IJSEM Papers in Press. Published May 29, 2015 as doi:10.1099/ijs.0.000365
International Journal of Systematic and Evolutionary Microbiology Rhizobium anhuiense sp. nov., isolated from effective nodules of Vicia faba and Pisum sativum grown in Southern China --Manuscript Draft-Manuscript Number:
IJS-D-15-00037R2
Full Title:
Rhizobium anhuiense sp. nov., isolated from effective nodules of Vicia faba and Pisum sativum grown in Southern China
Short Title:
Rhizobium anhuiense sp. nov.
Article Type:
Note
Section/Category:
New taxa - Proteobacteria
Corresponding Author:
xinhua sui, Ph,D China Agricultural University CHINA
First Author:
Yu Jing Zhang, MD
Order of Authors:
Yu Jing Zhang, MD Wen Tao Zheng, MD Isobel Everall J. Peter W Young, PhD Xiao Xia Zhang, PhD Chang Fu Tian, PhD Xin Hua Sui, Ph,D En Tao Wang, PhD Wen Xin Chen, PhD
Manuscript Region of Origin:
CHINA
Abstract:
Four rhizobia like strains, isolated from root nodules of Pisum sativum and Vicia faba grown in Anhui and Jiangxi Provinces of China, were grouped into the genus Rhizobium but were distinct from all recognized Rhizobium species by phylogenetic analysis of 16S rRNA and housekeeping genes. The combined sequence of housekeeping genes atpD, recA and glnII for strains CCBAU 23252T was 86.9% to 95% similar to those of known Rhizobium species. All the four strains had nodC and nifH genes and could form effective nodules with Pisum sativum and Vicia faba, and ineffective nodules with Phaseolus vulgaris, but did not nodulate Glycine max, Arachis hypogaea, Medicago sativa, Trifolium repens or Lablab purpureus in cross-nodulation tests. Fatty acid composition, DNA-DNA relatedness and a series of phenotypic tests also separated these strains from the closely related species. Based on all the evidence, we propose a novel species Rhizobium anhuiense sp. nov., and designate CCBAU 23252T (=CGMCC 1.12621T =LMG 27729T) as the type strain. This strain was isolated from a root nodule of Vicia faba and has a GC content of 61.1 mol% (Tm).
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1
Rhizobium anhuiense sp. nov., isolated from effective nodules of Vicia faba and
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Pisum sativum grown in Southern China
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Yu Jing Zhang1, Wen Tao Zheng1, Isobel Everall2, J. Peter W. Young2, Xiao Xia
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Zhang3, Chang Fu Tian1, Xin Hua Sui1*, En Tao Wang1,4, Wen Xin Chen1
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1.
State Key Lab for Agro-Biotechnology, Ministry of Agriculture Key Lab of Soil
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Microbiology, College of Biological Sciences, China Agricultural University,
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Beijing, 100193, China.
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2.
Department of Biology, University of York, York, YO10 5DD, UK
9
3.
Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences, Beijing 100081, China
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11 12
4.
Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, 11340 México D. F., Mexico.
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(Wen Tao Zhang is now at Beijing Century Kingdo Petro_Tech Co. Ltd, Beijing, 100029,
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China; Isobel Everall is now at Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10
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1SA, UK)
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Running title: Rhizobium anhuiense sp. nov.
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Contents category: New taxa - Proteobacteria
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*Corresponding author: Dr. Xin Hua Sui. Tel: 86 10 62734009; Fax: 86 10
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62734008; E-mail: [email protected]. 1
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Abbreviations: ML, maximum-likelihood; NJ, neighbour-joining; ANI, average
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nucleotide identity.
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Accession numbers for type strain: 16S rRNA gene: KF111868; atpD gene:
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KF111890; recA gene: KF111980; glnII gene: KF11913; rpoB gene: KR183848; gyrB
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gene: KR183844; dnaK gene: KR183840; nifH gene: KF111936 and nodC gene:
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KF111957.
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Three supplementary tables and six supplementary figures are available with the
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online version of this paper.
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2
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Abstract
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Four rhizobia like strains, isolated from root nodules of Pisum sativum and Vicia faba
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grown in Anhui and Jiangxi Provinces of China, were grouped into the genus
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Rhizobium but were distinct from all recognized Rhizobium species by phylogenetic
33
analysis of 16S rRNA and housekeeping genes. The combined sequence of
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housekeeping genes atpD, recA and glnII for strains CCBAU 23252T was 86.9% to 95%
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similar to those of known Rhizobium species. All the four strains had nodC and nifH
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genes and could form effective nodules with Pisum sativum and Vicia faba, and
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ineffective nodules with Phaseolus vulgaris, but did not nodulate Glycine max,
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Arachis hypogaea, Medicago sativa, Trifolium repens or Lablab purpureus in
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cross-nodulation tests. Fatty acid composition, DNA-DNA relatedness and a series of
40
phenotypic tests also separated these strains from the closely related species. Based on
41
all the evidence, we propose a novel species Rhizobium anhuiense sp. nov., and
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designate CCBAU 23252T (=CGMCC 1.12621T =LMG 27729T) as the type strain.
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This strain was isolated from a root nodule of Vicia faba and has a GC content of 61.1
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mol% (Tm).
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3
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Leguminous plants pea (Pisum sativum) and broad bean (Vicia faba) were
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originally domesticated in the Middle East (Bond, 1976). They are essential foods,
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vegetables and raw materials for other bean products. Vicia faba has been cultivated
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for around 6,000 years and is currently grown in 57 countries (George, 1999). Both
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Vicia faba and Pisum sativum are introduced plants widely grown in China.
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Mutualisms between Rhizobium leguminosarum symbiovar viciae and Pisum sativum
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or Vicia faba have been widely studied. Plants of Vicia faba strongly select rhizobia
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with a specific nodD type, while Pisum sativum plants are more promiscuous in
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selection of their symbionts (Laguerre et al., 2003, Mutch & Young, 2004). In
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addition to R. leguminosarum, three related species Rhizobium pisi, Rhizobium fabae,
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and Rhizobium laguerreae have been described for broad bean rhizobia
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(Ramírez-Bahena et al., 2008, Saïdi et al., 2014; Tian et al., 2008), demonstrating the
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existence of diverse rhizobial species associated with this plant.
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In the present study, root nodules of Vicia faba and Pisum sativum were collected
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from the fields of Anhui and Jiangxi Provinces in China and twenty-three fast
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growing, acid-producing bacteria were isolated from the surface-sterilized effective
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(red colored) nodules using the standard method on YMA plates incubated at 28C
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(Vincent, 1970). Eighteen isolates were clustered as a group that was distant from
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recognized species of the genus Rhizobium according to sequences of the
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housekeeping gene recA (Fig S1). Therefore, four isolates listed in Table S1 (CCBAU
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23242, CCBAU 23252T, CCBAU 33508, CCBAU 33602) were selected as
4
67
representatives to continue further sequencing studies. The isolates were preserved in
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20% (w/v) glycerol at −80C and also stored by lyophilization.
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The genomic DNA was extracted from each of the four strains using standard
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methods (Tan et al., 1997). The 16S rRNA gene (Tan et al., 1997), housekeeping
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genes atpD, recA and glnII (Vinuesa et al., 2005), nodulation gene nodC (Sarita et al.,
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2005) and nitrogen-fixing gene nifH (Laguerre et al., 2001) were amplified and
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sequenced, using the extracted DNA as template and primer sets P1/P6,
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atpD255F/atpD782R, recA41F/recA640R, glnII12F/glnII689R, nodC540/nodC1160
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and nifH1F/nifH1R, respectively. The acquired sequences were aligned with those of
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Rhizobium type strains extracted from the NCBI nucleotide sequence database. The
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genetic distances between sequences were calculated using the Clustal W program in
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the MEGA 5.0 software package (Tamura et al., 2011), and phylogenetic trees were
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constructed using the Maximum likelihood (ML) and Neighbor-joining (NJ) methods
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with 1000 bootstrap replications.
