international Journal of Systematic Bacteriology (1998), 48, 549-563 Printed in Great Britain

Burkholderia graminis sp. nov., a rhizospheric species, and reassessment of []phenazinium, [Pseudomonas] pyrrocinia and [Pseudomonas] glathei as Burkholderia

Vkronique Viallard,' Isabelle Poirier,' Benoit Cournoyer,' Jacqueline Haurat,' Sue Wiebkin,3 Kathy Ophel-Keller3 and Jacques Balandreaul

Author for correspondence : VCronique Viallard. Tel: + 33 4 72 44 80 00. Fax: + 33 4 72 43 12 23. e-mail : lemsl @biomserv.univ-lyonl .fr

1 Laboratoire d'Ecologie In a survey of soil and wheat or maize rhizoplane isolated using a Microbienne du Sol, medium containing azelaic acid and tryptamine as sole carbon and nitrogen UMR5557 CNRS-Universit6 Lyon I, 43 Bd du 11 sources, respectively, a large proportion of Burkholderia-li ke bacteria were Novembre 1918, 69 622 found. Among them, a homogeneous group of strains was identifiable based Vi Ileurbanne cedex, France on phenotypic properties, fatty acid composition, DNA-DNA hybridizations 2 ENSBANA, Campus and 16s rDNA sequences. According to molecular data, this group belongs to Universitaire de the genus Burkholderia but its weak similarity to previously described species Montmuzard, 2 Bd Gabriel, 21000 Dijon, France suggests that it belongs to a novel species. Closest 16s rDNA phylogenetic neighbours of this species are Burkholderia caryophylli and two previously 3 SARDI Field Crops Pathology Unit, Waite named Pseudomonas species which clearly appear to be part of the Research Precinct, Glen Burkholderia genus and were thus named Burkholderia glathei comb. nov. and Osmond, SA 5064, Burkholderia phenazinium comb. nov. Strains of the new species are oxidase- Austra Iia and catalase-positive, produce indole and gelatinase, and use L-xylose, lactose, rhamnose, trehalose, D-lyxose, L-arabitol, xylitol and D-raff inose as sole carbon source. This novel taxon is named Burkholderia graminis. In the course of this study, [Pseudomonas] pyrrocinia also proved to be a member of the Burkholderia genus.

Keywords : Burkholderia graminis sp. nov., genus Burkholderia, phenotypic analysis, genotypic analysis

INTRODUCTION by Doudoroff & Palleroni (14), along with other bacteria that utilize arginine and betaine as sole carbon Pseudomonas cepacia was first mentioned by Ballard et source, such as Pseudomonas mallei, Pseudomonas al. (3) in 1970. This term was used for bacteria pseudomallei, Pseudomonas caryophylli and Pseudo- responsible for bulbiferous Aliaceae root rot diseases monas gladioli, formerly Pseudornonas marginata (3 1). which had been described in decaying onions in 1950 This grouping was consistent with that of Ballard et al. by Burkholder (8). In 1966, Stanier et al. (47) had (3) and Palleroni et al. (37) using rRNA-DNA already described a Pseudomonas species (Pseudo- hybridization. rnonas rnultivorans), which later on was recognized to be identical to P. cepacia by Palleroni & Holmes (36), In 1992, Yabuuchi et al. (61) proposed to assign these who validly described P. cepacia in 198 1. bacteria to a new genus, Burkholderia. As well as the above-mentioned species (i.e. Burkholderia caryo- This species was ascribed to section I1 of Pseudomonas phylli, Burkholderia cepacia, Burkholderia gladioli, Burkholderia mallei and Burkholderia pseudornallei), The GenBank accession numbers for the sequences reported in this paper two other [Pseudomonas]species were also included on are U96927-U96941. the basis of 16s rRNA sequences, DNA-DNA hybrid-

00647 0 1998 IUMS 549 V. Viallard and others ization studies, cellular lipids and fatty acids, and of Lyon (France) on an alluvial soil where maize is grown phenotypic properties ; these were Pseudomonas sola- continuously; isolates have been obtained from the soil and nacearum and Pseudomonas pickettii (40). Later on, Li the rhizoplane (washed roots, macerated and diluted) of et al. (60) came to the conclusion that the latter two germinating, flowering or senescent maize root systems. species and [Alcaligenes] eutrophus were a separate Kapunda is an experimental field, 80 km north of Adelaide (South Australia), where wheat is grown either continuously lineage and Gillis et al. (20) proposed that they were a or in rotation with a lupin-based pasture; the soil is an separate genus. In 1995, Yabuuchi et al. (62) validly alphisol. Walpeup is an experimental wheat-growing station, described Ralstonia solanacearum, Ralstonia pickettii situated in Victoria (Australia), on a very poor sandy soil, in and Ralstonia eutropha. Both genera belong to the a fixed sand dune system. Soil samples of the two Australian same rRNA group I1 of [Pseudomonas] as defined by stations have been collected and used for growing wheat (cv. Palleroni et al. (37). Spear) in pots under glasshouse conditions (three plants per pot containing 1.5 kg soil). After 3-4 weeks, wheat plants In 1994, Urakami et al. (53) transferred the [Pseudo- were harvested and used to isolate bacteria from their monas] species Pseudomonas glumae (28) and Pseudo- rhizoplane, as above. A few strains were isolated directly on monas plantarii (2) to the genus Burkholderia and also PCAT medium from salt-affected and hydrophobic soils described a new species, Burkholderia vandii. To these near Adelaide. Also included in Table 1 are 18 reference Burkholderia species, Gillis et al. (20) added a nitrogen- strains of Burkholderia, Pseudomonas, Ralstonia and Alca- fixing bacterium discovered in Vietnam rice fields ligenes. Among the eleven type strains of Burkholderia (49-5 1) known as Burkholderia vietnamiensis. They species, only type strains of B. mallei and B. pseudomallei also transferred to the genus Burkholderia the species were not grown in this laboratory. [Pseudomonas] andropogonis ( = woodsii) (48) and Biochemical characterization. All tests were performed at [Pseudomonas]cocovenenans (see also 63). 28 "C. The Biolog GN system was used as recommended by the manufacturer to test the oxidation of 95 carbon The genus Burkholderia currently comprises eleven substrates. Results were read automatically with a spectro- species ; Burkholderia andropogonis, B. caryophylli, B. photometer after 24 or 48 h incubation at 28 "C. To test the cepacia, Burkholderia cocovenenans, B. gladioli, Burk- reproducibility of the method, eight isolates were run in holderia glumae, B. mallei, Burkholderia plantarii, B. duplicate. Numerical analysis of the results was made using pseudomallei, B. vandii and B. vietnamiensis. This genus the GN Microlog 2N software which calculates Microlog belongs to the P-subclass of the (59). distances derived from the number of differences between Most of the above-mentioned species appear in section strains. This software also permits clustering analysis using I1 of Pseudomonas in Bergey's Manual (39, whereas the UPGMA (unweighted mean pair group method) al- the others are in section V. The latter section included gorithm of Sneath & Sokal(44). species whose DNA or rRNA relatedness had not been Carbon substrate assimilation tests were performed using characterized in 1984. auxanographic API 50CH strips (bioMCrieux) as recom- mended by the manufacturer. Nine isolates were tested in During a field survey of B. cepacia populations in some duplicate. Numerical analysis was performed on data French and Australian agricultural soils (39), a large obtained after 7 d incubation. Interstrain distances were diversity of strains was isolated on PCAT agar (7), calculated using the coefficient of Dice and a phenogram was which is considered selective for B. cepacia. In com- built using UPGMA. parison with type strains from Burkholderia, Pseudo- The API 20NE microtube system (bioMCrieux) was used as monas and Ralstonia species, these isolates were a standardized method to test oxidase activity, nitrate characterized by phenotypic (Biolog, API, MIDI- reduction, gelatin and aesculin hydrolysis, glucose fermen- FAME) and genotypic (DNA-DNA hybridizations, tation, arginine dihydrolase activity and production of 16s rDNA sequencing) analyses. It became obvious indole, P-galactosidase and urease. that the isolates belonged to at least three, and possibly MIDI-FAME. The MIDI-FAME technique is based on the five or six, different Burkholderia species. One very conversion of fatty acids to methyl esters by mild alkaline homogeneous group (group A) existed among the methanolysis, followed by GLC analysis. Isolates were isolates and could not fit into any of the described grown overnight (1 6-1 8 h) on trypticase soy agar. Cells were Burkholderia or Pseudomonas species ; a novel species, removed from the plate using a plastic inoculating loop, Burkholderia graminis, is proposed. carefully scraped to avoid including medium in the sample. Cells were then transferred to glass tubes. In the first step, cells were saponified; 1 ml methanolic base (45 g NaOH, METHODS 150 ml methanol, 150 ml distilled water) was added before vortexing for 5-10 s and heating to 100 "C for 5 min. After Bacterial strains and medium. Strains used in this study are vortexing again, tubes were heated for a further 25 min at listed in Table 1. Unless otherwise stated, strains were 100 "C. Cells were then methylated as follows: after cooling isolated using PCAT medium [composition (in g 1-l) : in cold water, 2 ml methylation reagent (325 ml hydrochloric MgSO,, 0.1 ; azelaic acid, 2; tryptamine, 0.2; K,HPO,, 4; acid, 275ml methanol) was added and the tubes were KH,PO,, 4; yeast extract, 0.02 (pH 5.7)] (7). Only strains vortexed for 5-lOs, heated at 80°C for lOmin, and then forming white or beige opaque shining colonies with an rapidly cooled. Finally, fatty acids were extracted by entire margin were considered. They came mostly from three addition of 1.25 ml of a 50/50 mixture of HPLC grade fields: La C6te Saint AndrC, Kapunda and Walpeup. La hexane and methyl-tert-butyl ether to each tube. After C6te Saint AndrC is an experimental field located 40 km east 10 min on a rotary shaker, the aqueous phase was discarded,

