Rhizobium cauense sp. nov., isolated from root nodules of the herbaceous legume Kummerowia stipulacea grown in campus lawn soil

Systematic and Applied Microbiology 35 (2012) 415–420

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Short communication

Rhizobium cauense sp. nov., isolated from root nodules of the herbaceous legume Kummerowia stipulacea grown in campus lawn soil夽 Tian Yan Liu a , Ying Li Jr. a , Xiao Xiao Liu a , Xin Hua Sui a , Xiao Xia Zhang c , En Tao Wang a,b , Wen Xin Chen a , Wen Feng Chen a,∗ , Joanna Puławska d a

State Key Laboratory of Agrobiotechnology and College of Biological Sciences, China Agricultural University, Beijing 100193, China Departamento de Microbiología, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, México D.F. 11340, Mexico c Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences, Beijing 100080, China d Research Institute of Horticulture, Pomology Division, Pomologiczna 18, 96-100 Skierniewice, Poland b

a r t i c l e

i n f o

Article history: Received 13 April 2012 Received in revised form 28 July 2012 Accepted 10 August 2012 Keywords: Rhizobium cauense sp. nov. Kummerowia stipulacea Polyphasic taxonomy

a b s t r a c t Three bacterial isolates (CCBAU 101002T , CCBAU 101000 and CCBAU 101001) originating from root nodules of the herbaceous legume Kummerowia stipulacea grown in the campus lawn of China Agricultural University were characterized with a polyphasic taxonomic approach. Comparative 16S rRNA gene sequence analysis showed that the isolates shared 99.85–99.92% sequence similarities and had the highest similarities to the type strains of Rhizobium mesoamericanum (99.31%), R. endophyticum (98.54%), R. tibeticum (98.38%) and R. grahamii (98.23%). Sequence similarity of four concatenated housekeeping genes (atpD, glnII, recA and rpoB) between CCBAU 101002T and its closest neighbor (R. grahamii) was 92.05%. DNA–DNA hybridization values between strain CCBAU 101002T and the four type strains of the most closely related Rhizobium species were less than 28.4 ± 0.8%. The G + C mol% of the genomic DNA for strain CCBAU 101002T was 58.5% (Tm). The major respiratory quinone was ubiquinone (Q-10). Summed feature 8 (18:1ω7cis/18:1ω6cis) and 16:0 were the predominant fatty acids. Strain CCBAU 101002T contained phosphatidylcholine and phosphatidylethanolamine as major polar lipids, and phosphatidylglycerol and cardiolipin as minor ones. No glycolipid was detected. Unlike other strains, this novel species could utilize dulcite or sodium pyruvate as sole carbon sources and it was resistant to 2% (w/v) NaCl. On the basis of the polyphasic study, a new species Rhizobium cauense sp. nov. is proposed, with CCBAU 101002T (=LMG 26832T = HAMBI 3288T ) as the type strain. © 2012 Elsevier GmbH. All rights reserved.

The root nodule bacteria or rhizobia are generally referred to as the symbiotic nitrogen fixing bacteria isolated from the root nodules of some legumes. Currently, 98 species within 13 genera of Alpha- and Betaproteobacteria have been reported at the time of writing [2]. However, some non-symbiotic bacterial species were also reported in these genera based upon their phylogenetic relationships, such as the transfer of Agrobacterium species into the genus Rhizobium Frank 1889 [28]. Among these defined species, Rhizobium taibaishanense [27], Ensifer (former Sinorhizobium) kummerowiae [26], E. meliloti [8], Shinella kummerowiae [9] and Bradyrhizobium spp. [8,24] have been described or recorded in the rhizobia isolated from the root nodules of Kummerowia spp., a widely distributed annual herbaceous legume in China. These

