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Genetic diversity of rhizobia nodulating native Vicia spp. in Sweden
Accepted Manuscript Title: Genetic diversity of rhizobia nodulating native Vicia spp. in Sweden Author: Osei Yaw Ampomah Kerstin Huss-Danell PII: DOI: Reference:
S0723-2020(16)00021-7 http://dx.doi.org/doi:10.1016/j.syapm.2016.02.002 SYAPM 25751
To appear in: Received date: Revised date: Accepted date:
12-1-2016 8-2-2016 9-2-2016
Please cite this article as: O.Y. Ampomah, K. Huss-Danell, Genetic diversity of rhizobia nodulating native Vicia spp. in Sweden, Systematic and Applied Microbiology (2016), http://dx.doi.org/10.1016/j.syapm.2016.02.002 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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Genetic diversity of rhizobia nodulating native Vicia spp. in Sweden
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Osei Yaw Ampomaha1* and Kerstin Huss-Danella
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Sciences (SLU), SE-90183 Umeå, Sweden
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Biotechnology, S-10691, Stockholm, Sweden
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*Corresponding author. Royal Institute of Technology (KTH), Division of Glycoscience, School
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of Biotechnology, S-10691, Stockholm, Sweden.
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Telephone: +46 706084168
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Email: [email protected]
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Present address: Royal Institute of Technology (KTH), Division of Glycoscience, School of
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Department of Agricultural Research for Northern Sweden, Swedish University of Agricultural
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Abstract
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Despite the recognition that Rhizobium leguminosarum sv. viciae is the most common symbiont
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of Vicia species worldwide, there is no available information on rhizobia nodulating native Vicia
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species in Sweden. We have therefore studied the genetic diversity and phylogeny of root nodule
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bacteria isolated from V. cracca, V. hirsuta, V. sepium, V. tetrasperma and V. sylvatica growing
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in different locations in Sweden as well as an isolate each from V. cracca in Tromsø, Norway,
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and V. multicaulis in Siberia, Russia. Out of 25 isolates sampled from the six Vicia species in 12
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different locations, there were 14 different genotypes based on the atpD, recA and nodA gene
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phylogenies. All isolates were classified into Rhizobium leguminosarum sv. viciae group based
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on the concatenated atpD and recA phylogeny and the nodA phylogeny.
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Keywords: Diversity, Nodulation, Phylogeny, Rhizobia, Vicia.
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Introduction
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Nitrogen (N) is often a limiting nutrient for sustainable plant growth in many ecosystems. In N
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poor soils, legumes have an advantage as they are able to fix N biologically from the atmosphere
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through symbiotic interaction with soil bacteria collectively called rhizobia. The interaction
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between rhizobia and legumes is a major source of N input into the terrestrial ecosystem and
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therefore of great agricultural and ecological significance [9,20]. This symbiotic association is
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characterized by host specificity with each symbiovar of rhizobia interacting with only a defined
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range of host legumes when they are restrictive as occurs in the case of Vicia [6]. Despite a great
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wealth of information being uncovered over the years as regards to the species of rhizobia that
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are associated with specific legume hosts, the picture is far from complete because of the
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extraordinary biological complexity of the interaction, and for the fact that a lot of native
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legumes in temperate regions still remain unexplored with regards to their associated root nodule
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bacteria [40]. As a step to address this knowledge gap we recently conducted a survey to gather
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first-hand information on the distribution and nodulation of native legumes in Sweden [4].
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The genus Vicia is known to to comprise species from over 200 legumes all over the world [32].
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Even though Vicia spp. are common in Sweden and they are highly represented in our native
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legume collection, there is no information on the rhizobial strains associated with the Vicia genus
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in this country.
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Studies aimed at identifying rhizobia associated with Vicia spp. that have been carried out in
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different continents or geographic locations including Asia, America and some parts of Europe
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have all concluded that Rhizobium leguminosarum sv. viciae (Rlv) is the most common symbiont
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of Vicia spp. [10,22,25,27,29,31,38,52]. Even though different Vicia species can share rhizobia,
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evidence exists that host plants could be selective in nodulating with some Rlv genotypes [21].
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Bacterial species, including rhizobia, are classically defined by the 16S rRNA sequence
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information [24]. However, recent studies show that the 16S rRNA is not suited to distinguish
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between closely related rhizobial species or to assess intraspecific diversity for which several
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housekeeping genes are currently used [34, 37]. To obtain information on the diversity and
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phylogeny of rhizobia nodulating native Vicia spp. in Sweden, we have sampled nodules from
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different Vicia spp. growing naturally in different geographic locations and have used the atpD
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and recA housekeeping genes which have higher resolution [46] to assess their diversity and
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phylogeny. In addition, we have assessed the diversity of the isolates at the nodulation or
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symbiotic level using the nodA gene which is plasmid borne.
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Materials and Methods
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Plant sampling and nodule collection
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Vicia species studied were V. cracca L., V. hirsuta (L.) Gray, V. multicaulis Ledeb., V. sepium
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L., V. sylvatica L. and V. tetrasperma (L.) Schreb. Nodules from all these species were collected
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from different sites in Sweden except V. multicaulis which was sampled from Yakutsk, Siberia,
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Russia. For V. cracca, an additional sample was taken from Storskogåsen, Tromsø in northern
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Norway (Fig. 1, Table 1). The procedure below was followed in our sample collection. Briefly,
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plants were dug out from the soil with a small spade to a depth of about 15 cm, and the soil
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clump with the intact plant transported in plastic bags to the laboratory or stored in a cold room
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on the same day. Nodules were then detached from the roots and preserved over silica gels until
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root nodule bacteria were isolated [39].
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Nodule bacteria isolation
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To isolate nodule bacteria, nodules stored over silica gels were rehydrated in water and kept in a
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refrigerator overnight [39]. Surface sterilization and crushing of nodules was done as described
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in Ampomah and Huss-Danell [2]. Isolates spread on yeast mannitol agar (YMA) plates were
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kept at 28 °C and observed for 2-4 days for the appearance of colonies. Well isolated colonies
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typical of rhizobia were picked and streaked unto new YMA plates. Subsequent purifications
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were done until pure cultures of isolates were obtained. Pure cultures of isolates were stored at
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-80 °C in yeast mannitol liquid medium containing 20% (v/v) glycerol.
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Nodulation tests
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A total of 25 isolates from the six Vicia species sampled from 12 sites were studied (Table 1;
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Fig.1). For each site at least one isolate was randomly selected from the collection of nodule
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bacteria kept at -80 °C. Nodulation tests for all the selected isolates were carried out on their
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original host from which they were isolated except for V. multicaulis, V. sylvatica and V.
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tetrasperma as we did not have seeds to raise plants for the nodulation test. For these species,
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tests were done on V. sativa or V. cracca as host. Seed sterilization and germination of V. cracca
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and V. sativa was done as described in Ampomah and Huss-Danell [2]. A minor modification
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was made for V. hirsuta and V. sepium in that seeds were initially soaked in concentrated H2SO4
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for 9 min instead of 6 min. Seedlings from surface sterilized seeds were inoculated with cell
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suspensions of the appropriate rhizobia in sterilized CYG germination pouches (www.mega-
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international.com). Inoculated plants were kept in a greenhouse with a supplemental light of 16/8
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h light/dark photoperiod and set to 24/15 °C and a relative humidity of 70 %. Non-inoculated
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plants served as negative controls. Sterile Broughton and Dilworth nitrogen free nutrient solution
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[39] was supplied when necessary to the plants in the pouches. Nodulation was observed over a
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period of four weeks.
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DNA extraction
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DNA from the isolates was obtained by lysis of single colonies streaked out on tryptone yeast
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(TY) medium [36]. Briefly, colonies suspended in 100 µl of 0.05M NaOH were boiled for 4 min
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to lyse the cells. 900 µl of sterile ultrapure water was added to the lysate and then stored at -20
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°C prior to PCR.
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PCR amplification and sequencing of the atpD, recA and nodA
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The genetic diversity of the isolates was determined by sequencing of their housekeeping genes
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atpD and recA. The primer pairs atpD273 and atpD782 [15,46], and recA6 and recA504 [15],
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were used to amplify the atpD and recA genes, respectively. PCR running conditions were
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according to the procedure described by Weisburg et al. [48]. About 10 µl aliquots of amplified
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PCR products from the atpD and recA were examined by electrophoresis in 1% agarose stained
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with SYBR safe. The remnants of the PCR products were purified using the NucleoSpin Extract
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II column (Machery-Nagel, Duren, Germany) according to manufacturer’s recommendation and
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sequenced at Macrogen Inc., the Netherlands.
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The primers pair and protocol for amplification of the nodA was nodA-1 and nodA-2 [19] using
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the conditions described therein. Amplified products were visualized on a 1% agarose gel stained
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with SYBR safe. The remnants of the PCR products were purified as described above and
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sequenced at Macrogen Inc., the Netherlands.
