Bradyrhizobium Daqingense Sp. Nov., Isolated from Soybean Nodules

International Journal of Systematic and Evolutionary Microbiology (2013), 63, 616–624 DOI 10.1099/ijs.0.034280-0

Bradyrhizobium daqingense sp. nov., isolated from soybean nodules

Jing Yu Wang,13 Rui Wang,13 Yan Ming Zhang,1 Hong Can Liu,2 Wen Feng Chen,1 En Tao Wang,1,3 Xin Hua Sui1 and Wen Xin Chen1

Correspondence 1State Key Laboratory for Agro-Biotechnology, College of Biological Sciences, China Agricultural Xinhua Sui University, Beijing 100193, PR China [email protected] 2Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, PR China 3Departamento de Microbiologı´a, Escuela Nacional de Ciencias Biolo´gicas, Instituto Polite´cnico Nacional, 11340 Me´xico DF, Mexico

Thirteen slow-growing rhizobial strains isolated from root nodules of soybean (Glycine max L.) grown in Daqing city in China were classified in the genus Bradyrhizobium based on 16S rRNA gene sequence analysis. Multilocus sequence analysis of IGS, atpD, glnII and recA genes revealed that the isolates represented a novel clade in this genus. DNA–DNA relatedness lower than 42.5 % between the representative strain CCBAU 15774T and the type strains of the closely related species Bradyrhizobium liaoningense USDA 3622T, Bradyrhizobium yuanmingense CCBAU 10071T and Bradyrhizobium betae LMG 21987T, further confirmed that this group represented a novel species. CCBAU 15774T shared seven cellular fatty acids with the three above-mentioned species, but the fatty acids 15 : 0 iso and summed feature 5 (18 : 2v6,9c and/or 18 : 0 anteiso) were unique for this strain. The respiratory quinone in CCBAU 15774T was ubiquinone-10 and the cellular polar lipids were phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, cardiolipin and unknown aminolipid, polar lipid and phospholipid. In addition, some phenotypic features could be used to differentiate the novel group from the related species. On basis of these results, we propose the name Bradyrhizobium daqingense sp. nov., with CCBAU 15774T (5LMG 26137T5HAMBI 3184T5CGMCC 1.10947T) as the type strain. The

DNA G+C content of the type strain is 61.2 mol% (Tm).

The genus Bradyrhizobium was proposed by Jordan (1982) incubation at 28 uC. At the time of writing, the genus for the slow-growing root nodule bacteria, in which contained 12 defined species: Bradyrhizobium japonicum colonies are generally less than or equal to 1 mm on yeast (Jordan, 1982), B. elkanii (Kuykendall et al., 1992), B. extract-mannitol agar (YMA) medium after 5–10 days liaoningense (Xu et al., 1995), B. yuanmingense (Yao et al., 2002), B. betae (Rivas et al., 2004), B. canariense (Vinuesa et al., 2005a), B. denitrificans (van Berkum et al., 2006), B. 3These authors contributed equally to this work. iriomotense (Islam et al., 2008), B. jicamae, B. pachyrhizi Abbreviations: AL, aminolipid; BOX-PCR, BOX-repeat-based PCR; CL, (Ramı´rez-Bahena et al., 2009), B. lablabi (Chang et al., cardiolipin; GL, glycolipid; IGS, 16S–23S rRNA intergenic spacer; MLSA, 2011) and B. cytisi (Chahboune et al., 2011). With the multilocus sequence analysis; MP, maximum parsimony; NJ, neighbour- exception of B. betae, all species are symbiotic nitrogen- joining; NP, unknown phospholipid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PL, polar lipid. fixing bacteria associated with different legumes. The GenBank sequence accession numbers of 16S rRNA, IGS, atpD, Soybean [Glycine max (L.) Merrill] originated in China and glnII, recA, nodC and nifH are: HQ231274, HQ231312, HQ231289, has been planted for over 5000 years (Liu et al., 2008) as HQ231301, HQ231270, HQ231326 and HQ231323 for CCBAU one of the most important crops for edible oil and protein T 15774 ; HQ664956, HQ664959, HQ664967, HQ664973, production. Previously, B. japonicum, B. elkanii, B. HQ664970, HQ664988 and HQ664977 for CCBAU 15772; HQ664955, HQ664958, HQ664966, HQ664972, HQ664969, liaoningense, B. yuanmingense, Ensifer fredii and Ensifer HQ664989 and HQ664976 for CCBAU 15768; and HQ664954, sojae (Han et al., 2009; Li et al., 2011a; Man et al., 2008; HQ664957, HQ664960, HQ664971, HQ664968, HQ664978 and Wang et al., 2009; Xu et al., 1995) have been isolated from HQ664974 for CCBAU 15779, respectively. root nodules of soybean grown in different regions of Seven supplementary figures and two supplementary tables are China. Clear biogeographic patterns have been revealed in available with the online version of this paper. the soybean rhizobia and soil types seemed to be the main

