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1 LSTM – Laboratoire des Symbioses Tropicales et Méditerranéennes, UMR 113, Campus International de Baillarguet TA 10/J, 34398 Montpellier Cedex 05, France
2 Institut National de la Recherche Agronomique, UMR 77, Pathologie Végétale, BP 60057, 49071 Beaucouzé cedex, France
3 Institut des Régions Arides, Nahal-Gabès, Tunisia
Correspondence
Jean-Claude Cleyet-Marel
cleyet{at}ensam.inra.fr
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains STM 196T, ORS 1419T, STM 201T and STM 370T are respectively AY785319, AY785323, AY785320 and AY785325, and the accession numbers for the atpD gene sequences of strains STM 196T, ORS 1419T and STM 201T are AY785335, AY785339 and AY785336.
Details of culture media compositions, sequence alignments in PIR format and extended phylogenetic trees for 16S rRNA gene and atpD analysis are available as supplementary material in IJSEM Online.
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TOPABSTRACTMAIN TEXTREFERENCES |
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, but the genus name Phyllobacterium was originally coined by Knösel (1962) for bacteria developing within leaf nodules of tropical ornamental plants. The genus description was based mainly on phenotypic features, and the genus included two species, Phyllobacterium myrsinacearum (the type species) and Phyllobacterium rubiacearum (Knösel, 1984). Later, on the basis of molecular characteristics (DNA–DNA hybridizations and fatty acid composition), Mergaert et al. (2002) merged the two species under the emended description of the type species P. myrsinacearum. At present, the genus Phyllobacterium lies within the family Phyllobacteriaceae in the order Rhizobiales of the class Alphaproteobacteria, in the vicinity of Mesorhizobium, Allorhizobium, Aminobacter, Aquamicrobium, Defluvibacter and Pseudaminobacter. The genus includes P. myrsinacearum and two recently described species, Phyllobacterium trifolii (Valverde et al., 2005) and Phyllobacterium catacumbae (Jurado et al., 2005).
During the past 15 years, many bacteria have been assigned to the genus Phyllobacterium on the basis of molecular phylogeny (by using the 16S rRNA gene or ssu), fatty acid composition and phenotypic characterization. Identified in different environments (using molecular probes or after cultivation), a large majority of them are plant-associated bacteria and occupy diverse ecological niches: in the rhizosphere of Picea abies and Lotus spp. (Elo et al., 2000; Oger et al., 2004), in tight connection with roots in Saccharum officinarum, Beta vulgaris and Brassica napus (Lambert et al., 1990; Lilley et al., 1996; Bertrand et al., 2001), endophytic in Picea spp., Zea mays, Gossypium hirsutum and Trifolium pratense (Chanway et al., 1994; McInroy & Kloepper, 1995; Hallmann et al., 1997; Sturz et al., 1998) and in root nodules of Trifolium pratense (Sturz et al., 1997; Valverde et al., 2005) and Dalbergia louvelli (Rasolomampianina et al., 2005). They have also been found as free-living bacteria in soil (Jurado et al., 2005), in water (Mergaert et al., 2001) and associated with unicellular organisms (Gonzalez-Bashan et al., 2000; Alavi et al., 2001). Their great variety of habitats suggests that phyllobacteria have developed important adaptive capacities to the environment. In addition, their non-pathogenic status and their ability to ‘communicate’ with plant tissues has made them attractive for examination of their plant-growth-promoting potential. Indeed, several strains have been characterized as plant-growth-promoting bacteria on different plants. Strain W3 stimulates initial root growth and de novo root development in Picea spp. (Chanway et al., 1994), strain STM 196 (a synonym of isolate 29-15) was recognized as a plant-growth-promoting bacterium in plant culture of oilseed rape (Brassica napus) (Bertrand et al., 2001; Larcher et al., 2003) and Arabidopsis thaliana (Mantelin et al., 2006) and strain BOG-1-98 promotes growth of black mangrove seedlings in artificial sea water when co-inoculated with Bacillus licheniformis (Rojas et al., 2001). Interestingly, some strains have been isolated from root nodules (Sturz et al., 1997; Rasolomampianina et al., 2005), although the capacity of the isolates to induce nodulation was not clearly demonstrated. However, in early studies, van Veen et al. (1988) reported root-nodule formation in Vicia sativa by P. myrsinacearum after introduction of a Rhizobium leguminosarum symbiotic plasmid (pSym), indicating that chromosomal genes involved in nodule formation are functionally present in the bacterium. Furthermore, Valverde et al. (2005) recently isolated a Phyllobacterium strain that induced infective nodules on Trifolium pratense and Lupinus albus roots.
