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REGULAR ARTICLE
Diazotrophic diversity, nifH gene expression and nitrogenase
activity in a rice paddy field in Fujian, China
Lotta Mårtensson & Beatriz Díez &
Ingvild Wartiainen & Weiwen Zheng &
Rehab El-Shehawy & Ulla Rasmussen
Received: 21 October 2008 / Accepted: 17 March 2009
#
Springer Science + Business Media B.V. 2009
Abstract The diazotrophic communities in a rice paddy
field were characterized by a molecular polyphasic
approach including DNA/RNA-DGGE fingerprinting,
real time RT-PCR analysis of nifH gene and the
measurement of nitrogen fixation activities. The inves-
tigation was performed on a diurnal cycle and compar-
isons were made between bulk and rhizosphere / root
soil as well as between fertilized / unfertilized soils.
Real time RT-PCR showed no significant difference in
the total quantity of nifH expression under the
conditions investigated. The functional diversity and
dynamics of the nifH gene expressing diazotroph
community investigated using RT-PCR-DGGE
revealed high diurnal variations, as well as variation
between different soil types. Most of the sequence
types recovered from the DGGE gels and clone
libraries clustered within nifH Cluster I and III (65
different nifH sequences in total). Sequence types most
similar to Azoar cu s spp., Metylococcus spp., Rhizobium
spp., Methylocystis spp., Desulfovibrio spp., Geobacter
spp., Chlorobium spp., were abundant and indicate that
these species may be responsible for the observed
diurnal variation in the diazotrophic community struc-
ture in these rice field samples. Previously described
diazotrophic cyanobacterial genera in rice fields, such
as Nostoc and Cyanothece, were present in the samples
but not detectable in RT-PCR assays.
Keywords Acetylene reduction assay
.
Microbial
diversity
.
nifH gene activity
.
Real time RT-PCR
.
Rice
paddy
.
RT-PCR-DGGE
Introduction
Intense research has been performed for many years on
the application and utilization of diazotrophs as bio-
fertilizers for rice production (Kennedy et al. 2004)
and, although their positive effect on rice growth and
productivity is well documented, we are far from a
complete understanding and efficient exploitation of
diazotrophic organisms as a natur al nitrogen source.
Several N
2
fixing microorganisms have been isolated
from rice fields (Park et al. 2005; Vaishampayan et al.
2001; Xie et al. 2003) and strains of Azotobacter,
Clostridium, Azospirillum, Herbaspirillum, Burkhol-
deria and Azoarcus, as well as cyanobacteria have,
Plant Soil
DOI 10.1007/s11104-009-9970-8
Responsible Editor: Euan K. James.
L. Mårtensson
:
B. Díez
:
I. Wartiainen
:
R. El-Shehawy
:
U. Rasmussen (*)
Department of Botany, Stockholm University,
SE-106 91 Stockholm, Sweden
e-mail: ulla.rasmussen@botan.su.se
W. Zheng
Biotechnology Center,
Fujian Academy of Agricultural Sciences,
Fuzhou, Fujian 350003, Peoples Republic of China
with positive results, been tested and found suitable
for use as bio-fertilizers (Choudhury and Kennedy
2004). The identification of diazotrophs in rice paddy
soil has so far, mainly been performed using
cultivation-based analysis followed by morphological-
and/or molecular identification of individual isolates
(Khan et al. 1994; Xie and Yokota 2005). However,
only a limited number of microorganisms can be
recovered from the soil by traditional cultivation-
based techniques, resulting in underestimation of the
amount and the significance of diazotrophs present in
different soils (Garcia-Pichel et al. 2001;Smitetal.
2001).
In analysis of the indigenous diazotophic commu-
nity in diverse environments, the nifH gene, encoding
the dinitrogenase reductase part of the nitrogenase
complex, is a commonly used marker for studying the
diazotrophic assemblage and gene activity in different
ecosystems (Zehr et al. 2003). Polymerase chain
reaction (PCR) of the nifH gene combined with
cloning and sequencing or denaturing gradient gel
electrophoresis (DGGE) and sequencing gives infor-
mation on the diazotrophic composition in an envi-
ronment. Numerous PCR primers targeting the nifH
genes have been designed with different range
specificity, from “universal ” covering different diaz-
otropic taxa (Demba Diallo et al. 2008; Poly et al.
