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RESEARCH LETTER
Occurrence and diversity of nitrogen-¢xing Sphingomonas bacteria
associated with rice plants grown in Brazil
Sandy Sampaio Videira1,2, Jean Luiz Simões de Araujo2, Luciana da Silva Rodrigues1,2, Vera Lúcia Divan
Baldani2 & José Ivo Baldani2
1
Instituto de Agronomia, CPGA-CS, Universidade Federal Rural do Rio de Janeiro, Seropédica, RJ, Brazil; and 2Laboratório de Genética e Bioquı́mica,
Embrapa Agrobiologia, Seropédica, RJ, Brazil
Received 1 December 2008; accepted
9 December 2008.
First published online 13 February 2009.
DOI:10.1111/j.1574-6968.2008.01475.x
Editor: Yaacov Okon
Keywords
Sphingomonas ; ARDRA; RISA; diazotrophic;
16S rRNA gene; nif H sequences.
Abstract
So far, the occurrence of nitrogen-fixing Sphingomonas bacteria has been restricted
to three strains of Sphingomonas azotifigens. In this work, a group of 46
Sphingomonas-like isolates, which originated from two rice varieties grown in two
soils in Brazil, were characterized based on morphological, physiological and
genetic analyses. The PCR genus specifically applied indicated that all 46 isolates
belonged to the Sphingomonas genus and confirmed the results based on the yellow
pigment of the colonies grown on potato agar medium and the BIOLOG data. It
was also observed that 22 isolates are nitrogen-fixing bacteria as determined by the
acetylene reduction method and confirmed by nifH gene detection. The genetic
diversity based on the 16S rRNA analysis (amplified rDNA restriction analysis)
showed that the isolates formed two distinct groups at a similarity value of 60%.
Furthermore, five clusters at 60% similarity were observed with the 16S–23S
intergenic space (ribosomal intergenic space analysis) analysis. Sequencing of the
16S rRNA gene and nifH fragments showed that most of the 22 nitrogen-fixing
isolates formed clusters apart from that of the S. azotifigens. This is the first report
on the occurrence of nitrogen-fixing Sphingomonas bacteria associated with rice
grown in Brazil.
Introduction
The genus Sphingomonas comprises over 55 species, with the
Sphingomonas paucimobilis identified as the type species
(Yabuuchi et al., 1990). The genus Sphingomonas sensu
stricto includes species with broader characteristics such as
the ability to degrade aromatic and xenobiotic compounds,
plant pathogens as well as species that are opportunistic
pathogens to humans, but not animals (Yabuuchi & Kosako,
2005).
Despite the higher number of Sphingomonas species
described, knowledge of the occurrence of nitrogen-fixing
species within the genus is very recent. So far, only one
species, named Sphingomonas azotifigens, is known to fix
nitrogen (Xie & Yokota, 2006). Nevertheless, this species was
created based only on three strains previously isolated from
paddy soil and rhizosphere of rice plants grown in Japan
(Oyaizu-Masuchi & Komagata, 1988). In addition, the
suggestion that the Sphingomonas trueperi type strain
FEMS Microbiol Lett 293 (2009) 11–19
(NBRC 100456T), formerly known as ‘Pseudomonas
azotocolligans’ (Anderson, 1955), also fixes nitrogen could
not be confirmed by the nifH gene PCR amplification (Xie &
Yokota, 2006). The presence of the amplified nifD gene PCR
product was also detected in a single isolate of S. trueperi
obtained from rice, but no nitrogenase activity could be
shown (Adhikari et al., 2001). Other authors also suggested
the occurrence of nitrogen-fixing S. paucimobilis bacteria in
the rhizosphere of rice (Engelhard et al., 2000), millet and
sorghum (Hebbar et al., 1992) and rice seeds (Mano et al.,
2006), but no further studies were conducted to determine
the nitrogen-fixation ability of the isolates.
