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

Share Embed

Descrição do Produto

international Journal of Systematic Bacteriology (1998), 48, 549-563

Printed in Great Britain

Burkholderia graminis sp. nov., a rhizospheric Burkholderia species, and reassessment of [Pseudomonas]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: e-mail : lemsl @biomserv.univ-lyonl .fr


Laboratoire d'Ecologie Microbienne du Sol, UMR5557 CNRS-Universit6 Lyon I, 43 Bd du 11 Novembre 1918, 69 622 Vi Ileurbanne cedex, France


ENSBANA, Campus Universitaire de Montmuzard, 2 Bd Gabriel, 21000 Dijon, France


SARDI Field Crops Pathology Unit, Waite Research Precinct, Glen Osmond, SA 5064, Austra Iia

+ 33 4 72 44 80 00. Fax: + 33 4 72 43 12 23.

In a survey of soil and wheat or maize rhizoplane bacteria isolated using a medium containing azelaic acid and tryptamine as sole carbon and nitrogen sources, respectively, a large proportion of Burkholderia-like bacteria were found. Among them, a homogeneous group of strains was identifiable based on phenotypic properties, f a t t y acid composition, DNA-DNA hybridizations and 16s rDNA sequences. According to molecular data, this group belongs to the genus Burkholderia but its weak similarity to previously described species suggests that it belongs t o a novel species. Closest 16s rDNA phylogenetic neighbours of this species are Burkholderia caryophylli and two previously named Pseudomonas species which clearly appear to be part of the Burkholderia genus and were thus named Burkholderia glathei comb. nov. and Burkholderiaphenazinium comb. nov. Strains of the new species are oxidaseand 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 t h e 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


Pseudomonas cepacia was first mentioned by Ballard et al. (3) in 1970. This term was used for bacteria responsible for bulbiferous Aliaceae root rot diseases which had been described in decaying onions in 1950 by Burkholder (8). In 1966, Stanier et al. (47) had already described a Pseudomonas species (Pseudornonas rnultivorans), which later on was recognized to be identical to P. cepacia by Palleroni & Holmes (36), who validly described P. cepacia in 1981. This species was ascribed to section I1 of Pseudomonas The GenBank accession numbers for the sequences reported in this paper are U96927-U96941.

by Doudoroff & Palleroni (14), along with other bacteria that utilize arginine and betaine as sole carbon source, such as Pseudomonas mallei, Pseudomonas pseudomallei, Pseudomonas caryophylli and Pseudomonas gladioli, formerly Pseudornonas marginata (3 1). This grouping was consistent with that of Ballard et al. (3) and Palleroni et al. (37) using rRNA-DNA hybridization. In 1992, Yabuuchi et al. (61) proposed to assign these bacteria to a new genus, Burkholderia. As well as the above-mentioned species (i.e. Burkholderia caryophylli, Burkholderia cepacia, Burkholderia gladioli, Burkholderia mallei and Burkholderia pseudornallei), two other [Pseudomonas]species were also included on the basis of 16s rRNA sequences, DNA-DNA hybrid549

00647 0 1998 IUMS

Downloaded from www.microbiologyresearch.org by IP: On: Mon, 07 Mar 2016 04:37:59

V. Viallard and others

ization studies, cellular lipids and fatty acids, and phenotypic properties ; these were Pseudomonas solanacearum and Pseudomonas pickettii (40). Later on, Li et al. (60) came to the conclusion that the latter two species and [Alcaligenes] eutrophus were a separate lineage and Gillis et al. (20) proposed that they were a separate genus. In 1995, Yabuuchi et al. (62) validly described Ralstonia solanacearum, Ralstonia pickettii and Ralstonia eutropha. Both genera belong to the same rRNA group I1 of [Pseudomonas] as defined by Palleroni et al. (37). In 1994, Urakami et al. (53) transferred the [Pseudomonas] species Pseudomonas glumae (28) and Pseudomonas plantarii (2) to the genus Burkholderia and also described a new species, Burkholderia vandii. To these Burkholderia species, Gillis et al. (20) added a nitrogenfixing bacterium discovered in Vietnam rice fields (49-5 1) known as Burkholderia vietnamiensis. They also transferred to the genus Burkholderia the species [Pseudomonas] andropogonis ( = woodsii) (48) and [Pseudomonas]cocovenenans (see also 63). The genus Burkholderia currently comprises eleven species; Burkholderia andropogonis, B. caryophylli, B. cepacia, Burkholderia cocovenenans, B. gladioli, Burkholderia glumae, B. mallei, Burkholderia plantarii, B. pseudomallei, B. vandii and B. vietnamiensis. This genus belongs to the P-subclass of the Proteobacteria (59). Most of the above-mentioned species appear in section I1 of Pseudomonas in Bergey's Manual (39, whereas the others are in section V. The latter section included species whose DNA or rRNA relatedness had not been characterized in 1984. During a field survey of B. cepacia populations in some French and Australian agricultural soils (39), a large diversity of strains was isolated on PCAT agar (7), which is considered selective for B. cepacia. In comparison with type strains from Burkholderia, Pseudomonas and Ralstonia species, these isolates were characterized by phenotypic (Biolog, API, MIDIFAME) and genotypic (DNA-DNA hybridizations, 16s rDNA sequencing) analyses. It became obvious that the isolates belonged to at least three, and possibly five or six, different Burkholderia species. One very homogeneous group (group A) existed among the isolates and could not fit into any of the described Burkholderia or Pseudomonas species; a novel species, Burkholderia graminis, is proposed. METHODS Bacterial strains and medium. Strains used in this study are listed in Table 1. Unless otherwise stated, strains were isolated using PCAT medium [composition (in g 1-l) : MgSO,, 0.1 ; azelaic acid, 2; tryptamine, 0.2; K,HPO,, 4; KH,PO,, 4; yeast extract, 0.02 (pH 5.7)] (7). Only strains forming white or beige opaque shining colonies with an entire margin were considered. They came mostly from three fields: La C6te Saint AndrC, Kapunda and Walpeup. La C6te Saint AndrC is an experimental field located 40 km east


of Lyon (France) on an alluvial soil where maize is grown continuously; isolates have been obtained from the soil and the rhizoplane (washed roots, macerated and diluted) of germinating, flowering or senescent maize root systems. Kapunda is an experimental field, 80 km north of Adelaide (South Australia), where wheat is grown either continuously or in rotation with a lupin-based pasture; the soil is an alphisol. Walpeup is an experimental wheat-growing station, situated in Victoria (Australia), on a very poor sandy soil, in a fixed sand dune system. Soil samples of the two Australian stations have been collected and used for growing wheat (cv. Spear) in pots under glasshouse conditions (three plants per pot containing 1.5 kg soil). After 3-4 weeks, wheat plants were harvested and used to isolate bacteria from their rhizoplane, as above. A few strains were isolated directly on PCAT medium from salt-affected and hydrophobic soils near Adelaide. Also included in Table 1 are 18 reference strains of Burkholderia, Pseudomonas, Ralstonia and Alcaligenes. Among the eleven type strains of Burkholderia species, only type strains of B. mallei and B. pseudomallei were not grown in this laboratory. Biochemical characterization. All tests were performed at 28 "C. The Biolog G N system was used as recommended by the manufacturer to test the oxidation of 95 carbon substrates. Results were read automatically with a spectrophotometer after 24 or 48 h incubation at 28 "C. To test the reproducibility of the method, eight isolates were run in duplicate. Numerical analysis of the results was made using the G N Microlog 2N software which calculates Microlog distances derived from the number of differences between strains. This software also permits clustering analysis using the UPGMA (unweighted mean pair group method) algorithm of Sneath & Sokal(44).

