The endemic Genista versicolor from Sierra Nevada National Park in Spain is nodulated by putative new Bradyrhizobium species and a novel symbiovar (sierranevadense)

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The endemic Genista versicolor from Sierra Nevada National Park in Spain is nodulated by putative new Bradyrhizobium species and a novel symbiovar (sierranevadense) José F. Cobo-Díaz a,1 , Pilar Martínez-Hidalgo b,1 , Antonio J. Fernández-González a , Eustoquio Martínez-Molina b , Nicolás Toro a , Encarna Velázquez b,∗ , Manuel Fernández-López a a Departamento de Microbiología del Suelo y Sistemas Simbióticos, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Granada, Spain b Departamento de Microbiología y Genética, Universidad de Salamanca, Salamanca, Spain

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Article history: Received 23 July 2013 Received in revised form 27 September 2013 Accepted 30 September 2013 Keywords: Bradyrhizobium Genista Symbiovar Rhizobia Biodiversity Phylogeny

a b s t r a c t Genista versicolor is an endemic legume from Sierra Nevada National Park which constitutes one of the UNESCO-recognized Biosphere Reserves. In the present study, a collection of strains nodulating this legume was analysed in characteristic soils of this ecosystem. Most strains nodulating G. versicolor belonged to rrs group I within the genus Bradyrhizobium and only one strain, named GV137, belonged to rrs group II from which only a single species, B. retamae, has been described in Europe to date. Strain GV137, and some strains from rrs group I, belonged to putative new species of Bradyrhizobium, although most strains from group I belonged to B. canariense, according to the ITS fragment and atpD gene analysis. This result contrasted with those obtained in Genista tinctoria in Northeast Europe whose endosymbionts were identified as B. japonicum. The analysis of the symbiotic nodC and nifH genes carried by G. versicolornodulating strains showed that most of them belonged to symbiovar genistearum, as did those isolated from G. tinctoria. Nevertheless, strain GV137, belonging to rrs group II, formed a divergent lineage that constituted a novel symbiovar within the genus Bradyrhizobium for which the name sierranevadense is proposed. This finding showed that the Genisteae are not restrictive legumes only nodulated by symbiovar genistearum, since Genista is a promiscuous legume nodulated by at least two symbiovars of Bradyrhizobium, as occurs in Retama species. © 2013 Published by Elsevier GmbH.

Introduction The Sierra Nevada National Park is a mountain system located in southeast Spain, and currently constitutes one of the nine Biosphere Reserves in the Global Change in Mountain Regions Project from UNESCO (GLOCHAMOST, Global and Climate Change in Mountain Sites). It occupies 86,208 ha and is a unique refuge for biodiversity due to its strategic biogeographical location, isolation, altitudinal range and the diversity of ecological niches. All these characteristics have led to the description of 2100 plant species, of which 116 are threatened. Within the whole network of Spanish National

∗ Corresponding author at: Departamento de Microbiología y Genética, Lab. 209, Edificio Departamental de Biología, Campus Miguel de Unamuno, 37007 Salamanca, Spain. Tel.: +34 923 294532; fax: +34 923 224876. E-mail address: [email protected] (E. Velázquez). 1 These authors contributed equally to this work.

Parks, Sierra Nevada represents the Mediterranean high mountain ecosystem. There are several plant endemisms in Sierra Nevada National Park, among which can be highlighted the shrub Genista versicolor Boiss. (formerly G. baetica Spach) [12], a legume from the tribe Genisteae able to growth in siliceous soils at 1600–2700 m above sea level (asl), thus it always appears above forest formations. G. versicolor is a highly branched, thorny plant that can reach a height of 100 cm. It provides cover and protection for tree seeds and seedlings and, therefore, facilitates the forest formations in altitude. This legume contributes to soil formation at high altitudes because it enriches the soil with nitrogen, like other legumes, due to its ability to establish nitrogen-fixing symbioses. It has been reported that some species of the genus Genista establish symbiosis with the genus Bradyrhizobium [9,22], which currently contains 17 species (Euzéby, J.P. LPSN – list of prokaryotic names with standing in nomenclature, http://www.bacterio. cict.fr/index.html). Within this genus, according to the nomenclature of Menna et al. [13], the species currently known as

0723-2020/$ – see front matter © 2013 Published by Elsevier GmbH. http://dx.doi.org/10.1016/j.syapm.2013.09.008

Please cite this article in press as: J.F. Cobo-Díaz, et al., The endemic Genista versicolor from Sierra Nevada National Park in Spain is nodulated by putative new Bradyrhizobium species and a novel symbiovar (sierranevadense), Syst. Appl. Microbiol. (2013), http://dx.doi.org/10.1016/j.syapm.2013.09.008