616 034280 G 2013 IUMS Printed in Great Britain Bradyrhizobium daqingense sp. nov. determinant of the biogeography of these bacteria (Han Gram-negative, non-spore-forming rods. Colonies on et al., 2009; Li et al., 2011b; Man et al., 2008; Zhang et al., YMA medium were mucoid, white, translucent and 2011). circular, 1 mm in diameter after 7 days of incubation at 28 uC and they produced an alkaline pH on YMA medium Heilongjiang Province, in the north-east of China, is the supplied with bromothymol blue (0.025 %), indicating main soybean-producing area, accounting 44 % (4.01 that these isolates belonged to the genus Bradyrhizobium million ha) of the culturing area and 40 % of the yield in (Vincent, 1970). China in 2009 (http://zzys.agri.gov.cn/nongqing.aspx). In general, Heilongjiang has extremely fertile black soil with Similar to other studies (Cho & Tiedje, 2000; Healy et al., low acidity or neutral pH, in which B. japonicum is the 2005; Rademaker et al., 2000), BOX-PCR fingerprinting predominant microsymbiont of soybean, whereas B. was used to reveal the genetic diversity of the nodule elkanii, B. liaoningense and Ensifer fredii have been isolates. In this study, the BOXAIR primer (Versalovic et al., occasionally isolated (Wang et al., 2009; Xu et al., 1995). 1994) and method of Nick & Lindstrom (1994) were used In contrast to other places in Heilongjiang Province, in analysis of BOX-PCR. The PCR products were subjected Daqing city, in the south-west of the province, has saline, to electrophoresis in agarose gel (1.5 %, w/v) supplied with alkaline soil, high precipitation and lower terrain, making ethidium bromide. Four BOX-PCR patterns were obtained it possible to harbour soybean rhizobia that differ from among the 13 isolates (Fig. S1, Table 1), which shared more those in other places of Heilongjiang. than 84 % similarity. From them, isolates CCBAU 15768, CCBAU 15772, CCBAU 15774T and CCBAU 15779, In a study of bradyrhizobia associated with soybean in representing four BOX fingerprints, were chosen for the Heilongjiang province, a group of 13 isolates (Table 1) subsequent analyses. originating from Daqing city showed same 16S–23S rRNA intergenic spacer (IGS)-RFLP patterns and differed from The 16S rRNA genes of the four representative strains were the defined species of Bradyrhizobium (data not shown), amplified with the primers P1 and P6 by the PCR protocol implying that they may represent a novel species of the of Tan et al. (1997). The IGS fragments were amplified with genus. To clarify their taxonomic position, we performed the primers FGPS1490 and FGPL132 (Laguerre et al., 1996) the present study and a novel species, Bradyrhizobium as described by Kwon et al. (2005). The atpD, recA and glnII genes were amplified by using the method of Vinuesa daqingense, was defined based upon the results. et al. (2005b), with primer sets atpD255F/atpD782R, These 13 isolates were obtained from surface-sterilized root recA41F/recA640R and glnII12F/glnII689R, respectively. nodules collected in August 2009 on YMA medium as The amplification of partial nodC and nifH genes were described elsewhere (Bergersen, 1961). All the isolates were performed with the primers nodC540/nodC1160 (Sarita

Table 1. Sequence similarities of 16S rRNA gene, combined atpD, recA and glnII, IGS, nifH and nodC, DNA relatedness among CCBAU 15774T and related strains, DNA G+C contents and BOX types

2, Not done.

