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INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Jan. 1982, p. 136-139 Vol. 32, No. 1 0020-7713/82/0201 36-04$02.00/0

NOTES

Transfer of japonicum Buchanan 1980 to Bradyrhizobium gen. nov., a Genus of Slow-Growing, from Leguminous Plants D. C. JORDAN Microbiology Department, College of Biological Science, University of Guelph, Guelph, Onfurio, Canctdu NIG2WI

Recent data indicate that the slow-growing, non-acid-producing root nodule bacteria of leguminous plants should be separated from the fast-growing, acid- producing strains and placed in a new genus. The separation is warranted by numerical , deoxyribonucleic acid base ratio determinations, nucleic acid hybridization, ribosomal ribonucleic acid cistron similarities, serology, composition of extracellular gum, utilization and metabolism, bacteriophage and antibiotic susceptibilities, protein composition, and types of intracellular inclusion bodies in the bacteroid forms. The name proposed for the new genus is Bradyrhizobium. The type species of the genus is B. japonicum (Buchanan 1980) comb. nov. (basonym: Rhizobium japonicum Buchanan 1980), the type strain of which is ATCC 10324.

In the 8th edition of Bergey’s Manual of during the IV International Symposium on Ni- Determinative Bacteriology, Jordan and Allen trogen Fixation. This proposal was subsequently (14) subdivided the genus Rhizobium Frank 1889 approved by all the remaining members of the into two groups on the basis of flagellar arrange- Subcommittee, as was the suggestion that the ment, growth rate in yeast extract-mannitol- name of the new genus should incorporate the mineral salts medium, guanosine plus cytosine name “rhizobium” and should be prefixed by an content of the deoxyribonucleic acid (DNA), adjective signifying sluggish or slow. Both pro- and the genera of host plants nodulated. This posals received the unanimous support of the 23 subdivision was based on ‘t Mannetje’s (30) specialists who attended an open meeting of the numerical taxonomy data and the need for addi- Subcommittee held on 6 December 1980 at the tional investigations of the relationship between Australian National University. the slow- and fast-growing groups. The need to A suitable name for the new genus is Brady- separate these two groups within the existing rhizobium (Gr. adj. bradus slow; M. L. neut. n. genus Rhizobium stems from recent findings Rhizobium a bacterial generic name; M. L. neut . involving numerical taxonomy (10, 20, 30), DNA n. Bradyrhizobium the slow [growing] rhizobi- base ratios (3, 34), nucleic acid hybridization (7, um). This name has the advantage of emphasiz- 12), ribosomal ribonucleic acid cistron similar- ing the considerable agronomic importance of ities (4), serology (9, 13, 32, 33), composition of the root nodule bacteria and pays tribute to the extracellular gum (5, 6, 15, 16), carbohydrate long-standing and extensive literature on these utilization (28) and metabolism (18), bacterio- . It was such considerations phage (21) and antibiotic susceptibilities (29), which led the Judicial Commission of the Inter- protein composition based on polyacrylamide national Committee on the Nomenclature of gel electrophoresis (25), and types of intracellu- Bacteria (opinion 34, 1970, 11) to indicate the lar inclusion bodies in the bacteroids within the generic name of the nodule bacteria as Rhizobi- root nodules (2). um Frank 1889 nom. gen. cons. rather than as The proposal to establish a new genus for the Phytomyxa Schroeter 1886, which holds priori- slow-growing, non-acid-producing was ty. The utilizajion of “rhizobium” in the name initially approved by those members present at a of the proposed new genus also acknowledges meeting of the International Subcommittee on the hypothesis of Norris (22, 23), which is not Agrobacterium and Rhizobium held at the Aus- universally accepted, that the slow-growing, tralian National University on 3 December 1980 non-acid-producing strains of the root-nodule

