IJSEM Papers in Press. Published March 9, 2015 as doi:10.1099/ijs.0.000183
International Journal of Systematic and Evolutionary Microbiology Bradyrhizobium erythrophlei sp. nov. and Bradyrhizobium ferriligni sp. nov. , isolated from effective nodules of Erythrophleum fordii --Manuscript Draft-Manuscript Number:
IJSEM-D-14-00107R2
Full Title:
Bradyrhizobium erythrophlei sp. nov. and Bradyrhizobium ferriligni sp. nov. , isolated from effective nodules of Erythrophleum fordii
Short Title:
Bradyrhizobium erythrophlei and Bradyrhizobium ferriligni
Article Type:
Note
Section/Category:
New taxa - Proteobacteria
Corresponding Author:
xinhua sui, Ph,D China Agricultural University CHINA
First Author:
Yao Yao
Order of Authors:
Yao Yao Xin Hua Sui, Ph,D Xiao Xia Zhang En Tao Wang Wen Xin Chen
Manuscript Region of Origin:
CHINA
Abstract:
Six slow-growing rhizobial strains isolated from effective nodules of Erythrophleum fordii were classified into the genus Bradyrhizobium based on 16S rRNA gene sequence. The results of multilocus sequence analysis of recA, glnII and gyrB genes and IGS sequence phylogeny indicated that the six strains were two novel species represented by CCBAU 53325T and CCBAU 51502T, which were consistent with the results of DNA-DNA hybridization. CCBAU 53325T had 17.65%-25.59% and CCBAU 51502T had 22.69%-44.58% DNA-DNA relatedness with five closely related type strains including B. elkanii USDA 76T, B. pachyrhizi LMG 24246T, B. lablabi CCBAU 23086T, B. jicamae LMG 24556T, and B. japonicum USDA 6T. In addition, analysis of phenotype characteristics and fatty acid profile also distinguished the test strains from the defined species of Bradyrhizobium. Two novel species, Bradyrhizobium erythrophlei sp. nov. represented by the type strain CCBAU 53325T (= HAMBI 3614T = CGMCC 1.13002T = LMG 28425T) and Bradyrhizobium ferriligni sp. nov. represented by the type strain CCBAU 51502T (= HAMBI 3613T = CGMCC 1.13001T) were proposed.
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1
Bradyrhizobium erythrophlei sp. nov. and Bradyrhizobium ferriligni sp. nov. ,
2
isolated from effective nodules of Erythrophleum fordii
3
Yao Yao1, Xin Hua Sui1*, Xiao Xia Zhang2, En Tao Wang1,3, Wen Xin Chen1
4
1
5
Microbiology; College of Biological Sciences, China Agricultural University, Beijing,
6
100193, China.
7
2
8
Agricultural Sciences, Beijing 100081, China
9
3
State Key Lab for Agro-Biotechnology; Ministry of Agriculture Key Lab of Soil
Institute of Agricultural Resources and Regional Planning, Chinese Academy of
Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, 11340
10
México D. F., Mexico.
11
Running title: Bradyrhizobium erythrophlei and Bradyrhizobium ferriligni
12
Subject category: New Taxa (a-proteobacteria)
13
*Corresponding author: Dr. Xinhua Sui. Tel: 8610 62734009; Fax: 86 10 62734008;
14
E-mail: [email protected].
15
Abbreviations: ML, maximum-likelihood; MLSA, multilocus sequence analysis.
1
16
Accession numbers for the test strains: Forty nucleotide sequences obtained in this
17
study were deposited in the GenBank database (Supplementary Table S1). Accession
18
numbers of the type strains CCBAU 51502T and CCBAU 53325T are KJ818109 and
19
KF114576 for nodC gene, KJ818108 and KF114598 for nifH gene, KJ818105 and
20
KF114622 for IGS, KJ818096 and KF114645 for 16S rRNA gene, KJ818112 and
21
KF114669 for recA gene, KJ818099 and KF114693 for glnII gene, and KJ818102 and
22
KF114717 for gyrB gene.
