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Environmental Microbiology

Genomic identiﬁcation and characterization of the

elite strains Bradyrhizobium yuanmingense BR 3267

and Bradyrhizobium pachyrhizi BR 3262

recommended for cowpea inoculation in Brazil

a b c

Jakson Leite , Samuel Ribeiro Passos , Jean Luiz Simões-Araújo ,

d d d,∗

Norma Gouvêa Rumjanek , Gustavo Ribeiro Xavier , Jerri Édson Zilli

Departamento de Solos, Universidade Federal Rural do Rio de Janeiro, 23851-970 Seropédica, RJ, Brazil

Departamento de Ciências Ambientais, Universidade Federal Rural do Rio de Janeiro, 23851-970 Seropédica, RJ, Brazil

Laboratório de Genética e Bioquímica, Embrapa Agrobiologia, 23851-970 Seropédica, RJ, Brazil

Laboratório de Ecologia Microbiana, Embrapa Agrobiologia, 23851-970 Seropédica, RJ, Brazil

a r a

t i c l e i n f o b s t r a c t

Article history: The leguminous inoculation with nodule-inducing bacteria that perform biological nitrogen

Received 3 October 2016 ﬁxation is a good example of an “eco-friendly agricultural practice”. Bradyrhizobium strains

Accepted 12 January 2017 BR 3267 and BR 3262 are recommended for cowpea (Vigna unguiculata) inoculation in Brazil

Available online 31 March 2017 and showed remarkable responses; nevertheless neither strain was characterized at species

level, which is our goal in the present work using a polyphasic approach. The strains pre-

Associate Editor: Fernando Andreote

sented the typical phenotype of Bradyrhizobium with a slow growth and a white colony on

yeast extract-mannitol medium. Strain BR 3267 was more versatile in its use of carbon

Keywords:

sources compared to BR 3262. The fatty acid composition of BR 3267 was similar to the type

Vigna unguiculata

strain of Bradyrhizobium yuanmingense; while BR 3262 was similar to Bradyrhizobium elkanii and

Rhizobia

Bradyrhizobium pachyrhizi. Phylogenetic analyses based on 16S rRNA and three housekeep-

MLSA

ing genes placed both strains within the genus Bradyrhizobium: strain BR 3267 was closest

DDH

to B. yuanmingense and BR 3262 to B. pachyrhizi. Genome average nucleotide identity and

ANI

DNA–DNA reassociation conﬁrmed the genomic identiﬁcation of B. yuanmingense BR 3267

and B. pachyrhizi BR 3262. The nodC and nifH gene analyses showed that strains BR 3267 and

BR 3262 hold divergent symbiotic genes. In summary, the results indicate that cowpea can

establish effective symbiosis with divergent bradyrhizobia isolated from Brazilian soils.

Published by Elsevier Editora Ltda. on behalf of Sociedade Brasileira de Microbiologia.

This is an open access article under the CC BY-NC-ND license (http://creativecommons. org/licenses/by-nc-nd/4.0/).

∗

Corresponding author.

E-mail: [email protected] (J.É. Zilli).

https://doi.org/10.1016/j.bjm.2017.01.007

1517-8382/Published by Elsevier Editora Ltda. on behalf of Sociedade Brasileira de Microbiologia. This is an open access article under the

CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

704 b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 9 (2 0 1 8) 703–713

close strains into different species. Examples are the def-

Introduction

inition of the species Bradyrhizobium diazoefﬁciens based on

3,18

B. japonicum and Bradyrhizobium pachyrhizi from B. elkanii.

Bacteria collectively known as rhizobia form an important

Furthermore, new methods for genome-to genome compari-

part of the soil microbiota and perform biological nitrogen

son, like Average Nucleotide Identity (ANI) and Genome Blast

ﬁxation (BNF) through nitrogenase activity when in sym-

Distance Phylogeny (GBDP) have been introduced and are con-

biosis with leguminous plants. This ecological phenomenon 19,20

tributing to improve bacterial taxonomy.

has great biotechnological impact on biomass and grain pro-

Therefore, the goal of our study was to characterize at a

duction. For example, in the soybean crop production in

ﬁner the taxonomic level both BR 3267 and BR 3262 strains

Brazil it is estimated to save over US$ 10 billion annually

using a polyphasic approach.

by the use of Bradyrhizobium inoculation instead of chemical

fertilization.1

The Brazilian Ministry of Agriculture has a list of rhizobial Materials and methods

strains recommended for more than 50 leguminous grain-

producing crops, forage and green manure. In recent years Strains used

efforts have been made to better characterize these strains

recommended for inoculation in Brazil and at least ﬁve new The cowpea strains BR 3267 and BR 3262, and the type

3–6 T

species have been described. strain B. elkanii USDA 76 were obtained from the Johanna

Brazil is the world’s third leading cowpea producer, with Döbereiner Biological Resource Center (CRB-JD, Embrapa Agro-

an estimated production of 500,000 tons per year. The crop biologia, Seropédica-Rio de Janeiro, Brazil). The type strains

−1 T T

yield varies from 400 to 2000 kg ha , depending on the system B. pachyrhizi PAC 48 (=LMG 24246 ) and Bradyrhizobium yuan-

7 T

and the region of cultivation. This yield has been rising in mingense CCBAU 10071 (=LMG 21827) were obtained from the

recent years with improvements in cultivation management, LMG Culture Collection (Belgium). The strains were grown on

such as the inoculation of seeds with nitrogen-ﬁxing bacteria, yeast extract-mannitol agar medium (YMA) and were incu-

