Phylogeny and genetic diversity of native rhizobia nodulating common bean (Phaseolus vulgaris L.) in Ethiopia

Systematic and Applied Microbiology 35 (2012) 120–131

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Phylogeny and genetic diversity of native rhizobia nodulating common bean (Phaseolus vulgaris L.) in Ethiopia Aregu Amsalu Aserse a,b,∗ , Leena A. Räsänen a , Fassil Assefa b , Asfaw Hailemariam c , Kristina Lindström a a b c

University of Helsinki, Department of Food and Environmental Sciences, POB 56, FIN-00014 Helsinki, Finland Addis Ababa University, Cellular, Microbial and Molecular Biology Program Unit, POB 1176, Addis Ababa, Ethiopia Ministry of Agriculture and Rural Development, National Soil Testing Centre, POB 147, Addis Ababa, Ethiopia

a r t i c l e

i n f o

Article history: Received 2 September 2011 Received in revised form 22 November 2011 Accepted 24 November 2011 Keywords: Phaseolus vulgaris Rhizobium Diversity Phylogeny AFLP MLSA

a b s t r a c t The diversity and phylogeny of 32 rhizobial strains isolated from nodules of common bean plants grown on 30 sites in Ethiopia were examined using AFLP fingerprinting and MLSA. Based on cluster analysis of AFLP fingerprints, test strains were grouped into six genomic clusters and six single positions. In a tree built from concatenated sequences of recA, glnII, rpoB and partial 16S rRNA genes, the strains were distributed into seven monophyletic groups. The strains in the groups B, D, E, G1 and G2 could be classified as Rhizobium phaseoli, R. etli, R. giardinii, Agrobacterium tumefaciens complex and A. radiobacter, respectively, whereas the strains in group C appeared to represent a novel species. R. phaseoli, R. etli, and the novel group were the major bean nodulating rhizobia in Ethiopia. The strains in group A were linked to R. leguminosarum species lineages but not resolved. Based on recA, rpoB and 16S rRNA genes sequences analysis, a single test strain was assigned as R. leucaenae. In the nodC tree the strains belonging to the major nodulating groups were clustered into two closely linked clades. They also had almost identical nifH gene sequences. The phylogenies of nodC and nifH genes of the strains belonging to R. leguminosarum, R. phaseoli, R. etli and the putative new species (collectively called R. leguminosarum species complex) were not consistent with the housekeeping genes, suggesting symbiotic genes have a common origin which is different from the core genome of the species and indicative of horizontal gene transfer among these rhizobia. © 2011 Elsevier GmbH. All rights reserved.

Introduction Phaseolus vulgaris L. (common bean) is the third most important legume crop growing worldwide, superseded only by Glycine max (soybean) and Arachis hypogaea (peanut). It is one of the most widely distributed food legumes in many parts of the tropics, subtropics and temperate regions [62]. In eastern and southern Africa, Phaseolus is a major staple food legume, nourishing more than 100 million people. Its production covers over four million hectares annually in more than 20 countries of the region [79]. In this region, common bean is considered to be the second and the third principal source of dietary protein and calories, respectively [11]. The common bean has two major centers of genetic diversity, from which the Mesoamerican center, including Mexico, Colombia, Ecuador

Abbreviations: AFLP, amplified fragment length polymorphism fingerprints; MLSA, multilocus sequence analysis; BT, bootstrap support; NJ, neighbour-joining; ANI, average nucleotide identity; sv., symbiovar. ∗ Corresponding author at: University of Helsinki, Department of Food and Environmental Sciences, POB 56, FIN-00014 Helsinki, Finland. Tel.: +358 191 59278. E-mail address: aregu.aserse@helsinki.fi (A.A. Aserse). 0723-2020/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.syapm.2011.11.005

and northern Peru is probably the primary one. The Andean center which encompasses regions from Southern Peru to northern Argentina is the second one [20,28]. Common bean establishes symbiotic associations with a wide range of root-nodule nitrogen-fixing bacteria called rhizobia. Initially, based on the cross-inoculation-group concept, all beannodulating rhizobia were classified as Rhizobium leguminosarum symbiovar (sv.) phaseoli [27]. The symbiovar is the new name of the term biovar proposed by [57]. Latter, due to the advancement of new molecular biological techniques and the isolation of different strains from various areas of the world, different common bean nodulating rhizobia were described. R. tropici [41] was described as a new bean-nodulating species based on multilocus enzyme electrophoresis (MLEE), DNA–DNA hybridization and a sequence analysis of 16S rDNA. Likewise, Segovia at al. [60] proposed R. etli as a separate species based on its difference in 16S rRNA gene compared with R. leguminosarum. Subsequently, R. giardinii and R. gallicum were reported as common bean symbionts [2]. Recently, salt tolerant Sinorhizobium (syn. Ensifer) meliloti sv. mediterranense was isolated from bean growing regions in a Tunisian oasis [44]. Sequence analyses of 16S rRNA gene and DNA–DNA hybridization have been used as regular protocols in bacterial taxonomic

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studies. Nevertheless, the use of 16S rRNA gene solely as a phylogenetic marker has proven difficult because of its presence in multiple copies in a genome of some bacteria [25], susceptibility to genetic recombination and horizontal gene transfer [69] and low divergence among closely related species [1,21,72]. The DNA–DNA hybridization technique has better resolving power and strains should show 70% or greater DNA–DNA relatedness to be classified in the same species [64]. However, this technique is labour intensive and time consuming. Moreover, DNA–DNA hybridization techniques have been reported to vary between different laboratories [58] and this leads again to conflicting result for the same sets of strains. For example, S. xinjiangensis and S. fredii were reported to have 39% relatedness by Peng et al. [46] but later 74–89% similarity was reported by Martens et al. [36]. To overcome these mentioned drawbacks, protein-coding genes have been proposed as alternative phylogenetic markers to discriminate closely related species [64]. These genes have a faster evolution rate than the 16S rRNA gene but are conserved enough to retain genetic information. Multilocus sequence analysis (MLSA), using the sequences of multiple housekeeping genes have been reported to have higher discriminative power than 16S rRNA gene sequence analysis and DNA–DNA hybridization for species identification and delineation within the genus Ensifer (Sinorhizobium) [36]. Several studies revealed that MLSA can also be successfully used for phylogenetic studies of other rhizobia at species level [42,55]. Apart from its reliability, MLSA is also a rapid and economical method for species identification [6]. In Ethiopia, common bean is widely cultivated as a sole crop or intercropped with mainly sorghum and maize at altitudes between 1400 and 2000 m above sea level. The Hararghe highlands in east and the Rift valley zone that covers the southeastern parts of Ethiopia are the major common bean growing areas in the country [5], where it is also widely consumed. Bean export market has increased rapidly, supplying beans to canning industries in European countries and to the neighboring Kenya [17]. The yield of common bean, however, is extremely low due to low soil fertility, mainly due to nitrogen deficiency [7] and smallholder farming systems with minimal to zero fertilizer inputs [78]. The fertility problem can partly be solved by improving the capacity of common bean to fix nitrogen using the application of effective rhizobial inoculants. The presence of native bean-nodulating rhizobia in the country has been reported [8,9]. Nevertheless, detailed information about their taxonomy and diversity in Ethiopia is very scant. Therefore, in the present study, the MLSA technique was used to investigate the taxonomic position of native rhizobia nodulating common bean in Ethiopia. For this purpose we determined the phylogeny of housekeeping genes coding for recombination protein (recA), glutamine synthase II (glnII), RNA polymerase beta subunit (rpoB) and the 16S rRNA gene. The diversity of the strains was assessed using the Amplified Fragment Length Polymorphism (AFLP) fingerprinting technique. The phylogeny of symbiotic genes for nodulation (nodC) and nitrogen fixation (nifH) as well as the capacity of strains to induce effective nodules was also studied for the test strains. Materials and methods Isolation and sampling sites Nodule samples were collected randomly from common bean plants (local varieties of P. vulgaris) grown in 28 sites in the southern (14), eastern (9), central (3) and western (2) regions of Ethiopia. The nodules were collected in August and September 2007 when the common bean was at a flowering stage. In addition, a few isolates were obtained from the nodules induced by rhizobia present in soil samples taken from two sites in Addis Ababa (central Ethiopia)

