Accepted Manuscript Title: Phylogenetic multilocus sequence analysis of native rhizobia nodulating faba bean (Vicia faba L.) in Egypt Author: Sameh H. Youseif Fayrouz H. Abd El-Megeed Amr Ageez Edward C. Cocking Saleh A. Saleh PII: DOI: Reference:
S0723-2020(14)00140-4 http://dx.doi.org/doi:10.1016/j.syapm.2014.10.001 SYAPM 25648
To appear in: Received date: Revised date: Accepted date:
11-7-2014 1-10-2014 3-10-2014
Please cite this article as: S.H. Youseif, F.H.A. El-Megeed, A. Ageez, E.C. Cocking, S.A. Saleh, Phylogenetic multilocus sequence analysis of native rhizobia nodulating faba bean (Vicia faba L.) in Egypt, Systematic and Applied Microbiology (2014), http://dx.doi.org/10.1016/j.syapm.2014.10.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Phylogenetic multilocus sequence analysis of native rhizobia nodulating faba bean (Vicia faba L.) in Egypt
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Sameh H. Youseif , Fayrouz H. Abd El-Megeed , Amr Ageez , Edward C. Cocking and
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Saleh A. Saleh
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National Gene Bank and Genetic Resources, Agricultural Research Center, Giza, Egypt
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Agricultural Genetic Engineering Research Institute, Agricultural Research Center, Giza, Egypt
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Fayrouz H. Abd El-Megeed National Gene Bank and Genetic Resources, Agricultural Research Center, Giza, Egypt E-mail addresses: [email protected] Address: 9 Gamaa st. Giza, Egypt. Post office Box: 12619
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Amr Ageez Agricultural Genetic Engineering Research Institute, Agricultural Research Center, Giza, Egypt E-mail addresses: [email protected] Address: 9 Gamaa st. Giza, Egypt. Post office Box: 12619
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Edward C. Cocking Centre for Crop Nitrogen Fixation, University of Nottingham, Nottingham, UK E-mail addresses: [email protected] Address: University of Nottingham, Nottingham NG7 2RD, UK
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Saleh A. Saleh
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National Gene Bank and Genetic Resources, Agricultural Research Center, Giza, Egypt E-mail addresses: [email protected] Address: 9 Gamaa st. Giza, Egypt. Post office Box: 12619
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Sameh H. Youseif *Corresponding author. National Gene Bank and Genetic Resources, Agricultural Research Center, Giza, Egypt Tel.:+2 0106 1147472 E-mail addresses: [email protected] Address: 9 Gamaa st. Giza, Egypt. Post office Box: 12619
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Centre for Crop Nitrogen Fixation, University of Nottingham, Nottingham NG7 2RD
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Abstract
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The taxonomic diversity of forty-two Rhizobium strains, isolated from nodules of faba bean
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grown in Egypt, was studied using 16S rRNA sequencing, multilocus sequence analyses
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(MLSA) of three chromosomal housekeeping loci and one nodulation gene (nodA). Based on the
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16S rRNA gene sequences, most of the strains were related to Rhizobium leguminosarum, R. etli,
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and R. radiobacter (syn. A. tumefaciens). A Maximum Likelihood (ML) tree built from the
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concatenated sequences of housekeeping proteins encoded by glnA, gyrB and recA, revealed the
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existence of three distinct genospecies (I, II and III) affiliated to the defined species within the
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genus Rhizobium/ Agrobacterium. Seventeen strains in genospecies I could be classified as R.
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leguminosarum sv. viciae. Whereas, a single strain of genospecies II was linked to R. etli.
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Interestingly, twenty-four strains of genospecies III were identified as A. tumefaciens. Strains of
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R. etli and A. tumefaciens have been shown to harbor the nodA gene and formed effective
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symbioses with faba bean plants in Leonard jar assemblies. In the nodA tree, strains belonging to
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the putative genospecies were closely related to each other and were clustered tightly to R.
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leguminosarum sv. viciae, supporting the hypothesis that symbiotic and core genome of the
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species have different evolutionary histories and indicative of horizontal gene transfer among
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these rhizobia.
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Introduction Faba bean (Vicia faba L.) is a major food and feed legume because of the high nutritional
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value of its seeds [10]. In 2010, the world production of faba bean was 4.0 million tons, and most
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of its global production is located in China, Ethiopia, France and Egypt [11]. Faba bean is most
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commonly included in the diets of inhabitants of the Middle East, the Mediterranean region and
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China. They use faba bean seeds as green or dried, fresh or canned [7]. Faba bean is one of the
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most important legume crops grown in Egypt. The cultivated faba bean area in Egypt has been
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estimated to be 0.08 million ha and produced about 0.26 million tons [12].
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Faba bean commonly establishes effective nitrogen fixation symbiosis with fast-growing
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rhizobia of the species Rhizobium leguminosarum [52]. R. leguminosarum species comprises
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three symbiovars [22]: viciae (nodulating pea and vetch), trifolii (nodulating clover), and
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phaseoli (nodulating beans), which show a common chromosomal background and different host
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range specificities [24, 36]. Initially, all faba bean-nodulating rhizobia were classified as R.
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leguminosarum symbiovar (sv.) viciae [1]. Later, R. fabae was described as a new faba bean-
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nodulating species based on multilocus sequence analyses, sequence analysis of 16S rDNA and
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several phenotypic characteristics [48]. To date R. leguminosarum sv. viciae is distributed widely
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around the world, while R. fabae has been discovered in Chinese fields [49]. Recently,
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Rhizobium laguerrereae was identified as a faba bean nodulating microsymbiont in several
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countries including Spain, Peru and Tunisia [37].
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Previously, twenty-eight rhizobial strains were isolated from faba bean root nodules
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grown in Egypt [40]. Based on partial 16S rDNA sequencing, a majority of these strains was
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identified as Rhizobium leguminosarum sv. viciae. Nevertheless, the use of 16S rRNA gene
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sequence analyses for taxonomical purposes has proven difficult and cannot be done with great
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certainty [53]. Protein-encoding housekeeping genes have been proposed as alternative
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phylogenetic markers to help in distinguishing closely related species [45]. Although
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housekeeping genes have a higher evolution rate than 16S rRNA genes, they are conserved
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enough to provide informative phylogenetic data [6]. Therefore, in the present study, the MLSA
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technique was used to study the taxonomic position of native rhizobia nodulating faba bean in
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Egypt. For this purpose, the phylogenies of housekeeping genes coding for recombination protein
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(recA), glutamine synthetase (glnA), DNA gyrase B subunit (gyrB) and the 16S rRNA gene were
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determined. The phylogeny of the symbiotic gene for nodulation (nodA), as well as the capacity
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of strains to induce effective nodules, was also investigated.
Materials and Methods
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Bacterial isolation
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Rhizobium strains were isolated from surface-sterilized root nodules according to the standard
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routine laboratory techniques described by Vincent [54]. At flowering stage, nodules were
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collected from field-grown faba bean from different sites that represented sixteen governorates in
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Egypt. Strain numbers, geographical locations and their respective soil characteristics are listed
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in Table 1. Root nodules were surface sterilized by washing for 30 s with 95% ethanol, immersed
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in 3% sodium hypochlorite and finally were washed six times using sterile distilled water.
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Following sterilization, nodules were individually crushed aseptically in 1 ml sterile distilled
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water, streaked on the surface of Yeast Extract Mannitol Agar (YEM) plates supplemented with
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0.025 g L_1 of congo red and finally were incubated at 28 ˚C for 3-5 days [44].
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Plant nodulation assay
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The nodulation assays were performed in Leonard jar assemblies [44] containing sterilized sandy
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soil. The soil was analyzed according to Page et al. [34]. The main physical and chemical
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properties of the soil used are presented in supplementary Table S1. Each Leonard jar assembly
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was planted with three surface-sterilized seeds of faba bean cultivar Giza 716 and irrigated with
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nitrogen free nutrient solution [57]. Each seed was inoculated with 1.0 ml (about 1 X108 cells) of
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log-phase rhizobial cultures. The uninoculated plants were used as negative controls. Faba bean
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plants were cultivated using a randomized complete block design with three replicates in a
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controlled greenhouse at 24°C for 12 h (light) and 12°C for 12 h (dark). All treatments received
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20 ppm N as a starter N dose. After 35 days of planting, plants were uprooted and assayed for the
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number and dry weight of nodules, as well as the dry weight of shoots. Data were subjected to
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analysis of variance using MSTAT analysis software [43].
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Phenotypic characterization
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The tolerance ability of different rhizobial isolates to grow under stress conditions (salinity, high
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temperatures, acidity and alkalinity) was assessed as described by Youseif et al. [59]. The salt
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tolerance of rhizobia was tested on YEM agar medium containing 0.1%, 0.5%, 1%, 2%, 3%,
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3.5%, 4%, 5% and 6% (w/v) NaCl. Temperature tolerance of rhizobia was examined by
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inoculating rhizobia on YEM agar plates and incubating at 28, 37, 40, 42, 45 and 48 ˚C.
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Nevertheless, pH tolerance was evaluated by growing rhizobia on YEM agar medium adjusted to
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a pH range from pH 5 to pH 11. After 2-3 days of incubation at 28˚C, plates were examined for
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bacterial growth.
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DNA isolation, PCR amplifications and gene sequencing
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Genomic DNA of rhizobial cells was isolated using Wizard® Genomic DNA purification Kit
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(Promega® Corporation, Madison, USA). PCR was performed using the standard reaction
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mixture (50 µl) containing: 1X PCR buffer, 1.5 mM MgCl2, 5% dimethyl sulfoxide, 200 mM of
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each dNTPs, 15 pmol of each primer, 1 U of Taq polymerase enzyme (Promega® Corporation,
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Madison, USA) and 50 ng of DNA template. PCR conditions and primer sequences used for
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gene amplification and sequencing are shown in Table S2.