◦ 21

a practice applied to approximately 100,000 hectares. bated at 28 C for seven days until they reached sufﬁcient

The strains BR 3267 and BR 3262 are considered as “elite” colony growth levels to observe morphological features and

for inoculation of cowpea plants in Brazil and several stud- purity.

ies under controlled and ﬁeld conditions have shown that both

strains make signiﬁcant contributions to crop yields, including Phenotypic and physiologic characterizations

more than 50% N accumulation via BNF. BR 3267 strain was

isolated from the semiarid northeastern region of the country, Inoculum preparation for both BR 3267 and BR 3262 strains

◦

using cowpea as trap plants, while BR 3262 strain was isolated was carried out using YMA medium at 28 C. Carbon source

from an Atlantic Forest area in southeastern Brazil using the utilization was assessed with Biolog GN2 microplates (Biolog

1,9

same strategy. Although these strains were isolated more Inc., Hayward, CA) following the manufacturer’s instruc-

than a decade ago, they were only partially characterized tions, except that cell concentration was adjusted to 5 on

through assessment of the growth rate, colony morphology the McFarland scale. Plates were incubated in the dark at

11 ◦

and 16S rRNA phylogeny, but not classiﬁed at the species 28 C for ten days. The biochemical features were assessed

level. using API 20 NE strips (bioMérieux, Marcy-L’Etoile, France)

Zilli and colleagues have characterized the 16S rRNA following a standard protocol that uses a saline solution

genes of the BR 3267 and BR 3262 strains and concluded (0.85% NaCl) for bacterial suspension. Additionally, the toler-

that both are members of the genus Bradyrhizobium. This ance to abiotic stress, such as temperature, pH and salinity

genus was created in the early 1980s to accommodate root (NaCl), was determined by examining the growth in YMA

nodule-inducing bacteria with slow growth on media con- medium. The temperature tolerance was evaluated at 15,

12 ◦

taining mannitol and yeast extract. Since the turn of 20, 25, 28, 30, 32 and 37 C, and the pH tolerance was

the century, two major subgroup divisions (I and II) have tested in a range from 4 to 10. The salinity tolerance was

◦

been recognized within this genus based on DNA–DNA examined at 28 C in YMA medium supplemented with

12–14

hybridization. The Bradyrhizobium japonicum and Bradyrhi- 0.1, 0.3, 0.5, 1.0, 1.5, 2.0 or 2.5% (w/v) of NaCl. The resis-

zobium elkanii were the ﬁrst species assigned to subgroup I tance to antibiotics was determined using YMA medium

15,16 10

and II, respectively. According to Zilli and colleagues, BR and the disk diffusion method for ampicillin (25 ␮g), chlor-

3267 clustered within the subgroup B. japonicum and BR 3262 amphenicol (50 ␮g), erythromycin (30 ␮g), gentamicin (10 ␮g),

within the subgroup B. elkanii. However, in the light of cur- kanamycin (30 ␮g), neomycin (10 ␮g), penicillin (10 ␮g), strep-

rent knowledge, those results can be considered inconclusive tomycin (10 ␮g) and tetracycline (30 ␮g). All tests were run in

because new Bradyrhizobium species was recently described. triplicate.

Thus, further investigation employing the latest molecular

techniques is needed for the correct positioning of these

Fatty acid composition

strains.

In the past ﬁve years, the use of housekeeping genes

The BR 3267 and BR 3262 and type strains B. elkanii USDA

as powerful phylogenetic markers for bacteria has led to T T T

76 , B. pachyrhizi PAC 48 and B. yuanmingense CCBAU 10071

the description of over 15 new species within the genus

type strains were characterized based on whole-cell fatty

Bradyrhizobium and enabled the separation of genetically

acids, derived using the methyl esters’ method (FAME) and

b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 9 (2 0 1 8) 703–713 705

analyzed with a Hewlett Packard gas chromatograph ﬁtted DNA–DNA relatedness and average nucleotide identity

with a fused silica capillary column (25 m × 0.2 mm inter- (ANI)

nal diameter). The type strains B. elkanii USDA 76 , B.

T T

pachyrhizi PAC 48 and B. yuanmingense CCBAU 10071 were The DNA–DNA hybridization (DDH) among bacterial strains

chosen because they were the closest related strains to BR was determined based on the thermal denaturation temper-

3262 and BR 3267 respectively, based on EzTaxon identi- atures of hybrid and homologous genomic DNA, as described

29,30

ﬁcation search (http://www.ezbiocloud.net/eztaxon/identify). by Gonzalez and Saiz-Jimenez, except that the DNA con-

ChemStation A.09.01 software [1206] and a MIDI Micro- centration was adjusted to 2 ␮g per reaction. The experiments

bial Identiﬁcation System 4.0 (Sherlock TSBA Library, MIDI were performed in 96-well optical plates with appropriate