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on common bean variety RED WOLAYTA trap plants in the greenhouse. The cultivation and planting protocol was as described by Wolde-meskel et al. [77]. The seeds were surface sterilized with 95% ethanol for 3 min followed by 0.2% acidified mercuric chloride for 3 min. After washing several times with sterile distilled water, seeds were sown in pots containing the collected soils and plants were grown for 4 weeks. Soils taken from the sampling sites were analyzed for pH and electric conductivity following the methods described before [3,38]. The pH of the soils varied from moderately acidic (pH 6.0) to moderately alkaline (pH 8.8), but were commonly around neutral (Table S1). The districts, geographic coordinates, electric conductivity of the soils, and mean annual rainfall and temperature of the sampling sites are summarized in Table S1. Several nodules per plant per site were taken and kept in a vial containing the desiccant silica gel until isolation [63]. Strains were isolated from the nodules according to the standard routine laboratory techniques described by Vincent [70]. Nodules were surface-sterilized with 95% ethanol for 3 min followed by 0.2% acidified mercuric chloride for 3 min and washing several times with sterile distilled water. Sterilized nodules from each vial were crushed together in a drop of sterile distilled water. The nodule suspension was streaked onto yeast extract mannitol (YEM) and/or tryptophan yeast extract (TY) agar media. Bacterial colonies appeared after incubation at 30 ◦ C for 3–5 days. For each sample, a single representative colony was taken among morphologically similar colonies and was further streaked several times on YEM agar medium to check the purity of the cultures. When necessary, to separate mixed cultures, bacterial cultures grown until late log phase in YEM or TY broth were diluted in surfactant Tween 80 buffer (0.05 M phosphate, pH 6.6, 0.1% Tween 80). The first dilution was incubated at 30 ◦ C for half an hour in a shaker-incubator and separate strains were recovered from dilutions 10−4 and/or 10−5 after growing on YEM agar medium containing 0.025 g kg−1 Congo red [34]. Pure cultures of the strains were maintained and preserved in 20% glycerol–YEM broth at −80 ◦ C. Nodulation tests All isolated strains were tested for their ability to induce nitrogen-fixing nodules on the common bean host, variety RED WOLAYTA at National Soil Testing Center, Addis Ababa, Ethiopia. The seeds were surface sterilized as described above. Sterilized seeds were wrapped in sterilized Whatman paper in a Petri-plate and germinated at 28 ◦ C for 2–3 days. The nodulation experiment was done in triplicate pouches. Three seedlings were transferred into each pouch and each seedling was inoculated with 1 ml of bacterial cultures grown in YEM broths to exponential phase. Noninoculated treatments were used as negative controls. A positive control was fertilized with 0.05% KNO3 w/v solution. The seedlings were grown in a glasshouse with a 12 h day and 12 h night regime. Plants were watered alternatively with sterilized Jensen’s nitrogen free medium and distilled sterilized water according to the procedure described by Vincent [70]. After six weeks, plants were uprooted and the color of the leaves, nodulation status of roots and appearance of the plants were checked. Nodulation capacity was recorded as positive when nodules were found and negative if not found. Green plants and pink nodules indicated effective nitrogen fixation. Ineffective nitrogen fixation was considered when plants looked yellowish and had white nodules. DNA isolation Total genomic DNA of the strains was extracted from bacterial cultures grown in YEM or TY until late log phase. Extraction of the

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DNA was carried out following the phenol–chloroform procedure as modified by Li et al. [32]. AFLP fingerprinting Basically the AFLP protocol was as described by Vos et al. [73] with a modification made by Li et al. [32]. AFLP data were examined using the ABI GeneScan analysis software (PE Biosystems) and transformed to the Bionumerics software, version 5.0 (Applied Maths, Kortrijk, Belgium) for further analysis. Peaks equivalent to sizes between 35 and 500 bp were considered for comparison of the AFLP patterns of the strains. Pairwise similarities of the AFLPfingerprints were calculated using the Dice coefficient of the matrix with 1.0% and 0.5% position and optimization tolerance, respectively. A dendrogram was constructed using the Unweighted Pair Group Method using Arithmetic Means (UPGMA). PCR amplification and gene sequencing The genes encoding 16S rRNA, recA, glnII, rpoB, nodC and nifH were amplified and sequenced. The primers for amplification were found in literatures [19,32,36,56,59,68,71,72,75] and are listed in Table S2 together with PCR cycling conditions used. Amplification of the housekeeping and symbiotic genes was carried out following the instructions given by the manufacturer (Finnzymes) in 50 ␮l master mix containing 200–500 ng DNA, 1× Phusion HF buffer, 25 pmol each of the primers, 1000 pmol dNTPs, and 1 U Phusion DNA polymerase. The quality of the PCR product was checked and the size determined by gel electrophoresis. pGEM DNA marker was used to estimate the length of the amplicons. PCR products were sequenced using the normal Sanger method by the Institute of Biotechnology, University of Helsinki. Primer fD1 was used for sequencing the partial 16S rRNA genes whereas all the other genes were sequenced using the respective pair of primers that were used for amplification. Sequence analysis The quality of the sequences was checked and edited using Gap4 as implemented in the Staden-package 1.7.0 [65] and BioEdit 7.0.0 programs [23]. For preliminary identification, 16S rRNA gene sequences of the test strains were compared to the Genbank database by using the nucleotide blast program. For further study recA, glnII, rpoB, nodC and nifH gene sequences of the reference species that related to our strains were retrieved from EMBL database (http://www.ebi.ac.uk/Tools/sss/fasta/nucleotide.html) as well. Accession numbers of all sequences of newly isolated strains deposited in the EMBL database and the reference strains are listed in Table S3. Sequences of the type strains Bradyrhizobium japonicum DSMZ 30131, B. elkanii USDA76 and B. betae LMG 21987 were used as outgroups for each gene analysis. Sequences of each gene were aligned using ClustalW as implemented in Mega5 [66] and manually corrected when necessary. The index of substitution saturation (Iss and Issc ) for the third codon position for each housekeeping gene sequence was estimated by the test of Xia et al. [81] using the DAMBE program [80]. After trimming to the same length, core gene sequences including those of 16S rRNA were concatenated. For each housekeeping and concatenated sequence, both neighbor joining (NJ) and maximum likelihood (ML) phylogenetic trees were constructed using MEGA version 5. The NJ nodC and nifH trees were also constructed using the same method. The NJ analyses were performed using the Kimura’s 2-parameter distance correction model [29]. For the ML analyses, the best fit models estimated by the Akaike information criteria, using MEGA version 5 were as follows: recA, T92+G (Tamura 3-parameter plus Gamma rate distribution); glnII and rpoB, TN93+G (Tamura-Nei

