1 Identification at Species and Symbiovar Levels of Strains Nodulating P

1 Identification at species and symbiovar levels of strains nodulating P. vulgaris in saline 2 soils of Marrakech region (Morocco) and analysis of otsA gene putatively involved in 3 osmotolerance 4 5 Faghire M.1,2, Mandri B.1,2, Oufdou K.1, Bargaz A.1,2, Ghoulam C.2, Ramírez-Bahena, 6 M.H.3,4, Velázquez E.4,5, Peix A.*3,4 7 8 1 Laboratory of Biology and Biotechnology of Microorganisms, Faculty of Sciences-Semlalia, 9 Cadi Ayyad University P.O. Box 2390, Marrakesh, MOROCCO. 10 2 Unit of Plant Biotechnology and Symbiosis Agrophysiology, Faculty of Sciences and 11 Techniques, P.O. Box 549, Gueliz, Marrakesh, MOROCCO. 12 3 Instituto de Recursos Naturales y Agrobiología, IRNASA-CSIC, c/Cordel de Merinas 40-52, 13 37008 Salamanca, SPAIN. 14 4 Unidad Asociada Universidad de Salamanca-IRNASA(CSIC) Salamanca, Spain 15 5 Departamento de Microbiología y Genética, Lab. 209. Universidad de Salamanca. Edificio 16 Departamental de Biología, Campus M. Unamuno. Salamanca, SPAIN. 17

18 Abstract 19 Salinity is an increasing problem in Africa affecting rhizobia-legume symbioses. In Morocco, 20 Phaseolus vulgaris is cultivated in saline soils and its symbiosis with rhizobia depends on the 21 presence of osmotolerant strains in these soils. Here we identified 32 osmotolerant rhizobial 22 strains nodulating P. vulgaris at species and symbiovar levels by analysing core and 23 symbiotic genes, respectively. The most abundant strains were closely related to R. etli and R. 24 phaseoli and belong to symbiovar phaseoli. A second group of strains was identified as R. 25 gallicum sv gallicum. The remaining strains, identified as R. tropici, belong to the CIAT 899T 26 nodC group which has not been described as symbiovar yet. In representative strains, the otsA 27 gene involved in the accumulation of trehalose and putatively in osmotolerance was analysed. 28 The results showed that the phylogeny of this gene is not completely congruent with those of 29 other core genes since genus Ensifer is closer related to some Rhizobium species than several 30 species of Rhizobium among them. Although the role of otsA gene in osmotolerance is not 31 well established, it can be an useful protein-coding gene for phylogenetic studies in genus 32 Rhizobium since at species level the phylogenies of otsA and other core genes are coincident. 33 34 Key words: Rhizobia, salt stress, Phaseolus vulgaris, phylogeny, otsA, trehalose 35 36 37 * Corresponding author: 38 Alvaro Peix. Instituto de Recursos Naturales y Agrobiología, IRNASA-CSIC, c/Cordel de 39 Merinas 40-52, 37008 Salamanca, Spain. E-mail: [email protected] 40 41 42 43 44 45 46 47 Genebank Accession numbers of gene sequences: 16S rRNA: JQ085247-JQ085247 for strains 48 RHM14, RHM15, RHM19, RHM47, RHM54 and RHM67 respectively; atpD: JQ085253- 49 JQ085258 for strains RHM14, RHM15, RHM19, RHM47, RHM54 and RHM67 respectively; 50 recA: JQ085274- JQ085279 for strains RHM14, RHM15, RHM19, RHM47, RHM54 and 51 RHM67 respectively; nodC: JQ085259- JQ085264 for strains RHM14, RHM15, RHM19, 2

52 RHM47, RHM54 and RHM67 respectively; otsA: JQ085265- JQ085273 for strains RHM14, 53 RHM15, RHM19, Rhizobium gallicum R602spT, R. loessense CCBAU7190BT, R. lusitanum 54 P1-7T, R. mongolense USDA1844T, R. phaseoli ATCC14482T, R. yanglingense SH22623T. 55

56 Introduction 57 58 Common bean (Phaseolus vulgaris) is one of the most important grain food legumes in the 59 world [56, 62] and Morocco is the main producer of green bean in Africa with 187497 tonnes 60 per year in 2009 [14]. Common bean was brought to Africa via Spain, the Canary Islands or 61 Portugal after the discovery of America in the 16th century. American rhizobial strains 62 nodulating this legume were distributed in other continents harbored in bean seeds since at 63 least R. etli has been found in bean testa [34]. Other American species, R. tropici and R. 64 leucaenae (formerly R. tropici type A), nodulating P. vulgaris could have also been 65 transported by bean seeds to Europe and Africa. In Africa, strains identified as R. etli, R. 66 tropici and R. gallicum have been found in common bean nodules [4, 5, 10, 26, 31, 35], some 67 of them isolated in Morocco from soils affected by salinity. High concentrations of sodium 68 chloride in soils is an increasing problem which significantly limits the crop production in 69 arid and semi-arid areas [61], particularly when nitrogen nutrition is provided by symbiotic 70 nitrogen fixation [11, 18]. In P. vulgaris symbiosis, it has been reported that a best symbiotic