23
2
24
Abstract
25
Six slow-growing rhizobial strains isolated from effective nodules of
26
Erythrophleum fordii were classified into the genus Bradyrhizobium based on 16S
27
rRNA gene sequence. The results of multilocus sequence analysis of recA, glnII and
28
gyrB genes and IGS sequence phylogeny indicated that the six strains were two novel
29
species represented by CCBAU 53325T and CCBAU 51502T, which were consistent
30
with the results of DNA-DNA hybridization. CCBAU 53325T had 17.65%-25.59%
31
and CCBAU 51502T had 22.69%-44.58% DNA-DNA relatedness with five closely
32
related type strains including B. elkanii USDA 76T, B. pachyrhizi LMG 24246T, B.
33
lablabi CCBAU 23086T, B. jicamae LMG 24556T, and B. japonicum USDA 6T. In
34
addition, analysis of phenotype characteristics and fatty acid profile also distinguished
35
the test strains from the defined species of Bradyrhizobium. Two novel species,
36
Bradyrhizobium erythrophlei sp. nov. represented by the type strain CCBAU 53325T
37
(= HAMBI 3614T = CGMCC 1.13002T = LMG 28425T) and Bradyrhizobium
38
ferriligni sp. nov. represented by the type strain CCBAU 51502T (= HAMBI 3613T =
39
CGMCC 1.13001T) were proposed.
40
3
41
Ironwood (Erythrophleum fordii Oliver) is an evergreen tree species of
42
Caesalpiniaceae and is indigenous to the tropical and subtropical regions located in
43
the south of China and the north of Vietnam (Sein & Mitlöner, 2011). Erythrophleum
44
fordii is in the list of protected and endangered plant species in China, and its trunk is
45
an extremely valuable hard-wood with seeds hardly to germinate in nature
46
environment (Shi et al., 2005; Fang & Fang, 2006; Zhao et al., 2009). It was reported
47
recently that ironwood could establish nitrogen fixation symbiosis with rhizobia
48
(Chen & Wang, 2011) and some slow-growing rhizobial species, including
49
Bradyrhizobium
50
yuanmingense, have been isolated from the nodules of E. fordii as the dominant
51
microsymbionts (Lu et al., 2011; Yao et al., 2014).
elkanii,
Bradyrhizobium
pachyrhizi
and
Bradyrhizobium
52
In a previous study, 167 rhizobial isolates were obtained from effective root
53
nodules of E. fordii and three isolates CCBAU 53325T, CCBAU 53266, CCBAU
54
53267 from Guangxi Province of China were tentatively identified as potential novel
55
species belonging to the genus Bradyrhizobium (Yao et al., 2014). In a subsequent
56
research with additional isolates, CCBAU 51502T, CCBAU 51501 and CCBAU
57
51497 from Guangdong Province were found to be a distinct group based upon
58
sequence analyses of housekeeping genes recA, glnII and gyrB, which was different
59
from all the defined Bradyrhizobium species and the novel group reported in Yao et al.
60
(2014). Therefore, we further systematically studied these six isolates by using a
61
polyphasic approach.
4
62
These six strains were isolated from root nodules of E. fordii collected in
63
Guangdong and Guangxi provinces of China by the standard method (Vincent, 1970;
64
Yao et al., 2014). All of the six strains were slow-growing, alkali-produced bacteria
65
and formed mucoid colonies with a diameter less than 1 mm after 7 days of
66
incubation on YMA medium at 28 °C supplied with bromothymol blue indicator
67
(Vincent, 1970).
68
In the current study, total genomic DNA was extracted from each isolate
69
following the protocol of Terefework et al. (2001). The primers and procedures were
70
adopted from the previous reports for 16S rRNA gene (Tan et al., 1997), 16S-23S
71
intergenic sequences (IGS) (Navarro et al., 1992), partial recA, glnII and gyrB genes
72
(Vinuesa, et al., 2005; Rivas et al., 2009), and partial nodC and nifH genes (Laguerre
73
et al., 2001). All the sequences acquired in this study were aligned by the Clustal W
74
software with those of related Bradyrhizobium species extracted from NCBI database
75
using the BLAST search program. These sequences used for phylogenetic analysis
76
were processed by MEGA 5.1 software (Tamura et al., 2011) to construct
77
phylogenetic trees with the Maximum Likelihood method and the Kimura
78
2-parameter model (Kimura, 1983) with 1000 bootstrap replications for all the single
79
gene sequences, as well as for the multilocus sequence analysis (MLSA) using the
80
combined sequences of recA, glnII and gyrB. The pairwise distances in the ML trees
81
were calculated by the Kimura 2-parameter model in MEGA 5.1.