2001; Zehr et al. 1997 ; Wu et al. 2009), to genus and
group specific (Huang et al. 1999; Olson et al. 1998).
Recently, quantitative PCR (qPCR) has become a
frequently used method to quantify specific genes or
microbial groups in different environmental habitats
(Lim et al. 2008; Novinscak et al. 2007 ). How ever,
few studies have previously been performed on soil
where qPCR is combined with reverse trans criptase
(RT) PCR, enabling in vitro quantification of specific
gene expression (Cook and Britt 2007; Jacobsen and
Holben 2007) and qRT-PCR applied to the field
emerge as a powerful tool for quantification of an
active N
2
-fixing population within a complex com-
munity (Church et al. 2005) or to monitor diurnal
gene expression (El-Shehawy et al. 2003; Zehr et al.
2007).
In the present study, we combined DGGE and
cloning with real time RT-PCR and acetyl ene reduc-
tion activity (ARA) to identify the active diazotrophic
community composition and to quantify nifH expres-
sion in rice paddy soil samples collected at a diurnal
cycle.
Materials and methods
Soil characteristics and soil sampling
Soil samples were collected from two rice paddy
fields each approximately 325 m
2
in size, located in
Yongtai (25°39 ’N; 119°12’E), Fujian province,
South-East China. Since November 2003 one field
was fertilized with N, P, K and urea, according to
local custom and with two rice crops annually. The
other field was fertilized only with P and K (referred
to as F (fertilized) and UF (unfertilized) respectively).
Fertilizer was added to the field two days before
transplanting and 10, 45 and 75 days respectively
after transplanting. Soil samples analyzed in this
study were collected in May 2005, at which time the
rice was in the active tillering phase. Composite soil
samples were collected according to Nakatsu et al.
(2000) and Smalla et al. (2001). The samples were
collected in triplicate from bulk soil (B) (0–1cm
depth) three times at 15:00, 20:00 and 04:00 in
fertilized (F) and unfertilized (UF) soil. Concomitant-
ly, rhizosphere/root samp les (RH) were collected from
18 individua l rice plants (three from each field at each
sampling time). Excess soil (not in contact with roots)
was manually removed and the roots and soil from
0–5 cm depth (of each plant) were sampled individ-
ually and mixed later during DNA/RNA extraction. In
the field all samples were immediately submerged in
liquid nitrogen and transported to the laboratory
where they were kept in −80°C until further pro-
cessed. In July 2004 and May 2005, bulk soil was
collected and analysed for chemical composition of
the soil at the Soil and Fertilizer Institute of Fujian
(Table 1 ). Temperature and light intensity at the
sampling times were measured (Table 2).
RNA/DNA extraction and purification
Equal amounts of each of the three replicates from the
rhizosphere/root samples were mixed in the laborato-
ry, while the triplicates of the bulk soil were mixed
directly in the field. Two grams from each mixed
sample was used for crude RNA/DNA extraction
according to the method described by Hurt et al.
(2001). RNA was purified from crude RNA/DNA by
the QIAGEN RNA/DNA mini-kit (Qiagen, Hilden,
Germany) when used for RT-PCR-DGGE analysis,
and further purified using the FastRNA Pro Soil
Plant Soil
Direct Kit (Promega) prior to qRT-PCR analysis.
Additionally, all RNA samples were DNase treated
(Qiagen) and tested for DNA contamination by PCR/
qPCR. The extracted DNA and RNA were stored at
−80°C until further analysis.
Primer selection
A variety of nifH PCR primers had previously been
tested and optimized using DNA extracted from
laboratory cultures of cyanobacteria, methanotrophs
and rhizo bia as well as environmental DNA from
paddy soil (Wartiainen et al. 2008), and in this study
RT-PCR-DGGE and quantitative nifH gene RT-PCR
was performed with the primers PolF/PolR (Poly et al.
2001) and PolFI/AQER-GC30 (Wartiainen et al.
2008) as described in the following paragraphs.