550 International Journal of Systematic Bacteriology 48 Analysis of Burkholderia graminis sp. nov.

Table 1. Strains used in this study ...... Type strains are indicated by a superscript T. ATCC, American Type Culture Collection, Rockville, MD, USA; LMG, Culture Collection, Laboratorium voor Microbiologie, State University of Ghent, Ghent, Belgium; SA, South Australia; CSA, C6te Saint Andr6, France.

Strain Source GenBank no. Origin (reference)

AUS3-AUS9 SA, salty pasture soil* AUSlO-AUS 12, AUS28-AUS29, AUS3 1-AUS34 Kapunda (SA), wheat pasture rotation* AUS35 U9694 1 Kapunda (SA), wheat pasture rotation* AUS36-AUS3 8 Kapunda (SA), wheat pasture rotation* AUS13-AUS15, AUS26-AUS27, AUS30 Kapunda (SA), continuous wheat* AUS 16-AUS20 Kapunda (SA), native vegetation* AUS2 1 Walpeup , continuous wheat * AUS22-AUS23 Walpeup, native vegetation* AUS39-AUS40, AUS42-AUS44 SA, hydrophobic soils* C3AlM U96940 CSA, maize senescent root system* C3BlM U96938 CSA, maize senescent root system* C4BlM, C4ClM, C5AlM CSA, maize senescent root system* C4DlMT U96939 CSA, maize senescent root system* C3DlSn, C3D3Sn CSA, bare soil* C4A 1B CSA, wheat* C4AlMj, C3AlMj, C3ClMj CSA soil, young maize (3 weeks old) in pots* C4A3P, C4A7P CSA, pasture* m33d, m45 CSA young maize (1 month)* m35b U96937 CSA young maize (1 month)* m130 Brazil, maize rhizosphere PHQB 17 France, continuous wheat (23) 526 ATCC 53267 ' Blue Circle ', USA, maize rhizosphere Burkholderia sp. strain N2P5 U37342 Phenanthrene-enriched soil (33) Burkholderia sp. strain N3P2 u37344 Phenanthrene-enriched soil (33) Burkholderia sp. strain N2P6 u37343 Phenanthrene-enriched soil (33) Burkholderia sp. strain CRE7 U37340 Phenanthrene-enriched soil (33) [Pseudomonas] sp. strain LB400 U86373 (30) Burkholderia sp. strain JB1 X92188 Garden soil (46) Burkholderia sp. strain GSOY U16140 (17) Burkholderia sp. strain E264 U91838 (6) B. andropogonis ATCC 23061T X67037 Sorghum (48) B. caryophylli ATCC 2541gT X67039 Carnation (3) B. cepacia ATCC 25416T U96927 Onion sour skin (3) B. cepacia ATCC 17759 X87275 Forest soil (30) B. cocovenenans ATCC 33664T U96934 Fermented coconut (54) B. gladioli ATCC 1024gT X67038 Gladiolus sp. (43) B. glumae ATCC 33617T U9693 1 Rice (29) ' B. norimbergensis' YO9879 (28) B. plantarii LMG 9035T U96933 Rice pathogen (2) B. pseudomallei strainl026b U9 1839 (6) B. vandii LMG 16020T U96932 Orchid rhizosphere (53) B. vietnamiensis TVV70 U96929 Rice rhizosphere, Vietnam (20) B. vietnamiensis TVV75T LMG 10929T U96928 Rice rhizosphere, Vietnam (20) [P.]glathei ATCC 29195T U96935 Fossil lateritic soil (64) [P,]phenazinium LMG 2247T U96936 Soil (4) [P.]pyrrocinia ATCC 1595gT U96930 Unknown (25) R. eutropha ATCC 17697T M32021 Soil (12) R. eutropha CH34 LMG 1195 Waste water zinc factory (32) R. pickettii ATCC 275 11 X67042 Clinical (40) R. solanacearum ATCC 11696T X67036 Tomato pathogen (45) A. xylosoxidans ATCC 2706IT C 1in ic a 1 N. polysaccharea ATCC 4376gT LO6 167 Throat of a healthy child (1 3) * Isolated in this study.