In the past two decades, polyphasic taxonomic studies (Vandamme et al., 1996), especially using methods for analysing micro-organisms at the molecular level, have played a crucial role in improving the classification of many bacterial groups like the pseudomonads, rhizobia, Burkholderia, Caulobacter and Acetobacter (de Lajudie et al., 1994; Gillis et al., 1995; Abraham et al., 1999; Catara et al., 2002; Cleenwerck et al., 2002). The 16S rRNA phylogeny has had a major influence on our current perception of evolutionary relationships among bacteria and rhizobia in particular (Willems & Collins, 1993; Young & Haukka, 1996), but other genes (atpD, recA or the glutamine synthetase I gene) are now also being examined and integrated in phylogenetic studies (Turner & Young, 2000; Gaunt et al., 2001), and multilocus sequence analysis has been proposed for the delineation of genera and species (Martínez et al., 2004; Gevers et al., 2005).
Mergaert et al. (2002) emended the description of the genus Phyllobacterium, but the diversity and classification of bacteria assigned to Phyllobacterium still remain poorly documented. Here we report on a polyphasic taxonomic study that included 18 Phyllobacterium sp. strains originating from different ecological niches and geographical origins (Table 1): two reference strains described by Knösel (1984), nine additional strains assigned to P. myrsinacearum (Mergaert et al., 2002) and isolated from Saccharum officinarum in Europe (Lambert et al., 1990), two root-associated bacterial strains isolated from Brassica napus in France (Bertrand et al., 2001) and five strains isolated from naturally occurring nodules of three wild legume species growing in the infra-arid zone of Tunisia. On the basis of published data and our molecular and phenotypic results (16S rRNA gene and atpD phylogenies, DNA–DNA hybridizations and numerical taxonomy of phenotypic characteristics), we describe four novel species of the genus Phyllobacterium and we propose an emended description of the genus.
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. Naturally occurring root nodules were collected in natura and either used directly for bacterial isolation or stored dried in CaCl2. Upon utilization, nodules were rehydrated in sterile water and surface sterilized by immersion in 3 % (w/v) calcium hypochlorite for 5 min. The nodules were then rinsed aseptically eight times in sterile water and then crushed in a drop of sterile water and the suspension was streaked on yeast extract/mannitol (YM) agar medium in Petri dishes (Vincent, 1970). Colonies appeared after incubation for 1 week at 28 °C under aerobic conditions and were checked for purity by repeated streaking on YM agar and by microscopic examination of living cells. Isolates were stored at –80 °C in YM broth adjusted to 20 % glycerol (v/v). To complete phylogenetic data, the following type strains were used, provided by the BCCM/LMG Bacteria Collection: Agrobacterium tumefaciens LMG 140T (=ORS 1351T), Chelatobacter heintzii LMG 2122T (=STM 2150T), Mesorhizobium amorphae LMG 18977T (=STM 291T), Mesorhizobium chacoense LMG 19008T (=STM 2154T), Mesorhizobium plurifarium LMG 11892T (=ORS 1032T), Ochrobactrum anthropi LMG 3331T (=STM 2148T), Rhodobacter sphaeroides LMG 2827T (=STM 2152T), Sinorhizobium adhaerens LMG 20216T (=STM 2072T), Sinorhizobium morelense LMG 21331T (=STM 2064T) and Sinorhizobium xinjiangense LMG 17930T (=STM 2071T). All the strains were grown at 28 °C and, except for C. heintzii, O. anthropi and R. sphaeroides, strains were grown in YM medium. The compositions of the culture media are given as supplementary material in IJSEM Online.