夽 The accession numbers of the 16S rRNA, atpD, glnII, recA and rpoB genes for the type strain CCBAU 101002T in GenBank are JQ308326, JQ308329, JQ308332, JQ308335 and JQ340069, respectively. ∗ Corresponding author. Tel.: +86 10 6273 4009; fax: +86 10 6273 4008. E-mail address: [email protected] (W.F. Chen). 0723-2020/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.syapm.2012.08.006

findings implied that the root nodules of Kummerowia may be a reservoir of diverse rhizobial species. In the present study, some bacterial isolates recovered in August 2009 from the root nodules of Kummerowia stipulacea grown in the campus lawn of China Agricultural University (CAU) were identified using phenotypic and genetic techniques. The fresh root nodules of K. stipulacea were sampled, surface sterilized (0.2% HgCl2 for 3 min followed by seven rinses in sterile de-ionized water) and used for isolation of endophytic bacteria using a standard procedure and yeast extract mannitol agar (YMA) medium [22]. The isolates were cultured at 28 ◦ C for 3–7 days and were purified by streaking three times on YMA plates. A total of 70 isolates were obtained in which half were fast growing bacteria (colony diameter >1 mm in 3 days) and the others were slow growing bacteria (colony diameter <1 mm in 7 days). All the isolates were kept on YMA slants at room temperature for a short period (one month), or stored longer at −80 ◦ C in YM broth containing 20% (w/v) glycerol. All the 70 isolates were subjected to PCR analyses based on 16S rRNA gene restriction fragment length polymorphism (RFLP), as

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Table 1 Genetic relationship between Rhizobium cauense sp. nov. CCBAU 101002T and related strains. Strains

R. cauense CCBAU 101001 R. cauense CCBAU 101000 R. mesoamericanum CCGE 501T R. endophyticum CCGE 2052T R. grahamii CCGE 502T R. tibeticum CCBAU 85039T R. leguminosarum USDA 2370T