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The ML tree of the 16S rRNA gene sequences (1318 nt) showed that the four
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isolates had identical 16S rRNA genes and had same sequence as Rhizobium
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leguminosarum USDA 2370T, Rhizobium laguerreae FB 206T, R. indigoferae CCBAU
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71042T and Rhizobium sophorae CCBAU 03386T, a novel species published in 2014
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(Jiao et al.) (Fig. 1, Table S2). The similarities between the isolates and type strains of
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Rhizobium species varied from 95.6% to 100%. The NJ tree generated based upon
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the16S rRNA gene sequences in this study was consistent with ML tree (data not
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shown). 5
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Since the novel strains have 16S rDNA sequences identical with those of R.
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leguminosarum USDA 2370T, R. laguerreae FB 206T, R. indigoferae CCBAU 71042T,
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R. sophorae CCBAU 03386T, we used housekeeping genes to further clarify the
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relationships among the novel isolates and the defined Rhizobium species. The
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housekeeping genes recA, atpD and glnII were partially sequenced and a phylogenetic
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tree was constructed based upon the combined sequences (Fig. 2). The similarities of
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concatenated partial sequences of recA, atpD and glnII genes (1249nt) between the
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four isolates and type strains of Rhizobium species ranged from 85.1% to 95.6%. The
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strain CCBAU 23252T showed closest sequence similarity of 95% with R.
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leguminosarum USDA 2370T (Table S2). This result agreed with the phylogenetic
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trees generated separately for each of the recA, atpD or glnII gene sequences (Figs. S1
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-S3) and supported that these isolates represented a novel species, taking the
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Rhizobium species defined recently (Ramírez-Bahena et al., 2008, Tian et al., 2008,
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Saïdi et al., 2014) as references.
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In order to compare strains using Average Nucleotide Identity (ANI), genomic
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DNA for two representative strains CCBAU 23252T (from Anhui province) and 33602
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(from Jiangxi province) was extracted using PowerSoil DNA isolation kits (MoBio,
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Carlsbad, CA), and then fragmented, barcoded, quantitated and run as part of a batch
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of eight genomes on a 318 chip on an Ion Torrent PGM using the manufacturer’s
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recommended protocols (Thermo Fisher, Waltham, MA). Each genome was
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assembled using the Newbler GS De Novo assember version 2.8 (Roche diagnostics)
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with default parameter values. ANI was calculated within the JSpecies software 6
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(Richter et al., 2009). The Nucleotide MUMmer algorithm (NUCmer) was used, with
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default parameter settings, to calculate the ANI by subtracting the similarity errors
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from the alignment length (Richter et al., 2009, Kurtz et al., 2004). Genomes were
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compared with each other, and with complete genome assemblies downloaded from
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NCBI for the strains R. leguminosarum symbiovar viciae 3841 (GCA_000009265),
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and R. leguminosarum symbiovar trifolii WSM 1325 (GCA_000023185), which were
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widely used in genetic research representing R. leguminosarum species. The ANI
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between strains CCBAU 23252T and CCBAU 33602 was 98.34-98.37% (Table 1),
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which was above the 95–96% boundary for species definition (Richter et al., 2009),
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indicating that they belong to the same species. The ANI between these strains and
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representatives of the most closely related species R. leguminosarum were below 92%
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(Table 1), implying that these two novel strains cannot be included in the closest
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species.
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The genes nodC and nifH are essential for rhizobia to establish effective
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nitrogen-fixing symbiosis with the host legumes. A phylogenetic tree of part of the
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nifH gene (350 nt) showed that the sequences of this gene in the four isolates were
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different, but very similar: CCBAU 23252T and CCBAU 33602, both from Vicia faba,
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shared a similarity of 98.5%; CCBAU 23242 and CCBAU 33508, both from Pisum
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sativum, were two single lineages (Fig. S4). All of them were clustered at similarities
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of 95.2-97.9% with the five reference strains of R. leguminosarum, R. pisi, R. fabae, R.
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laguerreae and R. indigoferae, which mainly originated from V. faba. The ML tree of
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the partial nodC genes (450nt) of all strains showed a similar phylogenetic 7
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relationship with respect to that of the nifH gene, at 93.5-99.3% similarities among the
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tested strains and at 90.2-99.1% similarities with the closest reference strains R.
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leguminosarum, R. pisi, R. fabae, R. laguerreae and R. multihospitium (Fig. S5),
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together with the host range, implying all these strains belonged to symbiovar viciae.
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DNA–DNA hybridization using the renaturation-rate technique (De Ley et al.,
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1970) between the novel bacteria and type strains of related species has been
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recommended as a standard method for species delineation (Graham et al., 1991).
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Three replicates were tested in present study. The DNA-DNA relatedness between the
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reference isolate CCBAU 23252T and the other isolates of the novel group ranged
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from 79.51% to 97.54%. Meanwhile, the values were only 26.8% to 37.92% between
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CCBAU 23252T and the most closed type strains R. leguminosarum USDA 2370T, R.
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indigoferae CCBAU 71042T, R. sophorae CCBAU 03386T (Table S2). These values
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further illustrated that the four isolates represented a new genomic species in the
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genus Rhizobium.
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BOX-PCR fingerprinting was performed by using the procedure of Versalovic et
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al. (1994) and distinguished the four isolates from each other (Fig. S6), illustrating
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that they were not clones of the same strain. The G+C contents of genomes of the four
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isolates, measured by the thermal denaturation method for strains CCBAU 23242 and
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CCBAU 33508 (De Ley et al., 1970) or calculated by the genome data for strains
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CCBAU 23252T and CCBAU 33602 (Richter et al., 2009), varied between 61.1 and
8
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64.8 mol% (Tm), which is within the range for the genus Rhizobium (Young et al.,
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2001).
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Cellular fatty acid profiles were determined to provide additional description of
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the novel species. Strain CCBAU 23252T and the type strains for nine related species,
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R. leguminosarum USDA 2370T, R. laguerreae LMG 27434T, R. indigoferae CCBAU
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71042T, R. pisi DSM 30132T, R. etli CFN 42T, R. vallis CCBAU 65647T, R. phaseoli
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ATCC 14482T, R. sophorae CCBAU 03386T and R. sophoriradicis CCBAU 03470T
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were chosen to compare their fatty acid compositions. The strains were streaked on
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YMA medium and cultivated at 28C to the late exponential phase. About 40–100 mg
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of wet cells was collected into sample tubes (13×100 mm). Fatty acids were then
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extracted following the method described by Sasser (1990) and identified by the MIDI
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Sherlock Microbial Identification System (Sherlock license CD v 6.0) in the TSBA6
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database. All strains were tested in the present study except R. sophorae CCBAU
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03386T and R. sophoriradicis CCBAU 03470T, which were tested by using the same
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procedure at the same period in our lab and recent publication (Jiao et al., 2014).
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Thirty-three kinds of fatty acids were detected in total. Summed feature 8 (C18:1 ω6c
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and C18:1 ω7c, 45.73–68.17%), summed feature 2 (C14:0 3OH/C16:1 iso I, 4.4–12.76%)
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and C18:0 (4.39–9.1%) were the dominant components in all strains (Table S3), which
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were different from the previously reported results for the genera Bradyrhizobium and
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Mesorhizobium (Wang et al., 2013, Zheng, et al., 2013), but similar to those reported
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for Rhizobium species (Tighe et al., 2000). In comparison to the type strains of related
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Rhizobium species, C18:1 ω5c was only detected in CCBAU 23252T in this study, 9
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indicating that it was different from the other species. From the fatty acid
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compositions, CCBAU 23252T was most similar to R. pisi DSM 30132T and R. etli
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CFN 42T; however, some differences were observed: C18:1 ω7c 11-methyl was absent
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in R. etli CFN 42T and C20:2 ω6, 9c was absent in R. pisi DSM 30132T, whereas both
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were present in CCBAU 23252T. These distinctions provide additional evidence that
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CCBAU 23252T belongs to a novel species in the genus Rhizobium.
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The phenotypic features of the two representative isolates and the type strains of
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closely related species were assayed by using the Biolog GN2 MicroPlate to test the
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utilization of sole carbon sources, according to the manufacturer’s instructions; a
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previously described method (Gao et al., 1994) was used for other characteristics such
185
as growth temperature and pH ranges, tolerance to NaCl and antibiotics and
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generation time etc. The detailed differences between the novel isolates and the type
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strains are shown in Table 2, and the shorten different features compared the two
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novel strains and the most closed strain R. leguminosarum USDA 2370 are
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summarized in the description of the novel species.