and Gamma rate distribution); concatenated, GTR+G+I (General Time Reversible with invariant site and a Gamma rate distribution). For ML, statistical support of the trees was calculated by bootstrap analyses using 100 replications and for NJ, 1000 replications. The percentage similarity of the genes was estimated using the Kimura-2 distance matrix correction model as implemented in MEGA version 5. The incongruence length difference (ILD) tests using PAUP4 were used to check if the core gene markers had congruent signals among each other. Results Nodulation Totally 32 rhizobial strains were isolated from 30 sites located mainly in southern, eastern and central Ethiopia (Table 1 and S1). Four of the strains (HBR24, HBR21, HBR23 and HBR22) were obtained from soil samples taken from Addis Ababa using trap host plants in the greenhouse. Most of the isolates (27/32) could induce many pink nodules on the common bean variety RED WOLAYTA host plant. The plants inoculated with these strains appeared to be healthy and leaves looked deep green. There was no clear difference, in terms of height and overall appearance of the plants between the treatments inoculated with the 27 rhizobial strains. Three strains (HBR45, HBR75 and HBR78) induced only few, small and white nodules. No nodules were found on plants inoculated with strains HBR33 and HBR52. Those plants inoculated with ineffective and non-nodulating strains appeared yellowish and were not different from the negative control. Genetic diversity and identification All 32 rhizobial strains produced AFLP fingerprints containing 10–25 bands. Cluster analysis of the AFLP fingerprints revealed six genomic groups and six strains remained unclustered at the similarity level of 50% (Fig. 1). This cut-off value was chosen arbitrarily to define clusters of similar AFLP patterns but it has also been used commonly for other bacteria [26] and rhizobia [18,32,76]. Based on the similarity of partial 16S rRNA gene sequences (length 688–903 bp), strains in each of the AFLP clusters represented known rhizobial species (Table 1). AFLP cluster 1 with nine members, was similar to R. phaseoli ATCC 14482T (99%). Cluster 2 consisted of five strains that were related to R. etli CFN42T (99–100%). Cluster 3 with two strains was also related to R. etli CFN42T with a 99% similarity value. Strains in cluster 4 were closely related to R. leguminosarum strain USDA 2370T (99–100% similarity). AFLP cluster 5 consisted of five strains, of which four represented R. etli CFN42T (99–100%), whereas one strain (HBR45) had 100% 16S rRNA gene sequence similarity to A. radiobacter LMG 140T . All the three strains in cluster 6 were related to Agrobacterium tumefaciens C58 with 100% similarity. Six strains with a single position each in AFLP grouping were similar to known species as follows: HBR79, HBR24 and HBR23, HBR21, HBR12 and HBR78 were related to R. phaseoli ATCC 1482T (99%), R. etli CFN42T (99%), R. giardinii H152T (100%), R. leucaenae CFN 299 T (99%) and A. radiobacter LMG 140T (100%), respectively (Fig. 1 and Table 1). In our analysis, no clear relationship was detected between the majority of AFLP clustering and geographic origin of the strains. Except cluster 3 that contained only two strains isolated from Sodo (southern Ethiopia) district, all the other clusters included stains isolated from different regions (Fig. 1 and Table S1). In general, all strains in the same clusters produced distinct AFLP fingerprints, except HBR1 and HBR18 in cluster 1, which were isolated from different regions but having similar fingerprints. However, strains in the same clusters (except HBR45 in cluster 5) showed identical or nearly identical partial 16S rRNA gene sequence similarities with

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Table 1 List of rhizobial strains isolated from root nodules of Phaseolus vulgaris (common bean) in Ethiopia and their AFLP groupings and phylogenetic positions. Strain

AFLP cluster

Geographic origin

HBR9 HBR11 HBR17 HBR10 HBR20 HBR53 HBR1 HBR13 HBR18 HBR26 HBR51 HBR50 HBR7 HBR31 HBR14 HBR19 HBR22

1 1 1 1 1 1 1 1 1 2 2 2 2 2 3 3 4

Abeshegie – Alawla (SE) Harar (EE) Awassa – Tugaweransa (SE) Arbaminch – Ambokersha (SE) Awassa – Leku (SE) Elubabur – Dembi (WE) Arbaminch – Genetamecha (SE) Harar (EE) Wolayta Sodo – Adello (SE) Nazereth – Kurfana Soloke (CE) Assosa – Amba 13 (WE) Abeshegie Jejeba (SE) Konso – Gata (SE) Mieaso (EE) Sodo – Shaya (SE) Sodo – Awelgama sitalo (SE) Addis Ababa (CE)

HBR42

4

Chiro – Efabas (EE)

HBR2 HBR4 HBR5 HBR3 HBR45 HBR33 HBR52 HBR75 HBR23 HBR79 HBR78 HBR21 HBR12 HBR24

5 5 5 5 5 6 6 6 S S S S S S

Konso – Goho (SE) Ziway – Kemogerbi (CE) Konso – Mechela (SE) Wolayta sodo – Selamber (SE) Tulo – Terkam Feta (EE) Mieaso – Gulfa (EE) Zeway – Adamitulu (CE) Tulo – Efabas (EE) Addis Ababa (CE) Deder – Welta Geba (EE) Deder – Kobo (EE) Addis Ababa (CE) Sodo – Wachigoesho (SE) Addis Ababa (CE)

Identity based on the partial 16S rRNA gene sequence

Phylogenetic assignments and Groupsa

Nod

Fix

Closest species (accession number)

Identity (%)

Length (bp)b

R. phaseoli ATCC 14482T (EF141340) R. phaseoli ATCC 14482T (EF141340) R. phaseoli ATCC 14482T (EF141340) R. phaseoli ATCC 14482T (EF141340) R. phaseoli ATCC 14482T (EF141340) R. phaseoli ATCC 14482T (EF141340) R. phaseoli ATCC 14482T (EF141340) R. phaseoli ATCC 14482T (EF141340) R. phaseoli ATCC 14482T (EF141340) R. etli CFN42T (EU488751) R. etli CFN42T (EU488751) R. etli CFN42T (EU488751) R. etli CFN42T (EU488751) R. etli CFN42T (EU488751) R. etli CFN42T (EU488751) R. etli CFN42T (EU488751) R. leguminosarum USDA 2370T (AM181757) R. leguminosarum USDA 2370T (AM181757) R. etli CFN42T (EU488751) R. etli CFN42T (EU488751) R. etli CFN42T (EU488751) R. etli CFN42T (EU488751) A. radiobacter LMG140T (AM181758) A. tumefaciens C58 (AJ012209) A. tumefaciens C58 (AJ012209) A. tumefaciens C58 (AJ012209) R. etli CFN42T (EU488751) R. phaseoli ATCC 14482T (EF141340) A. radiobacter LMG140T (AM181758) R. giardinii H152T (AM181755) R. leucaenae CFN 299T (EU488741) R. etli CFN 42T (EU488751)