Strain Similarity with CCBAU 15774T (%) DNA G+C content

(mol%; Tm) 16S rRNA MLSA* IGS nifH nodC DNA–DNA relatedness (%)

B. daqingense sp. nov.D CCBAU 15774T (BOX type I) 100 100 100 100 100 100 61.2 CCBAU 15772 (BOX type II) 100 99.8 99.9 92.6 92.9 94.3±1.78 61.4 CCBAU 15768 (BOX type III) 100 100 100 100 100 94.0±2.05 60.1 CCBAU 15779 (BOX type IV) 100 100 100 99.7 100 93.6±1.69 60.6 B. liaoningense USDA 3622T 99.9 94.4 89.9 100 2 42.5±2.06 B. yuanmingense CCBAU 10071T 99.5 94.2 93.3 91.5 92.1 26.8±2.48 B. betae LMG 21987 T 99.3 94.1 88.8 22 22.37±1.93 B. canariense BTA-1T 99.5 92.4 89.7 78.5 68.6 2 B. japonicum USDA 6T 99.7 92.6 89.4 100 100 2 B. cytisi CTAW 11T 98.9 91.2 2 77.7 70.1 2 B. iriomotense LMG 24129T 98.8 91.9 92.4 79.8 66.8 2 B. denitrificans LMG 8443T 98.7 89.4 72.3 80.3 22

*atpD, recA and glnII sequences were included in MLSA. DNine other strains were also defined in this group: CCBAU 15766, 15769, 15775, 15776, 15777 and 15778 in BOX type I; CCBAU 15767 in BOX type II; and CCBAU 15770 and 15910 in BOX type IV. http://ijs.sgmjournals.org 617 J. Y. Wang and others et al., 2005) and nifH1F/nifH1R (Laguerre et al., 2001), criteria employed in the recent description of respectively. All the amplified fragments were directly Bradyrhizobium species (Ramı´rez-Bahena et al., 2009; sequenced as described previously (Hurek et al., 1997) and Rivas et al., 2004; Vinuesa et al., 2005a). Rhizobium the acquired sequences were aligned with those of defined lupini DSM 30140T described by Ludwig et al. (1995) Bradyrhizobium species using the CLUSTAL W program in should be reclassified in the genus Bradyrhizobium and the MEGA 4.0 software package (Kumar et al., 1994, 2008). Agromonas oligotrophica (Ohta & Hattori, 1983) might also Phylogenetic trees were inferred using the neighbour- be reclassified into this genus. As to the relationships joining (NJ) (Laguerre et al., 1996; Saitou & Nei, 1987) and among the two phylogenetic groups of Bradyrhizobium and maximum-parsimony (MP) (Felsenstein, 1985) methods. the four genera inserted between them, we think more Bootstrap analysis was based on 1000 replications. The evidence and a thorough discussion are needed, which are distances were calculated according to Jukes–Cantor for outside the scope of the present study. 16S rRNA genes and IGS (Jukes & Cantor, 1969) or Kimura two-parameter (Kimura, 1983) for housekeeping As previously described (Willems et al., 2001b, 2003), the genes (atpD, recA and glnII) and symbiotic genes (nodC IGS region was a valuable molecular marker to determine and nifH). For deduced amino acid sequences, the pairwise relatedness among the novel isolates and the closely related evolutionary distances used in the NJ trees were P- species. In the NJ phylogenetic tree based on the IGS sequences, the four representative isolates formed a group distances in MEGA 4.0. that was distinct from the defined Bradyrhizobium species According to the standards for definition of bacterial (Fig. S2). CCBAU 15774T, CCBAU 15779 and CCBAU species (Graham et al., 1991; Wayne et al., 1987), the 16S 15768 had identical IGS sequences which shared 99.9 % rRNA gene phylogeny is used to reveal the relationships of similarity with CCBAU 15772. The similarities of IGS the isolates with defined species. In the present study, the sequences between the four isolates and type strains of NJ tree generated from 16S rRNA gene sequences (Fig. 1) Bradyrhizobium species were in the range of 72.1–93.3 %; had similar phylogenetic topology to that obtained with the this implies that they represent a novel Bradyrhizobium MP method (figure not shown). Similar to the previous genospecies, according to the hypothesis that two bradyr- reports (Ramı´rez-Bahena et al. 2009; Rivas et al., 2004; hizobial strains with IGS sequence similarity less than Willems et al., 2001a), the Bradyrhizobium species were 95.5 % usually belong to separate genospecies (Willems divided into two phylogenetic groups, which were et al., 2003). separated by several species of Rhodopseudomonas, Afipia, Oligotropha and Nitrobacter (Fig. 1). The majority (eight) Multilocus sequencing analysis of several selected genes is a of the defined Bradyrhizobium species, represented by the reliable method for rhizobial species or genospecies type species B. japonicum, together with Agromonas description (Rivas et al., 2009; Sahgal & Johri, 2006). In oligotrophica formed a phylogenetic group; the remaining this study, we obtained partial sequences of the atpD, recA four Bradyrhizobium species represented by B. elkanii and glnII genes and constructed phylogenetic trees with formed another phylogenetic group (Fig. 1). Although the combined sequences of these three housekeeping genes, close phylogenetic relationships among the Bradyrhizobium with NJ and MP methods. The NJ tree (Fig. S3) and the species and the other related genera mentioned above have MP tree (not shown) of the combined genes showed been found for a long time (Willems & Collins, 1992), similar phylogenetic relatedness, indicating that the four several novel members of the genus Bradyrhizobium have representative isolates formed a group sharing 99.8–100 % been described based upon their close phylogenetic sequence similarity with the combined housekeeping genes. relationships with the defined Bradyrhizobium species, The novel group was closely related to B. liaoningense T T and their relationships with members of the genera USDA 3622 , B. yuanmingense CCBAU 10071 and B. T Rhodopseudomonas, Afipia, Oligotropha and Nitrobacter betae LMG 21987 with similarities of 94.4, 94.2 and were not considered (Ramı´rez-Bahena et al., 2009; Rivas 94.1 %, respectively, whereas these values were between et al., 2004; Vinuesa et al., 2005a). 88.6 and 93.5 % with other members of the genus Bradyrhizobium. The corresponding combined deduced In our case, the four representative strains for the novel amino acid sequence tree (Fig. S4) for the three group had identical 16S rRNA gene sequences, having housekeeping genes showed similar phylogenetic related- similarities of 98.7–99.9 % with the members in the major ness with that of their genes, except that similarity levels phylogenetic group, including the eight Bradyrhizobium were higher, i.e. 98.2, 98.5 and 98.5 %, respectively. species, 99.6 % with Rhizobium lupini and 98.9 % with Meanwhile these data were between 97.5 and 94.2 % with Agromonas oligotrophica; 97.8–98.4 % similarity with spe- other members of the genus Bradyrhizobium. cies of the genera Rhodopseudomonas, Afipia, Oligotropha and Nitrobacter; and 97.4–97.8 % similarity with the four The symbiotic genes (nod and nif) of rhizobia are adaptive species in the minor Bradyrhizobium phylogenetic group. genes and, in many cases, have different evolutionary The novel group of four strains represented by CCBAU histories from housekeeping genes (Wang et al., 2007). A 15774T could be allocated to the genus Bradyrhizobium comparison of their phylogenies with those derived from based upon their closest phylogenetic relationships with housekeeping genes may reveal lateral gene transfer events the members in the major phylogenetic group and the among rhizobia (Haukka et al., 1998). In the nifH gene

