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Environmental Microbiology Reports (2013)
doi:10.1111/1758-2229.12122
Endophytic Bradyrhizobium spp. isolates from
sugarcane obtained through different
culture strategies
Luc Felicianus Marie Rouws,1* Jakson Leite,2
Gustavo Feitosa de Matos,2 Jerri Edson Zilli,1
Marcia Reed Rodrigues Coelho,1
Gustavo Ribeiro Xavier,1 Doreen Fischer,3
Anton Hartmann,4 Verônica Massena Reis1 and
José Ivo Baldani1
1
Embrapa Agrobiologia, BR 465 km 7, 23891-460,
Seropédica, Rio de Janeiro, Brazil.
2
Instituto de Agronomia, Departamento de Solos,
Universidade Federal Rural do Rio de Janeiro, BR 465
km 7, 23890-000, Seropédica, Rio de Janeiro, Brazil.
3
Department Environmental Genomics and
4
Department Microbe-Plant Interactions, Helmholtz
Zentrum München, German Research Center for
Environmental Health, Ingolstaedter Landstrasse 1,
85764 Neuherberg, Germany.
Summary
Brazilian sugarcane has been shown to obtain part
of its nitrogen via biological nitrogen fixation (BNF).
Recent reports, based on the culture independent
sequencing of bacterial nifH complementary DNA
(cDNA) from sugarcane tissues, have suggested
that members of the Bradyrhizobium genus could
play a role in sugarcane-associated BNF. Here we
report on the isolation of Bradyrhizobium spp. isolates and a few other species from roots of sugarcane cultivar RB867515 by two cultivation strategies:
direct isolation on culture media and capture of
Bradyrhizobium spp. using the promiscuous legume
Vigna unguiculata as trap-plant. Both strategies
permitted the isolation of genetically diverse
Bradyrhizobium spp. isolates, as concluded from
enterobacterial repetitive intergenic consensus polymerase chain reaction (PCR) fingerprinting and 16S
ribosomal RNA, nifH and nodC sequence analyses.
Several isolates presented nifH phylotypes highly
similar to nifH cDNA phylotypes detected in fieldgrown sugarcane by a culture-independent approach. Four isolates obtained by direct plate cultivation
Received 30 July, 2013; accepted 27 October, 2013. *For correspondence. E-mail [email protected]; Tel. + 55 21 3441-1606;
Fax + 55 21 2682-1230.
© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd
were unable to nodulate V. unguiculata and, based
on PCR analysis, lacked a nodC gene homologue.
Acetylene reduction assay showed in vitro nitrogenase activity for some Bradyrhizobium spp. isolates, suggesting that these bacteria do not require a
nodule environment for BNF. Therefore, this study
brings further evidence that Bradyrhizobium spp.
may play a role in sugarcane-associated BNF under
field conditions.
Introduction
Sugarcane production in Brazil traditionally occurs with
low nitrogen (N) fertilizer inputs, maintaining productivity.
This observation led scientists to study the nutrient
balance of the sugarcane culture, and 15N isotope-aided
studies in Brazil have indicated that sugarcane obtains at
least 40 kg N ha−1 year−1 from biological N fixation (BNF)
(Lima et al., 1987; Urquiaga et al., 1992; 2012). Accordingly, several sugarcane-associated diazotrophic bacteria
have been isolated from sugarcane, the first of which
were obtained from rhizosphere soil and belonged to the
genus Beijerinckia (Döbereiner, 1961).
In the 1970s, the group of Johanna Döbereiner
started using semi-solid N-free culture media to isolate
diazotrophic bacteria from non-leguminous plants. This is
usually done by inoculating serum vials with N-free
semi-solid media with a small volume of plant extract. In
these media, an oxygen (O2) concentration gradient is
formed and mobile diazotrophic bacteria, which are
present in the extract, can migrate to a zone of appropriate O2 concentration for BNF, forming characteristic
pellicles. Since their development, these media have
become standard for the isolation of diazotrophic bacteria
from non-legumes, enabling the isolation of several new
bacterial species (reviewed by Baldani and Baldani,
2005). Several diazotrophic bacteria have been isolated
from internal tissues of sugarcane, and these bacteria
have been classified as endophytes (Döbereiner, 1992).
The first sugarcane endophyte to be isolated was
Gluconacetobacter diazotrophicus (Cavalcante and
Döbereiner, 1988), which was found in roots and shoots.
Later, bacteria from genera such as Herbaspirillum
and Burkholderia were also identified as sugarcane
endophytes (James, 2000; Perin et al., 2006).
