Soil Biology & Biochemistry 78 (2014) 128e137

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Soil Biology & Biochemistry

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Diversity and distribution of nodulated with in two ecoregions of China

* Ying Cao a, En-Tao Wang b, Liang Zhao a, Wei-Min Chen a, Ge-Hong Wei a, a State Key Laboratory of Soil Erosion and Dryland Farming on the Loess Plateau, College of Life Sciences, Northwest A&F University, 712100 Yangling, Shaanxi, China b Departamento de Microbiología, Escuela Nacional de Ciencias Biologicas, Instituto Politecnico Nacional, 11340 Mexico, D.F., Mexico article info abstract

Article history: To reveal the diversity and geographic distribution of bean (Phaseolus vulgaris L.) rhizobia in China, a total Received 21 May 2014 of 226 strains of nodule were isolated from bean plants grown in fields of nine sites in northern Received in revised form (ecoregion I) and southern (ecoregion II) regions of China, with soil pH ranged from 8.06 to 4.97. Based 30 July 2014 upon the phylogenies of housekeeping and symbiotic genes, 96.9% of the isolates were cor- Accepted 31 July 2014 responding to four defined species and four unnamed genospecies, while the remaining were five Bra- Available online 12 August 2014 dyrhizobium genospecies. Rhizobium leguminosarum, Rhizobium etli and Rhizobium sp. II or IV were dominant in different ecoregions, with varied relative abundances. Therefore, diverse and distinctive Keywords: Rhizobia bean rhizobial communities exist in different ecoregions of China, demonstrating that the introduction of fi Common bean bean plants has driven the evolution of rhizobia to t the necessity to nodulate the host plant under the Phylogeny local conditions, mainly the soil pH and the nutrient availability. The predominance of R. etli and the high Biogeography similarities in the symbiotic genes (nodC and nifH) of all the Rhizobium genospecies with R. etli evidenced Evolution that symbiotic genes were transferred from R. etli, possibly introduced to China accompanying the seeds, Alpha diversity to the related indigenous rhizobia. These findings enlarged the diversity of bean rhizobia, evidenced the Systematic biogeographic patterns of these rhizobia, and demonstrated the possible evolution and emerging of novel rhizobia under the combined selection of host plant and soil conditions. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction et al., 2005) in different regions: predominated by R. etli in the South and Middle Americas (Amarger, 2001), Europe (García-Fraile Bean or common bean (Phaseolus vulgaris L.), an annual legu- et al., 2010) and Jordan (Tamimi and Young, 2004); by minous herb, is originated in the Americas (van Schoonhoven and R. leguminosarum in the Andean region and Nepal (Bernal and Voysest, 1991). For its high concentration of protein, fiber, and Graham, 2001; Adhikari et al., 2013; Ribeiro et al., 2013); by complex carbohydrates, it is cultivated worldwide (Kaplan, 1981) R. tropici in regions with acid soils and high temperature (Martínez- and plays an important role in feeding human being. Although bean Romero et al., 1991; Anyango et al., 1995; Grange and Hungria, is considered to have poor nitrogen fixing capacity (Mnasri et al., 2004); and by R. phaseoli, R. etli and a novel Rhizobium group in 2007), it can acquire their nitrogen through an association with Africa (Aserse et al., 2012b). rhizobia. Meanwhile, the bean is a relatively promiscuous host, The great diversity of bean rhizobia might be a result of the nodulating with Rhizobium etli, Rhizobium tropici, Rhizobium legu- worldwide cultivation of this plant, since microbial distribution is minosarum bv. phaseoli, Rhizobium gallicum, Rhizobium giardinii, influenced by environmental variation with time and space Rhizobium lusitanum, Rhizobium phaseoli, Rhizobium azibense (Martiny et al., 2006). In previous studies, soil pH has been revealed (Mnasri et al., 2014), Rhizobium freirei (Dall'Agnol et al., 2013), as a major factor to affect legumeerhizobia symbiotic process and Rhizobium mesoamericanum (Lopez-L opez et al., 2012), Sino- nitrogen fixation (Wolff et al., 1991); while the temperature may rhizobium meliloti (Zurdo-Pineiro~ et al., 2009), have affected the evolution of Bradyrhizobium communities americanum (Mnasri et al., 2012), and Bradyrhizobium sp. (Han (Stepkowski et al., 2012). In China, biogeographic patterns have been observed in the soybean rhizobia which were correlated to the soil pH and salinity (Han et al., 2009; Li et al., 2011). Furthermore, * Corresponding author. Fax: þ86 2987091175. several strains belonged to R. leguminosarum bv. phaseoli and E-mail address: [email protected] (G.-H. Wei). Bradyrhizobium sp. have been reported as microsymbionts of bean http://dx.doi.org/10.1016/j.soilbio.2014.07.026 0038-0717/© 2014 Elsevier Ltd. All rights reserved. Y. Cao et al. / Soil Biology & Biochemistry 78 (2014) 128e137 129 in different regions of China, depending on the soil pH (Han et al., According to the result of RFLP analyses of 16S rRNA, nodC and 2005). It has been known that the extreme ecological conditions nifH genes, the isolates were designed into genotypes. Repre- could drive the rhizobia to develop active mechanisms to adapt the sentative isolates of different genotypes were selected for the environments, such as accumulating intracellular compatible sol- subsequent multilocus sequence analysis. The 16S rRNA, nifH and utes (glutamate and trehalose) for resistance of high salinity nodC genes were amplified again and three housekeeping genes (Faghire et al., 2012). atpD (ATP synthase b-subunit), glnII (glutamine synthase II) and Bean was directly introduced from the Americas to China in the recA (recombinase A protein) were amplified by using primer 15th century (van Schoonhoven and Voysest, 1991). To date, pro- pairs atpD255F/atpD782R, glnII12F/glnII689R and recA41F/ duction of bean in China is the highest among the Asian countries, recA640R, and the corresponding protocols (Turner and Young, up to 15,702,000 tons over 2011 (http://faostat.fao.org/). However, 2000; Vinuesa et al., 2005; Han et al., 2008). The amplified the diversity of bean rhizobia in China has not been fully studied genes were sequenced directly with the method of Sanger yet. Considering the long history and a vast area of bean cultivation, (Sanger et al., 1977) in Beijing AuGCT DNA-SYN Biotechnology as well as its promiscuous feature for microsymbionts, diverse Co., Ltd. rhizobia and varied community compositions of bean rhizobia might be expected in China. The study on diversity and distribution 2.3. Phylogenetic analysis of bean rhizobia collected from different geographical regions might improve our knowledge about the rhizobial diversity, evo- The acquired nucleotide sequences were applied for BLAST lution and biogeography. Thus, the goals of this study were (i) to (Altschul et al., 1990) to obtain the closely related sequences in the identify the diversity of bean rhizobia isolated from different GenBank database. The sequences obtained in this study together ecoregions; and (ii) to assess the geographic distribution of rhizo- with the related sequences from the database were aligned by bial species and its determinants. using the program ClustalW in the package of MEGA version 5 (Tamura et al., 2011). Sensitivity and quality analyses of data were 2. Materials and methods firstly involved in assessing phylogeny (Grant and Kluge, 2003). For detecting the quality of data, the index of substitution saturation (Iss 2.1. Bacterial strains and sampling sites and Iss.c) was measured with DAMBE (Xia, 2009, 2013). Combined sequences of housekeeping genes were subject to define the MLST In this study, root nodules were collected from bean plants (Multilocus sequences type). Meanwhile, it was necessary to grown at 9 different sites located in northern and southern regions analyze the sensitivity of concatenated sequences. To evaluate of China (Table S1). Nodules were sterilized by immerging in 95% whether the data sets containing different evolutionary histories ethanol and 0.2% HgCl2 following by rinsing six times with distilled are incongruent, incongruently length difference (ILD) test (Farris water as described previously (Kamicker and Brill, 1986). The et al., 1994, 1995) was performed with the program of surface-sterilized nodules were crushed and streaked on the PAUP*4.0b10. ILD analyses of combined housekeeping genes se- yeastemannitol agar (YMA) (Vincent, 1970) and incubated at 28 C quences were conducted with 1000 replicates of random addition for three days to two weeks, until the single colonies occurred. heuristic search with TBR branch swapping (Yoder et al., 2001). Single colonies were picked up and repeatedly streaked on the Phylogenetic trees were constructed by using the MEGA version same medium until the colony morphology was homogeneous. The 5(Tamura et al., 2011) with the neighbor joining (NJ) method. The purified isolates were suspended in the YM broth supplemented robust of the NJ tree was evaluated by bootstrap analyses with 1000 with 20% (w/v) of glycerol and stored at 70 C. replications (Sy et al., 2001). Genospecies were defined according to Soil samples were collected at the same sites from the surface the MLSA relationships using 97% of sequence similarity as the layer (0e10 cm in depth) together with the bean root system. threshold as suggested previously (Lopez-Guerrero et al., 2012; Physicochemical properties of the soils, including pH and contents Ribeiro et al., 2013). of nitrogen, phosphorus and potassium were measured with the routine methods (Rayment and Higginson, 1992). 2.4. Statistical analysis and community diversity estimation