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To further identify the host range of these isolates, cross-nodulation tests were
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conducted in vermiculite with N-free plant nutrient solution (Vincent, 1970). The four
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isolates could nodulate Pisum sativum and Vicia faba. Cross-sections of the nodules
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were pink (the color of leghemoglobin), indicating that the nodules were effective for
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fixing nitrogen. The isolates can also form nodules with Phaseolus vulgaris, but the
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inside of the nodules was white, illustrating that they were ineffective. The strains 10
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could not nodulate Medicago sativa, Trifolium repens, Lablab purpureus, Arachis
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hypogaea or Glycine max.
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Based on all the phenotypic, genotypic and phylogenetic characteristics obtained
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in this study, we identified our four strains different form the recognized Rhizobium
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species and propose that the four isolates represent a novel species, Rhizobium
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anhuiense.
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Description of Rhizobium anhuiense sp. nov.
203 204
Rhizobium anhuiense (an.hui.en′se. N.L. neut. adj. anhuiense originating from Anhui Province of China, from where the type strain was isolated).
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The cells are Gram-staining-negative, non-spore-forming, aerobic rods. Colonies
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are translucent, cream white and convex, with a diameter of 3–4 mm on YMA
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medium after incubation for 3 days at 28C. Cells are 0.41–0.48 µm 0.89–1.48 µm
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in size and have a generation time of 3.9 hours when growing in YM medium at 28C.
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They can grow at 15C to 37C, with an optimum temperature of 28C. The pH for
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growth ranges from 5 to 8, with optimum of pH 7.0. The bacteria grow weakly in the
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presence of 1.0% (w/v) NaCl. N-acetyl-D-galactosamine, N-acetyl-D-glucosamine,
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adonitol, D-cellobiose, D-galactose, gentiobiose, m-inositol, α-D-lactose, lactulose,
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maltose, D-mannitol, β-methyl-D-glucoside, D-raffinose, D-trehalose, xylitol,
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succinic acid mono-methyl ester, D-galactonic acid lactone, D-gluconic acid,
215
D-glucosaminic acid, β-hydroxybutyric acid, quinic acid, L-alanine, L-alanyl-glycine,
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L-glutamic acid, hydroxy-L-proline, L-ornithine, L-proline, γ-aminobutyric acid, 11
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urocanic acid, uridine and α-D-glucose-1-phosphate can be utilized as sole carbon
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sources. Succinic acid cannot be utilized as sole carbon sources. Summed feature 8
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(18:1 ω6c/18:1 ω7c), summed feature 2 (C14:0 3OH/C16:1 iso I) and C18:0 were the
220
dominant cellular fatty acids. CCBAU 23252T (= CGMCC 1.12621T = LMG 27729T)
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was designated as the type strain. The DNA G+C content of the type strain is 61.1 mol%
222
(Tm).
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Acknowledgements
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We are grateful to Dr. Claudine Vereecke at LMG for providing some type
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strains. This work was supported by the National Natural Science Foundation of
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China (31170003, 31470135 & 31211130109), by 863 Project (2013AA102802-04)
227
and by Royal Society International Exchange IE222202.
228
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migration and recombination in Bradyrhizobium species cohesion and delineation.
301
Mol Phylogenet Evol 34, 29-54.
302
Wang, R., Chang, Y.L., Zhang, W.T., Zhang, D., Zhang, X. X., Sui, X.H.,
303
Wang, E. T., Chen, W.X. (2013). Bradyrhizobium arachidis sp. nov., isolated from 15
304
effective nodules of Arachis hypogaea grown in China. Syst Appl Microbiol 36,
305
101-105.
306
Young, J.M., Kuykendall, L.D., Martınez-Romero, E., Kerr, A., Sawada, H.
307
(2001). A revision of Rhizobium Frank 1889, with an emended description of the
308
genus, and the inclusion of all species of Agrobacterium Conn 1942 and
309
Allorhizobium undicola de Lajudie et al. 1998 as new combinations: Rhizobium
310
radiobacter, R. rhizogenes, R. rubi, R. undicola and R. vitis. Int J Syst Evol Microbiol
311
51, 89–103.
312
Zheng W.T., Li Y. Jr, Wang R., Sui X.H., Zhang X.X., Zhang J.J., Wang
313
E.T., Chen W.X. (2013). Mesorhizobium qingshengii sp. nov., isolated from effective
314
nodules of Astragalus sinicus grown in the Southeast of China. Int J Syst Evol
315
Microbiol 63, 2002-2007.
16
316 317 318
Table 1. Average Nucleotide Identity (ANI) of CCBAU 23252T, CCBAU 33602 and most closely related strains with available genome sequences R. leguminosarum
R. leguminosarum 3841 R. leguminosarum WSM 1325 R. anhuiense CCBAU 23252T R. anhuiense CCBAU 33602
R. leguminosarum
R. anhuiense
R. anhuiense T
3841
WSM 1325
CCBAU 23252
100
94.15
91.18
91.13
94.16
100
91.12
91.13
91.18
91.11
100
98.37
91.12
91.12
98.34
100
319 320
17
CCBAU 33602
321
Table 2. Different features of Rhizobium anhuiense sp. nov. and its close relatives
322
1, CCBAU 23252T; 2, CCBAU 33602; 3, R. etli CFN 42T; 4, R. pisi DSM 30132T; 5, R.
323
leguminosarum USDA 2370T; 6, R. vallis CCBAU 65647T; 7, R. indigoferae CCBAU 71042T; 8, R.
324
phaseoli ATCC 14482T; 9, R. laguerreae LMG 27434T; 10, R. sophorae CCBAU 03386T; 11, R.