82
In the ML tree for 16S rRNA gene sequences (Fig. 1), all the type strains of the 5
83
Bradyrhizobium species were divided into two groups, which were similar to that in
84
the previous study (Menna et al., 2009). The six test isolates were grouped together
85
with eight type strains of Bradyrhizobium in the group II represented by B. elkanii, at
86
sequence similarity values of 97.3%-99.8%. CCBAU 51502T was the most divergent
87
lineage within the group II, sharing similarities of 98.0% and 97.9% with another two
88
isolates (CCBAU 51501 and CCBAU 51497, respectively) within the same novel
89
group, and of 97.3%-97.9% with the type strains in the group II. The other three
90
strains CCBAU 53325T, CCBAU 53266 and CCBAU 53267 shared 99.9%-100%
91
similarities with each other, and presented similarities of 99.3%-99.8% with the type
92
strains in group II. In addition, the six test isolates had the similarities from 94.6% to
93
97.7% with the type strains in Bradyrhizobium group I.
94
Since the similarities of 16S rRNA gene sequence among the defined
95
Bradyrhizobium species are very high, more characteristics have been suggested to
96
differentiate these closely related species (Willems et al., 2001). In the IGS sequence
97
analysis, the three isolates from Guangxi, CCBAU 53325T, CCBAU 53266 and
98
CCBAU 53267, shared the sequence similarities of 99.7%-99.8% with each other and
99
shared 75.2%-92.9% with the type strains of defined Bradyrhizobium species
100
(Supplementary Fig. S1). Since Willems et al. (2003) have hypothesized that two
101
bradyrhizobial strains with IGS sequence similarity
International Journal of Systematic and Evolutionary Microbiology Bradyrhizobium erythrophlei sp. nov. and Bradyrhizobium ferriligni sp. nov. , isolated from effective nodules of Erythrophleum fordii --Manuscript Draft-Manuscript Number:
IJSEM-D-14-00107R2
Full Title:
Bradyrhizobium erythrophlei sp. nov. and Bradyrhizobium ferriligni sp. nov. , isolated from effective nodules of Erythrophleum fordii
Short Title:
Bradyrhizobium erythrophlei and Bradyrhizobium ferriligni
Article Type:
Note
Section/Category:
New taxa - Proteobacteria
Corresponding Author:
xinhua sui, Ph,D China Agricultural University CHINA
First Author:
Yao Yao
Order of Authors:
Yao Yao Xin Hua Sui, Ph,D Xiao Xia Zhang En Tao Wang Wen Xin Chen
Manuscript Region of Origin:
CHINA
Abstract:
Six slow-growing rhizobial strains isolated from effective nodules of Erythrophleum fordii were classified into the genus Bradyrhizobium based on 16S rRNA gene sequence. The results of multilocus sequence analysis of recA, glnII and gyrB genes and IGS sequence phylogeny indicated that the six strains were two novel species represented by CCBAU 53325T and CCBAU 51502T, which were consistent with the results of DNA-DNA hybridization. CCBAU 53325T had 17.65%-25.59% and CCBAU 51502T had 22.69%-44.58% DNA-DNA relatedness with five closely related type strains including B. elkanii USDA 76T, B. pachyrhizi LMG 24246T, B. lablabi CCBAU 23086T, B. jicamae LMG 24556T, and B. japonicum USDA 6T. In addition, analysis of phenotype characteristics and fatty acid profile also distinguished the test strains from the defined species of Bradyrhizobium. Two novel species, Bradyrhizobium erythrophlei sp. nov. represented by the type strain CCBAU 53325T (= HAMBI 3614T = CGMCC 1.13002T = LMG 28425T) and Bradyrhizobium ferriligni sp. nov. represented by the type strain CCBAU 51502T (= HAMBI 3613T = CGMCC 1.13001T) were proposed.