Genomic DNAs were purified from an 800 ml 2-day culture. Bacterial cells were harvested by centrifugation (15 min, 3200 g, 20 °C) and washed twice with phosphate buffer (0·01 M, pH 7). Cell pellets (1·5–3 g) were suspended in 3 ml TE buffer (10 mM Tris/HCl, 1 mM EDTA, pH 8). Extraction and purification of genomic DNA were then performed as described by Pitcher et al. (1989). For other bacteria, genomic DNA was purified from a 5 ml 2-day culture, according to Chen & Kuo (1993). DNA quality and concentration were determined by UV spectrophotometry.
The primers used for DNA amplification and sequencing are described in Table 2. Nearly full-length 16S rRNA genes was amplified using the universal eubacterial primers FGPS6 and FGPS1509 (Sy et al., 2001) adapted from Weisburg et al. (1991). Partial amplification and sequencing of the atpD gene were performed as described by Gaunt et al. (2001). PCR amplifications were performed in a reaction mixture of 25 µl (total volume) containing: 50 or 75 ng genomic DNA for ssu and atpD amplification, respectively, 0·2 mM each dNTP, 0·8 µM each primer, 1·25 U GoTaq DNA polymerase (Promega) and the buffer supplied with the enzyme. PCR amplifications were performed using a GeneAmp PCR System 2400 thermocycler (Applied Biosystems). A touchdown PCR program (Don et al., 1991) was used for 16S rRNA gene amplification: 95 °C (5 min), 20 cycles of 94 °C (30 s), annealing temperature (30 s) from 60 to 50 °C and 72 °C (2 min), followed by 25 cycles of standard PCR (55 °C annealing temperature) and an additional cycle with a final 7 min chain elongation.
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), and then optimized by hand under the GeneDoc alignment editor program (Nicholas et al., 1997), especially for atpD, to obtain gene alignments in accordance with amino acid alignments. Five parts of the ssu sequences for which no unambiguous alignment could be achieved were removed from the final matrix. The ssu and atpD data matrices used in the phylogenetic analyses were finally 1410 and 447 bp, respectively, in length. Reduced alignments (without ambiguous regions) are available in PIR format as supplementary material in IJSEM Online.
Phylogenetic trees were generated by distance and maximum-likelihood methods using PAUP, version 4.0b10 (Swofford, 1998). We estimated the best likelihood model for each dataset using Winmodeltest (Posada & Crandall, 1998). We thus applied a GTR+G+I model for the ssu data matrix and estimated parameters (shape parameter of the gamma distribution of four rates at variable sites, base frequencies, proportion of invariable sites) from our data. A similar model (GTR+G+I, but with different parameters) was used for the atpD analyses, as a result of Winmodeltest choice tests. The distance trees were obtained by using a heuristic search implemented in PAUP. The distance matrix used was based on the maximum-likelihood model chosen with the parameters estimated previously. Bootstrap values of each node were calculated from 1000 replicates. Maximum-likelihood analysis was similarly conducted with a heuristic search using the model defined for each data matrix.
In the current bacterial taxonomy, genus delineation is mainly based upon the phylogenetic relationships of 16S rRNA genes. The 16S rRNA phylogeny reconstructed by the distance method placed the strains listed in Table 1 unambiguously in the Alphaproteobacteria and grouped all of them and P. myrsinacearum, P. catacumbae and P. trifolii in a clade with 88 % bootstrap support (Fig. 1; a complete version of this tree is available as Supplementary Fig. S1 in IJSEM Online). Although highly supported in the distance tree, the Phyllobacterium strains made a polyphyletic group in the maximum-likelihood tree (indicated in Fig. 1). The atpD phylogeny (Fig. 2; a complete version of this tree is available as Supplementary Fig. S2 in IJSEM Online) also grouped all studied strains with P. myrsinacearum, although not with high bootstrap support (less than 70 %). For technical reasons, we could not obtain atpD sequences for strains belonging to 16S rRNA cluster A. The failure to amplify atpD sequences from these strains could be due to a point mutation in the sequence corresponding to one of the two primers used for amplification. Although their phylogenetic position could therefore not be confirmed by the atpD phylogeny, we are confident that cluster A strains are closely related to P. myrsinacearum, since their position in the ssu phylogeny is highly supported in both phylogenetic analyses (distance and maximum-likelihood). Altogether, these results strongly support the conclusion that the strains investigated in this study form a monophyletic group with the previously defined Phyllobacterium species and that they may all be affiliated to Phyllobacterium.