Sequence similarity or relatedness (%) with CCBAU 101002T 16S rRNA

atpD

glnII

recA

rpoB

Concatenated atpD + glnII + recA + rpoB

DNA–DNA hybridization

99.85% 99.92% 99.31% 98.54% 98.23% 98.38% 96.97%

97.84% 96.61% 89.32% 93.05% 95.10% 90.94% 87.66%

99.15% 99.15% 89.07% 86.07% 85.82% 87.84% 82.41%

97.62% 96.54% 84.75% 84.43% 89.39% 83.46% 83.78%

98.40% 97.74% 95.92% 94.74% 95.92% 95.08% 87.41%

98.30% 97.60% 90.63% 90.28% 92.05% 90.16% 85.54%

100 93.2 ± 1.3 28.4 ± 0.8 22.4 ± 1.8 27.7 ± 1.8 23.8 ± 1.3 17.7 ± 0.8

reported previously [17]. Sequencing of the 16S rRNA and housekeeping genes (atpD, glnII, recA and rpoB) was performed for the isolates representing different RFLP patterns using the methods described by Zhang et al. [29]. In these analyses, total DNA was extracted as template for PCR from the bacterial cells using the GUTC method [18]. Primer pairs P1/P6 for the 16S rRNA gene [17], atpD225F/atpD782R for atpD [23], glnII12F/glnII689R for glnII [23], recA41F/recA640R for recA [23], and rpoB70F (5 -GTC GAA GAC GAA AAG GTC CA-3 )/rpoB949R (5 -ACT TCA CCG TCA GCA TTT CC-3 ) for rpoB were used to amplify and sequence these genes with the protocols described by Tan et al. [17], Vinuesa et al. [23], or Zhao et al. [30], respectively. Closely related sequences were retrieved from the GenBank database and aligned with the acquired sequences using the program ClustalW [4] integrated in the MEGA 5.05 software [16]. Phylogenetic trees were constructed separately for the 16S rRNA, atpD, glnII, recA and rpoB genes and for the concatenated housekeeping genes (atpD + glnII + recA + rpoB) according to the neighbor-joining (NJ), maximum likelihood (ML) or maximum parsimony (MP) algorithms using MEGA 5.05 [16]. The stability of the groupings was estimated by bootstrap analysis with 1000 pseudoreplications. Comparative 16S rRNA gene sequences revealed that the three isolates CCBAU 101002T , CCBAU 101000 and CCBAU 101001 formed a distinctive RFLP pattern (data not shown) and they shared 99.85–99.92% sequence similarities (Table 1), demonstrating their high genealogical relatedness. The BLAST query for the 16S rRNA gene sequence of strain CCBAU 101002T against the GenBank database revealed their clear affiliation to the genus Rhizobium, which showed the highest sequence similarities with the type strains of R. mesoamericanum (99.31%), R. endophyticum (98.54%), R. tibeticum (98.38%) and R. grahamii (98.23%) (Table 1). Sequence similarities of the 16S rRNA gene between strain CCBAU 101002T and other known Rhizobium species were less than 98.23%. In the analysis of concatenated housekeeping genes, they showed low sequence similarities (<92.05%) compared to the defined rhizobial species (Table 1). Taking the definition data of Rhizobium lusitanum as a reference [20], the three tested isolates clearly represented a novel species. Therefore, they were selected for further taxonomic study. The phylogenetic trees based on the ML (Fig. 1) and NJ (Supplementary Fig. S1) algorithms revealed that these three new isolates formed a distinct lineage, clustering with the abovementioned four species. Bootstrap resampling analysis supported a strong association between the novel isolates and these four defined species (Fig. 1 and Supplementary Fig. S1). However, the topological structure of the 16S rRNA MP tree (Supplementary Fig. S2) was not similar to the trees generated from MP and NJ. Rhizobium grahamii did not cluster with the novel group and the other three above-mentioned species, since it was placed in the outermost branch (Supplementary Fig. S2). Analyses of the concatenated housekeeping genes (atpD, glnII, recA and rpoB) confirmed this phylogenetic arrangement (Fig. 2). Strain CCBAU 101002T showed high similarities (97.60–98.30%) to

the other two isolates (CCBAU 101001 and CCBAU 101000) and had similarities of 90.16–92.05% with the type strains of R. tibeticum CCBAU 85039T , R. endophyticum CCGE 2052T (=ATCC BAA-2116T ), R. mesoamericanum CCGE 501T (=ATCC BAA-2123T ) and R. grahamii CCGE 502T (=ATCC BAA-2124T ) (Table 1). All the phylogenetic trees based on the ML (Fig. 2), NJ and MP algorithms (Supplementary Figs. S3 and S4) of the concatenated housekeeping genes demonstrated similar relationships and the novel isolates formed a distinctive lineage with high bootstrap value support. BOX-PCR fingerprinting defined by using the BOXAIR primer and the procedure described by Versalovic et al. [21] and profiles of whole cell soluble proteins, revealed through SDS-PAGE according to the method of Tan et al. [17], were used to evaluate the genetic relationships between the three isolates. The three isolates displayed three different BOX-PCR fingerprints and protein profiles (Supplementary Figs. S5 and S6), demonstrating that they are not clones of the same strain. Utilization of carbon and nitrogen sources, resistance to antibiotics, tolerance to NaCl and pH ranges, growth temperatures, reduction of H2 O2 and nitrate were carried out for the three novel isolates, the type strains of four closely related species (Table 2) and Rhizobium leguminosarum USDA 2370T , according to the methods described by Gao et al. [3]. Cell motility was detected using the hanging drop method [15]. The Gram stain and other biochemical properties, such as catalase, urease, l-phenylalanine deaminase, nitrate reduction, Nile blue reductase, H2 S production, and Voges–Proskauer (VP) reactions, among others, were determined as described by Smibert and Krieg [14]. Cell size was determined under the microscope after Gram staining. The three new isolates exhibited almost identical phenotypic characteristics, except for resistance to 5 ␮g mL−1 streptomycin (positive for CCBAU 101002T ), erythromycin (negative for CCBAU 101000) and chloramphenicol (negative for CCBAU 101000), and the reduction of nitrate (negative for CCBAU 101001). The phenotypic characteristics that differentiated the novel group from the type strains of related phylogenetic species are shown in Table 2. The utilization of dulcite and sodium pyruvate as sole carbon sources and tolerance to 2% NaCl (w/v) differentiated the three isolates of the novel group from their closest neighbors and the type species of the genus, R. leguminosarum USDA 2370T (Table 2). In the nodulation tests with a Leonard jar system filled with sterilized vermiculite moistened with N-free solution using the standard procedure of Vincent [22], strain 101002T did not form nodules on its original host (Kummerowia stipulacea) or any of the other tested legumes: Glycine max, Phaseolus vulgaris, Vigna unguiculata, Medicago sativa, Pisum sativum, Trifolium repens and Astragalus sinicus. Various attempts failed to amplify nodC or nifH gene sequences from the template DNA of strains CCBAU 101002T , CCBAU 101000 or CCBAU 101001 using the primer pairs of nodCF/nodCI or nifHF/nifHI, respectively, and the procedures described by Laguerre et al. [6]. These results demonstrated that the three strains were not symbiotic bacteria of K. stipulacea, although they were isolated from root nodules of this plant.