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Manuscript Including References (Word document) Click here to download Manuscript Including References (Word document): Manuscript-150224.docx
1
Bradyrhizobium erythrophlei sp. nov. and Bradyrhizobium ferriligni sp. nov. ,
2
isolated from effective nodules of Erythrophleum fordii
3
Yao Yao1, Xin Hua Sui1*, Xiao Xia Zhang2, En Tao Wang1,3, Wen Xin Chen1
4
1
5
Microbiology; College of Biological Sciences, China Agricultural University, Beijing,
6
100193, China.
7
2
8
Agricultural Sciences, Beijing 100081, China
9
3
State Key Lab for Agro-Biotechnology; Ministry of Agriculture Key Lab of Soil
Institute of Agricultural Resources and Regional Planning, Chinese Academy of
Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, 11340
10
México D. F., Mexico.
11
Running title: Bradyrhizobium erythrophlei and Bradyrhizobium ferriligni
12
Subject category: New Taxa (a-proteobacteria)
13
*Corresponding author: Dr. Xinhua Sui. Tel: 8610 62734009; Fax: 86 10 62734008;
14
E-mail: [email protected].
15
Abbreviations: ML, maximum-likelihood; MLSA, multilocus sequence analysis.
1
16
Accession numbers for the test strains: Forty nucleotide sequences obtained in this
17
study were deposited in the GenBank database (Supplementary Table S1). Accession
18
numbers of the type strains CCBAU 51502T and CCBAU 53325T are KJ818109 and
19
KF114576 for nodC gene, KJ818108 and KF114598 for nifH gene, KJ818105 and
20
KF114622 for IGS, KJ818096 and KF114645 for 16S rRNA gene, KJ818112 and
21
KF114669 for recA gene, KJ818099 and KF114693 for glnII gene, and KJ818102 and
22
KF114717 for gyrB gene.
23
2
24
Abstract
25
Six slow-growing rhizobial strains isolated from effective nodules of
26
Erythrophleum fordii were classified into the genus Bradyrhizobium based on 16S
27
rRNA gene sequence. The results of multilocus sequence analysis of recA, glnII and
28
gyrB genes and IGS sequence phylogeny indicated that the six strains were two novel
29
species represented by CCBAU 53325T and CCBAU 51502T, which were consistent
30
with the results of DNA-DNA hybridization. CCBAU 53325T had 17.65%-25.59%
31
and CCBAU 51502T had 22.69%-44.58% DNA-DNA relatedness with five closely
32
related type strains including B. elkanii USDA 76T, B. pachyrhizi LMG 24246T, B.
33
lablabi CCBAU 23086T, B. jicamae LMG 24556T, and B. japonicum USDA 6T. In
34
addition, analysis of phenotype characteristics and fatty acid profile also distinguished
35
the test strains from the defined species of Bradyrhizobium. Two novel species,
36
Bradyrhizobium erythrophlei sp. nov. represented by the type strain CCBAU 53325T
37
(= HAMBI 3614T = CGMCC 1.13002T = LMG 28425T) and Bradyrhizobium
38
ferriligni sp. nov. represented by the type strain CCBAU 51502T (= HAMBI 3613T =
39
CGMCC 1.13001T) were proposed.
40
3
41
Ironwood (Erythrophleum fordii Oliver) is an evergreen tree species of
42
Caesalpiniaceae and is indigenous to the tropical and subtropical regions located in
43
the south of China and the north of Vietnam (Sein & Mitlöner, 2011). Erythrophleum
44
fordii is in the list of protected and endangered plant species in China, and its trunk is
45
an extremely valuable hard-wood with seeds hardly to germinate in nature
46
environment (Shi et al., 2005; Fang & Fang, 2006; Zhao et al., 2009). It was reported
47
recently that ironwood could establish nitrogen fixation symbiosis with rhizobia
48
(Chen & Wang, 2011) and some slow-growing rhizobial species, including
49
Bradyrhizobium
50
yuanmingense, have been isolated from the nodules of E. fordii as the dominant
51
microsymbionts (Lu et al., 2011; Yao et al., 2014).