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Phylogenetic analyses
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Sequence reads were edited and assembled using the DNASTAR software (Lasergene, Madison,
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USA). Sequence similarity searches were performed at the National Center for Biotechnology
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Information (NCBI) server using BLASTN to identify 16S rRNA genes and BLASTX to
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annotate the proteins encoded by housekeeping and nodulation genes. The sequences were
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aligned using Clustal W version 1.8 [2], and subjected to phylogenetic analysis. Phylogenetic
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trees were constructed using the maximum likelihood methods (ML) in MEGA version 6 [46],
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and 1000 bootstrap replication to assess branching confidence. For 16S rRNA, phylogeny was
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constructed based on nucleotide sequences while for housekeeping (gyrB, glnA and recA) and
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nodulation (nodA) genes, phylogenies were constructed based on amino acids sequences. Using
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Mega 6 software, maximum likelihood fits with 48 different amino acid (aa) substitution models
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were tested. Models with the lowest BIC scores (Bayesian Information Criterion) are considered
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to describe the best substitution pattern. The JTT model was selected as it gives the lowest BIC
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scores.
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Results
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Nodulation, symbiotic effectiveness and phenotypic characteristics
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Forty-two local rhizobial isolates were isolated from root nodules of field grown faba bean in
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sixteen governorates representing most faba bean cultivation areas in Egypt (Table 1). The
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symbiotic effectiveness of rhizobial isolates was confirmed by using plant nodulation assay in
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Leonard jar assemblies (Table 2). All rhizobial isolates were able to induce symbiosis with faba
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beans, resulting in the number of nodules ranging from 13- 67 nodules/plant. Nodules dry weight
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varied from 37 mg/plant for isolate NGB-FR 137 to 181 mg/plant for isolate NGB-FR 128.
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Shoot dry weight, as an indirect measure of the nitrogen fixation benefit, was determined among
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tested isolates. Faba bean plants inoculated by strains NGB-FR 126 and 128 showed the highest
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shoot dry weights (2.14 and 2.01 g/plant, respectively) with significant increases over the
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uninoculated control (1.55 g/plant). All rhizobial isolates were phenotypically characterized
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under different stress conditions (Table S3). Most of the isolates appeared resistant to salinity
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and grew in a range from 0.1% to 5% NaCl. However, twenty-one isolates tolerated 5% NaCl.
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Considering tolerance to acidic and alkaline conditions, rhizobial isolates were resistant to a wide
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pH range started from 5 to 11. Under high temperatures, the isolates showed a variable tolerance
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to a wide range of temperatures (28-45 ºC). Most of isolates were sensitive to 42 ºC, however
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only eight Rhizobium isolates (NGB-FR 24, 26, 27, 38, 39, 51, 99 and 101) tolerated 45 ºC.
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16S rDNA ribosomal gene
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Nearly full-length 16S rDNA gene regions (1271-1522 bp) were successfully amplified and
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sequenced for all rhizobial strains. According to the sequences similarity, local rhizobial isolates
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were closely related to the defined species within the genus Rhizobium/ Agrobacterium (Table
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2). The maximum likelihood (ML) phylogenetic tree based on 16S rRNA sequences (Fig. 1),
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classified faba bean-nodulating rhizobia into five distinctive groups (A, B, C, D and E). Strains
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in group A were clustered at high bootstrap (BT) value (100%) along with R. leguminosarum sv.
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viciae strains (Access. No. EU730596 and NR_103919). They also were grouped with R.
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leguminosarum sv. viciae strain RL2 (Access. No. JQ303081) that was isolated also from faba
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bean root nodules grown in Egypt. Group B was a sister monophyletic cluster of group A and
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was linked along with R. leguminosarum RN2 (Access. No. KC884251). Strain NGB-FR 101,
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the only representative of group C showed high relatedness to R. etli EBRI 3 (Egyptian
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microsymbionts nodulating bean [41]. Strain NGB-FR 137 the sole representative of group D
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had a close phylogenetic affiliation to Rhizobium sp. USDA 1920, a symbiotic bacterium
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nodulating Medicago ruthenica [51]. Group E was a cluster of twenty-four strains along with
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five different A. tumefaciens references (Access. No. KJ499777, KF460525, HQ735085,
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KF439828 and KF730752) supported by a 100% bootstrap value.
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Protein-coding housekeeping genes
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Sequences for the glnA (947–974 bp), gyrB (648–792 bp) and recA (508–577 bp) genes were
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isolated from all Rhizobium strains. New sequences of the forty-two test strains were deposited
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into Genbank database (Table S4). Sequences of several housekeeping genes retrieved from the
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Genbank were shorter than our newly sequenced ones. Therefore, in the final alignments, parts
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of the new sequences were omitted. Consequently, the lengths of the alignments used were 879
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bp, 552 bp and 462 bp for glnA, gyrB and recA, respectively. DNA sequences were translated to
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proteins of 293 aa, 184 aa and 154 aa for glnA, gyrB and recA, respectively. The ML
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phylogenetic trees based on individual housekeeping proteins (aa) are presented in Figs. S1, S2
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and S3. Proteins encoded by different housekeeping loci showed variable informative positions
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(Table 3). According to the resultant analysis, gyrB protein had the greatest percentage of
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parsimony-informative characters (43%) of the positions used for the analysis, followed by 27%
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and 22% for glnA and recA proteins, respectively.
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Concatenated sequence analysis
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Aligned sequences from the glnA (293 aa), gyrB (184 aa) and recA (154 aa) protein-encoding
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genes were concatenated and 631(aa) sites were obtained (Table 3). The ML phylogenetic tree,
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based on the combined partial proteins sequences was constructed (Fig. 2). The results of the
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concatenated sequences were generally congruent with those of the individual protein-encoding
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genes (Figs S1, S2 and S3), but generated more robust and confident grouping. Based on the
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concatenated proteins analysis, faba bean-nodulating rhizobia were classified into three
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genospecies I, II and III (Fig. 2). Genospecies I including seventeen strains, clustered with R.
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leguminosarum sv. viciae strain 3841. This phylogeny was similar in all core protein analyses
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and was mostly consistent with 16S rRNA gene sequencing. One exception was for strain NGB-
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FR 137, that was placed in a distinct site according to 16S rRNA phylogeny.
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Strain NGB-FR 101, the only representative for genospecies II, was clustered on a well-
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supported branch (BT 85%) with type strain R. etli CFN 42 and R. etli Mim1 in the concatenated
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tree. This group was well separated from other genomic species in the concatenated tree, but its
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position varied in the individual ML gene trees.
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Interestingly, twenty-four strains classified as genospecies III were linked tightly with A.
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tumefaciens supported by high bootstrap value (96%). This grouping was commonly identified in
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gyrB, recA and 16S sequence trees, nevertheless in the glnA tree strain NGB-FR 39 was placed
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in a sister monophyletic position.
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Phylogeny of nodA symbiotic gene
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The partial sequences (488 to 574 bp) of the nodA nodulation gene were determined for
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all strains with the exception of strains NGB-FR 37 and NGB-FR 99. DNA sequences were
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translated to their respective proteins. Basic sequence alignment statistics for protein encoded by
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nodA is shown in Table 3. The nodA based-ML phylogenetic tree was constructed (Fig. 3).
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Unlike the grouping based on protein-encoding housekeeping genes and 16S rRNA sequences,
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all test strains within different genospecies had a very close phylogenetic affinity with R.
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leguminosarum sv. viciae.
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Discussion
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Phenotypic characteristics
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Faba bean is grown worldwide as a protein source for food and feed, and offers an
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important ecosystem service to cropping systems via its ability to symbiotically fix atmospheric
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N2 [21]. In Egypt, faba bean is considered as a cash crop and has been cultivated for over 3000
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years [42]. In this study, forty-two rhizobial strains were isolated from root nodules of field-
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grown faba bean, representative of a wide range of geographical regions in Egypt. All strains
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were able to nodulate faba bean plants and resulted in different nodulation patterns. The present
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study demonstrated a high phenotypic diversity among faba bean-nodulating rhizobia under
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stress conditions. The majority of strains was tolerant to salt stress, and 50% of isolated strains
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appeared highly salinity-tolerant and grew at 5% NaCl. Similar results were reported by
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Shamseldin et al. [40], who isolated faba bean-rhizobia under Egyptian soil conditions able to
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grow at 3 % NaCl. Most of the tested strains could grow under high temperatures and eight of
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them were able to grow at 45 ºC. The tolerance of rhizobia to stress conditions appeared to be
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more of a strain-specific than species-specific phenomenon, supporting the finding reported by
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Amarger et al. [3].
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Phylogenetic analyses of faba bean- nodulating rhizobia Sequence analysis of 16S rRNA genes has been used as standard practice in bacterial
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taxonomy [25]. To date, most of rhizobial strains isolated from Vicia faba were found to be
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closely related to R. leguminosarum [31, 38, 40, 47, 49]. In this study, based on the 16S rRNA
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gene analysis, only 38% of faba bean-nodulating rhizobia are closely related to R.
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leguminosarum, while 57% of tested strains are related to R. radiobacter (syn. A. tumefaciens)
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supported by highly bootstrap value (100%).
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The use of 16S rRNA gene as a sole molecular marker has been criticized due to its
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presence in multiple copies in a genome of some bacteria [17], its susceptibility to genetic
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recombination and horizontal gene transfer [53] and low phylogenetic power among closely
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related species [15, 38]. Considering the limitations of the 16S rRNA gene in bacterial
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taxonomy, the multilocus sequence analysis (MLSA) using protein-coding genes (housekeeping
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genes) has been preferred for studying closely-related species of bacteria, including rhizobia [4,
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9, 27, 35]. By using a concatenation analysis of several housekeeping genes, the phylogenetic
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signals can be enhanced to provide highly supported clades [6].