There is no additional information on the occurrence and
diversity of nitrogen-fixing bacteria within the Sphingomonas
genus, especially associated with rice plants. Most of the
studies on the occurrence of Sphingomonas are related to
pathogenic bacteria mainly associated with rice seeds
(Iizuka, 1960; Kim et al., 1998). Therefore, broader knowledge of the ecological distribution and diversity of the
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Correspondence: José Ivo Baldani,
Laboratório de Genética e Bioquı́mica,
Embrapa Agrobiologia, BR 465, km 07, CEP
23890-000, Seropédica, RJ, Brazil. Tel.: 155
21 3441 1555; fax: 155 21 2682 1230;
e-mail: [email protected]
12
Sphingomonas genus associated with rice plants may provide
new perspectives to explore its agricultural potential to
promote plant growth.
The aim of this work was to demonstrate the occurrence
and analyse the genetic diversity of nitrogen-fixing Sphingomonas genus isolated from rice plants grown in Brazil.
Materials and methods
Isolation of diazotrophic bacteria
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potato medium were isolated and subcultured for further
analysis.
Acetylene reduction assay (ARA)
The nitrogen-fixing ability of each isolate was tested using
the ARA, as described by Boddey (1987). Each bacterial
isolate was inoculated into 10-mL vials containing 5 mL
semi-solid JNFb medium, which were then sealed with
rubber septa. All isolates were incubated at 30 1C in the
dark. After 72 h, 10% (v/v) of the air phase was replaced
with acetylene (Burris, 1972) and the vials were reincubated.
After the addition of acetylene, three vials were sampled for
1 h to determine the amount of ethylene. Ethylene was
measured using a Perkin Elmer Autosystem XL gas chromatograph (Perkin Elmer) with a Poropak Q column and a
flame ionization detector connected to a chromatography
data computer system.
Biolog analysis
Carbon source utilization was determined using Biolog GN2
Microplates (Hayward, CA). The isolates were cultivated on
liquid dextrose, yeast, glutamate (DYGS) medium at 489 g
for 24 h at 30 1C. Afterwards, an aliquot was streaked onto
plates of Biolog Universal Growth (BUG) medium and
incubated for 48 h at 30 1C. The pure colonies were carefully
removed from the plates with a sterile swab and resuspended
in sterile saline (0.85% NaCl). The cell concentration was
adjusted to 52% of transmittance ( 3%) using a spectrophotometer coupled with a 590 nm filter (Biolog Inc., 1999).
The cell suspension (50 mL) of each isolate was transferred to
Biolog GN 96-well microplates (Biolog Inc.), closed and
incubated for 24 h at 30 1C. The purpure colour resulting
from the utilization of the different carbon sources was
considered as a positive growth. The results were compared
with the Biolog Microlog database (release 4.01B).
Isolation of DNA
Genomic DNA from the isolates was extracted using the
QUIamp DNA Mini Kits according to the QIAGENs
protocol. The purity was assessed from the A260/A280 and
A260/A230 extinction ratios.
Genus-specific PCR amplification
The primers 108f (5 0 -GCG TAA CGC GTG GGA ATC TG3 0 ) and 420r (5 0 -TTA CAA CCC TAA GGC CTT C-3 0 ),
specifically designed for the genus Sphingomonas (Leys et al.,
2004), were used to determine the taxonomical position of
the isolates. The PCR reactions were performed in a
final volume of 50 mL containing 50 ng of template DNA,
1 PCR buffer, 1.25 Tween 20 2%, 3.75 mM MgCl2;
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The bacteria were isolated from rice plant varieties IR 42 and
IAC 4440 grown in pots with two hydromorphic soil types
originating from Goiás (GO) and Rio de Janeiro (RJ) States
with at a least 10-year history of rice cultivation. The rice
seeds were disinfected according to Baldani et al. (2000) and
pregerminated on agar plates for 2 days at 30 1C. Only those