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Phylogenetic analysis of sequenced atpD, recA and nodA genes
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For phylogenetic analysis, nucleotide alignments of the sequenced atpD, recA and nodA were
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constructed with ClustalX 1.83, imported into Bioedit 4.8.4 [17] and manually corrected.
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Phylogenetic trees were constructed with aligned sequences using the neighbour-joining method
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in MEGA4 software [42] with 1000 bootstrap replication. Concatenated sequences of the atpD
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and recA were used to construct a phylogenetic tree and to decipher the genetic diversity of the
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isolates at the chromosomal level. Included in the phylogenetic analysis were sequences of the
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type or reference strains of Rhizobium leguminosarum sv. viciae strains USDA 2370 and 3841.
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Other Rhizobium species, including type strains of Rhizobium lentis, Rhizobium bangladeshense,
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Rhizobium binae, all of which nodulate Lens culinaris [34], and Rhizobium anhuiense which
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nodulates V. faba and Pisum sativum [51], were also included in the phylogenetic analysis. In
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addition, published sequences in the NCBI (www.ncbi.nlm.nih.gov) database for some isolates
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collected from Europe and America were included in the phylogenetic analysis. Sequences from
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the Sinorhizobium meliloti 1021 was used as outgroup in the phylogenetic trees.
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Accession numbers
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The nucleotide sequences obtained in this study were deposited in the GenBank under accession
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numbers from KM577280 to KM577304 for the atpD gene fragments, KM577305 to KM577329
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for the recA and KM591222 to KM591246 for the nodA.
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Results
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Nodulation test
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Since we did not have seeds for raising V. sylvatica, V. tetrasperma and V. multicaulis plants, we
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inoculated rhizobia isolated from these species on V. cracca or V. sativa (Table 1). On the other
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hand, rhizobia isolated from V. cracca, V. sepium and V. hirsuta were inoculated on their original
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host of isolation. Inoculation of the isolates on their respective or heterologous hosts resulted in
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formation of pink nodules on the host confirming that the isolates were indeed capable of
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inducing nodule formation. Non-inoculated control plants did not have any nodules after the end
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of the four week observation period.
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Phylogeny and diversity of isolates based on concatenated atpD and recA sequences
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To ascertain the phylogeny and genetic diversity of rhizobia nodulating the different Vicia
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species, we constructed a phylogenetic tree using a concatenated sequence from these genes.
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Amplification of the housekeeping genes atpD and recA for the isolates were successful as each
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isolate gave single band of expected size (data not shown). Partial atpD (313 bp) and recA (282
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bp) sequences were combined and a phylogenetic tree based on the concatenated sequences
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constructed with the inclusion of some other Rhizobium reference or type strains. Results from
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the phylogenetic tree showed that all the isolates belong to the Rhizobium leguminosarum group
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and was supported by a high bootstrap value of 94 % (Fig. 2). The isolates were further dispersed
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into four distinct clades under 12 chromosomal genotype backgrounds (Fig. 2, Table 1). Eleven
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of the 13 isolates from V. cracca grouped in the same clade with a high bootstrap value of 97 %.
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The isolates in this V. cracca associated clade were further divided into four different
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chromosomal backgrounds. One of the isolates of V. cracca, KHDVB 1902.3 which was from
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Degeberga in southern Sweden and not present in the V. cracca clade, formed a very tight cluster
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with several isolates from the other different Vicia species sampled from Bokenäset in southern
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Sweden. These sites are about 40 km apart. The phylogenetic tree suggested that KHDVB
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1902.3 has a similar chromosomal background as isolates KHDVB 1942.4 and KHDVB 1942.9
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from V. sylvatica, KHDVB 2018.1 and KHDVB 2009.7 from V. sepium and isolate KHDVB
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2103.2 from V. tetrasperma, as well as Rhizobium sp. MVPO1 which was isolated from V. sativa
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in Salamanca, Spain [1].
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The V. cracca isolate KHDVB 2171.3 grouped in the same clade as the V. multicaulis isolate
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KHDSib 8.11, the V. hirsuta isolate KHDVB 1556.1, Rhizobium sp. PEVF10 [38] isolated from
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V. faba in Ayacucho, Peru, and the reference strain R. leguminosarum sv. viciae 3841 originating
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from the United Kingdom. Among these, isolate KHDVB 1556.1 clustered with R.
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leguminosarum sv. viciae 3841 and Rhizobium sp. PEVF10 suggesting they have similar
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chromosomal background. Isolates KHDVB 2098.1 from V. tetrasperma and KHDVB 1554.5
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from V. hirsuta were the only isolates that were found in the same clade as the type strains of R.
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leguminosarum LMG14904 and USDA 2370. However, in this clade, they appeared to be more
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closely related to R. leguminosarum sv. trifolii ATCC 14480 which is associated with Trifolium
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(Fig. 2).
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Phylogeny and diversity of nodA
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The nodA gene present on the symbiotic plasmid encodes an acyl-transferase enzyme that
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transfers fatty acids unto the backbone of rhizobia Nod factor and is a determinant of host
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specificity. Amplification of the nodA yielded single band for each of the isolates (data not
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shown). The phylogenetic tree constructed with 440 bp of the nodA is shown in Fig. 3. The
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results show that all the isolates formed a monophyletic group with the Rlv type strain USDA
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2370, the reference strain R. leguminosarum sv. viciae 3841 and the three newly described
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rhizobial species from Bangladesh nodulating Lens [34]. This group was well separated from R.
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leguminosarum sv. trifolii ICMP 5377, R. etli CFN42 and R. leguminosarum sv. phaseoli ICMP
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2672. The isolates were placed in four main clades comprising six nodA genotype backgrounds
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with several isolates clustering within a clade, all with the exception of KHDSib 8.11 which was
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quite separated from the other isolates, suggesting that it has a unique nodA (Fig. 3). Nine out of
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the thirteen V. cracca isolates had identical nodA sequence and therefore formed a cluster within
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a clade, with the remaining four (OYAVB 169.1, KHDVB 2094.1, OYAVB 174.1 and KHDVB
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1030.3) being dispersed in clades containing the other Vicia species. Two of the V. cracca
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isolates, OYAVB 169.1 and KHDVB 1902.3, were present in the same clade with isolates from
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V. tetrasperma, V. sepium and V. sylvatica that were mainly from Bokenäset in southern Sweden.
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Within this clade all the four V. sylvatica isolates clustered together indicating that they have
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identical nodA. Similarly, both isolates from V. sepium and V. hirsuta had identical nodA. With
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V. tetrasperma, even though all the three isolates were from the same site, two of them (KHDVB
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2094.1 and KHDVB 2103.2) clustered together indicating identical nodA while the other
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(KHDVB 2098.1) was present in a different clade, clustering with the V. hirsuta isolates
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(KHDVB 1556.1 and KHDVB 1554.5) and some V. cracca isolates (KHDVB 1030.3 and
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OYAVB 174.1).
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Genotype assignment of isolates based on the phylogenies from concatenated atpD and recA
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sequences and nodA sequences
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There were 14 genotypes of Rlv from the 25 isolates based on the phylogeny of the concatenated
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atpD and recA sequences and the nodA sequences (Table 1). This consisted of eight isolates
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having genotypes that were different from any other isolate. The rest were identical to one or
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more isolates with four isolates having the same genotype background. In all, seven genotypes
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were associated with V. cracca, three genotypes with V. tetrasperma, two genotypes each with V.
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sylvatica and V. hirsuta, and one genotype each with V. sepium and V. multicaulis. None of the
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hosts shared rhizobia with the same genotype background except for V. cracca, V. tetrasperma
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and V. sepium. Here, one of the V. cracca isolates KHDVB 1902.3 had identical genotype with
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the V. tetrasperma isolate KHDVB 2103.2 and both V. sepium isolates KHDVB 2009.7 and
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KHDVB 2018.1
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Discussion
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To provide information on rhizobia associated with native Vicia spp. in Sweden, we have
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assessed the genetic diversity and phylogeny of root nodule bacteria associated with five native
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Vicia spp. sampled from different locations in Sweden, with the inclusion of an isolate each from
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V. cracca in Tromsø, Norway and V. multicaulis in Siberia, Russia. All the isolates were
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identified as Rlv and were effective in nodulation and fixing nitrogen with evidence from the
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pink looking nodules on the inoculated hosts. It is well established that Rlv forms effective
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symbiosis with species of the genera Pisum, Vicia, Lathyrus and Lens, all of which belong to the
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Viciae tribe [12,45]. The nodulation of V. sativa by isolates from V. sylvatica and V.
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tetrasperma, and V. cracca by an isolate of V. multicaulis was therefore not surprising.
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Moreover, Mutch and Young [31] had also earlier reported that most isolates from some Vicia
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species like V. cracca, V. sativa and V. hirsuta easily cross-nodulate forming effective symbiosis
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on their test host. These together with our results support the cross-nodulation of Rlv on Vicia
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species.