618 International Journal of Systematic and Evolutionary Microbiology 63 Bradyrhizobium daqingense sp. nov.

Bradyrhizobium daqingense CCBAU 15768 (HQ664955) Bradyrhizobium daqingense CCBAU 15779 (HQ664954) 0.01 Bradyrhizobium daqingense CCBAU 15774T (HQ231274) Bradyrhizobium daqingense CCBAU 15772 (HQ664956) B. liaoningense USDA 3622T (AF208513) B. yuanmingense CCBAU 10071T (AF193818) B. canariense BTA-1T (AJ558025 ) R. lupini DSM 30140 T (X87273) 94B. japonicum USDA 6T (AB231927) 96 Agromonas oligotrophica JCM 1494T (D78366) B. denitrificans LMG 8443T (X66025) B. japonicum USDA 110 (NC004463) B. iriomotense LMG 24129T (AB300992) B. cytisi CTAW11T (EU561065) B. betae LMG 21987T (AY372184) Rhodopseudomonas palustris 7 (D89811) 100 Rhodopseudomonas palustris ATCC 17001T (D25312) 99 Nitrobacter sp. LL (L11662) W (L11661) 100 Nitrobacter winogradskyi Afipia clevelandensis ATCC 49720T (M69186) Afipia felis ATCC 53690T (M65248) 73 Oligotropha carboxidovorans DSM 1227T (FR733697) B. jicamae PAC 68T (AY624134) CCBAU 23086T (GU433448) 100 B. lablabi B. elkanii USDA 76T (U35000) 98 B. pachyrhizi PAC 48T (AY624135) R. leguminosarum USDA 2370T (U29386) Azorhizobium caulinodans ORS 571T (D11342)