99 99 99 99 99 99 99 99 99 99 99 100 100 100 99 99 99

779 897 835 803 842 736 826 718 826 903 705 736 688 702 822 900 787

R. phaseoli (B) R. phaseoli (B) R. phaseoli (B) R. phaseoli (B) R. phaseoli (B) R. phaseoli (B) R. phaseoli (B) R. phaseoli (B) R. phaseoli (B) Rhizobium sp. (C) R. etli (D) Rhizobium sp. (C) Rhizobium sp. (C) Rhizobium sp. (C) R. etli (D) R. etli (D) Rhizobium sp. (A)

+ + + + + + + + + + + + + + + + +

+ + + + + + + + + + + + + + + + +

100

731

Rhizobium sp. (A)

+

+

100 100 99 100 100 100 100 100 100 99 100 100 99 99

740 789 739 780 786 803 889 815 822 721 770 771 840 772

R. etli (D) R. etli (D) R. etli (D) Rhizobium sp. (C) A. radiobacter (G2) A. tumefaciens complex (G1) A. tumefaciens complex (G1) A. tumefaciens complex (G1) Rhizobium sp. (C) Rhizobium sp. A. radiobacter (G2) R. giardinii (E) R. leucaenae R. etli (D)

+ + + + + − − + + + + + + +

+ + + + − − − − + + − + + +

SE, South Ethiopia; WE, West Ethiopia; EE, East Ethiopia and CE, Central Ethiopia; S, separate position in the AFLP dendrogram. R, Rhizobium; A, Agrobacterium. Nod, Nodulation; +, nodules found; −, no nodules found. Fix, Nitrogen fixation deduced from the color of leaves of the plant. −, light green to yellow; +, deep green. a Phylogenetic assignments and groups are based on the concatenated tree (Fig. 2). b The length of 16s rRNA gene sequences of the test strains used for identification of them using Genbank database (NCBI) blast program.

their respective related type strains (Table 1). The taxonomic positions of the strains were further investigated with recA, glnII and rpoB gene sequences analysis. Sequence analysis of individual housekeeping genes Sequences for the recA (506–577 bp), glnII (619–881 bp) and rpoB (846–925 bp) genes were obtained for all the test strains except for HBR12, for which we could not amplify the glnII fragment. In addition, the rpoB and glnII fragments for a number of reference type strains were sequenced for which such sequences were not available in the Genbank database (Table S3). Totally, 204 new sequences, including partial 16S rRNA gene sequences of the 32 test strains were deposited into Genbank database (Table S3). Sequences of several housekeeping genes retrieved from the Genbank were shorter than our newly sequenced ones. As result, in the final alignments parts of the new sequences were omitted. Consequently, the lengths of the alignments used were 384 bp, 508 bp and 509 bp for recA, glnII, and rpoB, respectively. As shown in Table 2 the housekeeping genes had more informative positions than the 16S rDNA sequences. In our analysis rpoB was the second longest gene sequence used and had the greatest number (212) of parsimony-informative characters, which was about 41.5% of the positions used for the analysis. The shortest gene but the second

most informative was recA with 140 bp (36.5%) informative positions. The third information rich gene was glnII with 109 (about 30.7%) parsimony informative sites (Table 2). All three codon positions were included in the analysis of all the individual housekeeping genes since there was no significant saturation shown at the third codon positions (Iss < Issc ; p < 0.001 data not shown). The NJ and ML trees constructed for each housekeeping gene gave similar groupings. However, the bootstrap support (BT) in each group of the ML trees was lower than the equivalent in the NJ. The NJ trees for recA, glnII and rpoB gene alignments are depicted in supplementary Fig. 1A–C. Although variation in topology was observed between the three NJ gene trees, most of our strains were similarly clustered into seven distinct groups (A, B, C, D, E, F and G) in every housekeeping gene trees. Concatenated sequence analysis Aligned sequences from the recA (384 bp), glnII (508 bp), rpoB (509 bp) and 16S rRNA (688 bp) genes were concatenated and 2089 bp positions were obtained (Table 2). A number of references strains and also strain HBR12 were excluded from the combined analysis since no sequences were obtained for their rpoB or glnII genes (Table S3). ILD tests between the gene partitions showed no congruence (p < 0.01; data not shown) between any combinations of the four genes. However, the phylogenetic tree made from the

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Table 2 Gene-locus alignment statistics calculated using Mega version 5. Gene locus

Number of sequences

Number of alignment sites

Number of conserved sites

Number of variable sites

Number and percentage of parsimony informative sites

Number of singletons

a 16S rRNA gene recA glnII rpoB recA + glnII + rpoB + 16S rDNA nodC nifH

61/15U 72/48U 71/50U 61/45U 60/48U 49/19U 53

688 384 508 509 2089 368 412

587 227 324 270 1412 187 230

101 157 184 239 677 181 182

95/14 140/36.5 156/30.7 212/41.5 603/28.8 154/42.8 152/36.9

6 17 28 27 74 27 30

U, Unique taxes resulted from the alignments using DAMBE program for each gene locus from the total sequences used in this analysis. The number of sequences for test strains, references and three out-group strains included in this analysis are indicated in supplementary Table S3. a Sequences alignment used only for concatenated analysis.

concatenated alignment was supported by high bootstrap values and the conflicting signals existing in individual housekeeping gene trees were leveled out in the combined one. Again the topology of NJ and ML phylogenetic trees were very similar like the case in the individual gene trees. The percentage of average nucleotide identity (ANI) calculated from concatenated sequences was plotted in Fig. 2 within and between the highly supported groups (A–E, G1, G2). The pair wise concatenated sequence similarity matrix between test and reference strains is presented in Table S4. Phylogenetic groups Generally, the concatenated genes analysis gave consistent grouping with individual housekeeping genes, but in the concatenated tree (Fig. 2) the groups were more robust than in the single-gene trees (Fig. 1A–C). In group A two test strains clustered with R. leguminosarum WSM2304 (BT 95%). Pair wise nucleotide identities within this group varied from 96% to 99% (ANI 97.1%). Similar to the concatenated tree, R. leguminosarum WSM2304 was clustered distantly with the two test strains in recA and rpoB trees as well. However, in the glnII tree, R. leguminosarum WSM2304 was placed in a separate position far from the test strains. In the concatenated tree, group A was linked to a lineage that contained two sub-clusters of reference strains; one of the sub-clusters was composed of R. leguminosarum species and R. indigoferae CCBAU 71042T . The second sub-cluster was occupied by Rhizobium sp. CCBAU 83309 isolated from multiple legumes, Rhizobium sp. CCBAU 83476 and Rhizobium sp. CCBAU 83479 isolated from G. max in China (Table S3). In the concatenated tree, group B was a cluster of nine test strains, R. etli CIAT652 and R. phaseoli ATCC 14482T (BT 77%; ANI about 98%). All the test strains had identical average nucleotide sequence similarity. R. etli CIAT652 had also 100% sequence similarity with the nine test strains in all core genes analysis except with its 16S rRNA gene sequence. A strain HBR79 was linked to the outskirt of group B. In the recA and glnII trees, HBR79 was tightly clustered with R. etli CCBAU 83475 isolated from G. max and R. etli sv. phaseoli strains (KIM5s, IE4874, IE1009, IE2755) isolated from P. vulgaris in Mexico and these clusters were linked to the group B in both tree as well. Nevertheless, in the rpoB tree HBR79 was aberrantly clustered with the three Rhizobium sp. isolated from G. max in China (Fig. S1C and Table S3). Group C was a cluster of six test strains (BT 96%; ANI 99.6%) without including any reference species in both concatenated as well as in the individual gene trees. The group D (BT 96%; ANI 98.1%) was a sister monophyletic cluster of group C with bootstrap support 96% in the concatenated tree. This group contained seven test strains and R. etli CFN42T in all trees, but one of the test strains, HBR51 had separate position in the glnII tree.