136 VOL. 32. 1982 NOTES 137

bacteria from tropical leguminous plants repre- alkaline reaction in mineral salts-mannitol medi- sent a survival of the ancestral type which um after 28 days at 27°C. Do not produce 3- ultimately gave rise to the fast-growing, acid- ketolactose, and exhibit negative hypertrophy- producing strains associated with the temperate- initiating ability (14). zone leguminous plants. Stimulate nodule production on roots of tropi- In 1964, Graham (lo), after a taxonomic study cal and some temperate-zone leguminous plants of 83 strains of nodule .bacteria, recommended (Leguminosae)such as those of the genera Gly- that the slow-growing strains be grouped in the cine, Vigna, and Macroptilium. In addition to genus Phytomyxa. This name, however, was + certain fast-growing strains, to be placed in the proposed by Schroeter (26) to represent the root genus Rhizobium, cause root nodule production nodule bacteria in general. It has no priority as a on (L. uliginosus and L. pedunculatus), generic name for the slow growers and it is , , Cicer, Sesbania, Leu- illogical, considering that the name was original- caena, Mimosa, Lablab, and Acacia. A highly ly proposed under the assumption that these specific nodulation of the nonleguminous plant bacteria were related to the slime molds. Parasponia (Trema) by a strain of Bradyrhizo- The genus Bradyrhizobium represents an ex- bium sp. also occurs (31). Fix atmospheric nitro- ceedingly heterogeneous group of nodule bacte- gen (dinitrogen) when in the symbiotic state ria within which the taxonomic relationships are within root nodules, although ineffective nod- not well understood. It is proposed that, until ules (unable to fix nitrogen) are occasionally such time as further species or biovars, or both, formed. Bacteria present in nodules as slightly are established within the genus, there be only swollen rods (containing polyphosphate inclu- one designated species, Bradyrhizobium japoni- sions) with rare branching or as coccus forms (in cum (Buchanan 1980) comb. nov. (basonym: Arachis nodules). Rhizobium japonicum Buchanan 1980 [l]). It is The guanine-plus-cytosine content of the suggested that, for the present, the members of DNA is 62 to 66 mol% (thermal denaturation). the genus Bradyrhizobium other than B. japoni- The type species is B. japonicum (Buchanan cum be referred to as Bradyrhizobium sp. with 1980) comb. nov. the name of the appropriate host plant in paren- Brudyrhizobium japonicum (Buchanan 1980) theses immediately following, for example, Bra- comb. nov. The cells are gram-negative, non- dyrhizobium sp. (Vigna) or Bradyrhizobium sp. sporeforming, short rods 0.5 to 0.9 pm by 1.2 to (Lupinus). The species formerly designated R. 3.0 pm. Motile by one polar or subpolar flagel- lupini (14) is not being specified in the genus lum. Bradyrhizobium since its only major distinguish- The colonies are circular, opaque, rarely ing characteristic was a high degree of nodula- translucent, white, and convex, and tend to be tion affinity for Ornithopus and Lupinus spp. granular in texture. Growth on carbohydrate The taxonomic position of the nodule bacteria media usually accompanied by extracellular from Lotononis is uncertain. These organisms slime. are similar to other strains of Bradyrhizobium