Fig. 1. Phylogenetic tree based on the 16S rRNA sequences showing the relationships of the strains of Bradyrhizobium daqingense sp. nov. and members of related species. The tree was constructed by the NJ method. Bootstrap values greater than 70 % are indicated at nodes. Bar, 1 % substitution. The sequence of Rhizobium leguminosarum USDA 2370T was used as an outgroup. phylogenetic tree (Fig. S5), the four representatives were three representatives were about 94 %, while 22.4–42.5 % divided into two different branches; CCBAU 15774T, similarities were detected between CCBAU 15774T and the CCABU 15768 and CCBAU 15779 were grouped with B. three reference strains used (Table 1), values that are much japonicum USDA 6T and B. liaoningense USDA 3622T at lower than the DNA–DNA threshold value (70 %) that 99.7–100 % similarities, whereas CCBAU 15772 clustered delineates species (Wayne et al., 1987). These data with B. yuanmingense CCBAU 10071T at 97.7 % similarity. indicated that the four representative isolates represented The nodC gene phylogenetic tree (Fig. S6) showed a similar a unique genomic species that differ from the defined relationship to that shown using the nifH gene sequences. species of Bradyrhizobium. We can infer from this result that lateral gene transfer may Fatty acid profiling is a popular method for characterizing have occurred between CCBAU 15772 and B. yuan- microbial communities from natural systems (Schutter & mingense and that nifH and nodC genes in B. daqingense Dick, 2000) and is a useful tool for identifying root-nodule have co-evolved. bacteria (Tighe et al., 2000). In this study, CCBAU 15774T Since DNA–DNA hybridization is considered as a standard was comparatively assayed together with B. liaoningense method for species definition (Graham et al., 1991; Wayne USDA 3622T, B. yuanmingense CCBAU 10071T and B. et al., 1987), the four representative isolates and closely betae LMG 21987T. All four tested strains were grown to related reference strains (B. liaoningense USDA 3622T, B. the late-exponential phase in the TY (tryptone-yeast yuanmingense CCBAU 10071T and B. betae LMG 21987T) extract) broth at 28 uC. Cultures were then centrifuged to were chosen for the hybridization assays. Total DNA was get the bacterial cells, which were then washed three times extracted by the method of Marmur (1961). DNA–DNA with 0.8 % NaCl solution. Fatty acid methyl esters were relatedness was determined with the renaturation rate prepared and separated using a previously described method (De Ley et al., 1970). The G+C content of the method (Sasser, 1990) and identified with the MIDI DNA was measured using the thermal denaturation Sherlock Microbial Identification System (Sherlock CD v method (Marmur & Doty, 1962) and DNA of Escherichia 6 . 0), using the TSBA6 database. Seventeen kinds of fatty coli K-12 as the standard. The DNA G+C content of the acids were detected in CCBAU 15774T, of which seven were four representative strains was 60.1–61.4 mol% (Table 1), common to all tested strains. Summed feature 8 (18 : 1v6c which is within the range for Bradyrhizobium. The DNA– and/or 18 : 1v7c; 72.22 %) and 16 : 0 (12.48 %) were the DNA similarities among CCBAU 15774T and the other predominant fatty acids in CCBAU 15774T, similar to that http://ijs.sgmjournals.org 619 J. Y. Wang and others

Table 2. Differential features for type strains of B. daqingenese sp. nov. and related reference species

Strains: 1, B. daqingense CCBAU 15774T;2,B. liaoningense USDA 3622T;3,B. yuanmingense CCBAU 10071T;4,B. canariense BTA-1T;5,B. japonicum USDA 6T;6,Agromonas oligotrophica JCM 1494T;7B. denitrificans LMG 8443T;8,B. iriomotense LMG 24129T;9,B. cytisi CTAW 11T; 10, T B. betae LMG 21987 . +, Positive; 2, negative; W, weak, NR, not reported. Data obtained in this study unless indicated otherwise.