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endosymbionts of some Genista species belong to Bradyrhizobium group I and symbiovar genistearum [9]. Nevertheless, there are few studies to date concerning the endosymbionts of Genista, and some species of this genus have never been studied, as occurs with the Spanish endemic G. versicolor. Therefore, the objective of this study was to identify at the species and symbiovar levels the rhizobial strains nodulating G. versicolor in the Sierra Nevada by analysing their core and symbiotic genes. The results showed that at least three putative new species of Bradyrhizobium and one symbiovar phylogenetically unrelated to those currently described in this genus formed nodules with G. versicolor in Sierra Nevada National Park.

Materials and methods

RAPD fingerprinting RAPD patterns were obtained as previously described [19] using the primer M13 (5 -GAGGGTGGCGGTTCT-3 ) and the REDExtractN-AmpTM Plant PCR Kit (Sigma, USA). PCR conditions were: preheating at 95 ◦ C for 9 min; 35 cycles of denaturing at 95 ◦ C for 1 min; annealing at 45 ◦ C for 1 min and extension at 75 ◦ C for 2 min, and a final extension at 72 ◦ C for 7 min. A total of 10 ␮L of each PCR product were electrophoresed in 2% agarose gel in TBE buffer (100 mM Tris, 83 mM boric acid, 1 mM EDTA, pH 8.5) at 6 V/cm, stained in a solution containing 0.5 g/mL ethidium bromide, and photographed under UV light. Standard XIV (Roche, USA) was used as a size marker. A dendrogram was constructed based on the matrix generated using the UPGMA method and the Pearson coefficient with Bionumerics version 4.0 software (Applied Maths, Austin, TX).

Sampling and strain isolation Sampling was carried out at an average altitude of approximately 1940 m above sea level in three plots of 50 m × 50 m (1: N 36◦ 57 20.3 , W 03◦ 26 38.5 – 1889 m asl; 2: N 36◦ 57 38.1 , W 03◦ 26 15.5 – 1944 m asl; 3: N 36◦ 57 59.0 , W 03◦ 25 34.5 – 1982 m asl). In each of the three sampling plots, three sites were selected with a minimum distance of 10 m between each one and without the influence of plant roots. At each site, 1 kg of soil was taken and stored at 4 ◦ C. Seeds of G. versicolor were also collected in July and August 2010 from the same sites where the soil was sampled. These seeds were scarified and sterilised by immersion in concentrated sulphuric acid for 30 min and subsequently washed five times with sterile distilled water and left to soak for 3 h. After this time, the seeds were again washed with sterile distilled water and then transferred into sterile Petri plates containing sterile filter paper. The seeds were germinated in these plates for 5 days at 20 ◦ C in darkness, and a minimum of 1 mL of sterile water was maintained in each plate during this period. The germinated seeds were transferred to 12 cm diameter pots (five seedlings per pot). These pots were prepared by mixing washed sterile sand with a mixture of the soils sampled at the different sites in a ratio of 3:1. The sand was autoclaved twice to avoid proliferation of strains with resistant forms (spores), and then it was subsequently mixed with the soil and poured into the pots. To collect the root nodules, 50 G. versicolor plants were used after two months growth in a chamber with a day/night cycle of 16/8 h and 22/16 ◦ C. After this time, plants were removed from the pots, the roots were washed with water, and the nodules were collected individually. The rhizobia isolation was carried out on yeast mannitol agar (YMA) according to Vincent [28]. A representative isolate of each group obtained after symbiotic gene analysis was inoculated in order to analyse the ability of the strains to nodulate G. versicolor, Glycine max (soybean), Adenocarpus decorticans, Retama sphaerocarpa and Vigna unguiculata (cowpea). The seeds of G. versicolor were scarified and sterilised, as previously explained, and then they were sown in pots with sterile vermiculite. The seeds of A. decorticans and R. sphaerocarpa were scarified mechanically and sterilised by immersion in 28.5% NaClO for 10 min, while those of G. max and V. unguiculata were sterilised by immersion in ethanol for 3 min. After sterilisation, all the seeds were washed five times with sterile distilled water and left to soak for 3 h. Five seeds of G. versicolor, A. decorticans and R. sphaerocarpa, and two seedlings of G. max or V. unguiculata were placed per pot and each plant was inoculated with a 1 mL suspension containing approximately 107 CFU/mL/strain. Then, a surface layer of sterilised perlite was added to prevent loss of moisture and fungal contamination. The pots were left in a chamber with a light–dark cycle of 16/8 h and a temperature of 22/16 ◦ C for two months in the case of G. versicolor, A. decorticans and R. sphaerocarpa, whereas G. max and V. unguiculata plants were grown for 1 month.

