International Journal of Systematic Bacteriology (1 998), 48, 1277-1 290 Printed in Great Britain
Allorhizobium undicola gen. nov., sp. nov., nitrogen-fixing bacteria that efficiently nodulate Neptunia natans in Senegal
Philippe de Lajudie,l** Etike Laurent-Fulele,’ Anne WiIlerns,*~~ Urbain Torck,’ Renata Coopman,2 Matthew D. Collin~,~Karel Kersters,’ Bernard Dreyfuslt and Monique Gillis’
Author for correspondence: Monique Gillis. Tel: + 32 9 264 51 17. Fax: + 32 9 264 5092/5346. e-mail : Moniek.Gillis(g rug.ac.be
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, Senegal, West Africa studied. Polyphasic taxonomy was performed, including SDS-PAGE of total proteins, auxanography using API galleries, host-plant specificity, PCR-RFLP of 2 Laboratorium voor the internal transcribed spacer region between the 165 and the rRNA Microbi olog ie, Un iversit eit 235 Gent, K.-L. coding genes, 16s rRNA gene sequencing and DNA-DNA hybridization. It was Ledeganckstraat, 35, B- demonstrated that this group is phenotypically and phylogenetically separate 9000 Ghent, Belgium f rom the known species of Rhizobium, Sinorhizobium, Mesorhizobium, 3 Microbiology Department, Agrobacterium, Bradyrhizobium and Azorhizobium. Its closest phylogenetic Reading Laboratory, Institute of Food Research, neighbour, as deduced by 16s rRNA gene sequencing, is Agrobacterium vitis Ear I ey Gate, Whitekn ig hts (96.2 YO 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 992T(= LMG 11875’).
Keywords: Allorlzizohiurn undicola, Neptunia natans, tropical rhizobia, polyphasic taxonomy, nitrogen fixation
INTRODUCTION Bacteria enter natural wounds caused by splitting of the epidermis and emergence of young lateral roots. Neptuniu natans L.f. (Druce), previously Neptunia Bacteria spread first intercellularly, then through oleraceu 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, internodal tissue and nodes with bright-red nodules indicating that they are root nodules rather than true and adventitious roots (Allen & Allen, 1981 ; Schaede, stem nodules (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 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), Neptuniaplena (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 arietinum, Lupinus albus, Lupinus ,. , . . , ...... , ...... , ., , , ...... , , , ...... , ...... angust ifolius, Viciafaba, Trifolium subterraneum, Gly- t Present address: LSTM ORSTOMKIRAD-ForCt, Baillarguet, BP 5035, 34032 Montpellier Cedex 1, France. cine mux and Mucroptilium atropurpureum. N. natans was recently reported to be nodulated by Abbreviations: ITS, internal transcribed spacer; YMA, yeast mannitol Mesorhi- agar; YEB, yeast extract peptone medium; TY, tryptone yeast extract zobium plurifarium strains isolated from Acacia (de mediurn. Lajudie et al., 1998). N. natans nodule isolates have The EMBL accession number for the 165 rRNA gene sequence of strain LMG been reported to be fast growers (Dreyfus et al., 1984) 11875 reported in this paper is Y17047. but have not yet been taxonomically characterized.
~ ~~ ~~ 00750 0 1998 IUMS 1277 P. de Lajudie and others
Phylogenetically, rhizobia belong in the alpha-2 sub- Following the proposition of Sawada et a/. (1993) to class of the Proteobacteria (Stackebrandt 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 (1 994) re- have been recognized, i.e. Rhizobium, Bradyrhizobium, quested a Judicial Opinion to decide whether Agro- Azorhizo bium, Sin orh izobium and Mesorh izob ium (for bacterium radiobacter or Agrobacterium tumefaciem a review see Young & Haukka, 1996). Polyphasic should be the type species of Agrobacterium. Because taxonomy has revealed that members of the genus this decision is still pending, we use here the no- Rhizohiurn 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 Azorhizobium and Brudy- isolated from N.natuns 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 Agrobacterium, grows naturally. We performed whole-cell protein the original species (Agrobacterium 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- METHODS bacterium tumefaciens and Agrobacterium radiobacter) 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 a/., 1994) from naturally constitutes a second group and a third taxon occurring nodules on adventitious roots of N. natans. All corresponds to the species Agrobacterium vitis ; Agro- strains used are listed in Table 1. They were checked for purity by repeated streaking and by microscopical exam- bucterium rubi was considered to have a separate 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 Rhizo b ium, Bradyr h izobiurn, Azo r h izohium , Mesor h izobiunz , & Yamasato, 1993) revealed four phylogenetic sub- Sinorhizobium and Agrobacteriunz species. Mycoplana, lineages on the Agrobacteriun-Rhizobium branch : (i) Ochrobactrum and Phyllobacterium representatives were a first sublineage contains Agrobacterium bv. 1, Agro- included in the auxanographic tests. bacterium rubi and Agrobacterium vitis; Rhizobium Growth and culture conditions. All Rhizobium and Brady- gaiegae and the recently proposed new species rhizobium strains were maintained on yeast mannitol agar Rhizobium giardinii (Amarger et al., 1997) also belong (YMA), containing (g 1-l): mannitol, 10; sodium glutamate, to this sublineage but have somewhat separate 0.5; K,HPO,, 0.5; MgSO,. 7H,O, 0.2; NaC1, 0.05; CaCl,, positions ; (ii) a second phylogenetic sublineage con- 0.04; FeCl,, 0.004; yeast extract (Difco), 1 ; agar, 20; pH 6.8. tains Rhizobium leguminosarum (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-I bacterium bv. 2 and a recently proposed new species, of 0.01 M phosphate buffer, pH 7-2: peptone (Oxoid), 5; (Amarger 1997); (iii) a third yeast extract (Oxoid), 1 ; beef extract (Oxoid), 5; sucrose, 5 Rhizobium gallicum et a/., 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 YO(v/v) glycerol. For protein deserve a separate genus status, for which the name and DNA preparations we used tryptone yeast extract Sinorh izob ium had priority . Sinorh izob ium contains medium (TY) containing (g l-l, pH 6.8-7) : tryptone (Oxoid), Sino rh izob ium me lilo ti, Sino r h izob ium fredii, Sino r h i- 5; yeast extract (Oxoid), 0.75; KH,PO,, 0.454; Na,HPO, . zobium xinjiangense, Sinorh izobium terangae, Sino- 12H,O, 2.388; CaCl,, 1 ; agar, 20. For protein preparation, rhizobium saheli (de Lajudie et al., 1994 ; Truper & de' TY with LabM agar was used. Mycoplana, Ochrobactrum Clari, 1997) and Sinorhizobium medicae (Rome et al., and Phyllobacterium strains were maintained on nutrient 1996); (iv) the fourth sublineage consists of species agar containing (g 1-') : beef extract (Oxoid), 1 ; yeast extract recently transferred in the new genus Mesorhizobium (Oxoid), 2; peptone (Oxoid), 5; NaCI, 5; pH 7.4; agar, 20. (Jarvis et al., 1997), namely Mesorhizobium loti, Morphological tests. Cell dimensions and morphology were Mesorhizobium huakuii, Mesorhizobium ciceri, Meso- determined on living cells by phase-contrast microscopy. rh izobium t ianshanense, Mesorh izobium mediterraneum Plant infection tests. The seeds were scarified and surface (Jarvis et al., 1997) and Mesorhizobiumplurifarium (de sterilized with concentrated sulfuric acid. The duration of Lajudie et al., 1998). treatment (min) in H,SO, for the different plant species was
~~ ~~~ 1278 International Journal of Systematic Bacteriology 48 Allorhizobium undicola gen. nov., sp. nov.
