Indian Journal of Biotechnology
Vol 13, October 2014, pp 540-543
Diversity of diazotrophs in tropical rice
field under the influence of organic
and nitrogen fertilization
Alaknanda Sarkar, K Manjunath and
Pranjali Vishwakarma*
Department of Microbiology and Biotechnology, Bangalore
University Jnana Bharthi Campus, Bangalore 560 056, India
Received 10 February 2013; revised 3 July 2013;
accepted 28 August 2013
The diversity of diazotrophic community of two different rice
field of typical tropical rice agroecosystem under long-term
application of nitrogen and organic fertilizer was studied. Most
probable number (MPN) result revealed that organic fertilizer
amended rice field (O) have higher diazotroph population than
nitrogen fertilizer amended rice field (N). NifH gene based
restriction fragment length polymorphism (RFLP) analysis revealed
presence of uncultured diazotroph, Anabaena, Rhizobium, Nostoc,
Azospirllium, Methylobacter, Leptolyngbya and Sinorhizobium.
Diversity index of diazotroph was found higher in O soil. The study
suggested that application of nitrogen fertilizer adversely affected
community composition of diazotroph in comparison to organic
fertilizer and the differences were found significant.
Keywords: Diazotroph, diversity, fertilizer, nifH gene, RFLP, rice
Biological nitrogen fixations by diazotrophs play a
significant role in nitrogen cycling and have drawn the
attention of researchers because they represent one of
the largest sources of nitrogen input to soil1. Diazotrophs
are present in the aerobic soil layers, rhizosphere,
roots and stem bases of rice agro-ecosystems. They
have the capacity to fix molecular nitrogen under
micro-aerophilic conditions as a growth promoting
substrate in nitrogen deficient environment. All the
known diazotrophs possess nifH gene, which encodes
the iron protein subunit of nitrogenase enzyme, and
has been frequently used as a molecular marker to
detect diazotrophs in environmental samples2,3. The
sequence-based nifH phylogeny correlates well with
the 16S rRNA gene based phylogeny and is
considered the ideal marker to characterize soil
diazotrophic communities and to assign specific
generic and species affiliations to diazotrophs3.
——————
*Author for correspondence:
E-mail: [email protected]
In general, diazotrophs are well studied in rice
fields as bio-fertilizers. Biological nitrogen fixation
by diazotrophic bacteria is an important process
affecting fertility of agro-ecosystem. It has been well
documented that physico-chemical factors and
management practices can influence microbial
populations. Therefore, it is inevitable that
amendment of synthetic nitrogen fertilizer could
affect the activity of diazotrophs. Nitrogen fixed by
free living nitrogen fixers is also typically important
for soil C:N ratio and these processes are fundamental
to the formation of soil aggregates4, which in turn
affect soil physical properties. In such circumstances,
we hypothesise that load of nitrogen fertilizer can lead
to alteration in diazotrophic community. The present
study is an attempt to assess the composition and
structure of N2-fixing bacteria associated with soil
under the effect of organic and nitrogen fertilizers. To
accomplish this, the molecular diversity of nifH gene
was investigated using RFLP.
Study sites were two rice field of the Cauvery plain
(Table 1). Chemical fertilizer in the form of urea was
applied at the rate of 30 Kg N ha-1 in two split doses
in nitrogen fertilizer amended rice field (N). Rice
straw fermented for 5 months was used as organic
compost in organic fertilizer amended rice field (O).
Field moist samples in triplicates, collected at
reproductive stages of rice plant, were mixed, sieved
and stored in polyethylene bags at 4°C and −20°C for
subsequent analysis.
Enrichment of diazotrophs from soil samples was
done in semisolid N-free NFb medium5. The number
of diazotrophs was obtained by MPN technique using
McCrady’s table. In brief, 10 g fresh soil was
suspended in saline solution (NaCl 0.8%) and serially
diluted in the range from 10-1 to 10-9 were inoculated
in triplicates in N-free NFb medium. After 7 d of
incubation at 37°C, population size was estimated by
MPN technique.