International Journal of Systematic Bacteriology 48 551 V. Viallard and others

10%

C3D 1Sn B. an dropogon isT C4A1 B C4A1 Mj AUS44 au540 AUS18 C3D3Sn au542 [P.] glatheiT B. cocovenenansT C4A1 Mj C3C1 MJ. B. plantariiT C3A1 MJ B. glumae' C3D3Sn B. gladioli' B. vandi7 B. caryophylliT B. plantarii'" [P.] phenaziniumT E B. cocovenenansT - AUS22 B. glumaeT 4 AUS44 8. gladioliT au539 [P.] phenazinium' - AUS18 AUS5-AUS7 AUS10-AUS1 1 AUSl5 AUS17 AUSl9-AUS21 AUS22-AUS23 - AUS28* A AUS33 - au528 A

b= AUS8US40 B. vietnamiensisT B. cepaciaT* [P.] pyrrociniaT AUSl2-AUS14 PHQB17 AUS26 AUS27" AUS29* AUS31 -AUS32 r AUS34 AUS30* B. cepaciaT* AUS36 [P.] pyrrociniaT AUS37 AUS38 AUS12-AUS14 m33d au524 m35b AUS26-AUS27 m45 C3B1 M* au529 M130 AUS30-AUS32 526 au534 AUS36-AUS38 B. caryophylliT m33d B. andropogonisT m35b* B. pickettii' m45 A. xylosoxidansT C3B1 M* R. eutropha CH34 R. solanacearumT RauwtrophaT AUS42 . Fig, I. UPGMA dendrograms obtained with the phenotypic analyses. (a) Biolog characteristics are used; bar represents 10% Microlog Distance. (b) API characteristics; bar represents 10% Dice Distance. See Table 1 for information about strains and GenBank accession numbers. *, Strains tested in duplicate. A, Phenon A; B, phenon B.

552 International Journal of Systematic Bacteriology 48 Analysis of Burkholderia graminis sp. nov.

3 ml NaOH (0.3 M) was added and the combination was of B. cepacia, B. andropogonis, B. caryophylli, B. gladioli, B. mixed before centrifugation. The upper phase was carefully pseudomallei and those of other members of the P-sub- removed and used for analysis. Analysis was carried out with division of the Proteobacteria like Ralstonia solanacearum, a Hewlett Packard HP 5890 Gas Chromatograph equipped R. pickettii and R. eutropha. The GenBank database also with a phenyl methyl silicone fused silica capillary column contained sequences of unknown bacteria which according (HP Ultra 2-25 m x 0.2 mm x 0.33 mm film thickness) and a to the BLAST software (1) appeared to be related to our flame ionization detector. Hydrogen was used as the carrier isolates: strains CRE7, N3P2, N2P5, N2P6 (33), strain JB1 gas. The temperature programme was initiated at 170 "C (46), ' Burkholderia norimbergensis' (27), strain GSOY (1 7), and increased at 5 "C min-l to a final temperature of 270 "C. strain E264 (6) and strain LB400 (29). Neisseria poly- saccharea (L06167) was used as an outgroup. Only well- DNA manipulation. DNA was extracted according to the defined sequences with less than five undetermined nucleo- procedure of Brenner et al. (5). To further purify DNA, tides were used. Sequences were aligned using CLUSTAL v (24) extra chloroform/isoamyl alcohol extraction steps were between positions 98 and 1496 (E. coli numbering). Align- added. Mean G+C contents (molY0) of five phenon A ment was refined manually using the SUNMASE algorithm strain DNAs were determined by HPLC (38). (1 5). A stretch of uncertain alignment (position 209-220) was removed before calculating painvise evolutionary dis- PCR and 165 rDNA sequencing. Five isolates from the various tances according to Jukes & Cantor (26) with the DIFFCOUNT field conditions were chosen as representatives of the two program. Phylogenetic trees were constructed using the major clusters obtained after phenotypic analysis : strains neighbour-joining method of Saitou & Nei (42) with the AUS35, C4D1MTand C3AlM (representative of phenon A) PHYLO-WIN graphic tool (19). The topology of this distance and m35b and C3BlM (representative of phenon B). The tree was tested by 1000 bootstrap resamplings of data (16). 16s rDNA of these strains was sequenced. Parsimony analysis was done using the PHYLO-WIN program. The 16s rDNA sequences of B. cepacia, B. cocovenenans, B. DNA-DNA hybridization. Native DNAs of strains ATCC vandii, B. glumae, B. plantarii, B. vietnamiensis, [Pseudo- 25416T and C4DlMT were labelled in vitro by nick-trans- monas] phenazinium, [Pseudomonas] glathei and [Pseudo- lation (4 1) with tritium-labelled nucleotides (Amersham). monas]pyrrocinia type strains and B. vietnamiensis TVV70 The procedure used for the hybridization experiments (S1 were also determined. nuclease/ trichloracetic acid method) has been described by The oligonucleotides used to amplify the 16s + intergenic Crosa et al. (1 1). DNA fragments (500 bp long) obtained by spacer region of the rRNA gene were 5' ATGGA(GA)AG- sonication were used. (TC)TTGATCCTGGCTCA 3' and 5' CCGGGTTTCCCC- ATTCGG 3' derived from the rrn sequences of Frankia sp. (34). PCR was performed in a final volume of 50 pl, under a thin layer of paraffin oil, directly on a reaction mixture RESULTS containing 1 pl cell suspension (about lo9cells ml-l) in 50 YO Phenotypic analysis of Burkholderia and related (w/v) glycerol, 5 p1 buffer [ 10 pM Tris/HCl, pH 8.2; 1.5 mM strains MgC1,; 50 mM KCl; 0.01 YO(w/v) gelatin], 20 pM of each dNTP (Pharmacia), 0.5 pM of each primer and 2.5 U TaqI General characteristics of sugar metabolism. The pheno- DNA polymerase (Gibco-BRL). Amplifications were car- ried out on a dry block thermocycler using the following typic comparisons between the strains isolated in this programme: 3 min at 95 "C followed by 35 cycles of study and the type strains of the different Burkholderia denaturation (1 min at 95 "C), annealing (1 min at 55 "C) species were based on the oxidation of 95 carbon and extension (2 min at 72 "C), and then a final extension of substrates (Biolog) and the assimilation of 49 carbon 10min at 72 "C. To check for amplification efficiency, sources (API) (Fig. 1, Table 2). In the two analyses, amplification products (5 pl) were run on a 2% horizontal most of our isolates (about 70%) were contained in agarose gel in TBE buffer at 4Vcm-l. The PCR ampli- only two phenons, named A and B. Reproducibility fication products were visualized by ethidium bromide was good with API in which nine strains were tested in staining. duplicate; results were extremely similar for the repli- cates (96100%). In Biolog tests, eight strains were Sequencing of the 16s rRNA gene only (positions 58-1541 run in duplicate and only 80-91 YO of results were of the Escherichia coli 16s rDNA sequence) was performed identical. by Genome Express, Grenoble, France. Sequences of both strands were determined using the following five oligo- Ralstonia strains were easily differentiated using both nucleotides (which correspond to positions 20-43, 5 18-532, Biolog and API. With both methods, B. andropogonis 885-904,152 1-1 540 and 880-899 of the E. coli small-subunit had only a weak similarity to other Burkholderia rDNA sequence, respectively) : 5' TGGCTCAGAACGA- strains. In the phenotypic analysis, it was difficult to ACGCTGGCGGC 3'; 5' AGCCTTGCGGCCGTACTC- CC 3'; 5' CAGCAGCCGCGGTAA 3'; 5' AAGGAG- assign B. caryophylli and B. andropogonis a clear GGGATCCAGCCGCA 3'; and 5' GCCTGGGGAGC- generic status ; they were located on the same branch as TCGGCCGCA 3'. Ralstonia in the Biolog analysis, whereas they were closer to Burkholderia after their API scoring. Strains Phylogenetic analyses. All DNA sequences were deposited AUS8 and AUS42 were also given different positions in the EMBL/GenBank database (Table 1). They were in the two analyses. This might be a consequence of the compared with previously published 16s rDNA sequences presence, among the strains analysed, of a large

