not correlate with the taxa found galleries. Based on the findings of this polyphasic by polyphasic taxonomy (Kersters & De Ley, 1984). study, we conclude that this group of rhizobia belongs For nomenclatural reasons, Agrobacterium tumefa- to the Agrobacterium rRNA sublineage, with Agro- ciens must be retained as the type species of Agro- bacterium vitis as its closest phylogenetic neighbour, bacterium. Consequently, no definite renaming of the and deserves a separate genus and species status, for species was proposed by Kersters & De Ley (1984), which the name Allorhizobium undicola, gen. nov., sp. although a temporary division into four groups was nov., is proposed. suggested, reflecting the polyphasic results. Agrobac- terium bv. 1 (containing the type strains of Agro- bacterium tumefaciens and Agrobacterium radiobacter) METHODS constitutes the first group, Agrobacterium bv. 2 in- Bacterial strains. Rhizobium strains were isolated as pre- cluding the type strain of Agrobacterium rhizogenes viously described (de Lajudie et al., 1994) from naturally constitutes a second group and a third taxon occurring nodules on adventitious roots of N. natans. All corresponds to the species Aprobacterium vitis; Agro- strains used are listed in Table 1. They were checked for bacterium rubi was considered to have a separate purity by repeated streaking and by microscopical exam- ination. The identity of the nodulating strains was verified position and represents the fourth group. Later, by plant infection tests on the original host plants. We comparison of the sequences of the 16s rRNA genes included type or representative strains of the different (Sawada et al., 1993; Willems & Collins, 1993; Yanagi Rhizobium, Bradyrhizobium, Azorhizobium, Mesorhizobium, & Yamasato, 1993) revealed four phylogenetic sub- Sinorhizobium and Agrobacterium species. Mycoplana, lineages on the Agrobacterium-Rhizobium branch: (i) Ochrobactrum and Phyllobacterium representatives were a bst sublineage contains Agrobacterium bv. 1, Agro- included in the auxanographic tests. bacterium rubi and Agrobacteriunz vitis; Rhizobium Growth and culture conditions. All Rhizobium and Brady- gaZegae and the recently proposed new species rhizobium strains were maintained on yeast mannitol agar Rhizobium giardiniì (Amarger et al., 1997) also belong (YMA), containing (g 1-l):mannitol, 10; sodium glutamate, to this sublineage but have somewhat separate 05; K,HPO,, 05; MgS04.7H,0, 0.2; NaCl, 005; CaCI,, positions; (ii) a second phylogenetic sublineage con- 0.04; FeCI,, 0.004; yeast extract (Difco), 1; agar, 20; pH 6.8. tains Rhizobium Iegunzinosarum (type species of Rhi- Azorhizobium and Agrobacterium strains were maintained zobium), Rhizobium tropici, Rhizobium etli, Agro- on yeast extract peptone medium (YEB) containing in g 1-1 bacterium bv. 2 and a recently proposed new species, of 0.01 M phosphate buffer, pH 7.2: peptone (Oxoid), 5; yeast extract (Oxoid), 1; beef extract (Oxoid), 5 ; sucrose, 5 Rhizobium gnllicum (Amarger et al., 1997); (iii) a third and MgSO,. 7H,O, 0.592. All strains were stored at - 80 "C sublineage was considered sufficiently different to on the same medium plus 15 % (v/v) glycerol. For protein deserve a separate genus status, for which the name and DNA preparations we used tryptone yeast extract Sinorhizobium had priority. Sinorhizobium contains medium (TY) containing(g tl,pH 6.8-7) : tryptone (Oxoid), Sinorhizobium meliloti, Sinorhizobium fredii, Sinorhi- 5; yeast extract (Oxoid), 0.75; KH,PO,, 0.454; Na,HPO,. zobiurn xinjiangense, Sinorhizobium terangae, Sino- 12H,O, 2.388; CaCl,, 1; agar, 20. For protein preparation, rhizobium saheli (de Lajudie et al., 1994; Triiper & de' TY with LabM agar was used.