325
sophoriradicis CCBAU 03470T 1
2
3
4
5
6
7
8
9
10#
11#
α-Cyclodextrin
-
-
-
-
-
+
-
-
-
-
-
Dextrin
+
+
w
-
w
+
+
+
-
-
-
Glycogen
-
-
-
-
w
+
-
-
-
-
-
N-Acetyl-D-Galactosamine
+
-
-
-
-
+
-
-
-
-
-
N-Acetyl-D-Glucosamine
+
+
-
w
-
+
-
-
-*
-
-
Adonitol
+
+
-
+
-
+
-
+
-
-
-
L-Arabinose
+
+
+
w
w
+
+
+
-*
+
+
D-Arabitol
+
+
+
+
w
+
+
+
-
w
-
D-Cellobiose
+
+
+
+
-
+
+
-
-
+
-
i-Erythritol
-
-
-
-
w
+
-
-
-
+
-
D-Fructose
+
+
+
+
w
+
w
+
-
-
-
L-Fucose
+
+
+
+
w
+
w
-
-*
-
-
D-Galactose
+
+
+
+
-
+
-
w
-
w
-
Gentiobiose
+
+
+
+
-
+
-
-
-
-
-
α-D-Glucose
+
+
+
+
+
+
+
+
-
w
-
m-Inositol
+
+
+
+
-
+
-
-
-
-
-
α-D-Lactose
+
+
+
+
-
+
-
+
-
-
-
Lactulose
+
+
+
-
-
+
-
+
-
-
-
Maltose
+
+
+
+
-
+
w
-
-
w
-
D-Mannitol
+
+
+
+
-
+
w
-
-
-
-
D-Mannose
+
+
+
+
w
+
w
+
-
-
-
D-Melibiose
-
-
w
+
-
+
-
-
-
-
-
D-Psicose
+
+
+
-
w
+
-
-
+
-
-
β-Methyl-D-Glucoside
+
+
+
+
-
+
-
-
-
-
-
D-Raffinose
+
+
-
+
-
+
-
-
-
-
-
L-Rhamnose
+
+
+
+
+
+
w
+
-*
-
-
D-Sorbitol
+
+
+
+
w
+
w
-
+
-
-
Sucrose
+
+
+
+
+
+
-
+
-*
+
-
D-Trehalose
+
-
+
+
-
+
-
-
-
-
-
Turanose
+
+
+
+
w
+
w
-
-
-
-
Xylitol
+
+
+
-
-
+
w
-
-
w
w
Cis-Aconitic Acid
+
+
+
+
w
+
-
+
-
-
-
Characteristic Utilization of sole carbon source：
18
Succinic Acid Mono- Methyl Ester
+
-
w
-
-
+
-
-
-
-
-
Citric Acid
-
-
-
-
-
+
-
-
-
-
-
Formic Acid
+
+
+
-
w
+
-
-
-
-
-
D-Galactonic Acid Lactone
+
+
+
-
-
+
w
+
-
+
-
D-Gluconic Acid
+
+
+
-
-
+
-
-
-
-
-
D-Glucosaminic Acid
+
-
-
-
-
+
-
-
-
-
-
β-Hydroxybutyric Acid
+
+
-
-
-
+
w
-
-
-
-
γ-Hydroxybutyric Acid
-
-
-
w
-
+
-
-
-
-
-
p-Hydroxy-phenlyacetic Acid
-
-
-
+
-
+
-
-
-
-
-
α-Ketoglutaric Acid
-
-
-
+
-
+
w
-
-
-
-
D,L-Lactic Acid
+
+
+
w
w
+
-
-
-
-
-
Malonic Acid
-
-
-
-
-
+
-
-
-
-
-
Propionic Acid
-
-
-
+
-
+
-
-
-
-
-
Quinic Acid
+
+
-
-
-
+
-
-
-
-
-
Succunic Acid
-
-
+
-
+
+
+
+
-
-
-
Bromosuccinic Acid
+
+
-
-
+
+
-
-
-
+
-
L-Alaninamide
+
+
-
-
+
+
w
-
-
w
-
L-Alanine
+
+
-
w
-
+
-
-
-
-
-
L-Alanyl-Glycine
+
+
-
-
-
+
-
-
-
-
-
L-Asparagine
-
-
w
-
-
+
-
+
-
-
-
L-Glutamic Acid
+
+
-
-
-
+
-
-
-
-
-
Glycyl-L-Aspartic Acid
-
+
-
+
-
+
-
-
-
-
-
Glycyl-L-Glutamic Acid
-
+
-
-
-
+
-
-
-
-
-
L-Histidine
+
+
+
-
w
+
-
-
-*
-
-
Hydroxy-L-Prolin
+
+
-
-
-
+
-
-
-
-
-
L-Ornithine
+
-
-
-
-
+
-
-
-
-
-
L-Proline
+
+
-
w
-
+
-
-
-*
-
-
D,L-Camitine
+
+
+
+
w
+
-
-
-
-
-
γ-Aminobutyric Acid
+
+
-
-
-
+
-
-
-
-
-
Urocanic Acid
+
+
+
-
-
+
w
-
-
+
-
Uridine
+
+
+
+
-
+
-
+
-
w
-
Thymidine
-
-
-
-
-
+
-
-
-
-
-
2-Aminoethanol
-
-
+
-
-
+
-
-
-
-
-
α-D-Glucose-1-Phosphate
+
+
+
-
-
+
-
-
-
-
-
D-Glucose-6-Phosphate
-
-
+
-
-
+
-
-
-
-
-
+
+
+
-
+
+
+
+
+
+
+
Growth at: pH 8.0
326
+, growth or resistant; -, no growth or sensitive; w. growth weakly; #All data were obtained with three replicates
327
in this study, except that of R. sophorae CCBAU 03386Tand R. sophoriradicis CCBAU 03470T (Jiao et al.,
328
2014); *, The results were not congruent with that of R. laguerreae LMG 27434T (Jiao et al., 2014).
19
329
Figures Legends
330
Fig. 1. Maximum likelihood tree constructed from 16S rRNA gene sequences
331
showing the phylogenetic relationships of R. anhuiense sp. nov. Bootstrap
332
values of more than 50% for 1000 replicates of the data set are provided on
333
the branches. The sequence of Bradyrhizobium japonicum USDA 6T was used
334
as an outgroup. Bar: 1% substitution.
335
Fig. 2. Maximum likelihood tree based on the partial concatenated sequences of the
336
atpD and recA genes of R. anhuiense sp. nov. and the species in the genus
337
Rhizobium. Bootstrap values of more than 50% for 1000 replicates of the data
338
set are provided on the branches. The accession numbers of the atpD and
339
recA genes of the strains are shown in the brackets. Bar: 2% substitution.
20
Figure 1 Click here to download Figure: fig1.docx
R. etli CFN 42T (CP000133) R. sophoriradicis CCBAU 03470T (KJ831225) 62 R. pisi DSM 30132T (AY509899) 98 R. fabae CCBAU 33202T (DQ835306) R. phaseoli ATCC 14482T (EF141340) CCBAU 23252T (KF111868) 50 CCBAU 23242 (KF111865) R. anhuiense sp. nov. CCBAU 33508 (KF111874) 100 CCBAU 33602 (KF111886) R. indigoferae CCBAU 71042T (AY034027) R. leguminosarum USDA 2370T (JQ085246) R. laguerreae FB206T (JN558651) 64 R. sophorae CCBAU 03386T (KJ831229) R. vallis CCBAU 65647T (FJ839677) 62 R. calliandrae CCGE 524T (JX855162) 97 57 R. paranaense PRF35 T (EU488753) 66 R. jaguaris CCGE525T (JX855169) R. mayense CCGE 526T (JX855172) T 58 R. rhizogense ATCC 11325 (AY945955) R. lusitanum p1-7T (AY738130) R. leucaenae CFN 299T (X67234) R. miluonense CCBAU 41251T (EF061096) 85 R. tropici IIB CIAT 899T (HQ850704) 62 R. multihospitium CCBAU 83401T (EF035074) 50 R. hainanense I66T (U71078) R. grahamii CCGE 502T (JF424608) 98 R. tibeticum LMG 24453T (EU256404) T 99 R. alamii GBV 016 (AM931436) 50 R. mesosinicum CCBAU 25010T (DQ100063) R. sullae IS123T (Y10170) 70 R. gallicum R602spT (CP006877) 100 R. yanglingense SH 22623T (AF003375) R. loessense CCBAU 7190BT (AF364069) 58 R. mongolense USDA 1844T (U89817) Bradyrhizobium japonicum USDA 6T (NC017249) 91
0.01
Fig. 1
1
Figure 2 Click here to download Figure: fig2.docx
CCBAU 23242 (KF111887, KF111983, KF111910)
92
CCBAU 33602 (KF111909, KF111999, KF111932)
100
R. anhuiense sp. nov.
CCBAU 23252T (KF111890, KF111980, KF111913) 0.02
96
72
100
CCBAU 33508 (KF111896, KF111986, KF111919)
R. leguminosarum USDA 2370T (AJ294405, AJ294376, EU155089) R. indigoferae CCBAU 71042T (GU552925, EF027965, JN580717)
69
53
R. sophorae CCBAU 03386T (KJ831235, KJ831252, KJ831241) 83
R. laguerreae FB206T (JN558661, JN558681, JN558671)
93
R. vallis CCBAU 65647T (GU211768, GU211770, GU211771) R. fabae CCBAU 33202T (EF579929, EF579941, EF579935)
75 100
R. pisi DSM 30132T (EF113129, EF113134, JN580715) R. sophoriradicis CCBAU 03470T (KJ831231, KJ831248, KJ831237)
94 77
R. phaseoli ATCC 14482T (EF113151, EF113136, JN580716) R. etli CFN 42T (CP000133) R. tibeticum LMG 24453T (KF206621, KF206877, EU409190) R. multihospitium CCBAU 83401T (EF490019, EF490029, EF490040)
99
R. tropici B LMG 9503T (AM418789, AJ294373, AF169584)
67
R. lusitanum p1-7T (DQ431671, DQ431674, EF639841) R. alkalisoli LMG24763T (EU672461, EU672490, EU672475)
63
R. cellulosilyticus LMG 23642T (AM286426, AM286427, KF206788) R. endophyticum CCGE 2052T (HM142760, HM142767, JF424619) R. sullae IS123T (DQ345069, FJ816279, FJ816280) R. gallicum R602spT (CP006877)
100
100
R. mongolense USDA 1844T (AY907372, AY907358, AY929453) B. japonicum USDA 6T (AM418753, AM168341, AF169582)
Fig. 2
Supplementary Material Files-normal Click here to download Supplementary Material Files: s-material 423 normal.pdf
International Journal of Systematic and Evolutionary Microbiology Rhizobium anhuiense sp. nov., isolated from effective nodules of Vicia faba and Pisum sativum grown in Southern China --Manuscript Draft-Manuscript Number:
IJS-D-15-00037R2
Full Title:
Rhizobium anhuiense sp. nov., isolated from effective nodules of Vicia faba and Pisum sativum grown in Southern China
Short Title:
Rhizobium anhuiense sp. nov.