Carbon substrate assimilation tests were performed using auxanographic API 50CH strips (bioMCrieux) as recommended by the manufacturer. Nine isolates were tested in duplicate. Numerical analysis was performed on data obtained after 7 d incubation. Interstrain distances were calculated using the coefficient of Dice and a phenogram was built using UPGMA. The API 20NE microtube system (bioMCrieux) was used as a standardized method to test oxidase activity, nitrate reduction, gelatin and aesculin hydrolysis, glucose fermentation, arginine dihydrolase activity and production of indole, P-galactosidase and urease. MIDI-FAME. The MIDI-FAME technique is based on the conversion of fatty acids to methyl esters by mild alkaline methanolysis, followed by GLC analysis. Isolates were grown overnight (16-1 8 h) on trypticase soy agar. Cells were removed from the plate using a plastic inoculating loop, 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, 150 ml methanol, 150 ml distilled water) was added before vortexing for 5-10 s and heating to 100 "C for 5 min. After vortexing again, tubes were heated for a further 25 min at 100 "C. Cells were then methylated as follows: after cooling in cold water, 2 ml methylation reagent (325 ml hydrochloric acid, 275ml methanol) was added and the tubes were vortexed for 5-lOs, heated at 80°C for lOmin, and then rapidly cooled. Finally, fatty acids were extracted by addition of 1.25 ml of a 50/50 mixture of HPLC grade hexane and methyl-tert-butyl ether to each tube. After 10 min on a rotary shaker, the aqueous phase was discarded, International Journal of Systematic Bacteriology 48

Downloaded from www.microbiologyresearch.org by IP: On: Mon, 07 Mar 2016 04:37:59

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


AUS3-AUS9 AUSlO-AUS 12, AUS28-AUS29, AUS3 1-AUS34 AUS35 AUS36-AUS3 8 AUS13-AUS15, AUS26-AUS27, AUS30 AUS 16-AUS20 AUS2 1 AUS22-AUS23 AUS39-AUS40, AUS42-AUS44 C3AlM C3BlM C4BlM, C4ClM, C5AlM C4DlMT C3DlSn, C3D3Sn C4A 1B C4AlMj, C3AlMj, C3ClMj C4A3P, C4A7P m33d, m45 m35b m130 PHQB 17 526 Burkholderia sp. strain N2P5 Burkholderia sp. strain N3P2 Burkholderia sp. strain N2P6 Burkholderia sp. strain CRE7 [Pseudomonas] sp. strain LB400 Burkholderia sp. strain JB1 Burkholderia sp. strain GSOY Burkholderia sp. strain E264 B. andropogonis B. caryophylli B. cepacia B. cepacia B. cocovenenans B. gladioli B. glumae ' B. norimbergensis' B. plantarii B. pseudomallei strainl026b B. vandii B. vietnamiensis TVV70 B. vietnamiensis TVV75T [P.]glathei [P,]phenazinium [P.]pyrrocinia R . eutropha R . eutropha CH34 R . pickettii R. solanacearum A . xylosoxidans N . polysaccharea

U9694 1

U96940 U96938 U96939


ATCC 53267


23061T 2541gT 25416T 17759 33664T 1024gT 33617T

LMG 9035T LMG 16020T LMG 10929T ATCC 29195T LMG 2247T ATCC 1595gT ATCC 17697T LMG 1195 ATCC 275 11 ATCC 11696T ATCC 2706IT ATCC 4376gT

Origin (reference)

GenBank no.

U37342 u37344 u37343 U37340 U86373 X92188 U16140 U91838 X67037 X67039 U96927 X87275 U96934 X67038 U9693 1 YO9879 U96933 U9 1839 U96932 U96929 U96928 U96935 U96936 U96930 M32021 X67042 X67036 LO6 167

SA, salty pasture soil* Kapunda (SA), wheat pasture rotation* Kapunda (SA), wheat pasture rotation* Kapunda (SA), wheat pasture rotation* Kapunda (SA), continuous wheat* Kapunda (SA), native vegetation* Walpeup , continuous wheat * Walpeup, native vegetation* SA, hydrophobic soils* CSA, maize senescent root system* CSA, maize senescent root system* CSA, maize senescent root system* CSA, maize senescent root system* CSA, bare soil* CSA, wheat* CSA soil, young maize (3 weeks old) in pots* CSA, pasture* CSA young maize (1 month)* CSA young maize (1 month)* Brazil, maize rhizosphere France, continuous wheat (23) ' Blue Circle ', USA, maize rhizosphere Phenanthrene-enriched soil (33) Phenanthrene-enriched soil (33) Phenanthrene-enriched soil (33) Phenanthrene-enriched soil (33) (30) Garden soil (46) (17) (6) Sorghum (48) Carnation (3) Onion sour skin (3) Forest soil (30) Fermented coconut (54) Gladiolus sp. (43) Rice (29) (28) Rice pathogen (2) (6) Orchid rhizosphere (53) Rice rhizosphere, Vietnam (20) Rice rhizosphere, Vietnam (20) Fossil lateritic soil (64) Soil (4) Unknown (25) Soil (12) Waste water zinc factory (32) Clinical (40) Tomato pathogen (45) C1inica1 Throat of a healthy child (13)

* Isolated in this study.

International Journal of Systematic Bacteriology 48 Downloaded from www.microbiologyresearch.org by IP: On: Mon, 07 Mar 2016 04:37:59


V. Viallard and others 10%


C3D 1Sn C4A1B AUS44 AUS18 [P.] glatheiT C4A1M j C3C1MJ. C3A1MJ C3D3Sn B. vandi7 B. plantarii'" B. cocovenenansT B. glumaeT 8. gladioliT [P.] phenazinium' AUS5-AUS7 AUS10-AUS1 1 AUSl5 AUS17 AUSl9-AUS21 AUS22-AUS23 AUS28* AUS33


B. an dropogonisT C4A1M j

4 -

au540 C3D3Sn au542 B. cocovenenansT B. plantariiT B. glumae' B. gladioli' B. caryophylliT [P.] phenaziniumT AUS22 AUS44 au539 AUS18

A -


US40 AUS8 B. vietnamiensisT B. cepaciaT* [P.] pyrrociniaT AUSl2-AUS14 PHQB17 AUS26 AUS27" AUS29* AUS31-AUS32 r AUS34 AUS30* AUS36 AUS37 AUS38 m33d m35b m45 C3B1M* M130 526


B. cepaciaT* [P.] pyrrociniaT AUS12-AUS14 au524 AUS26-AUS27

au529 AUS30-AUS32 au534 AUS36-AUS38 m33d m35b* m45 C3B1M*

B. caryophylliT B. andropogonisT B. pickettii' A. xylosoxidansT R. eutropha CH34 R. solanacearumT AUS42

RauwtrophaT .

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.


International Journal of Systematic Bacteriology 48 Downloaded from www.microbiologyresearch.org by IP: On: Mon, 07 Mar 2016 04:37:59

Analysis of Burkholderia graminis sp. nov.

3 ml NaOH (0.3 M) was added and the combination was mixed before centrifugation. The upper phase was carefully removed and used for analysis. Analysis was carried out with a Hewlett Packard HP 5890 Gas Chromatograph equipped with a phenyl methyl silicone fused silica capillary column (HP Ultra 2-25m x 0.2 mm x 0.33 mm film thickness) and a flame ionization detector. Hydrogen was used as the carrier gas. The temperature programme was initiated at 170 "C and increased at 5 "C min-l to a final temperature of 270 "C. DNA manipulation. DNA was extracted according to the procedure of Brenner et al. (5). To further purify DNA, extra chloroform/isoamyl alcohol extraction steps were added. Mean G + C contents (molY0) of five phenon A strain DNAs were determined by HPLC (38). PCR and 165 rDNA sequencing. Five isolates from the various field conditions were chosen as representatives of the two major clusters obtained after phenotypic analysis : strains AUS35, C4D1MTand C3AlM (representative of phenon A) and m35b and C3BlM (representative of phenon B). The 16s rDNA of these strains was sequenced.