in Most strains grow on a mineral salts medium that they are monotrichous, grow slowly, and containing yeast extract and glucose, galactose, produce an alkaline reaction and no serum zone gluconate, glycerol, fructose, arabinose, or man- in litmus milk. However, they appear as large, nitol. Maltose is utilized by about 10% of the banded ovoids, some strains are red pigmented, strains, but lactose, rhamnose, raffinose, treha- and peptone is utilized. The cells fail to react lose, sucrose, dulcitol, and dextrin are rarely with antisera derived from other slow-growing utilized. Organic acids such as fumarate, malate, nodule bacteria or from species of Rhizobium, succinate, citrate, and pyruvate are utilized pro- and the guanine-plus-cytosine content of the vided the basal medium has sufficient Ca2+ and DNA is 68 to 69 mol% (thermal denaturation) Mg2+ to overcome the inhibitory chelating ef- (8). The strains are highly specialized, nodulat- fects of these acids. Cellulose and starch are not ing Lotononis spp. and Macroptilium atropur- utilized. pureum effectively and selected species of Aes- Some strains can utilize ammonium salts or chynomene and Crotolaria ineffectively. nitrate as a sole source of nitrogen. Certain Description of Bradyrhizobiurn gen. nov. Gram- amino acids (glutamate, histidine, aspartate, and negative, aerobic, nonsporeforming, short rods proline) serve as sole nitrogen sources, but in 0.5 to 0.9 pm by 1.2 to 3.0 pm. Motile by one this respect they are inferior to vitamin-free polar or subpolar flagellum. Produce slow casein hydrolysate. Casein and agar are not growth on yeast extract-mannitol medium, colo- hydrolyzed. Peptone is poorly utilized. nies not exceeding 1 mm in diameter within 5 to Usually acid tolerant, most strains growing at 7 days at the optimal temperature of 25 to 30°C. pH 4.5. Over 30% of the strains will grow at pH Moderate turbidity is produced only after 3 to 5 4.0 and a few as low as pH 3.5. Fails to grow days or longer in agitated broth. Produce an above pH 9.0. An alkaline reaction is produced 138 NOTES INT. J. SYST.BACTERIOL. in litmus milk, without the production of a clear, recently have been obtained from root nod- upper “serum zone.” ules collected in China. Recent studies (H. H. Keyser, Fails to grow in media containing 2% NaCl B. B. Bohlool, and T. S. Hu, unpublished data) indi- H2S. cate that these bacteria form effective root nodules on and does not produce Penicillinase produc- wild (Glycine soja) and on Glycine max cv. tion is common. Peking, a black-seeded, genetically unimproved line of NADP+-linked 6-phosphogluconate dehydro- soybeans from China. However, ineffective nodules genase (EC 1.1.1.43), the key enzyme in the are produced on all commercial soybean cultivars thus pentose phosphate pathway, is absent or virtual- far examined. The taxonomic status of these strains is ly so (18). Glucose is metabolized largely by the currently unknown. Entner-Doudoroff route. Usually has no requirement for extracellular REPRINT REQUESTS vitamins, with the rare exception of biotin. Acid- Address reprint requests to: D. C. Jordan, Microbiology ic heteropolysaccharides of the extracellular Department, College of Biological Science, University of slime are heterogeneous in both structure and Guelph, Guelph, Ontario, Canada, N1G 2W1. composition (5, 6) and contain D-galacturonic acid and, frequently, methylated sugars; neutral LITERATURE CITED glycans of the slime also are heterogeneous. Nitrogenase activity by free-living cells oc- 1. Buchanan, R. E. 1980. In. V. B. D. Skerman, V. McGowan, and P. H. A. Sneath (ed.). Approved lists of curs in certain strains but only in media contain- bacterial names. Int. J. Syst. Bacteriol. 