seeds free from contaminants were planted on pots of 5 L
with 4 kg of each soil previously fertilized with P and K and
the pH adjusted to 6.0 with liming. The pots were maintained flooded (2-mm water layer) outside of the greenhouse at Embrapa Agrobiologia, Brazil. Tissue samples were
composed of the roots (1/3 upper part) and the aerial part
(1/3 lower part). Aerial part samples (10 g) were surface
sterilized with chloramine T (1%) for 5 min (Döbereiner
et al., 1995) or only washed with tap water. Aerial part
samples (10 g) were surface sterilized with alcohol 70%. The
samples were macerated with a mortar and pestle in sterile
phosphate-buffered saline, and 10-fold serial dilutions were
made. Vials containing 5 mL of semi-solid, semi-selective
N-free JNFb medium commonly used for isolation of
Herbaspirillum spp. (Baldani et al., 1992) were used. It
contains (g L1): malic acid, 5; K2HPO4, 0.6; KH2PO4, 1.8;
MgSO4 7H2O, 0.2; NaCl, 0.1; CaCl2 2H2O, 0.02; KOH, 4.5;
and (mL L1), micronutrient solution, 2; bromothymol blue
solution (0.5% in KOH), 2; FeEDTA solution (solution
1.64%), 4; vitamin solution, 1; and pH adjusted to 5.8
(Döbereiner et al., 1995). The medium was supplemented
with root a extract obtained from the same rice varieties to
allow the recovery of different bacterial species colonizing
the plants, especially those that did not use malate as carbon
source. Appropriate serial dilutions (100 mL) of root and
aerial samples were inoculated at the bottom of this medium
and incubated at 30 1C for 10 days (Cavalcante &
Döbereiner, 1988; Baldani et al., 1992). Initially, white
pellicles were formed, which later become yellow with loss
of the blue colour of the JNFb medium. This characteristic
was considered a presumptively positive growth of Sphingomonas spp. The flasks that presented the characteristic
pellicle were used for isolation of the bacteria and purification on potato agar medium (Döbereiner et al., 1995;
Baldani et al., 2000). Yellow colonies appearing on the
S.S. Videira et al.
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Occurrence of diazotrophic Sphingomonas bacteria in Brazil
200 mM of each dNTP; 2 U of Taq DNA polymerase
(InvitrogenTM); and 0.5 pmol of each primer. The PCR
conditions, carried out in the PTC 100 thermocycler (MJ
Research), were as follows: initial denaturation for 2 min
at 94 1C, 30 cycles for 1 min at 94 1C, annealing for 1 min
at 56 1C, extension for 2 min at 72 1C, and final extension at
72 1C for 5 min. The products were separated by agarose gel
electrophoresis (1.2%) and stained with ethidium bromide
(0.5 mg mL1) for 30 min. Gels were visualized and photographed using the Kodak Gel Logic 100 System.
Amplified rDNA restriction analysis (ARDRA)
Ribosomal intergenic space analysis (RISA)
The 16S–23S rRNA intergenic space of the isolates was
amplified using the primers pHr (5 0 -TGC GGC TGG ATC
ACC TCC TT-3 0 ) (Massol-Deya et al., 1995) and p23Suni322-anti (5 0 -GGT TCT TTT CAC CTT TCC CTC-3 0 )
(Honeycut et al., 1995). The PCR mix reactions were
performed in a final volume of 50 mL containing: 50 ng of
template DNA, 1 PCR buffer, 1.25 Tween 20 2%, 2 mM
MgCl2, 200 mM of each dNTP, 2 U Taq DNA polymerase
(InvitrogenTM) and 0.12 pmol of each primer. The PCR
conditions, carried out in the PTC 100 thermocycler (MJ
Research), were as follows: initial denaturation for 2 min at
94 1C, 30 cycles of denaturation for 1 min at 94 1C, annealing
for 1 min at 60 1C; extension for 2 min at 72 1C, and final
extension at 72 1C for 5 min. The products were separated by
FEMS Microbiol Lett 293 (2009) 11–19
Data analysis of ARDRA and RISA patterns
Gel images were analysed using GELCOMPAR II software
(Applied Maths, Kortrijk, Belgium). Pairwise similarity
matrices were obtained using the Dice coefficient. Cluster
analysis of similarity matrices was performed by the
unweighted pair group method using arithmetic averages.