ID, Inc., Newark, DE, USA) were used for the fatty acid optical adhesives in three replicates and including wells

identiﬁcation. without DNA as negative control. For DNA hybridization, a

Bio-Rad MyCicler thermocycler was used, and for ﬂuorimet-

ric measurements during the denaturation stage an Applied

DNA extraction, PCR ampliﬁcation and sequencing of PCR

fragments Biosystems 7500 Real-Time system was used. Thermal con-

ditions for hybridization consisted on a denaturation step of

◦ ◦

99 C for 10 min, followed by an annealing period of 8 h at 79

Pure cultures of Bradyrhizobium strains were grown on YM

◦ 29,31

C (optimum temperature for renaturation – TOR). It was

medium for 4 days at 28 C under stirring of 120 rpm. Then,

◦

followed by progressive 10-min steps, each at 1.8 C below the

2 mL of cell suspension were centrifuged (12,000 × g), and

® ◦

previous one, until 25 C when it was hold for 30 min before

the total genomic DNA was extracted using a Wizard

◦

refrigeration to 4 C. The ﬂuorescence was measured using a

Genomic DNA Puriﬁcation Kit (Promega, USA) following the

denaturation ramp settled at the step and hold mode. Heating

manufacturer’s instructions. The recA gene was ampliﬁed

◦ −1

rate was 0.2 C s with ﬂuorescence decreasing measure-

by PCR using the primers TSrecAf and TSrecAr, and the

◦

ment at each 0.2 C step, during a 12 s hold, and between 25

glnII gene was ampliﬁed with the primers TSglnIIf and

◦ 29

22 and 99.9 C. The DDH experiments were performed between

TSglnIIr. The primers NodCfor540 and NodCrev1160 were

strains BR 3267 and BR 3262 and their phylogenetically related

used to amplify the symbiotic nodC gene, as described by

23 type strains based on the 16S rRNA gene similarities. BR3267

Sarita et al. For the nifH, the primers NifHF/NifHI were

24 was compared against B. yuanmingense CCBAU 10071 , and BR

used following the methods described by Laguerre et al.

3262 was compared against B. pachyrhizi PAC 48 and B. elkanii

DNA sequences of the PCR products were obtained for T

USDA 76 .

both strands with the same primers used for gene ampli-

The ANI estimation was performed with a JSpecies plat-

ﬁcations. The 16S rRNA sequences of the BR 3267 (Gene

form version 1.2.1, and MUMmer was used for genome

Bank A.no. AY649439) and BR 3262 (A.no. AY649430) strains

10 alignment. All system requirements (BLAST and MUMmer)

obtained in a previous study were retrieved from GenBank

were downloaded and installed locally in a system Linux. Bac-

(http://www.ncbi.nlm.nih.gov/genbank/). For the gyrB gene,

terial genome were retrieved from NCBI database as fasta

the sequences were retrieved from draft genome sequences

ﬁle and used to ANI calculation, considering JSpecies default

of the BR 3267 and BR 3262 strains (Ac. no. KT005410 and

parameters. The sequenced genome of strain BR 3262 (Ac.no. KT005409, respectively).

LJYE00000000.1) was compared to genomes of the strains

USDA 76 (B. elkanii – GenBank A.no. ARAG00000000.1) and

Phylogenetic analysis T

PAC48 (B. pachyrhizi – GenBank A.no. LFIQ00000000.1). The

genome of the strain BR 3267 (A.no. LJYF00000000.1) was

Forward and reverse readings of the 16S rRNA, recA, glnII,

compared to that of the strain CCBAU 35157 (B. yuanmingense

nodC and nifH gene fragments were edited and assem-

– A.no. AJQL00000000.1).

bled using DNABASER (http://www.dnabaser.com). Multiple

sequence alignments were performed using ClustalW through

MEGA 6.0. Maximum likelihood (ML) phylogenetic recons- Results

tructions were completed using MEGA 6 for single gene

sequences (16S rRNA, recA, glnII, gyrB, nodC and nifH), and a

Phenotypic and physiologic features

multilocus sequence analysis (MLSA) was performed using

the concatenated sequences of the housekeeping genes recA,

Both BR 3267 and BR 3262 strains can grow in a pH range of

glnII and gyrB. The distance matrices were calculated using

4–10, tolerate up to 0.5% NaCl in the medium and present

26 ◦

the Kimura two-parameter substitution model, and the

normal growth in a temperature range from 15 to 32 C,

◦

robustness of the tree nodes was evaluated with a boot-

and BR 3267 could also grow up to 37 C (Table 1). Likewise,

strap analysis using 500 pseudoreplicates. The software

both strains showed positive reactions to enzyme urease and

default parameters were considered in all of the analy-

nitrate reductase. Their susceptibilities to different antibiotics

ses. Sequences of rhizobial type strains used for alignment

were variable (Table 1). BR 3267 was sensitive to streptomycin

and phylogenetic analysis were retrieved from GenBank

and tetracycline, while BR 3262 was tolerant. With respect to

(http://www.ncbi.nlm.nih.gov/genbank/), and a Microvirga vig-

the carbon sources evaluated with the Biolog kit, BR 3262 strain

nae BR 3299 strain was used as the outgroup in the ML

was able to use 33 of the 95 sources tested, while BR 3267 strain

analysis of the 16S rRNA gene.

was able to use more than 50.

706 b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 9 (2 0 1 8) 703–713

Table 1 – Phenotypic features of the cowpea Bradyrhizobium strains BR 3267 and BR 3262 as characterized by API 20NE

and Biolog GN2 microplates. Data obtained from 5 days duplicate read mean values (+ = positive; − = negative, w = weak).