Quantitative nifH gene-RT-PCR (qRT-PCR)
cDNA was synthesized from 200 ng RNA using the
iScript cDNA synthesis Kit (B ioRad, Hercules, CA,
USA), accordi ng to the manufacturer’s instructions,
and stored at −20°C until further processed. qPCR
protocols were optimized for PCR efficiency using
the universal diazotrophic primer pairs PolF and PolR
(Poly et al. 2001). The optimal conditions for qPCR
were found with 20 ng cDNA as template 59°C
annealing temperature and 40 cycles using iQ SYBR
®
Green qPCR Kit (Bio-Rad, Hercules, CA, USA) and
according to the manufac turer’s instructio n. The
quantitative PCR was carried out on a BioRad iCycler
(Bio-Rad, Hercules, CA, USA). Following each run, a
melt curve analysis step was performed to verify that
primer-dimers were absent. To enable agarose gel
electrophoresis, a 7 min final elongation step at 72°C
was added at the end of the melt curve.
The relative cDNA quantities of nifH, and hence
mRNA, were determined using serial dilutions of DNA
(25 ng/µl) from Anabaena strain PCC 7120 as an
external standard. Strong linear correlations (correla-
tion coefficient (r
2
)1.000)weremaintainedbetween
log values of template DNA and qPCR threshold
cycles over the range of DNA concentrations exam-
ined. The efficiency of the real time PCR reactions
were 86.8%±0.25, and the specificity of the reactions
was confirmed b y gel electrophoresis where all
samples produced the predicted 361 bp PCR fragment
(data not shown). Finally, water was used instead of
templates as a negative control to exclude that primers
and PCR buffers used in this study were contaminated
with nifH genes, as indicated by a study on commercial
PCR primers and polymerases (Goto et al. 2005).
Samples were analysed in three separate runs, with two
replicates of each sample in each run.
Table 2 Temperature and light intensity at the sampling times
Time
15:00 20:00 04:00
Temperature (°C)
Air 34.0 25.0 24.0
Water 34.0 28.4 25.0
Water surface 32.5 28.2 25.0
Light intensity (lux)
Air 21600 0 0
Water surface 14000 0 0
Organic matter (%) Total (%) Available Exchangeable
(mg/kg) (mg/kg)
Time and soil type N P K N P K Ca Mg
July 2004
a
Unfertilized 3.61 0.21 0.03 1.47 299 5.2 37 674 68.4
Fertilized 3.56 0.23 0.03 1.32 549 6.6 39 742 80.8
May 2005
b
Unfertilized 2.78 0.18 0.03 1.91 184 10.8 41.5 442 81.1
Fertilized 3.03 0.19 0.04 1.66 228 26.5 83.6 445 83.0
Table 1 Chemical and trace
element composition in
the soil
a
Between the first and
second growth season
b
Middle of first (spring)
growth season
Plant Soil
Diazotrophic nifH RT-PCR-DGGE
A two-step RT-PCR protocol for nifH PCR amplifi-
cations and DGGE fingerprinting analysis were
performed as previously described by Wartiainen et
al. (2008). The RT reaction was set up according to
the manufacturer’s protocol (Eppendorf, Germany),
with a reaction temperature of 42ºC for 30 minutes.
The cDNA was stored at −20°C until further handling.
A direct PCR approach with GC-clamp primers gave
no products using the cDNA as template; therefore a
nested PCR protocol was performed on the cDNA
samples. In the first PCR reaction, a 370 base pair
fragment of the nifH gene was amplified using the
PCR primers PolF and PolR (Poly et al. 2001). The
second PCR was perfor med with PCR primers PolFI/
AQER-GC30 and 1µl of the PCR products from first
PCR as template. The PCR cond itions we re as
previously described (Wartiainen et al. 2008). PCR
amplif icatio ns were repeated three times and the
different reactions were analyzed separately by
denaturing gradient gel electrophoresis as described
in Wartiainen et al. (2008). All possible DGGE bands
were excised from the gels and submerged in 20μl
DNAase/RNAase free H
2
O (ultraPURE, Gibco) and
stored at 4°C over-night. 1μl from each excised band
were reamplified using the same conditions as above
and further tested by DGGE, for verification of band
migration patterns. Bands with correct migrations
patterns were further amplified and sequenced.