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Fig. 1. Maximum likelihood (ML) phylogenetic tree inferred from comparison of 16S rRNA gene sequences showing the position of Rhizobium cauense sp. nov. (boldface) within the genus Rhizobium. The Tamura 3-parameter + G model was used to construct the tree. Bootstrap values (expressed as a percentage of 1000 replications) equal to or higher than 50% are given at the branching points. Bradyrhizobium japonicum USDA 6T was used as an out-group. Bar, 2% sequence divergence.

In the fatty acid analysis, strain CCBAU 101002T and the type strains of five species, R. leguminosarum USDA 2370T , R. mesoamericanum CCGE 501T , R. grahamii CCGE 502T , R. endophyticum CCGE 2052T and R. tibeticum CCBAU 85039T were cultivated

aerobically on YMA at 28 ◦ C and harvested at late log phase growth (after approximately 2 days incubation). Fatty acid methyl esters were prepared and separated using the method described by Sasser [13], and identified with the MIDI Sherlock Microbial

Fig. 2. Maximum likelihood (ML) phylogenetic tree inferred from comparison of the concatenated house-keeping genes (atpD, glnII, recA and rpoB) showing the position of the novel species R. cauense (boldface) with other related species in the genus Rhizobium. The General Time Reversible + G + I model was used to construct the tree. Bootstrap values (expressed as a percentage of 1000 replications) equal to or higher than 50% are given at the branching points. Bradyrhizobium japonicum UDSA 6T was used as an out-group. Bar, 5% sequence divergence.

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Table 2 Distinctive phenotypic characteristics of Rhizobium cauense sp. nov. CCBAU 101002T and related type strains. Characteristics

1

2

3

4

5

6

7

8

Utilization of sole carbon sources Dulcite + Meso-erythritol − + Inositol + Sodium pyruvate + d-Raffinose Salicin − d-Ribose − − Sodium acetate + Sodium-d-gluconate Sodium succinate + + d-Sorbitol − Sodium tartrate d-Xylose + l-Arginine − + l-Proline

+ − + + + − − − + + + − + − +

+ − + + + − − − + + + − + − +

− + + − − − − − − + + + + − +

− − + − + − + + + + + + + + +

− + − − − − − + + − + − + − −

− + + − + + + − + − − − − − −

− − + − + + − + + + + − + − −

Utilization of sole nitrogen sources l-Cysteine + d-Glutamic acid − + l-Valine + l-Methionine − l-Threonine

+ − + + −

+ − + + −

+ − + + −

+ + + + +

− − − − −

− − − − +

+ − + + −

pH 9 pH 10 Tolerance to 2% NaCl (w/v)

+ − +

+ − +

+ − +

− − −

+ − −

+ − −

+ + −

+ + −

Resistance to (␮g mL−1 ) Ampicillin (50) Ampicillin (100) Kanamycin (5) Neomycin (5) Streptomycin (5) Erythromycin (5) Erythromycin (50) Chloramphenicol (5) Chloramphenicol (50) Reduction of nitrate

+ + + + + + + + + +

+ + + + − − − − − −

+ + + + − + + + + +

− − − − − − − + − +

− − − + − + + + − −

+ + + − − + + + − +

+ + + − − + + + + +

+ + + + − + + + − −

Strains: 1, R. cauense sp. nov. CCBAU 101002T ; 2, R. cauense sp. nov. CCBAU 101000; 3, R. cauense sp. nov. CCBAU 101001; 4, R. leguminosarum USDA 2370T ; 5, R. mesoamericanum CCGE 501T ; 6, R. grahamii CCGE 502T ; 7, R. endophyticum CCGE 2052T ; 8, R. tibeticum CCBAU 85039T . +, positive; −, negative.