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The phylogeny of the atpD and recA genes located on the chromosome is known to support the
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16S rRNA based classification of rhizobia [15]. Moreover, the atpD and recA have higher
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resolution and are able to resolve intraspecific diversity in comparison to the 16S rRNA [46].
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Since Rlv is known to be the common symbiont of Vicia spp. worldwide, we relied on the atpD
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and recA to resolve the diversity and phylogeny of our isolates. Our results showed that indeed
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all the isolates belonged to Rlv group and fell under 12 different genotype backgrounds based on
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the phylogenetic tree from the concatenated atpD and recA gene sequences.
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Some of the factors that influence the occurrence and diversity of rhizobia include the population
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of resident soil bacteria, soil characteristics (e.g. pH, salinity, temperature, moisture), land use
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management and the host plant genotype [28]. Several studies have also shown that natural soil
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populations of rhizobia are diverse and that diversity between sites varies more greatly than
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within sites [14,35,41,44]. Our results showed that the V. cracca isolates fell under six different
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genotype backgrounds based on the concatenated atpD and recA sequences. Recently, an
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extensive study on the genetic diversity and population structure of V. cracca isolates in Belgium
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and France by van Cauwenberghe et al. [44] also showed strong genetic differentiation among
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both V. cracca populations that were separated by few kilometers, and among regions that were
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over 50 to 350 km apart. Even though we had not more than two V. cracca isolates per site in
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this study, most of the genotypes were restricted to unique sites. However, it is not uncommon to
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find isolates with similar chromosomal backgrounds across different geographic sites
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[11,16,47,50] as we have also observed in this study. The major chromosomal background
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among the isolates in this study, group XII (Table 1), had isolates originating from nodules on V.
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sylvatica, V. cracca, V. sepium and V. tetrasperma, all from sites in southern Sweden. It is
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worthwhile to note that Rhizobium sp. MVPO1 from Spain also shares similar chromosomal
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background as our isolates in group XII. Kumar et al. [23] have also reported the presence of
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specific genospecies of Rlv in different continents, supporting the successful spread and
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establishment of rhizobia strains into different environments. It appears that Rlv with this
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chromosomal background (group XII) may be dominant in the southern Sweden and thus easily
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picked up by the different Vicia species growing in those areas. A test to confirm this may be
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done by trapping rhizobia with different Vicia species from soils taken from selected locations in
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this region and sequencing some housekeeping genes from the isolates occupying the nodule.
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Among our isolates, only KHDVB 1556.1 which was isolated from V. hirsuta had similar
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chromosomal background as the reference strain R. leguminosarum sv. viciae 3841 originating
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from the United Kingdom. Interestingly, Alvarez-Martinez et al. [1] had earlier reported that an
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isolate Rhizobium sp. PEVF10 originating from Peru in South America also shares similar
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chromosomal background as R. leguminosarum sv. viciae 3841 and our phylogenetic tree
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confirmed that indeed these three (KHDVB 1556.1, Rhizobium sp. PEVF10 and R.
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leguminosarum sv. viciae 3841) have similar chromosomal background with reference to their
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atpD and recA sequences. The genetic evidence showing that isolates from different continents
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share similar chromosomal background strengthens the concept of the intercontinental spread of
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rhizobia.
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Over the years, the taxonomy of rhizobia has seen some revisions largely arising from the
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isolation of novel strains from previously unexplored areas as well as from native or wild
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legumes of non-agronomic importance. For instance it was thought that rhizobia were restricted
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to the α-proteobacteria group. This perception changed when bacteria from the β-proteobacteria
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group like Burkholderia sp. were found to nodulate with Aspalathus and Machaerium plants [30]
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and Ralstonia taiwanensis sp. nov., with Mimosa species [7]. Further studies have now shown
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that Mimosa are naturally nodulated by Burkholderia sp.[5,8,26]. In comparison to cultivated
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Vicia spp. like V. faba or V. sativa, not much work has been done to identify or characterize
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rhizobia associated with wild Vicia spp. For instance, there is only one report in the literature
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describing rhizobia associated with V. multicaulis [27] and no documented report for V.
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sylvatica. Even though it is widely believed that Rlv is associated with Vicia spp., a novel
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rhizobia named Rhizobium multihospitium was isolated by Han et al. [18] from V. hirsuta in
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Xinjiang, an isolated region in China. De Meyer et al. [10] also isolated Rhizobium sp. from V.
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hirsuta as well as Bradyrhizobium sp. from V. cracca in Belgium. These findings are indications
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that Vicia spp. may also nodulate with rhizobial species other than Rlv and affirms the
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importance of investigating nodule bacteria associated with non-cultivated legumes. However,
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we did not find any rhizobia group besides Rlv nodulating with the native legumes studied and
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our study provides the first evidence that V. sylvatica is indeed nodulated by Rlv.
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It was not surprising for all the isolates to fall under a monophyletic group which was well
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separated from the other Rhizobium species or type strains known to nodulate outside the Vicia
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group since the nodA is involved in host specificity (Fig. 3). Thus the phylogeny of the nodA
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confirmed that the isolates belonged to the symbiovar viciae. Even though four main clades were
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observed, most of the isolates clustered within a clade, with the exception of KHDSib 8.11 which
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was unique from the others (Fig. 3). This unique nodA from KHDSib 8.11 possibly reflects a
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different nodA backbone needed for the nodulation of V. multicaulis. The only documented
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report on rhizobia nodulating V. multicaulis [27] indicated an Rlv genotype background, but
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there was no information on the nodulation genes from those isolates which makes it impossible
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to compare with our work. A test to verify this is to check the possibility of some of the isolates
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from this work or the Rlv type strains to nodulate V. multicaulis. Another explanation is that,
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perhaps this nodA may be unique to that environment (Siberia) since the other isolates studied
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were from Scandinavia. Recently we isolated a Mesorhizobium huakuii strain with a unique
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nodA from Thermopsis lupinoides in Kamchatka, Russia [3] and it is likely rhizobia found in
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such unexplored regions may have genes adapted to nodulate legumes in such environment.
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The isolates nodulating V. cracca were under four different nodA backgrounds even though most
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of them (9 out of the 13) had identical housekeeping genes background. The dominance of one
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particular nodA background from all the isolates nodulating V. cracca suggests that V. cracca
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may have a preference for isolates harbouring that nodA type. Apart from the observation that the
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majority of the V. cracca isolates had identical nodA and formed a V. cracca clade, some of the
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other isolates originating from the other Vicia hosts also clustered together with respect to the
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nodA, an observation in support of the cross-nodulation of isolates from these hosts. In general,
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there was no pattern or association of nodA to sites as similar nodA genotype was found in
311
different locations even at significant distance apart as reported in some other studies [2,43].
312
However, it appears that nodA genotype I is more predominant in northern Sweden (Fig. 3, Table
313
1).
314
With rhizobia, it is well known that phylogeny of the housekeeping genes are usually not
315
congruent with the nodulation genes since the nodulation genes are located on a plasmid and are
316
more subject to lateral gene transfer [27,33,43,44,49]. Our concatenated atpD and recA
317
sequences were also not congruent with the nodA phylogeny. However, the phylogenetic analysis
318
of the nodulation genes confirms that the isolates belong to the symbiovar viciae. An interesting
319
observation in this work was that isolate OYAVB 164.10 from Tromsø in Northern Norway and
320
isolates KHDVB 1598.3 and KHDVB 1619.10 from Haparanda in northern Sweden had similar
321
genetic background despite being over 475 km apart. It is possible that these sites share rhizobia
322
with similar genetic background as a result of introduction of rhizobia through seeds or perhaps
323
through other human activities. A similar observation was also reported by Duodu et al. [13]
324
who also showed that some field isolates of rhizobia nodulating Trifolium spp. that were sampled
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15 Page 15 of 30
from Umeå in northern Sweden and Tromsø in northern Norway had similar genetic background
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as well as nodEF profiles.
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Conclusions
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In this work we have provided information on the genetic diversity and phylogeny of rhizobia
329
that are associated with native Vicia species in Sweden. We have shown that Rlv is the main
330
symbiont of Vicia species in Sweden. Future work will include testing the symbiotic efficiency
331
of some of these indigenous isolates on Vicia species of agronomic importance in Sweden like
332
Vicia sativa with the aim of identifying potential isolates that could be used as inoculants.
333
Acknowledgements
334
Funding for this research was received from the Swedish Research Council Formas to KHD. We
335
thank K. Danell for help in field work and A-S Hahlin for assistance in laboratory work. We are
336
grateful to Mats Högström for GIS support and map and to Bente Eriksen, Olga
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Khitun och V.V. Petrovskii for their kind help to identify Vicia multicaulis.