The 16s rDNA sequences of B. cepacia, B. cocovenenans, B. vandii, B. glumae, B. plantarii, B. vietnamiensis, [Pseudomonas] phenazinium, [Pseudomonas] glathei and [Pseudomonas]pyrrocinia type strains and B. vietnamiensis TVV70 were also determined. The oligonucleotides used to amplify the 16s +intergenic spacer region of the rRNA gene were 5' ATGGA(GA)AG(TC)TTGATCCTGGCTCA 3' and 5' CCGGGTTTCCCCATTCGG 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 containing 1 pl cell suspension (about lo9cells ml-l) in 50 YO (w/v) glycerol, 5 p1 buffer [ 10 pM Tris/HCl, pH 8.2; 1.5 mM 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 DNA polymerase (Gibco-BRL). Amplifications were carried out on a dry block thermocycler using the following programme: 3 min at 95 "C followed by 35 cycles of denaturation (1 min at 95 "C), annealing (1 min at 55 "C) and extension (2 min at 72 "C), and then a final extension of 10min at 72 "C. To check for amplification efficiency, amplification products (5 pl) were run on a 2 % horizontal agarose gel in TBE buffer at 4Vcm-l. The PCR amplification products were visualized by ethidium bromide staining. Sequencing of the 16s rRNA gene only (positions 58-1541 of the Escherichia coli 16s rDNA sequence) was performed by Genome Express, Grenoble, France. Sequences of both strands were determined using the following five oligonucleotides (which correspond to positions 20-43, 5 18-532, 885-904,152 1-1 540 and 880-899 of the E. coli small-subunit rDNA sequence, respectively) : 5' TGGCTCAGAACGAACGCTGGCGGC 3'; 5' AGCCTTGCGGCCGTACTCCC 3'; 5' CAGCAGCCGCGGTAA 3'; 5' AAGGAGGGGATCCAGCCGCA 3'; and 5' GCCTGGGGAGCTCGGCCGCA 3'. Phylogenetic analyses. All DNA sequences were deposited in the EMBL/GenBank database (Table 1). They were compared with previously published 16s rDNA sequences

of B. cepacia, B. andropogonis, B. caryophylli, B. gladioli, B. pseudomallei and those of other members of the P-subdivision of the Proteobacteria like Ralstonia solanacearum, R. pickettii and R. eutropha. The GenBank database also contained sequences of unknown bacteria which according to the BLAST software (1) appeared to be related to our isolates: strains CRE7, N3P2, N2P5, N2P6 (33), strain JB1 (46), ' Burkholderia norimbergensis' (27), strain GSOY (17), strain E264 (6) and strain LB400 (29). Neisseria polysaccharea (L06167) was used as an outgroup. Only welldefined sequences with less than five undetermined nucleotides were used. Sequences were aligned using CLUSTAL v (24) between positions 98 and 1496 (E. coli numbering). Alignment was refined manually using the SUNMASE algorithm (15). A stretch of uncertain alignment (position 209-220) was removed before calculating painvise evolutionary distances according to Jukes & Cantor (26) with the DIFFCOUNT program. Phylogenetic trees were constructed using the neighbour-joining method of Saitou & Nei (42) with the PHYLO-WIN graphic tool (19). The topology of this distance tree was tested by 1000 bootstrap resamplings of data (16). Parsimony analysis was done using the PHYLO-WIN program. DNA-DNA hybridization. Native DNAs of strains ATCC 25416T and C4DlMT were labelled in vitro by nick-translation (4 1) with tritium-labelled nucleotides (Amersham). The procedure used for the hybridization experiments (S1 nuclease/ trichloracetic acid method) has been described by Crosa et al. (1 1). DNA fragments (500 bp long) obtained by sonication were used.

RESULTS Phenotypic analysis of Burkholderia and related strains General characteristics of sugar metabolism. The


typic comparisons between the strains isolated in this study and the type strains of the different Burkholderia species were based on the oxidation of 95 carbon substrates (Biolog) and the assimilation of 49 carbon sources (API) (Fig. 1, Table 2). In the two analyses, most of our isolates (about 70%) were contained in only two phenons, named A and B. Reproducibility was good with API in which nine strains were tested in duplicate; results were extremely similar for the replicates ( 9 6 1 0 0 % ) . In Biolog tests, eight strains were run in duplicate and only 80-91 YO of results were

identical. Ralstonia strains were easily differentiated using both Biolog and API. With both methods, B. andropogonis had only a weak similarity to other Burkholderia strains. In the phenotypic analysis, it was difficult to assign B. caryophylli and B. andropogonis a clear generic status ;they were located on the same branch as Ralstonia in the Biolog analysis, whereas they were closer to Burkholderia after their API scoring. Strains AUS8 and AUS42 were also given different positions in the two analyses. This might be a consequence of the presence, among the strains analysed, of a large

International Journal of Systematic Bacteriology 48 Downloaded from www.microbiologyresearch.org by IP: On: Mon, 07 Mar 2016 04:37:59


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 ( 3 9 , 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: Lsorbose, 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:

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- Fuco se D-Tagatose l 5-Ketogluconate Xylitol L-Arabitol Cellobiose Rhamnose Aesculin* , D-Raffinose P-Gentiobiose Amygdalin Lactose L-Xylose Arbutin Erythritol Melibiose Salicio Maltose Starch I Glycogen ~






+ + + + + + + + + + + + + + - + + + + + + + -

+ + + + + + + + + + + + + + + + + + +

+ + + + + + + + + + + + + + + + + + +

+ + + + + + + + + + + + + + + + + -

+ + + + + + + + + + + + + + + + -



+ + + + + + + + + + + + + +

+ + + + + + + + + + + + +

D+ D+


+ + + D+ + +






9 1 0 1 1 1 2 1 3

+ + + + + + + + + + + + + + + + + + + + + + + + + + + + - + - + + - + + - -


+ + + + + + + + + + + + + + + +

+ + + + + + + + + + + + + -




+ + + + + + + + + + + + + + + + + + - - + -

+ + + + + + + + + -



+ +

* Hydrolysis of aesculin. number of poorly related strains contrasting with a large number of closely related ones. The closely related strains constitute a few well-defined clusters, whereas the poorly related strains form an unstable cloud which is very sensitive to small phenotypic differences. 554

In contrast, two phenons called A and B were clearly differentiated using both API and Biolog (Fig. 1) and they contained roughly the same strains for slightly different levels of relatedness. The similarity of phenon A strains was more than 97 YOusing API but only 82 % using Biolog; for phenon B strains, similarities were International Journal of Systematic Bacteriology 48

Downloaded from www.microbiologyresearch.org by IP: On: Mon, 07 Mar 2016 04:37:59

Analysis of Burkholderia graminis sp. nov. Euclidian distance 30





0 I


B. plantarii7 B. vandii7 B. pickettii7 [ R ] g/athei7 B. andropogonis' I B. cocovenenans' B. gladioli7 [ R ] phenaziniuml R. eutropha' B. caryophy/li7 B. g/umaeT

4 41



AUS35 AUS33 C4B1M C4D1M rn B. vietnamiensisT AUS27 AUS30 AUS 29 B. cepacia' m35b C3B1M [ R ] pyrrocinia' M130 526 rn45

....................... .................................................................... ...................................................... Fig. 2. Similarity dendrograrn obtained with MIDI-FAME data. Euclidian distances are shown. A, Phenon A; B, phenon B.

96 YOusing API and 78 % using Biolog. Phenon A did not contain any reference strain. According to the Biolog analysis, phenon B contained both B. vietnamiensis and B. cepacia along with [P.]pyrrocinia. Strains of phenon A. In oxidation and assimilation tests, the same 19 isolates were tightly clustered in phenon A and a twentieth strain, AUS22, clustered with phenon A when analysed by Biolog, but not by API. The strains showed a maximum 18 % Microlog distance in painvise comparisons based on the oxidation tests (Biolog) whereas this distance was less than 2.8% with the API system. No representative strain of any of the twelve Burkholderia or [Pseudomonas] species tested was present in this group. The group comprised strains isolated from all the different soils studied, both in Australia (15 strains) and in France (four strains). Australian strains came from Kapunda (nine strains), Walpeup (two strains), saltaffected pastures (three strains) and a hydrophobic soil (one strain). Kapunda and Walpeup strains have been isolated from wheat roots grown on a soil coming from the continuous wheat treatment (one in Kapunda and one in Walpeup) and the wheatlpasture rotation in

Kapunda (five strains). Strains isolated from wheat growing on Kapunda (three strains) or Walpeup (two strains) soils collected under native vegetation were exclusively of the A phenotype. Strains isolated in France all came from the roots of senescent maize plants after harvest. Strains of phenon B. API phenon B contained 20 strains, which were all also found in Biolog phenon B. The Biolog test was also done on two more strains, i.e. ml30 and 526. Moreover, with Biolog, B. vietnamiensis type strain TW75T clustered with phenon B; this explained the apparent discrepancy in strain numbers between Biolog (23 strains) and API (20 strains) in phenon B. The maximum distances between phenon B strains were 22% (Biolog) and 4 % (API). Phenon B included one (API) or two (Biolog) type strains of Burkholderia (B. cepacia and/or B. vietnamiensis) plus the type strain of [P.]pyrrocinia. In consequence, the latter strain was further characterized by molecular techniques.