2
L. F. M. Rouws et al.
Studies have been conducted under greenhouse
and field conditions in order to explore the potential of
associative diazotrophic bacteria to increase growth of
sugarcane and resulted in the selection of a consortium
of five bacterial species (G. diazotrophicus, Herbaspirillum seropedicae, Herbaspirillum rubrisubalbicans,
Azospirillum amazonense and Burkholderia tropica)
(Oliveira et al., 2006). This mixed inoculant has also been
tested in large scale field experiments, where it led to
increased yields (Schultz et al., 2012). However, 15N
isotope dilution experiments showed that the growth promotion effect of the inoculant was not due to BNF because
non-inoculated plants showed similar 15N dilution levels.
Production of growth-promoting substances, such as
auxins, by the bacteria in the inoculants, which has been
demonstrated in vitro, may be the determinant responsible
factor for the growth promotion (Lee et al., 2004). Therefore, a question remains which bacterium or bacterial
consortium is responsible for sugarcane-associated BNF.
In an effort to get more insight into the natural N-fixing
bacterial community present in sugarcane plants, Ando
and colleagues (2005) evaluated nifH gene diversity in
sugarcane tissues and found phylotypes similar to the
genera Serratia, Klebsiella and Bradyrhizobium. More
recently, culture-independent studies have focused on
the presence and phylogenetic composition of sugarcaneassociated 16S ribosomal RNA (rRNA) and nifH
messenger RNAs (mRNAs). Tissues from sugarcane
plants grown in Latin America, Africa and Japan (Burbano
et al., 2011; Thaweenut et al., 2011; Fischer et al.,
2012) presented relatively high abundances of nifH
mRNA phylotypes related to rhizobia (including genera
Rhizobium and Bradyrhizobium). This suggests that
rhizobia can express nifH genes in association in sugarcane and thus, they may be of quantitative importance
during sugarcane-associated BNF. In accordance with
this observation, the isolation of (fast-growing) Rhizobium
spp. isolates from sugarcane was recently reported in the
south of Brazil (Beneduzi et al., 2013). However, isolates
of the slow-growing Bradyrhizobium genus have not been
described, and the data provided by molecular techniques
can serve here as a guide to isolation strategies (Bomar
et al., 2011).
All culture methods, including the N-free semi-solid
media used to isolate these diazotrophic bacteria, are
selective for certain groups of bacteria. For example, if
slow-growing and fast-growing diazotrophs are present in
the same inoculum that is introduced in a vial with semisolid medium, the fast-growing bacteria may outcompete
the slow-growing ones, preventing the isolation of the
latter (Davis et al., 2011). Also, certain diazotrophic bacteria might possibly require the presence of a host plant to
efficiently fix N, a requirement that is not met under traditional in vitro conditions, although the addition of sugar-
cane extract to culture media has been applied to mimic
endophytic nutritional conditions (Baldani and Baldani,
2005). These factors may explain why Bradyrhizobium
spp. have not been isolated from sugarcane.
In the present study, two alternative cultivation strategies were applied with the aim of isolating endophytic
Bradyrhizobium spp. from sugarcane. The first strategy
used direct inoculation of diluted sugarcane extracts on
plates containing yeast extract (YE) as the N source and
preferential carbon sources for Bradyrhizobium spp.
(referred to as ‘direct plate isolates’). The second strategy
relied on the use of the nodule-forming trap-plant Vigna
unguiculata that is considered promiscuous for nodulation with Bradyrhizobium spp. (referred to as ‘trap-plant
isolates’).
Results and discussion
Isolation of sugarcane-inhabiting bacteria that induce
nodules on V. unguiculata plants
Unlignified white shoot roots (Smith et al., 2005) and
shoot pieces (stem base) were obtained from 5-monthold sugarcane plants (cultivar RB867515) growing at the
Embrapa Agrobiologia experimental field station in
Seropédica, Brazil. After surface disinfection and placing
disinfected roots on yeast mannitol agar (YMA) plates, or
plating an aliquot of the water from the last washing step,
no bacterial growth was ever observed on YMA (Vincent,
1970), showing that the disinfection methodology was
effective.
A pilot experiment was conducted in a growth chamber
with pots containing sterile sand to select an effective
trap-plant species. Thirty days after inoculation of
Macroptilium atropurpureum (siratro) plants with sugarcane root extract, sporadic small white and apparently
inactive nodules were observed, whereas V. unguiculata
(cowpea) formed abundant, apparently active (reddish)
nodules (data not shown). In contrast, inoculation with
sugarcane shoot extract did not induce any nodules
on either plant species (data not shown). For being
the best performing plant species in this pilot experiment, V. unguiculata was selected as a trap-plant to
capture sugarcane root-inhabiting rhizobia under in vitro
conditions.
Fifteen V. unguiculata plants were individually grown in
glass tubes with slopes of N-free agarized Norris solution
and inoculated with 1 ml sugarcane root extract each.