Phenotypic feature BR 3267 BR 3262 Phenotypic feature BR 3267 BR 3262 Phenotypic feature BR 3267 BR 3262

Enzymatic reaction pH 4 + + Ester

−

Catalase + pH 10 + + Methyl pyruvate + −

−

Hydrolysis of gelatine + 1% NaCl − − Mono-methyl-succinnate + −

Hydrolysis of esculin + + Carbohydrates Amino acids

ˇ-galactosidase w − d-Arabitol + − d-Alanine − +

Resistance to (g) ␣-d-Glucose + − l-Alanine − +

Ampicillin (25) − + d-Mannose + − Glycyl-l-aspartic acid + −

Chloramphenicol (50) − + d-Psicose + − l-Leucine + −

Kanamycin (30) + − d-Sorbitol + − l-Phenylalanine + −

Neomycin (10) + − Carboxylic acids l-Proline + −

Penicillin (10) − + d-Galacturonic acid + − d-Serine + −

Streptomycin (10) − + d-Glucosaminic acid + − l-Threonine + −

Tetracycline (30) − + ␣-Ketovaleric acid + − ␥-Aminobutyric acid + −

Erythromycin (30) + + d,l-Lactic acid + − Polymer

Gentamicin (10) + + Malonic acid + − Dextrin − +

Growth at Quinic acid + − Amide

◦

15 C + + d-Saccharic acid + − Glucuronamide + −

◦

32 C + + Sebacic acid + − Amine

◦ −

−

37 C+Succinic acid + Phenylethylamine + −

Both strains BR 3267 and BR 3262 were positive to: Oxidase, Urease, Nitrate reduction, Hydrolysis of esculin, l-Arabinose, l-Fucose, d-Galactose,

-Mannitol, l-Rhamnose, Tween 40, Tween 80, Acetic acid, Citric acid, Formic acid, d-Galactonic acid lactone, d-Gluconic acid, ␣-Hydroxybutyric

acid, ␤-Hydroxybutyric acid, ␥-Hydroxybutyric acid, p-Hydroxyphenylacetic acid, ␣-Ketobutyric acid, ␣-Ketoglutaric acid, Propionic acid, Glyc-

erol, l-Alanyl-glycine, l-Asparagine, l-Aspartic acid, l-Glutamic acid, Glycyl-l-glutamic acid, l-Pyroglutamic acid, l-Serine, Succinamic acid,

l-Alaninamide and Bromo succinic acid; and negative to: Tryptophan deaminase, Glucose fermentation, Arginine dihydrolase, N-Acetyl-

d-galactosamine, N-Acetyl-d-glucosamine, Adonitol, d-Cellobiose, i-Erythritol, d-Fructose, Gentiobiose, m-Inositol, ␣-d-Lactose, Lactulose,

Maltose, d-Melibiose, ␤-Methyl-d-Glucoside, d-Rafﬁnose, Sucrose, d-Trehalose, Turanose, Xylitol, ␣-Cyclodextrin, Glycogen, cis-Aconitic acid,

d-Glucuronic acid, Itaconic acid, 2,3-Butanediol, d,l-␣-Glycerol phosphate, Glucose-1-phosphate, Glucose-6-phosphate, l-Histidine, Hydroxy-

l-proline, l-Ornithine, Urocanic acid, Inosine, Uridine, Thymidine, Putrescine and 2-Aminoethanol.

Fatty acid composition phylogenetic linage together with B. pachyrhizi PAC48 (100%

16S rRNA similarity), B. elkanii USDA 76 (99.9%) and Bradyrhizo-

The fatty acid composition was analyzed for the BR 3262 bium tropiciagri CNPSo 1112 (99.2%), but with nodes lower than

T T

and BR 3267 strains and for B. elkanii USDA 76 , B. pachyrhizi <60% of bootstrap. The Bradyrhizobium ferriligni CCBAU 51502

T T

PAC 48 and B. yuanmingense CCBAU 10071 , the closest type was the closest taxon to the clade of B. tropiciagri–B. elkanii–B.

strains based on EzTaxon Identify search of the 16S rRNA gene pachyrhizi – BR 3262, as shown by the high ML bootstrap sup-

(http://www.ezbiocloud.net/eztaxon/identify). The fatty acid port (91%, Fig. 1); it shares 97.8% of 16S rRNA similarity with

compositions of BR 3267 and BR 3262 were similar to those of BR 3262.

other strains within Bradyhizobium genus, especially because We used aligned sequences from the recA (359 bp), glnII

of the presence of 16:00 in approximately 15% and a summed (505 bp) and gyrB (552 bp) housekeeping genes, which resulted

feature 8 (18:1 w7c) between approximately 70 and 80%. BR in a concatenated sequence of 1416 bp, to better assess the

3262 has a composition close to those of B. elkanii USDA 76 phylogenetic afﬁliation of the BR 3267 and BR 3262 strains. ML

and B. pachyrhizi PAC 48 , with the presence of 19:0 cyclo w8c of phylogenetic trees of single and concatenated genes (Fig. 2)

15.98% and a summed feature 8 (18:1 w7c) of 69.32. Instead, BR were generated and showed high congruence; therefore, we

3267 and B. yuanmingense CCBAU 10071 did not present 19:0 presented only the concatenated one. The concatenated phy-

cyclo w8c and had a summed feature 8 of 86.42 and 84.63%, logenetic analysis revealed a clear afﬁliation between BR 3267

respectively. and B. yuanmingense CCBAU 10071 , with high conﬁdence

bootstrap values of the 100%, in a lineage that was distinct

from the closest soybean isolates Bradyrhizobium daqingense Phylogenetic analyses

T48

CCBAU 15774 and Bradyrhizobium huanghuaihaiense CCBAU

T 49

23303 . On the other hand, BR 3262 was phylogenetically

The Maximum Likelihood (ML) phylogenetic analysis per-

close to B. pachyrhizi PAC48 , which formed a lineage with

formed on the 16S rRNA (1227 bp) gene placed the cowpea

high conﬁdence values (bootstrap ML of 98%) and sepa-

strains BR 3267 and BR 3262 into subgroups I and II, respec-

rated from the closed taxa B. ferriligni CCBAU 51502 and B.