Sequencing and phylogeny of nif H and 16S rRNA
gene fragments
A 390-bp fragment of the nifH gene was amplified using the
universal degenerated pair of primers: 19f (5 0 -GCI WTY
TAY GGI AAR GGI GG-3 0 ) and 407r (5 0 -AAI CCR CCR CAI
ACI ACR TC-3 0 ) designed by Ueda et al. (1995). The PCR
mix reactions were performed in a final volume of 50 mL
containing: 50 ng of template DNA, 1 PCR buffer,
1.25 Tween 20 2%, 2 mM MgCl2, 200 mM of each dNTP,
2 U Taq DNA polymerase (InvitrogenTM) and 0.1 pmol of
each primer. The PCR conditions, carried out in the PTC
100 thermocycler (MJ Research), were as follows: initial
denaturation for 2 min at 94 1C, 35 cycles of denaturation
for 1 min at 94 1C, annealing for 1 min at 55 1C, extension
for 2 min at 72 1C, and final extension at 72 1C for 5 min.
The purified PCR products were sequenced directly using an
automatic sequencer MegaBACE 1000 (Amershan Biosystem) and the same set of primers.
Fragments of the 16S rRNA gene (c. 1500 bp) were
amplified from genomic DNA as described above. The
purified PCR products were sequenced directly using the
sequencing primers 27f (AGA GTT TGA TCC TGG CTC
AG) (Furushita et al., 2003); 108r (CAG ATT CCC ACG
CGT TAC GC) and 420r (TTA CAA CCC TAA GGC CTT C)
(Leys et al., 2004); Amp2 (AAG GAG GTG ATC CAR CCG
CA) (Wang et al., 1993); 16S1203f (GAG GTG GGG ATG
ACG TCA AGT CCT C); and 16S1110r (TGC GCT CGT
TGC GGG ACT TAA CC) (Soares-Ramos et al., 2003).
The sequences were compared with those from the
GenBank using the BLASTN program (Altschul et al., 1997)
and aligned using the CLUSTALW software (Thompson et al.,
1994); the distances were calculated according to Kimura’s
two-parameter model (Kimura, 1980). A phylogenetic tree
was inferred using the Neighbour-Joining method (Saitou &
Nei, 1987). Bootstrap analysis was based on 1000 resamplings (Felsenstein, 1985). The MEGA 4 software (Tamura
et al., 2007) was used for all analyses.
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The 16S rRNA gene of the isolates was amplified using the
universal pair of primers 27f (5 0 -AGA GTT TGA TCC TGG
CTC AG-3 0 ) (Furushita et al., 2003) and Amp2 (5 0 -AAG
GAG GTG ATC CAR CCG CA-3 0 ) (Wang et al., 1993). The
PCR mix reactions were performed in a final volume of
50 mL containing: 50 ng of template DNA, 1 PCR buffer,
1.25 Tween 20 2%, 2 mM MgCl2, 200 mM of each dNTP,
2 U Taq DNA polymerase (InvitrogenTM) and 0.1 pmol of
each primer. The PCR conditions, carried out in the PTC
100 thermocycler (MJ Research), were as follows: initial
denaturation for 2 min at 94 1C, 35 cycles of denaturation
for 45 s at 93 1C, annealing for 45 s at 62 1C, extension for
2 min at 72 1C, and final extension at 72 1C for 5 min.
Four restriction enzymes, HinfI, RsaI (Gibco BRLs),
MboI and AluI (InvitrogenTM), were used to digest the
amplified DNA fragments. The enzymes were selected based
on the analyses of 16S rRNA gene sequences of Sphingomonas
strains deposited in the GenBank and applying the PDRAW 32
software (http://www.acaclone.com). Five microlitres of the
PCR product was digested with 10 U of each enzyme
following the manufacturers’ protocols. Restriction fragments obtained after incubation for 3 h at 37 1C were
analysed by agarose gel electrophoresis (3%) in 1 TAE
buffer at 50 V for 3 h.
agarose gel electrophoresis (1.2%) and stained with ethidium bromide (0.5 mg mL1) for 30 min. MspI, AluI, HaeIII
(InvitrogenTM) and RsaI (Gibco BRL) were used to digest
the amplified DNA fragments. Eight microlitres of the PCR
product was digested with 5 U of each enzyme following
the manufacturers’ protocol. Restriction fragments were
resolved as described for ARDRA.
14
S.S. Videira et al.