After 3–4 weeks of incubation, nodules on 13 plants could
be observed. After surface disinfection of these nodules
and crushing them on YMA plates, a total of 112 bacterial
isolates was obtained. These trap-plant isolates received
names as follows: P followed by (number of V.
unguiculata plant) − (isolate number) (Fig. S1). The vast
© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology Reports
Bradyrhizobium spp. isolates from sugarcane
majority (109 isolates) grew slowly and alkalinized the
culture medium, characteristics typical for the genus
Bradyrhizobium (Kuykendall, 2005). Three isolates (P5–2,
P5–21 and P5–25) were fast growing and showed a
neutral/acidic reaction on YMA. Three non-inoculated
control plants, which were included as negative control,
did not form any nodules. Positive controls, inoculated
with the recently described V. unguiculata-nodulating
strain Microvirga vignae BR3299 (Radl et al., 2013),
nodulated abundantly (data not shown). These data
indicated that V. unguiculata nodule-inducing bacteria
occur as endophytes in roots of sugarcane cultivar RB
867515.
Enterobacterial repetitive intergenic consensus
polymerase chain reaction fingerprinting reveals genetic
diversity among nodule-inducing bacteria obtained
from sugarcane
In order to identify genotypic redundancy among the 112
trap-plant isolates, enterobacterial repetitive intergenic
consensus polymerase chain reaction (ERIC-PCR)
fingerprinting was applied (Fig. S1) (Woods et al., 1993).
This analysis revealed 34 distinct ERIC profiles (six from
Bradyrhizobium spp. type strains) when all isolates/
strains were grouped with a low level of correlation (28%
similarity). Some identical fingerprint profiles were
shared by up to 36 isolates, demonstrating that these
genotypes in the collection were redundant. These isolates sharing identical ERIC-PCR profiles were obtained
from nodules of the same plant, but also from nodules of
independent V. unguiculata plants inoculated with the
same aliquot of sugarcane root extract. Some ERICPCR profiles (13) represented fewer isolates, and
several profiles represented unique isolates in the collection (Fig. S1). The relative abundance of certain
ERIC-PCR profiles may reflect the abundance of the
respective bacterial genotype in the sugarcane roots, differences in colonization efficiency of the V. unguiculata
trap-plants by different bacterial genotypes, or differences in bacterial growth rate. Based on the ERIC-PCR
profiles, 23 representative isolates were selected for
more detailed analyses.
Bradyrhizobium isolates from sugarcane root extract by
direct plate cultivation
The use of trap-plants to obtain novel rhizobia has the
drawback that it may select certain groups of bacteria
that are effective nodule inducers on the chosen plant
species. Therefore, as a complementary isolation strategy, sugarcane root extracts were also directly inoculated on modified YMA plates, containing mannitol
(1 g l−1) or a mixture of sodium gluconate (0.5 g l−1) and
3
arabinose (0.5 g l−1) as carbon source (Tong and
Sadowsky, 1994), which permitted the isolation of 24
slow-growing and one fast-growing isolate. These direct
plate isolates received names formed by M (obtained on
mannitol plates) or AG (obtained on arabinose/gluconate
plates) followed by sequential numbers. Nine isolates
(eight slow-growing and one fast-growing) were selected
for further characterization.
Classification of isolates based on 16S rRNA gene
sequence analyses
The partial 16S rRNA gene sequence was determined
for 23 representative trap-plant isolates that were
selected based on the ERIC-PCR profiles. Partial 16S
rRNA gene sequences were also determined for eight
slow-growing direct plate isolates that were positive for
the nifH gene, as revealed by PCR, and for one fastgrowing direct plate isolate with colony morphology compatible to the genus Rhizobium. The dendrogram in
Fig. 1 shows the phylogenetic relationships among
these isolates and several reference strains from the
genera Bradyrhizobium, Rhizobium, Methylobacterium
and Herbaspirillum. These analyses showed that 22 of
the 23 trap-plant isolates and six out of nine direct plate
isolates belong to the genus Bradyrhizobium (Fig. 1;
Table 1). These isolates were grouped into the two
Bradyrhizobium super clades, known as the as Bradyrhizobium japonicum and Bradyrhizobium elkanii lineages (Aserse et al., 2012) (Fig. 1).
The Bradyrhizobium spp. direct plate isolates M21,
AG41, AG48 and M12 grouped in the B. japonicum
super clade and the direct plate isolates M3 and AG14
grouped in the B. elkanii super clade (Fig. 1). Apart from
Bradyrhizobium spp., one slow-growing plate isolate
(AG46) was most similar to the genus Methylobacterium,
and one (AG47) was classified as Herbaspirillum sp.
The fast-growing plate isolate M30 and the trap-plant
isolate P5-2 were similar to the genus Rhizobium (Fig. 1;
Table 1). Burbano and colleagues suggested an important role of Rhizobium rosettiformans in sugarcane rootassociated BNF in Africa and South America, based on
the detection of nifH transcript phylotypes related to this
bacterial species in sugarcane roots (Burbano et al.,
2011). Nevertheless, R. rosettiformans were not isolated
from sugarcane roots in the present study because
basic local alignment search tool (BLAST) analyses with
the 16S rRNA gene sequences of strains P5-2 and M30
showed only 95% identity to the R. rosettiformans 16S
rRNA gene (accession number EU781656).