2.2. DNA extraction and sequencing of multilocus genes Sequence-based markers are used to evaluate species abun- dance and biogeography by non-parametric multivariate methods For extraction of DNA, the isolates were cultured in TY broth (Li et al., 2012). CCA (Canonical Correspondence Analysis) (ter (Tryptone 5 g; Yeast extract 3 g; CaCl2 0.6 g; Distilled water 1 L; pH Braak, 1986), as a constrained ordination method, was utilized to 7.0) with agitation at 28 C up to the early stationary stage and cells understand the relationship between environmental variables of were collected by centrifugation as reported elsewhere (Cava et al., sampling sites and genospecies abundance defined based on the 1989). Genomic DNA of each isolate was prepared using the CTAB results of MLSA. Before the CCA, a linear or unimodal ordination method (Wilson, 2001) as template for amplifying ribosomal RNA model was determined by DCA (Detrended Canonical Analysis) gene and symbiotic genes. The 16S rRNA gene was amplified with (Leps and Smilauer, 2003). The maximal value of the lengths of the primer P1 and P6 (Chen et al., 1997) and the related PCR protocol gradient in four ordination axes was below 3, suggesting that the (Chun and Goodfellow, 1995). Gene nodC was amplified using the linear gradient analysis model was more suitable, but the unimodal primer pairs nodCF/nodCI and nodCFu/nodCI for Rhizobium and also could be used. The analysis of species abundance and envi- Bradyrhizobium, respectively (Laguerre et al., 2001). Gene nifH was ronmental data was performed by CANOCO 5 (Microcomputer amplified using the primer pair nifHF/nifHI(Laguerre et al., 2001). Power, Ithaca, NY). The PCR products digested with three restriction endonucleases The community diversity of rhizobia in each of the ecoregions MspI, HhaI and HaeIII separately at 37 C for 4 h which were were estimated by the ShannoneWeaver index (H) and species resolved by electrophoresis in 2% (w/v) agarose gel (Amreso) in richness (d), calculated with the following formula: H ¼ (N log 0.5 TBE (TriseborateeEDTA) buffer. Then the gels were stained N S ni log ni)C/N and d ¼ (S 1)/log N. Here N is the number of with ethidium bromide (0.5 mg/ml) and the RFLP patterns were total isolates in the ecoregion, ni presents the number of isolates in scanned with Bio-red Gel Imager. The RFLP patterns for these genospecies I; C is a constant of 2.3; S is the number of genospecies authenticated genes of rhizobia were defined. detected in the ecoregion. 130 Y. Cao et al. / Soil Biology & Biochemistry 78 (2014) 128e137