Table 1. Sphingomonas isolates and reference strains used in the study
Isolates/reference strains
Rice variety
Soil sites
Plant source
SAG 62, BR12245, BR12246, SAG 96
SAG 67, BR12247, SAG 169, SAG 192, BR12200
BR12248, SAR 172, BR12258
SAR 73, BR12249, BR12255, BR12260
BR12250, SAR 77, BR12263, SAR 99, BR12256,
SAR 101, SAR 102, BR12259, SAR 248
SRG 81, SRG 82, BR12257, SRG 104, SRG 179,
SRG 180, SRG 195
BR12251, BR12262
BR12252, BR12253, SRG 106, SRG 184
BR12254, SRR 108, BR12264, SRR 110, BR12190, BR12261
SRR 95, BR12195
Sphingomonas azotifigens (NBRC 15497)
Sphingomonas trueperi (ATCC 12417)
Sphingomonas paucimobilis (ATCC 29837)
IAC4440
IAC4440
IAC4440
IAC4440
IAC4440
GO
GO
RJ
RJ
RJ
WR
SR
WR
SR
AP
IR42
GO
WR
IR42
IR42
IR42
IR42
GO
GO
RJ
RJ
SR
AP
WR
AP
Nucleotide sequence accession numbers
The nifH and 16S rRNA gene sequences from the 22
nitrogen-fixing Sphingomonas isolates were submitted to
GenBank and were assigned the following accession numbers: FJ455034–FJ455055 (nifH sequences) and DQ340853,
DQ340858, DQ340863 and FJ455056–FJ4550375 (16S
rRNA gene sequences). The specific accession number of
each isolate is indicated in Fig. 4 (nifH sequences) and Fig. 5
(16S rRNA sequences).
Results and discussion
Ecological and physiological aspects
A total of 46 isolates presenting characteristic yellow
colonies (Jenkins et al., 1979) were recovered from the IR42
and IAC4440 rice varieties grown in the GO and RJ soils
(Table 1). The colony morphology was highly similar among
the isolates grown on DYGS or Potato agar media (data not
shown). Comparison among the isolates present in Table 1
showed that the isolate number obtained from each rice
variety was quite similar. However, the number of isolates
that originated from washed roots was much higher than
those from the surface-sterilized roots, but did not differ
from the aerial part (Table 1). So far, the nitrogen-fixing
Sphingomonas bacteria have been isolated from paddy soil or
washed roots of rice plants (Oyaizu-Masuchi & Komagata,
1988), but not from the root interior and the aerial part, as
demonstrated here. In contrast, most of the pathogenic
Sphingomonas bacteria associated with plants have been
isolated from the surface or within the seeds (Mano et al.,
2006). A higher number of isolates were observed in plants
grown in soil originating from RJ (26) in contrast to those
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from GO soil (20). Independent of the number and origin of
the isolates, it is important to emphasize that both soil sites
were located at least 1000 km from each other, suggesting the
ubiquitous occurrence of this nitrogen-fixing bacterium
associated with rice in Brazil. Nitrogen-fixing Sphingomonaslike bacteria have been found to be associated with Palmaceae plants grown in the North region of Brazil (Fernandes
et al., 2001) and Drosera villosa growing in oligotrophic
habitats in Brazil (Albino et al., 2006), but no further studies
were carried out to confirm these results. More recently, a
nifH fragment similar to S. azotifigens was identified in a
library constructed with DNA obtained from the rhizosphere of sorghum cultivar IS 5322-C cultivated in Brazil
(Coelho et al., 2008).
Fifty percent of the isolates showed nitrogenase activity
when evaluated in the N-free semi-solid JNFb medium
(Table 1). Nitrogenase activities of the bacterial isolates
varied from 7 to 180 nmol ethylene h1 mg1 protein (data
not shown). This amount is comparable with the values
obtained for Azospirillum species (Eckert et al., 2001; Peng
et al., 2006). The number of the nitrogen-fixing isolates
originated from the GO soil was much higher than the RJ soil,
although they did not differ for the non-nitrogen-fixing
isolates. Similarly, there was no effect of rice variety on the
number of nitrogen and non-nitrogen-fixing isolates (Table 1).
Based on these results, only the isolates showing positive
nitrogenase activity were used for further characterization.