Sequence analysis of rrs, atpD, nodC and nifH genes, and the 16S–23S intergenic spacer (ITS) The rrs gene and the ITS were amplified and sequenced according to Rivas et al. [18] and Peix et al. [14], respectively. The atpD gene was amplified and sequenced as described by Vinuesa et al. [30]. The nodC and nifH genes were amplified with the primers and conditions described by Laguerre et al. [11]. PCR amplifications were performed with a REDExtract-N-Amp PCR Kit (Sigma) following the manufacturer’s instructions. The bands corresponding to the different genes were purified either directly from the gel by elution of the excised band or filtration through silicagel columns using the Qiaquick DNA Gel Extraction Kit (Qiagen, Germany) following the manufacturer’s instructions. The sequence reaction was performed in an ABI PRISM 3100 sequencer using a BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems Inc., USA), as supplied by the manufacturer. The sequences obtained were compared to those held in GenBank by using the BLASTN programme [1]. They were aligned by using Clustal W software [26]. Distances calculated according to Kimura’s two-parameter model [10] were used to infer phylogenetic trees with the maximum likelihood method [6] and the MEGA5 software [25]. Confidence values for nodes in the trees were generated by bootstrap analysis using 1000 permutations of the data sets.

Results and discussion Bacterial isolates and nodulation assays A total of 57 slow growing rhizobial strains with the typical morphology of the genus Bradyrhizobium on YMA medium were isolated from G. versicolor nodules. Nodulation assays were performed with strains GV101, GV135 and GV137 as representatives of the different nodC groups found in this study (see below) on G. versicolor, G. max, V. unguiculata, A. decorticans and R. sphaerocarpa. Two months after inoculation, all the strains were able to form nodules with G. versicolor, A. decorticans and R. sphaerocarpa but none of them nodulated with G. max. For V. unguiculata, after inoculation for one month, strain GV101 showed a positive nodulation (Nod+) phenotype but without nitrogen fixation capacity (Nif−), according to the white colour of the nodules. A total of 8.3% of the V. unguiculata plants nodulated by strain GV135 showed the Nod− phenotype, whereas 25% were Nod+/Fix− and the remaining plants (66.7%) showed a thickening on the roots suggesting bacterial infection but without nodule formation. Finally, strain GV137 was Nod− on V. unguiculata plants.

Please cite this article in press as: J.F. Cobo-Díaz, et al., The endemic Genista versicolor from Sierra Nevada National Park in Spain is nodulated by putative new Bradyrhizobium species and a novel symbiovar (sierranevadense), Syst. Appl. Microbiol. (2013), http://dx.doi.org/10.1016/j.syapm.2013.09.008

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Fig. 1. Dendrogram obtained for the strains of Genista versicolor using Pearson’s coefficient and UPGMA analysis of the RAPD profiles.

RAPD fingerprinting analysis The G. versicolor-nodulating isolates were analysed by RAPD fingerprinting that allows the differentiation between strains of the same Bradyrhizobium species, for which this technique provides an estimation of the genetic diversity [3,5,16]. In a recent study [16], we showed that Bradyrhizobium strains with more than 70% similarity in RAPD pattern analysis had identical ITS and atpD genes, therefore, in this current study, the same similarity was considered as the cut-off for selecting strains for gene analysis. The G. versicolor isolates were distributed into eight RAPD groups with similarity percentages lower than 70% (Fig. 1) and a representative isolate from each group was selected for phylogenetic analysis. Analysis of the rrs gene The current phylogenetic classification of rhizobia is predominantly based on rrs gene sequences and thus the identification of nodule isolates should be based on the same gene. In Bradyrhizobium, as mentioned earlier, two well-differentiated groups (Fig. 2) named I and II were defined by Menna et al. according to the rrs gene [13].