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Table 1. Strains used ...... ATCC, American Type Culture Collection, Rockville, MD, USA; BR and FL, strains from the CNPBS/EMBRAPA, Centro Nacional dc Pesquisa em Biologia do Solo, Seropedica 2385 1, Rio de Janeiro, Brazil/Emprasa Brasiliera de Pesquisa Agropequaria ; CFN, Centro de Investigacion sobre Fijacion de Nitrogeno, Universidad Nacional Autonoma de Mexico, Cuernavaca, Mexico; CIAT, Rhizobium Collection, Centro International de Agricultura Tropical, Cali, Columbia; HAMBI, Culture Collection of the Department of Microbiology, University of Helsinki, Helsinki, Finland ; IAM, Institute for Applied Microbiology, University of Tokyo, Tokyo, Japan; LMG, Collection of Bacteria of the Laboratorium voor Microbiologie, K.-L. Ledeganckstraat, 35, B-9000 Ghent, Belgium ; NCPPB, National Collection of Plant-pathogenic bacteria, Harpenden Laboratory, Hertfordshire, UK; NZP, Culture Collection of the Department for Scientific and Industrial Research, Biochemistry Division, Palmerston North, New Zealand ; ORS, ORSTOM Collection, Institut FranCais de Recherche Scientifique pour le Diveloppement en Cooperation, BP 1386, Dakar, Senegal; Pan., Panagopoulos, C., Crete, Greece; USDA, US Department of Agriculture, Beltsville. hlD, USA; UPM, Universidad Politecnica Madrid, Spain.
Strain* LMG no. Other strain Host plant or origin Geographical origin Reference or source designation
Allorlzizobium undicola ORS 991 11874 Neptunia natans Senegal (North This work Kaolack) ORS 992“‘ 11875’ Neptunia natans Senegal (Kaolack) This work ORS 995 11876 Neptunia natans Senegal (South This work Kaolack) ORS 9% 11877 Neptunia natans Senegal (South This work Kaolack) ORS 997 11878 Neptunia natans Senegal (North This work Kaolack) ORS W8 11879 Neptunia natans Senegal (Dakar-Be1 This work Air)
Mesorh izc ) hium plurifarium ORS 1001 7836 Acacia senegal Senegal de Lajudie et al. ( 1998) ORS 1014tl 7849tl Acacia senegal Senegal de Lajudie et al. (1998) ORS 1002 7854 Acacia senegal Senegal de Lajudie et al. (1 998) ORS 13 792 1 Acacia sp. Senegal de Lajudie et al. ( 1998) ORS 1018 11881 Acacia senegal Senegal de Lajudie et al. (1998) ORS 1037 11895 Acacia senegal Senegal de Lajudie et al. (1998) ORS 1040 11898 Acacia senegal Senegal de Lajudie et al. (1 998) HAMBI 1487 14925 Acacia senegal Soudan de Lajudie et a/. (1 998) Mesorhizobium loti
3F3C1 4269 Wisteria ,frutescens Jarvis et al. (1986) NZP 2230 6126 Lotus maroccanus Morocco Jarvis et al. (1 986) NZP 2213’ 6 125T ORS 664T Lotus tenuis New Zealand Jarvis et al. (1986) NZP 2037 6123 ORS 652 Lotus divaricatus New Zealand Jarvis et al. (1986) NZP 2014 6124 Lotus corniculatus Jarvis et al. (1986) Mesorhizl>hiumciceri UPM-Ca7’ 17150’ ORS 2738T Cicer arietinum L. Spain Nour et al. (1994) 522 17149 Cicer arietinum L. Russia Nour et al. (1994) Mesorh izohium mediterruneum Ca-36” 1714ST ORS 2739T Cicer arietinum L. Spain Nour et a/. (1 995) UPM-C’a142 14990 Cicer arietinuin L. Spain Nour et al. (I 995) Mesorhizohium sp. (Cicer) genospecies 4 IC-60 14995 Cicer arietinum L. India Nour et al. (1995)
[Contimud overleaf
International Journal of Systematic Bacteriology 48 1279 P. de Lajudie and others ~. -~
Table I (cont.)
Strain* LMG no. Other strain Host plant or origin Geographical origin Reference or source designation
Mesorhizobium huakuii IAM l415ST 14107”’ ORS 1752T Astragalus sinicus Nanjing, China Chen er al. (1991) Mesorhizobiun? tianshanense A- 1BS’ 15767’ ORS 2640T Clycyrrhiza Xinjiang, China Chen et ul. (1995) pa I1 idijlo r u Sinorhizobium .fredii USDA 205’ 62 1 7’ ORS 669’ Glycine max Honan, China Jarvis et nl. (1986) USDA 191 8317 Soil Shanghai, China, Jarvis et al. (1986) 1978 USDA 208 6219 Glycine max Honan, China LMG Sinorh izo b ium melilo ti NZP 4009 6130 Medicago sativa Australia LMG NZP 4027T 61331 ORS 665T Medicago sativa Virginia, USA LMG 102F34 ORS 620 ORS L5-30 ORS 621 ORS RCR 201 1 ORS 634 ORS 3DOa30 4266 Mcdicugo sntiva Turkey, 1952 LMG Sinorhizobium medicae HAMBI 1808 (m75) 16579 Medicago sativa Eardly et al. (1990) HAMBI 1809 (m102) 16580 ORS 504 Medicago sativa Eardly et al. (1990) HAMBI 1837 (m 158) 1658 1 Eardly et al. (1990) Sinorh izobium terangae ORS 15 7833 Seshania sp. Senegal de Lajudie et ul. (1 994) ORS 51 7843 Sesbania rostrata Senegal de Lajudie et al. ( 1994) ORS 604 11865 Sesbania aculeata Senegal de Lajudie ct cil. ( 1994) ORS 1007 7847 Acacia laeta Senegal de Lajudie et al. (1994) ORS 1009’ 78341 Acacia laeta Senegal de Lajudie et (11. ( 1994) ORS 1073 11926 Acacia senegal Senegal de Lajudie et al. ( 1994) Sinorhizobium suheli ORS 609T 7837T Sesban in can nab ina Senegal de Lajudie et al. ( 1994) ORS 609t2 8309t2 Seshaniu cannabina Senegal de Lajudie et al. ( 1994) ORS 611 7842 Sesbania gmndiflora Senegal de Lajudie et al. (1 994) ORS 61 1 83 10 Scsban ia grandiflo ra Senegal de Lajudie et al. (1994) Siizorhizobium sp . BR 816 ORS 2645 ORS NGR 234 ORS 644 Lablab purpureus Trinick (1980) Rhizobium leguminosarum CNPAF 146 9504 LMG NZP 561 6122 Trijolium repens Australia B. Jarvis ATCC 14482 8819t1 LMG Rhizobium tropici group a CNPAF 119 9502 Phaseolus vulgaris Brazil LMG L. CFN 299 9517 ORS 651 Phaseolus vulgaris Brazil Martinez-Romero L. rt al. (1991)
1280 International Journal of Systematic Bacteriology 48 Allorhizobium undicola gen. nov., sp. nov. ____~____
Table 1 (cont.)