Comparison of the carbon source utilized by the isolates
with the Biolog database revealed that all 22 isolates
presented similarity 4 90% to the genus Sphingomonas.
All isolates utilized a-cyclodextrine, dextrine, glycogen,
Tween 40, Tween 80, N-acetyl-glucosamine, gentiobiose,
a-D-glucose, maltose, succinic acid, D-galactose, L-arabinose
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Isolates identified with boldface are non-nitrogen-fixing bacteria.
GO, Goiás; RJ, Rio de Janeiro; WR, washed root; SR, surface-sterilized root; AP, aerial part.
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Occurrence of diazotrophic Sphingomonas bacteria in Brazil
and cellobiose as carbon substrates, but not L-aspartate,
fructose and pyruvate. However, few variations did occur
among these isolates in utilizing some of the carbon sources
present in the Biolog Kit, suggesting metabolic diversity
among the isolates obtained from both rice varieties and soil
types.
Molecular analyses
Fig. 1. Agarose gel electrophoresis of Sphingomonas genus-specific
DNA fragments. Lines: 1 and 16, 1-kb DNA ladder plus (Invitrogen); 2,
negative control; 3, Sphingomonas paucimobilis ATCC 29837; 4, Sphingomonas trueperi ATCC 12417; 5, Sphingomonas azotifigens NBRC
15497; lines 6–10, nitrogen-fixing Sphingomonas isolates BR12245,
BR12249, BR12253, BR12195 and BR12200, respectively; 11–15, nonnitrogen-fixing Sphingomonas isolates SAG 96, SAR 172, SAR 100, SRR
108 and SRR 95, respectively.
Fig. 2. Unweighted pair group method using
arithmetic averages cluster analysis of combined
HinfI, AluI, RsaI and MboI restriction patterns
from amplified 16S rRNA gene of Brazilian
isolates and reference strains (see Table 1).
FEMS Microbiol Lett 293 (2009) 11–19
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A PCR amplification product of the expected size (350 bp)
was observed with the genus-specific primers described by
Leys et al., 2004, therefore confirming that all the isolates
characterized based on the colony morphology and Biolog
test belonged to the genus Sphingomonas (Fig. 1). A similar
product size was observed for the non-nitrogen-fixing
isolates, indicating that non-nitrogen-fixing Sphingomonas
also colonize the root and aerial part of rice plants as
observed previously (Kim et al., 1998; Takeuchi et al., 2001).
Restriction analyses of the 16S rRNA gene from 22
nitrogen-fixing isolates and reference strains showed the
formation of three clusters at 60% similarity (Fig. 2). Cluster
I was formed only by the non-nitrogen-fixing S. paucimobilis,
cluster II was formed by six isolates, together with S. trueperi
and S. azotifigens strains, while cluster III contained most
of the Brazilian rice isolates (Fig. 2). Among the
restriction enzymes HinfI, RsaI, MboI and AluI used to
determine the 16S rRNA genetic diversity of the isolates,
only the endonuclease MboI allowed to discriminate the
isolates. The other three restriction enzymes generated a low
number of polymorphic bands, mostly of the same size,
therefore suggesting that these enzymes are not suitable for
use in the genetic diversity study of this genus. On the other
hand, analyses of the 16S–23S rRNA intergenic space
indicated a higher degree of genetic diversity among the
isolates (Fig. 3). The dendrogram showed the formation of
five clusters at 60% similarity with the reference strains
forming clusters distant from the Brazilian isolates:
S. paucimobilis (cluster I) and S. trueperi and S. azotifigens
(cluster II). Cluster V included most of the isolates (17) with
three subclusters. Subclusters Va and Vc were formed by
isolates from both soil sites while the subcluster Vb was
formed only by isolates obtained from the RJ soil. Cluster III
was also formed by isolates originating from RJ soil in
contrast to cluster IV, which was formed by isolates originating from the GO. Therefore, apparently, there is an influence
of the soil type and to a lesser extent, of rice variety on the
diversity of the isolates. The influence of soil type on genetic
diversity has already been observed for the nitrogen-fixing
Azospirillum amazonense associated with rice plants grown
in two different soils (Azevedo et al., 2005). Reis Junior et al.