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Conflict of interest: None declared
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Table
Host
Nodulation test
V. cracca
Nod+
KHDVB 654.3 KHDVB 1598.3 KHDVB 1619.10 KHDVB 1680.5 KHDVB 1681.2 KHDVB 1703.2 KHDVB 1707.1 OYAVB 169.1 OYAVB 174.1 KHDVB 1030.3 KHDVB 1902.3 KHDVB 1554.5 KHDVB 1556.1 KHDVB 2171.3 KHDVB 1942.4 KHDVB 1942.9 KHDVB 1942.14 KHDVB 1942.2 KHDVB 2094.1 KHDVB 2098.1 KHDVB 2103.2 KHDVB 2009.7 KHDVB 2018.1 KHDsib 8.11
V. cracca V. cracca V. cracca V. cracca V. cracca V. cracca V. cracca V. cracca V. cracca V. cracca V. cracca V. hirsuta V. hirsuta V. cracca V. sylvatica V. sylvatica V. sylvatica V. sylvatica V. tetrasperma V. tetrasperma V. tetrasperma V. sepium V. sepium V. multicaulis
Nod+ Nod+ Nod+ Nod+ Nod+ Nod+ Nod+ Nod+ Nod+ Nod+ Nod+ Nod+ Nod+ Nod+ Nod+ (on V. sativa) Nod+ (on V. sativa) Nod+ (on V. sativa) Nod+ (on V. sativa) Nod+ (on V. sativa) Nod+ (on V. sativa) Nod+ (on V. sativa) Nod+ Nod+ Nod+ (on V. cracca)
ce pt
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OYAVB 164.10
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Collection site
M an
Isolate
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Table 1. Nodulation phenotype, site of isolation and genetic background of isolates from the different Vicia species studied
Norway: Tromsø, Storskogåsen, Prestvannet (A) Sorsele, Ammarnäs, Tjärnen (B) Haparanda, Nikkala, Saantasaari (C) Haparanda, Nikkala, Saantasaari (C) Råneå, Högsön (D) Råneå, Högsön (D) Storuman, Barsele (E) Storuman, Barsele (E) Umeå, Ängersjö (F) Umeå, Ängersjö (F) Timrå, Gökböle (G) Kristianstad, Degeberga (H) Lund, Höjeå (I) Lund, Höjeå (I) Simrishamn, St Olof-Onslunda (J) Kristianstad, Bokenäset (K) Kristianstad, Bokenäset (K) Kristianstad, Bokenäset (K) Kristianstad, Bokenäset (K) Kristianstad, Bokenäset (K) Kristianstad, Bokenäset (K) Kristianstad, Bokenäset (K) Kristianstad, Bokenäset (K) Kristianstad, Bokenäset (K) Russia: Siberia, Jakutsk (n.a.)
atpDrecA genotype II
nodA genotype I
Genotype
III II II III III I I IV I I XII IX VII V XII XII XI XII X VIII XII XII XII VI
I I I I I I I II VI VI II VI VI I III III III III II VI II II II IV
4 1 1 4 4 5 5 2 3 3 6 13 14 7 9 9 10 9 11 12 6 6 6 Page 25 of 30 8
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Letters in parenthesis indicates the collection site shown in Figure 1. n.a means not available on the map in Figure 1.
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Figure legend
Figure captions
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Fig.1. Sites from Sweden and Norway where Vicia plants were sampled for this study.
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Fig. 2. Neighbour-joining phylogenetic tree based on concatenated atpD and recA sequences, showing the relationships between the isolates from the different Vicia species sampled from
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different sites and some reference and type strains. The tree was constructed using MEGA4 software. The numbers at branch points are the significant bootstrap values (expressed as
an
percentage based on 1000 replicates; only values greater than 70 % are shown). The horizontal
M
branch lines are proportional and indicate the p-distances. The scale bar represents the number of nucleotide substitutions per 100 nucleotides. Accession numbers from the Genbank are in
d
parenthesis.
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Fig. 3. Neighbour-joining phylogenetic tree based on nodA sequences, showing the relationships
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between the isolates from the different Vicia species sampled from different sites and some reference and type strains. The tree was constructed using MEGA4 software. The numbers at branch points are the significant bootstrap values (expressed as percentage based on 1000 replicates; only values greater than 70 % are shown). The horizontal branch lines are proportional and indicate the p-distances. The scale bar represents the number of nucleotide substitutions per 100 nucleotides. Accession numbers from the Genbank are in parenthesis.
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Figure
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Figure
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KHDVB 1703.2 (KM577289,KM577314) KHDVB 1707.1 (KM577290, KM577315) I KHDVB 1030.3 (KM577283, KM577308) OYAVB 174.1 (KM577282, KM577307) 86 KHDVB 1598.3 (KM577285, KM577310) KHDVB 1619.10 (KM577286, KM577311) II 97 OYAVB 164.10 (KM577280, KM577305) KHDVB 654.3 (KM577284, KM577309) III KHDVB 1680.5 (KM577287, KM577312) 86 KHDVB 1681.2 (KM577288, KM577313) OYAVB 169.1 (KM577281, KM577306) IV KHDVB 2171.3 (KM577292, KM577317) V KHDSib 8.11 (KM577293, KM577318) VI KHDVB 1556.1 (KM577302, KM577327) VII Rhizobium sp. PEVF10 (EF113143, EF113128) 100 Rhizobium leguminosarum sv. viciae 3841 (AM236080, AM236080) VIII KHDVB 2098.1 (KM577299, KM577324) 88 88 KHDVB 1554.5 (KM577301, KM577326) IX T 100 Rhizobium leguminosarum sv. trifolii ATCC 14480 (EF113150, EF113135)
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Fig 2
97
an
72
Ac c
99
ep te
d
M
94
99
99 89
T
Rhizobium leguminosarum LMG14904 (AM418783, AM182125) 98 Rhizobium leguminosarum sv. viciae USDA 2370T (AJ294405, AJ294376) KHDVB 2094.1 (KM577298, KM577323) X KHDVB 1942.14 (KM577296, KM577321) XI KHDVB 1902.3 (KM577291, KM577316) KHDVB 1942.4 (KM577294, KM577319) KHDVB 1942.9 (KM577295, KM577320) KHDVB 1942.2 (KM577297, KM577322) XII KHDVB 2103.2 (KM577300, KM577325) KHDVB 2009.7 (KM577303, KM577328) KHDVB 2018.1 (KM577304, KM577329) Rhizobium sp. MVPO1 (FJ596008, FJ596035) T
Rhizobium anhuiense CCBAU 23252 (KF111890, KF111980) T Rhizobium fabae CCBAU 33202 (EF579929, EF579941) T 99 Rhizobium pisi DSM 30132 (EF113149, EF113134) T Rhizobium phaseoli ATCC 14482 (EF113151, EF113136) T Rhizobium tropici B USDA 9030 (AJ294397, AJ294373) T Rhizobium etli USDA 9032 (AJ294404, AJ294375) T Rhizobium bangladeshense BLR175 (JN648967, JN649057) T Rhizobium lentis BLR27 (JN648941, JN649031) T Rhizobium binae BLR195 (JN648968, JN649058) Sinorhizobium meliloti 1021 (SMc02501, SMc00760)
0.01
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Figure KHDVB 1707.1 (KM591232) KHDVB 2171.3 (KM591234)
cr
KHDVB 1703.2 (KM591231)
Fig 3
KHDVB 1681.2 (KM591230) 100 KHDVB 1680.5 (KM591229) KHDVB 1598.3 (KM591227)
75
KHDVB 654.3 (KM591226)
an
OYAVB 164.10 (KM591222)
us
KHDVB 1619.10 (KM591228)
I
Rhizobium leguminosarum sv. viciae 3841 (pRL100185) 100 Rhizobium legumiosarum CTG-10vf (KC620562) 85
Rhizobium lentis BLR9 (JN648983)
T
M
100 Rhizobium bangladeshense BLR175 (JN648991) T
Rhizobium leguminosarum sv. viciae USDA 2370 (JN558711) 70
KHDVB 1902.3 (KM591233) KHDVB 2018.1 (KM591246)
d
KHDVB 2009.7 (KM591245) KHDVB 2094.1 (KM591240)
II
KHDVB 2103.2 (KM591233)
ep te
100
OYAVB 169.1 (KM591223) KHDVB 1942.4 (KM591236) KHDVB 1942.9 (KM591237) KHDVB 1942.14 (KM591238)
100
III
KHDVB 1942.2 (KM591239)
Ac c
KHDSib 8.11 (KM591235)
IV
Rhizobium binae BLR228 (JN648992) KHDVB 1556.1 (KM591244)
100
KHDVB 1554.5 (KM591243) KHDVB 2098.1 (KM591241)
V
OYAVB 174.1 (KM591224) 74 KHDVB 1030.3 (KM591225)
VI
Rhizobium leguminosarum sv. trifolii ICMP 5377 (EF115496) T
100
Rhizobium etli CFN42 (RHE_PD00310) Rhizobium leguminosarum sv. phaseoli ICMP 2672 (DQ100403) Sinorhizobium meliloti 1021 (SMa0869)
0.05
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S0723-2020(16)00021-7 http://dx.doi.org/doi:10.1016/j.syapm.2016.02.002 SYAPM 25751
To appear in: Received date: Revised date: Accepted date:
12-1-2016 8-2-2016 9-2-2016
Please cite this article as: O.Y. Ampomah, K. Huss-Danell, Genetic diversity of rhizobia nodulating native Vicia spp. in Sweden, Systematic and Applied Microbiology (2016), http://dx.doi.org/10.1016/j.syapm.2016.02.002 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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Genetic diversity of rhizobia nodulating native Vicia spp. in Sweden
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Osei Yaw Ampomaha1* and Kerstin Huss-Danella
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Sciences (SLU), SE-90183 Umeå, Sweden
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Biotechnology, S-10691, Stockholm, Sweden
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*Corresponding author. Royal Institute of Technology (KTH), Division of Glycoscience, School
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of Biotechnology, S-10691, Stockholm, Sweden.