Together, these data showed that both direct plate cultivation and the trap-plant strategy permit for the isolation
of diverse Bradyrhizobium spp. isolates from sugarcane
roots.
© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology Reports
4
L. F. M. Rouws et al.
P8-5 (KF113089)
P4-5 (KF113077)
P5-27 (KF113083)
AG14 (KF113098)
P10-5 (KF113094)
P10-6 (KF113095)
P7-17 (KF113086)
P15-7 (KF113097)
P5-5 (KF113081)
P5-26 (KF113082)
P7-40 (KF113088)
P8-11 (KF113090)
M3 (KF113103)
P5-32 (KF113084)
92% Bradyrhizobium pachyrhizi PAC48T (AY624135)
Bradyrhizobium elkanii USDA 76T (U35000)
90%
Bradyrhizobium jicamae PAC68T (AY624134)
100%
Bradyrhizobium lablabi CCBAU 23086T (GU433448)
Bradyrhizobium retamae Ro19T (KC247085)
Bradyrhizobium daqingense CCBAU 15774T (HQ231274)
Bradyrhizobium yuanmingense CCBAU 10071T (AF193818)
69% Bradyrhizobium liaoningense USDA 3622T (AF208513)
Bradyrhizobium canariense BTA-1T (AJ558025)
Bradyrhizobium japonicum USDA 6T (AB231927)
B. elkanii
superclade
Bradyrhizobium iriomotense LMG 24129T (AB300992)
P9-21 (KF113092)
P4-7 (KF113078)
P9-20 (KF113091)
P4-29 (KF113079)
P7-6 (KF113085)
Bradyrhizobium huanghuaihaiense CCBAU 23303T (HQ231463)
58%
Bradyrhizobium arachidis CCBAU 051107T (HM107167)
M21 (KF113105)
P1-6 (KF113075)
AG41 (KF113099)
P7-35 (KF113087)
P2-1 (KF113076)
52%
P9-22 (KF113093)
M12 (KF113104)
AG48 (KF113102)
P11-1 (KF113096)
Bradyrhizobium cytisi CTAW11T (EU561065)
71% Bradyrhizobium rifense CTAW71T (EU561074)
Bradyrhizobium betae LMG 21987T (AY372184)
83% Bradyrhizobium denitrificans LMG 8443T (X66025)
100%
Bradyrhizobium oligotrophica LMG 10732T (JQ619230)
Bradyrhizobium sp. STM 3843 (EU781679)
88%
Bradyrhizobium sp. STM 3844 (EU781680)
100%
96%
Bradyrhizobium sp. STM 3964 (EU781684)
100%
Methylobacterium oryzae CBMB20T (AY683045)
70%
Methylobacterium organophilum ATCC 27886T (AB175638)
99%
Methylobacterium nodulans ORS 2060T (AF220763)
100%
AG46 (KF113100)
Rhizobium miluonense CCBAU 41251T (EF061096)
P5-2 (KF113080)
Rhizobium tropici CIAT 889T (U89832)
100%
M30 (KF113106)
Rhizobium leguminosarum USDA 2370T (U29386)
100% Herbaspirillum_seropedicae Z67T (Y10146)
AG47 (KF113101)
99%
99%
4
B. japonicum
superclade
outgroup
0.10
Fig. 1. Phylogenetic relationships of partial 16S rRNA gene sequences of Bradyrhizobium spp. and some other isolates from sugarcane
cultivar RB867515. The evolutionary history was inferred using RAxML method using the GTRMIX rate distribution model. Bootstrap values
(expressed as percentages of 1000 replications) are shown at the branch. Evolutionary analyses were conducted in ARB (Ludwig et al., 2004).
Neighbour-joining analysis of the same dataset led to a highly similar tree topology. GenBank accession numbers are shown between
parentheses. See Appendix S1 for experimental details.
Functional and genetic nodulation traits of
sugarcane isolates
The individual capability to nodulate V. unguiculata plants
in vitro was checked for all trap-plant isolates and all direct
plate isolates in two independent experiments with three
replicates. All of the trap-plant isolates, except the
Rhizobium sp. isolate P5-2, were able to nodulate this
host plant (Table 1). Possibly, P5-2 represents a passenger that entered a V. unguiculata nodule formed by
© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology Reports
Bradyrhizobium spp. isolates from sugarcane
5
Table 1. Genetic and phenotypic symbiotic characteristics of selected bacterial isolates from roots of sugarcane cultivar RB867515f.