Extraction of DNA from soil samples were
carried out using Himedia soil DNA extraction Kit
as per manufacturer’s protocol and purified using
the QIA quick® gel extraction kit (QIAGEN).
PCR amplification of nifH gene was carried out
using
a
degenerate
primer
pair
PolF
(50-TGCGAYCCSAARGCBGACTC-30) and PolR
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541
Table 1—Soil characteristic of rice field supplemented with
organic (O) and nitrogen (N) fertilizers
Parameter
Latitude
Longitude
Soil type
Rainfall (Annual)
Soil temperature
pH
Total N%
Organic C%
Rice field
O
12058´
77038´
Red clay loam
1171.1mm
26-27
6.5-7.0
0.12
0.85
N
12058´
77038´
Red clay loam
1171.1mm
25-26
7.1-7.3
2.12
0.16
(50-ATSGCCATCATYTCRCCGGA-30)6.
PCR
condition was slightly modified and optimized as
described by Shukla et al7. The Thermocycler
(Q-CyclerII Server Gradient Thermal Cycler, Quanta
Biotech Ltd, UK) program included initial denaturation
at 94°C for 1 min, followed by 30 cycles at 94°C for
1 min, 57°C for 1 min, 72°C for 2 min and final
extension at 72°C for 5 min. Amplicons were resolved
by electrophoresis on 1% agarose gel.
PCR products were pooled and were cloned using
Qiagen pDrive Cloning Kit (Qiagen, Avenue
Stanford, USA) according to the manufacturer’s
protocol into Escherichia coli DH5α. The blue and
white colonies were screened on Luria-Bertani plates
with 100 µg mL−1 ampicillin and X-gal (0.5 mM). For
each soil sample, 100 clones were screened, making a
total of 200 clones to check the presence of insert by
using plasmid specific primer M13 forward and M13
reverse (Vishwakarma et al8). M13 PCR products
with nifH inserts were used for producing RFLP
pattern by digesting at 37°C overnight; briefly,
digestion mixture contained 0.4 U of tetrameric
endonuclease enzymes HaeIII (New England Biolabs,
Beverly, MA) in 1× buffer B (New, England,
Biolabs), bovine serum albumin (10 mg mL−1) in the
final volume of 20 µL. Restriction patterns were
manually verified by analyzing 20 µL reaction
mixture on 3% metaphor agarose gel stained with
ethidium bromide in the gel documentation system
(Fig. 1). The reported sequences of nifH gene
(Azospirllium,
Aazospira,
Rhodopseudomonas,
Nostoc, Rhodobacter, Rhizobium, Methylobacter,
Leptolyngbya, Burkholderia, Anabena, Azonexus,
Beijerinkia, Sinorhizobium and uncultured nifH
clones) available in the NCBI database were
downloaded and simulated RFLP patterns were
generated using Lasergene Software SeqManTMII
Fig. 1 (a & b)—RFLP pattern generated based on M13 PCR
product containing nifH gene insert using restriction endonuclease
HaeIII: a. O rice field; & (b) N rice field.
(DNAStar) by selecting same HaeIII enzyme from the
library of software9,10. A binary type data set was
generated for both RFLP pattern of clone and
simulated data of downloaded sequences. The average
linkage method of binary data resulted in a
dendrogram giving the topology of relationship
amongst the isolates using NTSYS 2.0 package.
The diversity of the diazotrophic community based
on nifH gene RFLP patterns was analysed using the
Shannon-Wiener index:
s
H´= - ∑ Pi In Pi
i=1
where s is the number of operational taxonomic units
(OTUs) and Pi is the relative abundance of clones
assigned to the ith OTU to total clones analyzed.
Statistical treatment of the data included two-way
analyses of variance (ANOVA) and linear regression
using software of the SPSS package.