Strain * LMG no. Other strain Host plant or origin Geographical origin Reference or source designation
Rhizobiuni tropici group b CIAT 899’ 9503’ ORS 1163’ Phaseolus vulgaris Columbia Martinez-Romero L. et al. (1991) C-05 9518 Phaseolus vulgaris Martinez-Romero L. et al. (1991) Rh izob iun I c tli CFN 42” ORS 645’ Phaseolus vulgaris Mexico Segovia et al. L. (1993) RhizohiuiiI galegae HAMBI 540T 62 14T ORS 66gT Galega orientalis Finland LMG HAMBI 1147 6215 Gakga orientalis Russia LMG HAMBI 1428/2 15 143 Galega orientalis Russia LMG Agrohuctclriunz bv. 2 ATCC 1 1325T 150‘ Agrobnc*tt,riunzbv. 1 ATCC 19358‘ 140’ ORS 1351 LMG M2/ 1 147 Ditch water Belgium Kersters et al. (1973) B61 1 87T Lycopersicon USA Kersters et al. Iycopersicon (1 973) ICPB TT111 196 Crown gall USA Kersters et al. (1 973) B2a 268 Lycopersicon Kersters et al. lycopersicon (1 973) IICHR 28 303 Chrysanthemum Germany, 1927 Kersters et al.
,frutescens (1 973) CDC A6597 383 Vagina South Carolina, LMG USA Agrohucic~riumruhi ICPB I‘R2 159 ORS 1353 Rubus sp. USA, 1942 Kersters et al. (1973) ATCC 13335T 156T Rubus ursinus USA, 1942 LMG Agrohrrctc~riunivitis Pan. AG61 257 ORS 2643 Vitis vinifera Crete, Greece LMG Pan. AG63 258 Vitis vinlfera Crete, Greece LMG NCPPH 1771 233 Vitis vinifera Iran Kersters et al. (1973) Azorhirohiun? caulinodans ORS 571“‘ 6465’ Seshania rostrata Senegal Dreyfus et al. (1988) FY 12 11352 Sesbania rostrata Senegal Rinaudo et al. (1991) * Original strain number, or as received.
as follows: Acacia senegal, 14; Acacia seyal, 30; Acacia The seeds were incubated in sterile Petri dishes on 1 % water tortilis subsp. raddiana, 150; Sesbania rostrata, 30-60 ; agar for 2448 h to allow germination and then transferred Sesbania pubescens, 60; Sesbania grandflora, 60 ; Neptunia to tubes containing Jensen seedling slant agar (Vincent, natans, 30 ; Medicago sativa, 25 ; Macroptilium atropur- 1970) for root nodulation trials (8-10 plants were routinely pureuni (Siratro), 3. For Vigna unguiculata (niebe), seeds tested with each strain). Root nodules appeared around were left for 3 min in 96 % alcohol followed by 5 min in 1 % 10-20 d after inoculation, and 3 weeks later they were fully HgCl,. After acid or HgC1, treatment, the seeds were washed developed. Nitrogen-fixing potential was estimated by visual with water until all traces of acid or HgC1, were removed. observation of plant vigour and foliage colour of 30- to 45-
~ International Journal of Systematic Bacteriology 48 1281 P. de Lajudie and others d-old plants and also by measuring the fresh and dry weights DNA-DNA hybridization. DNA-DNA hybridizations were of aerial parts ; infected plants were compared with control performed with the initial renaturation rate method (De uninoculated plants. Ley, 1970). Renaturations using approximately 50 mg DNA ml-' were carried out at 79.8 "C, which is the optimum PAGE of total bacterial proteins. PAGE was performed using renaturation temperature, in 2 x SSC (1 x SSC = 0.15 M small modifications of the procedure of Laemmli (1970), as NaCl, 0.015 M sodium citrate, pH 7). described previously (de Lajudie et al., 1994). The norma- lized densitometric traces of the protein electrophoretic Analysis of the 165 rRNA genes. The nearly complete 16s patterns were grouped by numerical analysis, using the rRNA gene of strain LMG 11875, a representative of the GelCompar 2.2 software package (Vauterin & Vauterin, new group, was determined. Lyophilized cells were re- 1992). Similarity between pairs of traces was expressed by suspended in 500 ml TES buffer (0.05 M Tris/HCl, 0.005 M the Pearson product-moment correlation coefficient (r) EDTA, 0.05 M NaCl, pH 8.0) and DNA was extracted by converted for convenience to a percentage value (Pot et al., the method of Lawson et al. (1989). A large fragment of the 1989, 1994). 16s rRNA gene (corresponding to positions 28-1521 of the Escherichia coli 16s rRNA gene) was amplified by PCR. The PCR-RFLP of the ITS of 165-235 rRNA genes. Strains were PCR products were purified using a Prep-A-Gene kit (Bio- grown at 28 "C for 3648 h on YMA, according to the Rad) and sequenced using a Tag DyeDeoxy Terminator method of Vincent (1970). Total DNA was purified with Cycle Sequencing Kit (Applied Biosystems) and an auto- Chelex 100 (Sigma). Cells resuspended in a 5 YOsuspension matic DNA sequencer (model 373A; Applied Biosystems). of Chelex 100 were boiled for 15 min. After centrifugation, The new sequence was aligned, together with reference the supernatant was used directly for PCR amplification. sequences obtained from the EMBL database, using the For strains for which the above procedure did not result in program PILEUP of the Genetics Computer Group package optimum DNA amplification, total DNA was purified using (Devereux et al., 1984). Altogether a continuous stretch of the phenokhloroform method as described by Boucher et 1348 base positions (including gaps) was used for further al. (1987). Primers FGPL 132'-38 and FGPS 1490-72, as analysis. This corresponded to positions 84-1480 of the described by Normand et al. (1996), were used for PCR Escherichia coli 16s rRNA gene. Distances, modified ac- amplification. These primers are derived from conserved cording to the Kimura-2 model, were calculated using the regions of the 23s and 16s rRNA genes, respectively, and DNADIST program of the Phylogeny Inference Package can be used to amplify the ITS of all prokaryotic DNAs (Felsenstein, 1982), and the program NEIGHBOR of the same tested so far. The oligonucleotides were purchased from package was used to produce an unrooted phylogenetic tree. Pharmacia. PCR amplification was carried out in a 100 yl The stability of the groupings was verified by bootstrap reaction volume containing template DNA (50 yg), reaction analysis (500 replications) using the programs DNABOOT, buffer (Appligene), 20 mM of each dNTP (Pharmacia), DNADIST, NEIGHBOR and CONSENSE (Felsenstein, 1982). Un- 0.1 mM of each of the primers and 1 U Tag polymerase corrected distances were calculated using the DISTANCES (Appligene). Amplifications were carried out in a GeneAmp program of the GCG package and used to calculate PCR System 2400 (Perkin Elmer) using the following similarity values. programme: initial denaturation for 5 min at 94 OC, 35 cycles of denaturation (30 s at 94 "C), annealing (30 s at Auxanographic