tively, within the genus Bradyrhizobium (Fig. 1). The closest

T elkanii USDA76 . Additionally, pairwise comparisons of the

strain to BR 3267 was B. yuanmingense CCBAU 10071 (99.5%

concatenated sequences revealed that BR 3267 shared 99%

16S rRNA similarity), with 67% ML bootstrap support, followed

T sequence identity with B. yuanmingense CCBAU 10071 , fol-

by Bradyrhizobium subterraneum 58 2-1 (99.6% 16S rRNA simi-

lowed by Bradyrhizobium liaoningense USDA 3622 (95%) and

larity) in a separated branch with 62% bootstrap. In contrast,

B. daqingense CCBAU 15774 (94.5%). Likewise, BR 3262 was

BR 3262 was placed within subgroup II of Bradyrhizobium in a

b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 9 (2 0 1 8) 703–713 707

T 65 Bradyrhizobium manausense BR 3351 (HQ641226) Bradyrhizobium guangdongense CCBAU 51649T (KC508867) Bradyrhizobium ganzhouense RITF806T (JQ796661) Bradyrhizobium cytisi CTAW11T (EU561065) Bradyrhizobium rifense CTAW71T (EU561074) Bradyrhizobium vignae 7-2T (KP899563) Bradyrhizobium huanghuaihaiense CCBAU 23303T (HQ231463) Bradyrhizobium arachidis CCBAU 051107T (HM107167) T 72 Bradyrhizobium kavangense 14-3 (KP899562) T 60 Bradyrhizobium iriomotense EK05 (AB300992) 95 Bradyrhizobium ingae BR 10250T (KF927043) Bradyrhizobium guangxiense CCBAU 53363T (KC508877) Bradyrhizobium diazoefficiens USDA 110T (NC_004463) Bradyrhizobium betae LMG 21987T (AY372184) BR 3267 (AY649439) 60 Bradyrhizobium yuanmingense CCBAU 10071T (AF193818) 59 Bradyrhizobium yuanmingense CCBAU 35157 (AJQL01000001.1) Bradyrhizobium subterraneum 58 2-1T (KP308152) T 7365 Bradyrhizobium liaoningense USDA 3622 (AF208513) Bradyrhizobium daqingense CCBAU 15774T (HQ231274) Bradyrhizobium japonicum USDA 6T (U69638) Bradyrhizobium canariense BTA-1T (AJ558025) Bradyrhizobium ottawaense OO99T (JN186270) Bradyrhizobium denitrificans LMG 8443T (X66025) 93 Bradyrhizobium oligotrophicum LMG 10732T (JQ619230) Bradyrhizobium retamae Ro 19T (KC247085) Bradyrhizobium valentinum LmjM3T (JX514883) T 90 Bradyrhizobium neotropicale BR 10247 (KF927051) Bradyrhizobium embrapense CNPSo 2833T (AY904773) 81 Bradyrhizobium erythrophlei CCBAU 53325T (KF114645) Bradyrhizobium viridifuturi SEMIA 690T (FJ025107) Bradyrhizobium lablabi CCBAU 23086T (GU433448) Bradyrhizobium icense LMTR 13T (KF896156) Bradyrhizobium paxllaeri LMTR 21T (AY923031) Bradyrhizobium jicamae PAC68T (AY624134) BR 3262 (AY649430) Bradyrhizobium pachyrhizi PAC48T (AY624135) 85 Bradyrhizobium elkanii USDA 76T (U35000) Bradyrhizobium tropiciagri CNPSo 1112T (AY904753) 50 Bradyrhizobium ferriligni CCBAU 51502T (KJ818096) Microvirga vignae BR 3299T (JX504804)

0.02

Fig. 1 – Maximum Likelihood phylogenetic tree based on 16S rRNA gene sequences showing relationships between the

strains BR 3262 and BR 3267 and the type species (T) of the genus Bradyrhizobium. Bootstrap values were inferred from 500

replicates and are indicated at the tree nodes when ≥50%. GenBank accession numbers are provided in parenthesis.

Microvirga vignae BR3299 was included as the outgroup. The bar represents two estimated substitutions per 100 nucleotide positions.

similar to B. pachyrhizi PAC48 (98.6%), followed by B. elka- DNA–DNA relatedness and average nucleotide identity

T T

nii USDA 76 (96.9%) and B. ferriligni CCBAU 51502 (95.5%).

In an independent analysis of the recA, glnII and a con- After obtaining the phylogenetic results, we established the

catenated recA-glnII sequences (data not shown), BR 3267 contrasts for the analysis of the DNA–DNA homology follow-

and BR 3262 were far from the newest proposal species ing the protocol described by Gonzales and Saiz-Jimenez. BR

T 50 T

Bradyrhizobium kavangense 14-3 , Bradyrhizobium vignae 7- 3262 was compared with B. elkanii USDA 76 and B. pachyrhizi