30:225-420. ing selected carbon sources and under reduced 2. Craig, A. S., R. M. Greenwood, and K. I. Williamson. oxygen tension (17, 19, 24). Although typically 1973. Ultrastructural inclusions of rhizobial bacteroids of chemoorganotrophic, some strains possess an Lotus nodules and their taxonomic significance. Arch. Mikrobiol. 89:23-32. active uptake hydrogenase which enables them 3. De Ley, J., and A. Rassel. 1965. DNA base composition, to grow chemolithotrophically in an atmosphere flagellation and taxonomy of the genus Rhizobium. J. of hydrogen, carbon dioxide, and low levels of Gen. Microbiol. 41:85-91. oxygen (11). 4. De Smedt, J., and J. De Ley. 1977. Intra- and intergeneric similarities of Agrobacterium ribosomal ribonucleic acid Normally causes formation of effective root cistrons. Int. J. Syst. Bacteriol. 27:222-240. nodules on species of Glycine and on Macroptili- 5. Dudman, W. I. 1976. The extracellular polysaccharides of urn atropurpureum. Rhizobium juponicum: compositional studies. Carbohydr. The guanine-plus-cytosine content of the Res. 46:97-110. 6. Dudman, W. I. 1978. Structural studies of the extracellu- DNA is 61 to 65 mol% (thermal denaturation). lar polysaccharides of Rhizobium juponicum strain 71A, Type strain: ATCC 10324 (27). In addition to CC708 and CB 1795. Carbohydr. Res. 66:9-23. the characteristics representative of all strains of 7. Gibbins, A. M., and K. F. Gregory. 1972. Relatedness the species, this strain grows on a mineral salts among Rhizobium and Agrobucterium species determined by three methods of nucleic acid hybridization. J. Bacter- medium containing yeast extract and gluconate, iol. 11 1:129-141. glycerol, arabinose, or mannitol. It fails to uti- 8. Godfrey, C. A. 1972. The carotinoid pigment and deoxyri- lize effectively glucose, galactose, fructose, bonucleic base ratio of a Rhizobium which nodulates maltose, lactose, rhamnose, raffinose, trehalose, Lotononis bainesii. J. Gen. Microbiol. 72:399-402. 9. Graham, P. H. 1963. Antigenic affinities of the root- sucrose, dulcitol, and dextrin. Ammonium salts nodule bacteria of . Antonie van Leeuwenhoek J. and certain amino acids are utilized as sole Microbiol. Serol. 29:281-291. sources of nitrogen. Growth occurs over the pH 10. Graham, P. H. 1964. The application of computer tech- range 4.0 to 9.0. There is no demonstrated niques to the taxonomy of the root-nodule bacteria of legumes. J. Gen. Microbiol. 35511-517. requirement for extracellular biotin. Nitrogen- 11. Hanus, F. J., R. J. Maier, and H. J. Evans. 1979. Autotro- ase activity can be demonstrated in free-living phic growth of H,-uptake positive strains of Rhizobium cells, and growth occurs chemolithotrophically japonicum in an atmosphere supplied with hydrogen gas. in an atmosphere of hydrogen, carbon dioxide, Proc. Natl. Acad. Sci. U.S.A. 76:1788-1792. 12. Heberlein, G. T., J. De Ley, and R. Tijtgat. 1967. Deoxyri- and low levels of oxygen. bonucleic acid homology and taxonomy of Agrobucter- ium, Rhizobium, and Chromobucteriurn. J. Bacteriol. I am indebted to the following individuals for valuable 94: 116-1 24. discussion and advice: M. Vincent (University of Sydney, J. 13. Humphrey, B., J. M. Vincent, and V. Skerdleta. 1973. Sydney, Australia), B. D. W. Jarvis (Massey University, Group antigens in slow-growing Rhizobium. Arch. Mikro- Palmerston North, New Zealand), B. W. Strijdom (Plant biol. 89:79-82. Protection Research Institute, Pretoria, South Africa), W. F. 14. Jordan, D. C., and 0. N. Allen. 