Strains GV115, GV102, GV159, GV101, GV104 and GV144, representing RAPD groups I, III, IV, V, VII and VIII, respectively, belonged to Bradyrhizobium phylogenetic group I and were closely related (identity higher than 99%) to B. canariense, a species able to nodulate other Genisteae legumes [29]. Strain GV135, representing RAPD group VI, also belonged to Bradyrhizobium phylogenetic group I and was related to the species B. cytisi and B. rifense isolated from nodules of Cytisus villosus from the tribe Genisteae [2,3]. These results contrasted with those obtained by Kalita and Małek [9] in Northeast Europe for Genista tinctoria that was nodulated by strains related to B. japonicum sv. genistearum BGA-1 (see Fig. 2). Strain GV137 belonged to Bradyrhizobium phylogenetic group II in which the recently described species B. retamae nodulating Retama spp. members of the tribe Genisteae was also included [7]. Nevertheless, the rrs gene of this strain was closely related (higher than 99% identity) to that of B. jicamae, a species nodulating Pachyrhizus erosus in America [17]. Therefore, the rrs gene sequences of our isolates from groups I and II presented identity values higher than 99% with respect to those of some validly described species of Bradyrhizobium. Nevertheless, in the genus Bradyrhizobium, high identities in the rrs gene (even 100%) are commonly found among different species, which

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Bradyrhizobium canariense GV104 (KF483529) 86 Bradyrhizobium canariense GV115 (KF483530) 56 Bradyrhizobium canariense GV102 (KF483528) Bradyrhizobium canariense GV101 (KF483527) 75 Bradyrhizobium canariense BTA-1T (AJ558025) 59 Bradyrhizobium sp. GV144 (KF483533) Bradyrhizobium sp. GV159 (KF4835234) Bradyrhizobium sp. J.UKR5.1 (EU882023) (Genista tinctoria) Bradyrhizobium genosp. beta BRE-1 (FJ428214) Bradyrhizobium sp. JAN10 (EU882024) (Genista tinctoria) Bradyrhizobium japonicum sv genistearum BGA-1 (AJ558024) Bradyrhizobium japonicum sv glycinearum LMG 6138T (X66024) Bradyrhizobium liaoningense LMG 18230T (AF345256) Bradyrhizobium daqingense CCBAU 15774T (HQ231274) 98 Bradyrhizobium yuanmingense CCBAU 11071T (AF193818) Bradyrhizobium sp. GV135 (KF483531) 59 Bradyrhizobium genosp. alpha BC-C1 (AJ558030) Bradyrhizobium cytisi CTAW11T (EU561065) 51 Bradyrhizobium rifense CTAW71T (EU561074) Bradyrhizobium arachidis CCBAU 051107T (HM107167) Bradyrhizobium huanghuaihaiense CCBAU 23303T (HQ231463) 86 Bradyrhizobium iriomotense EK05T (AB300992) Bradyrhizobium betae LMG 21987T (AY372184) Bradyrhizobium diazoefficiens USDA 110T (BA000040) Bradyrhizobium oligotrophicum LMG 10732T (JQ619230) Bradyrhizobium sp. ORS285 (AF230722) 93 97 Bradyrhizobium sp. ORS278 (AF239255) 47 Bradyrhizobium sp. BTAi1 (NC_009485) 99 Bradyrhizobium denitrificans LMG 8443T (X66025) Bradyrhizobium retamae Ro19T (KC247085) Bradyrhizobium jicamae PAC68T (AY624134) Bradyrhizobium sp. GV137 (KF483532) 99 Bradyrhizobium lablabi CCBAU 23086T (GU433448) 83 Bradyrhizobium pachyrhizi PAC48T (AY624135) 98 Bradyrhizobium elkanii USDA 76T (U35000) Bosea thiooxidans DSM 9653T (AJ250796) 0.01 Fig. 2. Maximum likelihood phylogenetic-rooted tree based on rrs gene sequences showing the taxonomic affiliation of the strains representative of the different RAPD groups. Bootstrap values calculated for 1000 replications are indicated. Bar, 1 nt substitution per 100 nt. Accession numbers from GenBank are given in brackets.

means that other phylogenetic markers should be analysed in order to assign isolates to a species and to identify new ones. Within those considered, the ITS fragment and/or the atpD gene have been widely sequenced in Bradyrhizobium species and have allowed the differentiation of species nodulating legumes from the tribe Genisteae [2,3,7,9,13,16,17,20,24,27,29–31].

Analysis of ITS sequences ITS region analysis has been reported as a more powerful tool than rrs gene analysis for species delineation within the genus Bradyrhizobium, in which ITS sequence similarities higher than 95.5% indicate a genospecies level relatedness [20,31]. Menna et al. [13] showed that ITS analysis allows the differentiation of two groups congruently with rrs gene analysis, although when the species B. denitrificans and B. oligotrophicum are considered in this analysis a third group is obtained, as was pointed out by RamírezBahena et al. [16]. Some photosynthetic bradyrhizobia also belong to this group and within these strain BTAi1 is very closely related to the species B. denitrificans (Fig. 3). The strains GV115, GV102, GV101, GV104 clustered with B. canariense BTA-1T (Fig. 3), as occurred in the rrs gene analysis. These strains were divided into two close ITS subgroups with identities higher than 98% (gaps not considered) that were coincident with those reported by Safronova et al. [23] within B. canariense, which can be differentiated by the presence of an insert in the strains from the ITS-I B. canariense subgroup. Strains GV101 and GV104 belonged to the ITS-I subgroup and strains GV102 and GV115 to the