Phenon A characteristics

Like all the Burkholderia species and [P.]glathei, [P.]phenazinium and [P.]pyrrocinia type strains tested, phenon A strains oxidized Tween 40, L-arabinose, D-arabitol, D-fructose, D-galactose, a-D-glucose, myo-inositol, D-mannitol, D-mannose, D-sorbitol, methyl pyruvate, monomethyl succinate, D- and L-alanine, L-asparagine, Lproline, L-serine, and cis-aconitic, formic, D-gluconic, P-hydroxybutyric, DL-lactic, malonic, D-saccharic, succinic, bromosuccinic, L-aspartic and L-glutamic 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. Burkholderia characteristics of phenon A.

Like all the Burkholderia species and [P.]glathei, [P.] phenazinium and [P.]pyrrocinia type strains tested in this study, phenon A strains assimilated (Table 2) the following as sole carbon source: glycerol, D- and L-arabinose, ribose, galactose, D-glucose, D-fructose, D-mannose, inositol, mannitol, sorbitol, D-arabitol and gluconate. All the Burkholderia species and [P.] glathei, [P.]phenaziniumand [P.]pyrrociniatype strains tested did not use L-sorbose, methyl P-D-xyloside, methyl a-D-mannoside, methyl a-D-glucoside, erythritol, maltose, inulin, melezitose, starch, glycogen or D-turanose as sole carbon source. Particular properties of phenon A. The 19 strains of phenon A formed thin, brownish-yellow, translucent colonies on LB agar. On PCAT agar, growth needed 2 or 3 d at 28 "C. On this medium, colonies were white, opaque and creamy. These isolates had catalase, oxidase, urease and P-galactosidase activities. They reduced nitrate to nitrite. They did not ferment glucose and did not hydrolyse aesculin or produce indole or gelatinase. All 19 strains used L-xylose, lactose, rhamnose, trehalose, D-lyxose, L-arabitol, xylitol and raffi-

lnternational Journal of Systematic Bacteriology 48 Downloaded from www.microbiologyresearch.org by IP: On: Mon, 07 Mar 2016 04:37:59


V. Viallard and others

[P.J phenaziniumT



Strain N2P6





Strain N2P5



B. caryophylliT Strain E264 B. pseudomallei strain 1026b B. vandii' B. plantarir' B. gladioliT


L B. glumaeT B. cepacia ATCC 17759 B. cepaciaT Strain CRE7 B. ~ietnarniensis~


[ R ]pyrrocinia'

C3B1M m35b Strain GSOY B. andropogonisT


Strain JB1 'Burkholderia norimbergensis' R. eutropha' R. pickettiiT

R. solanacearumT


Neisseria polysacchareaT

Fig. 3. Phylogenetic neighbour-joining tree obtained with the 16s rDNA sequences of members of Burkholderia, [Pseudomonas] and Ralstonia species. Numbers a t 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 W 7 0 , C4D1MT and C3AlM show the same 165 sequences.

nose; none of them used dulcitol or D-tagatose as sole carbon source. This represented a combination of characteristics unique among Burkholderia species (Table 2). Fatty acid composition

Fatty acid analysis of the 19 phenon A and B strains analysed showed the presence of 3-OH 16 :0, a characteristic feature of the Burkholderia genus. This compound represented 3-5 % of total fatty acids. The two Ralstonia strains analysed contained no trace of 3-OH 16 :0, confirming the taxonomic value of this charac556

teristic (20). Among seven analysed phenon A strains, AUS28 was slightly different (Fig. 2). The six remaining strains had a rather similar fatty acid composition including (mean values) : 16: lcu7c (23 YO); 16:0 (20 %) ;and a large (40 YO) mixed unresolved peak corresponding to 18: h 9 c , 18: lcol2t, 18: lcu7c. There was no obvious difference between phenon A and B strains. Phenon B strains were clearly assigned to two different groups; one contained one maize strain (m33d), B. vietnamiensis and three Kapunda strains, and the other one contained maize strains together with B. cepacia pyrrocinia. and [P.] International Journal o f Systematic Bacteriology 48

Downloaded from www.microbiologyresearch.org by IP: On: Mon, 07 Mar 2016 04:37:59

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)

Phenon A :* C4D1MT C3AlM C4BlM C5AlM AUS28 AUS33 AUS35 Phenon B:* PHQB17 C3BlM AUS27 AUS30 Other strains : C4ClM C4A3P C4A7P C3AlMj C3ClMj C4AlB C3D 1Sn AUS18 N2P5 N3P2 Type strains : B. cepacia B. andropogonis B. caryophylli B. cocovenenans B. gladioli B. glumae B. plantarii B. vandii B. vietnamiensis [P.]glathei [P.]phenazinium [P.]pyrrocinia R. pickettii R. solanacearum

B. cepacia ATCC 2541Q

100 89 84 83 86 77 75

17 16 17 17 14 15 17

15 13 15 15

60 53 53 54

30 26 29 27 24 15 19 30 24 30

18 15 20 15 17 14 16 13

13 3 8 8 12 11 12 13 19 17 16 12 3 3


100 ND

5 ND

27 21 26 ND

41 14 18 48 ND ND

m, Not determined.

* See Fig. 1(a, b).

Phylogenetic analysis

The 16s rDNA sequence (about 1500 bp) was determined for the following strains: AUS35, C4D1MT, C3AlM (which belong to phenon A), and m35b and C3BlM (phenon B). The 16s rDNA sequences were also determined for the type strains of B. cepacia, B. cocovenenans, B. plantarii, B. vietnamiensis, B. glumae, pyrrocinia. These [P.]glathei, [P.]phenazinium and [P.] sequences were aligned and compared with the closely

related sequences present in the GenBank database. Phylogenetic trees were inferred using the neighbourjoining and parsimony algorithms and rooted with N . polysaccharea (Fig. 3). The whole genus Burkholderia formed a homogeneous cluster; the maximum difference between the 16s sequences was 6.2 % (between B. andropogonis type strain and strain LB400) and this cluster was stable (bootstrap value of 96%). As well as members of

International Journal of Systematic Bacteriology 48 Downloaded from www.microbiologyresearch.org by IP: On: Mon, 07 Mar 2016 04:37:59


V. Viallard and others acknowledged Burkholderia species, the genus contained [P.]pyrrocinia, [P.]phenazinium, [P.]glathei and phenon A strains. This was supported by parsimony analysis. This genus was clearly distinct from the genus Ralstonia, which was also coherent. B. gladioli and B. cocovenenans appeared to be highly related (99.9 YO similarity). Similarly, B. glumae, B. vandii and B. plantarii also appeared to be closely related, with a high level of similarity (994-99-5 YO). Strains C4DlMTand C3AlM had the same 16s rDNA sequences. They differed from AUS35 by only two nucleotides. The distance between C4D lMT and the nearest sequence (N3P2) was 1.8%. The distance between C4D1MT and [P.] phenazinium was 2.4%. Strains m35b and C3BlM had distances of 1.2 and 0.7 % from B. cepacia, respectively. The B. andropogonis 16s sequence was different enough to put this species on a separate branch. Strains JB 1 and ' Burkholderia norimbergensis' were even further away from core Burkholderia species. DNA-DNA hybridization