A single test strain HBR21 formed a separate group E together with type strain R. giardinii H152 with 100% BT and 99.5% ANI support in tree built with the concatenated sequences. This group was well separated from all other genomic species in the concatenated tree, but its position was varied in the individual gene trees. Based on the recA and rpoB trees, another single test strain HBR12 was found to form the group F with R. leucaenae CFN 299T in between other closely related type strains (Figs. S1A and B). In addition, R. leucaenae strains isolated from P. vulgaris in Brazil were clustered with the HBR12 in the recA tree. Among these, reference strain H52 had the highest recA similarity to the HBR12 (97.3%). Group G was a cluster of five test strains and Agrobacterium reference species and these were clearly classified into sub-group G1 (ANI 96.4%) and sub-group G2 (ANI 99%) with bootstrap support value 100% in the combined tree. The latter includes two test strains, A. radiobacter LMG140T and A. radiobacter NCPPB 2437 in all trees. A. tumefaciens C58 was clustered in sub-group G1 together with three test strains in the concatenated and glnII trees but it was placed in separate position in both recA and rpoB trees under the group G. Phylogeny of symbiotic genes The nucleotide sequences of 412 bp nifH and 540–641 bp nodC genes were obtained for all test strains that belonged to the genus Rhizobium in the concatenated tree. In addition, the nifH gene was sequenced for type species R. indigoferae CCBAU 71042T , R. miluonense CCBAU 41251T , R. fabae CCBAU 33202T , R. pisi DSM30132T and R. phaseoli ATCC 14482T (Table S3). However, except the nifH for HBR78, nodC and nifH amplification was unsuccessful for test strains that were related to Agrobacterium in group G of the concatenated tree. The nifH gene amplification also failed for R. giardinii sv. giardinii H152T and R. leucaenae CFN299T . Basic sequence alignment statistics for both genes is shown in Table 2. The nodC and nifH NJ phylogenetic trees are displayed in Figs. 3 and 4, respectively. In the nodC tree, most of Ethiopian native strains were placed into two well-supported closely related monophyletic clades I and II (BT 99%). Pairwise nodC gene sequence similarities among strains in clade I varied from 99.2% to 100 (ANI 93%). The similarities within clade II ranged from 99.5 to 100 (ANI 99.6%) and the ANI value between the two clades was about 98%. A tree made from amino acid sequences deduced from nodC DNA sequences (not shown) gave also very similar topology as that of the nodC gene tree shown in Fig. 3. Average amino acid sequence similarities were 99, 99.5 and 97.2% for clade I, clade II and between them, respectively. Interestingly most strains belonging to group D and all of those belonging to group C in the concatenated tree were clustered together in clade I of the nodC tree. This clade also contained strains HBR22 and HBR79 that were clustered in the group A and close to the group B, respectively in the concatenated tree.

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R. etli CFN 42T , R. phaseoli ATCC 14482T and R. etli CIAT 652 and most of these strains had identical nifH sequences (Fig. 4). R. hainanense CCBAU 57015T isolated from Desmodium sinuatum in China was distantly linked to this clade. The nodC and nifH phylogeny of strain HBR12 was somehow consistent with the recA and rpoB genes tree. In the nodC tree, this strain was grouped with R. lusitanum P1-7T , R. tropici CIAT 899T and R. leucaenae CFN299T (BT 100%) and shared 100% sequence similarity with each other. In the nifH gene tree, strain HBR12 was tightly clustered (BT 100%) with R. tropici CIAT899T , R. lusitanum P1-7T , R. multihospitium CCBAU 83401T and R. miluonense CCBAU 41251T . Nevertheless, in the nodC tree R. miluonense CCBAU 41251T was located on the outskirts (BT 99%) of the lineage that comprised clades I and II. R. multihospitium CCBAU 83401T was also placed distantly from HBR12 with R. fabae CCBAU 33202T , R. leguminosarum USDA 2370T and R. pisi DSM 30132T . Discussion

Fig. 1. The genetic diversity of common bean rhizobia isolated from different regions of Ethiopia revealed by AFLP fingerprinting analysis. The dendrogram was constructed by the UPGMA method using the Bionumerics program. AFLP groups were defined at the 50% similarity levels and clusters are indicated by numbers after the curved lines. Vertical dashed line indicates cluster cutoff value at which the level of similarity for the groups was delineated. The cophenetic correlation value is shown at each branch.

The clade II contained the majority of the test strains that belonged to group B in the concatenated tree. This clade also included strains HBR10, HBR14 and HBR17 that had identical sequences with each other and with unclassified Rhizobium sp. CCBAU 65118 isolated from Vigna unguiculata in China (Table S3). The nodC phylogeny of strain HBR21 was consistent with that of the concatenated tree. In the nodC tree it also clustered closely with R. giardinii sv. giardinii H152T (BT100%; ANI 99.7%) but showed 100% sequence identity with strain Rhizobium sp. RPA02 obtained from nodules of Prosopis alba in Sudan. In the nifH phylogenetic tree all test strains except HBR12 were tightly clustered in one clade along with reference strains, such as

In this study 32 rhizobial strains isolated from root nodules of common bean in Ethiopia were classified taxonomically. AFLP fingerprint analysis showed that each isolate was genetically unique, with only one exception, as two isolates from separate locations had identical fingerprints. In cluster analysis the strains were grouped into six AFLP clusters and six separate positions. Identification to species level was done by using partial 16S gene sequences, but this method could not discriminate all putative species. MLSA with recA, glnII, rpoB and partial 16S rRNA genes was however powerful and by including appropriate reference strains and species; the isolates could be reliably positioned into eight taxonomic classes within the genus Rhizobium–Agrobacterium. A few exceptional test and reference strains were clustered differently depending on the housekeeping gene considered. These aberrant placements might be due to the different evolutionary history of a particular gene or lateral gene transfer [12]. In general, from this result we understood that a single housekeeping gene cannot be taken directly for predicting bacterial phylogeny. Thus, we combined sequences of recA, glnII, rpoB and partial 16S rRNA gene. ILD tests showed no congruence (p < 0.01) between any combinations of the four core genes used. The same situation was also reported from various housekeeping gene sequences analysis of Bradyrhizobium [45]. According to Dowton and Austin [16] this test is unreliable measure of congruence when the alignments differ markedly in the size of the sequences. In our case, the sequence alignments varied from 384 (recA) to 688 bp (16S rRNA) (Table 2). However, our phylogenetic tree made from the concatenated alignments was supported by high bootstrap values and robust. This indicates that the aberrant clustering observed in individual gene trees was resolved in the concatenated tree. Therefore, this tree is the most probable species tree. According to Konstantinidis et al. [30] the classical cut off 70% DNA–DNA relatedness for species delineation corresponds to 96% ANI value estimated from whole-genome analysis of conserved core genes of several groups of micro-organisms. The authors suggested also that sequence similarity values calculated from concatenation of a randomly selected six to eight genes would be likely to give a precise estimation of whole-genome relatedness, even when the genes used are among the worst-performing ones. Moreover, they demonstrated that concatenation analysis of as few as three best performing genes with full length (at least 3300 positions) can be enough to predict the whole-genome based phylogeny than using six to eight genes. In our study, based on the concatenated sequence analysis of four core genes, the newly isolated strains were clustered into seven monophyletic groups (Fig. 2) and