Identification System (Sherlock license CD v 6.0), using the TSBA6 database. The relative amounts of the major fatty acids of isolate CCBAU 101002T were compared with those of the type strains of the most related Rhizobium species (Table 3). Some fatty acids detected in strain CCBAU 101002T [summed feature 8 (18:1ω7cis/18:1ω6cis), 16:0, 19:0 cyclo ω8cis and summed feature 2 (12:0 aldehyde/unknown 10.928)] are in accordance with characteristic fatty acids found also in members of the genus Rhizobium [19], except for 20:3 ω6, 9, 12 cis that was not detected in the test strains in this study. The detection of many other fatty acids only found in strain CCBAU 101002T revealed its difference from other known species (Table 3 and Supplementary Table S1). Isolation, purification and identification of the respiratory quinone of the representative isolate CCBAU 101002T was carried out according to the procedure described by Lee et al. [7]. CCBAU 101002T contained ubiquinone Q-10 as the major respiratory quinone, in agreement with members of the order Rhizobiales [5]. For polar lipid analysis, cells of strain CCBAU 101002T were grown in YM broth at 28 ◦ C with shaking (170 rpm) to the late log phase of growth. Polar lipids from 200 to 250 mg freezedried cells were extracted according to Minnikin et al. [12], then separated in a TLC Silica gel-60 F254 plate (Merck KGaA, Germany) by two-dimensional thin-layer chromatograms using chloroform/methanol/water (14:6:1, v/v) in the first dimension and chloroform/methanol/acetic acid (13:5:2, v/v) in the second dimension. The following spray reagents, molybdenum blue [6] (results recorded immediately), ninhydrin (0.4% in n-butyl alcohol saturated with distilled water, w/v; developed at 100 ◦ C for 5 min) and anisaldehyde (concentrated H2 SO4 /glacial acetic acid/95% ethanol/p-anisaldehyde = 15:3:270:15, v/v; developed at 110 ◦ C for 4–6 min) were used for detection of phospholipids, aminolipids and glycolipids, respectively. Spots were identified by comparing with the standard mixtures of polar lipids [phosphatidylinositol (PI), phosphatidylcholine (PC) and phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and cardiolipin (CL)] purchased from Sigma–Aldrich Company. The polar lipid profile of strain CCBAU 101002T contained the major components PC and PE, and the minor components PG and CL (Fig. 3). An unknown trace phospholipid was also found (Fig. 3). No glycolipid was detected. Total DNA was extracted and purified by the method of Marmur [11] and genomic DNA–DNA reassociation analysis was carried out using the procedure described by De Ley et al. [1]. Two independent

Table 3 Cellular fatty acid compositions of Rhizobium cauense sp. nov. CCBAU 101002T and the type strains of closely related Rhizobium species. Fatty acid

1

2

12:0 2-OH 15:0 2-OH 15:0 3-OH 15:0 iso 15:0 iso 3-OH 15:0 anteiso 16:0 16:0 3-OH 16:1 ω5cis 18:0 18:0 3-OH 18:1 11-methyl ω7cis 18:1 ω9cis 19:0 cyclo ω8cis Summed feature 2a Summed feature 3 Summed feature 8