DNA of C4D1MT was hybridized to DNA of seven phenon A strains (C4D1MT,AUS28, AUS33, AUS35, C3A 1M, C4B 1M and C5Al M) ; reassociation values (Table 3) were 75-89% within phenon A. With nine Burkholderia species type strains, low levels of homology (3--19%) were obtained. [P.] pyrrocinia, [P.] phenazinium and [P.]glathei exhibited 12, 16 and 17% homology, respectively, with C4D lMT. R. solanacearum and R. pickettii type strains gave hybridization values of 3 %. Labelled DNA of C4D1MT was also hybridized with DNA of isolates phenotypically closely related to phenon A such as C4ClM, C4A3P, C4A7P, C3AlMj, C3ClMj, AUS18, C4AlB and C3DlSn; hybridization values were 15-30 %. Strains N2P5 and N3P2, which exhibited a high degree of 16s sequence similarity with C4D lMT, showed hybridization values of 24 and 30 YO,respectively. Hybridization of B. cepacia ATCC 25416TDNA with DNA of some phenon B isolates (C3BlM, AUS27, AUS30 and PHQB 17) showed reassociation levels of 53-60 YO.Labelled DNA of B. cepacia ATCC 25416T showed low levels of reassociation (5-27%) with B. caryophylli, B. plantarii, B. glumae, B. gladioli, [P.] glathei and [P.]phenazinium, whereas [P.]pyrrocinia and B. vietnamiensis appeared to be more closely related to B. cepacia (reassociation levels of 48 and 4 1YO,respectively). DISCUSSION Isolation medium

This study was initiated with the aim of characterizing the in situ intraspecific diversity of B. cepacia. To obtain a set of isolates representative of natural 558

populations of this species, use of PCAT medium, which was described by Burbage & Sasser (7) as being specific for Burkholderia cepacia, was evaluated. According to Burbage & Sasser, no growth was observed on PCAT for 12 isolates of Agrobacterium, Erwinia, Xanthomonas and Pseudomonas whereas all B. cepacia strains tested grew well. In our study, bacteria isolated from some French and Australian soils using PCAT have been further characterized to investigate the selectivity of this medium. All the strains obtained are indeed related to Burkholderia, on the basis of their phenotype or genotype. However, they belong to various species. Most of Burkholderia species type strains can grow on PCAT in spite of the fact that none of them has been isolated on PCAT. Nevertheless, on PCAT, growth of B. vandii type strain is scarce and B. andropogonis and B. caryophylli type strains do not grow. The type strains of B. mallei and B. pseudomallei do not grow on PCAT, whereas all recent isolates can (D. Vidal & F. Thibaut, personal communication). The closely related R. solanacearum and R. eutropha type strains do not grow on PCAT. Thus, there seems to be some taxonomic meaning to growth on PCAT but its level of specificity deserves more research. This is of increasing importance as there is a strong need for a selective medium to isolate Burkholderia strains from clinical and environmental specimens (58, 10, 22, 52, 56). Burkholderia vs Ralstonia

Strains isolated using PCAT were further characterized by Biolog and API methods. In terms of methodologies, both API and Biolog methods are applicable for highlighting groups of related strains against a background of more distantly related ones. Biolog has a lower reproducibility and is not very accurate when considering distantly related microorganisms. API 50CH appears more reliable but is less efficient when dealing with closely related phenotypes because of the smaller number of characters treated. The presence of 3-OH 16:O among fatty acids is confirmed as a phylogenetically meaningful characteristic to distinguish Burkholderia from Ralstonia. B. andropogonis

With phenotypic and molecular methods, there is a very clear-cut distinction between Burkholderia and the few Ralstonia reference strains studied. Nevertheless, some ambiguity arises about B. andropogonis and B. caryophylli. When using Biolog, type strains of both species cluster with Ralstonia, whereas when using API, B. caryophylli stays with Burkholderia while B. andropogonis clusters with Ralstonia, as already seen by Gillis et al. (20). When molecular data are taken into account, B. caryophylli again appears as a real Burkholderia, whereas B. andropogonis has a 16s sequence that is somewhat different (often more than 4 YO)from those of other Burkholderia species; moreover, in the neighbour-joining tree, B. andropogonis is International Journal of Systematic Bacteriology 48

Downloaded from www.microbiologyresearch.org by IP: On: Mon, 07 Mar 2016 04:37:59

Analysis of Burkholderia graminis sp. nov. situated on a branch that is separate from all other Burkholderia species. This confirms its marginal status in the genus, which is already obvious according to the phenotypic results obtained. Nevertheless, its sequence similarity to the other Burkholderia species is much greater than that to any Ralstonia species. In consequence, there is no obvious reason to place B. andropogonis in a separate genus for the time being. B. phenazinium, B. glathei and B. pyrrocinia comb. nov.

In the course of this study, Biolog and API diagnostic software and the fatty acid analysis pointed to the possible presence among the isolates of strains related to [P.] phenaziniurn (4), [P.] glathei (64) and [P.] pyrrocinia (26). The possibility that these [Pseudomonas] species would constitute exceptions to the selectivity of PCAT prompted a re-evaluation of their taxonomic status. DNA-DNA hybridization with B. cepacia is high (18, 14 and 48 YOfor [P.]phenaziniurn, [P.]glathei and [P.]pyrrocinia, respectively). According to their 16s rDNA sequences, these [Pseudornonas] are closer to B. cepacia ATCC 25416T (respective similarities of 96.4, 96.8 and 99.2%) than to the recognized Burkholderia species B. andropogonis (sequence similarity of 95 YO).In consequence, they actually belong to the Burkholderia genus and should be renamed B. phenaziniurn, B. glathei and B. pyrrocinia comb. nov. It is worth noting that, based on phenotypic properties, Gillis et al. (20) have already suggested that [P.]phenazinium could belong to the Burkholderia genus, although there was a lack of rRNA-DNA hybridization data. Similarly, considering its special type of tyrosine-inhibited arogenate/NADP dehydrogenase activity, Byng et al. (9) had already suggested that [P.] pyrrocinia could be closely related to Burkholderia. The Burkholderia genus revisited

The number of Burkholderia species validly described thus amounts to 14. These are B. andropogonis, B. caryophylli, B. cepacia, B. cocovenenans, B. gladioli, B. glathei, B. glurnae, B. mallei, B. phenaziniurn, B. plantarii, B. pseudornallei, B. pyrrocinia, B. vandii and B. vietnarniensis. Many of these species have very similar 16s rDNA sequences, and the species status of them remains uncertain as long as it has not been confirmed by DNA-DNA hybridization studies. For instance, B. vandii and B. glurnae are very closely related to B. plantarii (similarities of 99.5 and 99.4 YO, respectively). The distinction of these three species is nevertheless supported by DNA-DNA hybridization experiments conducted by Urakami et al. (53). These results show the limitations of 16s rRNA sequencing for differentiation of closely related species (18). The species B. gladioli and B. cocovenenans also exhibit a high level of similarity in their 16s rDNA sequences (99.9 YO)and, thus far, no DNA-DNA hybridization has confirmed their status of separate species.