International Journal of Systematic Bacteriology 48 553 V. Viallard and others

Table 2. Phenotypic characteristics of phenon A strains as opposed to type strains of Burkholderia and [Pseudornonas] species

Strains used: 1, phenon A (19 isolates); 2, [P.]phenazinium; 3, [P.]glathei; 4, B. cepacia; 5, [P.]pyrrocinia; 6, B. vietnamiensis; 7, B. glumae; 8, B. plantarii; 9, B. gladioli; 10, B. caryophylli; 11, B. andropogonis; 12, B. vandii; 13, B. cocovenenans; 14, B. mallei; and 15, B. pseudomallei. For B. mallei and B. pseudomallei, results are those of Yabuuchi et al. (61); where these results differ from those of Palleroni (39, the results of Palleroni are given in parentheses. All strains in the Table can use the following compounds as sole carbon source : glycerol, galactose, D-glucose, D-mannose, inositol and mannitol. None can grow on: L- sorbose, methyl a-D-mannoside, methyl p-D-glucoside, melezitose, inulin and D-turanose. + , a 90 % of strains are positive; - , 2 90 YOof strains are negative; D + , 11-89 YOof strains are positive and the type strain is positive; D -, 11-89 YOof strains are positive and the type strain is negative; D, 11-89 YOof strains are positive; ND,not reported.

Assimilation of: Strain

1 2 3 4 5 6 7 8 910111213 14 15

Gluconate + ++++++++++++ D-Arabinose + ++++++++++++ D-Arabitol + ++++++++++++ Fructose + ++++++++++++ L- Arabinose + ++++++++++++ N-Acetylglucosamine + +++++++++- ++ Ribose + ++++++++++++ Sorbitol + ++++++++++++ L-Fucose + +++++++++- ++ D-Xylose + +++++++++- ++ 2-Ketogluconate + +++++++++-++ Trehalose + -- +++++++- ++ D-Lyxose + -+++++++- +- + Dulcitol - +++++++-- ++ I Adonitol + ++++- +- +++-- Sucrose D+ +-+++--- +--- D- Fuco se D+ - +-- +-+++-- + D-Tagatose - - +++++- +--- + l 5-Ketogluconate + ++++--- +--- + Xylitol + ++++---- +-- + L-Arabitol + +- ++----+--- Cellobiose D+ Rhamnose + Aesculin* , D-Raffinose + P-Gentiobiose D- Amygdalin Lactose + ~ L-Xylose + Arbutin Erythritol Melibiose Salicio Maltose Starch I Glycogen * Hydrolysis of aesculin. number of poorly related strains contrasting with a In contrast, two phenons called A and B were clearly large number of closely related ones. The closely differentiated using both API and Biolog (Fig. 1) and related strains constitute a few well-defined clusters, they contained roughly the same strains for slightly whereas the poorly related strains form an unstable different levels of relatedness. The similarity of phenon cloud which is very sensitive to small phenotypic A strains was more than 97 YOusing API but only 82 % differences. using Biolog; for phenon B strains, similarities were

554 International Journal of Systematic Bacteriology 48 Analysis of Burkholderia graminis sp. nov.

Euclidian distance Kapunda (five strains). Strains isolated from wheat 30 20 10 0 growing on Kapunda (three strains) or Walpeup (two I I 1 I strains) soils collected under native vegetation were B. plantarii7 exclusively of the A phenotype. Strains isolated in B. vandii7 France all came from the roots of senescent maize B. pickettii7 plants after harvest. [R] g/athei7 B. andropogonis' Strains of phenon B. API phenon B contained 20 strains, IB. cocovenenans' which were all also found in Biolog phenon B. The B. gladioli7 Biolog test was also done on two more strains, i.e. [R] phenaziniuml ml30 and 526. Moreover, with Biolog, B. vietnamiensis R. eutropha' type strain TW75T clustered with phenon B; this B. caryophy/li7 explained the apparent discrepancy in strain numbers 4IB. g/umaeT between Biolog (23 strains) and API (20 strains) in phenon B. The maximum distances between phenon B 41 AUS35 strains were 22% (Biolog) and 4% (API). Phenon AUS33 B included one (API) or two (Biolog) type strains of C4B1 M Burkholderia (B. cepacia and/or B. vietnamiensis) plus C4D1 M the type strain of [P.]pyrrocinia. In consequence, the latter strain was further characterized by molecular rn techniques. B. vietnamiensisT AUS27 Phenon A characteristics AUS30 AUS 29 Burkholderia characteristics of phenon A. Like all the B. cepacia' Burkholderia species and [P.]glathei, [P.]phenazinium m35b and [P.]pyrrocinia type strains tested, phenon A strains i C3B1 M oxidized Tween 40, L-arabinose, D-arabitol, D-fruc- [R]pyrrocinia' tose, D-galactose, a-D-glucose, myo-inositol, D-man- M130 nitol, D-mannose, D-sorbitol, methyl pyruvate, mono- 526 methyl succinate, D- and L-alanine, L-asparagine, L- rn45 proline, L-serine, and cis-aconitic, formic, D-gluconic, ...... P-hydroxybutyric, DL-lactic, malonic, D-saccharic, Fig. 2. Similarity dendrograrn obtained with MIDI-FAME data. succinic, bromosuccinic, L-aspartic and L-glutamic Euclidian distances are shown. A, Phenon A; B, phenon B. acids. Among these characteristics, the oxidation of D-arabitol, myo-inositol, D-mannitol, D-mannose and D-sorbitol differentiated the studied strains from the three Ralstonia strains tested. 96 YOusing API and 78 % using Biolog. Phenon A did not contain any reference strain. According to the Like all the Burkholderia species and [P.]glathei, [P.] Biolog analysis, phenon B contained both B. viet- phenazinium and [P.]pyrrocinia type strains tested in namiensis and B. cepacia along with [P.]pyrrocinia. this study, phenon A strains assimilated (Table 2) the following as sole carbon source: glycerol, D- and Strains of phenon A. In oxidation and assimilation L-arabinose, ribose, galactose, D-glucose, D-fructose, tests, the same 19 isolates were tightly clustered in D-mannose, inositol, mannitol, sorbitol, D-arabitol phenon A and a twentieth strain, AUS22, clustered and gluconate. All the Burkholderia species and [P.] with phenon A when analysed by Biolog, but not by glathei, [P.]phenaziniumand [P.]pyrrociniatype strains API. The strains showed a maximum 18 % Microlog tested did not use L-sorbose, methyl P-D-xyloside, distance in painvise comparisons based on the oxi- methyl a-D-mannoside, methyl a-D-glucoside, dation tests (Biolog) whereas this distance was less erythritol, maltose, inulin, melezitose, starch, glycogen than 2.8% with the API system. No representative or D-turanose as sole carbon source. strain of any of the twelve Burkholderia or [Pseudo- monas] species tested was present in this group. The Particular properties of phenon A. The 19 strains of group comprised strains isolated from all the different phenon A formed thin, brownish-yellow, translucent soils studied, both in Australia (15 strains) and in colonies on LB agar. On PCAT agar, growth needed 2 France (four strains). Australian strains came from or 3 d at 28 "C. On this medium, colonies were white, Kapunda (nine strains), Walpeup (two strains), salt- opaque and creamy. These isolates had catalase, affected pastures (three strains) and a hydrophobic soil oxidase, urease and P-galactosidase activities. They (one strain). Kapunda and Walpeup strains have been reduced nitrate to nitrite. They did not ferment glucose isolated from wheat roots grown on a soil coming from and did not hydrolyse aesculin or produce indole or the continuous wheat treatment (one in Kapunda and gelatinase. All 19 strains used L-xylose, lactose, rham- one in Walpeup) and the wheatlpasture rotation in nose, trehalose, D-lyxose, L-arabitol, xylitol and raffi- lnternational Journal of Systematic Bacteriology 48 555 V. Viallard and others