10 ng of each template and 1.6µM primers were
mixed and the samples were sequenced on an ABI
3130XL system (Applied Biosystems, Warrington,
UK). The number/presence of DGGE bands were
estimated using the QuantityOne software (Bio-Rad,
Hercules, CA, USA).
Cyanobacteria specific nifH PCR-DGGE
Direct amplification of RNA with cyanobacte rial
specific PCR primers was not successful, therefore
DGGE on DNA amplified with the cyanobacterial
specific CNF (with a 40 bp GC clamp at the 5’ end
(Nübel et al. 1997)) and CNR primers (Olson et al.
1998) were performed as previously described (Díez
et al. 2007). 1µl of DNA was used as templat e in 50
µl reactions. The PCR conditions and thermal cycling
used were as described above: except that the
annealing temperature used was 50°C. All samples
were amplified in duplicates, pooled and concentrated
to 25µl through evaporation at 37ºC using increased
air flow in the sample tubes prior to DGGE analysis.
DGGE was performed as described above but on a
6% (w/v) polyacrylamide gel submerged in 1xTAE
buffer at 60°C with a linear 50 to 70% denaturant
gradient and stained in SYBRGold prior to illumina-
tion. An aliquot of the eluted DNA was subjected to
an additional PCR amplification using the same
primers and sequenced on an ABI 3130XL system
(Applied Biosystems, Warrington, UK). The number/
presence of DGGE bands were estimated using the
QuantityOne software (Bio-Rad, Hercules, CA,
USA).
Diazotrophic nifH clone libraries
Clone libraries were constructe d from cDNA samples
UFB04:00 and FB04:00. The PCR reaction was
performed using the PolF/PolR primers (see above).
Four PCR products from each sample were pooled and
concentrated by ethanol precipitation prior to ligation
into the prepared vector (pCR 2.1) supplied with a
TOPO TA cloning kit (Invitrogen) following the
manufacturer’s recommendations. Dou ble-stranded
plasmid DNA from selected clones (with correct insert
size verified by PCR) was extracted using a QIAprep
Spin miniprep kit (Qiagen, Halden, Germany) and
sequencedonanABI3130XLsystem(Applied
Biosystems, Warrington, UK).
Phylogenetic reconstructions
A consensus of each forward and reverse sequence
pair was created using Pregap 4 and Gap 4 in the
Staden package under windows. Partial nifH gene
sequences obtained from excised DGGE bands and
the RNA clone libraries were aligned in Bioedit
version 7.0.4.1 (http://www.mbio.ncsu.edu/bioedit/
bioedit.html) using ClustalW. All sequences were
subjected to BLASTN searches (www.ncbi.nlm.nih.
gov/blast) (Altschul et al. 1997) and the closest
relatives from GeneBank were included for phyloge-
netic analysis. Only sequences from published studies
or culture collections were included. The neighbor-
joining method and Kimura two-parameter (K2P)
were used in PAUP (version 4.0b10, Sinauer Asso-
ciates Inc., Sunderland, MA) to estimate phylogenetic
reconstructions. A total of 1000 bootstrap replicates
Plant Soil
were perfor med. The nifH gene sequences of several
archaeal sequences were used as outgroup.
The sequences generated in this study have been
deposited in the EMBL Nucleotide Sequence Database
under accession numbers: AM946232-AM946263
(nifH-RT-PCR-DGGE bands A1-A32 ), AM946264-
AM946280 (nifH-PCR-DGGE bands B1-B16), and
AM946281-AM946313 (nifH- RT-PCR clones).
Acetylene Reduction Assay (ARA)
Nitrogen fixation activity was measured using the
acetylene reduction assay (ARA) (Hardy et al. 1968).