elkanii,
Bradyrhizobium
pachyrhizi
and
Bradyrhizobium
52
In a previous study, 167 rhizobial isolates were obtained from effective root
53
nodules of E. fordii and three isolates CCBAU 53325T, CCBAU 53266, CCBAU
54
53267 from Guangxi Province of China were tentatively identified as potential novel
55
species belonging to the genus Bradyrhizobium (Yao et al., 2014). In a subsequent
56
research with additional isolates, CCBAU 51502T, CCBAU 51501 and CCBAU
57
51497 from Guangdong Province were found to be a distinct group based upon
58
sequence analyses of housekeeping genes recA, glnII and gyrB, which was different
59
from all the defined Bradyrhizobium species and the novel group reported in Yao et al.
60
(2014). Therefore, we further systematically studied these six isolates by using a
61
polyphasic approach.
4
62
These six strains were isolated from root nodules of E. fordii collected in
63
Guangdong and Guangxi provinces of China by the standard method (Vincent, 1970;
64
Yao et al., 2014). All of the six strains were slow-growing, alkali-produced bacteria
65
and formed mucoid colonies with a diameter less than 1 mm after 7 days of
66
incubation on YMA medium at 28 °C supplied with bromothymol blue indicator
67
(Vincent, 1970).
68
In the current study, total genomic DNA was extracted from each isolate
69
following the protocol of Terefework et al. (2001). The primers and procedures were
70
adopted from the previous reports for 16S rRNA gene (Tan et al., 1997), 16S-23S
71
intergenic sequences (IGS) (Navarro et al., 1992), partial recA, glnII and gyrB genes
72
(Vinuesa, et al., 2005; Rivas et al., 2009), and partial nodC and nifH genes (Laguerre
73
et al., 2001). All the sequences acquired in this study were aligned by the Clustal W
74
software with those of related Bradyrhizobium species extracted from NCBI database
75
using the BLAST search program. These sequences used for phylogenetic analysis
76
were processed by MEGA 5.1 software (Tamura et al., 2011) to construct
77
phylogenetic trees with the Maximum Likelihood method and the Kimura
78
2-parameter model (Kimura, 1983) with 1000 bootstrap replications for all the single
79
gene sequences, as well as for the multilocus sequence analysis (MLSA) using the
80
combined sequences of recA, glnII and gyrB. The pairwise distances in the ML trees
81
were calculated by the Kimura 2-parameter model in MEGA 5.1.
82
In the ML tree for 16S rRNA gene sequences (Fig. 1), all the type strains of the 5
83
Bradyrhizobium species were divided into two groups, which were similar to that in
84
the previous study (Menna et al., 2009). The six test isolates were grouped together
85
with eight type strains of Bradyrhizobium in the group II represented by B. elkanii, at
86
sequence similarity values of 97.3%-99.8%. CCBAU 51502T was the most divergent
87
lineage within the group II, sharing similarities of 98.0% and 97.9% with another two
88
isolates (CCBAU 51501 and CCBAU 51497, respectively) within the same novel
89
group, and of 97.3%-97.9% with the type strains in the group II. The other three
90
strains CCBAU 53325T, CCBAU 53266 and CCBAU 53267 shared 99.9%-100%
91
similarities with each other, and presented similarities of 99.3%-99.8% with the type
92
strains in group II. In addition, the six test isolates had the similarities from 94.6% to
93
97.7% with the type strains in Bradyrhizobium group I.
94
Since the similarities of 16S rRNA gene sequence among the defined
95
Bradyrhizobium species are very high, more characteristics have been suggested to
96
differentiate these closely related species (Willems et al., 2001). In the IGS sequence
97
analysis, the three isolates from Guangxi, CCBAU 53325T, CCBAU 53266 and
98
CCBAU 53267, shared the sequence similarities of 99.7%-99.8% with each other and
99
shared 75.2%-92.9% with the type strains of defined Bradyrhizobium species
100
(Supplementary Fig. S1). Since Willems et al. (2003) have hypothesized that two
101
bradyrhizobial strains with IGS sequence similarity