c53 [P.J phenaziniumT 99* Strain N2P6 0.0073 s/s

I Strain N2P5 114 B. caryophylliT Strain E264 B. pseudomallei strain 1026b B. vandii' B. plantarir'

B. gladioliT L B. glumaeT B. cepacia ATCC 17759 47 B. cepaciaT Strain CRE7 B. ~ietnarniensis~ [R]pyrrocinia' C3B1 M f- m35b Strain GSOY B. andropogonisT ,oo Strain JB1 'Burkholderia norimbergensis' R. eutropha' R. pickettiiT R. so lana cea rumT I Neisseria polysacchareaT

Fig. 3. Phylogenetic neighbour-joining tree obtained with the 16s rDNA sequences of members of Burkholderia, [Pseudomonas] and Ralstonia species. Numbers at nodes represent the percentages of bootstrap samplings (heuristic search based on 1000 resamplings; only values greater than 50% are shown). Asterisks indicate branches also found in the parsimony analysis with bootstrap values greater than 50 %. Analyses correspond to positions 98-209 and 22Cb1496 (E. coli numbering). The scale bar represents 0,0073 fixed mutations per nucleotide position. See Table 1 for GenBank accession numbers. B. vietnamiensis type strain and B. vietnamiensis W70, C4D1MT and C3AlM show the same 165 sequences. nose; none of them used dulcitol or D-tagatose as sole teristic (20). Among seven analysed phenon A strains, carbon source. This represented a combination of AUS28 was slightly different (Fig. 2). The six re- characteristics unique among Burkholderia species maining strains had a rather similar fatty acid com- (Table 2). position including (mean values) : 16: lcu7c (23 YO); 16 :0 (20 %) ;and a large (40 YO)mixed unresolved peak corresponding to 18: h9c, 18: lcol2t, 18: lcu7c. There Fatty acid composition was no obvious difference between phenon A and B Fatty acid analysis of the 19 phenon A and B strains strains. analysed showed the presence of 3-OH 16 :0, a charac- Phenon B strains were clearly assigned to two different teristic feature of the Burkholderia genus. This com- groups; one contained one maize strain (m33d), B. pound represented 3-5 % of total fatty acids. The two vietnamiensis and three Kapunda strains, and the other Ralstonia strains analysed contained no trace of 3-OH one contained maize strains together with B. cepacia 16 :0, confirming the taxonomic value of this charac- and [P.]pyrrocinia.

556 International Journal of Systematic Bacteriology 48 Analysis of Burkholderia grarninis sp. nov.

Table 3. Results of DNA-DNA hybridization experiments

Source of unlabelled DNA Reassociation (%) at 72 "C with tritium-labelled DNA from:

C4DllW (phenon A) B. cepacia ATCC 2541Q

Phenon A :* C4D1MT 100 17 C3AlM 89 16 C4BlM 84 17 C5AlM 83 17 AUS28 86 14 AUS33 77 15 AUS35 75 17 Phenon B:* PHQB17 15 60 C3BlM 13 53 AUS27 15 53 AUS30 15 54 Other strains : C4ClM 30 18 C4A3P 26 15 C4A7P 29 20 C3AlMj 27 15 C3ClMj 24 17 C4AlB 15 14 C3D 1Sn 19 16 AUS18 30 13 N2P5 24 ND N3P2 30 ND Type strains : B. cepacia 13 100 B. andropogonis 3 ND B. caryophylli 8 5 B. cocovenenans 8 ND B. gladioli 12 27 B. glumae 11 21 B. plantarii 12 26 B. vandii 13 ND B. vietnamiensis 19 41 [P.]glathei 17 14 [P.]phenazinium 16 18 [P.]pyrrocinia 12 48 R. pickettii 3 ND R. solanacearum 3 ND m, Not determined. * See Fig. 1(a, b).

Phylogenetic analysis related sequences present in the GenBank database. Phylogenetic trees were inferred using the neighbour- The 16s rDNA sequence (about 1500 bp) was de- joining and parsimony algorithms and rooted with N. termined for the following strains: AUS35, C4D1MT, polysaccharea (Fig. 3). C3AlM (which belong to phenon A), and m35b and C3BlM (phenon B). The 16s rDNA sequences were The whole genus Burkholderia formed a homogeneous also determined for the type strains of B. cepacia, B. cluster; the maximum difference between the 16s cocovenenans, B. plantarii, B. vietnamiensis, B. glumae, sequences was 6.2 % (between B. andropogonis type [P.]glathei, [P.]phenazinium and [P.]pyrrocinia. These strain and strain LB400) and this cluster was stable sequences were aligned and compared with the closely (bootstrap value of 96%). As well as members of