Soil was sampled in triplicates from fertilized and
unfertilized bulk- and rhizosphere soil (at 15:00,
20:00 and 04:00) and incubated individually with
10% acetylene for three hours. The assay included
negative controls using dH
2
O incubated with 10%
acetylene and rhizosphere soil sampled at 20:00
incubated without acetylene for three hours (both
negative controls in three replicates). The measure-
ments were done at Fujian Institute of Testing
Technology, Fuzhou, China, using a gas chromato-
graph (Agilent Teqnologies 6890 N Network GC
System) equipped with a GDX502 column and a
flame ionisation detector.
Statistic analysis of the data
Two-factor ANOVA was performed to detect signif-
icant differences (P<0.05) among data obtained from
qRT-PCR and ARA.
Results
Soil characteristics
The soil texture was analysed in April and July 2004,
and the soil was c haracterised as a silt loam (results
not shown). The chemical analysis revealed small
differences in total amount of nitrogen, phosphorus
and potassium (NPK) betw een sampling times and
soil types (Table 1). The amount of available N in the
fertilized soil was reduced from 549 mg/kg in July
2004 to 228 mg/ kg in May 2005. At the same time
the amount of available N in unfertilized soil
decreased from 299 mg/kg to 184 mg/kg. The amount
of available P and K increased whereas the trace
elements Ca and M g were no t affected by the
different management regimes (Table 1).
Quantitative nifH gene expression and N
2
-fixation
activity
Results from the real time RT-PCR analysis
revealed no significant differences between the
samples (Fig. 1a) (P<0 .05), indicating that the
community of the N
2
-fixing bacteria is stable in
quantity in both fertilized and unfertilized bulk and
rhizosphere/root soil and is not affected either by the
type of soil or by the day/night cycle. Fig. 1a
represents a reproducible pattern obtained from the
real time PCR analysis. This result was further
confirmed by measuring the total N
2
-fixation activity
in the samples by the ARA. Also, the ARA activities
showed no significant difference between samples
(P<0.05) (Fig. 1b).
Functional diversity and distribution of the active
diazotrophic communities along a diurnal cycle
The RT-PCR-DGGE profiles of the nifH expressing
communities from all samples are shown in Fig. 2a.
High functional diversity was observed at different
sampling times within both fertilized and unfertilized
soil, as well as between rhizosphere/root and bulk
soil.
Phylogenetic rec ons tructions of the excised nifH
RT-PCR-DGGE bands (Fig. 2a) revealed similarities
to phylotypes with in nifH clusters I and III (Fig. 3).
Moreover, all the sequences obtained from the RNA
clone libraries were also affiliated within the same
clusters. From a total of 32 RT-PCR-DGGE bands
and 33 cDNA clones (20 clones from unfertilized
bulk (04:00) and 13 clones from fertilized bulk
(04:00)), 22 RT-PCR-DGGE bands and 20 cDNA
clones belo ng ed t o nifH Cluster I (Fig. 3). Among
those, 18 RT-PCR-DGGE bands and 19 cDNA
clones were more closely related to different mem-
bers of the proteobacteria family, from which 7 RT-
PCR-DGGE bands and at least 3 cDNA clones were
most simila r t o m em be rs of β-proteobacteria, such as
Azoarcus spp., Azospira oriza, Azotobacter spp., and
Ideonella sp .. Four RT-PCR-DGGE bands and 4
cDNA clones were most similar to γ-proteobacteria
such as Methylococcus, and 3 RT-PCR-DGGE bands
and 4 cDNA clones most similar to rhizobial α-
Plant Soil
proteobacteria like Rhizobium or Methylocystis
(Fig. 3). The remaining phylotypes were related to
the heterogeneous clade inclu din g Firmicutes like
Heliobacterium, δ-proteobacteria like Geobacter and
Actinobacteria like Frankia. In addition, 10 RT-
PCR-DGGE bands an d 10 cDNA clone s were
closely related to the nifH Cluster III that includes
nifH sequences from diverse anaerobic bacteria such
as Clostridia (low G+C, gram positive), sulfate
reducers such as Desulfobacter and Desulfovibrio
(δ-proteobacteria) and the green sulfur-oxidizing
bacterium Chlorobium (Chlorobia) (Fig. 3).