1st BLAST hit (16S rRNA)
GenBank
accession
number 1st
BLAST hit
nifH
PCRa
nodC
PCRb
Nodulationc
KF113075
KF113076
KF113077
KF113078
KF113079
KF113080
KF113081
KF113082
KF113083
KF113084
KF113085
KF113086
KF113087
KF113088
KF113089
KF113090
KF113091
KF113092
KF113093
KF113094
KF113095
KF113096
KF113097
Bradyrhizobium genosp. TUXTLAS-6 1020v
Bradyrhizobium genosp. TUXTLAS-6 1020v
Uncultured Bradyrhizobiumsp. clone
Bradyrhizobium japonicum SEMIA 6192
Bradyrhizobium japonicum SEMIA 6192
Rhizobium tropici BRUESC261
Bradyrhizobium elkanii USDA 4348
Bradyrhizobium elkanii USDA 4348
Uncultured Bradyrhizobium sp. clone
Bradyrhizobium elkanii USDA 4348
Bradyrhizobium sp. H10
Uncultured Bradyrhizobium sp. clone
Bradyrhizobium genosp. TUXTLAS-6 1020v
Bradyrhizobium sp. RST88bis
Bradyrhizobium elkanii USDA 4348
Uncultured Bradyrhizobium sp. clone
Bradyrhizobium japonicum SEMIA 6192
Bradyrhizobium japonicum SEMIA 6192
Bradyrhizobium genosp. TUXTLAS-6 1020v
Bradyrhizobium elkanii CCBAU 83387
Bradyrhizobium elkanii CCBAU 83387
Bradyrhizobium sp. RITF 1109
Bradyrhizobium elkanii CCBAU 83387
JF266658
JF266658
FJ193273
AY904772
AY904772
KF031510
JQ911631
JQ911631
FJ193273
JQ911631
AB601649
FJ193273
JF266658
FJ514041
JQ911631
FJ193273
AY904772
AY904772
JF266658
EF549397
EF549397
JQ697598
EF549397
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
–
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
–
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
KF113098
KF113099
KF113100
KF113101
KF113102
KF113103
KF113104
KF113105
KF113106
Bradyrhizobium elkanii CCBAU 53142
Bradyrhizobium sp. CCBAU 65749
Uncultured bact. clone YT-49
Herbaspirillum sp. sptzw31
Bradyrhizobium sp. DW3.1
Bradyrhizobium sp. RST88bis
Bradyrhizobium liaoningense z82b
Bradyrhizobium arachidis CCBAU 23155
Rhizobium miluonense CC-B-L1
EF394150
JF834148
JQ770097
GU377118
JQ349504
FJ514041
AB698736
GU433450
JN896360
+
faintd
+
faint
+
+
+
faint
NTe
+
–
+
–
–
+
–
–
NT
+
–
–
–
–
+
–
–
NT
Isolate
Nucleotides
sequenced
(16S rRNA)
GenBank
accession
number
Trap-plant isolates
P1–6
P2–1
P4–5
P4–7
P4–29
P5-2
P5–5
P5–26
P5–27
P5–32
P7–6
P7–17
P7–35
P7–40
P8–5
P8–11
P9–20
P9–21
P9–22
P10–5
P10–6
P11–1
P15–7
1345
1350
1350
1365
1356
1339
1344
1350
1355
1362
1325
1360
1358
1045
1349
1361
1360
1370
1361
1360
1359
1364
1354
Direct plate isolates
AG14
AG41
AG46
AG47
AG48
M3
M12
M21
M30
1354
1341
1351
984
1350
1359
1208
1084
1342
a. The plus sign indicates positive reaction with primer pair nifHF/nifHI.
b. The plus sign indicates positive reaction with primer pair NodCFor540/NodCRev1160.
c. The plus sign indicates induction of nodules in vitro on V. unguiculata with three replicates.
d. Faint PCR product obtained.
e. NT: not tested.
f. See Appendix S1 for experimental details.
another nodulating bacterial strain. To form a better understanding of their symbiotic characteristics, the strains
shown in Fig. 1 were submitted to PCR for the nodC gene
with primers NodCFor540 and NodCRev1160 (Sarita
et al., 2005), and the success of amplification was evaluated by gel electrophoresis. As shown in Table 1, all trapplant isolates, apart from P5-2, contain the nodC gene.
Among the direct plate isolates, only two (M3 and AG14)
were able to nodulate V. unguiculata, whereas the other
direct plate isolates identified as Bradyrhizobium spp.
(AG41, AG48, M12 and M21) were not. Accordingly, only
the Bradyrhizobium spp. isolates M3 and AG14 and the
Methylobacterium sp. isolate AG46 tested positive for the
nodC gene, although this latter isolate was not able to
nodulate V. unguiculata (Table 1). Figure 2 shows a
dendrogram representing the phylogenetic relationships
among the nodC gene sequences of the isolates. In
general terms, the nodC phylogenetic relationships are
congruent with the 16S rRNA phylogeny in that similar
clusters are formed for both genes, although there are
some exceptions as will be discussed later on (Figs 1
and 2).