International Journal of Systematic Bacteriology 48 557 V. Viallard and others acknowledged Burkholderia species, the genus con- populations of this species, use of PCAT medium, tained [P.]pyrrocinia, [P.]phenazinium, [P.]glathei and which was described by Burbage & Sasser (7) as being phenon A strains. This was supported by parsimony specific for Burkholderia cepacia, was evaluated. Ac- analysis. This genus was clearly distinct from the genus cording to Burbage & Sasser, no growth was observed Ralstonia, which was also coherent. B. gladioli and B. on PCAT for 12 isolates of Agrobacterium, Erwinia, cocovenenans appeared to be highly related (99.9 YO Xanthomonas and Pseudomonas whereas all B. cepacia similarity). Similarly, B. glumae, B. vandii and B. strains tested grew well. In our study, bacteria isolated plantarii also appeared to be closely related, with a from some French and Australian soils using PCAT high level of similarity (994-99-5 YO). have been further characterized to investigate the selectivity of this medium. All the strains obtained are Strains C4DlMTand C3AlM had the same 16s rDNA indeed related to Burkholderia, on the basis of their sequences. They differed from AUS35 by only two phenotype or genotype. However, they belong to nucleotides. The distance between C4D lMT and the various species. Most of Burkholderia species type nearest sequence (N3P2) was 1.8%. The distance strains can grow on PCAT in spite of the fact that none between C4D1MT and [P.]phenazinium was 2.4%. of them has been isolated on PCAT. Nevertheless, on Strains m35b and C3BlM had distances of 1.2 and PCAT, growth of B. vandii type strain is scarce and B. 0.7 % from B. cepacia, respectively. andropogonis and B. caryophylli type strains do not The B. andropogonis 16s sequence was different grow. The type strains of B. mallei and B. pseudomallei enough to put this species on a separate branch. do not grow on PCAT, whereas all recent isolates can Strains JB 1 and ' Burkholderia norimbergensis' were (D. Vidal & F. Thibaut, personal communication). even further away from core Burkholderia species. The closely related R. solanacearum and R. eutropha type strains do not grow on PCAT. Thus, there seems DNA-DNA hybridization to be some taxonomic meaning to growth on PCAT but its level of specificity deserves more research. This DNA of C4D1MT was hybridized to DNA of seven is of increasing importance as there is a strong need for phenon A strains (C4D1MT,AUS28, AUS33, AUS35, a selective medium to isolate Burkholderia strains from C3A 1M, C4B 1M and C5Al M) ; reassociation values clinical and environmental specimens (58, 10, 22, 52, (Table 3) were 75-89% within phenon A. With nine 56). Burkholderia species type strains, low levels of hom- ology (3--19%) were obtained. [P.] pyrrocinia, [P.] Burkholderia vs Ralstonia phenazinium and [P.]glathei exhibited 12, 16 and 17 % homology, respectively, with C4D lMT. R. solana- Strains isolated using PCAT were further charac- cearum and R. pickettii type strains gave hybridization terized by Biolog and API methods. In terms of values of 3 %. methodologies, both API and Biolog methods are Labelled DNA of C4D1MT was also hybridized with applicable for highlighting groups of related strains DNA of isolates phenotypically closely related to against a background of more distantly related ones. phenon A such as C4ClM, C4A3P, C4A7P, C3AlMj, Biolog has a lower reproducibility and is not very C3ClMj, AUS18, C4AlB and C3DlSn; hybridization accurate when considering distantly related micro- values were 15-30 %. organisms. API 50CH appears more reliable but is less efficient when dealing with closely related phenotypes Strains N2P5 and N3P2, which exhibited a high degree because of the smaller number of characters treated. of 16s sequence similarity with C4D lMT, showed The presence of 3-OH 16:O among fatty acids is hybridization values of 24 and 30 YO,respectively. confirmed as a phylogenetically meaningful charac- Hybridization of B. cepacia ATCC 25416T DNA with teristic to distinguish Burkholderia from Ralstonia. DNA of some phenon B isolates (C3BlM, AUS27, AUS30 and PHQB 17) showed reassociation levels of B. andropogonis 53-60 YO.Labelled DNA of B. cepacia ATCC 25416T showed low levels of reassociation (5-27%) with B. With phenotypic and molecular methods, there is a caryophylli, B. plantarii, B. glumae, B. gladioli, [P.] very clear-cut distinction between Burkholderia and glathei and [P.]phenazinium, whereas [P.] pyrrocinia the few Ralstonia reference strains studied. Never- and B. vietnamiensis appeared to be more closely theless, some ambiguity arises about B. andropogonis related to B. cepacia (reassociation levels of 48 and and B. caryophylli. When using Biolog, type strains of 4 1 YO,respectively). both species cluster with Ralstonia, whereas when using API, B. caryophylli stays with Burkholderia while B. andropogonis clusters with Ralstonia, as already DISCUSSION seen by Gillis et al. (20). When molecular data are Isolation medium taken into account, B. caryophylli again appears as a real Burkholderia, whereas B. andropogonis has a 16s This study was initiated with the aim of characterizing sequence that is somewhat different (often more than the in situ intraspecific diversity of B. cepacia. To 4 YO)from those of other Burkholderia species; more- obtain a set of isolates representative of natural over, in the neighbour-joining tree, B. andropogonis is