71 N2 fixation under salinity conditions could be achieved if both symbiotic partners, as well as 72 the different steps of their interaction are all tolerant to this stress [8]. Several rhizobial strains 73 have developed active adaptative mechanisms to survive in hyperosmolarity conditions 74 accumulating intracellular compatible solutes such as glutamate, trehalose or related 75 compounds [6, 9, 12, 51]. Therefore the analysis of strains nodulating common bean in saline 76 soils is crucial since the survival or success of the symbiosis can depend on the inoculation 77 with salt-tolerant rhizobial strains showing high symbiotic nitrogen fixation efficiency [7]. 78 This can be especially important in saline soils under arid climate such as Marrakech region 79 soils, since aridity is an additional constraint affecting crop yield. Moreover, up to date no 80 report is available on the genetic characterization of indigenous rhizobial strains of P. vulgaris 81 and their phylogenetic relationships in Marrakech region. 82 Therefore, the first objective of this work was to identify at species and symbiovar (sv) levels 83 the osmotolerant rhizobial strains nodulating common bean growing under arid climate in 84 saline soils in the Marrakech-Tensift-Al Haouz region in Morocco, by analysing the core 85 genes rrs, atpD, recA, and the symbiotic gene nodC. The second objective was to analyse the 86 otsA gene encoding a trehalose-6-phosphate synthase involved in the accumulation of 87 trehalose carried by our strains in order to compare it with the otsA gene from three 88 osmotolerant strains of rhizobia isolated in Tunisia (Africa) from P. vulgaris nodules. 89

90 Materials and methods 91 92 Isolation of rhizobia 93 Rhizobial strains used in this study were obtained from P. vulgaris plants growning in soils of 94 Marrakech-Tensift-Al Haouz region. The strains were isolated from large pink-colour nodules 95 of field-grown bean plants on YMA plates according to Vincent [58]. The isolated strains of 96 rhizobia were verified for nodulation through re-infectivity tests according to Ramírez- 97 Bahena et al. [37]. Noninoculated nitrogen-free and nitrogen-supplemented plants were used 98 as controls. Five replicates for each treatment were used; plants were grown in a greenhouse 99 and were harvested 6 weeks after planting to determine the number and the size of nodules. 100 101 Tolerance to NaCl tests 102 The tolerance to salinity of free-living rhizobia was determined on YEM agar Petri dishes 103 containing different NaCl concentrations (from 0 to 6.6% NaCl w/v). 104 105 RAPD fingerprinting 106 RAPD patterns were obtained as previously described [41] using the primer M13 (5’- 107 GAGGGTGGCGGTTCT –3’) and the GoTaq Flexi DNA polymerase (Promega). PCR 108 conditions were: preheating at 95 oC for 9 min; 35 cycles of denaturing at 95 oC for 1 min; 109 annealing at 45 oC for 1 min and extension at 75 oC for 2 min, and a final extension at 72 oC 110 for 7 min. 10 µl of each PCR products were electrophoresed on 1.5% agarose gel in TBE 111 buffer (100 mM Tris, 83 mM boric acid, 1 mM EDTA, pH 8.5) at 6 V/cm, stained in a 112 solution containing 0.5 g/ml ethidium bromide, and photographed under UV light. Standard 113 VI (Roche, USA) was used as a size marker. A dendrogram was constructed based on the 114 matrix generated using UPGMA method and the Pearson coefficient with Bionumerics 115 version 4.0 software (Applied Maths, Austin, TX) 116 117 3.2. Sequence analyses of rrs, recA, atpD, nodC and otsA genes 118 The rrs gene was amplified and sequenced as previously described [39], recA and atpD genes 119 according to Gaunt et al. [16], nodC gene according to Laguerre et al. [22] and otsA gene 120 according to Fernández-Aunión et al. [12]. PCR amplifications were performed with a 121 REDExtract-N-Amp PCR Kit (Sigma) following the manufacturer’s instructions. The band 122 corresponding to the different genes was purified either directly from the gel by room 123 temperature centrifugation using a DNA gel extraction device (Millipore Co., USA) for 10