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Although a limited variation in topologies was observed between the three individual ML
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gene trees (Fig. S1, S2 and S3), strains in this study were clustered into three distinct
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monophyletic genospecies (I, II and III) in every housekeeping gene tree as well as in the ML
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tree of the concatenated sequences (Fig. 2). Genospecies I was closely related to R.
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leguminosarum sv. viciae strain 3841 in all of the analyzed housekeeping genes. Whereas, strain
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NGB-FR 137 (related to the genus Rhizobium, based on the 16S rRNA sequence data), was
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integrated into R. leguminosarum sv. viciae - genospecies I, supporting the fact that the 16S
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rRNA gene is not adequate for identification at species level within the genus Rhizobium [38].
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Remarkably, NGB-FR 101 the only representative strain of genospecies II was related to
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R. etli CFN 42 and R. etli Mim1 with a 85% concatenated bootstrap value. R. etli was originally
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described as exclusively nodulating and fixing nitrogen with Phaseolus vulgaris [39].
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Nevertheless, isolation of R. etli strains from nodulated faba bean plants has been previously
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reported in Chinese [47] and Egyptian soils [40]. 9
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Another interesting finding in this study is that, 57% of tested strains were identified as
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genospecies III and were clustered together with R. radiobacter (syn. A. tumefaciens) supported
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by concatenated high bootstrap values (96%). Strains of the genus Agrobacterium (now, rather
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controversially, included in the genus Rhizobium [13, 58] have been frequently isolated from
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root nodules of various legumes including common bean [30], Sesbania [8], soybean [59], lentil
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[35] and faba bean [50]. In general, Agrobacteria failed to nodulate their hosts [4] and this is may
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be due to the loss of their nodulation genes during storage [20], supporting the idea that these
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isolates may be opportunistic bacteria able to colonize nodules induced by rhizobia [35]. In
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contrast, our results confirmed the nodulation ability of A. tumefaciens to nodulate faba bean
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under Leonard jar conditions. This finding in accordance with other publications supports the
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symbiotic effectiveness of Agrobacteria to nodulate their original hosts [8, 59].
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Symbiosis related gene: Implication of Sym plasmid transfer
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The nodulation (Nod) factors determine the legume-rhizobia symbiosis [24]. These genes
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are often found on the symbiotic plasmid pSym and enable the rhizobia to fix nitrogen in
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symbiosis with legume plants [23]. Because of the nature of the Nod factor acyl group attached
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by the nodA gene can contribute to the determination of host range [33], nodA has been used as a
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symbiotic marker in the analyses of specificity between rhizobia and different host plants [35,
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59]. In respect to the nodA protein-based phylogenetic tree (Fig. 3), nearly all strains in the
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putative genospecies I, II and III were grouped along with R. leguminosarum sv. viciae strain
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3841 into one separate branch supported by a highly bootstrap value (88%). However strain
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NGB-FR 101, the only representative of genospecies III (related to R. etli, based on the
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concatenated core gene analyses), formed a sister monophyletic cluster to R. leguminosarum sv.
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viciae, but was placed in a distinct position (100% BT). In bacteria, the chromosomal genetic
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background and the plasmid-borne genetic background are often not correlated due to horizontal
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transfer of plasmid-borne genes [32]. Moreover, different rhizobial species can share similar
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symbiotic genes, and different symbiosis genes can be harbored by similar genomic backgrounds
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[16, 24]. Thus, incongruence may exist between phylogeny based on symbiotic and
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chromosomal genes, and this phenomenon has been demonstrated to play a role in the evolution
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and structure of natural populations of rhizobia [5, 26, 55]. The nodA based-phylogenies suggest
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that, A. tumefaciens and R. etli in this study might have acquired their symbiosis-related genes by
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lateral gene transfer from R. leguminosarum (Fig. 3), with a potential candidate being R.
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leguminosarum sv. viciae. The transfer of Sym plasmid within R. leguminosarum sv. viciae
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populations in the field was previously published [23, 40, 49]. Gene transfer and rearrangement
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of symbiotic plasmids could be involved in the acquisition and evolution of rhizobial symbiotic
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functions [24]. In the case of Agrobacterium, the mobility of Sym genes has been exemplified in
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laboratory experiments [19, 29]. Nevertheless, it was recently found that naturally occurring
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agrobacterial symbionts possess a Sym-plasmid with symbiosis-specific genes and can
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effectively nodulate their original hosts and fix N2 to the same degree as standard rhizobia [8,
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60].
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In conclusion, forty-two Rhizobium strains isolated from faba bean grown in Egypt, were
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characterized using a polyphasic approach. Our results revealed that (1) the majority of the
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Egyptian faba bean rhizobia belonged to the species R. radiobacter (syn. A. tumefaciens), and R.
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leguminosarum (2) All these bacteria formed effective symbioses with faba bean plants,
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suggesting that they are true symbionts of faba bean in Egypt (3) Irrespective of their
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chromosomal background, the symbiosis related gene (nodA) of faba bean symbionts were
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highly related to each other with close affinity to R. leguminosarum sv. viciae, suggesting that
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symbiotic genes have a common origin that is incongruent with chromosomal loci.
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Acknowledgements
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This work has been financed by the Science and Technology Development Fund (STDF), Egypt,
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project ID: STDF 901.
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[54] Vincent, J.M. 1970 A manual for the practical study of root nodule bacteria. IBP Handbook No. 15, Black Well Scientific Publications, Oxford, p. 164. [55] Vinuesa, P., Silva, C., Werner, D., Martinez-Romero, E. (2005) Population genetics and phylogenetic inference in bacterial molecular systematics: the roles of migration and recombination in Bradyrhizobium species cohesion and delineation. Mol. Phylogenet. Evol. 34, 29–54. [56] Weisburg, W.G., Barns, S.M., Pelletie, D.A., Lane, D.J. (1991) 16S rDNA amplification for phylogenetic study. J. Bacteriol. 173, 697–703. [57] Werner, D., Wilcockson, J., Zimmermann, E. (1975), Adsorption and selection of rhizobia with ion-exchange papers. Arch. Microbiol. 105, 27-32. [58] Young, J.M., Kuykendall, L.D., Martínez-Romero E., Kerr, A., Sawada, H. (2001) A revision of Rhizobium Frank 1889, with an emended description of the genus, and the inclusion of all species of Agrobacterium Conn 1942 and Allorhizobium undicola de Lajudie et al. 1998 as new combinations: Rhizobium radiobacter, R. rhizogenes, R. rubi, R. undicola and R. vitis, Int. J. Syst. Evol. Microbiol. 51, 89-103. [59] Youseif, S.H., Abd El-Megeed, F.H., Ageez, A., Mohamed, Z.K., Shamseldin, A., Saleh, S.A. (2014) Phenotypic characteristics and genetic diversity of rhizobia nodulating soybean in Egyptian soils. Eur. J. Soil Biol. 60, 34-43
491 492 493 494
[60] Youseif, S.H., Abd El-Megeed, F.H., Khalifa, M.A., Saleh, S.A. (2014) Symbiotic effectiveness of Rhizobium (Agrobacterium) compared to Ensifer (Sinorhizobium) and Bradyrhizobium genera for soybean inoculation under field conditions. Res. J. Microbiol. 9, 151-162
M
an
us
cr
ip t
473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490
498 499 500 501 502 503 504 505
pt
497
Ac ce
496
ed
495
506 507 508
15
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Table 1 List of rhizobial local isolates used in this study, their respective geographical regions and soil characteristics pH
E.C (dS m-1)
N (ppm)
P (ppm)
K (ppm)
Clay Clay Clay Clay Clay Sandy Clay Clay Clay Clay Clay Clay Clay Clay Clay Clay Clay Clay Clay Clay Sandy Sandy Clay Clay Clay Sandy Sandy Calcareous Clay Clay Sandy Sandy Clay Clay Clay Clay Clay Clay Clay Clay Clay Clay
7.6 7.8 7.9 8.6 8.4 8.4 7.9 7.6 7.4 7.6 8.2 7.7 8.2 7.7 7.4 7.8 7.73 7.6 7.7 7.8 8.4 8.7 7.7 7.8 7.6 8.6 8.5 8.6 7.5 7.6 8.4 8.2 7.7 7.7 7.6 7.3 7.5 7.8 7.7 8.1 7.7 7.73
0.75 0.37 0.72 0.67 0.86 0.15 0.21 0.52 0.54 0.54 2.57 0.85 1.30 0.53 0.45 0.50 1.12 0.35 0.90 0.60 1.30 1.50 0.56 0.16 0.24 0.24 0.14 0.97 0.44 0.67 0.55 0.41 0.56 0.56 0.35 0.93 0.42 0.37 1.63 1.61 1.33 1.12
68 56 42 25 40 10 45 44 80 55 15 60 20 80 55 60 47 42 44 45 20 10 45 75 56 10 15 10 65 48 10 10 45 45 42 75 75 56 42 26.5 31.3 47
7.5 6.5 3.2 6.50 3.4 3.3 6.2 6.3 9.2 5.5 4.3 2.6 6.3 9.2 3.4 2.9 10.9 8.2 12.8 13 4.5 6.5 10.2 9.4 8.3 3.4 5.2 3.2 11.2 8.5 3.5 2.4 10.2 10.2 8.2 5.6 8.2 6.5 6.3 5.0 4.4 10.9
288 320 169 199 210 132 212 325 280 250 315 150 310 360 190 210 372 352 368 344 250 214 366 350 288 152 112 125 365 275 112 125 366 366 352 225 330 320 310 140 160 372
cr
ip t
Soil texture
ed
M
an
us
Behairah, Edko, Al-Zohoor village Behairah, Rasheed, Al-Borg village Dakahlia, Mansoura Dakahlia, Menyat El-Nasr, Brembala village Damietta/ Damietta Ismailia/ Al-Qassasen Kafr El-Sheikh/ Kafr El-Sheikh, Awerah village Kafr El Sheikh/ Highway, Ewira village Kafr El Sheikh/ International Highway Kafr El Sheikh/ Al-Hamool, Al-kheregeen village Sharkia/Al-Hosanyah Sharkia/Al-Hosanyah, Abo omar village Sharkia/Al-Hosanyah, Al-Tarek village Gharbia/ Al-Santa, Al-Gemizah Menoufia/ Al-Bagoor, Meet Al-Beydah village Menoufia/ Al-Bagoor, Al-Aatf village Port Said/ Port Said, Al-kaab area Giza/ Giza, Agricultural Research Center Menia/ Abo-Qourqas, Beni mousa village Assuit/ Assuit New Valley/ El-Kharga Agricultural Res. Station New Valley/ Al- Dakhla, Al-Moosheyah village Fayoum/ Ebsheway, Shakshook village Beni Suef/ Nasser, Dandil village Beni Suef/Seds, Haramashant village North Sinai/ Romana North Sinai/Beer Al-Aabd Behaira /Noubaria Fayoum/ Atsa, Al-Mahmoudia village Fayoum/ Youssef Al-Sedeek, Kahk village Fayoum/ Youssef Al-Sedeek, Mafarek Al-Sedeek Fayoum/ Youssef Al-Sedeek, Al-Khergeen village Fayoum/ Ebsheway, Shakshook village, location 1 Fayoum/ Ebsheway, Shakshook village, location 2 Giza/ Giza, Agricultural Research Center Behairah/ Edko Behairah/ Edko, 6th October village Behairah/ Rasheed, Al-Gedia village Port Said/ Sahl Port Said, Agricultural area 1 Port Said/ Sahl Port Said, Agricultural area 2 Port Said/ Sahl Port Said, Agricultural area 6 Port Said/ Port Said, Al-kaab area
pt
NGB-FR-2 NGB-FR-4 NGB-FR-10 NGB-FR-11 NGB-FR-15 NGB-FR-20 NGB-FR-24 NGB-FR-25 NGB-FR-26 NGB-FR-27 NGB-FR-37 NGB-FR-38 NGB-FR-39 NGB-FR-51 NGB-FR-61 NGB-FR-62 NGB-FR-65 NGB-FR-70 NGB-FR-76 NGB-FR-88 NGB-FR-99 NGB-FR-101 NGB-FR-107 NGB-FR-114 NGB-FR-122 NGB-FR-126 NGB-FR-128 NGB-FR-132 NGB-FR-137 NGB-FR-138 NGB-FR-139 NGB-FR-140 NGB-FR-141 NGB-FR-142 NGB-FR-144 NGB-FR-145 NGB-FR-146 NGB-FR-147 NGB-FR-148 NGB-FR-149 NGB-FR-150 NGB-FR-151
Governorate/ site
Ac ce
Isolate No.*
*NGB-FR: National Gene Bank-Faba bean Rhizobia.