558 International Journal of Systematic Bacteriology 48 Analysis of Burkholderia graminis sp. nov. situated on a branch that is separate from all other The inclusion of three [Pseudomonas]species does not Burkholderia species. This confirms its marginal status change the definition of the genus Burkholderia. in the genus, which is already obvious according to the Auxanographic studies show that none of the charac- phenotypic results obtained. Nevertheless, its sequence teristics common to all previously described Burk- similarity to the other Burkholderia species is much holderia species are different in any of the new species. greater than that to any Ralstonia species. In conse- A reservation should be made for B. mallei and B. quence, there is no obvious reason to place B. pseudornallei; these two species were not grown for this andropogonis in a separate genus for the time being. study and literature data (61, 35) were used in which contradictory results have been reported. Beyond this B. phenazinium, B. glathei and B. pyrrocinia comb. reservation, the genus Burkholderia can be defined as nov. follows. All species can grow with the following substrates as sole carbon source : glycerol, D-arabinose, In the course of this study, Biolog and API diagnostic galactose, D-glucose, D-mannose, inositol, mannitol, software and the fatty acid analysis pointed to the sorbitol and gluconate. Nevertheless, contrary to all possible presence among the isolates of strains related other studies, Yabuuchi et al. (61) found that B. to [P.] phenaziniurn (4), [P.] glathei (64) and [P.] gladioli could not grow on D-arabinose. The following pyrrocinia (26). The possibility that these [Pseudo- compounds are not used by any Burkholderia species: monas] species would constitute exceptions to the methyl P-D-xyloside, L-sorbose, methyl a-D-manno- selectivity of PCAT prompted a re-evaluation of their side, methyl a-D-glucoside, maltose, inulin, melezitose taxonomic status. DNA-DNA hybridization with B. and D-turanose. Nevertheless, Yabuuchi et al. (61) and cepacia is high (18, 14 and 48 YOfor [P.]phenaziniurn, Gillis et al. (20) reported growth of B. cepacia and B. [P.]glathei and [P.]pyrrocinia, respectively). Accord- vietnarniensis, respectively, on maltose. The results ing to their 16s rDNA sequences, these [Pseudornonas] reported herein support those of Gillis et al. (20) on are closer to B. cepacia ATCC 25416T (respective many strains of Burkholderia and Ralstonia and show similarities of 96.4, 96.8 and 99.2%) than to the that utilization of a few substrates is probably enough recognized Burkholderia species B. andropogonis (se- to distinguish members of these two genera; D- quence similarity of 95 YO).In consequence, they mannose is used by all Burkholderia and no Ralstonia actually belong to the Burkholderia genus and should and most Ralstonia do not use sorbitol and mannitol, be renamed B. phenaziniurn, B. glathei and B. pyrro- whereas most Burkholderia can. cinia comb. nov. It is worth noting that, based on phenotypic properties, Gillis et al. (20) have already Phenon A taxonomic status suggested that [P.]phenazinium could belong to the Burkholderia genus, although there was a lack of Among soil isolates studied, two phenons (A and B) rRNA-DNA hybridization data. Similarly, consider- are dominant. Phenon A strains have been isolated ing its special type of tyrosine-inhibited aro- from a large diversity of rhizospheres, very different genate/NADP dehydrogenase activity, Byng et al. (9) soil types from France and South Australia, and had already suggested that [P.] pyrrocinia could be rhizospheres of native Australian plants, wheat, maize closely related to Burkholderia. and pasture grasses. Phenotypic analyses revealed that all these strains are very similar. Three strains have had The Burkholderia genus revisited their rDNA sequenced; two have exactly the same 16s rDNA sequence, and the third one differs only by two The number of Burkholderia species validly described bases. DNAs of six phenon A strains, when hybridized thus amounts to 14. These are B. andropogonis, B. with the DNA of C4D1MT (chosen to represent this caryophylli, B. cepacia, B. cocovenenans, B. gladioli, B. new phenon), have a very high reassociation value glathei, B. glurnae, B. mallei, B. phenaziniurn, B. (7549% at 72 "C). Phenon A thus fulfils the pre- plantarii, B. pseudornallei, B. pyrrocinia, B. vandii and requisites of a new species (55). In the phylogenetic B. vietnarniensis. Many of these species have very tree, the three sequenced strains form a cluster inside similar 16s rDNA sequences, and the species status of the Burkholderia genus. Moreover, their sequences are them remains uncertain as long as it has not been highly homologous (956 and 95.8 YO)to the sequence confirmed by DNA-DNA hybridization studies. For of the genus type strain B. cepacia ATCC 25416T. instance, B. vandii and B. glurnae are very closely Thus, the phylogenetic position confirms the pheno- related to B. plantarii (similarities of 99.5 and 99.4 YO, typic analyses (API, Biolog) and places this geno- respectively). The distinction of these three species is species into the genus Burkholderia. nevertheless supported by DNA-DNA hybridization experiments conducted by Urakami et al. (53). These Proposal of Burkholderia graminis results show the limitations of 16s rRNA sequencing for differentiation of closely related species (18). The Phenon A strains are phenotypically different from all species B. gladioli and B. cocovenenans also exhibit a the above Burkholderia species tested (B.andropogonis, high level of similarity in their 16s rDNA sequences B. caryophylli, B. cepacia, B. cocovenenans, B. gladioli, (99.9 YO)and, thus far, no DNA-DNA hybridization B. glathei, B. glumae, B. phenaziniurn, B. plantarii, B. has confirmed their status of separate species. pyrrocinia, B. vandii and B. vietnarniensis).For security

In termtiona I lourna I of Systematic Bacteriology 48 559 V. Viallard and others reasons, it was not possible to compare their pheno- currently subject to more taxonomic work which will types to those of the two remaining Burkholderia be published later. species, B. mallei and B. pseudomallei. Nevertheless, they differ from the description given for these two Conclusions pathogenic species (35, 61) by many characteristics. C4D lMT homology with other Burkholderia species On the basis of this polyphasic approach, the genus type strains is 3-19%. At the level of 16s rDNA Burkholderia appears to be more complex than pre- sequences, this strain differs from all other known viously thought. The phenotypic data show that 20 of species of Burkholderia (similarity is never more than the 60 strains isolated do not belong to any phenon 97.6%). In conclusion, phenon A strains can be containing a known species. Moreover, they seem distinguished by all the different approaches used : extremely diverse. phenotypic properties, MIDI-FAME analysis, DNA- Contrasting with this large background diversity, two DNA reassociation values and 16s ribosomal sequence analysis. All criteria defined by Vandamme et al. (55) groups of isolates appear to be clearly dominant in the are verified and, consequently, phenon A strains situations studied, in France as well as in Australia. should be considered as a new species. The name One group, containing phenon B isolates, is not Burkholderia graminis sp. nov. is proposed for this new resolved at the taxonomic level and will be subject to species, referring to its known habitat, the rhizosphere further studies; it seems to constitute a branching of grasses. cluster, centred around B. cepacia, B. vietnamiensis and [P.]pyrrocinia. The latter taxon is more properly described as B. pyrrocinia. Strains related to B. graminis The other group corresponds to the very homogeneous In the GenBank database there are some 16s rDNA phenon A. High DNA-DNA hybridization values and sequences which are highly similar to the C4D1MT the three very similar 16s DNA sequences confirm that sequence and could eventually belong to B. graminis. these strains constitute a genospecies (21, 55). The These belong to strains N2P5, N3P2 and N2P6 (33), name B. graminis is proposed for this group. The most and to strain LB400 (29). DNAs of N2P5 and N3P2 closely related species are [P.]phenazinium and [P.] have hybridization values of 24 and 30 %, respectively, glathei, which should be renamed B. phenazinium and with C4D1MT DNA. This is not enough to consider B. glathei. them as belonging to B. graminis, but they are very closely related. According to its 16s sequence, LB400 Description of Burkholderia graminis sp. nov. is not closer to C4D1MT. Moreover, B. graminis and B. phenazinium belong to the same branch on the Burkholderia graminis (gra'miais. M.L. adj. graminis neighbour-joining tree (with 99 % of bootstrap data referring to its isolation from the rhizosphere of samplings) and this situation is confirmed by the grasses). parsimony tree. Nevertheless, the DNA-DNA re- association value between B. graminis C4D 1MT and B. Motile cells, 1.0-13 pm in length, 0-3-0-8pm in width. phenazinium is 16% confirming that the two species On LB agar, colonies are thin, brownish-yellow, are related but different (57). B. graminis thus appears translucent. Grows in 3 d on PCAT (7) at 28 "C, to be a completely new taxon. forming white colonies, more or less opaque and creamy, with an entire margin. Oxidase, catalase, urease and arginine dihydrolase are produced. Re- Phenon B taxonomic status duces nitrates to nitrites but does not denitrify. Like all Burkholderia, can assimilate the following as sole Phenon B is a heterogeneous cluster containing pheno- carbon source : glycerol, D- and L-arabinose, ribose, typically related strains belonging to different geno- galactose, D-glucose, D-fructose, D-mannose, inositol, species. When measured, their DNA-DNA homology mannitol, sorbitol, D-arabitol, gluconate and 2-keto- (determined by reassociation) with B. cepacia ATCC gluconate. Like all Burkholderia, cannot use L-sorbose, 25416T is higher than 47% but never close to 70% methyl a-D-xyloside, methyl a-D-mannoside, methyl (data not shown), showing that they are closely related a-D-glucoside, indin, melezitose, starch, glycogen or but different. The two isolates sequenced, m35b and D-turanose as sole carbon source. Contrary to other C3BlM, show a high similarity to B. cepacia (98.8 and Burkholderia species, does not acidify glucose, does 99.3 %, respectively) and B. vietnamiensis (98.7 and not hydrolyse aesculin nor produce indole or gela- 99%, respectively), but they differ more from each tinase. Grows on L-xylose, lactose, rhamnose, tre- other (99% similarity) than B. cepacia does from B. halose, D-lyxose, L-arabitol, xylitol and raffinose, but vietnamiensis (99.4 YO).The fatty acid composition of not on dulcitol or D-tagatose as sole carbon source. phenon B strains also shows the heterogeneity of this The G + C content is 62-5-63.0 mol YO.Isolated from group (Fig. 2). Phenon B thus constitutes a complex of the rhizosphere of wheat, corn and pasture grasses. related species, not resolved enough by the set of data The proposed type strain is C4D1MT (G + C content reported in this article. It contains [P.]pyrrocinia and 63 mol %), which has been deposited in the ATCC and strains of clinical origin (data not shown). It is given the accession number ATCC 700544.