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HBR22 A HBR42 ANI: 97.1% R. leguminosarum WSM 2304 100 Rhizobium sp. CCBAU 83479 53 100 Rhizobium sp. CCBAU 83476 ANI:95.5 % Rhizobium sp. CCBAU 83309 74 R. leguminosarum 3841 95 86 R. leguminosarum WSM 1325 R. leguminosarum USDA 2370T 100 T 100 R. indigoferae CCBAU 71042 62 R. leguminosarum ATCC 14480 R. fabae CCBAU 33202T ANI:98.5 % R. pisi DSM 30132T 100 100 T 100 R. phaseoli ATCC 14482 B 100 HBR53, HBR1, HBR9,HBR10,HBR11,HBR13,HBR17,HBR18,HBR20 ANI: 77 R. etli CIAT 652 98.0% 95

60 96

91

96

Between C&D C ANI: 99.6% ANI: 96.2%

HBR23

70

R. etli CFN 42T HBR51

HBR5 99 HBR24 88 D HBR4, HBR14 100 ANI: 98.2% 50 HBR19 90 HBR2 100 R. rhizogenes LMG 150T 97 R. rhizogenes K84 R. lusitanum P1-7T R. leucaenae CFN 299 100 R. multihospitium CCBAU 83401T 81 R. tropici CIAT 899T 98 R. miluonense CCBAU 41251T 83 R. hainanense CCBAU 57015T 55 HBR21

98

100

T 100 R. giardinii H152

HBR33, HBR52 HBR75

E

ANI: 99.5%

G

G1

ANI: 96.4% A. tumefaciens C58 T HBR45, A. radiobacter LMG140

100 100 96 100

A N I:95. 1%

HBR79

92 HBR7,HBR50 68 HBR31 HBR26 100 99 HBR3

100

HBR78

A. radiobacter NCPPB 2437 B. elkanii USDA 76T B. betae LMG 21987T B. japonicum DSMZ 30131T

G2 ANI: 99.0%

Between subgroups ANI: 94.1%

0.02 Fig. 2. Phylogenetic tree based on four concatenated genes (recA, glnII, rpoB and partial 16S rRNA) sequences, showing the relationships among Phaseolus vulgaris symbionts and recognized species of the genus Rhizobium–Agrobacterium. The tree was constructed by the neighbor-joining method using MEGA version 5. Bootstrap values (using 1000 replicates) are indicated at the branching points. Bar, % estimated substitutions. Reference strains are highlighted with bold and type strains with superscript T. The average nucleotide similarity (ANI) within and between groups are indicated after the lines that delineate the groups.

average sequence identity (ANI) ranged from 96.4 to 99.6% in each group (intraspecies level). Most Ethiopian bean nodulating rhizobia were clustered in four of the groups (A, B, C and D) and ANI within the clade that encompasses all of these four groups was 95.1%. However, ANI within each group in this clade was much higher than 95.1%. Therefore, the gaps in sequence heterogeneity values within a group (intraspecies) and between the groups (interspecies) were high and sufficient to create species boundaries (Fig. 2). In most cases, the ANI value within a group was higher than the cut off value (96%) proposed by Konstantinidis et al. [30]. In one case this might be attributed to the inclusion of the more conserved 16S rRNA gene sequences in our analysis. Secondly, it might be due to the fact that in our analysis four concatenated genes gave 2098 positions which is shorter than the minimum proposed (3300 bases) by Konstantinidis et al. [30]. Based on the analysis of seven concatenated genes the ANI value of 97.3% was deduced as cut off point for species delineation in Sinorhizobium [36]. Analysis of four concatenated genes (including

the 16S rRNA gene) was reported to give reliable phylogenetic positions of the Mesorhizobium genospecies [14]. These indicate that setting a universal cut-off value for species delineation might not be appropriate since different bacterial groups might have different evolutionary and speciation rates. However, addition of more reference strains for each group and/or analysis of more variable housekeeping genes could give a better correlation with the 96% ANI value as was suggested by Martens et al. [36]. In our analysis, in addition to having high ANI values, strains belonging to a single species formed clearly separated, closely clustered and highly supported monophyletic groups. Therefore, in most cases tree topology was a good indicator and helpful to delineate strains at species level (Table 1). The identification to species level revealed that the majority of the Ethiopian bean rhizobia belonged to the species R. etli, R. leguminosarum and R. phaseoli, with a sister clade to R. etli consisting of Ethopian strains forming a putative new species (group C). All these bacteria formed effective symbioses with bean plants, suggesting

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HBR3 +

R. leguminosarum BAPVT02 (GQ863540), R. giardinii sv. phaseoli H251 (AF217264)

I ANI:99.3%

HBR22, HBR23, HBR26, HBR31, HBR50, HBR79, HBR7, R. etli sv. phaseoli RP338 (DQ413011.1)

96

HBR42 9974 HBR10, HBR14, HBR17, Rhizobium sp. CCBAU 65118 (EU056342.1) 59 HBR (11, 13, 18, 19, 1, 20, 53), HBR9, R. etli CFN 42T (NC 004041.2), R. phaseoli ATCC 14482T(HM441255) 70

II ANI: 99.6%

ANI Between I & II: 97..9 %

97 HBR (24, 2, 4, 51, 5), R. etli sv. phaseoli CIAT 652 (NC 010996)

127

R. miluonense CCBAU 41251T (JN580680)

R. giardinii sv. giardinii H152T (AF217267.1) 43

100 HBR21, Rhizobium sp. RPA02 R. hainanense CCBAU 57015T (DQ010039) HBR12, R. lusitanum sv. phaseoli P1-7T (HM852098), R. tropici sv. phaseoli CIAT 899T (JN580681)

61

100 R. leucaenae sv. phaseoli CFN299 (X98514) R. leguminosarum sv. trifolii ATCC14480 (FJ895269) R. multihospitium CCBAU 83401T (EF050781)

78

R. fabaeT (JN580683)

100

R. leguminosarum sv. viciae USDA 2370T

74 98

Rhizobium sp. CCBAU 83309 (EF05077), R. pisi DSM 30132T (JN580682)

B. elkanii USDA 76T (HQ233221) 99

B. japonicum DSMZ 30131T (AB354632)

0.05 Fig. 3. Phylogenetic tree based on nodC gene sequences, showing the relationships among Phaseolus vulgaris symbionts and recognized species of the genus Rhizobium–Agrobacterium. Reference strains are indicated with bold and type strains with superscript T. The tree was constructed by the neighbor-joining method using MEGA version 5. Numbers at nodes indicate levels of bootstrap support (using 1000 replicates). Scale bar % indicates the number of substitutions per site. The accession numbers for the sequences are indicated within parentheses.