In general, the distri bution of phylotypes between
different soil types, or at d ifferent times within the
soil types, appears to be indiscriminate. Twenty one
of the 32 phylotypes were recovered from more than
one DGGE profile (soil sample) (Fig. 2c). Many
Firmicutes an d δ-proteobacteria phylotypes from
cluster I and III of nifH gene were present both in
fertilized and unfertilized samples (Fig. 2a, c; Fig. 3).
None of the phylotypes recovered from DGGE bands
or clone lib raries were related to cyanobacteria.
Furthermore, many of the sequences recovered from
our DGGE bands and clone libraries showed simi-
larities less than 92% when compared to the closest
related sequences present in the NCBI GenBank
database.
Diversity and distribution of the cyanobacterial
community along a diurnal cycle
DNA-based PCR-DGGE was performed using
cyanobacteria specific nifH primers (CNF/CNR
(Olson et al. 1998)). The result sh owed no major
differences in the DNA-PCR-DGGE profile between
the individual samples (Fig. 2b). The phylo ty pes
identified were closely related to the heterocystous
filamentous cyanobacteria genera Nostoc an d Ana-
baena (DGGE bands B1, B 6, B11, Fig. 2b), to the
non-heterocystous unicellular genus Cyanothece
(DGGE bands B5 and B9, Fig. 2b), which were
relatively abundant in those s amples (Fig. 2d), and to
Dermocarpa sp. (DGGE band B10, Fig. 2b) . The
Cyanothec e was also the most represented genus
identified by 16S r RNA-DGGE in the same samples
(data not shown). In addition, phylotypes closely
Fig. 1 nifH gene expression
and activity along a diurnal
cycle a Starting quantities
(SQ) of nifH mRNA per
reaction visualized as mean
of duplicates with standard
deviations b Nitrogenase
activity per gram
(dry weight) and hour,
measured using the
acetylene reduction assay
(ARA), visualized as mean
of triplicates with standard
deviations. Abbreviations:
Fertilized bulk (FB),
fertilized rhizosphere/roots
(FRH), unfertilized bulk
(UFB), unfertilized
rhizosphere/roots (UFRH)
Plant Soil
related to members of the Firmicutes from Cluster I
(DGGE bands B12 and B13, Fig. 2b) as well as
members of Cluster III (DGGE bands B3, B4, B7,
B8,B14,B15,Fig.2b) and one possibly related to
Cluster II (DGGE band B16, Fig. 2b) w ere identified
(Fig. 3) . Many of thos e phylotype s shared less than
90% similarity with sequences present in the NCBI
GenBank d atab ase .
Fig. 2 nifH-DGGE
fingerprinting profiles on
the PCR amplified products
of RNA/DNA samples
extracted from rice paddy
along a diurnal cycle a
cDNA amplified in a
semi-nested approach using
general diazotroph nifH
primers PolF/PolR (Poly
et al. 2001) and PolFI/
AQER-GC30 (Wartiainen
et al. 2008) b DNA
amplified using
cyanobacteria specific nifH
primers CNF/CNR (Olson
et al. 1998). For clarification
the first four lanes have
been moved from the right
part of the gel using Adobe
Illustrator. Marks and
numbers indicate the
excised bands from which
sequences were determined.
c–d) Binary matrix indicat-
ing presence (black boxes)
or absence (empty boxes) of
bands in: c the PolF/PolR
DGGE gel and d the
CNF/CNR DGGE gel.
Numbers on the left
correspond to numbers
assigned to each band in the
DGGE. Bands that were
sequenced are numbered
accordingly with the
numbers on the DGGE.
Abbreviations: Fertilized
bulk (FB), fertilized
rhizosphere/roots (FRH),
unfertilized bulk (UFB),
unfertilized rhizosphere/
roots (UFRH)
Plant Soil
Fig. 3 Estimated nifH
phylogeny from the Paup
analysis. Neighbour-joining
tree based on
1000 bootstrap replicates
using Kimura two
parameter (K2P). Branch
lengths are drawn
proportional to the mean
estimated change (scale bar
is 0.05 substitutions per
site). All sequences
generated in this study are
indicated in bold face.
DGGE band A1–A3 2
indicate R T-PCR-DGGE
products amplified with
general diazotroph primers
PolF/PolR (Poly et al.