1.11 – 4.80 1.02 2.54 2.61 10.17 T 2.09 3.56 1.22 – T 4.26 1.69 1.51 54.62

– T 1.86 – – – 5.94 – – 7.29 1.85 2.65 1.21 3.97 5.99 T 67.87

3 – – – – – – 1.70 5.05 – 5.51 2.59 – – 1.53 5.14 – 78.48

4

5

6

– 1.03 – – – – 3.44 1.79 – 5.3 3.07 T – 16.33 5.59 T 59.90

– T – – – – 3.63 2.86 – 4.14 T 2.41 – 10.41 5.18 1.53 67.85

– – – – – – 3.80 1.79 – 6.15 2.18 – – 4.73 4.68 – 76.66

Strains: 1, R. cauense CCBAU 101002T ; 2, R. leguminosarum USDA 2370T ; 3, R. mesoamericanum CCGE 501T ; 4, R. grahamii CCGE 502T ; 5, R. endophyticum CCGE 2052T ; 6, R. tibeticum CCBAU 85039T . Values are percentages of total fatty acids and only the contents above 1% in strain CCBAU 101002T are shown. −, not detected. T, trace contents lower than 1%. The details of the fatty acid compositions are shown in Supplementary Table S1. Summed feature 2: 12:0 aldehyde/unknown 10.928; Summed feature 3: 16:1ω6cis/16:1ω7cis; Summed feature 8: 18:1ω7cis/18:1ω6cis. a Summed features are groups of two or three fatty acids that cannot be separated by GLC with the MIDI system.

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Fig. 3. Two-dimensional thin layer chromatography (TLC) of polar lipids of Rhizobium cauense strain CCBAU 101002T . PG, phosphatidylglycerol; PE, phosphatidylethanolamine; PC, phosphatidylcholine; CL, cardiolipin; Un, unknown.

determinations were carried out for each reassociation experiment and mean values with the variation for the hybridization results are given in Table 1. The hybridization values between isolate CCBAU 101002T and isolates CCBAU 101000 and CCBAU 100001 ranged from 93.2 ± 1.3 to 100.0%, confirming that these strains were members of the same species. The DNA–DNA hybridization results between strain CCBAU 101002T and the nearest phylogenetic neighbors, R. tibeticum CCBAU 85039T , R. mesoamericanum CCGE 501T , R. endophyticum CCGE 2052T and R. grahamii CCGE 502T ranged from 22.4 ± 1.8% to 28.4 ± 0.8% (Table 1). The DNA–DNA hybridization between CCBAU 101002T and the type species R. leguminosarum USDA 2370T was 17.7 ± 0.8%. All these values were far lower than the 70% cut-off point recommended for the bacterial species definition, which confirmed that these novel isolates represented a distinct species [25]. The G + C content of strain CCBAU 101002T determined from the mid-point value (Tm) [10] with Escherichia coli K-12 as a standard was found to be 58.5 mol%, which was consistent with what has been observed for species G + C content among species in the genus Rhizobium (56.9–60.9 mol%) [5]. On the basis of the data obtained above, this study has shown the existence of one novel Rhizobium species, for which the name Rhizobium cauense is proposed. Description of Rhizobium cauense sp. nov. Rhizobium cauense (ca.u.en se. N.L. neut. adj. cauense pertaining to CAU, the acronym of the China Agricultural University, where the strains of this species were isolated). Cells are Gram-negative, aerobic, non-spore forming, motile, rod-shaped (1.15–1.72 ␮m × 3.05–7.54 ␮m). Good growth occurs on King B, NA (nutrient agar), Luria–Bertani (LB) broth and YMA medium. Colonies on YMA medium have a diameter of approximately 2 mm after 48 h growth at 28 ◦ C, and are circular, convex and pearly. The optimum temperature for growth is 28 ◦ C. Strains cannot grow at temperatures of 4 and 37 ◦ C, and cannot tolerate 60 ◦ C for 10 min. They produce an acidic reaction on YMA. Growth occurs at pH 6.0–9.0, with an optimum at pH 7.0–8.0. Strains do not produce 3-ketolactose from lactose. Growth is not affected by 2% NaCl, but is inhibited at 2.5% (w/v). Catalase, oxidase and urease assays are positive. Strains can hydrolyze