16
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Table 2 Symbiotic properties of faba bean nodulating rhizobia used in this study and their phylogenetic homology Identity based on 16S rRNA gene sequence
A. tumefaciens ATCC 13332 (HQ735085) A. tumefaciens ATCC 13332 (HQ735085) A. tumefaciens ATCC 13332 (HQ735085) A. tumefaciens ATCC 13332 (HQ735085) A. tumefaciens ATCC 13332 (HQ735085) Rhizobium sp. IRBG74 (HG518322) A. tumefaciens ATCC 13332 (HQ735085) A. tumefaciens ATCC 13332 (HQ735085) A. tumefaciens ATCC 13332 (HQ735085) A. tumefaciens ATCC 13332 (HQ735085) Rhizobium sp. IRBG74 (HG518322) A. tumefaciens ATCC 13332 (HQ735085) Rhizobium sp. IRBG74 (HG518322) A. tumefaciens CAF69 (EU399912) A. tumefaciens CAF428 (EU399933) A. tumefaciens ATCC 13332 (HQ735085) R. leguminosarum bv. viciae GLR1 (KC679411) R. leguminosarum bv. viciae 3841(NR_103919) R. leguminosarum bv. viciae 3841(NR_103919) R. leguminosarum bv. viciae GLR1 (KC679411) A. tumefaciens CAF69 (EU399912) R. etli EBRI 3 (AY221175) A. tumefaciens CAF69 (EU399912) A. tumefaciens CAF69 (EU399912) A. tumefaciens ATCC 13332 (HQ735085) R. leguminosarum bv. viciae 3841(NR_103919) R. leguminosarum bv. viciae 3841(NR_103919) A. tumefaciens ATCC 13332 (HQ735085) Rhizobium sp. USDA 1920 (U89823) R. leguminosarum bv. viciae 3841(NR_103919) R. leguminosarum bv. viciae 3841(NR_103919) R. leguminosarum bv. viciae GLR1 (KC679411) A. tumefaciens ATCC 13332 (HQ735085) A. tumefaciens CAF69 (EU399912) A. tumefaciens CAF69 (EU399912) R. leguminosarum bv. viciae 3841(NR_103919) R. leguminosarum bv. viciae 3841(NR_103919) R. leguminosarum bv. viciae 3841(NR_103919) R. leguminosarum bv. viciae 3841(NR_103919) R. leguminosarum bv. viciae 3841(NR_103919) R. leguminosarum bv. viciae 3841(NR_103919) R. leguminosarum bv. viciae 3841(NR_103919) -
Ac ce
pt
ed
NGB-FR-2 20 50 1.59 NGB-FR-4 23 68 1.67 NGB-FR-10 43 125 1.90 NGB-FR-11 16 41 1.57 NGB-FR-15 18 43 1.56 NGB-FR-20 40 122 1.88 NGB-FR-24 30 98 1.65 NGB-FR-25 44 132 1.96 NGB-FR-26 34 97 1.85 NGB-FR-27 42 145 1.93 NGB-FR-37 23 51 1.57 NGB-FR-38 27 97 1.70 NGB-FR-39 46 161 1.80 NGB-FR-51 32 103 1.85 NGB-FR-61 30 106 1.68 NGB-FR-62 31 96 1.79 NGB-FR-65 33 109 1.80 NGB-FR-70 62 152 1.95 NGB-FR-76 44 118 1.71 NGB-FR-88 31 104 1.75 NGB-FR-99 46 150 1.88 NGB-FR-101 40 140 1.84 NGB-FR-107 20 84 1.81 NGB-FR-114 26 89 1.71 NGB-FR-122 34 112 1.77 NGB-FR-126 58 164 2.14 NGB-FR-128 67 181 2.01 NGB-FR-132 37 131 1.92 NGB-FR-137 14 37 1.57 NGB-FR-138 24 61 1.59 NGB-FR-139 15 52 1.64 NGB-FR-140 20 79 1.56 NGB-FR-141 24 92 1.76 NGB-FR-142 35 103 1.78 NGB-FR-144 25 86 1.68 NGB-FR-145 18 55 1.67 NGB-FR-146 17 69 1.61 NGB-FR-147 20 80 1.82 NGB-FR-148 22 77 1.74 NGB-FR-149 13 45 1.64 NGB-FR-150 15 38 1.59 NGB-FR-151 21 64 1.70 Un-inoculated 0 0 1.55 LSD at 0.05 level 13.2 26.5 0.27 * Putative genospecies are based on the concatenated tree (Fig. 2)
Closest species (accession number)
Identity (%) 99 100 100 100 99 99 100 100 100 100 100 99 100 100 100 100 99 99 99 99 100 99 100 100 100 99 98 99 99 100 100 99 99 100 100 100 100 100 100 100 100 100 -
ip t
length (bp) 1352 1324 1327 1316 1329 1343 1335 1338 1326 1315 1313 1271 1328 1325 1318 1322 1312 1414 1478 1316 1313 1318 1474 1316 1315 1505 1522 1499 1516 1308 1311 1312 1469 1318 1315 1320 1316 1316 1311 1316 1317 1314 -
cr
Shoots dry weight (g/ plant)
us
Nodules dry weight (mg/plant)
an
No. of nodules/ plant
M
Rhizobial isolates
Putative Genospecies* III III III III III III III III III III III III III III III III I I I I III II III III III I I III I I I I III III III I I I I I I I
17
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Table 3 Protein alignment statistics used in this study as calculated by MEGA version 6 No. of alignment sites
No. of conserved sites
No. of variable sites
No. and percentage of parsimony informative sites
No. of singletons
glnA
59
293
203
90
80/27
10
gyrB
70
184
102
82
recA
67
154
118
36
glnA+ gyrB+ recA
56
631
429
202
nodA
49
162
76
86
ip t
No. of sequences
3
34/22
2
181/29
21
57/35
29
pt
ed
M
an
us
cr
79/43
Ac ce
Protein
18
Page 18 of 21
Ac ce p
te
d
M
an
us
cr
ip t
Figure
Fig.1. ML tree based on 16S rRNA sequences, showing the relationships among faba beansymbionts and recognized species of the genus Rhizobium–Agrobacterium. Bootstrap values are indicated for each node (1000 replicates). Sequences retrieved in this study are shaded. Abbreviations used, A: Agrobacterium; R: Rhizobium; B: Bradyrhizobium; NGB-FR: National Gene Bank-Faba bean Rhizobia.
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Fig.2. ML tree based on three concatenated proteins (glnA, gyrB, recA) sequences, showing the relationships among faba bean-symbionts and recognized species of the genus Rhizobium– Agrobacterium. Bootstrap values are indicated for each node (1000 replicates). Sequences retrieved in this study are shaded. Abbreviations used, A: Agrobacterium; R: Rhizobium; B: Bradyrhizobium; NGB-FR: National Gene Bank-Faba bean Rhizobia.
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Fig.3. ML tree based on nodA protein sequences. Bootstrap values are indicated for each node (1000 replicates). Sequences retrieved in this study are shaded. Abbreviations used, A: Agrobacterium; R: Rhizobium; B: Bradyrhizobium; NGB-FR: National Gene Bank-Faba bean Rhizobia.