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Telephone: +46 706084168
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Present address: Royal Institute of Technology (KTH), Division of Glycoscience, School of
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Department of Agricultural Research for Northern Sweden, Swedish University of Agricultural
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Abstract
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Despite the recognition that Rhizobium leguminosarum sv. viciae is the most common symbiont
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of Vicia species worldwide, there is no available information on rhizobia nodulating native Vicia
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species in Sweden. We have therefore studied the genetic diversity and phylogeny of root nodule
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bacteria isolated from V. cracca, V. hirsuta, V. sepium, V. tetrasperma and V. sylvatica growing
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in different locations in Sweden as well as an isolate each from V. cracca in Tromsø, Norway,
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and V. multicaulis in Siberia, Russia. Out of 25 isolates sampled from the six Vicia species in 12
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different locations, there were 14 different genotypes based on the atpD, recA and nodA gene
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phylogenies. All isolates were classified into Rhizobium leguminosarum sv. viciae group based
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on the concatenated atpD and recA phylogeny and the nodA phylogeny.
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Keywords: Diversity, Nodulation, Phylogeny, Rhizobia, Vicia.
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Introduction
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Nitrogen (N) is often a limiting nutrient for sustainable plant growth in many ecosystems. In N
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poor soils, legumes have an advantage as they are able to fix N biologically from the atmosphere
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through symbiotic interaction with soil bacteria collectively called rhizobia. The interaction
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between rhizobia and legumes is a major source of N input into the terrestrial ecosystem and
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therefore of great agricultural and ecological significance [9,20]. This symbiotic association is
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characterized by host specificity with each symbiovar of rhizobia interacting with only a defined
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range of host legumes when they are restrictive as occurs in the case of Vicia [6]. Despite a great
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wealth of information being uncovered over the years as regards to the species of rhizobia that
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are associated with specific legume hosts, the picture is far from complete because of the
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extraordinary biological complexity of the interaction, and for the fact that a lot of native
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legumes in temperate regions still remain unexplored with regards to their associated root nodule
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bacteria [40]. As a step to address this knowledge gap we recently conducted a survey to gather
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first-hand information on the distribution and nodulation of native legumes in Sweden [4].
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The genus Vicia is known to to comprise species from over 200 legumes all over the world [32].
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Even though Vicia spp. are common in Sweden and they are highly represented in our native
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legume collection, there is no information on the rhizobial strains associated with the Vicia genus
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in this country.
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Studies aimed at identifying rhizobia associated with Vicia spp. that have been carried out in
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different continents or geographic locations including Asia, America and some parts of Europe
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have all concluded that Rhizobium leguminosarum sv. viciae (Rlv) is the most common symbiont
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of Vicia spp. [10,22,25,27,29,31,38,52]. Even though different Vicia species can share rhizobia,
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evidence exists that host plants could be selective in nodulating with some Rlv genotypes [21].
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Bacterial species, including rhizobia, are classically defined by the 16S rRNA sequence
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information [24]. However, recent studies show that the 16S rRNA is not suited to distinguish
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between closely related rhizobial species or to assess intraspecific diversity for which several
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housekeeping genes are currently used [34, 37]. To obtain information on the diversity and
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phylogeny of rhizobia nodulating native Vicia spp. in Sweden, we have sampled nodules from
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different Vicia spp. growing naturally in different geographic locations and have used the atpD
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and recA housekeeping genes which have higher resolution [46] to assess their diversity and
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phylogeny. In addition, we have assessed the diversity of the isolates at the nodulation or
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symbiotic level using the nodA gene which is plasmid borne.
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Materials and Methods
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Plant sampling and nodule collection
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Vicia species studied were V. cracca L., V. hirsuta (L.) Gray, V. multicaulis Ledeb., V. sepium
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L., V. sylvatica L. and V. tetrasperma (L.) Schreb. Nodules from all these species were collected
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from different sites in Sweden except V. multicaulis which was sampled from Yakutsk, Siberia,
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Russia. For V. cracca, an additional sample was taken from Storskogåsen, Tromsø in northern
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Norway (Fig. 1, Table 1). The procedure below was followed in our sample collection. Briefly,
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plants were dug out from the soil with a small spade to a depth of about 15 cm, and the soil
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clump with the intact plant transported in plastic bags to the laboratory or stored in a cold room
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on the same day. Nodules were then detached from the roots and preserved over silica gels until
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root nodule bacteria were isolated [39].
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Nodule bacteria isolation
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To isolate nodule bacteria, nodules stored over silica gels were rehydrated in water and kept in a
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refrigerator overnight [39]. Surface sterilization and crushing of nodules was done as described
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in Ampomah and Huss-Danell [2]. Isolates spread on yeast mannitol agar (YMA) plates were
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kept at 28 °C and observed for 2-4 days for the appearance of colonies. Well isolated colonies
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typical of rhizobia were picked and streaked unto new YMA plates. Subsequent purifications
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were done until pure cultures of isolates were obtained. Pure cultures of isolates were stored at
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-80 °C in yeast mannitol liquid medium containing 20% (v/v) glycerol.
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Nodulation tests
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A total of 25 isolates from the six Vicia species sampled from 12 sites were studied (Table 1;
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Fig.1). For each site at least one isolate was randomly selected from the collection of nodule
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bacteria kept at -80 °C. Nodulation tests for all the selected isolates were carried out on their
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original host from which they were isolated except for V. multicaulis, V. sylvatica and V.
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tetrasperma as we did not have seeds to raise plants for the nodulation test. For these species,
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tests were done on V. sativa or V. cracca as host. Seed sterilization and germination of V. cracca
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and V. sativa was done as described in Ampomah and Huss-Danell [2]. A minor modification
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was made for V. hirsuta and V. sepium in that seeds were initially soaked in concentrated H2SO4
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for 9 min instead of 6 min. Seedlings from surface sterilized seeds were inoculated with cell
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suspensions of the appropriate rhizobia in sterilized CYG germination pouches (www.mega-
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international.com). Inoculated plants were kept in a greenhouse with a supplemental light of 16/8
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h light/dark photoperiod and set to 24/15 °C and a relative humidity of 70 %. Non-inoculated
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plants served as negative controls. Sterile Broughton and Dilworth nitrogen free nutrient solution
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[39] was supplied when necessary to the plants in the pouches. Nodulation was observed over a
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period of four weeks.
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DNA extraction
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DNA from the isolates was obtained by lysis of single colonies streaked out on tryptone yeast
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(TY) medium [36]. Briefly, colonies suspended in 100 µl of 0.05M NaOH were boiled for 4 min
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to lyse the cells. 900 µl of sterile ultrapure water was added to the lysate and then stored at -20
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°C prior to PCR.
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PCR amplification and sequencing of the atpD, recA and nodA
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The genetic diversity of the isolates was determined by sequencing of their housekeeping genes
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atpD and recA. The primer pairs atpD273 and atpD782 [15,46], and recA6 and recA504 [15],
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were used to amplify the atpD and recA genes, respectively. PCR running conditions were
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according to the procedure described by Weisburg et al. [48]. About 10 µl aliquots of amplified
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PCR products from the atpD and recA were examined by electrophoresis in 1% agarose stained
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with SYBR safe. The remnants of the PCR products were purified using the NucleoSpin Extract
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II column (Machery-Nagel, Duren, Germany) according to manufacturer’s recommendation and
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sequenced at Macrogen Inc., the Netherlands.
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The primers pair and protocol for amplification of the nodA was nodA-1 and nodA-2 [19] using
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the conditions described therein. Amplified products were visualized on a 1% agarose gel stained
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with SYBR safe. The remnants of the PCR products were purified as described above and
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sequenced at Macrogen Inc., the Netherlands.