Article Type:
Note
Section/Category:
New taxa - Proteobacteria
Corresponding Author:
xinhua sui, Ph,D China Agricultural University CHINA
First Author:
Yu Jing Zhang, MD
Order of Authors:
Yu Jing Zhang, MD Wen Tao Zheng, MD Isobel Everall J. Peter W Young, PhD Xiao Xia Zhang, PhD Chang Fu Tian, PhD Xin Hua Sui, Ph,D En Tao Wang, PhD Wen Xin Chen, PhD
Manuscript Region of Origin:
CHINA
Abstract:
Four rhizobia like strains, isolated from root nodules of Pisum sativum and Vicia faba grown in Anhui and Jiangxi Provinces of China, were grouped into the genus Rhizobium but were distinct from all recognized Rhizobium species by phylogenetic analysis of 16S rRNA and housekeeping genes. The combined sequence of housekeeping genes atpD, recA and glnII for strains CCBAU 23252T was 86.9% to 95% similar to those of known Rhizobium species. All the four strains had nodC and nifH genes and could form effective nodules with Pisum sativum and Vicia faba, and ineffective nodules with Phaseolus vulgaris, but did not nodulate Glycine max, Arachis hypogaea, Medicago sativa, Trifolium repens or Lablab purpureus in cross-nodulation tests. Fatty acid composition, DNA-DNA relatedness and a series of phenotypic tests also separated these strains from the closely related species. Based on all the evidence, we propose a novel species Rhizobium anhuiense sp. nov., and designate CCBAU 23252T (=CGMCC 1.12621T =LMG 27729T) as the type strain. This strain was isolated from a root nodule of Vicia faba and has a GC content of 61.1 mol% (Tm).
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manuscript (contents category added) Click here to download Manuscript Including References (Word document): manuscript (contents category added).docx
1
Rhizobium anhuiense sp. nov., isolated from effective nodules of Vicia faba and
2
Pisum sativum grown in Southern China
3
Yu Jing Zhang1, Wen Tao Zheng1, Isobel Everall2, J. Peter W. Young2, Xiao Xia
4
Zhang3, Chang Fu Tian1, Xin Hua Sui1*, En Tao Wang1,4, Wen Xin Chen1
5
1.
State Key Lab for Agro-Biotechnology, Ministry of Agriculture Key Lab of Soil
6
Microbiology, College of Biological Sciences, China Agricultural University,
7
Beijing, 100193, China.
8
2.
Department of Biology, University of York, York, YO10 5DD, UK
9
3.
Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences, Beijing 100081, China
10
11 12
4.
Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, 11340 México D. F., Mexico.
13
(Wen Tao Zhang is now at Beijing Century Kingdo Petro_Tech Co. Ltd, Beijing, 100029,
14
China; Isobel Everall is now at Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10
15
1SA, UK)
16
Running title: Rhizobium anhuiense sp. nov.
17
Contents category: New taxa - Proteobacteria
18
*Corresponding author: Dr. Xin Hua Sui. Tel: 86 10 62734009; Fax: 86 10
19
62734008; E-mail: [email protected]. 1
20
Abbreviations: ML, maximum-likelihood; NJ, neighbour-joining; ANI, average
21
nucleotide identity.
22
Accession numbers for type strain: 16S rRNA gene: KF111868; atpD gene:
23
KF111890; recA gene: KF111980; glnII gene: KF11913; rpoB gene: KR183848; gyrB
24
gene: KR183844; dnaK gene: KR183840; nifH gene: KF111936 and nodC gene:
25
KF111957.
26
Three supplementary tables and six supplementary figures are available with the
27
online version of this paper.
28
2
29
Abstract
30
Four rhizobia like strains, isolated from root nodules of Pisum sativum and Vicia faba
31
grown in Anhui and Jiangxi Provinces of China, were grouped into the genus
32
Rhizobium but were distinct from all recognized Rhizobium species by phylogenetic
33
analysis of 16S rRNA and housekeeping genes. The combined sequence of
34
housekeeping genes atpD, recA and glnII for strains CCBAU 23252T was 86.9% to 95%
35
similar to those of known Rhizobium species. All the four strains had nodC and nifH
36
genes and could form effective nodules with Pisum sativum and Vicia faba, and
37
ineffective nodules with Phaseolus vulgaris, but did not nodulate Glycine max,
38
Arachis hypogaea, Medicago sativa, Trifolium repens or Lablab purpureus in
39
cross-nodulation tests. Fatty acid composition, DNA-DNA relatedness and a series of
40
phenotypic tests also separated these strains from the closely related species. Based on
41
all the evidence, we propose a novel species Rhizobium anhuiense sp. nov., and
42
designate CCBAU 23252T (=CGMCC 1.12621T =LMG 27729T) as the type strain.
43
This strain was isolated from a root nodule of Vicia faba and has a GC content of 61.1
44
mol% (Tm).
45
3
46
Leguminous plants pea (Pisum sativum) and broad bean (Vicia faba) were
47
originally domesticated in the Middle East (Bond, 1976). They are essential foods,
48
vegetables and raw materials for other bean products. Vicia faba has been cultivated
49
for around 6,000 years and is currently grown in 57 countries (George, 1999). Both
50
Vicia faba and Pisum sativum are introduced plants widely grown in China.
51
Mutualisms between Rhizobium leguminosarum symbiovar viciae and Pisum sativum
52
or Vicia faba have been widely studied. Plants of Vicia faba strongly select rhizobia
53
with a specific nodD type, while Pisum sativum plants are more promiscuous in
54
selection of their symbionts (Laguerre et al., 2003, Mutch & Young, 2004). In
55
addition to R. leguminosarum, three related species Rhizobium pisi, Rhizobium fabae,
56
and Rhizobium laguerreae have been described for broad bean rhizobia
57
(Ramírez-Bahena et al., 2008, Saïdi et al., 2014; Tian et al., 2008), demonstrating the
58
existence of diverse rhizobial species associated with this plant.
59
In the present study, root nodules of Vicia faba and Pisum sativum were collected
60
from the fields of Anhui and Jiangxi Provinces in China and twenty-three fast
61
growing, acid-producing bacteria were isolated from the surface-sterilized effective
62
(red colored) nodules using the standard method on YMA plates incubated at 28C
63
(Vincent, 1970). Eighteen isolates were clustered as a group that was distant from
64
recognized species of the genus Rhizobium according to sequences of the
65
housekeeping gene recA (Fig S1). Therefore, four isolates listed in Table S1 (CCBAU
66
23242, CCBAU 23252T, CCBAU 33508, CCBAU 33602) were selected as
4
67
representatives to continue further sequencing studies. The isolates were preserved in
68
20% (w/v) glycerol at −80C and also stored by lyophilization.
69
The genomic DNA was extracted from each of the four strains using standard
70
methods (Tan et al., 1997). The 16S rRNA gene (Tan et al., 1997), housekeeping
71
genes atpD, recA and glnII (Vinuesa et al., 2005), nodulation gene nodC (Sarita et al.,
72
2005) and nitrogen-fixing gene nifH (Laguerre et al., 2001) were amplified and
73
sequenced, using the extracted DNA as template and primer sets P1/P6,
74
atpD255F/atpD782R, recA41F/recA640R, glnII12F/glnII689R, nodC540/nodC1160
75
and nifH1F/nifH1R, respectively. The acquired sequences were aligned with those of
76
Rhizobium type strains extracted from the NCBI nucleotide sequence database. The
77
genetic distances between sequences were calculated using the Clustal W program in
78
the MEGA 5.0 software package (Tamura et al., 2011), and phylogenetic trees were
79
constructed using the Maximum likelihood (ML) and Neighbor-joining (NJ) methods
80
with 1000 bootstrap replications.