T51 T52 T

2 and B. subterraneum 58-2-1 isolated from African PAC 48 (the closest strains according to MLSA), and BR 3267

soils. was compared with B. yuanmingense CCBAU 10071 (also the

708 b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 9 (2 0 1 8) 703–713

T 57 Bradyrhizobium cytisi CTAW 11 (GU001575, GU001594, JN186292) 80 Bradyrhizobium ganzhouense RITF806T (JX277144, JX277110, KP420022) 67 Bradyrhizobium rifense CTAW71T (GU001585, GU001604, KC569466) 54 Bradyrhizobium canariense BTA-1T (AY591553, AY386765, AB353736) 85 Bradyrhizobium betae LMG 21987T (FM253174, AB353733, AB353735) Bradyrhizobium diazoefficiens USDA 110T (NC_004463, NC_004463, NC_004463) Bradyrhizobium japonicum USDA 6T (AM168341, AF169582, AM418801) Bradyrhizobium ottawaense OO99T (HQ587287, HQ587750, HQ873179) Bradyrhizobium liaoningense USDA 3622T (AY494833, AY494803, FM253223) Bradyrhizobium arachidis CCBAU 051107T (HM107233, HM107251, JX437675) Bradyrhizobium neotropicale BR 10247T (KJ661714, KJ661700, KJ661707) Bradyrhizobium iriomotense EK05T (AB300996, AB300995, HQ873308) T 98 Bradyrhizobium ingae BR 10250 (KF927061, KF927067, KF927079) Bradyrhizobium huanghuaihaiense CCBAU 23303T (HQ231595, HQ231639, JX437672) Bradyrhizobium daqingense CCBAU 15774T (HQ231270, HQ231301, JX437669) BR 3267 (KF828822, KF828821, KT005410) 100 Bradyrhizobium yuanmingense CCBAU 10071T (AY591566, AY386780, FM253226) 99 Bradyrhizobium manausense BR 3351T (KF785992, KF785986, KF786000) Bradyrhizobium guangdongense CCBAU 51649T (KC509269, KC509023, KC509072) 64 Bradyrhizobium guangxiense CCBAU 53363T (KC509279, KC509033, KC509082) Bradyrhizobium denitrificans LMG 8443T (FM253196, HM047121, FM253239) T 100 Bradyrhizobium oligotrophicum LMG 10732 (JQ619231, JQ619233,KC569467) T 95 Bradyrhizobium jicamae PAC68 (HM590776, FJ428204, AM418801) 99 Bradyrhizobium paxllaeri LMTR 21T (JX943617, KF896169, KF896195) T 99 Bradyrhizobium lablabi CCBAU 23086 (GU433522, GU433498, JX437670) Bradyrhizobium retamae Ro19T (KC247094, KC247108, KF962698) 95 T 99 Bradyrhizobium icense LMTR13 (JX943615, KF896175, KF896201) Bradyrhizobium valentinum LmjM3T (JX518589, JX518575,NZ_LLXX01000044.1) Bradyrhizobium erythrophlei CCBAU 53325T (KF114669, KF114693, KF114717) Bradyrhizobium tropiciagri CNPSo 1112T (FJ391168, FJ391048, HQ634890) Bradyrhizobium embrapense CNPSo 2833T (HQ634899, GQ160500, HQ634891) 98 Bradyrhizobium viridifuturi SEMIA 690T (KR149140, KR149131, KR149134) T 99 Bradyrhizobium elkanii USDA 76 (AY591568, AY599117, AB070584) Bradyrhizobium ferriligni CCBAU 51502T (KJ818112, KJ818099, KJ818102) BR 3262 (KF828817, KF828816, KT005409) T 98 Bradyrhizobium pachyrhizi PAC48 (HM590777, FJ428201, HQ873310)

0.02

Fig. 2 – Unrooted maximum likelihood phylogenetic tree based on three concatenated sequences (recA, glnII, gyrB) showing

relationships between the strains BR 3262 and BR 3267 and type species (T) of the genus Bradyrhizobium. The phylogenetic

tree was built using the Kimura two-parameter method. Bootstrap values were inferred from 500 replicates and are

indicated at the tree nodes when ≥50%. GenBank accession numbers are provided in parenthesis. The bar represents two

estimated substitutions per 100 nucleotide positions.

closest strain in the MLSA). The hybridization of BR 3262 B. 3262 and B. pachyrhizi PAC 48 was 94.6 and 95.3, respectively.

T T T

elkanii USDA 76 and B. elkanii USDA 76 B. pachyrhizi PAC 48 On another hand the ANI in the comparison of BR 3267 and B.

◦ ◦

presented Tm values equal to 10.1 C and 3.9 C, respectively, yuanmingense CCBAU 35157 was 96.5.

and the hybridization of BR 3267 × B. yuanmingense CCBAU

T ◦

10071 presented a value of 3.9 C (Fig. 3). Phylogeny based on the nodC and nifH symbiotic genes

For the ANI calculation, we used the available genomes of

T T

B. elkanii USDA 76 and B. pachyrhizi PAC 48 for comparison Maximum Likelihood phylogenetic analyses of the symbi-

with BR 3262 and, B. yuanmingense CCBAU 35157 for compar- otic genes nodC and nifH separated the BR 3267 and BR

ison with BR 3267 because no genome was available for the 3262 strains into two divergent phylogenetic lineages (Fig. 4A

type strain of B. yuanmingense CCBAU 10071 . The ANI between for nodC, Fig. 4B for nifH). For both nodC and nifH, BR 3267

strain BR 3262 and B. elkanii USDA 76 and between strain BR formed an independent branch that clustered together with

b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 9 (2 0 1 8) 703–713 709

100 BR 3267 CCBAU 10071 Hybrid: BR 3267/CCBAU 10071 Δ = 3.9 50 Tm

25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 100 BR 3262 PAC 48 Hybrid: BR 3262/PAC 48

50 Δ Tm = 3.9 Fluorescence (%)

25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 100 BR 3262

USDA 76 Hybrid: BR 3262/USDA 76 Δ 50 Tm = 10.1

25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100

Temperature (ºC)

Fig. 3 – Melting curves generated with Applied Biosystems 7500 Real-Time System for Tm determination between

homologous and hybrid DNA. (a) Comparison between strain BR 3267 and B. yuanmingense CCBAU 1007 ; (b) between BR

T T

3262 and B. pachyrhizi PAC 48 ; (C) between BR 3262 and B. elkanii USDA 76 .