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ferric citrate. Hydrolyzation of starch and gelatin, Voges–Proskauer (VP) test and production of H2 S are negative. Reduction of nitrate is variable. Strains are able to utilize: dulcite, d-galactose, dglucose, inositol, d-fructose, lactose, sodium-dl-malate, maltose, d-melibiose, d-mannose, trehalose, sodium pyruvate, d-raffinose, l-rhamnose, sodium-d-gluconate, d-sorbitol, sucrose, sodium succinate, d-xylose and l-proline as carbon sources without gas formation, but not adipic acid, d-arabinose, inulin, calcium malonate, dextrin, meso-erythritol, d-melezitose, salicin, d-ribose, sodium acetate, hippuric acid, sorbose, soluble starch, syringic acid, sodium tartrate, vanillic acid, l-arginine, d-glycine, dl-asparagine, l-glycine, l-methinonine and l-threonine, respectively. Strains are capable of utilizing dl-alanine, l-arginine, l-aspartate, l-cysteine, l-glutamic acid, hypoxanthine, l-isoleucine, l-lysine, l-valine and l-methionine as nitrogen sources, but not d-glutamic acid, dthreonine and l-threonine, respectively. Strains are resistant to the antibiotics (␮g mL−1 ) ampicillin (100), kanamycin (5) and neomycin (5), but sensitive to tetracycline (5) and gentamicin (5–300). Resistance is variable to 5 ␮g mL−1 erythromycin, chloramphenicol and streptomycin. The major respiratory quinone is ubiquinone (Q-10). Summed feature 8 (18:1ω7cis/18:1ω6cis) and 16:0 are the predominant fatty acids. Strain CCBAU 101002T contains phosphatidylcholine and phosphatidylethanolamine as its major polar lipids and phosphatidylglycerol and cardiolipin as its minor polar lipids. No glycolipid was detected. The type strain is CCBAU 101002T (=LMG 26832T = HAMBI 3288T ) isolated from a root nodule of Kummerowia stipulacea grown in West Campus lawn soil of the China Agricultural University (CAU), Beijing, China. The G + C content of strain CCBAU 101002T is 58.5 mol% (Tm). Acknowledgements This work was supported by the National Basic Research Program of China (973 Program, No. 2010CB126504) and the Basic Research Program of Chinese Universities (No. 2012QT002). Thanks are expressed to Yán Li, Margaret Baker and Junjie Zhang for their improvement of the manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.syapm.2012.08.006. References [1] De Ley, J., Cattoir, H., Reynaerts, A. (1970) The quantitative measurement of DNA hybridization from renaturation rates. Eur. J. Biochem. 12, 133–142. [2] Euzéby, J.P. (1997) List of bacterial names with standing in nomenclature: a folder available on the Internet. Int. J. Syst. Bacteriol. 47, 590–592, Last full update January 01, 2012, available from http://www.bacterio.net (List of Prokaryotic Names with Standing in Nomenclature). [3] Gao, J.L., Sun, J.G., Li, Y., Wang, E.T., Chen, W.X. (1994) Numerical taxonomy and DNA relatedness of tropical rhizobia isolated from Hainan Province, China. Int. J. Syst. Bacteriol. 44, 151–158. [4] Higgins, D., Thompson, J., Gibson, T., Thompson, J.D., Higgins, D.G., Gibson, T.J. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucl. Acids Res. 22, 4673–4680. [5] Jordan, D.C. (1984) Family III. Rhizobiaceae. In: Krieg, N.R., Holt, J.G. (Eds.), Bergey’s Manual of Systematic Bacteriology, Williams & Wilkins, Baltimore, pp. 234–242. [6] Laguerre, G., Nour, S.M., Macheret, V., Sanjuan, J., Drouin, P., Amarger, N. (2001) Classification of rhizobia based on nodC and nifH gene analysis reveals a close phylogenetic relationship among Phaseolus vulgaris symbionts. Microbiology 147, 981–993. [7] Lee, J.S., Shin, Y.K., Yoon, J.H., Takeuchi, M., Pyun, Y.R., Park, Y.H. (2001) Sphingomonas aquatilis sp. nov., Sphingomonas koreensis sp. nov., and Sphingomonas taejonensis sp. nov., yellow-pigmented bacteria isolated from natural mineral water. Int. J. Syst. Evol. Microbiol. 51, 1491–1498.

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