560 In ternationa I Jo urnaI of Systematic Bacteriology 48 Analysis of Burkholderia graminis sp. nov.

Description of Burkholderia phenazinium corn b. nov. 9. Byng, G. S., Whitaker, R. J., Gherna, R. L. & Jensen, R. A. (Bell and Turner 1973) (1980). Variable enzymological patterning in tyrosine bio- synthesis as a means of determining natural relatedness The description of B. phenazinium is the description among the Pseudomonadaceae. J Bacterioll44,247-257. given by Bell & Turner (4). The type strain is NCIB 10. Cimolai, N., Trombley, C., Davidson, A. G. F. & Wong, L. T. K. 11027T (= LMG 2247T, = ATCC 33666T), isolated (1995). Selective media for isolation of Burkholderia (Pseu- from soil, and it is able to use L-threonine as sole domonas) cepacia from the respiratory secretions of patients carbon source. with cystic fibrosis. J Clin Pathol48, 5. 11.' Crosa, 1. M. D., Brenner, D. 1. & Falkow, 5. (1973). Use of a Description of Burkholderia pyrrocinia corn b. nov. single-strand-specific nuclease for analysis of bacterial and (Imanaka, Kousaka, Tamura and Arima 1965) plasmid deoxyribonucleic acid homo- and heteroduplexes. J Bacteriol 115, 904-9 11. The description of B. pyrrocinia is the description given by Imanaka et al. (25). The type strain is ATCC 12. Davis, D. H., Doudoroff, M., Stanier, R. Y. & Mandel, M. 1 ( = LMG 1419 1 '), which is of unknown origin. (1969). Proposal to reject the genus Hydrogenomonas : 5958T taxonomic implications. Int J Syst Bacterioll9, 375-390. Description of Burkholderia glathei comb. nov. (Zolg 13. Dewhirst, F. E., Chen, C.-K. C., Paster, B. J. & Zambon, J. 1. (1992). Phylogeny of species in the family Neisseriaceae and Ottow 1975) isolated from human dental plaque and description of The description of B. glathei is the description given by Kingella orale sp. nov. Int J Syst Bacteriol43, 490-499. Zolg & Ottow (64). The type strain is ATCC 29195' 14. Doudoroff, M. & Palleroni, N. J. (1974). Genus Pseudomonas. ( = LMG 14 1 goT), isolated from a fossil lateritic soil in In Bergey's Manual of Determinative Bacteriology, 8th edn, Germany. pp. 217-243. Edited by R. E. Buchanan & N. E. Gibbons. Baltimore: Williams & Wilkins. ACKNOWLEDGEMENTS 15. Faulkner, D. V. & Jurka, 1. (1988). Multiple aligned sequence editor (MASE). Trends Biochem Sci 13, 321-322. The authors are grateful to Maria Fernandez for her help in 16. Felsenstein, J. (1985). Confidence limits on phylogenies : an discussing DNA-DNA hybridization results, to Cindy approach using the bootstrap. Evolution 39, 783-79 1. Morris for allowing us to use the Microlog (Biolog) software, to Bruce Hawke who performed the MIDI-FAME analyses 17. Floro, G., Buzzelli, J. A,, Griffin, W. & Stolz, J. F. (1997). and to HCheMeugnier who determined the G + C contents. Aerobic degradation of soy diesel by Burkholderia sp. This research has been financially supported by a CNRS- Unpublished (quoted in GenBank). CSIRO PICS (International Cooperative Scientific Pro- 18. Fox, G. E., Wisotzkey, J. D. & Jurtshuk, P. 1. (1992). How close gramme). J.B. is grateful to C. Pankhurst, CSIRO Soil is close: 16s rRNA-sequence identity may not be sufficient Division, Adelaide (South Australia) who made available to guarantee species identity. Int J Syst Bacteriol 42, his laboratory facilities during a sabbatical leave financially 166-1 70. supported by an OECD fellowship. 19. Galtier, N., Gouy, M. & Gautier, C. (1996). SEAVIEW and PHYLO-WIN : two graphic tools for sequence alignment and REFERENCES molecular phylogeny. Comput Appl Biosci 12, 543-548. 1. Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, 20. Gillis, M., Tran Van, V., Bardin, R. & 7 other authors (1995). D. J. (1990). Basic local alignment search tool. J Mol Biol Polyphasic in the genus Burkholderia leading to 215,403-410. an emended description of the genus and the proposition of 2. Azegami, K., Nishiyama, K., Watanabe, Y., Kadota, I., Ohuchi, Burkholderia vietnamiensis sp. nov. for N,-fixing isolates A. & Fukazawa, C. (1987). Pseudomonas plantarii sp. nov., from rice in Vietnam. Int J Syst Bacteriol45, 274-289. the causal agent of rice seedling blight. Int J Syst Bacteriol 21. Grimont, P. A. D. (1988). Use of DNA reassociation in 37, 144-152. bacterial classification. Can J Microbiol34, 54 1-546. 3. Ballard, R. W., Palleroni, N. J., Stanier, R. Y. & Mandel, M. 22. Hagedorn, C., Could, W. D., Bardinelli, T. R. & Gustavson, (1 970). Taxonomy of the aerobic pseudomonads Pseudo- D. R. (1987). A selective medium for enumeration and monas cepacia, P. marginata, P. alliicola and P. caryophylli. recovery of Pseudomonas cepacia biotypes from soil. Appl J Gen MicrobioE60, 199-214. Environ MicrobioE53, 2265-2268.

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562 International Journal of Systematic Bacteriology 48 Analysis of Burkholderia graminis sp. nov.

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