2.5. Reinfection test the twenty-four representatives. Group I covered the five isolates representing rRNA types E (isolate L6) and F (M8, NC4, L43, L13) and Twenty-four representative isolates for different genomic spe- reference strains of R. leguminosarum and R. laguerreae. Group II cies defined in the present study were involved in this analysis. The included twelve isolates representing the rRNA types H, J and G, common bean seeds were surface sterilized with 0.5% HgCl2 for together with reference strains of R. fabae, R. phaseoli, R. etli and 3 min and germinated on the 0.8% water-agar plates as described R. pisi. Group III was consisted of strain J15 (rRNA type I) and previously (Vincent, 1970). Seedlings were transferred into the pots R. vallis. Group IV was comprised of isolate C9 (rRNA type D), B. containing sterile vermiculite and each seedling was inoculated elkanii NBRC 14791T and B. pachyrhizi PAC48T, and C9 had 99.70% with 0.1 ml of 24 h or 72 h culture in YM broth for Rhizobium or similarity with B. elkanii. Group V included isolate L2 (rRNA type A) Bradyrhizobium isolates, respectively. Plants were grown green- and B. liaoningense. Y36 representing rRNA type B (also isolate house with sunlight at temperature of 28/20 C (day/night) and M28) was in group VI and was closed to B. betae. In group VII, L1 watered by nitrogen-free nutrient solution (Vincent, 1970). The (rRNA type A), Y21 (rRNA type C) and JX1 (rRNA type A) were highly control group was dealt with the same condition without rhizobia closed to B. diazoefficiens. inoculation. Nodulation was examined after the plants growing 35 days in the greenhouse and the effectiveness (nitrogen fixation) of 3.2. Analysis of housekeeping genes and definition of genospecies the nodules was estimated from the color of leaves: dark green leaves mean effective and yellow leaves mean no effective. In MLSA of atpD, glnII and recA genes of the 24 representative isolates, indexes of substitution saturation for all the three protein- 3. Results coding sequences on the third codon position revealed no signifi- cant saturation (Iss < Iss.c, p < 0.01). ILD test showed a congruence 3.1. Sequence analyses of 16S rRNA gene (p ¼ 0.01) among the phylogenetic relationships of atpD, glnII and recA. Topology of the phylogenetic tree reconstructed with the In PCR, about 1500 bp fragments of the 16S rRNA genes were concatenate dataset (Fig. 2) obtained higher bootstrap values than amplified from the isolates. Based on restriction patterns, ten rRNA the single gene trees (Figures available as Supplementary Figs. S1, genotypes were defined among the 226 isolates (Table 1). The most S2 and S3) which often have a clash. Most isolates in the rRNA abundant genotype was J containing 91 isolates (40.27%), followed group I (types E and F), except L43, were tightly clustered with by F and H accounting for 28.32% and 20.80% respectively. R. leguminosarum in the three single housekeeping gene trees as A total of twenty-four isolates representing the 10 rRNA geno- well as in the concatenate tree, with similarities 99% in the MLSA types were used in sequence analysis of 16S rRNA genes. According (Table 1, Fig. 2). Then, the 73 isolates represented by L6, L13, M8 and to the phylogeny of 16S rRNA genes (Fig. 1), seven groups belonging NC4 were identified as members of R. leguminosarum. Isolate L43 to the genera Rhizobium and Bradyrhizobium were revealed among was linked to R. laguerreae FB206T based on the atpD and recA

Table 1 Rhizobial strains used in this study and their phylogenetic relationships.