ITS-II subgroup. Subgroup ITS-I was more widely represented in G. versicolor nodules than ITS-II, as occurs in other hosts previously analysed [23,27]. Strains GV159 and GV144 were related to B. canariense but with identities lower than 97%, and they formed a second cluster within Bradyrhizobium phylogenetic group I (Fig. 3). Strain GV135, which was related to B. iriomotense EK05T , also belonged to this group, although with less than 95% identity in the ITS sequences. Finally, strain GV137 belonged to Bradyrhizobium phylogenetic group II with less than 92% identity with respect to the type strains from all the remaining species of this group, B. elkanii, B. pachyrhizi, B. jicamae, B. lablabi and B. retamae. Therefore, in spite of the closeness of the rrs genes from G. versicolor isolates with respect to the currently described Bradyrhizobium species, the analysis of the ITS region suggested that several of them could represent putative novel species. In order to confirm these results, the atpD gene was analysed, since its usefulness in delineation of Bradyrhizobium species has been well-established and previously analysed in several strains isolated from Genisteae [2,3,7,9,13,16,17,24,27,29,30].

Analysis of the atpD gene The atpD gene is a housekeeping gene that has been sequenced in all Bradyrhizobium species, as well as in two strains, JAN10 and JAN16, isolated from G. tinctoria in Europe [9]. The analysis of this gene (Fig. 4) confirmed that the isolates obtained in this study formed different branches divergent to those formed by strains B.

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Bradyrhizobium canariense GV102 (KF483544) Bradyrhizobium canariense sv genistearum BC-C2 (AY386704) 61 Bradyrhizobium canariense Oc4 (DQ646575) Bradyrhizobium canariense GV115 (KF483546)

55

Bradyrhizobium canariense MCLA12 (EF694746)

Group I

Bradyrhizobium canariense Oc1 (DQ646573) Bradyrhizobium canariense MCLA07 (EF694745) 77 Bradyrhizobium canariense sv genistearum BTA-1T (AY386708) Bradyrhizobium canariense GV104 (KF483545) 66

Bradyrhizobium canariense GV101 (KF483543) 55 Bradyrhizobium canariense sv genistearum BES-1 (AY386707)

Group II

Bradyrhizobium canariense sv genistearum BCO-1 (AY599098) Bradyrhizobium japonicum sv genistearum BGA-1 (AY386714) 81 Bradyrhizobium sp. RLA08 (EF694749) Bradyrhizobium sp. GV144 (KF483549) 100 Bradyrhizobium sp. GV159 (KF483550) Bradyrhizobium genosp. alpha BC-C1 (AY386703) Bradyrhizobium japonicum sv glycinearum USDA 6T (HQ143390)

37

Bradyrhizobium rifense CTAW71T (KC247123)

53

92 Bradyrhizobium cytisi CTAW11T (KC247124) Bradyrhizobium diazoefficiens USDA 110T (Z35330) Bradyrhizobium betae LMG 21987T (AJ631967)

56

Bradyrhizobium liaoningense LMG 18230T (AF345256) Bradyrhizobium genosp. beta BRE-1 (AY386715) Bradyrhizobium yuanmingense CCBAU 11071T (AJ534605) Bradyrhizobium arachidis CCBAU 051107T (HM107198)

87 94

Bradyrhizobium huanghuaihaiense CCBAU 23303T (HQ428043) Bradyrhizobium daqingense CCBAU 15774T (HQ231312)

62

Bradyrhizobium sp. GV135 (KF483547) Bradyrhizobium iriomotense EK05T (AB300993) Bradyrhizobium elkanii USDA 76T (AB100747)

76

Bradyrhizobium pachyrhizi PAC48T (AY628092) Bradyrhizobium sp. GV137 (KF483548)

99

Bradyrhizobium retamae Ro19T (KF638356)

73

Bradyrhizobium lablabi CCBAU 23086T (GU433583)

93

Bradyrhizobium jicamae PAC68T (AY628094)

88 82

Bradyrhizobium sp. LMG 15376 (ORS285) (AJ279297) Bradyrhizobium sp. ORS278 (CU234118) Bradyrhizobium oligotrophicum LMG 10732T

100

Bradyrhizobium denitrificans LMG 8443T (AJ279318)