124 min at 5000 g or by elution of the excised band and filtration through silicagel columns using 125 the Qiaquick DNA Gel Extraction Kit (Qiagen, Germany) in all cases following the 126 manufacturers’ instructions. The sequence reaction was performed on an ABI PRISM 3100 127 sequencer using a BigDye terminator v3.1 cycle sequencing kit (Applied Biosystems Inc., 128 USA) as supplied by the manufacturer. The sequences obtained (1475nt for rrs, 520nt for 129 recA, 500nt for atpD, 906nt for nodC and 840nt for otsA) were compared to those held in 130 GenBank by using the BLASTN program [2]. They were aligned by using Clustal W software 131 [53]. Distances calculated according to Kimura's two-parameter model [20] were used to infer 132 phylogenetic trees with the neighbor-joining method [46] and maximum likelihood with 133 MEGA5 software [52]. Confidence values for nodes in the trees were generated by bootstrap 134 analysis using 1000 permutations of the data sets. 135 136 Results and discussion 137 138 Isolation of strains and RAPD fingerprinting 139 A collection of 70 rhizobial strains was isolated from nodules of common bean from saline 140 soils in the Marrakech region. In agreement with the results of other authors that commonly 141 isolated Rhizobium radiobacter in P. vulgaris nodules [45], 38 of our strains were classified 142 in this species by rrs gene sequencing (data not shown). Since they were unable to reinfect P. 143 vulgaris in the nodulation tests, they were not further studied nor included in this study. The 144 remaining 32 strains were able to reinfect P. vulgaris forming typical and effective nodules. 145 The genetic diversity of these strains was assessed by RAPD fingerprinting, a strain 146 dependent technique suitable in analysing the intraspecific diversity of rhizobial populations 147 [19, 30, 32, 42, 54]. The numerical analysis of the patterns obtained (Figure 1) showed that 148 they belong to eight groups according to the dendrogram obtained, with a similarity 149 percentage lower than 70% (Figure 1 and Table 1) allowing us the selection of representative 150 strains for gene sequencing. 151 152 Identification at species level 153 P. vulgaris may be nodulated by different species of genera Rhizobium and Ensifer and thus 154 the rrs gene must be analysed in order to classify bean isolates since this gene is the current 155 basis of rhizobial classification at genus level [16]. Strains from RAPD groups 1, 2 and 8 have 156 identical rrs gene sequences and they were represented by strains RHM54 and RHM14 157 (belonging to the most dissimilar groups) whose sequences were 100% identical to that of R.

158 tropici CIAT 899T (Figures 2 and S1). Strains RHM15 and RHM47 representative of RAPD 159 group 7 have rrs gene sequences identical to that of the type strain of R. gallicum R602spT 160 and that of strain PhD12, both isolated in Europe from P. vulgaris. American strains of R. 161 gallicum isolated from a Milpa in Mexico (represented by strain IE4770) were related with 162 strains RHM15 and RHM47 but their rrs gene sequences were not identical. Finally, strains 163 from RAPD groups 3, 4, 5 and 6 have identical rrs gene sequences and they were represented 164 by strains RHM19 and RHM67 (belonging to the most dissimilar groups) in Figure 2. These 165 strains clustered with several species of genus Rhizobium, R. phaseoli, R. etli, R. pisi and R. 166 fabae. However they presented differences in the rrs gene sequence with respect to all these 167 species being R. etli CFN42T the most closely related with only two nucleotide differences at 168 the positions 170 and 1078 taking as reference the sequence of strain CFN42T whose genome 169 has been completely sequenced [17]. The rrs gene sequence of strain RHM19 was very 170 closely related to those of other strains previously isolated in Morocco, such as RP305 and 171 RP218 [31] and in Ethiopia, such as strain AK-3 [5]. Interestingly these sequences were also 172 related with those of several strains classified in the species R. etli (represented by strain 173 IE4804) that were isolated in a milpa of San Miguel in Mexico (Mesoamerica), a 174 domestication center of common bean [48, 49]. 175 The results of concatenated recA and atpD gene analyses (Figures 3 and S2) are completely 176 congruent with those of rrs gene analysis. The strains RHM15 and RHM47 were 177 unambiguously identified as R. gallicum since they carry recA and atpD genes with almost 178 100% identity with respect to those of the type strain of this species and with respect to those 179 of strain PhD12 isolated from P. vulgaris nodules in France [3]. They were also 180 phylogenetically related to several strains classified as R. gallicum (IE4868, IE2703, IE992 181 and IE4770) that were isolated in Mexico (Mesoamerica), a domestication centre of common 182 bean as mentioned above, for which the American origin of this species was hyphotesized 183 [48, 49]. However since the American isolates have housekeeping genes different to those of 184 European and African strains, they could possibly have different origin. This would support 185 the second hypothesis of Silva et al. [49] that suggests a possible worldwide distribution of R. 186 gallicum that would have acquired different symbiotic plasmids along the evolution in 187 different ecosystems. In this case, the Moroccan strains could have the same origin that 188 European strains rather than American ones. 189 The strains RHM14 and RHM54 were unambiguously identified as R. tropici with almost 190 100% identity with respect to the type strain of this species CIAT 899T. These genes were 191 also close to those of other American strain Br859 isolated in Brazil. R. tropici was isolated in