As observed previously by Gaunt et al. (2001), the placement of the P. myrsinacearum clade relative to other genera differs in the two trees (ssu and atpD) and is thus uncertain. The close relationship with Mesorhizobium seen in the ssu phylogeny was not supported by the atpD phylogeny, which suggested a deeper placement. In the atpD tree (Fig. 2), Phyllobacterium appears closer to the Sinorhizobium–Rhizobium clade than to Mesorhizobium. This uncertainty may originate from ancestral lateral gene transfer among alphaproteobacteria. More molecular and phylogenetic analyses should be performed to resolve this.
Within the Phyllobacterium clade, the studied strains are divided into four well-supported clusters (in the 16S rRNA phylogeny), obtained by both phylogenetic methods (distance and maximum-likelihood), three of which are confirmed by the atpD phylogeny (Figs 1 and 2). The P. myrsinacearum cluster includes recognized members of the species, together with the strains studied by Lambert et al. (1990), with 99 % internal sequence similarity [all isolates of Lambert et al. (1990) display the same ssu sequence]. The strains originating from Brassica napus (Bertrand et al., 2001) are genetically different, but share more than 98 % identity, and fall within cluster B. Clusters A and C consist of strains from Tunisia, having identical ssu gene sequences within each cluster. Cluster A is part of a larger cluster, supported by a high bootstrap value (86 %), that includes the type strains of the recently described species P. catacumbae and P. trifolii (Jurado et al., 2005; Valverde et al., 2005). In this clade, P. trifolii is a sister branch of the subclade including P. catacumbae and cluster A strains. Although the P. catacumbae type strain and the two cluster A strains can not be distinguished phylogenetically, the overall divergence of their sequences suggests that P. catacumbae is genuinely different from the two studied strains found in cluster A. This relationship may warrant further DNA–DNA hybridization studies, however. The relationships of the studied strains in the atpD phylogeny are similar to those constructed by 16S rRNA gene sequences (Fig. 1), and all nodes are recovered in both distance and maximum-likelihood trees (indicated in Fig. 2). In conclusion, according to the phylogenetic analyses, all the strains investigated in this study form a monophyletic group relative to the genus Phyllobacterium and form well-defined clusters.
To determine the species status of the three clusters identified in our phylogenetic approach, DNA–DNA hybridizations were performed (Table 3). Native DNAs were labelled in vitro by random priming with tritium-labelled nucleotides using the Megaprime DNA labelling system (Amersham Biosciences). The S1 nuclease/trichloroacetic acid method for hybridization has been described previously (Crosa et al., 1973; Grimont et al., 1980). Reassociation was performed at 70 °C in 0·42 M NaCl. DNA–DNA hybridization tests were carried out using labelled DNA from P. myrsinacearum STM 948T and from representative strains of each phylogenetic cluster. Each value is a mean of two to four replicate experiments. Thermal stability of reassociated DNA was determined by using the method of Crosa et al. (1973) when the percentage of DNA–DNA hybridization was between 65 and 75 % (
Tm given in Table 3).
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using a different method, thus confirming the synonymy of these two species. DNA hybridization values between representative strains of phylogenetic clusters A, B and C and the type strain of P. myrsinacearum ranged from 5 to 19 % (Table 3). Reciprocal hybridizations gave the same results. These values are not significant and indicate that these clusters may represent novel Phyllobacterium species. Genetic homogeneity inside clusters A and C was verified by high internal DNA hybridization values between members of each cluster. These results indicated unambiguously that clusters A and C represent two distinct genomospecies. In contrast, DNA reassociation between the two strains of cluster B was only 20 %; strains STM 201T and STM 196T therefore represent two additional genomospecies.
The DNA base composition was determined by the thermal denaturation temperature protocol (Marmur & Doty, 1962) and was calculated by using the equation of Owen & Lapage (1976). Escherichia coli K-12 [DNA G+C content of 50·6 mol% (Tm)] was used as a control. The DNA base compositions are indicated in Table 3. The DNA G+C contents of the studied strains are in the range 51–58·5 mol% (Tm), which corresponds to a lower range of values than those reported by Gillis & De Ley (1980) for recognized members of the genus Phyllobacterium, i.e. 60–61 mol% (Tm) for P. myrsinacearum strains. The DNA base composition is homogeneous between strains belonging to the same genomospecies.
