F-54501 Vandewre-les-Nancy, and URA CNRS 1977, Ecologie Microbienne, USBSE,The taxonomic position of nitrogen-fixing strains that were isolated from rhizosphere macerates of rice cultivated in the Binh Thanh region of Vietnam was determined by using polyphasic taxonomy. We determined the phylogenetic relationships of these organisms by performing DNA-rRNA hybridization experiments with a labeled rRNA probe from the type strain of Burkholderia cepacia, and we found that they belong to a single rRNA complex. Other members of this rRNA complex were also studied, and the N,-fixing strains were found to be closely related to B. cepacia. In addition, all members of the rRNA complex containing B. cepacia were studied by performing auxanographic and DNA-DNA hybridization experiments. Phenotypically and genotypically, the N,-fixing isolates constitute a single cluster together with two strains of clinical origin. These organisms constitute a new Burkholderia species, for which the name Burkholderia vietnamiensis is proposed; the type strain of this species is TVV75 (= LMG 10929). All members of this species can fix nitrogen. On the basis of our polyphasic taxonomy results and previously published data we concluded that the genus Burkholderia should be restricted to the following species: B. cepacia (the type species), Burkholderia mallei, Burkholderia pseudomallei, B. vietnamiensis, Burkholderia gladioli, Burkholderia caryophylli, Burkholderia plantarii, Burkholderia glumue, Burkholderia vandii, BurkhoMeria cocovenenans comb. nov., and Burkholderia andropogonis comb. nov. On the basis of genotypic and phenotypic results [Alcaligenes] eutrophus, [BurkhoZderia] solanacearum, and[Burkholderia] pickettii belong to two other clusters whose internal structures must be studied further.On the basis of the results of extensive phenotypic studies and DNA-rRNA and DNA-DNA hybridization experiments performed by Palleroni, Stanier, and their collaborators, the genus Pseudomonas was divided into five groups (24, 43, 46, 56). Additional phylogenetic data have shown that these groups are only very remotely related and that each of them contains species belonging to other genera (21-23, 70, 71). In addition to the former pseudomonads that have been shown to belong to the genus Xanthomonas and to a smaller group related to Pseudomonas diminuta and Pseudomonas vesicularis (21-23,43) classified in the recently described genus Brevundirnonas (52), three large groups of pseudomonads can be considered. Pseudomonas rRNA group I (43) is part of rRNA superfamily I1 (18) or the gamma subclass of the Proteobacteria (S), where it constitutes a separate rRNA complex (18,21,22,68); this group represents the authentic pseudomonads which are grouped with Pseudomonas aeruginosa, the type species (43). Although the internal relationships within this group have not been determined yet, it is evident that the genus Pseudomonas must be limited to this group and that all other Pseudomonas species have been generically misnamed as determined by phylogenetic da...

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Deoxyribonucleic acid (DNA)-ribosomal ribonucleic acid (rRNA) hybridizations were carried out between 23s 14C-or 3H-labeled rRNAs from type strains Pseudomonas jluorescens ATCC 13525, Pseudomonas acidovorans ATCC 15668, Pseudomonas solanacearum ATCC 11696, and Xanthomonas campestris NCPPB 528 and DNA from one or more strains of several taxonomically assigned or unassigned phytopathogenic Pseudomonas species. The strains were assigned to one of the following rRNA branches: (i) the P. Jluorescens rRNA branch (the authentic pseudomonads), containing also Pseudomonas agarici, Pseudomonas amygdali, Pseudomonas asplenii, Pseudomonas caricapapayae, Pseudomonas cichorii, Pseudomonas corrugata, Pseudomonas fuscovaginae, Pseudomonas marginalis (P. marginalis pathovar marginalis, P . marginalis pathovar alfalfae, and P. marginalis pathovar pastinacae) , Pseudomonas meliae, the Pseudomonas syringae group (including "Pseudomonas aceris, " "Pseudomonas antirrhini," "Pseudomonas apii,' ' "Pseudomonas berberidis," "Pseudomonas cannabina," 'LPseudomonas coronafaciens", "Pseudomonas eriobotryae," "Pseudomonas lapsa,') "Pseudomonas maculicola," "Pseudomonas oryzicola,9' "Pseudomonas panici," "Pseudomonas