Characterstic 1 2 3 4 5 6 7 8 9 10

a-Cyclodextrin 2222+ 22222 Dextrin 22+ 222WW2 + Glycogen 2222+ 22222 Tween 40 2 + 2 ++++++2 Tween 80 ++2 ++++++2 N-Acetyl-D-galactosamine 22+ 222222+ N-Acetyl-D-glucosamine 2222222+ 22 Adonitol 222222W 2 + 2 L-Arabinose ++2 ++++++2 D-Arabitol + 2 ++++++++ i-Erythritol 22+ 222222+ D-Fructose + 2 ++++++++ D-Galactose +++++++++2 Gentiobiose 22+ 2222+++ myo-Inositol 22+ 222++++ a-Lactose 22+ 2222222 Lactulose 2 ++222222+ Maltose 22+ 22222++ D-Mannitol + 2 ++++++2 + D-Mannose +++++ W +++2 Melibiose 222222222+ Methyl b-D-glucoside 2222222+ 22 D-Psicose 22+ WW2 +++2 Raffinose 22+ 222222+ L-Rhamnose + W 2 + WW++++ D-Sorbitol 222+++++22 Sucrose 22+ 2222222 Trehalose 22+ 2222W 2 + Turanose 2222W 2222+ Xylitol 2222222+ 22 Pyruvic acid methyl ester +++++++++2 cis-Aconitic acid 222+ 2 ++2 + 2 Citric acid 222++++++2 Formic acid +++++++++2 D-Galactonic acid lactone +++++ W 2 +++ D-Galacturonic acid ++2 ++ 2 +++2 D-Gluconic acid ++2 ++++++2 D-Glucosaminic acid 2 W + 22 WW++2 D-Glucuronic acid + 2 ++ W 2 ++ W + a-Hydroxybutyric acid ++ W 2 + 2 +++2 b-Hydroxybutyric acid ++2 ++++++2 c-Hydroxybutyric acid W + 2 ++++++2 p-Hydroxyphenylacetic acid 2 W 2222+ 2 W 2 Itaconic acid 2 + 2222+ W 22 a-Ketobutyric acid +++2 + 2 ++++ a-Ketovaleric acid + W 2 + W 22++2 DL-Lactic acid ++2 ++++++2 Malonic acid 22++ 22W 22+ Quinic acid + 22++++++2 D-Saccharic acid ++2 ++++++2 Sebacic acid +++++ 2 ++++ Succinic acid ++ W ++++++2

620 International Journal of Systematic and Evolutionary Microbiology 63 Bradyrhizobium daqingense sp. nov.

Table 2. cont.

Characterstic 1 2 3 4 5 6 7 8 9 10

Bromosuccinic acid ++2 ++++2 + 2 Glucuronamide ++2 +++++++ L-Alaninamide +++++++++2 D-Alanine 2 + 2 W + W +++2 L-Alanine 2 W + 2 + 2 W + 2 + L-Alanyl glycine 2 + 22 W 2 + 2 ++ L-Asparagine + 2 +++ 2 +++2 L-Aspartic acid W + 2 ++++++2 L-Glutamic acid + 2 ++++++++ Glycyl L-aspartic acid 2222222+ 2 + Glycyl L-glutamic acid 2222222++2 L-Leucine 2 + 2 ++ 2 + 2 ++ L-Ornithine 22222222+ 2 L-Phenylalanine 2 + 2 ++ 22++2 L-Proline 2 W 22 W 22+ 22 L-Pyroglutamic acid 2 W 2 +++++++ L-Serine 222222+ 222 L-Threonine 2 + 2222W 2 + 2 DL-Carnitine 222222222+ Urocanic acid 2 + 22+ 2 W 2 + 2 Uridine 22+ 222222+ Phenylethylamine 222222+ 222 2-Aminoethanol 22+ 22++22+ Glycerol W 22+ W ++++2 a-D-Glucose 6-phosphate 22+ 222222+ Opt. growth temperature (uC) 28 28 28 28* 28 25227* 41* 28* 28* 28* Sodium nitrate reduction + 2 + NR* 2 +* +* 2* 2* 2* Nodulation ability ++++* +* 2* +* +* +* 2*