tests. API galleries (API 50CH, API 50AO 55 "C) and extension (1 min at 72 "C) and a final extension and APL 50AA; bioM6rieux) were used to test the as- (5 min at 72 "C). PCR-amplified DNAs were visualized by similation of 147 organic compounds as sole carbon sources, electrophoresis of 4 pl of the amplified mixture on 1.4% and the results of auxanographic tests were scored as (w/v) horizontal agarose gel (type 11 ; Sigma) in TBE buffer described previously (de Lajudie et al., 1994). (83 mM Tris base, 89 mM boric acid, 2 mM EDTA, pH 8.0) 3-Ketolactose test. The 3-ketolactose test was performed at 4 V cm for 1 h. The gels were stained in an aqueous using the original method of Bernaerts & De Ley (1963) as solution of 1 mg ethidium bromide 1-1 and photographed modified by Bouzar (1 995). with Polaroid Type 667 positive film using a 260 nm UV et al. source. Aliquots of 6 yl of PCR products were digested in a 10 pl RESULTS final volume with restriction endonucleases as specified by Six isolates were purified from root nodules collected the manufacturer but with an excess of enzyme (5 U per reaction). The following enzymes were used: Ah I, Dde I, either on N. natans plants growing naturally in Hinf I, Pal I (Pharmacia), Cfo I (Boehringer Mannheim), waterlogged areas around the town of Kaolack in the Msp I (Gibco-BRL), Rsa I (Amersham or Gibco-BRL). Sine Saloum region of Senegal or on N. natans plants Restricted DNA was analysed by horizontal electrophoresis seeded in our experimental field at Dakar-Be1 Air in in 3 '10 (w/v) agarose gel (Nusieve 3: 1 ; FMC). Electro- Senegal. phoresis was run at 2.3 V cm-' for 3 h. Gels were stained and photographed as described previously. Host specificity Clustering was obtained using the Gelcornpar 2.2 software package (Vauterin & Vauterin, 1992). The six Neptunia isolates induced nodules on their original host, resulting in a very efficient nitrogen- DNA base composition. Cells were grown for 2-3 d in Roux flasks on TY medium. High-molecular-mass DNA was fixing symbiosis. They were also found to be effective prepared using the method of Marmur (1961). The G+C on Acacia species (Acacia tortilis subsp. raddiuna, content was determined by thermal denaturation (De Ley, A cue ia senegal, A cacia seyal) , Fa idherb ia alb ida , and 1970) and calculated by using the equation of Marmur & some strains were effective on Lotus arabicus, but Doty (1962), as modified by De Ley (1991). DNA from ineffective on Medicago sativa, Sesbania species Escherichia coli LMG 2093 was used as a reference. (Sesbania rostrata, Sesbania pubescens, Sesbania gran-
1282 lnternational Journal of Systematic Bacteriology 48 Allorhizobium undicola gen. nov., sp. nov.
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Table 2. Host specificity of N. natans isolates ...... All strains nodulated N. natans, Acacia seyal, Faidherbia albida and Acacia tortilis subsp. raddiana. No strain nodulated Sesbania rostrata, Sesbania pubescens or Sesbania grandzj?ora.
Strain Acacia Lotus Medicago Vigna Macvoptilium senegal avabicus sativa unguiculata atvopurpuveum
~~~ ORS 991 + -+ * NT NT ORS 992' NT - - - - ORS 995 NT -+ - NT NT ORS 996 NT + - NT NT ORS 997 + + - - - ORS 998 + - - NT NT
+ , Nodulation; -, no nodulation; &, 10-30 % plants were nodulated; NT, not tested.
Lh4G9517 LMG 140 T ORS 1351 T Agrobacterium bv. I LMG 9502 R tropici LMG 9518 LMG 159 ORS 1353 Ag. rubi LMG 9503 T CMT 899 T LMG 62 I4 T ORS 668 T R. galegae LMG 6465 T Az. caulinodans LMG 11352 CFN 42 T ORS 645 T R. efli LMG 7842 ORS 61 I LMG 7843 LMG 7837 T ORS609T ] S. saheli LMG 830912 ORS 609t2 LMG 7834 T ORS 1009 T S. terangae LMG6125T NZP 22 I3 T M. loti LMG 11926 ORSS1ORS 1073 LMG 7847 ORS 1007 I' 1 LMG 7834 T ORS 1009 T S. terangae HAMB11809 ORS 504 S. medicae LMG 1 I865 ORS 604 LMG 7833 BR 816 ORS 2645 Sinorhizobiumsp. LMG 16580 LMG 8309 T LMG 16579 S. medicae S. saheli HAMBl 1837 LMG 8310 :yy ] LMG 16581 g: LMG6126 NZP 2230 M. loti LMG 6217 T ORS 669 T S. fredii LMG 6214 T HAMBl540 LMG6215 R. galegae LS-30 ORS 621 LMG 15143 LMG 6133 T ORS 665 T LMG17150T M. cicen LMG 17149 RCR 201 1 ORS 634 S. meliloti M. huakuii LMG 14107 T L4M 14158T ATCC 11325T Agmbacteriumbiovar 2 102F34 620 LMG I50 T ORS LMG 784931 ORS 1014tl NGR 234 ORS 644 1 Sinorhizobium sp. LMG 14925 HAMBl 1487 M. p/urifarium LMG 7921 ORS13 ] LMG 257 ORS 2643 Ag. vitis LMG 11881 ORS 1018 LMG 11875 T ORS 992 T LMG 6124 NZP2014 LMG6123 NZP2037 j M. loti LMG 11876 ORS 995 LMG 4269 3F3CI LMG 11877 LMG 14995 IC-60 Mesorhizobiurn sp. (Cicar) ORS 996 A/. undicok 1 LMG 11878 ORS 997 Fzig;. ] Agrobacterium biovar 1 LMG 11879 ORS 998 74417 1771 ] A. vitis LMG 11874 ORS 991 NZP 4009 3DOa30 ] s meliloti LMG 6125 T ORS 664 T 1 M. loti UPM Ca7 T ORS 2738 T M. ciceri :g2:46] R. /egumrnosarum ATCC 14482 LMG I4107 T ORS 1752 T M. huakuii UPM Ca142 M. maditerraneum LMG 15767 T ORS 2640 T M. tianshanense B2a86 T ] Agmbacterium biovar 1 UPM Ca36 T ORS 2739 T M. mediterraneum LMG 11878 ORS 9w LMG 6123 ORS 652 M. loti LMG 11879 0RSm 1 LMG 11876 OR' 995 Allorhizobium undicola LMG 7836 ORS 100 1 LMG 11877 ORS 996 LMG 11895 LMG 11875 T ORS 992 T ORS 1037 M. plurifariurn LMG 11874 ORS 991 LMG 7854 ORS 1002 LMG 8317 USDA 191 I USDA208 ] ''fredii LMG 11898 ORS 1040 ATCC 133351 A. rubi CFN 299 ORS 651 1R. fropici ...... Fig. 1. Dendrogram showing the relationships between the ...... electrophoretic protein patterns from nodule isolates of N. Fig. 2. Dendrogram showing the relationships between the natans and reference strains of Mesorhizobium, Rhizobium, PCR-RFLP profiles of the ITS of N. natans nodule isolates and Bradyrhizobium, Azorhizobium, Sinorhizobium and representatives of different Mesorhizobium, Rhizobium, Agrobacterium species. The mean correlation coefficient (r) was Bra dyrhizobium, Azorhizo bium, Sin orhizo bium a nd represented as a dendrogram and calculated by the Agrobacterium species. The mean correlation coefficient (r)was unweighted pair group method with averages. Positions 10-320 represented as a dendrogram and calculated by the of the 400 point traces were used for calculation of similarities unweighted pair group method with averages. T indicates type between individual pairs of traces. T indicates type strain. The strain. The scale represents the r value converted to scale represents the r values converted to percentages. percentages.