The inclusion of three [Pseudomonas]species does not change the definition of the genus Burkholderia. Auxanographic studies show that none of the characteristics common to all previously described Burkholderia species are different in any of the new species. A reservation should be made for B. mallei and B. pseudornallei; these two species were not grown for this study and literature data (61, 35) were used in which contradictory results have been reported. Beyond this reservation, the genus Burkholderia can be defined as follows. All species can grow with the following substrates as sole carbon source : glycerol, D-arabinose, galactose, D-glucose, D-mannose, inositol, mannitol, sorbitol and gluconate. Nevertheless, contrary to all other studies, Yabuuchi et al. (61) found that B. gladioli could not grow on D-arabinose. The following compounds are not used by any Burkholderia species: methyl P-D-xyloside, L-sorbose, methyl a-D-mannoside, methyl a-D-glucoside, maltose, inulin, melezitose and D-turanose. Nevertheless, Yabuuchi et al. (61) and Gillis et al. (20) reported growth of B. cepacia and B. vietnarniensis, respectively, on maltose. The results reported herein support those of Gillis et al. (20) on many strains of Burkholderia and Ralstonia and show that utilization of a few substrates is probably enough to distinguish members of these two genera; Dmannose is used by all Burkholderia and no Ralstonia and most Ralstonia do not use sorbitol and mannitol, whereas most Burkholderia can. Phenon A taxonomic status

Among soil isolates studied, two phenons (A and B) are dominant. Phenon A strains have been isolated from a large diversity of rhizospheres, very different soil types from France and South Australia, and rhizospheres of native Australian plants, wheat, maize and pasture grasses. Phenotypic analyses revealed that all these strains are very similar. Three strains have had their rDNA sequenced; two have exactly the same 16s rDNA sequence, and the third one differs only by two bases. DNAs of six phenon A strains, when hybridized with the DNA of C4D1MT (chosen to represent this new phenon), have a very high reassociation value (7 5 4 9 % at 72 "C). Phenon A thus fulfils the prerequisites of a new species (55). In the phylogenetic tree, the three sequenced strains form a cluster inside the Burkholderia genus. Moreover, their sequences are highly homologous (956 and 95.8 YO)to the sequence of the genus type strain B. cepacia ATCC 25416T. Thus, the phylogenetic position confirms the phenotypic analyses (API, Biolog) and places this genospecies into the genus Burkholderia. Proposal of Burkholderia graminis

Phenon A strains are phenotypically different from all the above Burkholderia species tested (B. andropogonis, B. caryophylli, B. cepacia, B. cocovenenans, B. gladioli, B. glathei, B. glumae, B. phenaziniurn, B. plantarii, B. pyrrocinia, B. vandii and B. vietnarniensis).For security

Int e r m tionaI lo urnaI of Systematic Bacteriology 48 Downloaded from www.microbiologyresearch.org by IP: On: Mon, 07 Mar 2016 04:37:59


V. Viallard and others reasons, it was not possible to compare their phenotypes to those of the two remaining Burkholderia species, B. mallei and B. pseudomallei. Nevertheless, they differ from the description given for these two pathogenic species (35, 61) by many characteristics. C4D lMT homology with other Burkholderia species type strains is 3-19%. At the level of 16s rDNA sequences, this strain differs from all other known species of Burkholderia (similarity is never more than 97.6%). In conclusion, phenon A strains can be distinguished by all the different approaches used : phenotypic properties, MIDI-FAME analysis, DNADNA reassociation values and 16s ribosomal sequence analysis. All criteria defined by Vandamme et al. (55) are verified and, consequently, phenon A strains should be considered as a new species. The name Burkholderia graminis sp. nov. is proposed for this new species, referring to its known habitat, the rhizosphere of grasses. Strains related to B. graminis

In the GenBank database there are some 16s rDNA sequences which are highly similar to the C4D1MT sequence and could eventually belong to B. graminis. These belong to strains N2P5, N3P2 and N2P6 (33), and to strain LB400 (29). DNAs of N2P5 and N3P2 have hybridization values of 24 and 30 %, respectively, with C4D1MT DNA. This is not enough to consider them as belonging to B. graminis, but they are very closely related. According to its 16s sequence, LB400 is not closer to C4D1MT. Moreover, B. graminis and B. phenazinium belong to the same branch on the neighbour-joining tree (with 99 % of bootstrap data samplings) and this situation is confirmed by the parsimony tree. Nevertheless, the DNA-DNA reassociation value between B. graminis C4D 1MTand B. phenazinium is 16% confirming that the two species are related but different (57). B. graminis thus appears to be a completely new taxon. Phenon B taxonomic status

Phenon B is a heterogeneouscluster containing phenotypically related strains belonging to different genospecies. When measured, their DNA-DNA homology (determined by reassociation) with B. cepacia ATCC 25416T is higher than 47% but never close to 70% (data not shown), showing that they are closely related but different. The two isolates sequenced, m35b and C3BlM, show a high similarity to B. cepacia (98.8 and 99.3 %, respectively) and B. vietnamiensis (98.7 and 99%, respectively), but they differ more from each other (99% similarity) than B. cepacia does from B. vietnamiensis (99.4 YO).The fatty acid composition of phenon B strains also shows the heterogeneity of this group (Fig. 2). Phenon B thus constitutes a complex of related species, not resolved enough by the set of data pyrrocinia and reported in this article. It contains [P.] strains of clinical origin (data not shown). It is 560

currently subject to more taxonomic work which will be published later. Conclusions

On the basis of this polyphasic approach, the genus Burkholderia appears to be more complex than previously thought. The phenotypic data show that 20 of the 60 strains isolated do not belong to any phenon containing a known species. Moreover, they seem extremely diverse. Contrasting with this large background diversity, two groups of isolates appear to be clearly dominant in the situations studied, in France as well as in Australia. One group, containing phenon B isolates, is not resolved at the taxonomic level and will be subject to further studies; it seems to constitute a branching cluster, centred around B. cepacia, B. vietnamiensis and [P.] pyrrocinia. The latter taxon is more properly described as B. pyrrocinia. The other group corresponds to the very homogeneous phenon A. High DNA-DNA hybridization values and the three very similar 16s DNA sequences confirm that these strains constitute a genospecies (21, 55). The name B. graminis is proposed for this group. The most closely related species are [P.] phenazinium and [P.] glathei, which should be renamed B. phenazinium and B. glathei. Description of Burkholderia graminis sp. nov.

Burkholderia graminis (gra'miais. M.L. adj. graminis referring to its isolation from the rhizosphere of grasses). Motile cells, 1.0-13 pm in length, 0-3-0-8pm in width. On LB agar, colonies are thin, brownish-yellow, translucent. Grows in 3 d on PCAT (7) at 28 "C, forming white colonies, more or less opaque and creamy, with an entire margin. Oxidase, catalase, urease and arginine dihydrolase are produced. Reduces nitrates to nitrites but does not denitrify. Like all Burkholderia, can assimilate the following as sole carbon source : glycerol, D- and L-arabinose, ribose, galactose, D-glucose, D-fructose, D-mannose, inositol, mannitol, sorbitol, D-arabitol, gluconate and 2-ketogluconate. Like all Burkholderia, cannot use L-sorbose, methyl a-D-xyloside, methyl a-D-mannoside, methyl a-D-glucoside, indin, melezitose, starch, glycogen or D-turanose as sole carbon source. Contrary to other Burkholderia species, does not acidify glucose, does not hydrolyse aesculin nor produce indole or gelatinase. Grows on L-xylose, lactose, rhamnose, trehalose, D-lyxose, L-arabitol, xylitol and raffinose, but not on dulcitol or D-tagatose as sole carbon source. The G C content is 62-5-63.0 mol YO.Isolated from the rhizosphere of wheat, corn and pasture grasses. The proposed type strain is C4D1MT (G + C content 63 mol %), which has been deposited in the ATCC and given the accession number ATCC 700544.


In ternationaI JournaI of Systematic Bacteriology 48 Downloaded from www.microbiologyresearch.org by IP: On: Mon, 07 Mar 2016 04:37:59

Analysis of Burkholderia graminis sp. nov.