*Data from the following sources: B. canariense, Vinuesa et al. (2005b) and Zhang et al. (2011); Agromonas oligotrophica, Ohta & Hattori (1983); B. denitrificans, van Berkum et al. (2006); B. iriomotense, Islam et al. (2008); B. cytisi, Chahboune et al. (2011). of reference strains, whereas 15 : 0 iso and summed feature detected in CCBAU 15774T only (Fig. S7); one unidentified 5 (18 : 2v6,9c and/or 18 : 0 anteiso) were only present in glycolipid (GL) was detected only in B. liaoningense USDA CCBAU 15774T. Detailed data are available in Table S1. 3622T, B. yuanmingense CCBAU 10071T and B. japonicum USDA 6T (Table S2); one unknown aminolipid (AL) was Analysis of respiratory quinones and polar lipids have been detected in CCBAU 15774T (Fig. S7) and in B. yuanmingense suggested for characterization for prokaryotes (Tindall CCBAU 10071T only; and three unidentified ALs were found et al., 2010), but there are few publications on taxonomy in B. betae LMG 21987T (Table S2). of rhizobia (Choma & Komaniecka, 2003; Miller et al., 1990; Minder et al., 2001; Orgambide et al., 1993). The The strain CCBAU 15774T and nine closely related reference respiratory quinones for CCBAU 15774T, B. liaoningense strains, B. liaoningense USDA 3622T, B. yuanmingense USDA 3622T, B. yuanmingense CCBAU 10071T, B. betae CCBAU 10071T, B. canariense BTA-1T, B. japonicum USDA LMG 21987T and B. japonicum USDA 6T were analysed by 6T, Agromonas oligotrophica JCM 1494T, B. denitrificans LMG HPLC as described by Lee et al. (2001). Polar lipids were 8443T, B. iriomotense LMG 24129T, B. cytisi CTAW 11T and extracted, examined by two-dimensional TLC and iden- B. betae LMG 21987T, were characterized phenotypically by tified on the basis of procedures described by Minnikin using Gram-negative (GN2) MicroPlates (Biolog) according et al. (1984). The respiratory quinone for all tested strains to the manufacturer’s instructions. To prepare the inocu- was ubiquinone-10 (Q-10). Phosphatidylcholine (PC), lants, single colonies were subcultured on YMA plates for phosphatidylethanolamine (PE), phosphatidylglycerol (PG) optimum growth, and incubated to late-exponential phase at and cardiolipin (CL) were detected as the dominant cellular 28 uC. After inoculation, the microplates were incubated at polar lipids in all the five bradyrhizobial strains tested (Table 28 uC and were read at 590 nm at 4, 24, 48 and 96 h with a S2), which were similar to the results reported by Miller et al. computer-controlled MicroPlate reader. Strain CCBAU (1990) and Choma & Komaniecka (2003). One unknown 15774T could be distinguished from closely related species polar lipid (PL) and one unknown phospholipid (NP) were with 77 different features (Table 2), while 18 were common http://ijs.sgmjournals.org 621 J. Y. Wang and others to all tested strains; these are presented in the description of quinone was Q-10 and the main cellular polar lipids were the novel species. The generation time was 10 h for CCBAU PE, PG, PC and CL and unidentified AL, PL and NP. 15774T according to the method described by Gao et al. The type strain is CCBAU 15774T (5LMG 26137T5 (1994). HAMBI 3184T5CGMCC 1.10947T), isolated from an Host range of rhizobia is an important feature for root- effective nodule of Glycine max in Daqing city in and/or stem nodule bacteria, and cross-nodulation tests Heilongjiang province, China. The DNA G+C content of with selected hosts are required for the description of novel the type strain is 61.2 mol%. rhizobial species (Graham et al., 1991). Strains CCBAU 15774T and CCBAU 15772, representing the novel Bradyrhizobium group, were used for cross-nodulation Acknowledgements tests with eight legume species. The seeds of the legume This work was supported by National Natural Science Foundation of plants were surface-sterilized, germinated and inoculated China (project no. 31170003). E . T . W. is financially supported by with test strains according to a standard procedure grants SIP20100067, authorized by Instituto Polite´cnico Nacional, (Vincent, 1970). All the plants were maintained in glass and PICS08-3, authorized by ICyT DF of Mexico. tubes supplied with a nitrogen-free nutrient solution (Brunel et al., 1996), while non-inoculated plants were used as controls. Five replicates were prepared for each References treatment. Legumes were incubated in a greenhouse under natural sunlight for 45–60 