~ ~~~ International Journal of Svstematic Bacterioloav 48 1283 P. de Lajudie and others
1W Afipia felis ATCC 53690 (M65248) Bradyrhizobium elkanii USDA 76 (~35000) Azohizobium caulinodans LMG 6465 (x672213 Agrobacterium vitis IR h izo biu m ga lega e
-Mesorh/zobum transhanense A-I BS (U71079) Bartonella bacilhfomrs ATCC 35685 (21I 683) Im Phyllobactenum myrsrnacearum IAM 13584 (DI 2789) 21 Phyllobacterrum rub/acearum IAM I 3587 (D12790) - 1 Sinorhizobium saheli Rhizobium tropici a Rhizobium leguminosarum Srnorhizobwn fredrr LMG 6217 (X67231) Smorhrzobrum terangae LMG 7834 (X68388) Rhmbrum giardm HI 52 (u86344)
Mesorhizobium plurifarium
RhlZOblUfll Sp 113 (D14512) -58 100 Agrobacterrum VItIS LMG 8750 (X67225) Agrobactenum Vlt/S NCPPB 3554 (D14502) Agrobacterium rubi - Allorhizobium undicola LMG 11875 (Y17047) Blastobacter aggregatus ATCC 43293 (~73041) Agrobacterium biovar 1
Sinorhizobium melilo ti Agrobacterium biovar 2 Rhizobium tropici b.. Sinorhizobium fredii Allorhizobium undicola
Rhizobium tfOplCl LMG 9518 (X67233) ,-= Phyllobacterium Rhfzobium CFN 42 (U28916) Rhizobium leguminosarurn LMG 8820 (~67227) Rhmbium mongolense USDA 1844 (~89817) Ochrobactrum anthropi Rhizobium gallicum R602sp (~86343) Rhizobum hainanense 166(~71078) r rlv 1-4 Mycoplana 1% estimated substitubons -Azorhizo bium ca ulinodans
Fig. Dendrogram showing the phylogenetic relationships of 3. Fig. 4. Dendrogram obtained from an unweighted pair group strain LMG 11875 and representatives of the alpha subclass of method with averages cluster analysis of Canberra metric the Proteobacteria. The tree was calculated from a distance similarity coefficients based on 147 auxanographic matrix (modified according to the Kimura-2 model) using the characteristics. Reference strains included in this study were neighbour-joining method. Bootstrap values, expressed as a essentially the same as those we used in a previous report (de percentage of 500 replications, are given at the branching Lajudie et a/., 1994). Numbers of strains used were as follows: points. Numbers in parentheses are the accession numbers of Agrobacterium bv. 1, 9; Agrobacterium bv. 2, 3; Agrobacterium the sequences used. The bar represents one expected rubi, 1; Agrobacterium vitis, 3; Rhizobium galegae, 2; substitution per 100 nucleotide positions. Sinorhizobium meliloti, 3; Sinorhizobium fredii, 2; Sinorhizobium terangae, 20; Sinorhizobium saheli, 4; Rhizobium leguminosarum, 1 ; Rhizobium tropici a, 3; Rhizobium tropici b, 3; Mesorhizobium plurifarium, 26; diflora), Vigna urzguiculata and Mctcroptiliiim atro- Azorhizobium caulinodans, 1 ; Phyllobacterium, 5; purpureum (Table 2). Ochrobactrum anthropi, 10; Mycoplana, 2.
SDS-PAGE of total bacterial proteins rhizobiurn sp. (Cicer) formed separate clusters. This is We purified whole-cell proteins from the N. natcins illustrated in Fig. 1, which presents a limited den- isolates, performed SDS-PAGE in standardized con- drogram with a few representatives of the different ditions (de Lajudie et al., 1994) and compared the species. Only one strain each of Mestwhizohiiirn normalized patterns (Vauterin & Vauterin, 1992) with huakuii, Mesorhizobiunz mediterraneum, Agrohiic- those in our database, which contains profiles of strains terium bv. 2, Agrobacterium vitis and Agrobacterium of different species of Rhizobium, Sinorhizobiunz, rubi were included, and these each had separate Mesorh izobiurn, Agrobac terium and A zorh izobium. positions. Strains of Agrobacterium bv. 1 exhibited Sinorhizobium terangae, Sinorhizobium saheli, Siizo- diverse protein patterns and could be grouped into two r h izob iumfr edii, Sino r h izoh ium melil o ti, Sino r h iz ob iiim different clusters, as observed previously (de Lajudie et medicae, Rhizobium leg urn irzosar urn, Rhizo hium tropici, al., 1998). The strains of Mesorhizobium loti did not Rhizobiurn galegae, Azorhizobium cnulinodnrzs, Meso- group together and were found to fall into three rhizobium plur farium, Mesorhizobiurn cicer i, Meso- clusters. The six N. natnns isolates were related with a
1284 lnternational Journal of Systematic Bacteriology 48 Allorlzizobium undicola gen. nov., sp. nov.