Description of Burkholderiaphenazinium cornb. nov. (Bell and Turner 1973)

The description of B. phenazinium is the description given by Bell & Turner (4). The type strain is NCIB 11027T (= LMG 2247T, = ATCC 33666T), isolated from soil, and it is able to use L-threonine as sole carbon source. Description of Burkholderia pyrrocinia cornb. nov. (Imanaka, Kousaka, Tamura and Arima 1965)

The description of B. pyrrocinia is the description given by Imanaka et al. (25). The type strain is ATCC 15958T ( = LMG 1419 1'), which is of unknown origin. Description of Burkholderia glathei comb. nov. (Zolg and Ottow 1975)

The description of B. glathei is the description given by Zolg & Ottow (64). The type strain is ATCC 29195' ( = LMG 141 goT), isolated from a fossil lateritic soil in Germany. ACKNOWLEDGEMENTS The authors are grateful to Maria Fernandez for her help in discussing DNA-DNA hybridization results, to Cindy Morris for allowing us to use the Microlog (Biolog) software, to Bruce Hawke who performed the MIDI-FAME analyses and to H C h e Meugnier who determined the G C contents. This research has been financially supported by a CNRSCSIRO PICS (International Cooperative Scientific Programme). J.B. is grateful to C. Pankhurst, CSIRO Soil Division, Adelaide (South Australia) who made available his laboratory facilities during a sabbatical leave financially supported by an OECD fellowship.


REFERENCES 1. Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. (1990). Basic local alignment search tool. J Mol Biol 215,403-410. 2. Azegami, K., Nishiyama, K., Watanabe, Y., Kadota, I., Ohuchi, A. & Fukazawa, C. (1987). Pseudomonas plantarii sp. nov., the causal agent of rice seedling blight. Int J Syst Bacteriol 37, 144-152. 3. Ballard, R. W., Palleroni, N. J., Stanier, R. Y. & Mandel, M. (1970). Taxonomy of the aerobic pseudomonads Pseudomonas cepacia, P. marginata, P. alliicola and P. caryophylli. J Gen MicrobioE60, 199-214. 4. Bell, 5. C. & Turner, 1. M. (1973). Iodinin biosynthesis by a pseudomonad. Biochem Soc Trans 1, 751-753. 5. Brenner, D. J., McWorter, A. C., Leete Knuston, J. K. & Steigerwalt,A. G. (1982). Escherichia vulneris: a new species of Enterobacteriaceae associated with human wounds. J Clin Microbioll5, 1133-1 140.

6. Brett, P. J., DeShazer, D. & Woods, D. E. (1997). Characterization of Burkholderia pseudomallei and Burkholderia pseudomallei-like strains. Epidemiol Infect 118, 137-148. 7. Burbage, D. A. & Sasser, M. (1982). A medium selective for Pseudomonas cepacia. Phytopathol Abstr 72, 706. 8. Burkholder, W. H. (1950). Sour skin, a bacterial rot of onion bulbs. Phytopathology 40, 115-1 17.

9. Byng, G. S., Whitaker, R. J., Gherna, R. L. & Jensen, R. A. (1980). Variable enzymological patterning in tyrosine biosynthesis as a means of determining natural relatedness among the Pseudomonadaceae. J Bacterioll44,247-257. 10. Cimolai, N., Trombley, C., Davidson, A. G. F. & Wong, L. T. K. (1995). Selective media for isolation of Burkholderia (Pseudomonas) cepacia from the respiratory secretions of patients with cystic fibrosis. J Clin Pathol48, 5 . 11.' Crosa, 1. M. D., Brenner, D. 1. & Falkow, 5. (1973). Use of a single-strand-specificnuclease for analysis of bacterial and plasmid deoxyribonucleicacid homo- and heteroduplexes. J Bacteriol 115, 904-9 11. 12. Davis, D. H., Doudoroff, M., Stanier, R. Y. & Mandel, M. (1969). Proposal to reject the genus Hydrogenomonas: taxonomic implications. Int J Syst Bacterioll9, 375-390. 13. Dewhirst, F. E., Chen, C.-K. C., Paster, B. J. & Zambon, J. 1. (1992). Phylogeny of species in the family Neisseriaceae isolated from human dental plaque and description of Kingella orale sp. nov. Int J Syst Bacteriol43, 490-499. 14. Doudoroff, M. & Palleroni, N. J. (1974). Genus Pseudomonas. In Bergey's Manual of Determinative Bacteriology, 8th edn, pp. 217-243. Edited by R. E. Buchanan & N. E. Gibbons. Baltimore: Williams & Wilkins. 15. Faulkner, D. V. & Jurka, 1. (1988). Multiple aligned sequence editor (MASE). Trends Biochem Sci 13, 321-322. 16. Felsenstein, J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783-79 1. 17. Floro, G., Buzzelli, J. A,, Griffin, W. & Stolz, J. F. (1997). Aerobic degradation of soy diesel by Burkholderia sp. Unpublished (quoted in GenBank). 18. Fox, G. E., Wisotzkey, J. D. & Jurtshuk, P. 1. (1992). How close is close: 16s rRNA-sequence identity may not be sufficient to guarantee species identity. Int J Syst Bacteriol 42, 166-1 70. 19. Galtier, N., Gouy, M. & Gautier, C. (1996). SEAVIEW and PHYLO-WIN : two graphic tools for sequence alignment and molecular phylogeny. Comput Appl Biosci 12, 543-548. 20. Gillis, M., Tran Van, V., Bardin, R. & 7 other authors (1995). Polyphasic taxonomy in the genus Burkholderia leading to an emended description of the genus and the proposition of Burkholderia vietnamiensis sp. nov. for N,-fixing isolates from rice in Vietnam. Int J Syst Bacteriol45, 274-289. 21. Grimont, P. A. D. (1988). Use of DNA reassociation in bacterial classification. Can J Microbiol34, 54 1-546. 22. Hagedorn, C., Could, W. D., Bardinelli, T. R. & Gustavson, D. R. (1987). A selective medium for enumeration and recovery of Pseudomonas cepacia biotypes from soil. Appl Environ MicrobioE53, 2265-2268. 23 Hebbar, P., Davey, A. G. & Dart, P. J. (1992). Rhizobacteria of maize antagonistic to Fusarium moniliforme, a soil borne fungal pathogen : isolation and identification. Soil Biol Biochem 24, 979-987. 24 Higgins, D. G., Blessby, A. 1. & Fuchs, R. (1992). CLUSTAL v: improved software for multiple sequence alignment. Comput Appl Biosci 8, 189-191. 25. Imanaka, H., Kousaka, M., Tamura, G. & Arima, K. (1965). Studies on pyrrolnitrin, a new antibiotic. Taxonomy studies on pyrrolnitrin-producing strain. J Antibiot 18,205-206. 26. Jukes, T. H. & Cantor, C. R. (1969). Evolution of protein molecules. In Mammalian Protein Metabolism, vol. 111. Edited by H. N. Munro. New York: Academic Press. I.

International Journal of Systematic Bacteriology 48 Downloaded from www.microbiologyresearch.org by IP: On: Mon, 07 Mar 2016 04:37:59