that they are true symbionts of common bean in Ethiopia. A few strains were R. leucaenae or R. giardinii, and can be considered sporadic symbionts. Strains classified as Agrobacterium species were non-symbiotic and are considered other endophytes according to the classification proposed by Li et al. [32]. The true symbionts all represent species that form one monophyletic clade in the phylogenetic tree of core genes. For clarity, this clade is called “the R. leguminosarum species complex”. R. etli is a major species in the R. leguminosarum complex. All strains belonging to the two sisters monophyletic groups C and D in the concatenated tree showed 99–100% partial 16S rRNA sequences similarity to R. etli CFN42T . In the concatenated tree, the strains in group D clustered closely with type strain R. etli CFN42 with ANI support 98.2%, however, the strains within group C (ANI 99.6%) excluded any reference species. The gap in ANI between the strains within and between the two groups was clearly high enough to delineate them into two different species groups. Therefore, the strains in the group D can be assigned as members of the species R. etli. The strains in group C formed clearly separated sister clade to R. etli, this might warrant a new species status. However, we must ask ourselves how many named species are convenient in the R. leguminosarum complex. “Splitters” prefer more, “lumpers” less. According to the cross-disciplinary species concept [33], this clade shows signs of evolutionary speciation, but the scientific community should discuss the need for more names in this complex. Strain CIAT652, previously classified as R. etli, is in our analysis in fact R. phaseoli and should be transferred to this species. In previous study of recA, R. etli CIAT 652 was also found closely clustered with R. phaseoli ATCC 14482T but far from R. etli CFN42T [61]. Nine Ethiopian R. phaseoli isolates were identified as well. These were clustered in the same AFLP Cluster 1 but they had different AFLP

fingerprints and were isolated from different places. This indicates that they represent genetically distinct and diverse strains within the species R. phaseoli. The strain HBR79 was related to R. phaseoli ATCC 14482T with 96% concatenated sequence similarity. However, because of its close relatedness to R. etli reference strains both in recA and glnII trees and since it had a separate position in the AFLP clustering, the phylogenetic position of this strain at species level is not clear in our analysis. Another interesting observation is the position of strain WSM2304. This strain isolated from clover plants and accordingly classified as R. leguminosarum sv. trifolii, forms a clade with two bean isolates (group A). Previously based on recA gene tree, R. leguminosarum WSM2304 was found in separate position far from other R. leguminosarum species [61]. The strains in group A had 97.1% ANI support. However, R. leguminosarum WSM2304 had the same pairwise average sequence similarity (96.0%) with neighbor R. leguminosarum USDA 2370T and HBR22 belonging to group A. This value illustrates the lack of a clear gap in sequence similarity levels between the group A and neighbor lineage that contained R. leguminosarum species. Therefore, the strains in group A were not distinguished well from R. leguminosarum species and this ambiguity can be clarified with analysis of more housekeeping genes and more strains. On the other hand Rhizobium sp. CCBAU 83309, Rhizobium sp. CCBAU 83476 and Rhizobium sp. CCBAU 83479 that were clustered with a group of R. leguminosarum species could be assigned as R. leguminosarum species since they had high ANI (about 97%) with R. leguminosarum USDA 2370T . In our study, some reference type species clustered tightly together. R. leguminosarum species complex also harbors R. fabae and R. pisi, which group tightly together with a high pairwise

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67 78 74 60

100

HBR1 HBR4 R. etli sv. phaseoli CFN 42T (NC_004041.2) HBR20 HBR22 HBR2 R. etli sv. phaseoli IE4794 (AY907478) HBR51 HBR11 HBR50 R. etli sv. phaseoli CIAT 652 (CP001076) HBR7 HBR24 HBR26 HBR21 R. phaseoli sv. phaseoli ATCC 14482T (JN580788) 62 HBR53 R. etli sv. phaseoli IE1009 (AY9074889) HBR17 R. etli sv. phaseoli IE2755 (AY907498) HBR31 R. etli sv. phaseoli KIM 5S (AY929542) HBR23 Rhizobium sp. CCBAU 83475 (EU146000) HBR79 93 HBR10 R. etli sv. phaseoli IE4874 (AY907494) HBR13 HBR42 HBR5 HBR9 98 HBR14 HBR19 HBR78 HBR18 HBR3 R. hainanense T (AY934876) R. multihospitium CCBAU 83401T(EF490070) R. miluonense CCBAU41251T(JN580786) R. tropici CIAT 899T (M55225) 100 HBR12 R. lusitanum P1-7T (AY943644) R. rhizogenes LMG 150T (AY945954) R. etli CCBAU 65830 (EU622089) 100 R. etli CCBAU 65708 (EU622088) R. leguminosarum sv. viciae 3841 (NC_008381) R. indigoferae CCBAU 71042T (JN580790) 100 R. fabae CCBAU 33202T (JN580789) 47 R. leguminosarum sv. viciae USDA 2370T (DQ450935) 83 T 93 R. pisi DSM 30132 (JN580787) R. leguminosarum sv. trifolii WSM2304 (NC_011368.1) B. japonicum DSMZ30131T (HM047126) B. elkanii USDA76T (B094963)

0,05 Fig. 4. Phylogenetic tree based on nifH gene sequences, showing the relationships among Phaseolus vulgaris symbionts and recognized species of the genus Rhizobium–Agrobacterium. Reference strains are indicated with bold and type strains with superscriptT. The tree was constructed by the neighbor-joining method using MEGA version 5. Numbers at nodes indicate levels of bootstrap support (using 1000 replicates). Scale bar % indicates number of substitutions per site. The accession numbers for the sequences are indicated within parentheses.

sequence similarity and they might be considered as one species. R. miluonense CCBAU 41251T and R. hainanense CCBAU 57015T were also clustered tightly together and the pairwise sequence similarity between them was very high as well. The same situation was also seen among different Mesorhizobium species from four core gene analysis [14]. In some case, this might be due to the low number of reference strains included for each species in our analyses. However, it remains to be seen if these species are distantly related to each other when studying other housekeeping loci. The position of R. indigoferae differs from the previously published one [74]. For R. indigoferae CCBAU 71042T we found two different 16S rRNA gene sequences in the Genbank and one of the 16S rRNA gene sequence (AF364068) showed 97% similarity to R. leguminosarum USDA 2370T . However, the one we used in this study showed 100% similarity to R. leguminosarum USDA 2370T . In addition, these two type strains showed 99–100% similarity for their recA, glnII and rpoB sequences. We suspect that the original type strain has been replaced by another “false” strain at some stage during handling in collections. For Phaseolus nodulating rhizobia, the nodC and nifH genes were reported to have incongruent phylogeny with 16S rRNA gene, Laguerre et al. [31]. In agreement with this, the phylogenies of

symbiotic genes (nodC and nifH) and the core genes were not congruent for the Ethiopian true bean symbionts and related references. Despite the strains in groups A-D in the concatenated tree had a distinct phylogeny; they were interspersed into two closely linked sister clades in the nodC tree. In most species of Rhizobium, the nod and nif genes are located on transmissible plasmids [39]. Thus, the close relatedness of nodC gene among bean nodulating rhizobia observed in our study supports the view that explained the presence of lateral gene transfer among Rhizobium [2,31,40]. In addition, this reveals the production of similar Nacetyl-glucosaminyl transferase that is encoded by nodC and is used for the first step in Nod factor assembly [31,48]. Common bean nodulating rhizobia that have sv. phaseoli character, such as R. etli, R. phaseoli and R. giardinii, are reported to contain three copies of nifH gene in the their symbiotic plasmids. Accordingly, these bacteria usually show a similar nifH restriction fragment length polymorphism profiles [2,40]. In our analysis most of the bean nodulating rhizobia had very similar nifH gene sequences. This supports again the presence of interspecific symbiotic plasmid exchange and common evolutionary history of nifH gene among these species [2,31,40].