Photosynthetic and also non-photosynthetic Bradyrhizobium spp. isolates lacking nod genes (strains BTAi1
and ORS278) have previously been shown to nodulate
dalbergioid legumes of the genus Aeschynomene (Giraud
et al., 2007; Miché et al., 2010; Okubo et al., 2012). Some
recently described Aeschynomene isolates present 16S
rRNA genes (accession numbers EU781684, EU781679
and EU781680) similar to several isolates obtained in the
present study (Fig. 1). Photosynthetic Bradyrhizobium
spp. isolates have also been isolated from African wild
© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology Reports
6
L. F. M. Rouws et al.
P7-17 (KF196787)
AG14 (KF196798)
P8-5 (KF196790)
99
P10-5 (KF196794)
P7-40 (KF196789)
50
P8-11 (KF196791)
100 P15-7 (KF196797)
86
M3 (KF196799)
Bradyrhizobium elkanii USDA 76T (AB354631)
P5-27 (KF196785)
100
100
P5-32 (KF196786)
Bradyrhizobium pachyrhizi PAC 48T (HM047128)
Bradyrhizobium arachidis CCBAU 051107T (HM107267)
87
P11-1 (KF196796)
Bradyrhizobium daqingense CCBAU 15774T (HQ231326)
Bradyrhizobium japonicum IAM 12608T (AB354632)
100
99
Bradyrhizobium huanghuaihaiense CCBAU 23303T (HQ231507)
94
Bradyrhizobium yuanmingense CCBAU 10071T (AB354633)
P7-35 (KF196788)
99
100 P9-22 (KF196793)
P4-7 (KF196783)
96
P4-29 (KF196784)
100
P9-20 (KF196792)
P10-6 (KF196795)
Bradyrhizobium canariense BTA-1T (AJ560653)
Bradyrhizobium cytisi CTAW11T (EU597844)
86
Bradyrhizobium rifense CTAW71T (EU597853)
Bradyrhizobium jicamae PAC 68T (AB573869)
Bradyrhizobium lablabi CCBAU 23086T (GU433565)
Bradyrhizobium retamae Ro19T (KC247112)
Bradyrhizobium iriomotense LMG 24129T (AB301000)
Bradyrhizobium liaoningense LMG 18230T (GU263466)
Burkholderia mimosarum PAS44T (EU386155)
91
88
93
99
49
21
45
100
75
100
0.05
Fig. 2. Phylogenetic relationships of partial nodC gene sequences (450 basepair positions) of Bradyrhizobium spp. isolates from sugarcane
cultivar RB867515. The evolutionary history was inferred using the neighbour-joining method (Saitou and Nei, 1987) based on the Kimura
two-parameter model (Kimura, 1980). Burkholderia mimosarum PAS44T was used as outgroup. Bootstrap values (expressed as percentages
of 1000 replications) are shown at the branch. Bar, 0.02 substitutions per nucleotide position. Evolutionary analyses were conducted in MEGA
5. GenBank accession numbers are shown between parentheses. See Appendix S1 for experimental details.
wetland rice (Oryza breviligulata), a species which,
like sugarcane, is a member of the Poaceae family
(Chaintreuil et al., 2000). The infection processes of
dalbergioid legumes by rhizobia and of Poaceae by
endophytic bacteria share the so-called crack-entry
infection mechanism, which is distinct from the wellcharacterized Nod factor-dependent infection mechanism
via root hairs (Okubo et al., 2012). However, when inoculated on in vitro grown Aeschynomene sensitiva plants,
none of the isolates M12, M21, AG48 or AG14 induced
any nodules, whereas consistent nodulation was observed at the stem-base of this plant species 3 weeks
after inoculation with strain BTAi1, which served as a
positive control (data not shown) (Giraud et al., 2007).