54

96 Bradyrhizobium sp. LMG 11795 (BTai1) (AJ534588)

0.05 Fig. 3. Maximum likelihood phylogenetic unrooted tree based on 16S–23S rRNA internal transcribed spacer (ITS) sequences showing the taxonomic affiliation of the strains representative of the different RAPD groups. Bootstrap values calculated for 1000 replications are indicated. Bar, 5 nt substitution per 100 nt. Accession numbers from GenBank are given in brackets.

japonicum sv. genistearum JAN10 and JAN16 which were closely related to strain BGA-1 isolated from Genisteae in the Canary Islands [29]. Strains GV101, GV102 and GV104, which had slightly different ITS regions, had identical atpD genes and were identified

with B. canariense (100% identity). However, strain GV115, although clustering with these strains, had a slightly divergent ITS region occupying an intermediate position between B. canariense and B. cytisi with identities lower than 98 and 97%, respectively.

Please cite this article in press as: J.F. Cobo-Díaz, et al., The endemic Genista versicolor from Sierra Nevada National Park in Spain is nodulated by putative new Bradyrhizobium species and a novel symbiovar (sierranevadense), Syst. Appl. Microbiol. (2013), http://dx.doi.org/10.1016/j.syapm.2013.09.008

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71 Bradyrhizobium sp. JAN10 (EU882015) Genista tinctoria (Poland) Bradyrhizobium sp. RLA08 (AM168303) 94 Bradyrhizobium japonicum sv genistearum BGA-1 (AY386747) Bradyrhizobium genosp. beta BRE-1 (AY386748) 63 Bradyrhizobium sp. JAN16 (EU882016) Genista tinctoria (Poland) Bradyrhizobium japonicum sv glycinearum LMG 6138T (AM418753) Bradyrhizobium sp. GV144 (KF483541) 99 Bradyrhizobium sp. GV159 (KF483542) Bradyrhizobium iriomotense EK05T (AB300994) Bradyrhizobium sp. GV135 (KF483539) Bradyrhizobium yuanmingense CCBAU1007T (AY386760) Bradyrhizobium sp. GV137 (KF483540) Bradyrhizobium elkanii LMG 6134T (AM418752) Bradyrhizobium pachyrhizi PAC48T (FJ428208) 87 56 Bradyrhizobium lablabi CCBAU 23086T (GU433473) Bradyrhizobium retamae Ro19T (KC247101) Bradyrhizobium jicamae PAC68T (FJ428211) Bradyrhizobium oligotrophicum LMG 10732T (JQ619232) Bradyrhizobium cytisi CTAW11T (GU001613) 77 Bradyrhizobium sp.GV115 (KF483538) Bradyrhizobium canariense MCLA12 (FM253159) 75 90 Bradyrhizobium canariense BC-C2 (AY386736) 85 Bradyrhizobium canariense BES-1 (AY386738) Bradyrhizobium canariense BTA-1T (AY386739) 77 Bradyrhizobium canariense GV101 (KF483535) 76 Bradyrhizobium canariense GV102 (KF483536) 85 Bradyrhizobium canariense GV104 (KF483537) Bradyrhizobium canariense MCLA07 (AM168301) Bradyrhizobium diazoefficiens USDA 110T (NC_004463) Bradyrhizobium daquingense CCBAU 15774T (HQ231289) Bradyrhizobium huanghaihainense CCBAU 23303T (HQ231682) Bradyrhizobium genosp. alpha BC-C1(AY386735) Bradyrhizobium rifense CTAW71T (GU001617) 89 Bradyrhizobium liaoningense LMG18230T (AY386752) Bradyrhizobium betae LMG21987T (FM253129) 78 Bradyrhizobium arachidis CCBAU 051107T (HM107217) Bradyrhizobium sp.ORS285 (FJ347203) Bradyrhizobium sp. ORS 278 (NC_009445) 65 Bradyrhizobium denitrificans LMG 8443T (FM253153) 60 Bradyrhizobium sp. BTAi-1 (NC_009485) 99 0.01 Fig. 4. Maximum likelihood phylogenetic unrooted tree based on partial atpD gene sequences showing the taxonomic affiliation of the strains representative of the different RAPD groups. Bootstrap values calculated for 1000 replications are indicated. Bar, 1 nt substitution per 100 nt. Accession numbers from GenBank are given in brackets.