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S0723-2020(14)00140-4 http://dx.doi.org/doi:10.1016/j.syapm.2014.10.001 SYAPM 25648
To appear in: Received date: Revised date: Accepted date:
11-7-2014 1-10-2014 3-10-2014
Please cite this article as: S.H. Youseif, F.H.A. El-Megeed, A. Ageez, E.C. Cocking, S.A. Saleh, Phylogenetic multilocus sequence analysis of native rhizobia nodulating faba bean (Vicia faba L.) in Egypt, Systematic and Applied Microbiology (2014), http://dx.doi.org/10.1016/j.syapm.2014.10.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Phylogenetic multilocus sequence analysis of native rhizobia nodulating faba bean (Vicia faba L.) in Egypt
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Sameh H. Youseif , Fayrouz H. Abd El-Megeed , Amr Ageez , Edward C. Cocking and
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Saleh A. Saleh
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National Gene Bank and Genetic Resources, Agricultural Research Center, Giza, Egypt
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Agricultural Genetic Engineering Research Institute, Agricultural Research Center, Giza, Egypt
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Fayrouz H. Abd El-Megeed National Gene Bank and Genetic Resources, Agricultural Research Center, Giza, Egypt E-mail addresses: [email protected] Address: 9 Gamaa st. Giza, Egypt. Post office Box: 12619
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Amr Ageez Agricultural Genetic Engineering Research Institute, Agricultural Research Center, Giza, Egypt E-mail addresses: [email protected] Address: 9 Gamaa st. Giza, Egypt. Post office Box: 12619
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Edward C. Cocking Centre for Crop Nitrogen Fixation, University of Nottingham, Nottingham, UK E-mail addresses: [email protected] Address: University of Nottingham, Nottingham NG7 2RD, UK
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Saleh A. Saleh
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National Gene Bank and Genetic Resources, Agricultural Research Center, Giza, Egypt E-mail addresses: [email protected] Address: 9 Gamaa st. Giza, Egypt. Post office Box: 12619
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Sameh H. Youseif *Corresponding author. National Gene Bank and Genetic Resources, Agricultural Research Center, Giza, Egypt Tel.:+2 0106 1147472 E-mail addresses: [email protected] Address: 9 Gamaa st. Giza, Egypt. Post office Box: 12619
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Centre for Crop Nitrogen Fixation, University of Nottingham, Nottingham NG7 2RD
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Abstract
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The taxonomic diversity of forty-two Rhizobium strains, isolated from nodules of faba bean
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grown in Egypt, was studied using 16S rRNA sequencing, multilocus sequence analyses
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(MLSA) of three chromosomal housekeeping loci and one nodulation gene (nodA). Based on the
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16S rRNA gene sequences, most of the strains were related to Rhizobium leguminosarum, R. etli,
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and R. radiobacter (syn. A. tumefaciens). A Maximum Likelihood (ML) tree built from the
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concatenated sequences of housekeeping proteins encoded by glnA, gyrB and recA, revealed the
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existence of three distinct genospecies (I, II and III) affiliated to the defined species within the
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genus Rhizobium/ Agrobacterium. Seventeen strains in genospecies I could be classified as R.
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leguminosarum sv. viciae. Whereas, a single strain of genospecies II was linked to R. etli.
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Interestingly, twenty-four strains of genospecies III were identified as A. tumefaciens. Strains of
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R. etli and A. tumefaciens have been shown to harbor the nodA gene and formed effective
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symbioses with faba bean plants in Leonard jar assemblies. In the nodA tree, strains belonging to
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the putative genospecies were closely related to each other and were clustered tightly to R.
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leguminosarum sv. viciae, supporting the hypothesis that symbiotic and core genome of the
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species have different evolutionary histories and indicative of horizontal gene transfer among
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these rhizobia.
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Introduction Faba bean (Vicia faba L.) is a major food and feed legume because of the high nutritional
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value of its seeds [10]. In 2010, the world production of faba bean was 4.0 million tons, and most
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of its global production is located in China, Ethiopia, France and Egypt [11]. Faba bean is most
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commonly included in the diets of inhabitants of the Middle East, the Mediterranean region and
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China. They use faba bean seeds as green or dried, fresh or canned [7]. Faba bean is one of the
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most important legume crops grown in Egypt. The cultivated faba bean area in Egypt has been
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estimated to be 0.08 million ha and produced about 0.26 million tons [12].
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Faba bean commonly establishes effective nitrogen fixation symbiosis with fast-growing
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rhizobia of the species Rhizobium leguminosarum [52]. R. leguminosarum species comprises
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three symbiovars [22]: viciae (nodulating pea and vetch), trifolii (nodulating clover), and
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phaseoli (nodulating beans), which show a common chromosomal background and different host
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range specificities [24, 36]. Initially, all faba bean-nodulating rhizobia were classified as R.
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leguminosarum symbiovar (sv.) viciae [1]. Later, R. fabae was described as a new faba bean-
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nodulating species based on multilocus sequence analyses, sequence analysis of 16S rDNA and
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several phenotypic characteristics [48]. To date R. leguminosarum sv. viciae is distributed widely
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around the world, while R. fabae has been discovered in Chinese fields [49]. Recently,
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Rhizobium laguerrereae was identified as a faba bean nodulating microsymbiont in several
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countries including Spain, Peru and Tunisia [37].
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Previously, twenty-eight rhizobial strains were isolated from faba bean root nodules
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grown in Egypt [40]. Based on partial 16S rDNA sequencing, a majority of these strains was
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identified as Rhizobium leguminosarum sv. viciae. Nevertheless, the use of 16S rRNA gene
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sequence analyses for taxonomical purposes has proven difficult and cannot be done with great
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certainty [53]. Protein-encoding housekeeping genes have been proposed as alternative
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phylogenetic markers to help in distinguishing closely related species [45]. Although
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housekeeping genes have a higher evolution rate than 16S rRNA genes, they are conserved
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enough to provide informative phylogenetic data [6]. Therefore, in the present study, the MLSA
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technique was used to study the taxonomic position of native rhizobia nodulating faba bean in
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Egypt. For this purpose, the phylogenies of housekeeping genes coding for recombination protein
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(recA), glutamine synthetase (glnA), DNA gyrase B subunit (gyrB) and the 16S rRNA gene were
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determined. The phylogeny of the symbiotic gene for nodulation (nodA), as well as the capacity
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of strains to induce effective nodules, was also investigated.
Materials and Methods
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Bacterial isolation
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Rhizobium strains were isolated from surface-sterilized root nodules according to the standard
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routine laboratory techniques described by Vincent [54]. At flowering stage, nodules were
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collected from field-grown faba bean from different sites that represented sixteen governorates in
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Egypt. Strain numbers, geographical locations and their respective soil characteristics are listed
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in Table 1. Root nodules were surface sterilized by washing for 30 s with 95% ethanol, immersed
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in 3% sodium hypochlorite and finally were washed six times using sterile distilled water.
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Following sterilization, nodules were individually crushed aseptically in 1 ml sterile distilled
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water, streaked on the surface of Yeast Extract Mannitol Agar (YEM) plates supplemented with
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0.025 g L_1 of congo red and finally were incubated at 28 ˚C for 3-5 days [44].
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Plant nodulation assay
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The nodulation assays were performed in Leonard jar assemblies [44] containing sterilized sandy
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soil. The soil was analyzed according to Page et al. [34]. The main physical and chemical
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properties of the soil used are presented in supplementary Table S1. Each Leonard jar assembly
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was planted with three surface-sterilized seeds of faba bean cultivar Giza 716 and irrigated with
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nitrogen free nutrient solution [57]. Each seed was inoculated with 1.0 ml (about 1 X108 cells) of
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log-phase rhizobial cultures. The uninoculated plants were used as negative controls. Faba bean
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plants were cultivated using a randomized complete block design with three replicates in a
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controlled greenhouse at 24°C for 12 h (light) and 12°C for 12 h (dark). All treatments received
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20 ppm N as a starter N dose. After 35 days of planting, plants were uprooted and assayed for the
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number and dry weight of nodules, as well as the dry weight of shoots. Data were subjected to
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analysis of variance using MSTAT analysis software [43].
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Phenotypic characterization
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The tolerance ability of different rhizobial isolates to grow under stress conditions (salinity, high
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temperatures, acidity and alkalinity) was assessed as described by Youseif et al. [59]. The salt
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tolerance of rhizobia was tested on YEM agar medium containing 0.1%, 0.5%, 1%, 2%, 3%,
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3.5%, 4%, 5% and 6% (w/v) NaCl. Temperature tolerance of rhizobia was examined by
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inoculating rhizobia on YEM agar plates and incubating at 28, 37, 40, 42, 45 and 48 ˚C.
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Nevertheless, pH tolerance was evaluated by growing rhizobia on YEM agar medium adjusted to
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a pH range from pH 5 to pH 11. After 2-3 days of incubation at 28˚C, plates were examined for
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bacterial growth.
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DNA isolation, PCR amplifications and gene sequencing
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Genomic DNA of rhizobial cells was isolated using Wizard® Genomic DNA purification Kit
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(Promega® Corporation, Madison, USA). PCR was performed using the standard reaction
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mixture (50 µl) containing: 1X PCR buffer, 1.5 mM MgCl2, 5% dimethyl sulfoxide, 200 mM of
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each dNTPs, 15 pmol of each primer, 1 U of Taq polymerase enzyme (Promega® Corporation,
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Madison, USA) and 50 ng of DNA template. PCR conditions and primer sequences used for
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gene amplification and sequencing are shown in Table S2.