81
The ML tree of the 16S rRNA gene sequences (1318 nt) showed that the four
82
isolates had identical 16S rRNA genes and had same sequence as Rhizobium
83
leguminosarum USDA 2370T, Rhizobium laguerreae FB 206T, R. indigoferae CCBAU
84
71042T and Rhizobium sophorae CCBAU 03386T, a novel species published in 2014
85
(Jiao et al.) (Fig. 1, Table S2). The similarities between the isolates and type strains of
86
Rhizobium species varied from 95.6% to 100%. The NJ tree generated based upon
87
the16S rRNA gene sequences in this study was consistent with ML tree (data not
88
shown). 5
89
Since the novel strains have 16S rDNA sequences identical with those of R.
90
leguminosarum USDA 2370T, R. laguerreae FB 206T, R. indigoferae CCBAU 71042T,
91
R. sophorae CCBAU 03386T, we used housekeeping genes to further clarify the
92
relationships among the novel isolates and the defined Rhizobium species. The
93
housekeeping genes recA, atpD and glnII were partially sequenced and a phylogenetic
94
tree was constructed based upon the combined sequences (Fig. 2). The similarities of
95
concatenated partial sequences of recA, atpD and glnII genes (1249nt) between the
96
four isolates and type strains of Rhizobium species ranged from 85.1% to 95.6%. The
97
strain CCBAU 23252T showed closest sequence similarity of 95% with R.
98
leguminosarum USDA 2370T (Table S2). This result agreed with the phylogenetic
99
trees generated separately for each of the recA, atpD or glnII gene sequences (Figs. S1
100
-S3) and supported that these isolates represented a novel species, taking the
101
Rhizobium species defined recently (Ramírez-Bahena et al., 2008, Tian et al., 2008,
102
Saïdi et al., 2014) as references.
103
In order to compare strains using Average Nucleotide Identity (ANI), genomic
104
DNA for two representative strains CCBAU 23252T (from Anhui province) and 33602
105
(from Jiangxi province) was extracted using PowerSoil DNA isolation kits (MoBio,
106
Carlsbad, CA), and then fragmented, barcoded, quantitated and run as part of a batch
107
of eight genomes on a 318 chip on an Ion Torrent PGM using the manufacturer’s
108
recommended protocols (Thermo Fisher, Waltham, MA). Each genome was
109
assembled using the Newbler GS De Novo assember version 2.8 (Roche diagnostics)
110
with default parameter values. ANI was calculated within the JSpecies software 6
111
(Richter et al., 2009). The Nucleotide MUMmer algorithm (NUCmer) was used, with
112
default parameter settings, to calculate the ANI by subtracting the similarity errors
113
from the alignment length (Richter et al., 2009, Kurtz et al., 2004). Genomes were
114
compared with each other, and with complete genome assemblies downloaded from
115
NCBI for the strains R. leguminosarum symbiovar viciae 3841 (GCA_000009265),
116
and R. leguminosarum symbiovar trifolii WSM 1325 (GCA_000023185), which were
117
widely used in genetic research representing R. leguminosarum species. The ANI
118
between strains CCBAU 23252T and CCBAU 33602 was 98.34-98.37% (Table 1),
119
which was above the 95–96% boundary for species definition (Richter et al., 2009),
120
indicating that they belong to the same species. The ANI between these strains and
121
representatives of the most closely related species R. leguminosarum were below 92%
122
(Table 1), implying that these two novel strains cannot be included in the closest
123
species.
124
The genes nodC and nifH are essential for rhizobia to establish effective
125
nitrogen-fixing symbiosis with the host legumes. A phylogenetic tree of part of the
126
nifH gene (350 nt) showed that the sequences of this gene in the four isolates were
127
different, but very similar: CCBAU 23252T and CCBAU 33602, both from Vicia faba,
128
shared a similarity of 98.5%; CCBAU 23242 and CCBAU 33508, both from Pisum
129
sativum, were two single lineages (Fig. S4). All of them were clustered at similarities
130
of 95.2-97.9% with the five reference strains of R. leguminosarum, R. pisi, R. fabae, R.
131
laguerreae and R. indigoferae, which mainly originated from V. faba. The ML tree of
132
the partial nodC genes (450nt) of all strains showed a similar phylogenetic 7
133
relationship with respect to that of the nifH gene, at 93.5-99.3% similarities among the
134
tested strains and at 90.2-99.1% similarities with the closest reference strains R.
135
leguminosarum, R. pisi, R. fabae, R. laguerreae and R. multihospitium (Fig. S5),
136
together with the host range, implying all these strains belonged to symbiovar viciae.
137
DNA–DNA hybridization using the renaturation-rate technique (De Ley et al.,
138
1970) between the novel bacteria and type strains of related species has been
139
recommended as a standard method for species delineation (Graham et al., 1991).
140
Three replicates were tested in present study. The DNA-DNA relatedness between the
141
reference isolate CCBAU 23252T and the other isolates of the novel group ranged
142
from 79.51% to 97.54%. Meanwhile, the values were only 26.8% to 37.92% between
143
CCBAU 23252T and the most closed type strains R. leguminosarum USDA 2370T, R.
144
indigoferae CCBAU 71042T, R. sophorae CCBAU 03386T (Table S2). These values
145
further illustrated that the four isolates represented a new genomic species in the
146
genus Rhizobium.
147
BOX-PCR fingerprinting was performed by using the procedure of Versalovic et
148
al. (1994) and distinguished the four isolates from each other (Fig. S6), illustrating
149
that they were not clones of the same strain. The G+C contents of genomes of the four
150
isolates, measured by the thermal denaturation method for strains CCBAU 23242 and
151
CCBAU 33508 (De Ley et al., 1970) or calculated by the genome data for strains
152
CCBAU 23252T and CCBAU 33602 (Richter et al., 2009), varied between 61.1 and
8
153
64.8 mol% (Tm), which is within the range for the genus Rhizobium (Young et al.,
154
2001).
155
Cellular fatty acid profiles were determined to provide additional description of
156
the novel species. Strain CCBAU 23252T and the type strains for nine related species,
157
R. leguminosarum USDA 2370T, R. laguerreae LMG 27434T, R. indigoferae CCBAU
158
71042T, R. pisi DSM 30132T, R. etli CFN 42T, R. vallis CCBAU 65647T, R. phaseoli
159
ATCC 14482T, R. sophorae CCBAU 03386T and R. sophoriradicis CCBAU 03470T
160
were chosen to compare their fatty acid compositions. The strains were streaked on
161
YMA medium and cultivated at 28C to the late exponential phase. About 40–100 mg
162
of wet cells was collected into sample tubes (13×100 mm). Fatty acids were then
163
extracted following the method described by Sasser (1990) and identified by the MIDI
164
Sherlock Microbial Identification System (Sherlock license CD v 6.0) in the TSBA6
165
database. All strains were tested in the present study except R. sophorae CCBAU
166
03386T and R. sophoriradicis CCBAU 03470T, which were tested by using the same
167
procedure at the same period in our lab and recent publication (Jiao et al., 2014).
168
Thirty-three kinds of fatty acids were detected in total. Summed feature 8 (C18:1 ω6c
169
and C18:1 ω7c, 45.73–68.17%), summed feature 2 (C14:0 3OH/C16:1 iso I, 4.4–12.76%)
170
and C18:0 (4.39–9.1%) were the dominant components in all strains (Table S3), which
171
were different from the previously reported results for the genera Bradyrhizobium and
172
Mesorhizobium (Wang et al., 2013, Zheng, et al., 2013), but similar to those reported
173
for Rhizobium species (Tighe et al., 2000). In comparison to the type strains of related
174
Rhizobium species, C18:1 ω5c was only detected in CCBAU 23252T in this study, 9
175
indicating that it was different from the other species. From the fatty acid
176
compositions, CCBAU 23252T was most similar to R. pisi DSM 30132T and R. etli
177
CFN 42T; however, some differences were observed: C18:1 ω7c 11-methyl was absent
178
in R. etli CFN 42T and C20:2 ω6, 9c was absent in R. pisi DSM 30132T, whereas both
179
were present in CCBAU 23252T. These distinctions provide additional evidence that
180
CCBAU 23252T belongs to a novel species in the genus Rhizobium.