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Phylogenetic analysis of sequenced atpD, recA and nodA genes
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For phylogenetic analysis, nucleotide alignments of the sequenced atpD, recA and nodA were
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constructed with ClustalX 1.83, imported into Bioedit 4.8.4 [17] and manually corrected.
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Phylogenetic trees were constructed with aligned sequences using the neighbour-joining method
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in MEGA4 software [42] with 1000 bootstrap replication. Concatenated sequences of the atpD
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and recA were used to construct a phylogenetic tree and to decipher the genetic diversity of the
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isolates at the chromosomal level. Included in the phylogenetic analysis were sequences of the
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type or reference strains of Rhizobium leguminosarum sv. viciae strains USDA 2370 and 3841.
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Other Rhizobium species, including type strains of Rhizobium lentis, Rhizobium bangladeshense,
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Rhizobium binae, all of which nodulate Lens culinaris [34], and Rhizobium anhuiense which
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nodulates V. faba and Pisum sativum [51], were also included in the phylogenetic analysis. In
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addition, published sequences in the NCBI (www.ncbi.nlm.nih.gov) database for some isolates
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collected from Europe and America were included in the phylogenetic analysis. Sequences from
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the Sinorhizobium meliloti 1021 was used as outgroup in the phylogenetic trees.
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Accession numbers
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The nucleotide sequences obtained in this study were deposited in the GenBank under accession
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numbers from KM577280 to KM577304 for the atpD gene fragments, KM577305 to KM577329
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for the recA and KM591222 to KM591246 for the nodA.
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Results
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Nodulation test
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Since we did not have seeds for raising V. sylvatica, V. tetrasperma and V. multicaulis plants, we
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inoculated rhizobia isolated from these species on V. cracca or V. sativa (Table 1). On the other
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hand, rhizobia isolated from V. cracca, V. sepium and V. hirsuta were inoculated on their original
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host of isolation. Inoculation of the isolates on their respective or heterologous hosts resulted in
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formation of pink nodules on the host confirming that the isolates were indeed capable of
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inducing nodule formation. Non-inoculated control plants did not have any nodules after the end
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of the four week observation period.
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Phylogeny and diversity of isolates based on concatenated atpD and recA sequences
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To ascertain the phylogeny and genetic diversity of rhizobia nodulating the different Vicia
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species, we constructed a phylogenetic tree using a concatenated sequence from these genes.
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Amplification of the housekeeping genes atpD and recA for the isolates were successful as each
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isolate gave single band of expected size (data not shown). Partial atpD (313 bp) and recA (282
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bp) sequences were combined and a phylogenetic tree based on the concatenated sequences
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constructed with the inclusion of some other Rhizobium reference or type strains. Results from
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the phylogenetic tree showed that all the isolates belong to the Rhizobium leguminosarum group
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and was supported by a high bootstrap value of 94 % (Fig. 2). The isolates were further dispersed
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into four distinct clades under 12 chromosomal genotype backgrounds (Fig. 2, Table 1). Eleven
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of the 13 isolates from V. cracca grouped in the same clade with a high bootstrap value of 97 %.
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The isolates in this V. cracca associated clade were further divided into four different
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chromosomal backgrounds. One of the isolates of V. cracca, KHDVB 1902.3 which was from
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Degeberga in southern Sweden and not present in the V. cracca clade, formed a very tight cluster
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with several isolates from the other different Vicia species sampled from Bokenäset in southern
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Sweden. These sites are about 40 km apart. The phylogenetic tree suggested that KHDVB
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1902.3 has a similar chromosomal background as isolates KHDVB 1942.4 and KHDVB 1942.9
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from V. sylvatica, KHDVB 2018.1 and KHDVB 2009.7 from V. sepium and isolate KHDVB
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2103.2 from V. tetrasperma, as well as Rhizobium sp. MVPO1 which was isolated from V. sativa
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in Salamanca, Spain [1].
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The V. cracca isolate KHDVB 2171.3 grouped in the same clade as the V. multicaulis isolate
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KHDSib 8.11, the V. hirsuta isolate KHDVB 1556.1, Rhizobium sp. PEVF10 [38] isolated from
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V. faba in Ayacucho, Peru, and the reference strain R. leguminosarum sv. viciae 3841 originating
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from the United Kingdom. Among these, isolate KHDVB 1556.1 clustered with R.
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leguminosarum sv. viciae 3841 and Rhizobium sp. PEVF10 suggesting they have similar
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chromosomal background. Isolates KHDVB 2098.1 from V. tetrasperma and KHDVB 1554.5
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from V. hirsuta were the only isolates that were found in the same clade as the type strains of R.
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leguminosarum LMG14904 and USDA 2370. However, in this clade, they appeared to be more
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closely related to R. leguminosarum sv. trifolii ATCC 14480 which is associated with Trifolium
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(Fig. 2).
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Phylogeny and diversity of nodA
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The nodA gene present on the symbiotic plasmid encodes an acyl-transferase enzyme that
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transfers fatty acids unto the backbone of rhizobia Nod factor and is a determinant of host
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specificity. Amplification of the nodA yielded single band for each of the isolates (data not
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shown). The phylogenetic tree constructed with 440 bp of the nodA is shown in Fig. 3. The
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results show that all the isolates formed a monophyletic group with the Rlv type strain USDA
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2370, the reference strain R. leguminosarum sv. viciae 3841 and the three newly described
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rhizobial species from Bangladesh nodulating Lens [34]. This group was well separated from R.
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leguminosarum sv. trifolii ICMP 5377, R. etli CFN42 and R. leguminosarum sv. phaseoli ICMP
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2672. The isolates were placed in four main clades comprising six nodA genotype backgrounds
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with several isolates clustering within a clade, all with the exception of KHDSib 8.11 which was
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quite separated from the other isolates, suggesting that it has a unique nodA (Fig. 3). Nine out of
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the thirteen V. cracca isolates had identical nodA sequence and therefore formed a cluster within
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a clade, with the remaining four (OYAVB 169.1, KHDVB 2094.1, OYAVB 174.1 and KHDVB
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1030.3) being dispersed in clades containing the other Vicia species. Two of the V. cracca
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isolates, OYAVB 169.1 and KHDVB 1902.3, were present in the same clade with isolates from
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V. tetrasperma, V. sepium and V. sylvatica that were mainly from Bokenäset in southern Sweden.
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Within this clade all the four V. sylvatica isolates clustered together indicating that they have
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identical nodA. Similarly, both isolates from V. sepium and V. hirsuta had identical nodA. With
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V. tetrasperma, even though all the three isolates were from the same site, two of them (KHDVB
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2094.1 and KHDVB 2103.2) clustered together indicating identical nodA while the other
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(KHDVB 2098.1) was present in a different clade, clustering with the V. hirsuta isolates
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(KHDVB 1556.1 and KHDVB 1554.5) and some V. cracca isolates (KHDVB 1030.3 and
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OYAVB 174.1).
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Genotype assignment of isolates based on the phylogenies from concatenated atpD and recA
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sequences and nodA sequences
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There were 14 genotypes of Rlv from the 25 isolates based on the phylogeny of the concatenated
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atpD and recA sequences and the nodA sequences (Table 1). This consisted of eight isolates
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having genotypes that were different from any other isolate. The rest were identical to one or
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more isolates with four isolates having the same genotype background. In all, seven genotypes
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were associated with V. cracca, three genotypes with V. tetrasperma, two genotypes each with V.
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sylvatica and V. hirsuta, and one genotype each with V. sepium and V. multicaulis. None of the
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hosts shared rhizobia with the same genotype background except for V. cracca, V. tetrasperma
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and V. sepium. Here, one of the V. cracca isolates KHDVB 1902.3 had identical genotype with
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the V. tetrasperma isolate KHDVB 2103.2 and both V. sepium isolates KHDVB 2009.7 and
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KHDVB 2018.1
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Discussion
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To provide information on rhizobia associated with native Vicia spp. in Sweden, we have
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assessed the genetic diversity and phylogeny of root nodule bacteria associated with five native
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Vicia spp. sampled from different locations in Sweden, with the inclusion of an isolate each from
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V. cracca in Tromsø, Norway and V. multicaulis in Siberia, Russia. All the isolates were
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identified as Rlv and were effective in nodulation and fixing nitrogen with evidence from the
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pink looking nodules on the inoculated hosts. It is well established that Rlv forms effective
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symbiosis with species of the genera Pisum, Vicia, Lathyrus and Lens, all of which belong to the
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Viciae tribe [12,45]. The nodulation of V. sativa by isolates from V. sylvatica and V.
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tetrasperma, and V. cracca by an isolate of V. multicaulis was therefore not surprising.
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Moreover, Mutch and Young [31] had also earlier reported that most isolates from some Vicia
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species like V. cracca, V. sativa and V. hirsuta easily cross-nodulate forming effective symbiosis
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on their test host. These together with our results support the cross-nodulation of Rlv on Vicia
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species.