passiflorae," "Pseudomonas primulae," "Pseudomonas ribicolu," "Pseudomonas sesami," L'Pseudomonas striafaciens, )' P. syringae, "Pseudomonas ulmi,' ' and "Pseudomonas viburni"), Pseudomonas toluasii, Pseudomonas viridiflava, and "Pseudomonas washingtoniae;" (ii) the P . solanacearum rRNA branch, containing P. Additional approaches have been used to clarify the relationships among the taxa in the genus Pseudomonas. In general, deoxyribonucleic acid (DNA) base compositions (mean molar percent guanine plus cytosine [G+CJ) (46) and the results of DNA-DNA hybridization (58) agreed with the phenotypic classification scheme. DNA-ribosomal ribonucleic acid (rRNA) hybridization (59) revealed the existence of five rRNA homology groups of named Pseudomonas. These findings were supported by the results of immunological characterization of the host factor required for coliphage QP ribonucleic acid replication (24), the results of cellular fatty acid composition studies (36), and enzymic data (15).Recently, De Vos and De Ley (22) confirmed the existence of the same five rRNA groups by using another DNA-rRNA hybridization technique. However, by including many more strains and in particular many strains of a great variety of gram-negative taxa, these authors found that

The deoxyribonucleic acid (DNA):ribosomal ribonucleic acid (rRNA) hybridization technique was used to reveal the relationships and taxonomic positions of an additional 83 strains belonging to 43 saprophytic or pathogenic Pseudomonus species and 29 named and unnamed Pseudomonus-like strains. The DNA:rRNA hybrids were characterized by the following two parameters: (i) the temperature at which one-half of the hybrid was eluted and (ii) the percentage of rRNA binding (the amount of rRNA bound per 100 pg of filter-fixed DNA). We also used, for a limited number of strains, numerical analysis of carbon assimilation tests to delineate the finer taxonomic relationships of organisms. Of the 83 strains examined, 78 could be definitely assigned either to an rRNA branch or to an rRNA superfamily within the Proteobucteriu. Only 25 of our strains belong in the genus Pseudomonus sensu strict0 (our PseudomonusJluorescens rRNA branch). In general, about two-thirds of the named Pseudomonas species have been misclassified and are distributed over at least seven genera all through the Proteobacteriu. These organisms need to be reclassified and generically renamed according to their phylogenetic rglationships. However, more detailed phenotypic and genotypic studies are necessary before definite nomenclatural proposals can be made. A comprehensive list of the phylogenetic affiliations of the Pseudomonus species is included.From the original creation of Pseudomonas Migula 1894 until now, this genus has been a dumping ground for incompletely described, aerobic, polarly flagellated, gram-negat h e , rodlike bacteria. In the past and even today organisms isolated from a heterogeneous variety of ecological niches were and still are classified without sufficient grounds as new Pseudomonas species. One of the reasons for this situation is the incomplete generic definition.In the United States, the Berkeley group has reported in several papers (42,43,48) on the taxonomy of the genus Pseudomonas. The several hundred strains included in the important study of these workers involved only a relatively small number of species. Their new generic definition and subdivision of Pseudomonas was reflected in Bergey 's Manual of Determinative Bacteriology, 8th ed. (20). In an important reduction in the number of Pseudomonas species, more than 90% of the clinical, phytopathological, and saprophytic pseudomonads were relegated to four addenda to the genus Pseudomonas and were thus only marginally retained in this genus (20). Most of these organisms have no clear taxonomic status because their names were not retained on the Approved Lists of Bacterial Names (38, 46) nor were they on Validation Lists 17 to 23 (24-30). In Bergey's Manual of Systematic Bacteriology , Palleroni (41) listed 62 of these species in Section V of the genus Pseudomonas, although their natural relationships with other taxa are still largely unknown.We have shown in the past (11-15, 21, 45, 51, 54, 55) that the deoxyribonucleic acid (DNA):ribosomal ribonucleic acid (rRNA) hybridization...