Straina RFLP genotypeb The most related species (sequence similarity, %)c Ecoregion

rRNA nodC nifH 16S rRNA atpD glnII recA Combined

B. diazoeficiens L1 A e AB.dT (100) B. dT (99.28) B. dT (98.21) B. d (97.13) B. d (98.27) I Y21 C e BB.dT (100) B. dT (100) B. dT (100) B. d (99.43) B. d (99.67) II Bradyrhizobium sp. I C9 D e CB.eT (99.70) B. pT/B. eT (96.64) B. pT (97.54) B. pT (94.83) B. pT (96.45) II Bradyrhizobium sp. II. L2 A e AB.lT (99.84) B. sp. (100) B. sp. (100) B. sp. (94.25) B. sp. (98.35) I Bradyrhizobium sp. III. Y36 B eeB. dT (99.77) B. sp. (96.40) B. lT/B. jT (94.63) B. sp. (94.54) B. lT (94.55) II (2) Bradyrhizobium sp. IV. JX1 A e AB.dT (99.78) B. dT (98.80) B. jT (96.87) B. lT (94.54) B. d (95.71) II R. leguminosarum L6 (6) E A D R. laT (100) R. l (100) R. l (98.21) R. l (99.14) R. l (99.00) I L13 (61) F A D R. laT (100) R. l (100) R. l (99.78) R. l (100) R. l (99.92) I, II NC4 (4) E B D R. laT (100) R. l (100) R. l (99.11) R. l (100) R. l (99.67) II M8 (2) F B D R. laT (100) R. l (99.75) R. l (99.11) R. l (99.14) R. l (99.33) II R. etli complexd S6 (11) Subgroup 1 J C D R. pT (100) R. eT (97.04) R. eT (96.87) R. eT (97.41) R. eT (97.08) II L101 Subgroup 2 J A E R. eT (99.85) R. e (99.51) R. e (99.78) R. e (99.43) R. e (99.58) I H7 (20) Subgroup 2 H C D R. eT (99.85) R. e (99.51) R. e (99.78) R. e (99.43) R. e (99.58) I L7 (4) Subgroup 3 J C D R. pT (100) R. e (100) R. e (97.76) R. e (100) R. e (99.17) I NC3 (13) Subgroup 3 H A D R. pT (100) R. e (100) R. e (97.99) R. e (100) R. e (98.08) II NC1 (10) Subgroup 4 H B D R. fT (99.93) R. e (98.52) R. e (100) R. e (99.71) R. e (99.42) II R. phaseoli Y20 GBDR.fT (99.93) R. phT (100) R. phT (99.78) R. e (99.14) R. phT (99.50) II R. vallis J15 (6) I A D R. vT (99.92) R. v (96.79) R. v (98.21) R. v (97.70) R. v (97.58) II Rhizobium sp. I. L43 FAER.laT (100) R. l (97.04) R. fT (96.64) R. lT (97.99) R. l (96.42) I Rhizobium sp. II. L9 (7) J A D R. eT (99.85) R.v (96.05) R. eT (96.64) R. eT (96.84) R. eT (96.17) I Rhizobium sp. III. M1 (14) J D F R. fT (99.93) R. vT (99.01) R. e (96.64) R. e (97.70) R. e (95.83) II L46 (4) H A D R. eT (99.85) R. l (98.52) R. e (96.42) R. e (98.28) R. e (95.83) I Rhizobium sp. IV. C5 (29) J B D R. pT (99.93) R.v (95.80) R. fT (97.32) R. vT (95.40) R. v (94.58) II C6 (25) J A D R. pT (99.93) R. v (95.80) R. fT (97.32) R. vT (95.40) R. v (94.58) II

a Representative isolates (number of isolates). The letters in the isolate numbers indicate their geographic origins. L ¼ Shenyang and H ¼ Daqing are located in the temperate region of Liaoning and Heilongjiang Provinces (Ecoregion I); JX ¼ Jinxian, Y ¼ Yudu, M ¼ Mazuyan, C ¼ Chutan, NC ¼ Nanchang, J ¼ Jiangkou and S ¼ Sanjiang are located in Jiangxi, a subtropical Province (Ecoregion II). b The capital letters represent the genotype. “e” indicates amplification failed by unknown reason. c Determined based on the sequence similarities of the genes aligning in http://eztaxon-e.ezbiocloud.net/. The superscript T means type strain. B. l ¼ B. liaoningense;B. d ¼ B. diazoeficiens;B.e¼ B. elkanii;B.p¼ B. pachyrhizi;R.la¼ R. laguerreae;R.l¼ R. leguminosarum;R.ph¼ R. phaseoli;R.p¼ R. pisi;R.e¼ R. etli;R.v¼ R. vallis;R.f¼ R. fabae. d The representative isolates for the four subgroups were most similar to the reference strains CFN42T, CCBAU 83475, Mim2 and CIAT 652, respectively. Y. Cao et al. / Soil Biology & Biochemistry 78 (2014) 128e137 131