B. yuanmingense CCBAU 10071 and the soybean type strains enzymes reactions, which are apparently common character-

38,39

that represent the symbiovar glycinearum. In contrast, BR istics among strains of the genus Bradyrhizobium. It seems

3262 was grouped in an early branch linked to the related to be a common characteristic among Bradyrhizobium species

T T

strains B. pachyrhizi PAC48 , B. elkanii USDA 76 and B. ferriligni of subgroup I (close to B. japonicum) to be sensitive to strepto-

CCBAU51502 for both symbiotic genes (Fig. 4A and B). mycin and tetracycline, as showed by BR 3267. In contrast, BR

3262 was tolerant to these antibiotics, a common characteris-

tic of Bradyrhizobium species belonging to subgroup II (close to

Discussion B. elkanii). The 50 carbon sources used by BR 3267 points to

greater access to different soil carbon compounds. The results

The analyses in the culture medium showed that both strains, of the fatty acid composition analysis corroborate previous

BR 3267 and BR 3262, have similar behaviors in comparison ﬁndings that indicate that the main differences between sub-

to other members of the Bradyrhizobium genus regarding tol- groups I and II of Bradyrhizobium are the 19:0 cyclo w8c and

erance to temperature, pH and NaCl concentration in the the summed feature 8 (18:1 w7c), which indicate that BR 3267

culture medium. These traits indicate they are tolerant to dif- belongs to the subgroup of B. japonicum and BR 3262 to the

40,41

ferent edapho-climatic factors and make them well adapted subgroup of B. elkanii.

to the conditions of the tropical soils from which they were When the 16S rRNA genes of strains BR 3267 and BR 3262

isolated. This adaptability is corroborated by their good per- were ﬁrst studied, it was observed that although BR 3267

8,36,37

formance in cowpea inoculations. The BR 3267 and BR could be grouped with B. japonicum, there were differences

3262 strains were positive to urease and nitrate reductase with respect to the B. japonicum type strain pattern. At the time,

710 b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 9 (2 0 1 8) 703–713

B. japonicum USDA 6T (AB354632)

ABB. huanghuaihaiense CCBAU 23303T (HQ231551) B. ottawaense OO99T (HQ587980) B. liaoningense USDA 3622T (EU818925) 100B. huanghuaihaiense CCBAU 23303T (KF962706) B. japonicum USDA 6T (HM047126) B. diazoefficiens USDA 110TT(NC_004463) B. ottawaense OO99T (JN186287) B. daqingense CCBAU 15774TT (HQ231326) 100 99 T B. diazoefficiens USDA 110T (NC_004463) B. yuanmingense CCBAU 10071 (AB354633) 55 92 BR 3267 (KF828824) B. daqingense CCBAU 15774T (HQ231323) 84 B. arachidis CCBAU 051107T (KF962705) BR 3267 (KF828825) B. vignae 7-2T (KT362339) 97 T 69 B. yuanmingense CCBAU 10071 (EU818927) 53 T B. kavangense 14-3 (KT033402) T 84 59 B. arachidis CCBAU 051107 (HM107283) BR 3262 (KF828819) 99 BR 3262 (KF828820) Bradyrhizobium. sp. VUPME10 (HG940520) B. pachyrhizi PAC48 T (HM047124) 94 B. pachyrhizi PAC48 T (HQ588110) 98 99 B. elkanii USDA 76T (AB094963) B. elkanii USDA 76T (AB354631) 71 67 T T 99 B. ferriligni CCBAU 51502 (KJ818108) 99 B. ferriligni CCBAU 51502 (KJ818109) T B. tropiciagri CNPSo 1112T (KP234520) 89 B. tropiciagri CNPSo 1112 (HQ259540) T B. embrapense CNPSo 2833T (KP234518) 100 B. embrapense CNPSo 2833 (KP234521) 100 B. canariense BTA-1T (AJ560653) 95 B. viridifuturi SEMIA 690T (KR149137) B. cytisi CTAW11T (EU597844) T 100 B. erythrophlei CCBAU 53325 (KF114598) T B. rifense CTAW71 (EU597853) B. lablabi CCBAU 23086T (GU433546) T 99 B. erythrophlei CCBAU 53325 (KF114576) B. paxllaeri LMTR 21T (DQ085619) B. jicamae PAC68T (AB573869) B. icense LMTR 13T (KF89616) B. lablabi CCBAU 23086T (GU433565) 97 100 B. retamae Ro19T (KF670138) B. paxllaeri LMTR 21T (KF896160) 93 B. valentinum LmjM3T (KF806461) B. retamae Ro19T (KC247112) 82 100 T B. icense LMTR 13T (JX514897) B. jicamae PAC68 (HM047127) 99 T T 96 B. valentinum LmjM3 (JX514897) 66 B. guangdongense CCBAU 51649 (KC509130) B. neotropicale BR 10247T (KJ661727) B. guangxiense CCBAU 53363T (KC509140) T B. ingae BR 10250 (KF927054) B. denitrificans LMG 8443T (HM047125) B. iriomotense EK05T (AB301000) T 89 B. ganzhouense RITF806 (JX292065) T 100 B. manausense BR 3351 (KF786002) B. canariense BTA-1T (EU818926) B. ganzhouense CCBAU 51649TT (JX292035) B. cytisi CTAW11T (GU001618) Bradyrhizobium sp. ORS285 (AF284858) 100 95 B. rifense CTAW71T (GU001627)