correlation coefficient of 92% and formed a rather Agrobac ter ium rh izogenes, Agrobac t er ium tumgf aciens, homogeneous gel electrophoretic cluster, distinct from Agrobacteriunz vitis, Agrobacterium rubi, Azorhizohium all described species and clusters contained in our caulinodans, Ochrobactrum, Phyllohacterium and MJJ- database. The highest correlation coefficient between coplana available in our database (de Lajudie et al., the cluster of Neptunia strains and the other rhizobial 1994). The dendrogram obtained by numerical analysis species and groups was 70 YO. of these results (Fig. 4) showed that the Neptunia strains formed a very homogeneous group distinct PCR-RFLP of the ITS from every other species. They were related to Agro- bacterium vitis at a correlation coefficient of 0.695. We performed PCR-RFLP analysis of the ITS region Table 3 shows the results of the Neptunia strains and between the16S and 23s rRNA genes of the Neptunia their nearest phylogenetic relatives, namely Agro- strains and some representative strains of rhizobial bacterium vitis, Agrobacterium bv. 1, Agrobacterium species of R h izo b ium, Sino r h izob ium, Mesorhiz o b ium rubi and Rhizobium galegae. and Agrohiicterium. The size of the amplified ITS fragment varied from 1000 to 1450 bp depending on strains anti was 1380-1 390 bp for the Neptunia isolates. 3-Ketolactose test The results of RFLP analysis are shown in Fig. 2. All strains belonging to the same species, except The six strains from Neptunia were negative, as were Meso- the four control strains of bv. 2 tested rliizobizrm loti, grouped together. At a correlation Agrobacterium coefficient of 30%, three main branches could be (LMG 150', LMG 155, LMG 161, LMG 341). Control Agrobacterium bv. 1 strains LMG 64, LMG 146, LMG distinguished. The first branch consisted of the Rhi- and species, 196, LMG 201, LMG 296t2 were positive; Agro- zobium, Sinorhizobium Agrobacterium bv. 1 strain LMG 26 and sp. together with two sp. strains, BR 8 16 bacterium Agrobacterium Sinorhizobium strain LMG 294 were negative. and NGR 234. The second branch consisted of the Mesorhizohium species (Mesorhizobium loti, Meso- rhizobium huakuii, Mesorhizobium ciceri, Mesorhizo- DISCUSSION hium ticriishunense, Mesorhizohium mediterraneum, Mesorhizohium plurifurium). All isolates from N. We isolated six new strains from naturally occurring natans could be grouped in a third branch as a separate nodules of Neptunia natans plants in Senegal. These homogeneous group (internal correlation coefficient of strains grew rapidly on YMA, produced exopoly- 73 YO)distinct from every other described species. saccharides and exhibited a particular spectrum of carbon source utilization. We employed a polyphasic approach to the taxonomic characterization of this G C content of DNA and DNA-DNA hybridizations + new group of tropical rhizobia, using techniques with The G + (' content of ORS 992'' is 60.1 mol YO.We wide discriminative powers (16s rRNA gene sequenc- found a high degree of DNA-DNA binding (89%) ing and auxanography) and others at the species and between two strains (ORS 992T and ORS 997) of the infra-species levels (DNA-DNA hybridization, SDS- new N. ricitirns group. PAGE, PCR-RFLP of the ITS). Two screening methods, SDS-PAGE protein profile analysis and Analysis of the 165 rRNA genes numerical analysis of auxanographic data, indicated that these isolates constitute a homogeneous phenon, The determined sequence of the 16s rRNA gene of distinct from a wide range of rhizobia, agro- strain LMG 11875 consisted of 1433 bases. A search in bacteria and other related bacteria (Figs 1 and 4). the EMBL database revealed the new sequence to be These results suggested that this group constituted a most similar to the 16s rRNA gene sequence of separate species, which we further characterized using Agrobactt,rium vitis, thereby placing the new isolates in genotypic techniques with diverse taxonomic dis- the Rhizc,hiuni-Agrobat.tc.rium group of the alpha criminative powers. subclass of the Proteohucteria. A dendrogram showing the phylogenetic relationships of strain LMG 11875 PCR-RFLP analysis of the ITS of 16s-23s genes and reprcsentatives of the alpha subclass of the demonstrated that the new Neptunia isolates constitute Proteohcic tuiii is shown in Fig. 3. a homogeneous genotypic group (Fig. 2), and this was further supported by the high level of DNA-DNA %) Numerical analysis of auxanographic results binding (89 found between two representative strains. Our RFLP analysis of the 16s-23s ITS The six h eptunia isolates were tested for assimilation confirmed that the new group is also genotypically of 147 organic compounds as sole carbon source using distinct from representatives of all known species of the API 50 system, and the results were compared with the Rhizobium-Agrobacteriunz group (Fig. 2). To those of representative strains of Sinorhizohiumfredii, determine precisely the phylogenetic position of the Sino rli izoh iunz me 1ilo ti, Sin0r h izob ium t er angae , Sino - Nepturzia natans isolates, the 16s rRNA gene sequence rliizohiui77 saheli, Mesorhizohium loti, Mesorhizobium of a representative strain (LMG 11875) was deter- h uak uii. Mesorh izob iuni plur farium, Rhizob ium Iegu- mined. Phylogenetic analysis revealed that this strain is m in osar ui 11, Rh izoh iuni t ropici, Rh izobium galegae, related to the Agrobacterium lineage that contains
~~ International Journal of Systematic Bacteriology 48 1285 P. de Lajudie and others
Table 3. Results of carbon assimilation tests performed with Allorhizobium undicola and reference strains of Rhizobium galegae, Agrobacterium bv. 1, Agrobacterium vitis and Agrobacterium rub;
M = Number of strains studied; results recorded for strains Agrobacterium vitis LMG 257 and LMG 258, Allorhizobium undiicolo ORS 991, ORS 992T, ORS 995, ORS 996, ORS 997 and ORS 998, Rhizobium galegae LMG 6214T and LMG 6215, Agrobacterium bv. 1 LMG 140, LMG 147, LMG 187T,LMG 196, LMG 268, LMG 303, LMG 383 and Agrobacterium rubi LMG 156T. +, All strains are positive; -, all strains are negative; the values are the percentage of positive strains. The main discriminative results between Allorhizobium undicolcl and Agvobucteriurn vitis are given in bold face. The reaction of the type strain is given in parentheses. All strains grew in API 50 on glycerol, ribose, L-arabinose, D-xylose, D-galactose, D-glucose, D-fructose, D-mannose, D-cellobiose, D-maltose, lactose, rhamnose, D-turanose, D-lyxose, inositol, mannitol, D-arabitol, fumarate, DL-lactate, D-malate, L- (a)-alanine, L-proline and L-histidine and did not grow on erythritol, aesculin, inulin, starch, glycogen, isobutyrate, n-valerate, isovalerate, n-caproate, heptanoate, caprylate, pelargonate, caprate, maleate, oxalate, adipate, pimelate, suberate, azelate, sebacate, glycolate, laevulinate, citraconate, itaconate, mesaconate, phenylacetate, benzoate, o-hydroxybenzoate, D-mandelate, L- mandelate, phthalate, isophthalate, terephthalate, glycine, DL-norvaline, ~~-2-aminobutyrate,L-methionine, L-phenylalanine, L- tyrosine, D-tryptophan, L-tryptophan, DL-kynurenine, creatine, urea, acetamide, ethylamine, butylamine, amylamine, benzylamine, diaminobutane, spermine, histamine and tryptamine.