192 America from nodules of Leucaena leucocephala and it is also able to nodulate Phaseolus 193 [25]. Initially this species included two groups, A and B, but recently the strains from group A 194 have been reclassified into a new species named R. leucaenae [38]. It is interesting to 195 highlight that curiously the strains isolated in Morocco belong to R. tropici [31], whereas 196 those found in Europe belong to R. leucaenae [21, 55]. This can be due to a better adaptation 197 of each one of these species to different environmental conditions including the local varieties 198 of common bean in each site. 199 In contrast with the phylogenies obtained for the other groups, the strains RHM19 and 200 RHM67 have recA and atpD genes divergent to those of R. etli in spite of the closeness of rrs 201 genes. They were more closely related to R. phaseoli, a species distinguishable from the other 202 species of genus Rhizobium according to the housekeeping gene analysis [37]. The recA and 203 atpD genes of RHM19 and RHM67 were nearly identical (more than 99% similarity) with 204 respect to several strains isolated in the Mexican milpa already mentioned [49] and 100% 205 identical to those of a strain isolated in USA (strain KIM5s, see Figures 3 and S2). Although 206 these strains were named R. etli, the analysis of recA and atpD genes suggested that they do 207 not belong to this species [49]. Despite the fact that our strains are closer to R. phaseoli than 208 to R. etli on the basis of housekeeping gene analysis, the differences found in the rrs gene do 209 not allow including these strains in R. phaseoli. The genetic interchange among species could 210 explain the phylogenetic position of the strains represented by RHM19 and RHM67, 211 nevertheless before establishing conclusions a taxonomic revision of the complete group R. 212 phaseoli-R. etli would be desirable. 213 214 Identification at symbiovar level 215 Whereas the identification at species level is based on core gene analysis, symbiovars 216 identification is based on nodulation genes. Although nodA [33, 57] and nodB [36, 48, 49] 217 genes have been used with this purpose, currently nodC gene is commonly used to define 218 symbiovars [15, 19, 23, 29, 40, 59]. The analysis of symbiovars is in rhizobia as important as 219 that of species, since rhizobial host range, legume promiscuity, coevolution and biogeography 220 studies should be based on symbiovars rather than on species. For example, P. vulgaris is not 221 considered a promiscuous legume because it can be nodulated by several chromosomal 222 species of rhizobia, but rather because it nodulates with many different symbiovars (which 223 harbour different nodC genes). In this way, common bean is commonly nodulated by 224 symbiovars phaseoli, gallicum, giardinii, mediterranense [44] and by strains from other 225 groups that belong to undefined symbiovars [55]. Among these symbiovars, only sv phaseoli

226 seems to be exclusive for common bean nodulation [3, 55] and therefore the symbiovar 227 phaseoli plasmid should have evolved with native common bean plants in America [1, 24]. 228 Moreover, given that R. etli is the major endosymbiont of common bean in the American 229 domestication centres of this legume [1, 49] the combination R. etli sv phaseoli is with high 230 probability of American origin. Also, since R. phaseoli was considered to be R. etli for many 231 years, the combination R. phaseoli sv phaseoli is also likely indigenous of American 232 continent. Since the Moroccan strains represented by RHM19 and RHM67 and by the 233 previously isolated strains PR218 and PR305 [31] belong to the symbiovar phaseoli (Figures 234 4 and S3) and to the chromosomal group R. etli-R. phaseoli (see Figures 2 and 3), they 235 possibly have an American origin in agreement with Diouf et al. [10] that proposed that bean 236 rhizobia had been introduced in Africa from the Americas together with the seeds. 237 The group of strains identified as R. gallicum, represented by RHM15 and RHM47, belong to 238 the symbiovar gallicum and have nodC genes identical to that of strain RP421 previously 239 isolated in Morocco from P. vulgaris nodules [31] and to that of the type strain of R. gallicum 240 R602spT isolated in France [3] (Figure 4). However the strain PhD12, whose housekeeping 241 genes are identical to those of our strains, carries a nodC typical of symbiovar phaseoli. Since 242 the symbiovar phaseoli was not found in the R. gallicum strains isolated in Mesoamerica [49] 243 and considering that this symbiovar evolved with R. etli, it is possible that the transfer of 244 nodulation genes typical of this symbiovar to R. gallicum strains have occurred in European 245 soils. In any case the presence of symbiovar gallicum in different continents could indicate a 246 worldwide distribution of R. gallicum that would have acquired different symbiotic plasmids 247 along the evolution in different ecosystems as suggested Silva et al. [49]. 248 As for the group of strains identified as R. tropici, RHM14 and RHM54, they were identical 249 to the Moroccan strain RP261 [31] and to several American strains from R. tropici and from 250 R. leucaenae (Figure 4). They were also identical to nodC of R. lusitanum type strain, a 251 Portuguese species nodulating common bean that probably acquired its nodC gene from R. 252 leucaenae, since this last species has been found in common bean nodules in Portugal and 253 carries identical nodC gene [55]. Therefore the American origin of the R. tropici strains 254 nodulating P. vulgaris in Morocco seems to be clear in agreement with the suggestions of 255 Diouf et al. [10]. 256 257 Osmotolerance and analysis of otsA gene 258 The soils in which we isolated the strains from this study are affected by salinity as well as 259 arid climate, with variable values of electric conductivity (EC) ranging from 2.5-9