Partial nifH sequence characterization of
sugarcane isolates
Partial nifH gene sequences from the isolates were amplified with the primers nifHF and nifHI, and sequenced with
primer nifHI (Laguerre et al., 2001). Although sequencing
efforts of some faint nifH PCR products were not successful, phylogenetic analyses were conducted for the majority
of the isolates (Fig. 3; Table 1). The results of these analyses, which are presented in Fig. 3, showed the formation
of two major nifH clades, the largest of which contained all
nodule-inducing isolates described in this study. This
clade also accommodated one nifH complementary
DNA (cDNA) phylotype (accession number JF297300)
© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology Reports
Bradyrhizobium spp. isolates from sugarcane
7
P15-7 (KF113070)
M3 (KF113073)
P8-11 (KF113063)
99 P7-40 (KF113061)
P5-26 (KF113057)
79
P5-5 (KF113055)
P8-5 (KF113062)
P4-7(KF113053)
96
65
P7-17 (KF113059)
67
P4-5 (KF113052)
AG14 (KF113071)
Bradyrhizobium elkanii USDA 76T (AB094963)
76
P10-5 (KF113067)
98
79 41
Uncultured bacterium clone F8S 30 (JF297300)
P4-29 (KF113054)
P5-32 (KF113056)
96 P5-27 (KF113058)
38
P9-20 (KF113064)
Bradyrhizobium pachyrhizi PAC48T (HM047124)
P10-6 (KF113068)
59
Bradyrhizobium arachidis CCBAU 051107T (HM107283)
94
P11-1 (KF113069)
Bradyrhizobium yuanmingense CCBAU 10071T (EU818927)
67
P7-35 (KF113060)
74
100 P9-22 (KF113066)
Bradyrhizobium huanghuaihaiense CCBAU 23303T (HQ231551)
79
Bradyrhizobium liaoningense USDA 3622T (EU818925)
T
100 Bradyrhizobium daqingense CCBAU 15774 (HQ231323)
T
Bradyrhizobium japonicum USDA 6 (HM047126)
AG48 (KF113072)
Bradyrhizobium lablabi CCBAU 23086T (GU433546)
Bradyrhizobium cytisi CTAW11T (GU001618)
76
72
20
Bradyrhizobium rifense CTAW71T (U001627)
100
Bradyrhizobium canariense BTA-1T (EU818926)
Bradyrhizobium iriomotense LMG 24129T (AB300998)
20 30
Bradyrhizobium jicamae PAC 68T (HM047127)
M12 (KF113074)
11
Uncultured bacterium clone F8R 27 (JF297299)
35
Bradyrhizobium denitrificans LMG 8443T (HM047125)
83
Uncultured bacterium clone F13R 20 (JF297297)
53
98
59
Uncultured bacterium clone F7R 17 (JF297298)
Uncultured bacterium clone F4R 7 (JF297301)
Burkholderia mimosarum PAS44T (AY883420)
0.02
Fig. 3. Phylogenetic relationships of partial nifH genes sequences (314 bp) of Bradyrhizobium spp. isolates from sugarcane cultivar
RB867515. nifH cDNA phylotypes described by Fischer and colleagues (2012) were also included in the analysis. The evolutionary history
was inferred using the neighbour-joining method (Saitou and Nei, 1987) based on the Kimura two-parameter model (Kimura, 1980). Bootstrap
values (expressed as percentages of 1000 replications) are shown at the branch. Bar, 0.02 substitutions per nucleotide position. Evolutionary
analyses were conducted in MEGA 5. GenBank accession numbers are shown between parentheses. See Appendix S1 for experimental
details.
© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology Reports
8
L. F. M. Rouws et al.
obtained by culture-independent approach by Fischer and
colleagues (2012). The smaller clade contained the nifH
phylotypes of the non-nodulating Bradyrhizobium isolates
AG48 and M12, and also three nifH cDNA phylotypes
obtained from field-grown sugarcane tissues by Fischer
and colleagues (2012) in a culture-independent manner
(JF297299, JF297297, JF297298 and JF297301) (Fig. 3).
In order to permit the inclusion of the short nifH cDNA
phylotypes from Fischer and colleagues (2012), the nifH
analyses took into account only 314 base pair residues.
When these short sequences were omitted from the
analysis and the analysis was performed with 584 base
pair positions, the topology was almost identical (data not
shown).
For the majority of isolates, the phylogenetic relationships deduced from nifH gene sequences appeared to be
congruent with the relationships observed for the nodC
gene (Figs 2 and 3). However, there are some exceptions;
for example, the isolates P4–29, P9–20, P5–27 and
P5–32 form a cluster in the nifH dendrogram (Fig. 3),
although the isolates P4–29 and P9–20 on one side and
P5–27 and P5–32 on the other form distinct nodC clusters
(Fig. 2). Also, the nifH sequence of isolate P10-6 did not
cluster with any other sequence in the dataset, which was
also observed for the nodC gene of this isolate (Fig. 2).
Another interesting observation is that both the nodC and
the nifH gene from isolate P11-1 are highly similar to the
recently described species Bradyrhizobium arachidis
CCBAU 051107T (Wang et al., 2013), where this is not the
case for its 16S rRNA gene (Figs 1, 2 and 3). The lack of
congruence of phylogenetic relationships among 16S
rRNA and symbiotic genes has been reported on before
and is probably attributable to lateral gene transfer events
(Menna and Hungria, 2011). In general terms, the analyses conducted in the present study showed considerable
nodC and nifH sequence diversity in the Bradyrhizobium
spp. endophytic population from sugarcane roots.