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Phylogenetic analyses
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Sequence reads were edited and assembled using the DNASTAR software (Lasergene, Madison,
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USA). Sequence similarity searches were performed at the National Center for Biotechnology
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Information (NCBI) server using BLASTN to identify 16S rRNA genes and BLASTX to
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annotate the proteins encoded by housekeeping and nodulation genes. The sequences were
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aligned using Clustal W version 1.8 [2], and subjected to phylogenetic analysis. Phylogenetic
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trees were constructed using the maximum likelihood methods (ML) in MEGA version 6 [46],
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and 1000 bootstrap replication to assess branching confidence. For 16S rRNA, phylogeny was
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constructed based on nucleotide sequences while for housekeeping (gyrB, glnA and recA) and
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nodulation (nodA) genes, phylogenies were constructed based on amino acids sequences. Using
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Mega 6 software, maximum likelihood fits with 48 different amino acid (aa) substitution models
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were tested. Models with the lowest BIC scores (Bayesian Information Criterion) are considered
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to describe the best substitution pattern. The JTT model was selected as it gives the lowest BIC
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scores.
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Results
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Nodulation, symbiotic effectiveness and phenotypic characteristics
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Forty-two local rhizobial isolates were isolated from root nodules of field grown faba bean in
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sixteen governorates representing most faba bean cultivation areas in Egypt (Table 1). The
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symbiotic effectiveness of rhizobial isolates was confirmed by using plant nodulation assay in
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Leonard jar assemblies (Table 2). All rhizobial isolates were able to induce symbiosis with faba
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beans, resulting in the number of nodules ranging from 13- 67 nodules/plant. Nodules dry weight
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varied from 37 mg/plant for isolate NGB-FR 137 to 181 mg/plant for isolate NGB-FR 128.
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Shoot dry weight, as an indirect measure of the nitrogen fixation benefit, was determined among
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tested isolates. Faba bean plants inoculated by strains NGB-FR 126 and 128 showed the highest
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shoot dry weights (2.14 and 2.01 g/plant, respectively) with significant increases over the
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uninoculated control (1.55 g/plant). All rhizobial isolates were phenotypically characterized
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under different stress conditions (Table S3). Most of the isolates appeared resistant to salinity
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and grew in a range from 0.1% to 5% NaCl. However, twenty-one isolates tolerated 5% NaCl.
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Considering tolerance to acidic and alkaline conditions, rhizobial isolates were resistant to a wide
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pH range started from 5 to 11. Under high temperatures, the isolates showed a variable tolerance
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to a wide range of temperatures (28-45 ºC). Most of isolates were sensitive to 42 ºC, however
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only eight Rhizobium isolates (NGB-FR 24, 26, 27, 38, 39, 51, 99 and 101) tolerated 45 ºC.
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16S rDNA ribosomal gene
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Nearly full-length 16S rDNA gene regions (1271-1522 bp) were successfully amplified and
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sequenced for all rhizobial strains. According to the sequences similarity, local rhizobial isolates
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were closely related to the defined species within the genus Rhizobium/ Agrobacterium (Table
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2). The maximum likelihood (ML) phylogenetic tree based on 16S rRNA sequences (Fig. 1),
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classified faba bean-nodulating rhizobia into five distinctive groups (A, B, C, D and E). Strains
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in group A were clustered at high bootstrap (BT) value (100%) along with R. leguminosarum sv.
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viciae strains (Access. No. EU730596 and NR_103919). They also were grouped with R.
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leguminosarum sv. viciae strain RL2 (Access. No. JQ303081) that was isolated also from faba
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bean root nodules grown in Egypt. Group B was a sister monophyletic cluster of group A and
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was linked along with R. leguminosarum RN2 (Access. No. KC884251). Strain NGB-FR 101,
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the only representative of group C showed high relatedness to R. etli EBRI 3 (Egyptian
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microsymbionts nodulating bean [41]. Strain NGB-FR 137 the sole representative of group D
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had a close phylogenetic affiliation to Rhizobium sp. USDA 1920, a symbiotic bacterium
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nodulating Medicago ruthenica [51]. Group E was a cluster of twenty-four strains along with
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five different A. tumefaciens references (Access. No. KJ499777, KF460525, HQ735085,
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KF439828 and KF730752) supported by a 100% bootstrap value.
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Protein-coding housekeeping genes
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Sequences for the glnA (947–974 bp), gyrB (648–792 bp) and recA (508–577 bp) genes were
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isolated from all Rhizobium strains. New sequences of the forty-two test strains were deposited
192
into Genbank database (Table S4). Sequences of several housekeeping genes retrieved from the
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Genbank were shorter than our newly sequenced ones. Therefore, in the final alignments, parts
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of the new sequences were omitted. Consequently, the lengths of the alignments used were 879
195
bp, 552 bp and 462 bp for glnA, gyrB and recA, respectively. DNA sequences were translated to
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proteins of 293 aa, 184 aa and 154 aa for glnA, gyrB and recA, respectively. The ML
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phylogenetic trees based on individual housekeeping proteins (aa) are presented in Figs. S1, S2
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and S3. Proteins encoded by different housekeeping loci showed variable informative positions
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(Table 3). According to the resultant analysis, gyrB protein had the greatest percentage of
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parsimony-informative characters (43%) of the positions used for the analysis, followed by 27%
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and 22% for glnA and recA proteins, respectively.
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Concatenated sequence analysis
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Aligned sequences from the glnA (293 aa), gyrB (184 aa) and recA (154 aa) protein-encoding
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genes were concatenated and 631(aa) sites were obtained (Table 3). The ML phylogenetic tree,
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based on the combined partial proteins sequences was constructed (Fig. 2). The results of the
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concatenated sequences were generally congruent with those of the individual protein-encoding
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genes (Figs S1, S2 and S3), but generated more robust and confident grouping. Based on the
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concatenated proteins analysis, faba bean-nodulating rhizobia were classified into three
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genospecies I, II and III (Fig. 2). Genospecies I including seventeen strains, clustered with R.
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leguminosarum sv. viciae strain 3841. This phylogeny was similar in all core protein analyses
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and was mostly consistent with 16S rRNA gene sequencing. One exception was for strain NGB-
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FR 137, that was placed in a distinct site according to 16S rRNA phylogeny.
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Strain NGB-FR 101, the only representative for genospecies II, was clustered on a well-
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supported branch (BT 85%) with type strain R. etli CFN 42 and R. etli Mim1 in the concatenated
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tree. This group was well separated from other genomic species in the concatenated tree, but its
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position varied in the individual ML gene trees.
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Interestingly, twenty-four strains classified as genospecies III were linked tightly with A.
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tumefaciens supported by high bootstrap value (96%). This grouping was commonly identified in
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gyrB, recA and 16S sequence trees, nevertheless in the glnA tree strain NGB-FR 39 was placed
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in a sister monophyletic position.
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Phylogeny of nodA symbiotic gene
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The partial sequences (488 to 574 bp) of the nodA nodulation gene were determined for
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all strains with the exception of strains NGB-FR 37 and NGB-FR 99. DNA sequences were
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translated to their respective proteins. Basic sequence alignment statistics for protein encoded by
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nodA is shown in Table 3. The nodA based-ML phylogenetic tree was constructed (Fig. 3).
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Unlike the grouping based on protein-encoding housekeeping genes and 16S rRNA sequences,
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all test strains within different genospecies had a very close phylogenetic affinity with R.
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leguminosarum sv. viciae.
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Discussion
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Phenotypic characteristics
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Faba bean is grown worldwide as a protein source for food and feed, and offers an
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important ecosystem service to cropping systems via its ability to symbiotically fix atmospheric
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N2 [21]. In Egypt, faba bean is considered as a cash crop and has been cultivated for over 3000
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years [42]. In this study, forty-two rhizobial strains were isolated from root nodules of field-
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grown faba bean, representative of a wide range of geographical regions in Egypt. All strains
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were able to nodulate faba bean plants and resulted in different nodulation patterns. The present
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study demonstrated a high phenotypic diversity among faba bean-nodulating rhizobia under
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stress conditions. The majority of strains was tolerant to salt stress, and 50% of isolated strains
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appeared highly salinity-tolerant and grew at 5% NaCl. Similar results were reported by
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Shamseldin et al. [40], who isolated faba bean-rhizobia under Egyptian soil conditions able to
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grow at 3 % NaCl. Most of the tested strains could grow under high temperatures and eight of
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them were able to grow at 45 ºC. The tolerance of rhizobia to stress conditions appeared to be
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more of a strain-specific than species-specific phenomenon, supporting the finding reported by
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Amarger et al. [3].
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Phylogenetic analyses of faba bean- nodulating rhizobia Sequence analysis of 16S rRNA genes has been used as standard practice in bacterial
247
taxonomy [25]. To date, most of rhizobial strains isolated from Vicia faba were found to be
248
closely related to R. leguminosarum [31, 38, 40, 47, 49]. In this study, based on the 16S rRNA
249
gene analysis, only 38% of faba bean-nodulating rhizobia are closely related to R.
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leguminosarum, while 57% of tested strains are related to R. radiobacter (syn. A. tumefaciens)
251
supported by highly bootstrap value (100%).
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The use of 16S rRNA gene as a sole molecular marker has been criticized due to its
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presence in multiple copies in a genome of some bacteria [17], its susceptibility to genetic
254
recombination and horizontal gene transfer [53] and low phylogenetic power among closely
255
related species [15, 38]. Considering the limitations of the 16S rRNA gene in bacterial
256
taxonomy, the multilocus sequence analysis (MLSA) using protein-coding genes (housekeeping
257
genes) has been preferred for studying closely-related species of bacteria, including rhizobia [4,
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9, 27, 35]. By using a concatenation analysis of several housekeeping genes, the phylogenetic
259
signals can be enhanced to provide highly supported clades [6].
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Although a limited variation in topologies was observed between the three individual ML
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gene trees (Fig. S1, S2 and S3), strains in this study were clustered into three distinct
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monophyletic genospecies (I, II and III) in every housekeeping gene tree as well as in the ML
263
tree of the concatenated sequences (Fig. 2). Genospecies I was closely related to R.