260 mmhos/cm. For this reason we analyzed the strains osmotolerance, which is reported in Table 261 1. This characteristic was variable, but all the strains were able to growth in presence of NaCl 262 concentrations ranging from 1.5% to 3%. These values were similar to those previously 263 reported for strains isolated in other region of Morocco [8] where the climate is sub-humid 264 instead of arid, and lower than those reported for rhizobial strains isolated from P. vulgaris in 265 Egypt, which are tolerant up to 4% NaCl [47]. In the three species detected in this study we 266 found Moroccan strains able to grow in higher NaCl concentration than the corresponding 267 type strain. R. tropici CIAT899 was the most osmotolerant type strain (2.5%), whereas R. 268 gallicum R602sp grew up to 1% NaCl. The most significant case was R. etli, R. 269 leguminosarum and R. phaseoli whose type strains only grew in presence of 0.5% NaCl, 270 whereas the Moroccan strains phylogenetically related with these species (strains from RAPD 271 groups 3, 4, 5 and 6) were able to grow in higher concentrations, over 2% NaCl in all cases, 272 which demonstrate that this adaptative trait is independent of phylogenies and very variable 273 within a single species. In our case this implies an adaptation of American strains arriving 274 with the bean seeds to the edapho-climatic conditions in saline Moroccan soils characterized 275 by high temperatures, low relative humidity, high rate of evaporation and little rainfall. 276 Hence, it will be very interesting to carry out more studies to find out what has been the 277 adaptive modification that renders bacteria tolerant to salt. The salt stress can produce a 278 failure in the establishment of infection and nodulation in the rhizosphere [60, 61], but 279 osmotolerant rhizobia can stand large modifications in the osmolarity without decrease in the 280 number of viable cells [50]. When bacteria are subjected to severe osmotic stress the 281 accumulation of trehalose was observed [28] and this disaccharide play a major role in the 282 adaptation of rhizobia to osmotic stress conditions [27]. The genes encoding the trehalose-6- 283 phosphate synthase are involved in the accumulation of trehalose process and the otsA gene 284 has been sequenced in some osmotolerant strains of rhizobia isolated in Tunisia from P. 285 vulgaris nodules [12]. However only six species of genus Rhizobium were analysed in this 286 previous work and moreover all of them belong to a close phylogenetic group within this 287 genus. In the present work we have sequenced this gene in the strains isolated in Marrakech 288 (Morocco) as well as in the type strains of six rhizobial species closely related with African 289 isolates (Morocco and Tunisia). Moreover in the phylogenetic analysis of otsA gene we also 290 included the strains whose complete genomes are currently available except Agrobacterium 291 tumefaciens (currently R. radiobacter) C58 in whose genome the otsA gene is not present 292 (Figures 5 and S4). In all the remaining strains the otsA gene is present, but it has different 293 locations, harboured in the chromosome in Rhizobium strains whereas in Ensifer it is located

294 in a symbiotic plasmid. In the case of strain E. meliloti AK83 the otsA gene is located in 295 chromosome 3, which really correspond to a symbiotic plasmid since it contains nod genes. 296 These differences in the location of genes essential for environmental adaptation have been 297 previously found as occurs in the case of celC gene which is also located in Ensifer plasmids 298 but in Rhizobium chromosomes. However in the case of celC gene, that encodes a cellulase, 299 its phylogeny is completely congruent with that of other core genes since the celC genes of 300 Ensifer strains are divergent from those of genus Rhizobium sensu stricto [43]. In the case of 301 the otsA gene, the phylogeny is not completely congruent with those of other core genes, 302 given that species from genus Ensifer are more closely related to the group R. gallicum-R. 303 mongolense than to other species of Rhizobium. Nevertheless, at species level the phylogeny 304 of otsA gene is congruent with those of core genes since the strains of concrete species 305 clustered with the corresponding type strains. For example, RHM15 has otsA gene identical to 306 that of the type strain R. gallicum R602spT and to that of the osmotolerant strain 8a3 isolated 307 in Tunisia by Fernández-Aunión et al. [12]. Very closely related to this group are located the 308 species R. mongolense, R. yanglingense and R. loessense in agreement with the results of 309 other core genes supporting the hypothesis that they belong to the same species [13, 43, 49]. 310 The otsA gene of RHM14 was nearly identical to that of the type strain of R. tropici CIAT 311 899T in agreement with the results obtained after the analysis of rrs, recA and atpD genes. 312 Finally, RHM19 was closely related to the strain 12a3 isolated in Tunisia by Fernández- 313 Aunión et al. [12]. These two strains are more closely related to R. phaseoli than to R. etli in 314 agreement with the results of the housekeeping gene analysis. It is remarkable that the strain 315 CIAT 652, currently included in R. etli, was closer related to R. phaseoli ATCC 14482T than 316 to R. etli CFN42T and as was previously pointed out this strain probably belong to R. phaseoli 317 [43]. Both species R. etli and R. phaseoli were clearly separated between them and from the 318 cluster formed by R. leguminosarum that includes non-osmotolerant strains such as the type 319 strain USDA 2370T and osmotolerant ones such as 31c3 isolated in Tunisia by Fernández- 320 Aunión et al. [12]. 321 Therefore the phylogenetic analysis of otsA gene showed that it is closely related in strains 322 from the same species with independence of their osmotolerance. For example the type strains 323 of R. phaseoli and R. etli are not osmotolerant and have otsA genes nearly identical to those of 324 osmotolerant strains from the same species. Therefore the role of otsA gene in osmotolerance 325 is not clear and it cannot be used as a marker to identify osmotolerant strains although it can 326 be a good marker to be included as a protein-coding gene useful for phylogenetic studies in 327 genus Rhizobium.