Strains GV144 and GV159, which showed slightly different ITS fragment sequences, had identical atpD genes which were phylogenetically divergent with identities of lower than 97% with respect to the currently described species of the genus Bradyrhizobium, although they clustered with B. japonicum sv. genistearum BGA1 isolated on the Canary Islands [29] and strains JAN10 and JAN16 isolated from G. tinctoria in Poland [9]. Strain GV135 was also phylogenetically divergent and had identities of lower than 95% with respect to the remaining species of Bradyrhizobium, as well as to the other Genista isolates from Spain and Poland, and it clustered with B. iriomotense EK05T isolated in Japan from tumours of Entada koshunensis [8], as also observed in the ITS analysis. Finally, strain GV137 that belonged to Bradyrhizobium phylogenetic group II was divergent (lower than 93% identity) with respect to the species of this group, including the recently described new species B. retamae that was also isolated from a Genisteae legume [7]. These results indicated that several strains nodulating G. versicolor in Sierra Nevada National Park constituted new lineages within the genus Bradyrhizobium, and GV135 and GV137 at least

may represent two different novel species within this genus. In addition, the atpD gene analysis showed that, in agreement with the results from the ITS fragment, strain BTAi1 belonged to the species B. denitrificans, indicating that the name of this species should be changed.

Analysis of symbiotic genes The nodC gene encoding a chitin synthase is related to the host range of rhizobia and the promiscuity degree of the hosts [11,15,21]. Therefore, currently, this gene is commonly used in Bradyrhizobium to define symbiovars [7,23,29]. The available data showed that the strains isolated from Genista in Northeast Europe belong to the symbiovar genistearum [9], but their sequences were too short and they were not included in our analysis. The nifH gene encodes the dinitrogenase reductase of the nitrogenase complex and, like nodC, usually exists as a single copy in the strains of Bradyrhizobium whose genome has been sequenced recently. In the photosynthetic bradyrhizobia strains, the nifH gene has been found and some of the strains (Btai1 and ORS278) carry two copies of this

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7

53 Bradyrhizobium canariense GV104 (KF483553) Bradyrhizobium sp. GV144 (KF483557) 89 66

Bradyrhizobium sp. GV159 (KF483558) Bradyrhizobium rifense CTAW71T (EU597853) Bradyrhizobium genosp. beta BRE-1 (FJ499319) Bradyrhizobium canariense GV115 (KF483554) sv genistearum

Bradyrhizobium canariense GV101 (KF483551)

59

95 Bradyrhizobium canariense GV102 (KF483552) Bradyrhizobium japonicum sv genistearum BGA-1 (EF694757) 82 100

Bradyrhizobium genosp. alpha BC-C1 (AJ560654) Bradyrhizobium cytisi CTAW11T (EU597844)

Bradyrhizobium sp. GV135 (KF483555) Bradyrhizobium canariense BTA-1T (AJ560653)

76 97

Bradyrhizobium retamae Ro19T (KC247112)

sv retamae

Bradyrhizobium lablabi CCBAU 23086T (GU433565) Bradyrhizobium jicamae PAC68T (AB573869) Bradyrhizobium sp. GV137 (KF483556) 97

sv sierranevadense

Bradyrhizobium elkanii LMG 6134T (AB354631) Bradyrhizobium pachyrhizi PAC48T (HQ588110) Bradyrhizobium arachidis CCBAU 051107T (HM107267)

91

Bradyrhizobium yuanmingense CCBAU 10071T (AB354633) 89

Bradyrhizobium diazoefficiens USDA 110T (NC_004463) 99

Bradyrhizobium japonicum USDA 6T (AB354632)

sv glycinearum

100 Bradyrhizobium huanghuaihaiense CCBAU 23303T (HQ231507) Bradyrhizobium daqingense CCBAU 15774T (HQ231326) Bradyrhizobium iriomotense EK05T (AB301000) Bradyrhizobium sp. ORS285 (AF284858) 0.05 Fig. 5. Maximum likelihood phylogenetic unrooted tree based on nodC gene sequences showing the position of representative strains from different RAPD groups. Bootstrap values calculated for 1000 replications are indicated. Bar, 5 nt substitution per 100 nt. Accession numbers from GenBank are given in brackets.