264
leguminosarum sv. viciae strain 3841 in all of the analyzed housekeeping genes. Whereas, strain
265
NGB-FR 137 (related to the genus Rhizobium, based on the 16S rRNA sequence data), was
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integrated into R. leguminosarum sv. viciae - genospecies I, supporting the fact that the 16S
267
rRNA gene is not adequate for identification at species level within the genus Rhizobium [38].
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Remarkably, NGB-FR 101 the only representative strain of genospecies II was related to
269
R. etli CFN 42 and R. etli Mim1 with a 85% concatenated bootstrap value. R. etli was originally
270
described as exclusively nodulating and fixing nitrogen with Phaseolus vulgaris [39].
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Nevertheless, isolation of R. etli strains from nodulated faba bean plants has been previously
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reported in Chinese [47] and Egyptian soils [40]. 9
Page 9 of 21
Another interesting finding in this study is that, 57% of tested strains were identified as
274
genospecies III and were clustered together with R. radiobacter (syn. A. tumefaciens) supported
275
by concatenated high bootstrap values (96%). Strains of the genus Agrobacterium (now, rather
276
controversially, included in the genus Rhizobium [13, 58] have been frequently isolated from
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root nodules of various legumes including common bean [30], Sesbania [8], soybean [59], lentil
278
[35] and faba bean [50]. In general, Agrobacteria failed to nodulate their hosts [4] and this is may
279
be due to the loss of their nodulation genes during storage [20], supporting the idea that these
280
isolates may be opportunistic bacteria able to colonize nodules induced by rhizobia [35]. In
281
contrast, our results confirmed the nodulation ability of A. tumefaciens to nodulate faba bean
282
under Leonard jar conditions. This finding in accordance with other publications supports the
283
symbiotic effectiveness of Agrobacteria to nodulate their original hosts [8, 59].
284
Symbiosis related gene: Implication of Sym plasmid transfer
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The nodulation (Nod) factors determine the legume-rhizobia symbiosis [24]. These genes
286
are often found on the symbiotic plasmid pSym and enable the rhizobia to fix nitrogen in
287
symbiosis with legume plants [23]. Because of the nature of the Nod factor acyl group attached
288
by the nodA gene can contribute to the determination of host range [33], nodA has been used as a
289
symbiotic marker in the analyses of specificity between rhizobia and different host plants [35,
290
59]. In respect to the nodA protein-based phylogenetic tree (Fig. 3), nearly all strains in the
291
putative genospecies I, II and III were grouped along with R. leguminosarum sv. viciae strain
292
3841 into one separate branch supported by a highly bootstrap value (88%). However strain
293
NGB-FR 101, the only representative of genospecies III (related to R. etli, based on the
294
concatenated core gene analyses), formed a sister monophyletic cluster to R. leguminosarum sv.
295
viciae, but was placed in a distinct position (100% BT). In bacteria, the chromosomal genetic
296
background and the plasmid-borne genetic background are often not correlated due to horizontal
297
transfer of plasmid-borne genes [32]. Moreover, different rhizobial species can share similar
298
symbiotic genes, and different symbiosis genes can be harbored by similar genomic backgrounds
299
[16, 24]. Thus, incongruence may exist between phylogeny based on symbiotic and
300
chromosomal genes, and this phenomenon has been demonstrated to play a role in the evolution
301
and structure of natural populations of rhizobia [5, 26, 55]. The nodA based-phylogenies suggest
302
that, A. tumefaciens and R. etli in this study might have acquired their symbiosis-related genes by
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lateral gene transfer from R. leguminosarum (Fig. 3), with a potential candidate being R.
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leguminosarum sv. viciae. The transfer of Sym plasmid within R. leguminosarum sv. viciae
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populations in the field was previously published [23, 40, 49]. Gene transfer and rearrangement
306
of symbiotic plasmids could be involved in the acquisition and evolution of rhizobial symbiotic
307
functions [24]. In the case of Agrobacterium, the mobility of Sym genes has been exemplified in
308
laboratory experiments [19, 29]. Nevertheless, it was recently found that naturally occurring
309
agrobacterial symbionts possess a Sym-plasmid with symbiosis-specific genes and can
310
effectively nodulate their original hosts and fix N2 to the same degree as standard rhizobia [8,
311
60].
312
In conclusion, forty-two Rhizobium strains isolated from faba bean grown in Egypt, were
313
characterized using a polyphasic approach. Our results revealed that (1) the majority of the
314
Egyptian faba bean rhizobia belonged to the species R. radiobacter (syn. A. tumefaciens), and R.
315
leguminosarum (2) All these bacteria formed effective symbioses with faba bean plants,
316
suggesting that they are true symbionts of faba bean in Egypt (3) Irrespective of their
317
chromosomal background, the symbiosis related gene (nodA) of faba bean symbionts were
318
highly related to each other with close affinity to R. leguminosarum sv. viciae, suggesting that
319
symbiotic genes have a common origin that is incongruent with chromosomal loci.
320
Acknowledgements
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This work has been financed by the Science and Technology Development Fund (STDF), Egypt,
322
project ID: STDF 901.
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324 325 326 327 328 329 330 331 332 333 334 335 336
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[54] Vincent, J.M. 1970 A manual for the practical study of root nodule bacteria. IBP Handbook No. 15, Black Well Scientific Publications, Oxford, p. 164. [55] Vinuesa, P., Silva, C., Werner, D., Martinez-Romero, E. (2005) Population genetics and phylogenetic inference in bacterial molecular systematics: the roles of migration and recombination in Bradyrhizobium species cohesion and delineation. Mol. Phylogenet. Evol. 34, 29–54. [56] Weisburg, W.G., Barns, S.M., Pelletie, D.A., Lane, D.J. (1991) 16S rDNA amplification for phylogenetic study. J. Bacteriol. 173, 697–703. [57] Werner, D., Wilcockson, J., Zimmermann, E. (1975), Adsorption and selection of rhizobia with ion-exchange papers. Arch. Microbiol. 105, 27-32. [58] Young, J.M., Kuykendall, L.D., Martínez-Romero E., Kerr, A., Sawada, H. (2001) A revision of Rhizobium Frank 1889, with an emended description of the genus, and the inclusion of all species of Agrobacterium Conn 1942 and Allorhizobium undicola de Lajudie et al. 1998 as new combinations: Rhizobium radiobacter, R. rhizogenes, R. rubi, R. undicola and R. vitis, Int. J. Syst. Evol. Microbiol. 51, 89-103. [59] Youseif, S.H., Abd El-Megeed, F.H., Ageez, A., Mohamed, Z.K., Shamseldin, A., Saleh, S.A. (2014) Phenotypic characteristics and genetic diversity of rhizobia nodulating soybean in Egyptian soils. Eur. J. Soil Biol. 60, 34-43
491 492 493 494
[60] Youseif, S.H., Abd El-Megeed, F.H., Khalifa, M.A., Saleh, S.A. (2014) Symbiotic effectiveness of Rhizobium (Agrobacterium) compared to Ensifer (Sinorhizobium) and Bradyrhizobium genera for soybean inoculation under field conditions. Res. J. Microbiol. 9, 151-162
M
an
us
cr
ip t
473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490
498 499 500 501 502 503 504 505
pt
497
Ac ce
496
ed
495
506 507 508
15
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Table 1 List of rhizobial local isolates used in this study, their respective geographical regions and soil characteristics pH
E.C (dS m-1)
N (ppm)
P (ppm)
K (ppm)
Clay Clay Clay Clay Clay Sandy Clay Clay Clay Clay Clay Clay Clay Clay Clay Clay Clay Clay Clay Clay Sandy Sandy Clay Clay Clay Sandy Sandy Calcareous Clay Clay Sandy Sandy Clay Clay Clay Clay Clay Clay Clay Clay Clay Clay
7.6 7.8 7.9 8.6 8.4 8.4 7.9 7.6 7.4 7.6 8.2 7.7 8.2 7.7 7.4 7.8 7.73 7.6 7.7 7.8 8.4 8.7 7.7 7.8 7.6 8.6 8.5 8.6 7.5 7.6 8.4 8.2 7.7 7.7 7.6 7.3 7.5 7.8 7.7 8.1 7.7 7.73
0.75 0.37 0.72 0.67 0.86 0.15 0.21 0.52 0.54 0.54 2.57 0.85 1.30 0.53 0.45 0.50 1.12 0.35 0.90 0.60 1.30 1.50 0.56 0.16 0.24 0.24 0.14 0.97 0.44 0.67 0.55 0.41 0.56 0.56 0.35 0.93 0.42 0.37 1.63 1.61 1.33 1.12
68 56 42 25 40 10 45 44 80 55 15 60 20 80 55 60 47 42 44 45 20 10 45 75 56 10 15 10 65 48 10 10 45 45 42 75 75 56 42 26.5 31.3 47
7.5 6.5 3.2 6.50 3.4 3.3 6.2 6.3 9.2 5.5 4.3 2.6 6.3 9.2 3.4 2.9 10.9 8.2 12.8 13 4.5 6.5 10.2 9.4 8.3 3.4 5.2 3.2 11.2 8.5 3.5 2.4 10.2 10.2 8.2 5.6 8.2 6.5 6.3 5.0 4.4 10.9
288 320 169 199 210 132 212 325 280 250 315 150 310 360 190 210 372 352 368 344 250 214 366 350 288 152 112 125 365 275 112 125 366 366 352 225 330 320 310 140 160 372
cr
ip t
Soil texture
ed
M
an
us
Behairah, Edko, Al-Zohoor village Behairah, Rasheed, Al-Borg village Dakahlia, Mansoura Dakahlia, Menyat El-Nasr, Brembala village Damietta/ Damietta Ismailia/ Al-Qassasen Kafr El-Sheikh/ Kafr El-Sheikh, Awerah village Kafr El Sheikh/ Highway, Ewira village Kafr El Sheikh/ International Highway Kafr El Sheikh/ Al-Hamool, Al-kheregeen village Sharkia/Al-Hosanyah Sharkia/Al-Hosanyah, Abo omar village Sharkia/Al-Hosanyah, Al-Tarek village Gharbia/ Al-Santa, Al-Gemizah Menoufia/ Al-Bagoor, Meet Al-Beydah village Menoufia/ Al-Bagoor, Al-Aatf village Port Said/ Port Said, Al-kaab area Giza/ Giza, Agricultural Research Center Menia/ Abo-Qourqas, Beni mousa village Assuit/ Assuit New Valley/ El-Kharga Agricultural Res. Station New Valley/ Al- Dakhla, Al-Moosheyah village Fayoum/ Ebsheway, Shakshook village Beni Suef/ Nasser, Dandil village Beni Suef/Seds, Haramashant village North Sinai/ Romana North Sinai/Beer Al-Aabd Behaira /Noubaria Fayoum/ Atsa, Al-Mahmoudia village Fayoum/ Youssef Al-Sedeek, Kahk village Fayoum/ Youssef Al-Sedeek, Mafarek Al-Sedeek Fayoum/ Youssef Al-Sedeek, Al-Khergeen village Fayoum/ Ebsheway, Shakshook village, location 1 Fayoum/ Ebsheway, Shakshook village, location 2 Giza/ Giza, Agricultural Research Center Behairah/ Edko Behairah/ Edko, 6th October village Behairah/ Rasheed, Al-Gedia village Port Said/ Sahl Port Said, Agricultural area 1 Port Said/ Sahl Port Said, Agricultural area 2 Port Said/ Sahl Port Said, Agricultural area 6 Port Said/ Port Said, Al-kaab area
pt
NGB-FR-2 NGB-FR-4 NGB-FR-10 NGB-FR-11 NGB-FR-15 NGB-FR-20 NGB-FR-24 NGB-FR-25 NGB-FR-26 NGB-FR-27 NGB-FR-37 NGB-FR-38 NGB-FR-39 NGB-FR-51 NGB-FR-61 NGB-FR-62 NGB-FR-65 NGB-FR-70 NGB-FR-76 NGB-FR-88 NGB-FR-99 NGB-FR-101 NGB-FR-107 NGB-FR-114 NGB-FR-122 NGB-FR-126 NGB-FR-128 NGB-FR-132 NGB-FR-137 NGB-FR-138 NGB-FR-139 NGB-FR-140 NGB-FR-141 NGB-FR-142 NGB-FR-144 NGB-FR-145 NGB-FR-146 NGB-FR-147 NGB-FR-148 NGB-FR-149 NGB-FR-150 NGB-FR-151
Governorate/ site
Ac ce
Isolate No.*
*NGB-FR: National Gene Bank-Faba bean Rhizobia.