1974. Family 111. Rhizo- Dudman (Commonwealth Scientific and Industrial Research biuceae Conn, 1938, p. 261-264. In R. Buchanan and Organization, Canberra, Australia), R. A. Date (Common- E. N. E. Gibbons (ed.), Bergey’s manual of determinative wealth Scientific and Industrial Research Organization, St. bacteriology, 8th ed. The Williams & Wilkins Co., Balti- Lucia, Queensland, Australia), M. J. Trinick (Commonwealth more. Scientific and Industrial Research Organization, Wembly, 15. Kennedy, L. D. 1976. Isolation of 3-O-methyl-~-ribose Western Australia), and J. E. Beringer (Rothamsted Experi- Rhizobium mental Station, Harpenden, England). from polysaccharide. Carbohydr. Res. 52:259- 261. ADDENDUM IN PROOF 16. Kennedy, L. D., and R. W. Bailey. 1976. Monomethyl sugars in extracellular polysaccharides from slow-growing Fast-growing root nodule bacteria, physiologically rhizobia. Carbohydr. Res. 49:451-454. distinct from members of the genus Bradyrhizobium, 17. Kurtz, W. G. W., and T. A. LaRue. 1975. Nitrogenase VOL. 32, 1982 NOTES 139

activity in rhizobia in absence of host plant. Nature J. U. Kern's Verlag, Breslau. (London) 256407-409. 27. Skerman, V. B. D., V. McGowan, and P. H. A. Sneath 18. Martinez-De Drets, G., and A. Arias. 1972. Enzymatic (ed.). 1980. Approved lists of bacterial names. Int. J. Syst. basis for differentiation of Rhizobium into fast- and slow- Bacteriol. 30:225-420. growing groups. J. Bacteriol. 109:467-470. 28. Skotnicki, M. L., and B. G. Rolfe. 1978. Differential stim- 19. McComb, J. A., J. Elliot, and M. J. Dilworth. 1975. ulation and inhibition of growth of Rhizobium trifoliistrain Acetylene reduction by Rhizobium in pure culture. Na- T1 and other Rhizobium species by various carbon ture (London) 256:409-410. sources. Microbios 20:15-28. 20. Moffett, M. L., and R. R. Colwell. 1968. Adansonian 29. Strzelcowa, A. 1968. The use of the technique of van analysis of the Rhizobiaceae. J. Gen. Microbiol. 51:245- Schreven for the taxonomy of Rhizobiurn strains. Acta 266. Microbiol. Pol. 17:263-268. 21. Napoli, C., R. Sanders, R. Carlson, and P. Albersheim. 30. 't Mannetje, L. 1967. A re-examination of the taxonomy of 1980. Host-symbiont interactions: recognizing Rhizobi- the genus Rhizobium and related genera using numerical urn, p. 189-203. In W. E. Newton and W. H. Orme- analysis. Antonie van Leeuwenhoek J. Microbiol. Serol. Johnson (ed.), , vol. 2. University Park 33~477-491. Press, Baltimore. 31. Trinick, M. J. 1973. Symbiosis between Rhizobiurn and 22. Norris, D. 0. 1956. Legumes and the symbiosis. the non-legume Trerna aspera. Nature (London) 244:459- Emp. J. Exp. Agric. 24:247-270. 460. 23. Norris, D. 0. 1965. Acid production by Rhizobium. A 32. Vincent, J. M. 1977. Rhizobium-general microbiology, unifying concept. Plant Soil 22:143-166. p. 277-366. In R. W. F. Hardy and W. S. Silver (ed.), A 24. Pagan, J. D., J. J. Child, W. R. Scowcroft, and A. H. treatise on dinitrogen fixation, section 111. John Wiley & Gibson. 1975. Nitrogen fixation by Rhizobium cultured in Sons, Inc., New York. a defined medium. Nature (London) 256:406-407. 33. Vincent, J. M., and B. A. Humphrey. 1970. Taxonomical- 25. Roberts, G. P., W. T. Lev, L. E. Silver, and W. J. Brill. ly significant group antigens in Rhizobium. J. Gen. Micro- 1980. Use of two-dimensional polyacrylamide gel electro- biol. 63:379-382. phoresis to identify and classify Rhizobium strains. Appl. 34. Wagenbreth, D. 1961. Ein Beitrag zur systematischen Environ. Microbiol. 39:414-422. Einordnung der Knollchenbakterien durch Bestimmung 26. Schroeter, J. 1886. Die Pilze Schlesiens, p. 131-256. In F. des relativen Basengehaltes ihrer Desoxyribo-nuclein- Cohn, Kryptogamenflora von Schlesien. Band 3, Heft 3. sauren. Flora (Jena) 151:219-230.