328 329 Acknowledgments 330 This research was funded by the AECID Spanish-Moroccan projects n°A/018163/08 and 331 A/025374/09 as well as the IFS project F/2826-3F. MF is supported by an AECID fellowship 332 from Spanish Ministry of External Affairs and Cooperation (MAEC). MHRB is recipient of a 333 JAE-Doc researcher contract from CSIC 334 335 References 336 337 [1] Aguilar, O.M., Riva, O., and Peltzer, E. (2004) Analysis of Rhizobium etli and of its 338 symbiosis with wild Phaseolus vulgaris supports coevolution in centers of host 339 diversification. Proc. Natl. Acad. Sci. U S A. 101, 13548-13553. 340 341 [2] Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J. (1990) Basic local 342 alignment search tool. J. Mol. Biol. 215, 403–410. 343 344 [3] Amarger, N., Macheret, V., Laguerre, G. (1997) Rhizobium gallicum sp. nov. and 345 Rhizobium giardinii sp. nov., from Phaseolus vulgaris nodules. Int. J. Syst. Bacteriol. 47, 346 996-1006. 347 348 [4] Anyango, B., Wilson, K.J., Beynon, J.L., Giller, K.E. (1995) Diversity of rhizobia 349 nodulating Phaseolus vulgaris L. in two Kenyan soils with contrasting pHs. Appl. Environ. 350 Microbiol. 61, 4016–4021. 351 352 [5] Beyene, D., Kassa, S., Ampy, F., Asseffa, A., Gebremedhin, T., van Berkum, P. 2004. 353 Ethiopian soils harbor natural populations of rhizobia that form symbioses with common bean 354 ( Phaseolus vulgaris L.). Arch. Microbiol. 181, 129-136. 355 356 [6] Botsford, J. L. , Lewis, T. A. (1990). Osmoregulation in Rhizobium meliloti: production 357 of glutamic acid in response to osmotic stress. Appl. Environ. Microbiol. 56, 488-494. 358 359 [7] Bouhmouch, I., Brhada, F., Filali-Maltouf A., Aurag J. (2001) Selection of osmotolerant 360 and effective strains of Rhizobiaceae for inoculation of common bean (Phaseolus vulgaris) in 361 Moroccan saline soils. Agronomie 21, 591–599.

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589 Figure legends 590 591 Figure 1. Dendrogram obtained for the strains isolated in this study using Pearson’s 592 coefficient and UPGMA analysis of the RAPD patterns. 593 594 Figure 2. Neighbour-joining phylogenetic rooted tree based on rrs gene sequences (1475 nt) 595 showing the taxonomic location of representative strains from different RAPD groups. 596 Bootstrap values calculated for 1000 replications are indicated. Bar, 1 nt substitution per 100 597 nt. Accession numbers from Genbank are given in brackets. 598 599 Figure 3. Neighbour-joining phylogenetic tree based on concatenated recA and atpD genes 600 sequences (520 and 500 nt) showing the position of representative strains from different 601 RAPD groups. Bootstrap values calculated for 1000 replications are indicated. Bar, 2 nt 602 substitution per 100 nt. Accession numbers from Genbank are given in brackets.

603

604 Figure 4. Neighbour-joining phylogenetic tree based on nodC gene sequences (906 nt) 605 showing the position of representative strains from different RAPD groups. Bootstrap values 606 calculated for 1000 replications are indicated. Bar, 5 nt substitution per 100 nt. Accession 607 numbers from Genbank are given in brackets.

608

609 Figure 5. Neighbour-joining phylogenetic tree based on otsA gene sequences (840 nt) 610 showing the position of representative strains from different RAPD groups. Bootstrap values 611 calculated for 1000 replications are indicated. Bar, 5 nt substitution per 100 nt. Accession 612 numbers from Genbank are given in brackets.

613

614 615

Table 1

Table 1. Summary of the results obtained in the analysis of strains from this study