(2006) also observed an effect of the plant genotypes on the
genetic diversity of A. amazonense strains isolated from two
Brachiaria cultivars. In contrast, Rodrigues et al. (1986)
observed no effect of soil type or rice variety on the diversity
16
S.S. Videira et al.
Fig. 3. Unweighted pair group method using
arithmetic averages cluster analysis of combined
AluI, MspI, RsaI and HaeIII restriction patterns
from the amplified intergenic 16S–23S rRNA
gene space of Brazilian isolates and reference
strains (see Table 1).
of the nitrogen-fixing Herbaspirillum and Burkholderia
species isolated from these rice varieties grown at the same
soil sites. However, the analyses were based on colony
morphology and the ability of the isolates to use different
carbon sources.
The expected fragment (c. 390 bp) of the nifH gene was
obtained from all isolates showing positive nitrogenase
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activity and also from the A. amazonense strainY2 used as a
positive control. The only exception was the isolate
BR12257, which showed a nifH gene PCR product, but no
nitrogenase activity (data not shown). Other authors also
observed strains of different species showing the presence of
the nif genes, but with no nitrogenase activity (Achouak
et al., 1999; Berg et al., 2002; Ding et al., 2005).
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Fig. 4. Neighbour-Joining phylogenetic tree
based on partial nifH sequences of Brazilian
isolates, reference strains of Sphingomonas and
other nitrogen-fixing bacteria. The database
accession numbers are indicated after the
bacterial names. Bootstrap analyses were
performed with 1000 cycles. The dendrogram
was constructed using the MEGA4 program, and
the clustering was carried out using the
Neighbour-Joining method. Bootstrap values
4 50% are indicated at nodes. Scale Bar = 0.02
substitutions per nucleotide position.
17
Occurrence of diazotrophic Sphingomonas bacteria in Brazil
The phylogenetic analysis of the nifH nucleotide sequences
clustered the isolates into four different groups (Fig. 4). Most
of the nifH sequences obtained from rice Sphingomonas-like
isolates formed a distinct group with no close relation to the
reference sequences. Only the sequences of isolates BR12259
and BR12260 were closely related to S. azotifigens (Fig. 4). In
contrast, the phylogenetic tree based on the 16S rRNA gene
sequences showed that S. azotifigens and S. trueperi formed a
cluster together with a few isolates (Fig. 5). Interestingly, these
isolates (BR12195, BR12245, BR12263 and BR12246) also
formed a quite distant cluster in the nifH phylogenetic tree
(Fig. 4). The remaining isolates formed five clusters, confirming the high diversity among the Brazilian Sphingomonas
isolates observed by the nifH analysis. Previous works also
showed large nifH gene diversity among strains isolated from
sweet potato (Reiter et al., 2003), rice (Knauth et al., 2005) and
maize (Roesch et al., 2007). The ARDRA and RISA patterns
obtained, as well as the nifH and 16S rRNA gene phylogenetic
analyses, showed that the rice Sphingomonas isolates are quite
diverse and the nifH gene sequences were derived from
noncharacterized Sphingomonas sp.
FEMS Microbiol Lett 293 (2009) 11–19
In conclusion, this study confirms the occurrence of
nitrogen-fixing bacteria of the Sphingomonas genus in rice
plants grown in Brazil and shows a high degree of diversity
among the isolates that was influenced by the soil site, and to
a lesser extent, by the rice variety.
Acknowledgements
This work was partially supported by the projeto Instituto
do Milênio and CNPq. The authors also thank CNPq and
FAPERJ for the fellowships.
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Fig. 5. Neighbour-Joining phylogenetic tree
based on partial 16S rRNA gene sequences of
Brazilian isolates, reference strains of
Sphingomonas and other nitrogen-fixing
bacteria. The database accession numbers are
indicated after the bacterial names. Bootstrap
analyses were performed with 1000 cycles.
The dendrogram was constructed using the
MEGA4 program, and the clustering was carried
out using the Neighbour-Joining method.
Bootstrap values 4 50% are indicated at nodes.
Scale Bar = 0.02 substitutions per nucleotide
position.
18
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c
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