Fig. 1. Phylogenetic tree based on 16S rRNA gene sequences of rhizobia isolated from common beans in Liaoning, Heilongjiang and Jiangxi. The tree was constructed by the neighbor-joining method using MEGA version 5. Numbers on the branches are bootstrap value. Genbank access numbers are in parentheses. Superscript T indicates the strains are type strains. RFLP type is marked following the isolate. genes, and to R. vallis CCBAU 65647T in the glnII gene tree; while it housekeeping gene trees (97.58% similarity in MLSA) and was was a single lineage distantly related to R. laguerreae in the recognized as this species. The isolate C9 (rRNA group IV, genotype concatenate tree with similarity of 96.42%. So, we defined it as D) was always distantly related to B. elkanii/B. pachyrhizi (96.45% Rhizobium sp. I. similarity in MLSA with B. pachyrhizi); isolate L2 (group V, type A) Among the rRNA group II, isolates H7, L7, L101, NC1, NC3 and S6 was closely related to Bradyrhizobium sp. CCBAU 25642 (98.35% (types H and J) representing 59 isolates showed 98.08%e99.58% similarity in MLSA); and Y36 (group VI, type B) was distantly related similarities (Table 1)toR. etli CFN42T or other three reference to B. liaoningense (94.55% similarity in MLSA); therefore, they were strains in MLSA and we classified them as R. etli complex including identified as Bradyrhizobium sp. I, sp. II and sp. III respectively. The four subgroups because they were located in three branches isolates JX1 (group VII, type A) was distantly related to B. japonicum, intermingled with strains of other species (Table 1). The isolates B. liaoningense or B. diazoefficiens in the phylogenetic trees of the Y20 (type G), L43 (type F) and L9 (7 isolates in type J) were designed three genes and presented as a distinct lineage in the concatenate as R. phaseoli, Rhizobium sp. I and Rhizobium sp. II respectively tree (95.71% similarity with B. diazoefficiens in MLSA); therefore, it based upon their relationships with the reference strains (Fig. 2, was designed as Bradyrhizobium sp. IV. The remaining Bradyrhi- Table 1). M1 (type J) and L46 (type H) representing 18 isolates were zobium isolates L1 and Y21 presented close relationships with the also considered as a separate genospecies, Rhizobium sp. III, a B. diazoefficiens strains in all the trees (98.27% similarity in MLSA) lineage distantly related to the R. etli complex (95.83% similar- and were identified as member of this species. ities). Isolates C5 and C6, representing 54 isolates in type J, were a lineage distantly related to a R. vallis reference strain (94.58%) and 3.3. Typing and phylogeny of the symbiotic genes were designed as Rhizobium sp. IV. The rRNA group III isolate J15 representing 6 isolates in type I For RFLP analysis, the nodC gene was amplified from all the showed high similarities to R. vallis CCBAU 65751 in all the 4 Rhizobium isolates, but not the seven Bradyrhizobium isolates. 132 Y. Cao et al. / Soil Biology & Biochemistry 78 (2014) 128e137

Fig. 2. Phylogenetic trees based on the concatenated gene sequences (atpD, glnII and recA) showing the relationships among the rhizobia isolated from common beans in Liaoning, Heilongjiang and Jiangxi. The tree was constructed by the neighbor-joining method using MEGA version 5. Numbers on the branches are bootstrap value. Genbank access numbers are in parentheses. Superscript T indicates the strains are type strains. A threshold of 97% similarity was used for definition of genospecies.

Based upon the RFLP patterns, four nodC types were defined among Subgroup III isolates were isolated from both the ecoregions and the isolates, in which 124 (54.87%) belonged to type A; while 46, 35 had nodC gene sequences identical to that of R. etli bv. phaseoli and 14 isolates were assigned to types B, C and D accounting for RP338. Subgroup IV also contained isolates from two ecoregions 20.35%, 15.49% and 6.11% of all, respectively (Table 1). The nifH gene and they harbored nodC with sequence identical to those of three was amplified from most of the isolates, except the two Bradyrhi- reference strains for R. phaseoli, R. etli bv. phaseoli and zobium sp. III isolates M28 and Y36. Six nifH genotypes were R. leguminosarum bv. phaseoli. The Bradyrhizobium isolates L1 and defined based upon the RFLP patterns (Table 1) dominated by type L2 were a nodC lineage together with a B. japonicum reference D including 203 isolates (89.82%). The type A, B, C, E and F con- strain; while Y21 had nodC gene with sequence identical to those of tained 3, 1, 1, 2 and 14 isolates, respectively. two B. japonicum reference strains and of the type strain for In the sequence analysis, the nodC gene was amplified from B. diazoefficiens. three Bradyrhizobium isolates; therefore, 21 and 22 representative The nifH gene phylogenetic relationships of the test bacteria isolates were used, respectively, in the nodC and nifH gene phylo- (Figure available as Supplementary Fig. S4) were very similar to genetic analyses. All the Rhizobium isolates showed nodC genes those of the nodC phylogeny (Fig. 3), with two small differences: 1) very similar (96.4%e100% sequence similarity) to those of R. etli the isolates in nodC subgroups I and IV showed identical nifH genes; strains and could be divided into four subgroups at the similarity 2) the isolate C9, that was failed for nodC amplification, had a nifH level of 98.0% in the nodC gene tree. All the isolates of subgroup I gene almost identical with those of B. elkanii reference strains. were originated from Jiangxi Province (ecoregion II) and their nodC gene sequences were identical to those of six reference strains for 3.4. Geographic distribution of rhizobia and the environment R. vallis, R. leguminosarum bv. phaseoli, R. lusitanum, and R. etli. The determinants subgroup II isolates were originated from Heilongjiang and Liaon- ing Provinces (ecoregion I) and they had nodC gene sequences The physicochemical features of the soils were presented in identical to those of two reference strains for R. etli and R. gallicum. Table 2. The soils were sandy loam with neutral to slight alkaline pH Y. Cao et al. / Soil Biology & Biochemistry 78 (2014) 128e137 133

Fig. 3. Phylogenetic tree based on nodC gene sequences showing the rhizobia isolated from common beans in Liaoning, Heilongjiang and Jiangxi. The tree was constructed by the neighbor-joining method using MEGA version 5. Numbers on the branches are bootstrap value. Genbank access numbers are in parentheses. Superscript T indicates the strains are type strains.