Twenty-two conventional biochemical and physiological tests were performed. Gram determination was performed with 3 % KOH solution (Suslow et al., 1982). Oxidase activity was assessed with dimethyl p-phenylenediamine reagent (Kovacs, 1956). Oxidative or fermentative glucose metabolism was determined by using Hugh & Leifson medium (Hugh & Leifson, 1953) in Yvan Hall tubes. Urease activity and indole formation were tested by using commercial urea-indole medium (bioMérieux) and 
-galactosidase activity by using ONPG discs (bioMérieux). Hydrolysis of gelatin and Tween 80 was tested on gelatin and Tween 80 agar, respectively (Sands, 1990). The presence of DNase was tested on commercial DNA agar (Diagnostic Pasteur). Production of 3-ketolactose was assessed according to Bernaerts & De Ley (1963). Strains were also investigated for their ability to grow under different conditions. The ability of the strains to grow in Luria–Bertani (LB) medium, YM broth without CaCl2, YM broth with 1, 2 or 3 % NaCl and YM broth at pH 4, 5, 9 or 10 was determined in liquid medium as described previously (Nour et al., 1994). All the tests were incubated at 28 °C and the presence or absence of growth was recorded at 3, 6, 9, 12 and 15 days. Maximum growth temperature was tested on YM with agar (15 g l–1) at 35, 37 and 40 °C. Metabolic profiles based on 99 carbon sources were studied by using Biotype 100 strips with biomedium 1 (biomedium 2 was used for strains of cluster C), as recommended by the manufacturer (bioMérieux). Strips were incubated at 28 °C and read at 2, 4 and 6 days after inoculation.
A total of 121 tests, including 22 conventional and physiological characters and assimilation of 99 carbon sources, were used for numerical taxonomy analysis for the 18 Phyllobacterium strains listed in Table 1. The distance matrix was calculated using the Jaccard coefficient (Sneath & Sokal, 1973). Cluster analysis was performed by using the unweighted pair group method with arithmetic averages (Sneath & Sokal, 1973). Discriminatory tests were selected using the diagnostic ability coefficient deduced from the numerical analysis (Descamps & Véron, 1981). The dendrogram of phenotypic distances is shown in Fig. 3.
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. The major features that differentiate P. myrsinacearum from the four novel genomospecies are its ability to grow at pH 4 and to assimilate glutarate and L-tyrosine. Strains of cluster C were distantly related to the other Phyllobacterium strains and are distinguished by assimilation of few Biotype 100 substrates, even with the use of biomedium 2 (bioMérieux), which contains 31 growth factors, as the inoculation medium. Strains of cluster C are the only Phyllobacterium strains that assimilate mucate and saccharate on Biotype 100 (strain STM 201T is the other exception, as it assimilates D-saccharate). Strains of clusters A and C are the only Phyllobacterium strains able to grow at 37 °C. This phenotypic trait is probably related to their common geographical origin (southern Tunisia), where high temperatures occur. Assimilation of i-erythritol is an exclusive character of strains from cluster A. Like some strains of P. myrsinacearum, strains of cluster A are able to grow in 3 % NaCl. Strains of cluster B, isolated from roots of Brassica napus, share common properties that differentiate them from other Phyllobacterium strains; they cannot grow at 35 °C and do not assimilate D-glucuronate or D-galacturonate. Among the Phyllobacterium strains studied, strain STM 196T shares common features with strains of cluster C, which are unable to assimilate (–)-L-arabitol, D-tagatose and adonitol. Strain STM 201T is characterized by its inability to assimilate 1-O-methyl -D-glucopyranoside and its inability to grow in LB broth and in 2 % NaCl YM broth.
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) indicated that Phyllobacterium includes four novel genomospecies that can be clearly differentiated from each other by phylogenetic and phenotypic analyses. As a consequence, we propose the creation of four novel species: Phyllobacterium leguminum sp. nov. for cluster C strains, Phyllobacterium ifriqiyense sp. nov. for cluster A strains, Phyllobacterium brassicacearum sp. nov. for cluster B strain STM 196T and Phyllobacterium bourgognense sp. nov. for cluster B strain STM 201T, and an emended description of the genus Phyllobacterium. Among the novel species, P. leguminum was phylogenetically and phenotypically the most distant from P. myrsinacearum, but it shares sufficient common properties (both phenotypic and genotypic) that allow it to be included in the genus Phyllobacterium.