181
The phenotypic features of the two representative isolates and the type strains of
182
closely related species were assayed by using the Biolog GN2 MicroPlate to test the
183
utilization of sole carbon sources, according to the manufacturer’s instructions; a
184
previously described method (Gao et al., 1994) was used for other characteristics such
185
as growth temperature and pH ranges, tolerance to NaCl and antibiotics and
186
generation time etc. The detailed differences between the novel isolates and the type
187
strains are shown in Table 2, and the shorten different features compared the two
188
novel strains and the most closed strain R. leguminosarum USDA 2370 are
189
summarized in the description of the novel species.
190
To further identify the host range of these isolates, cross-nodulation tests were
191
conducted in vermiculite with N-free plant nutrient solution (Vincent, 1970). The four
192
isolates could nodulate Pisum sativum and Vicia faba. Cross-sections of the nodules
193
were pink (the color of leghemoglobin), indicating that the nodules were effective for
194
fixing nitrogen. The isolates can also form nodules with Phaseolus vulgaris, but the
195
inside of the nodules was white, illustrating that they were ineffective. The strains 10
196
could not nodulate Medicago sativa, Trifolium repens, Lablab purpureus, Arachis
197
hypogaea or Glycine max.
198
Based on all the phenotypic, genotypic and phylogenetic characteristics obtained
199
in this study, we identified our four strains different form the recognized Rhizobium
200
species and propose that the four isolates represent a novel species, Rhizobium
201
anhuiense.
202
Description of Rhizobium anhuiense sp. nov.
203 204
Rhizobium anhuiense (an.hui.en′se. N.L. neut. adj. anhuiense originating from Anhui Province of China, from where the type strain was isolated).
205
The cells are Gram-staining-negative, non-spore-forming, aerobic rods. Colonies
206
are translucent, cream white and convex, with a diameter of 3–4 mm on YMA
207
medium after incubation for 3 days at 28C. Cells are 0.41–0.48 µm 0.89–1.48 µm
208
in size and have a generation time of 3.9 hours when growing in YM medium at 28C.
209
They can grow at 15C to 37C, with an optimum temperature of 28C. The pH for
210
growth ranges from 5 to 8, with optimum of pH 7.0. The bacteria grow weakly in the
211
presence of 1.0% (w/v) NaCl. N-acetyl-D-galactosamine, N-acetyl-D-glucosamine,
212
adonitol, D-cellobiose, D-galactose, gentiobiose, m-inositol, α-D-lactose, lactulose,
213
maltose, D-mannitol, β-methyl-D-glucoside, D-raffinose, D-trehalose, xylitol,
214
succinic acid mono-methyl ester, D-galactonic acid lactone, D-gluconic acid,
215
D-glucosaminic acid, β-hydroxybutyric acid, quinic acid, L-alanine, L-alanyl-glycine,
216
L-glutamic acid, hydroxy-L-proline, L-ornithine, L-proline, γ-aminobutyric acid, 11
217
urocanic acid, uridine and α-D-glucose-1-phosphate can be utilized as sole carbon
218
sources. Succinic acid cannot be utilized as sole carbon sources. Summed feature 8
219
(18:1 ω6c/18:1 ω7c), summed feature 2 (C14:0 3OH/C16:1 iso I) and C18:0 were the
220
dominant cellular fatty acids. CCBAU 23252T (= CGMCC 1.12621T = LMG 27729T)
221
was designated as the type strain. The DNA G+C content of the type strain is 61.1 mol%
222
(Tm).
223
Acknowledgements
224
We are grateful to Dr. Claudine Vereecke at LMG for providing some type
225
strains. This work was supported by the National Natural Science Foundation of
226
China (31170003, 31470135 & 31211130109), by 863 Project (2013AA102802-04)
227
and by Royal Society International Exchange IE222202.
228
References
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strain R. leguminosarum DSM 30132 (5NCIMB 11478) as Rhizobium pisi sp. nov. Int
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304
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306
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307
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308
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309
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310
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311
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312
Zheng W.T., Li Y. Jr, Wang R., Sui X.H., Zhang X.X., Zhang J.J., Wang
313
E.T., Chen W.X. (2013). Mesorhizobium qingshengii sp. nov., isolated from effective
314
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315
Microbiol 63, 2002-2007.
16
316 317 318
Table 1. Average Nucleotide Identity (ANI) of CCBAU 23252T, CCBAU 33602 and most closely related strains with available genome sequences R. leguminosarum
R. leguminosarum 3841 R. leguminosarum WSM 1325 R. anhuiense CCBAU 23252T R. anhuiense CCBAU 33602
R. leguminosarum
R. anhuiense
R. anhuiense T
3841
WSM 1325
CCBAU 23252
100
94.15
91.18
91.13
94.16
100
91.12
91.13
91.18
91.11
100
98.37
91.12
91.12
98.34
100
319 320
17
CCBAU 33602
321
Table 2. Different features of Rhizobium anhuiense sp. nov. and its close relatives
322
1, CCBAU 23252T; 2, CCBAU 33602; 3, R. etli CFN 42T; 4, R. pisi DSM 30132T; 5, R.
323
leguminosarum USDA 2370T; 6, R. vallis CCBAU 65647T; 7, R. indigoferae CCBAU 71042T; 8, R.
324
phaseoli ATCC 14482T; 9, R. laguerreae LMG 27434T; 10, R. sophorae CCBAU 03386T; 11, R.
325
sophoriradicis CCBAU 03470T 1
2
3
4
5
6
7
8
9
10#
11#
α-Cyclodextrin
-
-
-
-
-
+
-
-
-
-
-
Dextrin
+
+
w
-
w
+
+
+
-
-
-
Glycogen
-
-
-
-
w
+
-
-
-
-
-
N-Acetyl-D-Galactosamine
+
-
-
-
-
+
-
-
-
-
-
N-Acetyl-D-Glucosamine
+
+
-
w
-
+
-
-
-*
-
-
Adonitol
+
+
-
+
-
+
-
+
-
-
-
L-Arabinose
+
+
+
w
w
+
+
+
-*
+
+
D-Arabitol
+
+
+
+
w
+
+
+
-
w
-
D-Cellobiose
+
+
+
+
-
+
+
-
-
+
-
i-Erythritol
-
-
-
-
w
+
-
-
-
+
-
D-Fructose
+
+
+
+
w
+
w
+
-
-
-
L-Fucose
+
+
+
+
w
+
w
-
-*
-
-
D-Galactose
+
+
+
+
-
+
-
w
-
w
-
Gentiobiose
+
+
+
+
-
+
-
-
-
-
-
α-D-Glucose
+
+
+
+
+
+
+
+
-
w
-
m-Inositol
+
+
+
+
-
+
-
-
-
-
-
α-D-Lactose
+
+
+
+
-