The population size of diazotrophs in O rice field
was significantly higher (F1,4=49.70; P=0.02)
compared to N rice field (Fig. 2). NH4+ N ion
concentration and NO3- N ion concentration were
higher in N rice field in comparison to O rice field
INDIAN J BIOTECHNOL, OCTOBER 2014
542
(Table 2). ANOVA indicated that these differences
were significant (F1,4=72.95; P=0.00 & F1,4=67.114;
P=0.01) for both the parameters, respectively. The
experimental result clearly indicated that, following
application of N-fertilizer, there was distinct effect on
diazotrophic population size. This result is supported
by studies showing inhibitory effect of nitrogen
+
-
Table 2—Parameters including NH4 N and NO3 N in O and
N rice fields
Parameter
NH4+ N (µg g-1)
NO3- N (µg g-1)
Rice field
O
2.36±0.176
0.70±0.11
N
7.53±0.12
4.67±0.47
Fig. 2—Variations in diazotrophs population size (×105 cells g–1 dw
soil) in O and N soils. [Vertical bars are ±SE]
fertilizer on diazotrophic bacteria11,12. Nitrogen
fertilizer affects plants association with the
diazotrophic bacterial population due to alteration of
the physiological state of rice plant and this could be
one of the possible reasons behind decrease in
diazotrophic population in N rice field13.
Based on RFLP, 27 OTUs were used for
phylogenetic analysis (rice field O=O1, O2, O3, O4,
O5, O6, O7, O8, O9, O10, O11. O12, O13, O14 &
O15; rice field N=N1, N2, N3, N4, N5, N6, N7, N8
N9, N10 & N11). Results revealed the presence of
Azospirllium, Nostoc, Rhizobium, Methylobacter,
Leptolyngbya, Burkholderia, Anabena, Azonexus,
Sinorhizobium and uncultured nifH clone (Fig. 3). In
rice field, Muthukumarswamy et al14 and Wartiainen
et al1 have also reported the presence of diazotrophs
using cultivation dependent and independent method,
respectively. Based on UPGMA tree relative
proportion of diazotrophic clone library in two rice
field was verified (Fig. 4). Uncultured diazotrophs,
Anabena, Rhizobium and Nostoc related nifH gene
were found common in both rice fields; while nifH
gene related to Azospirllium, Methylobacter,
Leptolyngbya and Sinorhizobium was found only in
O rice field. The Shannon-Winner diversity index
(H´) for diazotrophic community in O and N rice field
was found to be 1.89 and 1.12, respectively. Diversity
analysis revealed that O rice field was rich in
Fig. 3 (a & b)—Cluster analysis of RFLP pattern showing variation among diazotrophic community in rhizospheric soil: a. O rice field;
& b. N rice field.
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3
4
5
Fig. 4 (a & b)—Relative proportion of nifH gene among total
OTUs based on RFLP UPGMA tree: a. O rice field; & b. N ricr
field. The phylogenetic affliation of nifH gne are indicated as
different pattern and pie chart represents clone library.
diazotrophic community when compared to N rice
field. Our results are in agreement with the findings of
Tapia-Herandez et al15 and Muthukumarsway et al12,
where they demonstrated that addition of compost
(organic fertilizer) helped in the proliferation of
diazotrophic bacteria. High loads of nitrogen may
result in osmotic stress; therefore, highly sensitive
microbe can lead to death16. Further, moderate load of
nitrogen results in C deficiency and C starvation
resulting in low C/N ratio in soil. Therefore, it is
inevitable here that under high load of nitrogen
fertilizer and carbon deficiency, diversity of diazotroph
was found lower in N rice field. Further, Salgado et al17
reported that organic matter protected bacteria against
soil physiological factor. This could be one of the
reasons behind higher nifH diversity in O rice field.
In conclusion, the present study showed the
presence of diverse group of nifH genes in rice fields.
And application of organic fertilizer gave positive
effect on the diversity of nifH genes. In addition to it,
organic fertilizers are good for environment as
well. Therefore, use of organic fertilizer should
be encouraged. Further, extension of the present
study using quantitative reverse transcripts PCR
(qRT-RCR) to assess effect of fertilizers on nifH gene
would eventually help in formulation of strategies for
mitigation of excess use of synthetic fertilizers in soil.
Acknowledgement
Authors thank the Council for Scientific and
Industrial Research, New Delhi and the University
Grants Commission, New Delhi for financial assistance
to PV as Dr D S Kothari Post-Doc Fellowship and
Senior Research Fellowship, respectively.
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