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The phylogeny of the atpD and recA genes located on the chromosome is known to support the
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16S rRNA based classification of rhizobia [15]. Moreover, the atpD and recA have higher
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resolution and are able to resolve intraspecific diversity in comparison to the 16S rRNA [46].
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Since Rlv is known to be the common symbiont of Vicia spp. worldwide, we relied on the atpD
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and recA to resolve the diversity and phylogeny of our isolates. Our results showed that indeed
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all the isolates belonged to Rlv group and fell under 12 different genotype backgrounds based on
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the phylogenetic tree from the concatenated atpD and recA gene sequences.
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Some of the factors that influence the occurrence and diversity of rhizobia include the population
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of resident soil bacteria, soil characteristics (e.g. pH, salinity, temperature, moisture), land use
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management and the host plant genotype [28]. Several studies have also shown that natural soil
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populations of rhizobia are diverse and that diversity between sites varies more greatly than
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within sites [14,35,41,44]. Our results showed that the V. cracca isolates fell under six different
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genotype backgrounds based on the concatenated atpD and recA sequences. Recently, an
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extensive study on the genetic diversity and population structure of V. cracca isolates in Belgium
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and France by van Cauwenberghe et al. [44] also showed strong genetic differentiation among
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both V. cracca populations that were separated by few kilometers, and among regions that were
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over 50 to 350 km apart. Even though we had not more than two V. cracca isolates per site in
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this study, most of the genotypes were restricted to unique sites. However, it is not uncommon to
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find isolates with similar chromosomal backgrounds across different geographic sites
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[11,16,47,50] as we have also observed in this study. The major chromosomal background
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among the isolates in this study, group XII (Table 1), had isolates originating from nodules on V.
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sylvatica, V. cracca, V. sepium and V. tetrasperma, all from sites in southern Sweden. It is
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worthwhile to note that Rhizobium sp. MVPO1 from Spain also shares similar chromosomal
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background as our isolates in group XII. Kumar et al. [23] have also reported the presence of
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specific genospecies of Rlv in different continents, supporting the successful spread and
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establishment of rhizobia strains into different environments. It appears that Rlv with this
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chromosomal background (group XII) may be dominant in the southern Sweden and thus easily
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picked up by the different Vicia species growing in those areas. A test to confirm this may be
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done by trapping rhizobia with different Vicia species from soils taken from selected locations in
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this region and sequencing some housekeeping genes from the isolates occupying the nodule.
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Among our isolates, only KHDVB 1556.1 which was isolated from V. hirsuta had similar
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chromosomal background as the reference strain R. leguminosarum sv. viciae 3841 originating
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from the United Kingdom. Interestingly, Alvarez-Martinez et al. [1] had earlier reported that an
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isolate Rhizobium sp. PEVF10 originating from Peru in South America also shares similar
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chromosomal background as R. leguminosarum sv. viciae 3841 and our phylogenetic tree
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confirmed that indeed these three (KHDVB 1556.1, Rhizobium sp. PEVF10 and R.
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leguminosarum sv. viciae 3841) have similar chromosomal background with reference to their
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atpD and recA sequences. The genetic evidence showing that isolates from different continents
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share similar chromosomal background strengthens the concept of the intercontinental spread of
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rhizobia.
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Over the years, the taxonomy of rhizobia has seen some revisions largely arising from the
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isolation of novel strains from previously unexplored areas as well as from native or wild
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legumes of non-agronomic importance. For instance it was thought that rhizobia were restricted
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to the α-proteobacteria group. This perception changed when bacteria from the β-proteobacteria
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group like Burkholderia sp. were found to nodulate with Aspalathus and Machaerium plants [30]
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and Ralstonia taiwanensis sp. nov., with Mimosa species [7]. Further studies have now shown
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that Mimosa are naturally nodulated by Burkholderia sp.[5,8,26]. In comparison to cultivated
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Vicia spp. like V. faba or V. sativa, not much work has been done to identify or characterize
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rhizobia associated with wild Vicia spp. For instance, there is only one report in the literature
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describing rhizobia associated with V. multicaulis [27] and no documented report for V.
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sylvatica. Even though it is widely believed that Rlv is associated with Vicia spp., a novel
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rhizobia named Rhizobium multihospitium was isolated by Han et al. [18] from V. hirsuta in
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Xinjiang, an isolated region in China. De Meyer et al. [10] also isolated Rhizobium sp. from V.
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hirsuta as well as Bradyrhizobium sp. from V. cracca in Belgium. These findings are indications
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that Vicia spp. may also nodulate with rhizobial species other than Rlv and affirms the
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importance of investigating nodule bacteria associated with non-cultivated legumes. However,
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we did not find any rhizobia group besides Rlv nodulating with the native legumes studied and
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our study provides the first evidence that V. sylvatica is indeed nodulated by Rlv.
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It was not surprising for all the isolates to fall under a monophyletic group which was well
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separated from the other Rhizobium species or type strains known to nodulate outside the Vicia
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group since the nodA is involved in host specificity (Fig. 3). Thus the phylogeny of the nodA
291
confirmed that the isolates belonged to the symbiovar viciae. Even though four main clades were
292
observed, most of the isolates clustered within a clade, with the exception of KHDSib 8.11 which
293
was unique from the others (Fig. 3). This unique nodA from KHDSib 8.11 possibly reflects a
294
different nodA backbone needed for the nodulation of V. multicaulis. The only documented
295
report on rhizobia nodulating V. multicaulis [27] indicated an Rlv genotype background, but
296
there was no information on the nodulation genes from those isolates which makes it impossible
297
to compare with our work. A test to verify this is to check the possibility of some of the isolates
298
from this work or the Rlv type strains to nodulate V. multicaulis. Another explanation is that,
299
perhaps this nodA may be unique to that environment (Siberia) since the other isolates studied
300
were from Scandinavia. Recently we isolated a Mesorhizobium huakuii strain with a unique
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nodA from Thermopsis lupinoides in Kamchatka, Russia [3] and it is likely rhizobia found in
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such unexplored regions may have genes adapted to nodulate legumes in such environment.
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The isolates nodulating V. cracca were under four different nodA backgrounds even though most
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of them (9 out of the 13) had identical housekeeping genes background. The dominance of one
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particular nodA background from all the isolates nodulating V. cracca suggests that V. cracca
306
may have a preference for isolates harbouring that nodA type. Apart from the observation that the
307
majority of the V. cracca isolates had identical nodA and formed a V. cracca clade, some of the
308
other isolates originating from the other Vicia hosts also clustered together with respect to the
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nodA, an observation in support of the cross-nodulation of isolates from these hosts. In general,
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there was no pattern or association of nodA to sites as similar nodA genotype was found in
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different locations even at significant distance apart as reported in some other studies [2,43].
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However, it appears that nodA genotype I is more predominant in northern Sweden (Fig. 3, Table
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1).
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With rhizobia, it is well known that phylogeny of the housekeeping genes are usually not
315
congruent with the nodulation genes since the nodulation genes are located on a plasmid and are
316
more subject to lateral gene transfer [27,33,43,44,49]. Our concatenated atpD and recA
317
sequences were also not congruent with the nodA phylogeny. However, the phylogenetic analysis
318
of the nodulation genes confirms that the isolates belong to the symbiovar viciae. An interesting
319
observation in this work was that isolate OYAVB 164.10 from Tromsø in Northern Norway and
320
isolates KHDVB 1598.3 and KHDVB 1619.10 from Haparanda in northern Sweden had similar
321
genetic background despite being over 475 km apart. It is possible that these sites share rhizobia
322
with similar genetic background as a result of introduction of rhizobia through seeds or perhaps
323
through other human activities. A similar observation was also reported by Duodu et al. [13]
324
who also showed that some field isolates of rhizobia nodulating Trifolium spp. that were sampled
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15 Page 15 of 30
from Umeå in northern Sweden and Tromsø in northern Norway had similar genetic background
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as well as nodEF profiles.
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Conclusions
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In this work we have provided information on the genetic diversity and phylogeny of rhizobia
329
that are associated with native Vicia species in Sweden. We have shown that Rlv is the main
330
symbiont of Vicia species in Sweden. Future work will include testing the symbiotic efficiency
331
of some of these indigenous isolates on Vicia species of agronomic importance in Sweden like
332
Vicia sativa with the aim of identifying potential isolates that could be used as inoculants.
333
Acknowledgements
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Funding for this research was received from the Swedish Research Council Formas to KHD. We
335
thank K. Danell for help in field work and A-S Hahlin for assistance in laboratory work. We are
336
grateful to Mats Högström for GIS support and map and to Bente Eriksen, Olga
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Khitun och V.V. Petrovskii for their kind help to identify Vicia multicaulis.