We observed that the strains isolated by Lambert et al. (1990) are closely associated with P. myrsinacearum by both phylogenetic (Figs 1 and 2) and phenotypic (Table 4 and Fig. 3) characteristics. These results strengthen the conclusion of Mergaert et al. (2002), who proposed to assign the sugar-beet isolates to P. myrsinacearum based on results from two representative strains (LMG 8225 and LMG 8229).
Since Phyllobacterium strains are geographically widespread in a great variety of habitats, they could be soil residents that may occasionally become associated specifically with plant roots. In addition, the fact that some bacteria were isolated from either leaf or root nodules indicates that phyllobacteria might be a general phytosphere colonizer able to evolve and adopt different life styles. This important adaptive capacity to various environments developed by phyllobacteria was foreseen by Lambert et al. (1990) on the basis of the nutritional versatility of P. myrsinacearum strains and their antifungal activity. They reported that these features ‘probably support [their] competitive growth and abundant proliferation in the rich environment of the root surface where various compounds present in the root exudates attract diverse micro-organisms’.
Emended description of the genus Phyllobacterium (ex Knösel 1962) Knösel 1984
This description takes into account results from Valverde et al. (2005) and Jurado et al. (2005) together with those from this study. Cells are Gram-negative rods, motile by means of polar, subpolar or lateral flagella. Colonies grown on YM agar medium are circular, white or cream-coloured with regular margins. Most strains are highly mucoid on YM medium. The optimal growth temperature is 28 °C. Growth occurs in 1 % NaCl and does not occur at 40 °C. Glucose metabolism is oxidative. Cultures are oxidase- and urease-positive. They lack the following exoenzyme activities: -galactosidase, gelatinase, DNase (not tested for P. trifolii) and Tween 80 hydrolase (not tested for P. trifolii). Aesculin is hydrolysed (weak reaction for P. trifolii). Indole and 3-ketolactose are not produced (not tested for P. trifolii). Assimilation of 
-(+)-D-glucose, (+)-D-mannose, maltose, (+)-L-arabinose, D-mannitol and n-acetyl-D-glucosamine is positive. Additional features common to the 18 strains studied in this paper, representing five of the seven species of the genus Phyllobacterium, are presented in the legend to Table 4. The G+C content of the DNA is 51–61 mol% (Tm). The type species is Phyllobacterium myrsinacearum.
Description of Phyllobacterium leguminum sp. nov.
Phyllobacterium leguminum (le.gu'min.um. L. gen. pl. neut. n. leguminum of legumes, referring to its isolation from root nodules of legumes).
Strains share the general properties of the genus Phyllobacterium. Colonies on YM agar are white and highly convex. The optimal growth temperature is 28 °C. Growth on YM agar occurs at 37 °C but not at 40 °C. Strains are able to grow in YM broth, YM broth without CaCl2 and LB broth. Growth occurs in YM broth with 2 % NaCl, but not 3 %, and in YM broth at pH 5–10, but not at pH 4. Strains assimilate few substrates on Biotype 100 strips, which differentiates the species from other Phyllobacterium species. Assimilation of D-galacturonate, D-glucuronate, D-saccharate, mucate and 1-O-methyl -D-galactopyranoside are discriminatory characters. Assimilation of the following substrates is weak: (+)-D-trehalose, maltose, 1-O-methyl -D-glucopyranoside, (–)-D-ribose, glycerol, myo-inositol, (+)-D-turanose, 2-keto-D-gluconate, D-glucosamine, L-glutamate and L-proline. Can be differentiated from the other Phyllobacterium species by DNA–DNA hybridization, 16S rRNA gene or partial atpD gene sequencing and phenotypic tests (Table 4). The G+C content of the DNA is 57–58 mol% (Tm).
The type strain is strain ORS 1419T (=CFBP 6745T=LMG 22833T). Strains have been isolated from root nodules of Argyrolobium uniflorum and Astragalus algerianus.
Description of Phyllobacterium ifriqiyense sp. nov.