(6.52e8.06) in ecoregion I and red clay with acid to neutral pH R. leguminosarum (29.6%), R. etli complex (21.0%; not subgroup 2) (4.97e7.22) in ecoregion II. All the maxima values and greater and other minor groups (Table 2). variations of the nutrient contents were found in Ecoregion II. The diagram in Fig. 4 shows the possible influence on species In general, Rhizobium was the absolutely dominant group (about composition of the soil characteristics. In the DCA, species as 96.9%) to nodulate bean plants in both the ecoregions I and II; while response data were compositional and have a gradient 2.5 SD units the Bradyrhizobium was the minor group (Table 2). The diversity of long, so RDA was recommended although unimodal also could be bean rhizobia was greater in ecoregion II (H ¼ 1.57; d ¼ 1.77) than used. Judging from the result of the permutation test, we chose CCA those in ecoregion I (H ¼ 1.34; d ¼ 1.44) (Table 2). In this case, it is test to illustrate the relationship between species and environ- not clear that the differences in diversity parameters were related mental factors. Results of CCA (Fig. 4) indicated that soil pH and to the ecoregions or were caused by the difference in sampling total nitrogen had the greatest effect on the distribution of rhizobia sizes, since 162 isolates were from ecoregion II and only 64 isolates nodulating with bean. All the test environmental factors could were from ecoregion I in this study. Although R. leguminosarum, explain the variation of species accounted for 85.2% (p ¼ 0.048). The R. etli complex, Rhizobium sp. III and B. diazoefficiens were found in pH value was the most significant factor affecting the variability in both the ecoregions in different relative abundances, most of the abundance of species (p ¼ 0.049). The relative abundances of R. etli, minor groups were not shared by the two ecoregions. Therefore, Rhizobium sp. I, Rhizobium sp. II, Rhizobium sp. III, B. diazoefficiens the community compositions of bean rhizobia in the two ecor- and Bradyrhizobium sp. II were the most typical species corre- egions were distinct from each other. In ecoregion I, the bean sponding to a neutral or slightly alkaline soil with high potassium rhizobial community is characterized by the abundance of content. Bradyrhizobium sp. I, Bradyrhizobium sp. IV, R. vallis, R. R. leguminosarum (39.0%), R. etli complex (39.0%; mainly subgroup phaseoli and Rhizobium sp. IV were able to adapt to acidic soil with 2) and Rhizobium sp. II (10.94%) together with the minor groups. In high phosphorus, organic matter, nitrogen and low potassium, ecoregion II, the rhizobial community is characterized by the while R. leguminosarum and Bradyrhizobium sp. III were adapt to dominance of Rhizobium sp. IV (33.3%), followed by acid soil with low organic matter, nitrogen, potassium and 134 Y. Cao et al. / Soil Biology & Biochemistry 78 (2014) 128e137

Table 2 housekeeping genes, the combined analysis can illustrate the Soil characters and distribution of the rhizobial genospecies associated with com- phylogeny better than the separate analysis (Bininda-Emonds, mon bean in the two ecoregions. 2004; de Queiroz and Gatesy, 2007). Therefore, the 226 isolates Soil features Ecoregion I Ecoregion II were identified into 13 genospecies. It is worthy to notice that four Soil texture Sandy loam soil Red clay soil subgroups within R. etli were divided, which were intermingled Value of pH 6.52e8.06 4.97e7.22 with R. vallis, R. phaseoli and deeply branched genospecies Rhizo- Total phosphor (TP, mg kg 1) 220e740 310e2750 bium sp. III. These results imply that either the three species (R. etli, Total nitrogen (TN, mg kg 1) 510e1130 410e2240 R. vallis and R. phaseoli) are very similar species unable differenti- Available nitrogen (AN, mg kg 1) 35.70e75.33 27.13e195.37 Total potassium (TK, mg kg 1) 21,800e26,600 11,300e29,300 ated by the three housekeeping genes; or the taxonomic relation- Organic Carbon (TOC, %) 0.91e1.68 0.44e5.68 ships among the four subgroups of R. etli and the intermingled