0.05 0.02

Fig. 4 – Unrooted maximum likelihood phylogenetic tree based on nodC (A) and nifH (B) genes showing relationships

between the strains BR 3262 and BR 3267 and type species (T) and reference strains of the genus Bradyrhizobium. The

phylogenetic tree was built using the Kimura two-parameter method. Bootstrap values were inferred from 500 replicates

and are indicated at the tree nodes when ≥50%. GenBank accession numbers are provided in the parenthesis. The bar

represents ﬁve or two estimated substitutions per 100 nucleotide positions.

however, the species B. yuanmingense, which also belongs to pachyrhizi species, and BR 3267 is a member of the B. yuan-

subgroup I of Bradyrhizobium, had not yet been described; thus, mingense species. As previously shown, a Tm value equal to

◦

there was no species indication. It was observed on the other 5 C can be used as the threshold to delineate a species, where

hand that BR 3262 had a strong resemblance to B. elkanii by lower values indicate equal species and higher values indi-

29 ◦

the 16S rRNA gene, which supported that the strain should be cate different species. A Tm value of 5 C corresponds to a

assigned to that species. At that time, the species B. pachyrhizi, homology value of approximately 70% obtained by traditional

29,30

which in terms of base composition of the 16S rRNA gene dif- DNA–DNA hybridization techniques. Moreover, we calcu-

fers from strains B. elkanii USDA 76 only on the order of 0.1%, lated the ANI, a robust method that replaces the traditional

had not yet been described. 16S rRNA sequence analysis is not DNA–DNA hybridization techniques. This method involves

42,43

always ﬁne enough for species assignment ; in the genus comparing the sequences of two bacterial genomes, and a

19,53

Bradyrhizobium some species share almost identical 16S rRNA value of 95% is considered as the threshold. In this case,

gene sequences. Examples are the type strains of the species when the homology is greater than 95%, the two strains belong

B. japonicum and B. liaoningense, along with B. elkanii and B. to the same species; if the homology is lower, they belong to

pachyrhizi. Due to the high conservation of the 16S rRNA different species. Thus, the results of the comparisons again

14,15

gene among members of the Bradyrhizobium genus, several conﬁrmed that BR 3262 belongs to B. pachyrhizi (ANI greater

authors have recommended an MLSA of protein-encoding (i.e., than 95%) and the same BR 3267 to B. yuanmingense (ANI greater

6,22,44–47

housekeeping) genes. In our case, the MLSA results than 95%).

were congruent to the 16S rRNA analysis and clearly indicate Symbiotic genes related to nodulation (nod) and nitro-

that BR 3267 and BR 3262 as members of the species B. yuan- gen ﬁxation (nif) processes hold no taxonomic information

mingense and B. pachyrhizi, respectively (Fig. 2). The DNA–DNA for species deﬁnition. However, in our study, they showed

homology results conﬁrm the results obtained in the anal- good phylogenetic correspondence to the core genome ones.

ysis of housekeeping genes, i.e., BR 3262 belongs to the B. Interestingly, nodC and nifH gene phylogenetic analyses have

b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 9 (2 0 1 8) 703–713 711

been used to predict the host range nodulation capacity and Fixation. Hoboken, NJ: John Wiley & Sons, Inc.;

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2. Brasil Ministério da Agricultura Pecuária e Abastecimento.

strains with similar symbiotic behaviors concerning nodu- ◦

55 Instruc¸ão normativa n 13 de 24 de marc¸o de 2011. Aprova as

lation and nitrogen ﬁxation capacity with legumes. For

normas sobre especiﬁcac¸ões, garantias, registro, embalagem e

the nodC gene, for example, a BLAST search in the NCBI

rotulagem dos inoculantes destinados à agricultura, bem como as

database (http://blast.ncbi.nlm.nih.gov/) showed the Arachis relac¸ões dos microorganismos autorizados e recomendados para

hypogae strains CCBAU 53380 (Ac. no. KC509232) and CCBAU produc¸ão de inoculantes no Brasil, na forma dos Anexos I, II, III,

51663 (Ac. no. KC509217), isolated in China, as the Bradyrhizo- desta Instruc¸ão Normativa. Diário Oﬁcial da União da

República Federativa do Brasil; 2011:3–7. Sec¸ão 1.

bium strains that had the closest similarity (98%) with BR 3267,

3. Delamuta JRM, Ribeiro RA, Ormeno-Orrillo˜ E, Melo IS,

indicating that peanut might be a possible host for this latter

Martínez-Romero E, Hungria M. Polyphasic evidence

strain. Instead, in the nodC gene, BR 3262 had 95% similarity

supporting the reclassiﬁcation of Bradyrhizobium japonicum

with the cowpea strain Bradyrhizobium sp. VUMPE10 (Fig. 4A),

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Bradyrhizobium tropiciagri sp. nov. and Bradyrhizobium

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nodulation has not yet been well analyzed to identify true cow-

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