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Conflict of interest: None declared
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Table
Host
Nodulation test
V. cracca
Nod+
KHDVB 654.3 KHDVB 1598.3 KHDVB 1619.10 KHDVB 1680.5 KHDVB 1681.2 KHDVB 1703.2 KHDVB 1707.1 OYAVB 169.1 OYAVB 174.1 KHDVB 1030.3 KHDVB 1902.3 KHDVB 1554.5 KHDVB 1556.1 KHDVB 2171.3 KHDVB 1942.4 KHDVB 1942.9 KHDVB 1942.14 KHDVB 1942.2 KHDVB 2094.1 KHDVB 2098.1 KHDVB 2103.2 KHDVB 2009.7 KHDVB 2018.1 KHDsib 8.11
V. cracca V. cracca V. cracca V. cracca V. cracca V. cracca V. cracca V. cracca V. cracca V. cracca V. cracca V. hirsuta V. hirsuta V. cracca V. sylvatica V. sylvatica V. sylvatica V. sylvatica V. tetrasperma V. tetrasperma V. tetrasperma V. sepium V. sepium V. multicaulis
Nod+ Nod+ Nod+ Nod+ Nod+ Nod+ Nod+ Nod+ Nod+ Nod+ Nod+ Nod+ Nod+ Nod+ Nod+ (on V. sativa) Nod+ (on V. sativa) Nod+ (on V. sativa) Nod+ (on V. sativa) Nod+ (on V. sativa) Nod+ (on V. sativa) Nod+ (on V. sativa) Nod+ Nod+ Nod+ (on V. cracca)
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OYAVB 164.10
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Collection site
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Isolate
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Table 1. Nodulation phenotype, site of isolation and genetic background of isolates from the different Vicia species studied
Norway: Tromsø, Storskogåsen, Prestvannet (A) Sorsele, Ammarnäs, Tjärnen (B) Haparanda, Nikkala, Saantasaari (C) Haparanda, Nikkala, Saantasaari (C) Råneå, Högsön (D) Råneå, Högsön (D) Storuman, Barsele (E) Storuman, Barsele (E) Umeå, Ängersjö (F) Umeå, Ängersjö (F) Timrå, Gökböle (G) Kristianstad, Degeberga (H) Lund, Höjeå (I) Lund, Höjeå (I) Simrishamn, St Olof-Onslunda (J) Kristianstad, Bokenäset (K) Kristianstad, Bokenäset (K) Kristianstad, Bokenäset (K) Kristianstad, Bokenäset (K) Kristianstad, Bokenäset (K) Kristianstad, Bokenäset (K) Kristianstad, Bokenäset (K) Kristianstad, Bokenäset (K) Kristianstad, Bokenäset (K) Russia: Siberia, Jakutsk (n.a.)
atpDrecA genotype II
nodA genotype I
Genotype
III II II III III I I IV I I XII IX VII V XII XII XI XII X VIII XII XII XII VI
I I I I I I I II VI VI II VI VI I III III III III II VI II II II IV
4 1 1 4 4 5 5 2 3 3 6 13 14 7 9 9 10 9 11 12 6 6 6 Page 25 of 30 8
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Letters in parenthesis indicates the collection site shown in Figure 1. n.a means not available on the map in Figure 1.
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Figure legend
Figure captions
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Fig.1. Sites from Sweden and Norway where Vicia plants were sampled for this study.
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Fig. 2. Neighbour-joining phylogenetic tree based on concatenated atpD and recA sequences, showing the relationships between the isolates from the different Vicia species sampled from
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different sites and some reference and type strains. The tree was constructed using MEGA4 software. The numbers at branch points are the significant bootstrap values (expressed as
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percentage based on 1000 replicates; only values greater than 70 % are shown). The horizontal
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branch lines are proportional and indicate the p-distances. The scale bar represents the number of nucleotide substitutions per 100 nucleotides. Accession numbers from the Genbank are in
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parenthesis.
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Fig. 3. Neighbour-joining phylogenetic tree based on nodA sequences, showing the relationships
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between the isolates from the different Vicia species sampled from different sites and some reference and type strains. The tree was constructed using MEGA4 software. The numbers at branch points are the significant bootstrap values (expressed as percentage based on 1000 replicates; only values greater than 70 % are shown). The horizontal branch lines are proportional and indicate the p-distances. The scale bar represents the number of nucleotide substitutions per 100 nucleotides. Accession numbers from the Genbank are in parenthesis.
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Figure
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Figure
cr
KHDVB 1703.2 (KM577289,KM577314) KHDVB 1707.1 (KM577290, KM577315) I KHDVB 1030.3 (KM577283, KM577308) OYAVB 174.1 (KM577282, KM577307) 86 KHDVB 1598.3 (KM577285, KM577310) KHDVB 1619.10 (KM577286, KM577311) II 97 OYAVB 164.10 (KM577280, KM577305) KHDVB 654.3 (KM577284, KM577309) III KHDVB 1680.5 (KM577287, KM577312) 86 KHDVB 1681.2 (KM577288, KM577313) OYAVB 169.1 (KM577281, KM577306) IV KHDVB 2171.3 (KM577292, KM577317) V KHDSib 8.11 (KM577293, KM577318) VI KHDVB 1556.1 (KM577302, KM577327) VII Rhizobium sp. PEVF10 (EF113143, EF113128) 100 Rhizobium leguminosarum sv. viciae 3841 (AM236080, AM236080) VIII KHDVB 2098.1 (KM577299, KM577324) 88 88 KHDVB 1554.5 (KM577301, KM577326) IX T 100 Rhizobium leguminosarum sv. trifolii ATCC 14480 (EF113150, EF113135)
us
Fig 2
97
an
72
Ac c
99
ep te
d
M
94
99
99 89
T
Rhizobium leguminosarum LMG14904 (AM418783, AM182125) 98 Rhizobium leguminosarum sv. viciae USDA 2370T (AJ294405, AJ294376) KHDVB 2094.1 (KM577298, KM577323) X KHDVB 1942.14 (KM577296, KM577321) XI KHDVB 1902.3 (KM577291, KM577316) KHDVB 1942.4 (KM577294, KM577319) KHDVB 1942.9 (KM577295, KM577320) KHDVB 1942.2 (KM577297, KM577322) XII KHDVB 2103.2 (KM577300, KM577325) KHDVB 2009.7 (KM577303, KM577328) KHDVB 2018.1 (KM577304, KM577329) Rhizobium sp. MVPO1 (FJ596008, FJ596035) T
Rhizobium anhuiense CCBAU 23252 (KF111890, KF111980) T Rhizobium fabae CCBAU 33202 (EF579929, EF579941) T 99 Rhizobium pisi DSM 30132 (EF113149, EF113134) T Rhizobium phaseoli ATCC 14482 (EF113151, EF113136) T Rhizobium tropici B USDA 9030 (AJ294397, AJ294373) T Rhizobium etli USDA 9032 (AJ294404, AJ294375) T Rhizobium bangladeshense BLR175 (JN648967, JN649057) T Rhizobium lentis BLR27 (JN648941, JN649031) T Rhizobium binae BLR195 (JN648968, JN649058) Sinorhizobium meliloti 1021 (SMc02501, SMc00760)
0.01
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ip t
Figure KHDVB 1707.1 (KM591232) KHDVB 2171.3 (KM591234)
cr
KHDVB 1703.2 (KM591231)
Fig 3
KHDVB 1681.2 (KM591230) 100 KHDVB 1680.5 (KM591229) KHDVB 1598.3 (KM591227)
75
KHDVB 654.3 (KM591226)
an
OYAVB 164.10 (KM591222)
us
KHDVB 1619.10 (KM591228)
I
Rhizobium leguminosarum sv. viciae 3841 (pRL100185) 100 Rhizobium legumiosarum CTG-10vf (KC620562) 85
Rhizobium lentis BLR9 (JN648983)
T
M
100 Rhizobium bangladeshense BLR175 (JN648991) T
Rhizobium leguminosarum sv. viciae USDA 2370 (JN558711) 70
KHDVB 1902.3 (KM591233) KHDVB 2018.1 (KM591246)
d
KHDVB 2009.7 (KM591245) KHDVB 2094.1 (KM591240)
II
KHDVB 2103.2 (KM591233)
ep te
100
OYAVB 169.1 (KM591223) KHDVB 1942.4 (KM591236) KHDVB 1942.9 (KM591237) KHDVB 1942.14 (KM591238)
100
III
KHDVB 1942.2 (KM591239)
Ac c
KHDSib 8.11 (KM591235)
IV
Rhizobium binae BLR228 (JN648992) KHDVB 1556.1 (KM591244)
100
KHDVB 1554.5 (KM591243) KHDVB 2098.1 (KM591241)
V
OYAVB 174.1 (KM591224) 74 KHDVB 1030.3 (KM591225)
VI
Rhizobium leguminosarum sv. trifolii ICMP 5377 (EF115496) T
100
Rhizobium etli CFN42 (RHE_PD00310) Rhizobium leguminosarum sv. phaseoli ICMP 2672 (DQ100403) Sinorhizobium meliloti 1021 (SMa0869)
0.05
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