Genospecies Number %a Number %a species need further study. The definition of B. diazoefficiens, R. etli complex, R. phaseoli, R. leguminosarum 25 39.06 48 29.63 R. leguminosarum, R. vallis and eight unnamed genospecies among R. etli complexb 25 39.06 34 20.99 1) S6-CFN42T (0) (0) (11) (6.79) our isolates indicated that the rhizobia of bean harbor a huge di- 2) H7/L101-CCBAU 83475 (21) (32.81) (0) (0) versity in China. Among these species, R. etli, R. phaseoli and 3) L7/NC3-Mim2 (4) (6.25) (13) (8.03) R. leguminosarum were well known microsymbionts of bean, but 4) NC1-CIAT 652 (0) (0) (10) (6.17) B. diazoefficiens, R. vallis and the eight unnamed species were new R. phaseoli 0 0 1 0.62 R. vallis 0 0 6 3.70 records as bean rhizobia. It was interesting that the bean rhizobia, Rhizobium sp. I 1 1.56 0 0 like R. gallicum, R. giardinii, R. lusitanum etc., found in other regions Rhizobium sp. II 7 10.94 0 0 or the dominant groups adapted to some extreme conditions, like Rhizobium sp. III 4 6.25 14 8.64 R. tropici in acid soils (Martínez-Romero et al., 1991; Anyango et al., Rhizobium sp. IV 0 0 54 33.33 1995; Grange and Hungria, 2004) and Sinorhizobium species in Subtotal (Rhizobium) 62 96.88 157 96.91 e ~ B. diazoefficiens 1 1.56 1 0.62 alkaline saline soils (Mnasri et al., 2007; Zurdo-Pineiro et al., 2009; Bradyrhizobium sp. I 0 0 1 0.62 Mnasri et al., 2012), were not found in our study. These phenomena Bradyrhizobium sp. II 1 1.56 0 0 might demonstrated that the introduction of bean was an evolu- Bradyrhizobium sp. III 0 0 2 1.24 tional force for emerging of novel rhizobia, which might be Bradyrhizobium sp. IV 0 0 1 0.62 Subtotal (Bradyrhizobium) 2 3.12 5 3.09 developed by the symbiotic gene transfer from R. etli because this species was the dominant rhizobia in the center of origin for bean þ Total (Rhizobium Bradyrhizobium) 64 100.00 162 100.00 plant (Amarger, 2001). In this way, some rhizobia adapted to ShannoneWeaver index (H) 1.34 1.57 Species richness (d) 1.44 1.77 different soil types or ecoregions have developed their nodulation

a ability on bean plants. Therefore, our results evidenced that the Relative abundance is presented by the ratio: Number of isolate in the geno- fl species/Total number of isolates in the ecoregion. evolution of rhizobia was in uenced by both the environmental b The four subgroups in the complex are presented by the representative isolate factors and their hosts. most related reference strain. The close phylogenetic relationships of the symbiotic genes among the Rhizobium isolates obtained in this study (Fig. 3)were evidence to support the possible lateral gene transfer (Laguerre phosphorus. A parallel CCA was also performed to consider the four et al., 2001; Slater et al., 2009) among the bean-nodulating subgroups of R. etli as independent taxa, together with the other rhizobia in China. Although four groups were divided based upon Rhizobium and Bradyrhizobium genospecies. The result (available as the nodC phylogenetic relationships among the Rhizobium isolates Supplementary Fig. S5) was similar to that in Fig. 4. The only dif- ference was that the R. etli subgroups 1, 3 and 4 were distantly separated from subgroup 2.

3.5. Nodulation

All the twenty-four representative isolates successfully nodu- lated with beans and the control group did not have nodules. The isolates of Rhizobium species formed round and pink nodules which were deemed to be effective for nitrogen fixation; while the Bra- dyrhizobium species formed rod-like and white nodules which did not fix nitrogen based upon the yellow leaves of their host plants.