In vitro nitrogenase activity
Because sugarcane does not form nodules, it is important
to know if the Bradyrhizobium spp. isolates from sugarcane are capable of fixing N outside of the nodule environment. To test this, the acetylene reduction assay (ARA)
was used, which permits a semi-quantitative analysis of
nitrogenase activity. Cell suspensions of some isolates
were inoculated in vials with modified semi-solid JMV
medium. Under these conditions, the bacteria can migrate
to a depth with adequate O2 concentration for BNF to
occur. After 5 days of growth, nitrogenase activity was
evaluated. None of the isolates presented any nitrogenase activity when no N source was provided, and so
we conducted ARA assays in YE-supplemented media
(40 or 400 mg l−1 YE) (Table 2). Apart from the positive
Table 2. Acetylene-dependent ethylene productiona in semi-solid
culture medium by Bradyrhizobium spp. isolates from roots of sugarcane cultivar RB867515.
Isolate
Mean
ethylene
production
(nmol)a
Uninoculated
Ppe8
M3
M12
AG48
AG14
P9–20
P9–21
P5–2
P8–11
P10–5
P11–1
P15–7
YE 40 mg l−1
≤ 0.01
≤ 0.01
40.2
± 23.0
≤ 0.01
≤ 0.01
3.5
± 2.0
3.1
± 1.7
≤ 0.01
≤ 0.01
5.9
± 3.3
ND
NDb
≤ 0.01
≤ 0.01
≤ 0.01
≤ 0.01
≤ 0.01
≤ 0.01
≤ 0.01
≤ 0.01
≤ 0.01
≤ 0.01
Standard
error
Mean
ethylene
production
(nmol)
Standard
error
YE 400 mg l−1
≤ 0.01
≤ 0.01
128.8
± 74.0
≤ 0.01
≤ 0.01
7.0
± 4.0
51.6
± 29.8
≤ 0.01
≤ 0.01
17.7
± 10.2
3.5
± 2.0
≤ 0.01
≤ 0.01
≤ 0.01
≤ 0.01
≤ 0.01
≤ 0.01
≤ 0.01
≤ 0.01
≤ 0.01
≤ 0.01
a. See Appendix S1 for detailed experimental procedures.
b. ND, not determined.
control, B. tropica Ppe8 (Reis et al., 2004), the direct plate
isolates M12 and AG48 and the trap-plant isolates P9–20
and P9–21 showed significant AR activity (Table 2).
However, the majority of the trap-plant isolates and the
V. unguiculata-nodulating isolates M3 and AG14 did not
show any AR activity. Also, no ethylene was ever formed
when no acetylene was provided (data not shown).
These data demonstrate that at least some of these
Bradyrhizobium spp. isolates may present nitrogenase
activity outside the nodule environment. Several studies
carried out in the 1970s and 1980s showed that some
Bradyrhizobium spp. strains, such as strain 32H1, are
able of fixing N under in vitro conditions, while others are
not and that this capability depends on the carbon and/or
N source provided (Kurz and LaRue, 1975; Pagan et al.,
1975; Kaneshiro and Kurtzman, 1982). Altogether, some
sugarcane endophytic Bradyrhizobium spp. apparently
have the capability to fix N outside the nodule environment, a trait required for sugarcane-associated BNF.
Concluding remarks
The data presented here showed, by using two different
isolation strategies, that Bradyrhizobium spp. occur as
endophytes in roots of sugarcane cultivar RB867515 in
Seropédica, Brazil. It was also shown that the application
of differentiated isolation strategies (direct plate cultivation and trap-plant mediated cultivation) permits the cultivation of diverse novel diazotrophic bacteria from
sugarcane, especially Bradyrhizobium spp. This study
also emphasized that growth rate is an important feature
to be taken into account in efforts to cultivate novel bacterial groups. Although the ecological relevance of the
© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology Reports
Bradyrhizobium spp. isolates from sugarcane
presence of Bradyrhizobium spp. remains to be determined, the fact that some of these isolates presented
nitrogenase activity in vitro suggests that these bacteria
might possibly fix N in association to sugarcane plants.
Acknowledgements
The authors thank CNPq (Instituto Nacional de Ciência e
Tecnologia FBN and process 477231/2012-8) and Faperj
(process E-12/111–398/2012) for financial support. Bruno
José Rodrigues Alves and Robert Michael Boddey are
acknowledged for their support with the gas chromatography
experiments.
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Supporting information
Additional Supporting Information may be found in the online
version of this article at the publisher’s web-site:
Fig. S1. Genetic similarity (UPGMA) tree of ERIC-PCR patterns from Bradyrhizobium spp. type strains and 112 bacterial
isolates obtained from nodules of V. unguiculata plants inoculated with root extract of sugarcane cultivar RB867515. Boldfaced isolates indicated with dots were selected for more
detailed analyses. See Appendix S1 for experimental details.
Appendix S1. Detailed experimental procedures.
© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology Reports