Strains RAPD group Salinity Species Symbiovar tolerance (%) RHM46, RHM50 1 1.5 R. tropici unnamed* RHM54 1 2 R. tropici unnamed* RHM60 2 2 R. tropici unnamed* RHM66 2 3 R. tropici unnamed* RHM1, RHM19, RHM21, RHM25 3 2.5 R. phaseoli /R. etli phaseoli R19, R24 4 2 R. phaseoli /R. etli phaseoli R23 4 2.5 R. phaseoli /R. etli phaseoli R26 4 3 R. phaseoli /R. etli phaseoli RHM69, RHM72 4 2.5 R. phaseoli /R. etli phaseoli RHM70, RHM74 4 2 R. phaseoli /R. etli phaseoli RHM36, RHM37, RHM39, RHM40 5 2 R. phaseoli /R. etli phaseoli RHM41 5 2.5 R. phaseoli /R. etli phaseoli RHM67 6 2.5 R. phaseoli /R. etli phaseoli R15, RHM15 7 3 R. gallicum gallicum RHM47, RHM56 7 1.5 R. gallicum gallicum RHM62, RHM64 7 2.5 R. gallicum gallicum RHM3, RHM5, RHM14 8 2 R. tropici unnamed* Type strains: CFN42T 0.5 R. etli phaseoli ATCC 14482T 0.5 R. phaseoli phaseoli R602spT 1 R. gallicum gallicum CIAT 899T 2.5 R. tropici unnamed* CFN 299T 1 R. leucaenae unnamed* USDA 2370T 0.5 R. leguminosarum viciae * These strains belong to the nodC cluster formed by R. tropici CIAT 899T. Figure 1 Click here to download high resolution image Figure 2 language corrected 67 Rhizobium etli sv. phaseoli IE4804 (AY998046) 63 Rhizobium etli CFN42T (NC 007761) Rhizobium etli RP305 (DQ406700) RHM19 78 RHM67

77 Rhizobium etli RP218 (DQ406696) Rhizobium sp.Ak3-1 (AY210705) Rhizobium etli RP330 (DQ406703) 96 71 Rhizobium phaseoli ATCC14482T (EF141340) Rhizobium sp.Dz8-2 (AY210714)

T 62 Rhizobium fabae CCBAU33202 (DQ835306) 73 Rhizobium etli sv. mimosae Mim-2 (DQ648574) 66 Rhizobium etli CIAT652 (NC 010994) 67 Rhizobium pisi DSM30132T (AY509899) Rhizobium leguminosarum RP422 (DQ406712) Rhizobium leguminosarum 3841 (NC 008380) 99 98 Rhizobium leguminosarum sv. trifolii WSM1325 (NC 012850) 47 Rhizobium leguminosarum sv. viciae USDA2370T (U29386) 69 Rhizobium leguminosarum sv. trifolii WSM2304 (NC 011369) Rhizobium sp. Db4-14 (AY210708 ) Rhizobium gallicum sv. gallicum IE4770 (AY998045)

54 Rhizobium loessense CCBAU7190BT (AF364069) Rhizobium mongolense USDA1844T (U89817) 99 Rhizobium yanglingense SH22623T (AF003375)

99 92 Rhizobium gallicum RP421 (DQ406711) Rhizobium gallicum PhD12 (HQ735084) 72 RHM15 68 Rhizobium gallicum R602spT (U86343) RHM47

61 Rhizobium rhizogenes K84 (CP000628) 92 Rhizobium rhizogenes ATCC11325T (AY945955) 52 Rhizobium lusitanum P1-7T (AY738130) Rhizobium leucaenae CFN299T (L21837) 99 Rhizobium tropici RP261 (DQ406713) RHM54 99 RHM14 73 Rhizobium tropici CIAT899T (U89832) 94 Rhizobium tropici Br859 (HQ394213) Ensifer sp. NGR234 (CP001389)

84 Ensifer medicae WSM419 (NC 009636) 99 Ensifer medicae A321T (L39882) Ensifer meliloti AK83 (CP002781) 98 Ensifer meliloti SM11 (CP001830) 90 Ensifer meliloti BL225C (CP002740) Ensifer meliloti LMG 6133T (X67222) Rhizobium vitis NCPPB3554T (D14502) 99 Rhizobium vitis S4 (CP000633)

0.005 Figure 3 language corrected

70 RHM15 39 RHM47 98 Rhizobium gallicum PhD12 (HQ394250, AY929482) 97 Rhizobium gallicum R602spT (AY907357, AY907371) 7 Rhizobium yanglingense SH22623T (AY907359, AY907373) 87 Rhizobium gallicum bv. gallicum IE4770 (AY907452, AY907434) T 99 Rhizobium mongolense USDA1844 (AY907358, AY907372) 85 Rhizobium loessense LMG21975T (HQ735076, HM142761) 28 Rhizobium gallicum bv. gallicum IE4868 (AY907449, AY907427) Rhizobium lusitanum P1-7T (DQ431674, DQ431671) Rhizobium leucaenae USDA9039T (AJ294372, AJ294396) 99 99 Rhizobium radiobacter K84 (CP000628) 90 Rhizobium rhizogenes NCPPB2991T (AJ294374, AJ294398) T 97 61 Rhizobium tropici USDA9030 (AJ294373, AJ294397) RHM54 99 RHM14 Rhizobium etli CFN42T (NC 007761)

91 Rhizobium leguminosarum bv. trifolii WSM1325 (NC 012850) 99 Rhizobium leguminosarum 3841 (NC 008380) 99 T 94 Rhizobium leguminosarum bv. viciae USDA2370 (AJ294376, AJ294405) Rhizobium leguminosarum WSM2304 (NC 011369)

75 Rhizobium etli CIAT652 (NC 010994) 98 99 Rhizobium etli bv. mimosae Mim-2 (HQ394249, AY929507) Rhizobium phaseoli ATCC14482T (EF113136, EF113151)