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R. etli have been reported as one of the main bean-nodulating rhizobia in the center of the host origin of diversity, the Latin America [60]. Based on MLEE data west African soil was reported to have only R. etli and R. tropici [15]. In east Africa, Kenyan soils harbor bean-nodulating rhizobia related to R. leguminosarum, R. etli and R. tropici [4]. Previously in Ethiopia, indecisive findings based on MLEE and 16S rDNA sequences similarities showed only the presence of bean-nodulating rhizobia related to R. leguminosarum or R. etli [9]. In present study using MLSA we clarified that R. phaseoli, R. etli and the novel group of Rhizobium spp. are the main common bean-nodulating rhizobia in soils of Ethiopia. R. leguminosarum sv. phaseoli (now R. phaseoli) was first identified in Europe and it was suggested that the symbiotic plasmid may have been transferred to native R. leguminosarum from introduced seeds containing R. etli sv. phaseoli from America [47,60]. Therefore, the presence of R. etli in Ethiopian soil supports the suggestion of Diouf et al. [15] that bean rhizobia originating from the America had been introduced together with seeds and colonized soils of Africa. Ethiopia has been proposed to be one of the centers of diversity of pea [22] and this legume is known to grow in Ethiopia since antiquity [13]. As result, soils of Ethiopia believed to harbor symbionts of pea, R. leguminosarum. Therefore, these native rhizobia may have acquired molecular characteristics of R. phaseoli from the introduced R. etli sv. phaseoli. with the same processes mentioned above. R. tropici type A strains were reclassified as R. leucaenae species and these strains together with R. tropici type B, R. lusitanum, R. multihospitium and R. miluonense and R. rhizogenes are designated as “R. tropici group” [54]. In our analysis, both recA and rpoB gene trees showed the close relatedness of test strain HBR12 with R. leucaenae CFN299T in R. tropici group. In addition, the recA tree showed that HBR12 was more close to a number of R. leucaenae related strains isolated from P. vulgaris in Brazil [53]. Therefore, our strain HBR12 is classified as R. leucaenae. R. tropici was reported to have different nod gene and produces different Nod factor than other commonly known bean nodulating rhizobia, however, can effectively nodulate the same plant [50,51]. Accordingly, in nodC tree, R. leucaenae HBR12 including the closest related references fell outside the most common bean symbiont clades and the same is true for its nifH gene phylogeny. In our analysis the “R. tropici group” had identical or nearly identical nodC and/or nifH gene sequences. Similarly, the presence of identical or very similar nodD and nifH sequences were reported for R. tropici, R. lusitanum and R. multihospitium [24]. Hence, this may suggest that symbiotic genes in the R. tropici group may have the same origin. On the contrary, in our nodC tree, R. multihospitium was clustered with R. leguminosarum far from the R. tropici group and therefore this confusion should be clarified. Strain HBR21 had a separate position in the AFLP dendrogram and was separately clustered with the R. giardinii H152T in group E on the concatenated tree. The symbiotic genes nifKDH are believed to be absent in the symbiovar giardinii [2]. Accordingly, in our study amplification of the nifH fragment was not possible for the type strain R. giardinii H152 T that has sv. giardinii character. However, the nifH gene of our strain HBR21 was closely related to the major common bean nodulating rhizobia, indicating that this strain may belong to sv. phaseoli. On the contrary, strain HBR21 formed a different nodC clade together with the type strain of R. giardinii sv. giardinii which is contradictory to the report showing that each of the sv. phaseoli and giardinii formed distinct lineages on the nodC tree [31]. The sporadic symbionts of bean in Ethiopia are major players in other regions. R. leucaenae is prevalent in South America [53]. R. giardinii was originally isolated and described in France [2] and also inhabits soils of Latin America [60]. Therefore, the presence

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of R. giardinii and R. leucaenae in Ethiopian soils might further illustrate the migration of bean nodulating rhizobia to Africa via trade [15]. Other nodule endophytes of bean were also detected in this study. The test strains, which belonged to the same cluster 6 in the AFLP dendrogram, were also clustered into the same subgroup G1 in the concatenated tree together with A. tumefaciens C58. In the sub-cluster G2 the test strains were clustered with A. radiobacter LMG140T and A. tumefaciens NCPPB 2437. According to the International Committee on Systematics of Prokaryotes Subcommittee on the Taxonomy of Agrobacterium and Rhizobium, Agrobacterium biovar 1 species complex should be collectively called the “Agrobacterium tumefaciens species complex”. A. radiobacter LMG140T , A. tumefaciens NCPPB 2437 and A. tumefaciens C58 are members of this complex, however, the former two bacteria belonging to the bona fide species A. radiobacter, for which A. radiobacter strain LMG140 has been assigned as a type strain [35]. Therefore, our test strains within sub-group G1 are the members of A. tumefaciens species complex, whereas strains within sub-cluster G2 represent A. radiobacter species. Strains belonging to A. radiobacter (sub-group G2) and A. tumefaciens complex (sub-group G1) were ineffective in nitrogen fixation or did not form nodules at all. Amplification of nodC and nifH was not possible from these strains (except nifH of HBR78). Implies, these agrobacteria might lack symbiotic genes but could enter to the nodules by unknown mechanisms [43]. Alternatively, Agrobacterium strains might have had symbiotic genes when entering nodules, but may have lost the genes within a nodule or after subsequent isolation and cultivation processes. AFLP reflects the whole-genome fingerprint of the bacteria and is suggested as one of the approaches to determine genomic relationships between bacteria [26]. A high correlation between AFLP and DNA–DNA homology was demonstrated from the comparison of a large collection of Xanthomonas strains [52] and between species of Agrobacterium [49]. In our study AFLP clusters 1, 3, 4 and 6 supported the phylogenic grouping obtained from concatenated tree at groups B, D, A and G1. Nevertheless, the groupings in AFLP clusters 2 and 5 did not reflect fully the phylogenetic relationship of the strains resulted from concatenated sequences. Our result is in agreement with the finding that showed incongruence of AFLP clustering and phylogenetic relationship of Rhizobium species [67]. In general, the AFLP technique is not a good phylogenic tool [67], but it is a good method to study the diversity of closely related rhizobial strains since it provides high resolution between strains not distinguished by the housekeeping gene sequences [10]. In agreement of this, in our analysis R. phaseoli strains that had identical housekeeping gene sequences displayed different AFLP fingerprints. MLSA is a valuable method for the taxonomic study of Rhizobium as was suggested for other species [36,37]. In Ethiopia the true bean nodulating rhizobia revealed to be R. etli, R. phaseoli, R. leguminosarum related and the novel group. As these all were effective in nitrogen fixation on Variety RED WOLAYTA common bean host in greenhouse, it is recommend to check their effectiveness with other varieties and in field conditions.

Acknowledgments We are grateful to National Soil Testing Center of Ethiopia, for giving us the seeds and allowing us a greenhouse for nodulation tests. We thank to the National Meteorology Agency of Ethiopia, for providing the annual mean rainfall and temperature data of the sampling sites. This work was supported by the Academy of Finland, Societas Scientiarum Fennica, The Ella and Georg Ehrnrooth Foundation and the University of Helsinki.

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