days and then nodulation was Bergersen, F. J. (1961). The growth of Rhizobium in synthetic media. observed. The results showed that CCBAU 15774T and Aust J Biol 14, 349–360. CCBAU 15772 formed effective nodules with Glycine max Brunel, B., Rome, S., Ziani, R. & Cleyet-Marel, J. C. (1996). and Vigna unguiculata, as shown by red nodules and dark Comparison of nucleotide diversity and symbiotic properties of Rhizobium meliloti populations from annual Medicago species. FEMS green leaves; they also formed white (ineffective) nodules Microbiol Ecol 19, 71–82. with Medicago sativa; but did not form nodules with Chahboune, R., Carro, L., Peix, A., Barrijal, S., Vela´zquez, E. & Trifolium repens, Lotus corniculatus, Phaseolus vulgaris or Bedmar, E. J. (2011). Bradyrhizobium cytisi sp. nov., isolated from Pisum sativum under laboratory conditions. effective nodules of Cytisus villosus. Int J Syst Evol Microbiol 61, 2922– Based upon all the results obtained in our study, we believe 2927. that the 13 isolates represent a novel species in the genus Chang, Y. L., Wang, J. Y., Wang, E. T., Liu, H. C., Sui, X. H. & Chen, Bradyrhizobium and we propose the name Bradyrhizobium W. X. (2011). Bradyrhizobium lablabi sp. nov., isolated from effective nodules of Lablab purpureus and Arachis hypogaea. Int J Syst Evol daqingense sp. nov. Microbiol 61, 2496–2502. Cho, J. C. & Tiedje, J. M. (2000). Biogeography and degree of Description of Bradyrhizobium daqingense endemicity of Pseudomonas fluorescent strains in soil. Appl Environ sp. nov. Microbiol 66, 5448–5456. Choma, A. & Komaniecka, I. (2003). 9 The polar lipid composition of Bradyrhizobium daqingense (da.qing.en se. N.L. neut. adj. Mesorhizobium ciceri. Biochim Biophys Acta 1631, 188–196. daqingense of or belonging to Daqing city in China, where De Ley, J., Cattoir, H. & Reynaerts, A. (1970). The quantitative these novel bradyrhizobia were isolated). measurement of DNA hybridization from renaturation rates. Eur J Gram-negative, aerobic, non-spore-forming rods. Colonies Biochem 12, 133–142. on YMA medium are white, translucent and circular, Felsenstein, J. (1985). Confidence limits on phylogenies: an approach 1 mm in diameter, after 7 days incubation at 28 uC. The using the bootstrap. Evolution 39, 783–791. generation time for CCBAU 15774T is 10 h. The pH range Gao, J., Sun, J., Li, Y., Wang, E. & Chen, W. (1994). Numerical for growth is 6–9 and the optimum is pH 7.0. Growth taxonomy and DNA relatedness of tropical rhizobia isolated from occurs between 28 and 37 uC, and the optimum temper- Hainan Province, China. Int J Syst Bacteriol 44, 151–158. ature is 28 uC. Grows weakly in the presence of 1 % NaCl. Graham, P., Sadowsky, M., Keyser, H., Barnet, Y., Bradley, R., Cooper, J., De Ley, D., Jarvis, B., Roslycky, E. & other authors (1991). Can utilize acetic acid, a-ketoglutaric acid, a-D-glucose, L- fucose, monomethyl ester, propionic acid, succinic acid Proposed minimal standards for the description of new genera and species of root-and stem-nodulating bacteria. Int J Syst Bacteriol 41, and succinamic acid but not D-serine, cellobiose, DL-a- 582–587. glycerol phosphate, D-glucose 6-phosphate, hydroxy-L- Han, L., Wang, E., Han, T., Liu, J., Sui, X., Chen, W. & Chen, W. (2009). proline, inosine, L-histidine, putrescine, thymidine, c- Unique community structure and biogeography of soybean rhizobia aminobutyric acid or 2,3-butanediol. Differential features in the saline-alkaline soils of Xinjiang, China. Plant Soil 324, 291–305. of B. daqingenese sp. nov. and related reference species are Haukka, K., Lindstro¨ m, K. & Young, J. P. (1998). Three phylogenetic listed in Table 2. Seventeen fatty acids were detected and groups of nodA and nifH genes in Sinorhizobium and Mesorhizobium summed feature 8 (18 : 1v6c and/or 18 : 1v7c) and 16 : 0 isolates from leguminous trees growing in Africa and Latin America. were the predominant fatty acids in CCBAU 15774T. The Appl Environ Microbiol 64, 419–426. fatty acids 15 : 0 iso and summed feature 5 (18 : 2v6,9c and/ Healy, M., Huong, J., Bittner, T., Lising, M., Frye, S., Raza, S., or 18 : 0 anteiso) were unique in CCBAU 15774T. The Schrock, R., Manry, J., Renwick, A. & other authors (2005). Microbial

622 International Journal of Systematic and Evolutionary Microbiology 63 Bradyrhizobium daqingense sp. nov.

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