V. Viallard and others 27. Kuczius, R., Ludwig, W., Peiffer, 5. & Kleiner, D. (1997). Isolation and characterization of Burkholderia norimbergensis sp. nov., a mildly alkaliphilic sulfur oxidizer. Unpublished (quoted in GenBank). 28. Kurita, T. & Tabei, H. (1967). On the pathogenic bacterium of bacterial grain rot of rice. Ann Phytopathol Soc Jpn 33, 111. 29. Lau, P. C. K. & Bergeron, H. (1997). 16s rDNA sequence of Pseudornonas sp. LB400, a prototype biphenyl/polychlorinated biphenyl degrader. Unpublished (quoted in GenBank). 30. Ludwig, W., Rossello-Mora, R., Aznar, R. & 14 other authors (1995). Comparative sequence analysis of 23s rRNA from proteobacteria. Syst Appl Microbioll8, 164-188. 31. McCulloch, L. (1921). A bacterial disease of Gladiolus. Science 54, 115-116. 32. Mergeay, M., Nies, D., Schlegel, H. G., Gerits, J., Charles, P. & Van Gijsegem, F. (1985). Alcaligenes eutrophus CH 34 is a facultative chemolithotroph with plasmid-bound resistance to heavy metals. J Bacterioll62, 328-334. 33. Mueller, J. G., Devereux, R., Santavy,D. L., Lantz, S. E., Willis, 5. G. & Pritchard, P. H. (1996). Phylogeneticand physiological comparison of PAH-degrading bacteria from geographically diverse soils. Antonie Leeuwenhoek 71, 329-343. 34. Normand, P., Cournoyer, B., Simonet, P. & Nazaret, 5. (1992). Analysis of ribosomal rRNA operon in the actinomycete Frankia. Gene 111, 119-124. 35. Palleroni, N. J. (1984). Genus I. Pseudomonas Migula 1894. In Bergey’s Manual of Systematic Bacteriology, vol. 1, pp. 141-199. Edited by N. R. Krieg & J. G. Holt. Baltimore: Williams & Wilkins. 36. Palleroni, N. 1. & Holmes, R. (1981). Pseudomonas cepacia sp. nov. nom. rev. Int J Syst Bacteriol31, 479-481. 37. Palleroni, N. J., Kunisawa, R., Contopoulou, R. & Doudoroff, M. (1973). Nucleic acid homologies in the genus Pseudomonas. Int J Syst Bacteriol23, 333-339. 38. Peyret, M., Freney, J., Meugnier, H. & Fleurette, J. (1989). Determination of G + C content of DNA using HPLC for the identification of staphylococci and micrococci. Res Microbioll40, 467-475. 39. Poirier, 1. (1994). Taxonomie des souches bactkriennes isolkes sur milieu PCAT. Memoire pour diplome d’itudes approfondies, Universiti C1. Bernard Lyon I, France. 40. Ralston, E., Palleroni, N. J. & Doudoroff, M. (1973). Pseudomonas pickettii, a new species of clinical origin related to Pseudomonas solanacearum. Int J Syst Bacteriol23, 15-19. 41. Rigby, P. W. J., Dieckmann, M.,Rhodes, C. & Berg, P. (1977). Labelling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. Int J Syst Bacteriolll3, 237-25 1. 42. Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol4,406-425. 43. Severini, G. (1913). Una bacteriosi dell’Ixia maculata e del Gladiolus coluilli. Ann Bot (Rome) 11 , 413-424. 44. Sneath, P. H. A. & Sokal, R. R. (1973). Numerical Taxonomy: the Principles and Practice of Numerical Classijcation. San Francisco : W. H. Freeman. 45. Sneath, P. H. A. & Skerman, V. B. D. (1966). A list of type and reference strains of bacteria. Int J Syst Bacterioll6, 1-133. 46. Springael, D., van Thor, J., Goorissen, H. & 7 other authors (1996). RP4 ::Mu3A-mediated in vivo cloning and transfer 562

of a chlorobiphenyl catabolic pathway. Microbiology 142, 3283-3293. 47. Stanier, R. Y., Palleroni, N. 1. & Doudoroff, M. (1966). The aerobic pseudomonads : a taxonomic study. J Gen Microbiol 43, 159-271. 48. Stapp, C. (1935). Contemporary understanding of bacterial plant diseases and their causal organisms. Bot Rev 1, 405-418. 49. Tran Van, V. (1989). Isolement et identification des bactiries hatrices d’azote associies aux racines du riz poussant sur un sol de rizikre de Vietnam. Memoire pour diplome d’itudes approfondies UniversitC Cl. Bernard Lyon I, France. 50. Tran Van, V., Berge, O., Balandreau, J., Ngo KQ, S. & Heulin, T. (1996). Isolement et activite nitrogenasique de Burkholderia vietnamiensis, bactkrie fixatrice d’azote associie au riz (Oryza sativa L.) cultivk sur un sol sulfati acide du Vietnam. Agronomie 16,479-491. 51. Tran Van, V., Gillis, M., Hebbar, P., Fernandez, M., Segers, P., Martel, M. H., Berge, O., Meyer, J. M. & Heulin, T. (1993).

Isolation of a new species of nitrogen-fixing Proteobacteria, from the rice rhizosphere, belonging to the genus Burkholderia. In Nitrogen Fixation with Non-Legumes, pp. 299-309. Edited by N. A. Hegazi, M. Fayez & M. Monib. Cairo : American University in Cairo Press. 52. Tsuchiya, K., Homma, Y., Komoto, Y. & Suzui, T. (1995). Practical detection of Pseudomonas cepacia from rhizosphere antagonistic to plant pathogenes with a combination of selective medium and ELISA. Ann Phytopathol SOCJpn 61,318-324. 53. Urakami, T., Ito-Yoshida, C., Araki, H., Kijima, T., Suzuki, K. & Komagata, K. (1994). Transfer of Pseudomonasplantarii and Pseudomonas glumae to Burkholderia as Burkholderia spp. and description of Burkholderia vandii sp. nov. Int J Syst Bacteriol44, 235-245. 54. Vandamme, P., Johannes, A.G., Cox, H.C. & Berends, W. (1960). On toxoflavin, the yellow poison of Pseudomonas cocovenenans. Recl Trav Chim Pays-Bas Belg 79, 255-261. 55. Vandamme, P., Pot, B., Gillis, M., De Vos, P., Kersters, K. & Swings, J. (1996). Polyphasic taxonomy, a consensus approach to bacterial systematics. Microbiol Rev 60,407-438. 56. Walsh, A. L., Wuthiekanun, V., Smith, M. C. D., Suputtamongkol, Y. & White, N. 1. (1995). Selective broths for the isolation of Pseudomonas pseudomallei from clinical samples. Trans R SOCTrop Med Hyg 89, 124. 57. Wayne, L. D. G., Brenner, D. J., Colwell, R. R. & 9 other authors (1987). International Committee on Systematic Bacteriology. Report of the ad hoc committee on reconciliation of approaches to bacterial systematics.Int J Syst Bacteriol37, 463-464. 58. Welch, D. F., Muszynski, M. J., Pai, C. H. & 7 other authors (1987). Selective and differential medium for recovery of Pseudomonas cepacia from the respiratory tracts of patients with cystic fibrosis. J Clin Microbiol25, 1730-1734. 59. Woese, C. R. (1987). Bacterial evolution. Microbiol Rev 51, 22 1-27 1. 60. Li, X., Dorsch, M., Del Dot, T., Sly, L. I., Stackebrandt, E. & Hayward, A. C. (1993). Phylogenetic studies of the rRNA group I1 pseudomonads based on 16s rRNA gene sequences. J Appl Bacteriol74, 324-329. 61. Yabuuchi, E., Kosako, Y., Oyaizu, H., Yano, I., Hotta, H., Hashimoto, Y., Ezaki, T. & Arakawa, M. (1992). Proposal of International Journal of Systematic Bacteriology 48

Downloaded from www.microbiologyresearch.org by IP: On: Mon, 07 Mar 2016 04:37:59

Analysis of Burkholderia graminis sp. nov. Burkholderia gen. nov. and transfer of seven species of the genus Pseudomonas homology group I1 to the new genus, with the type species Burkholderia cepacia (Palleroni and Holmes, 1981) comb. nov. Microbiol Immunol 36, 12511275. 62. Yabuuchi, E., Kosako, Y., Yano, I., Hotta, H. & Nishiuchi, Y. (1995). Transfer of two Burkholderia and an Alcaligenes species to Ralstonia gen. nov. : proposal of Rulstonia pickettii (Ralston, Palleroni and Doudoroff, 1973) comb. nov., Ralstonia solanacearum (Smith, 1896) comb. nov. and

Ralstonia eutropha (Davis, 1969) comb. nov. Microbiol Immunol39, 897-904. 63. Zhao, N. X., Qu, C. F., Wang, E. T. & Chen, W. X. (1995). Phylogenetic evidence for the transfer of Pseudomonas cocovenenans (van Damme et al. 1960) to the genus Burkholderia as Burkholderia cocovenenans (van Damme et al. 1960) comb. nov. Int J Syst Bacteriol45, 600-603. 64. Zolg, W. & Ottow, J. C. G. (1975). Pseudomonas glathei sp. nov. a new nitrogen scavenging rod isolated from acid lateritic relicts in Germany. 2 Allg Mikrobiol 15, 287-299.

International Journal of Systematic Bacteriology 48 Downloaded from www.microbiologyresearch.org by IP: On: Mon, 07 Mar 2016 04:37:59


Lihat lebih banyak...


Copyright © 2017 DADOSPDF Inc.