Phyllobacterium ifriqiyense (if.ri'qi.yen.se. N.L. neut. adj. ifriqiyense pertaining to Ifriqiya, the earliest Arabic name of the North African territory that included Tunisia, where the first strains were isolated).
Strains have the general characteristics of the genus Phyllobacterium. The optimal growth temperature is 28 °C. Growth on YM agar occurs at 37 °C but not at 40 °C. Strains are able to grow in YM broth, YM broth without CaCl2 and LB broth. Growth occurs in YM broth with 1, 2 or 3 % NaCl and in YM broth at pH 5–10, but not at pH 4. Assimilation of the following substrates is variable or weak: -lactose, lactulose, 1-O-methyl -galactopyranoside, 1-O-methyl -galactopyranoside, cis-aconitate, succinate, DL--amino-n-valerate, trigonelline and L-serine. Assimilation of i-erythritol is an exclusive character of strains of this species. Lack of assimilation of maltotriose, citrate, protocatechuate and quinate on Biotype 100 strips are discriminatory characters for strains of P. ifriqiyense, shared with P. leguminum strains; other discriminatory characters within the genus are listed in Table 4. At the molecular level, the species is differentiated by DNA–DNA hybridization, by 16S rRNA gene sequencing and by its low DNA G+C content, 51–52 mol% (Tm).
The type strain is strain STM 370T (=CFBP 6742T=LMG 22831T). Strains have been isolated from root nodules of Astragalus algerianus and Lathyrus numidicus.
Description of Phyllobacterium brassicacearum sp. nov.
Phyllobacterium brassicacearum (bras.si.ca.ce.ar'um. N.L. gen. pl. fem. n. brassicacearum of the Brassicaceae, referring to the isolation of the type strain from Brassica napus and its growth-promoting effect on B. napus and Arabidopsis thaliana, members of the Brassicaceae).
Strains share the general properties of the genus Phyllobacterium. The optimal growth temperature is 28 °C. Growth on YM agar does not occur at 35 °C. Strains are able to grow in YM broth, YM broth without CaCl2 and LB broth. Growth occurs in YM broth with 2 % NaCl, but not 3 %, and in YM broth at pH 5–10, but not at pH 4. This species can be differentiated from other Phyllobacterium species by its auxanotrophic characteristics (Table 4), mainly its inability to assimilate (–)-L-arabitol, D-tagatose and adonitol, like strains of P. leguminum. The inability of the type strain to grow at 37 °C is one of the discriminatory characters between this species and P. leguminum. At the molecular level, DNA–DNA hybridization and 16S rRNA gene and/or partial atpD gene sequencing can be used to differentiate the strain. The G+C content of the DNA is 55·5 mol% (Tm).
The type strain, strain STM 196T (=CFBP 5551T=LMG 22836T), was isolated from the rhizoplane of Brassica napus.
Description of Phyllobacterium bourgognense sp. nov.
Phyllobacterium bourgognense (bour.gogn.en'se. N.L. neut. adj. bourgognense pertaining to Bourgogne, a region of central France, where the type strain was isolated).
Strains have the general characteristics of the genus Phyllobacterium. The optimal growth temperature is 28 °C. Growth on YM agar does not occur at 35 °C. Growth occurs in YM broth at pH 5–10, but not at pH 4. In contrast to other Phyllobacterium species, it does not grow in LB broth or in YM broth at 2 % NaCl. Can be differentiated from other Phyllobacterium species by its auxanotrophic characteristics (Table 4), mainly its inability to assimilate 1-O-methyl -D-glucopyranoside, DL-glycerate and ethanolamine and its ability to assimilate D-saccharate. Most of these characters are shared by P. leguminum, but these two species are easily differentiated by temperature and NaCl tolerance. At the molecular level, DNA–DNA hybridization and 16S rRNA gene and/or partial atpD gene sequencing can be used to differentiate the species. The G+C content of the DNA is 54 mol% (Tm).
The type strain, strain STM 201T (=CFBP 5553T=LMG 22837T), was isolated from the rhizoplane of Brassica napus.
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70 %) derived from 1000 replicates. Bar, 0·01 % sequence dissimilarity (evolutionary distance). The designation of the original sequenced strain is given after the species name. GenBank/EMBL/DDBJ accession numbers are indicated in parentheses; asterisks indicate sequences obtained in this study.