Substrate An.- vitis Al. undicola R. galegae Ag. bv. 1 Ag. vubi (n = 2) (n = 6) (n = 2) (n = 7) (n = 1)
Dulcitol, methyl a-D-glucoside, D-melezitose, D-tagatose, L- - + + arabitol, 5-ketogluconate, propionate, aconitate, L-lysine, L- citrulline, sarcosine, ethanolamine Methyl a-D-xyloside, xylitol - + + D-Tartrate, mesotartrate, rn-hydroxybenzoate - - - Arbutin + + + L-Ornithine - + + DL-Glycerate, adonitol, N-acetylglucosamine, D-melibiose, + + + gluconate, D-raffinose, L-fucose L-Tartrate, citrate + - - Glutarate + 50 - Butyrate + - - Malonate - - + L-Sorbose - 75 + D-Arabinose, 2-ketogluconate 50 + + Methyl a-D-mannoside 50 - - Amygdalin 50 + + L-Xylose 50 50 - P-Gen tio bio se 50 + + Sorbitol, acetate + + + Salicin + + + Trehalose, D-fucose + + + Succinate + + + Sucrose, ~~-3-hydroxybutyrate,L-malate + + + Pyruva te + 75 + 2-Ketoglutarate - 50 + p-Hydroxybenzoate + 75 + L-Leucine - 25 - L-Isoleucine, L-valine - 25 - D-(a)-Alanine, L-norleucine - 50 - L-Cysteine - 75 - L-Serine, L-threonine 50 + + Trigonelline + 25 - L- Aspartate + + + L-Glutamate 50 + + L-Arginine - + + Betaine 50 + + P-Alanine - 50 + DL-3-Aminobutyrate - - - ~~-4-Aminobu tyra te + 75 + DL-5-Aminovalerate - 25 + 2-Aminobenzoate, 3-aminobenzoate7 4-aminobenzoate - - - Glucosamine + 75 +
1286 International Journal of Systematic Bacteriology 48 Allorhizobium undicola gen. nov., sp. nov.
Table 4. Discriminatory utilization of carbohydrates as sole carbon source in Allorhizobium and other related genera and phylogenetic groups ...... -...... Results from this work, de Lajudie et al. (1994, 1998), Nour et al. (1994, 1995) and Chen et al. (1991, 1995). +, All strains are positive; - , all strains are negative; d, some strains are positive.
Carbohydrate Allorhizobium Agrobacterium Rhizobium Rhizobium Rhizobium* Sinorhizobiumt Mesovhizobiumf Azorhizobium bv. 1 vitis galegae
- Adonilol + + + + + + - D-Arabinox - + d + + d d - - L-FUCOS~: + + + + d d - N-Acetylgluco~ainine - + + + d + + - D-Melibiose - + + + + + d - - 1,-Riaffinosc + f + + + d - Trehalose d + + + + + + - Methyl xylosidi. - + - + + d - - Sucrose d + f + f + d - Xylitol - + - + + d d - L-Arabitol - + - - + d d - Cluconate - + + + + d d + Succinate d + + + d + + + ~~-glycera~r + + + d d d + * Results for Rhizobium tropici, Rhizohium leguminosarum and Agrobacterium bv. 2. 7 Results fcr Sinorhizobium fredii, Sinorhizobium terangae, Sinorhizohium saheli and Sinorhizobium meliloti. 1Results for Mesorhizobium loti, Mesorhizobium huakuii, Mesorhizobium ciceri, Mesorhizobium mediterraneum, Mesorhizobium tianshantws i’ and Mesorhizobium plurijarium.
~
Agrobactc&m tumefaciens bv. 1, Agrobacterium rubi, other genera [Agrobacterium (94.5-96.3 YOsequence Agrobacttbrium strains isolated from Ficus, Agro- similarity, Sinorhizobium (94-2% sequence similarity bacterium vitis and Rhizobium galegae (Fig. 3). Se- with Sinorhizobiumfredii) and Mesorhizobium (93.1 YO quence similarity values of members of this lineage sequence similarity with Mesorhizobium loti)] are also with strain LMG 11875 ranged from 94.5 to 95.2 %, closely related to the new group. The proposal of a new with the highest value found between this strain and Rhizobium species for the Neptunia isolates is thus Agrobactorium vitis strain LMG 8750 (96.3 YOsequence excluded on phylogenetic grounds. similarity. corresponding to at least 55 base dif- ferences). From the level of these similarity values, it (ii) The new group could be described as a new can be presumed that there is no significant DNA- Agrobacterium species, because phylogenetically it DNA binding between these groups of organisms; in belongs to the bv. 1 Agrobacterium lineage, which consequence, and following the recommendation of contains the type strain of the type species of the genus Stackebrandt & Goebel (1994), no additional DNA- Agrobacterium. However, it is generally recognized DNA hybridizations were performed. that the species delineation in this genus is unclear and needs revision. Although the new group is clearly The results of all the techniques used converge to the distinct from all other Agrobacterium species in the conclusion that the new isolates from N. natans bv. 1 lineage (Fig. 3), the peripheral position of nodules iorm a homogeneous group that can be in this lineage and the presence phenotypically and genotypically distinguished from Agrobacterium vitis of Rhizobium galegae, together with the low bootstrap other described species of rhizobia and agrobacteria. values (Fig. 3), indicate that this lineage may represent It is clear- that this taxon represents at least a new several genera, and it therefore seems unwise to create species. The genus allocation of this group is less a new Agrobacterium species in this group. In addition clear-cut and several possibilities are apparent : to these phylogenetic considerations, the inclusion of (i) The new group could be described as a new a non-tumorigenic species in the genus Agrobacterium Rhizobiurii species, because it lives in a nitrogen-fixing would undoubtedly raise opposition from phyto- symbiosis with leguminous plants and is phylo- pathologists and lead to considerable practical genetically related to Rhizobium galegae (Fig. 3, problems. sequence similarity 95.1 YO).However, it is clear that both the new group and Rhizobium galegae are (iii) The new group could be described as a new genus phylogenctically distinct from the lineage that contains of nitrogen-fixing legume symbionts. It is most closely the type species of the genus Rhizobium, Rhizobium related to Rhizobium galegae and Agrobacterium vitis, leguminosarum, and therefore represents the true genus but the 16s rRNA gene sequence similarity levels RIiizohiuin (Fig. 3, sequence similarity of the Rhizo- (approx. 95-5-96 Yo) and the low bootstrap values bium leguminosarum lineage with strain LMG 1 1875 (Fig. 3) suggest that none of these relationships is ranges from 92.9 to 93.5 YO).Furthermore, several particularly significant at present. In view of the data