gene, but the nodC gene has only been shown in strain ORS285 (Figs. 5 and 6). Within Bradyrhizobium, three symbiovars are currently described that nodulate different legumes and they are clearly distinguishable on the basis of nodC gene sequences (Fig. 5). The symbiovar glycinearum was defined by Vinuesa et al. [29] for strains nodulating soybean and, currently, this symbiovar is present in several species nodulating this legume, such as B. japonicum, B. huanghuaihainense, B. daqingense and the recently described species B. diazoefficiens whose type strain is USDA 110T [4]. The same authors defined the symbiovar genistearum for strains nodulating Genisteae, and it is present in the species B. canariense, B. cytisi and B. rifense. Recently, the new symbiovar retamae has been described to distinguish the type strain of the species B. retamae, isolated from Retama monosperma, from the tribe Genisteae [7]. The analysis of the nodC gene showed that all Bradyrhizobium group I strains isolated from G. versicolor belonged to the same phylogenetic group to which strains isolated from other Genisteae corresponding to symbiovar genistearum also belonged (Fig. 5). However, the nodC gene of strain GV137 from Bradyrhizobium group II belonged to a new phylogenetic lineage with less than 84% identity with respect to the remaining strains of genus Bradyrhizobium, according to the results of a search using the BLASTN programme. The nodC gene of strain GV137 showed identities of lower than 85% compared to the remaining symbiovars described in the genus Bradyrhizobium. Since the symbiovars currently described within this genus have internal identities higher

than 92% for the nodC gene, it can be concluded that strain GV137, which was able to nodulate Genista, Adenocarpus and Retama but not Glycine or Vigna, represented a novel symbiovar within Bradyrhizobium. The analysis of the nifH gene showed results congruent to those of the nodC gene, since our strains also clustered into two divergent phylogenetic groups, with one of them formed by the strains from the symbiovar genistearum and a second lineage formed by strain GV137 (Fig. 6). The strains from symbiovar genistearum formed a cluster equivalent to that formed after nodC gene analysis, although GV115 was the most phylogenetically divergent strain with an identity of approximately 94%. Strain GV137 from Bradyrhizobium group II, in agreement with the results of the nodC gene, was phylogenetically divergent from the remaining species of the genus Bradyrhizobium with less than 92% identity. Therefore, the analysis of the symbiotic genes nodC and nifH showed that the strains isolated in this study from G. versicolor belonged to two symbiovars of Bradyrhizobium, one of which corresponded to symbiovar genistearum and another to a putative new symbiovar. Since it has only been found to date in Sierra Nevada National Park, and considering that G. versicolor, the host from which it was first isolated, is an endemism of this Park, we propose the name sierranevadense for this novel symbiovar. The symbiovar is able to nodulate Genisteae species but not Glycine, as occurs with the symbiovars genistearum and retamae [7,29]. In summary, the results of this study showed that G. versicolor was nodulated by B. canariense in southern Spain, as well as by a putative new species of Bradyrhizobium. In addition, it is a

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Bradyrhizobium canariense GV102 (KF483560)

89 96 44 52

63

79

Bradyrhizobium sp. GV144 (KF483565)

Bradyrhizobium canariense GV101 (KF483559) Bradyrhizobium rifense CTAW71T (GU001627)

69 50

Bradyrhizobium canariense GV104 (KF483561)

sv genistearum

Bradyrhizobium sp.GV159 (KF483566) Bradyrhizobium cytisi CTAW11T (GU001618) Bradyrhizobium sp. GV135 (KF483563)

98

Bradyrhizobium canariense BTA-1T (EU818926) Bradyrhizobium canariense GV115 (KF483562)

98 79

Bradyrhizobium sp. ORS285 (FJ347420) Bradyrhizobium sp. ORS278 ( NC 009445)

52

Bradyrhizobium denitrificans LMG 8443T (HM047125)

100 100

Bradyrhizobium sp. BTAi-1 (CP000494)

Bradyrhizobium jicamae PAC68T (EU822944) Bradyrhizobium lablabi CCBAU 23086T (GU433546.)

44

Bradyrhizobium retamae Ro19T (KF670138)

97

Bradyrhizobium sp. GV137 (KF483564) 95

sv retamae

sv sierranevadense

Bradyrhizobium elkanii LMG 6134T (AB094963) Bradyrhizobium pachyrhizi PAC48T (EU822943)

68 98

Bradyrhizobium yuanmingense CCBAU 10071T (EU818927) Bradyrhizobium arachidis CCBAU 051107T (HM107283)

47

Bradyrhizobium daqingense CCBAU 15774T (HQ231323) Bradyrhizobium huanghuaihaiense CCBAU 23303T (HQ231551) 100 Bradyrhizobium liaoningense LMG18230T (EU818925) 67 Bradyrhizobium diazoefficiens USDA

110T

sv glycinearum

(K01620)

Bradyrhizobium japonicum LMG 6138T (HM04712) 0.02 Fig. 6. Maximum likelihood phylogenetic unrooted tree based on nifH gene sequences showing the position of representative strains from different RAPD groups. Bootstrap values calculated for 1000 replications are indicated. Bar, 2 nt substitution per 100 nt. Accession numbers from GenBank are given in brackets.

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