+
-
+
-
-
-
Lactulose
+
+
+
-
-
+
-
+
-
-
-
Maltose
+
+
+
+
-
+
w
-
-
w
-
D-Mannitol
+
+
+
+
-
+
w
-
-
-
-
D-Mannose
+
+
+
+
w
+
w
+
-
-
-
D-Melibiose
-
-
w
+
-
+
-
-
-
-
-
D-Psicose
+
+
+
-
w
+
-
-
+
-
-
β-Methyl-D-Glucoside
+
+
+
+
-
+
-
-
-
-
-
D-Raffinose
+
+
-
+
-
+
-
-
-
-
-
L-Rhamnose
+
+
+
+
+
+
w
+
-*
-
-
D-Sorbitol
+
+
+
+
w
+
w
-
+
-
-
Sucrose
+
+
+
+
+
+
-
+
-*
+
-
D-Trehalose
+
-
+
+
-
+
-
-
-
-
-
Turanose
+
+
+
+
w
+
w
-
-
-
-
Xylitol
+
+
+
-
-
+
w
-
-
w
w
Cis-Aconitic Acid
+
+
+
+
w
+
-
+
-
-
-
Characteristic Utilization of sole carbon source：
18
Succinic Acid Mono- Methyl Ester
+
-
w
-
-
+
-
-
-
-
-
Citric Acid
-
-
-
-
-
+
-
-
-
-
-
Formic Acid
+
+
+
-
w
+
-
-
-
-
-
D-Galactonic Acid Lactone
+
+
+
-
-
+
w
+
-
+
-
D-Gluconic Acid
+
+
+
-
-
+
-
-
-
-
-
D-Glucosaminic Acid
+
-
-
-
-
+
-
-
-
-
-
β-Hydroxybutyric Acid
+
+
-
-
-
+
w
-
-
-
-
γ-Hydroxybutyric Acid
-
-
-
w
-
+
-
-
-
-
-
p-Hydroxy-phenlyacetic Acid
-
-
-
+
-
+
-
-
-
-
-
α-Ketoglutaric Acid
-
-
-
+
-
+
w
-
-
-
-
D,L-Lactic Acid
+
+
+
w
w
+
-
-
-
-
-
Malonic Acid
-
-
-
-
-
+
-
-
-
-
-
Propionic Acid
-
-
-
+
-
+
-
-
-
-
-
Quinic Acid
+
+
-
-
-
+
-
-
-
-
-
Succunic Acid
-
-
+
-
+
+
+
+
-
-
-
Bromosuccinic Acid
+
+
-
-
+
+
-
-
-
+
-
L-Alaninamide
+
+
-
-
+
+
w
-
-
w
-
L-Alanine
+
+
-
w
-
+
-
-
-
-
-
L-Alanyl-Glycine
+
+
-
-
-
+
-
-
-
-
-
L-Asparagine
-
-
w
-
-
+
-
+
-
-
-
L-Glutamic Acid
+
+
-
-
-
+
-
-
-
-
-
Glycyl-L-Aspartic Acid
-
+
-
+
-
+
-
-
-
-
-
Glycyl-L-Glutamic Acid
-
+
-
-
-
+
-
-
-
-
-
L-Histidine
+
+
+
-
w
+
-
-
-*
-
-
Hydroxy-L-Prolin
+
+
-
-
-
+
-
-
-
-
-
L-Ornithine
+
-
-
-
-
+
-
-
-
-
-
L-Proline
+
+
-
w
-
+
-
-
-*
-
-
D,L-Camitine
+
+
+
+
w
+
-
-
-
-
-
γ-Aminobutyric Acid
+
+
-
-
-
+
-
-
-
-
-
Urocanic Acid
+
+
+
-
-
+
w
-
-
+
-
Uridine
+
+
+
+
-
+
-
+
-
w
-
Thymidine
-
-
-
-
-
+
-
-
-
-
-
2-Aminoethanol
-
-
+
-
-
+
-
-
-
-
-
α-D-Glucose-1-Phosphate
+
+
+
-
-
+
-
-
-
-
-
D-Glucose-6-Phosphate
-
-
+
-
-
+
-
-
-
-
-
+
+
+
-
+
+
+
+
+
+
+
Growth at: pH 8.0
326
+, growth or resistant; -, no growth or sensitive; w. growth weakly; #All data were obtained with three replicates
327
in this study, except that of R. sophorae CCBAU 03386Tand R. sophoriradicis CCBAU 03470T (Jiao et al.,
328
2014); *, The results were not congruent with that of R. laguerreae LMG 27434T (Jiao et al., 2014).
19
329
Figures Legends
330
Fig. 1. Maximum likelihood tree constructed from 16S rRNA gene sequences
331
showing the phylogenetic relationships of R. anhuiense sp. nov. Bootstrap
332
values of more than 50% for 1000 replicates of the data set are provided on
333
the branches. The sequence of Bradyrhizobium japonicum USDA 6T was used
334
as an outgroup. Bar: 1% substitution.
335
Fig. 2. Maximum likelihood tree based on the partial concatenated sequences of the
336
atpD and recA genes of R. anhuiense sp. nov. and the species in the genus
337
Rhizobium. Bootstrap values of more than 50% for 1000 replicates of the data
338
set are provided on the branches. The accession numbers of the atpD and
339
recA genes of the strains are shown in the brackets. Bar: 2% substitution.
20
Figure 1 Click here to download Figure: fig1.docx
R. etli CFN 42T (CP000133) R. sophoriradicis CCBAU 03470T (KJ831225) 62 R. pisi DSM 30132T (AY509899) 98 R. fabae CCBAU 33202T (DQ835306) R. phaseoli ATCC 14482T (EF141340) CCBAU 23252T (KF111868) 50 CCBAU 23242 (KF111865) R. anhuiense sp. nov. CCBAU 33508 (KF111874) 100 CCBAU 33602 (KF111886) R. indigoferae CCBAU 71042T (AY034027) R. leguminosarum USDA 2370T (JQ085246) R. laguerreae FB206T (JN558651) 64 R. sophorae CCBAU 03386T (KJ831229) R. vallis CCBAU 65647T (FJ839677) 62 R. calliandrae CCGE 524T (JX855162) 97 57 R. paranaense PRF35 T (EU488753) 66 R. jaguaris CCGE525T (JX855169) R. mayense CCGE 526T (JX855172) T 58 R. rhizogense ATCC 11325 (AY945955) R. lusitanum p1-7T (AY738130) R. leucaenae CFN 299T (X67234) R. miluonense CCBAU 41251T (EF061096) 85 R. tropici IIB CIAT 899T (HQ850704) 62 R. multihospitium CCBAU 83401T (EF035074) 50 R. hainanense I66T (U71078) R. grahamii CCGE 502T (JF424608) 98 R. tibeticum LMG 24453T (EU256404) T 99 R. alamii GBV 016 (AM931436) 50 R. mesosinicum CCBAU 25010T (DQ100063) R. sullae IS123T (Y10170) 70 R. gallicum R602spT (CP006877) 100 R. yanglingense SH 22623T (AF003375) R. loessense CCBAU 7190BT (AF364069) 58 R. mongolense USDA 1844T (U89817) Bradyrhizobium japonicum USDA 6T (NC017249) 91
0.01
Fig. 1
1
Figure 2 Click here to download Figure: fig2.docx
CCBAU 23242 (KF111887, KF111983, KF111910)
92
CCBAU 33602 (KF111909, KF111999, KF111932)
100
R. anhuiense sp. nov.
CCBAU 23252T (KF111890, KF111980, KF111913) 0.02
96
72
100
CCBAU 33508 (KF111896, KF111986, KF111919)
R. leguminosarum USDA 2370T (AJ294405, AJ294376, EU155089) R. indigoferae CCBAU 71042T (GU552925, EF027965, JN580717)
69
53
R. sophorae CCBAU 03386T (KJ831235, KJ831252, KJ831241) 83
R. laguerreae FB206T (JN558661, JN558681, JN558671)
93
R. vallis CCBAU 65647T (GU211768, GU211770, GU211771) R. fabae CCBAU 33202T (EF579929, EF579941, EF579935)
75 100
R. pisi DSM 30132T (EF113129, EF113134, JN580715) R. sophoriradicis CCBAU 03470T (KJ831231, KJ831248, KJ831237)
94 77
R. phaseoli ATCC 14482T (EF113151, EF113136, JN580716) R. etli CFN 42T (CP000133) R. tibeticum LMG 24453T (KF206621, KF206877, EU409190) R. multihospitium CCBAU 83401T (EF490019, EF490029, EF490040)
99
R. tropici B LMG 9503T (AM418789, AJ294373, AF169584)
67
R. lusitanum p1-7T (DQ431671, DQ431674, EF639841) R. alkalisoli LMG24763T (EU672461, EU672490, EU672475)
63
R. cellulosilyticus LMG 23642T (AM286426, AM286427, KF206788) R. endophyticum CCGE 2052T (HM142760, HM142767, JF424619) R. sullae IS123T (DQ345069, FJ816279, FJ816280) R. gallicum R602spT (CP006877)
100
100
R. mongolense USDA 1844T (AY907372, AY907358, AY929453) B. japonicum USDA 6T (AM418753, AM168341, AF169582)
Fig. 2
Supplementary Material Files-normal Click here to download Supplementary Material Files: s-material 423 normal.pdf