2001), DGGE band
B1-B16 indicates
PCR-DGGE products
amplified with
cyanobacteria specific
primers CNF/CNR (Olson
et al. 1998) and clone
UFA1-UFE5 and FA1-FC6
indicates clones (amplified
with PolF/PolR) from the
RNA clone libraries
unfertilized bulk (UF) and
fertilized bulk (F)
respectively. The tree was
rooted on the branch to
Methanocaldococcus
jannaschii
Plant Soil
Discussion
In recent years, nifH has been one of the most
important functional genes used when studying
structural and functional diversity in numerous envi-
ronments including diverse soil habit ats (Bürgmann
et al. 2003; Demba Diallo et al. 2008; Knauth et al.
2005; Poly et al. 2001; Wu et al. 2009). In the present
study, the nifH gene was used as a molecular marker
for studying the diazotrophic diversity and abun-
dance in the rice paddy in an area with intensive
farming practise, including two growth seasons a
year.
The investigation of the nifH transcript using qRT-
PCR with the universal nifH diazotrophic primers
showed that nifH gene expression together with
nitrogenase activity measurements did not reveal any
significant differences between the soil types or
between the sampling times, indicating a stable N
2
-
fixing community actively fixing nitrogen over the
day/night cycle. These results are in accordance with
the resent paper by Hsu and Buckley (2009) in which
a relationship b etween N
2
-fixation rates and soil N or
nitrate contents could not be found. Moreover, soil
analysis performed from the two fields (Table 1)
revealed that the amount of available nitr ogen in
unfertilized soil was only 44 mg/kg lower than in
fertilized soil, and probably not low enough to be a
discriminating factor. This might explain the absence
of a significant difference in N
2
-fixation expression
and activity betwee n the fertilized and unfertilized
soil types, and that the quantity of the nitrogen that is
being provided to the fertilized soil is probably not
affecting the N
2
-fixation activity of the microbial
community. The unfertilized soil had been without
nitrogen for 18 months, and it was therefore surprising
to find such small differences in available nitrogen
between the two different treatments. Although the
concentration of available P and K was twice as high in
fertilized than in unfertilized soil there was no
observed effect on either the quantity or the activity
of the diazotrophic activity in the samples. Thus, our
results are not in accord with previous nitrogen fixation
measurements from rice fields that showed diurnal
Fig. 3 (continued)
Plant Soil
patterns with peaks either at day (15:00) (Balandreau et
al. 1974) or during the night (Abdel Wahab 1980), as
well as seasonal variations (Quesada et al. 1998). This
could be the result of differences in the diversity and/
or activity of the different N
2
-fixing communities
present in our samp les.
Our phylogenetic analysis showed that several phylo-
types identified in the present study formed separate
clusters where the closest related known cultured bacteria
sequences in the database were members of the N
2
-fixing
β-proteobacteria, e.g. genus Azoar cus. Azoarcus spp.
has been described to be endophytically associated with
the rhizosphere of Kallar grass (Letptochloa fusca (L.)
Kunth) (Hurek et al. 2002),andassociatedwithrice
roots (Demba Diallo et al. 2008;Engelhardetal.2000;
Knauth et al. 2005;Wuetal.2009) and might be an
important active diazotroph in paddy fields (Wartiainen
et al. 2008). Additionally, other sequences recovered in
this study were closely related to known diazotrophic
bacteria previously reported from the rice soil e.g.
Methylococcus (Henckel et al. 1999; Mohanty et al.
2007)andIdeon ella (Coelho et al. 2008;Luetal.
2006).The findings indicate a significant importance of
those organisms for the paddy ecosystem. However, the
results are limited by the conditions used in the study ,
and PCR based methods on complex environments will
underestimate the diversity, and may even underestimate
dominating phylotypes. In a study of nifH pools in roots
of Oryza longistaminata, Demba Diallo et al. (2008)
found that the Poly primers (Poly et al. 2001)was
biased in amplification of nifH sequences from envi-
ronmental samples, while the Zehr primers (Zehr and
McReynolds 1989) detected a more complex nifH pool.