-. _~ lnterna tional Journal of Systematic Bacteriology 48 1287 P. de Lajudie and others presented above, we propose to create a new genus, wherein the bacteria occur as intracellular symbionts. Allorhizobium, with one new species, Allorhizobium All strains exhibit host specificity. No strain was found undicola, to describe the new N. natans isolates. to nodulate Seshunia rostrata, Sesbunia pubescens, A number of discriminative features between Allo- Sesban ia grandiflor a, Vigna ungu iculat a or Macrop t i- and its phylogenetic relatives, lium atropurpureum. The G + C content of the DNA is rlzizobium Rhizobium, 60.1 mol YO(by The type species is Sino rh izobium , Mesor/z izob ium, Azo rli izob ium, Rhizo- Tm). AlIorlzizobiuiPi At the molecular level the genus can be hiurn galegae and Agrobacteriurn species can be found undicola. in Tables 3 and 4. In particular, at least 12 features can recognized by SDS-PAGE whole-cell protein analysis, ITS PCR-RFLP and 16s rRNA gene sequencing. be used to discriminate between Allorhizobium undicola and its closest phylogenetic neighbour, Agrobacterium vitis : growth on adonitol, N-acetylglucosamine, D- Description of Allorhizobium undicola sp. nov. melibiose, D-raffinose, L-fucose, gluconate, butyrate, (un.di’c0.h. L. n. water; glutarate, DL-glycerate, L-tartrate, citrate and L- Allorhizobium undicola unda ornithine (Table 3). L. suff. cola dweller; L. n. undicolu water-dweller, re- ferring to the isolation of these strains from nodules of In recent years, polyphasic research into the genus the aquatic plant Neptunia natans). Rhizobium has resulted in its gradual subdivision, with the proposal of Bradvrhizobium (Jordan, 1982), Sino- Strains have all the characteristics of the genus rhizohium (Chen et al., 1988) and, more recently, Allorhizobium. They grow fast and form colonies of Mesorlzizobium (Jarvis et ul., 1997). The proposal of 0.5-3 mm diameter within 1-2 d on YMA. Colonies Allorlzizobium is a further step in this process. From are round, creamy, convex to drop-like, beige- the phylogenetic data (Fig. 3), it is evident that the coloured; margin and surfxe have a smooth aspect. taxonomic position of Rlzizobiunz galegae and Agro- Aerobic, Gram-negative, non-spore-forming rods that hncterium vitis should be revised because these species are 0.5-0.7 pm wide by 2-4 ym long. Motile in liquid are distinct from the phylogenetic groups containing medium. A wide range of carbohydrates, organic acids the type strain of their genus. However, our study and amino acids are utilized as sole carbon sources for mainly concerned the new Neptunia isolates and growth (Table 3). Discriminating features from related therefore we refrain from making any formal proposals species are given in Table 3. Strains are 3-ketolactose- for these other taxa just yet. The taxonomic revision of negative. Strains can induce nitrogen-fixing nodules on Rhizobium galegae and Agrobacterium vitis is a com- their original host N. natans and can also nodulate plex issue, linked with the revision of the genus Medicngo sativa, Acacia senegal, Acacia seyal, Acaciu Agrohacterium, and can only be attempted after tortilis subsp. raddiana, Lotus arabicus and Fuidherhia extensive study of the literature data and international albida, but nodules do not always fix nitrogen. No consultation. strain was found to nodulate Sesbnnia rostrutu, Sesbania pubescens, Sesbania grandiflora, Vigna un- or Description of Allorhizobium gen. nov. guiculuta Macroptiliunz atropurpureum. They can be differentiated by SDS-PAGE of their total AIlor/zizobium gen. nov. (Al.lo.rhi.zo’bi.um. Gr. adj. cellular proteins, and at the molecular level by PCR- allos other; M.L. neut. n. Rhizobium a bacterial RFLP profiles of the ITS and the sequence of their 16s generic name; M.L. neut. n. Allorhizobiunz the other rRNA gene. Rhizobium,to refer to the fact that it is phylogenetically separate from other rhizobia). The well-studied strain ORS 992T (=LMG 11875), Aerobic, Gram-negative, non-spore-forming rods that isolated from N. nutans in Senegal, is designated as the are 0.5-0.7 pm wide by 2-4 ym long. Strains grow fast type strain and its features are given in Tables 2 and 3. and form colonies of 0-5-3 mm diameter within 1-2 d The G + C content of ORS 992T is 60.1 mol %. All on yeast mannitol mineral salts agar. Pronounced Allorhizohium undicola strains have been deposited in turbidity develops after 1-2 d in agitated broth media. the Culture Collection of the Laboratorium voor Chemo-organotrophic, utilizing a wide range of carbo- Microbiologie, University of Gent, in the Culture hydrates, organic acids and amino acids as sole carbon Collection of the Laboratory of Soil Microbiology, sources for growth (Table 3). Discriminative features ORSTOM, Dakar, Senegal, and in the Culture Col- between Allorhizobium and other related genera and lection of LSTM, CIRAD-ORSTOM, Baillarguet, phylogenetic groups are shown in Table 4. 3-Keto- France. lactose is not produced from lactose. Growth on carbohydrate media is usually accompanied by extra- ACKNOWLEDGEMENTS cellular polysaccharide production. The organisms are typically able to invade the root hairs of some We thank F. Dazzo, E. James and J. Sprent for helpful discussions. We thank D. Monget and bioMirieux, temperate-zone (Medicago sutiva) and some tropical Montalieu-Vercieu, France, for kindly supplying API zone (Neptunia natans, Acacia senegal, Acacia seyal, galleries. We thank B. Pot for helpful discussion and Acacia tortilis subsp. raddiana, Lotus arabicus, software assistance and J. Bakhoum, P. Tendeng, D. Badji, Faidherbia albida) leguminous plants (family Legumi- 0. Camara and T. Badji for technical assistance. This work nosae) and induce the production of root nodules, was supported by the Commission of the European Corn-
~~ ~- 1288 lnterna tional Journal of Systematic Bacteriology 48 Allorhizobiuni undicolu gen. nov., sp. nov. munities (STD3 programme, contract TS2 01 69-F; natural populations of the nitrogen-fixing bacterium Rhizobium BRIDGE programme, contracts BIOT-CT91-0263 and meliloti. .4ppl Enviroiz Microbiol 56, 187-1 94. BIOT-CT9 1-0294; by French and Belgian Embassies Felsenstein, J. (1982). Numerical methods for inferring evol- through Programme d'Actions Integrees franco-belge utionary trees. Q Rev Biol57, 379-404. Tournesol 94085). M.G. is indebted to the Fund for James, E. K., Sprent, J., Sutherland, J., Mclnroy, S. & Minchin, F. Scientific Research - Flanders (Belgium), for research and personnel grants. A.W. is indebted to the Fund for Scientific (1992). The structure of nitrogen fixing root nodules on the aquatic mimosoid legume Neptuniaplena. Ann Bot 69, 173-1 80. Research - Flanders (Belgium) for a position as post- doctoral rehearch fellow. Jarvis, B. D. W., Gillis, M. & De Ley, J. (1986). Intra- and intergeneric similarities between the ribosomal ribonucleic acid REFERENCES cistrons of Rhizohium and Brac
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