16
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Table 2 Symbiotic properties of faba bean nodulating rhizobia used in this study and their phylogenetic homology Identity based on 16S rRNA gene sequence
A. tumefaciens ATCC 13332 (HQ735085) A. tumefaciens ATCC 13332 (HQ735085) A. tumefaciens ATCC 13332 (HQ735085) A. tumefaciens ATCC 13332 (HQ735085) A. tumefaciens ATCC 13332 (HQ735085) Rhizobium sp. IRBG74 (HG518322) A. tumefaciens ATCC 13332 (HQ735085) A. tumefaciens ATCC 13332 (HQ735085) A. tumefaciens ATCC 13332 (HQ735085) A. tumefaciens ATCC 13332 (HQ735085) Rhizobium sp. IRBG74 (HG518322) A. tumefaciens ATCC 13332 (HQ735085) Rhizobium sp. IRBG74 (HG518322) A. tumefaciens CAF69 (EU399912) A. tumefaciens CAF428 (EU399933) A. tumefaciens ATCC 13332 (HQ735085) R. leguminosarum bv. viciae GLR1 (KC679411) R. leguminosarum bv. viciae 3841(NR_103919) R. leguminosarum bv. viciae 3841(NR_103919) R. leguminosarum bv. viciae GLR1 (KC679411) A. tumefaciens CAF69 (EU399912) R. etli EBRI 3 (AY221175) A. tumefaciens CAF69 (EU399912) A. tumefaciens CAF69 (EU399912) A. tumefaciens ATCC 13332 (HQ735085) R. leguminosarum bv. viciae 3841(NR_103919) R. leguminosarum bv. viciae 3841(NR_103919) A. tumefaciens ATCC 13332 (HQ735085) Rhizobium sp. USDA 1920 (U89823) R. leguminosarum bv. viciae 3841(NR_103919) R. leguminosarum bv. viciae 3841(NR_103919) R. leguminosarum bv. viciae GLR1 (KC679411) A. tumefaciens ATCC 13332 (HQ735085) A. tumefaciens CAF69 (EU399912) A. tumefaciens CAF69 (EU399912) R. leguminosarum bv. viciae 3841(NR_103919) R. leguminosarum bv. viciae 3841(NR_103919) R. leguminosarum bv. viciae 3841(NR_103919) R. leguminosarum bv. viciae 3841(NR_103919) R. leguminosarum bv. viciae 3841(NR_103919) R. leguminosarum bv. viciae 3841(NR_103919) R. leguminosarum bv. viciae 3841(NR_103919) -
Ac ce
pt
ed
NGB-FR-2 20 50 1.59 NGB-FR-4 23 68 1.67 NGB-FR-10 43 125 1.90 NGB-FR-11 16 41 1.57 NGB-FR-15 18 43 1.56 NGB-FR-20 40 122 1.88 NGB-FR-24 30 98 1.65 NGB-FR-25 44 132 1.96 NGB-FR-26 34 97 1.85 NGB-FR-27 42 145 1.93 NGB-FR-37 23 51 1.57 NGB-FR-38 27 97 1.70 NGB-FR-39 46 161 1.80 NGB-FR-51 32 103 1.85 NGB-FR-61 30 106 1.68 NGB-FR-62 31 96 1.79 NGB-FR-65 33 109 1.80 NGB-FR-70 62 152 1.95 NGB-FR-76 44 118 1.71 NGB-FR-88 31 104 1.75 NGB-FR-99 46 150 1.88 NGB-FR-101 40 140 1.84 NGB-FR-107 20 84 1.81 NGB-FR-114 26 89 1.71 NGB-FR-122 34 112 1.77 NGB-FR-126 58 164 2.14 NGB-FR-128 67 181 2.01 NGB-FR-132 37 131 1.92 NGB-FR-137 14 37 1.57 NGB-FR-138 24 61 1.59 NGB-FR-139 15 52 1.64 NGB-FR-140 20 79 1.56 NGB-FR-141 24 92 1.76 NGB-FR-142 35 103 1.78 NGB-FR-144 25 86 1.68 NGB-FR-145 18 55 1.67 NGB-FR-146 17 69 1.61 NGB-FR-147 20 80 1.82 NGB-FR-148 22 77 1.74 NGB-FR-149 13 45 1.64 NGB-FR-150 15 38 1.59 NGB-FR-151 21 64 1.70 Un-inoculated 0 0 1.55 LSD at 0.05 level 13.2 26.5 0.27 * Putative genospecies are based on the concatenated tree (Fig. 2)
Closest species (accession number)
Identity (%) 99 100 100 100 99 99 100 100 100 100 100 99 100 100 100 100 99 99 99 99 100 99 100 100 100 99 98 99 99 100 100 99 99 100 100 100 100 100 100 100 100 100 -
ip t
length (bp) 1352 1324 1327 1316 1329 1343 1335 1338 1326 1315 1313 1271 1328 1325 1318 1322 1312 1414 1478 1316 1313 1318 1474 1316 1315 1505 1522 1499 1516 1308 1311 1312 1469 1318 1315 1320 1316 1316 1311 1316 1317 1314 -
cr
Shoots dry weight (g/ plant)
us
Nodules dry weight (mg/plant)
an
No. of nodules/ plant
M
Rhizobial isolates
Putative Genospecies* III III III III III III III III III III III III III III III III I I I I III II III III III I I III I I I I III III III I I I I I I I
17
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Table 3 Protein alignment statistics used in this study as calculated by MEGA version 6 No. of alignment sites
No. of conserved sites
No. of variable sites
No. and percentage of parsimony informative sites
No. of singletons
glnA
59
293
203
90
80/27
10
gyrB
70
184
102
82
recA
67
154
118
36
glnA+ gyrB+ recA
56
631
429
202
nodA
49
162
76
86
ip t
No. of sequences
3
34/22
2
181/29
21
57/35
29
pt
ed
M
an
us
cr
79/43
Ac ce
Protein
18
Page 18 of 21
Ac ce p
te
d
M
an
us
cr
ip t
Figure
Fig.1. ML tree based on 16S rRNA sequences, showing the relationships among faba beansymbionts and recognized species of the genus Rhizobium–Agrobacterium. Bootstrap values are indicated for each node (1000 replicates). Sequences retrieved in this study are shaded. Abbreviations used, A: Agrobacterium; R: Rhizobium; B: Bradyrhizobium; NGB-FR: National Gene Bank-Faba bean Rhizobia.
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Fig.2. ML tree based on three concatenated proteins (glnA, gyrB, recA) sequences, showing the relationships among faba bean-symbionts and recognized species of the genus Rhizobium– Agrobacterium. Bootstrap values are indicated for each node (1000 replicates). Sequences retrieved in this study are shaded. Abbreviations used, A: Agrobacterium; R: Rhizobium; B: Bradyrhizobium; NGB-FR: National Gene Bank-Faba bean Rhizobia.
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Fig.3. ML tree based on nodA protein sequences. Bootstrap values are indicated for each node (1000 replicates). Sequences retrieved in this study are shaded. Abbreviations used, A: Agrobacterium; R: Rhizobium; B: Bradyrhizobium; NGB-FR: National Gene Bank-Faba bean Rhizobia.
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