As tested and described in previous work (Wartiainen et
al. 2008), different nifH PCR primers were analyzed
prior to this study, both on environmental samples and
laboratory cultures of nitrogen fixing organisms such as
rhizobia, methanotrophs and cyanobacteria. The only
primers giving a single, correct sized PCR product from
all test organisms and environmental samples was the
primer pair described in this paper. Our results therefore
indicate that the Poly (Poly et al. 2001)PCRprimers
give reliable information, and are able to amplify nifH
sequences from a broad spectrum of diazotrophs from
this ecosystem. Our results are in agreement with the
findings by Wu et al. (2009) where the Poly primers
successfully were applied to resolve a high diversity of
proteobacteria associated with roots of modern rice
cultivars.
In the present study, no phylotypes most similar to
cyanobacteria were observ ed from the cDN A-DGGE
analysis although cyanobacteria are known diazo-
trophs in the rice paddy (Khan et al. 1994; Song et al.
2005). In order to examine if a bias was generated by
the nested PCR approach and/or from the DGGE
analysis, two clone libraries from cDNA samples
(UFB04:00 and FB04:00) were generated by direct
amplification with the PolF/PolR primers. In accor-
dance with the results from the RT-PCR-DGGE
analysis, neither cyanobacterial phylotypes nor mem-
bers of nifH phylogeny clusters II or IV were detected
in t h e two selected clone libraries. However, all
sequences recorded were closely related to and affiliated
with the p hylogene tic groups generated from the
sequenced DGGE bands. This strongly indicates that
no bias is generated with the nested PCR approach used
for the DGGE fingerprinting analysis. To confirm that
cyanobacteria were indeed present in the studied rice
field the RNA and DNA were analyzed by cyanobac-
teria specific PCR primers (Olson et al. 1998). As PCR
amplification of RNA using cyanobacteria specific
primers was unsuccessful, DNA was amplified, ana-
lyzed by DGGE and sequencing. The phylogenetic
analysis of these potential N
2
-fixing cyanobacterial
populations showed a diverse composition and distri-
bution of heterocystous and non-heterocystous as well
as unicellular and filamentous cyanobacteria throughout
the samples. Tentative phylogenetic affiliation related
our cyanobacterial DGGE phylotypes to members of the
Nostocales and the Chroococales. The unicellular
N
2
-fixing cyanobacteria of the genus Cyanothece,
which are known diazotrophs in paddy soils, were in
this study relatively abundant and thus might be
important nitrogen fixers in the ecosystem. Our
findings indicate that cyanobacteria are present, but
not detectable with the broad range PCR primers used
in this study. The fact that we were unable to amplify
cyanospecific nifH from RNA indica tes that the
cyanobacteria may be less active than heterotrophic
diazotrops in this paddy field and that the input from
the heterotrophic diazotrophic communities in the total
nitrogen fixation budget might have been underesti-
mated in previous studies. In accord, some recent
studies indicate that the density and significance of
cyanobacter ia in rice paddy soil may have been
overestimated when using culture- or microscopy-
based techniques (Ariosa et al. 2004). A more detailed
study should be undertaken to investigate the nitrogen
Plant Soil
fixation input from cyanobacteria and other potential
diazotrophic members of the community.
In summary, our analyses of the active diazotro-
phic community in the rice field revealed that a wide
range of nitrogen fixing bacteria was acti vely fixing
nitrogen, even under a normal fertilizer regime.
Coupling reduced nitrogen fertilizing and biological
nitrogen fixing activity to crop yield would be the next
step in investigating the potential of using and increas-
ing the biological nitrogen fixation in rice production.
Acknowledgments This work was supported by grants to UR
from The Swedish International Development Cooperation Agen-
cy Department for Research Cooperation (Sida/SAREC) and The
Swedish Research Link Programme. Grant from The Swedish
Research Council (FORMAS) to RE is acknowledged. We also
acknowledge the financial support from K. and A. Wallenberg
Foundation. Dr. L. Zhou and Mr Z. Lin are acknowledged for help
during the fieldwork, Ms C. Bin for help during fieldwork and
DGGE experiments and Ms S. Vintila, Department of Botany,
Stockholm University, for help with statistical analyses.
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Plant Soil