95 Rhizobium etli bv. phaseoli IE1009 (AY907458, AY907438) 86 Rhizobium etli bv. phaseoli IE4804 (AY907457, AY907437)

99 Rhizobium etli bv. phaseoli KIM5s (AY907360, AY907374)

94 RHM19 65 RHM67 Ensifer sp. NGR234 (CP001389, NC 012587)

99 Ensifer medicae WSM419 (NC 009636, CP000738) 92 Ensifer medicae A321T (AJ294381, AJ294401) T 99 Ensifer meliloti USDA1002 (AJ294382, AJ294400) Ensifer meliloti AK83 (CP002781) 99 Ensifer meliloti SM11 (CP001830) 22 Ensifer meliloti BL225C (CP002740) Ensifer meliloti 1021 (NC 003047) Rhizobium vitis NCPPB2562T (AB303680, AM418788) 99 Rhizobium vitis S4 (CP000633)

0.02 Figure 4 language corrected

Rhizobium leguminosarum sv. phaseoli H132 (AF217263) 99 Rhizobium etli CIAT652 (NC 010996) Rhizobium giardinii sv. phaseoli H251 (AF217264) Rhizobium etli sv. phaseoli RP218 (DQ413007) 99 Rhizobium gallicum sv. phaseoli PhD12 (AF217265) 94 RHM67 99 94 RHM19 Rhizobium etli sv. phaseoli CFN42T (NC 004041) 99 Rhizobium phaseoli sv. phaseoli ATCC14482T (HM441255) 99 Rhizobium etli sv. phaseoli RP305 (DQ413010) Ensifer fredii sv. mediterranense 16b1(AF481764) Ensifer meliloti sv. mediterranense GVPV12 (FJ462796) 99 98 99 Ensifer fredii sv. mediterranense GR-06 (AF217269)

T 99 Rhizobium giardinii sv. giardinii H152 (AF217267) Rhizobium etli sv. mimosae Mim2 (EU386150) Rhizobium gallicum sv. gallicum R602spT (AF217266) 99 74 RHM15 99 RHM47 Rhizobium gallicum sv. gallicum RP421 (DQ413005) Rhizobium leucaenae CFN299T (N98514) Rhizobium lusitanum P1-7T (HM852098)

86 T 99 Rhizobium tropici CIAT899 (HQ824719) RHM54 68 RHM14 Rhizobium tropici P3-8 (HM852105) Rhizobium tropici RP261 (DQ413013) Ensifer sp. NGR234 (U00090)

99 Ensifer meliloti sv. meliloti 1021 (M11268) Ensifer meliloti AK83 (CP002783)

99 Ensifer medicae WSM419 (CP000740) 99 Ensifer medicae USDA1037T (EF209422)

59 Ensifer meliloti BL225C (CP002741)

99 Ensifer meliloti SM11 (CP001831) 62 Ensifer meliloti sv. meliloti ATCC9930T (EF209423)

99 Rhizobium leguminosarum sv. trifolii ATCC14480 (FJ895269) 99 Rhizobium leguminosarum sv. trifolii WSM1325 (CP001623) Rhizobium leguminosarum sv. trifolii WSM2304 (NC 011368) 99 Rhizobium leguminosarum sv. viciae USDA2370T (FJ596038)

99 Rhizobium leguminosarum sv. viciae RP422 (DQ413006) 64 Rhizobium leguminosarum sv. viciae 3841 (NC 008381)

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44 Rhizobium gallicum 8a3 (FN433086) 99 Rhizobium gallicum R602spT 97 RHM15

100 Rhizobium yanglingense SH22623T Rhizobium loessense CCBAU7190BT 100 75 Rhizobium mongolense USDA1844T Ensifer medicae WSM419 (CP000740) Ensifer sp. NGR234 (U00090) 100 Ensifer meliloti 1021 (NC 003047) 27 59 Ensifer meliloti BL225C (CP002741) 100 88 Ensifer meliloti SM11 (CP001831) 49 72 Ensifer meliloti AK83 (NC 015591) Rhizobium lusitanum P1-7T Rhizobium rhizogenes K84 (CP000628) 99 RHM14 100 Rhizobium tropici CIAT899T (FN433087)

80 Rhizobium leguminosarum bv. trifolii NZP561 (EF444931) 94 Rhizobium leguminosarum bv. viciae 3841 (NC 008381) 100 Rhizobium leguminosarum bv. trifolii WSM1325 (CP001622) 34 Rhizobium leguminosarum bv. phaseoli 31c3 (FN433085) Rhizobium etli CFN42T (CP000133)

100 Rhizobium leguminosarum bv. trifolii WSM2304 (CP001191)

100 RHM19 54 Rhizobium etli 12a3 (FN433084)

84 Rhizobium etli CIAT652 (CP001074)

89 Rhizobium etli CNPAF512 (AEYZ01000267) 98 Rhizobium phaseoli ATCC14482T Rhizobium vitis S4 (CP000633)

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