4. Discussion

4.1. Diversity of rhizobia nodulating with bean in two ecoregions of China

In the present study, we used the phylogeny of the 16S rRNA gene to define the genus and MLSA of atpD, glnII and recA genes to define the specific positions of the rhizobial isolates. As the mo- Fig. 4. Canonical correspondence analyses (CCA) were used to evaluate the influence lecular evolutionary markers, housekeeping genes showing of main soil factors on rhizobial abundance. The arrows represent soil factors and the conserved and variable regions could provide relatively accurate triangles represent different species. TN: total nitrogen, AN: available nitrogen, TPh: total phosphorus, Org: organic material, TP: total potassium. Rhi sp I: Rhizobium sp. I, phylogenetic information to support 16S rRNA gene identification Rhi sp II: Rhizobium sp. II, Rhi sp III: Rhizobium sp. III, Rhi sp IV: Rhizobium sp. IV, Brady and their concatenated tree makes phylogeny of housekeeping sp I: Bradyrhizobium sp. I, Brady sp II: Bradyrhizobium sp. II, Brady sp III: Bradyrhizobium genes robust (Case et al., 2007; Aserse et al., 2012a). For the sp. III, Brady sp IV: Bradyrhizobium sp. IV. Y. Cao et al. / Soil Biology & Biochemistry 78 (2014) 128e137 135 in this study (Fig. 3), their nodC gene sequences were very similar, the previous studies. Soil pH, salinity and available phosphorus in comparison with their phylogenetic relationships of 16S rRNA content were estimated as the determinants for distribution of gene (Fig. 1) and of the housekeeping genes (Fig. 2). So, it is clear Bradyrhizobium and Sinorhizobium nodulated with soybean (Han that the nodC genes in the bean-nodulating Rhizobium species in et al., 2009; Li et al., 2011). Correspondingly, the Bradyrhizobium China have the same origin and they were divergently evolved core genome is bound up with lipid and secondary metabolism, rather recently, after this plant was introduced 700 years ago (van while Sinorhizobium core genome is connected with osmopro- Schoonhoven and Voysest, 1991). Previously, similar nodC and nifH tection and adaptation to alkaline pH (Tian et al., 2012), which trees have been reported among some bean-nodulating strains in might be reasons to explain their different geographic distribution R. etli, R. leguminosarum, R. gallicum, R. giardinii, and the interspe- associated with soybean. In our study, the dominance of cies sym plasmid transfer among these species has been estimated R. leguminosarum in both ecoregions (acid and alkaline soils), the (Herrera-Cervera et al., 1999). Based upon the high similarities of dominance of Rhizobium sp. IV/R. etli subgroups I and IV in acid soils symbiotic genes, biovar phaseoli has been described in each of (ecoregion II), and of Rhizobium sp. II/R. etli subgroups II in the these species. The nodC and nifH phylogenetic relationships of the alkaline soils (ecoregion I) made them good candidates for a Rhizobium isolates in our present study demonstrated that all of comparative core genome analysis to estimate the genetic basis of them could also be defined as biovar phaseoli. their geographic distribution. Three types (a, g and d)ofnodC gene alleles were distinguished Differentiation of host-associated microorganisms like rhizobia among R. etli bv. phaseoli in the center of origin for bean, and they is connected with the distribution of their hosts (Bala et al., 2003). showed distinct predominance in the different habitats (Aguilar Based upon the high similarities in the housekeeping and symbiotic et al., 2004). Allele g were abundant in strains isolated from genes observed among the R. etli complex isolates and the reference Spain (Mulas et al., 2011) and Nepal (Adhikari et al., 2013). Our strains (Table 1, Figs.1e3), we suppose that the R. etli-related strains results in the present study showed that most of the Rhizobium might have migrated along with the host seeds (Perez-Ram ırez isolates had the g allele of nodC which was originated in American et al., 1998). Therefore, the introduction of a host plant is also the regions. This also suggests that the symbiotic genes of bean- reason for the wide geographical distribution of rhizobia associated nodulating Rhizobium species in China have an American origin. with it. When similar microbial taxa are among the communities in The failure of amplifying the symbiotic genes from some of the different sites, geographic distance should predict genetic diver- Bradyrhizobium isolates and the fact that their nodC and/or nifH gence foremost in the light of biogeographic patterns driven by genes presented distinct phylogenetic relationships, distant from dispersal limitation primarily (Martiny et al., 2006). Furthermore, each other and from the Rhizobium isolates (Fig. 3) implied that the distance effect may impact on past divergence of community ob- Bradyrhizobium isolates harbored divergent symbiotic genes and tained by genetic drift or adaptation to past environments (Martiny each species have independently evolved since a long period ago. et al., 2006). A comparative study on the genetic diversity in Although the Bradyrhizobium isolates could form nodules with geographic distant rhizobial populations is needed to confirm this beans, the nodules were ineffective. Based upon the host sanction, estimation. plants were in favor of nitrogen fixing bacteria where both the Conclusively, diverse and distinctive bean rhizobial commu- fixing and non-fixing bacteria exist (Gubry-Rangin et al., 2010). nities were detected in two ecoregions in China, in which some Therefore, they may be opportunistic bacteria occupied the bean novel and unique groups were found. These results demonstrated nodules. that the introduction of bean plants has driven the evolution of rhizobia to fit the necessary to nodulate the host plant and to adapt 4.2. Community composition and geographic distribution of bean to the local conditions, mainly the soil pH and the nutrient avail- rhizobia ability. The relative predominance of R. etli and the high similarities in the symbiotic genes (nodC and nifH) evidenced that R. etli might In general, the community structure of bean rhizobia in China have been introduced to China accompanying the seeds, and then was different from those reported in other regions (Martínez- its symbiotic genes were transferred to the related indigenous Romero et al., 1991; Anyango et al., 1995; Amarger, 2001; Grange rhizobia. This evolutionary event might have helped the bean to and Hungria, 2004; Tamimi and Young, 2004; García-Fraile et al., successfully colonize the new continent during the last 700 years fi 2010; Aserse et al., 2012b). The most abundant group Rhizobium since its introduction directly from America. These ndingś sp. IV and the minor groups of Rhizobium sp. III, R. vallis, Rhizobium enlarged the diversity of bean rhizobia, evidenced the biogeo- sp. I, Bradyrhizobium spp. found in ecoregion II (Table 2) repre- graphic patterns of these rhizobia, and demonstrated the possible sented a totally new community composition, demonstrating that evolution and emerging of novel rhizobia under the combined se- the bean plants have been selecting some new microsymbionts lection of host plant and soil conditions. adapted to the local conditions. The dominance of R. leguminosarum and R. etli in ecoregion I was similar to several reports in the An- Acknowledgments dean, Mesoamerican region and Nepal (Bernal and Graham, 2001; Adhikari et al., 2013; Ribeiro et al., 2013), as well as in Yunnan This work was supported by the project from National Natural and Inner Mongolia of China (Han et al., 2005). Since these regions Science Foundation of China (31125007). ETW was supported by covered the tropic and temperate regions, we estimate that some the SIP projects authorized by IPN, Mexico. similar soil characters, not the climate factors, in these studied regions might be the determinants for the dominance of Appendix A. Supplementary data R. leguminosarum. These variations in predominant groups and community structures might be an evidence for the biogeographic Supplementary data related to this article can be found at http:// patterns of the bean rhizobia, like previously revealed for the dx.doi.org/10.1016/j.soilbio.2014.07.026. soybean rhizobia (Han et al., 2009; Li et al., 2011). Also, dominant rhizobia may have more ability to adapt to the local environments References (Rahi et al., 2012). In the present study, we found that pH of soil as a main envi- Adhikari, D., Itoh, K., Suyama, K., 2013. 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