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Molecular Identification and Characterization of a Rice Field Cyanobacterium for Its Possible Use as Biofertilizer in Acidic Environment

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Abstract

Initial screening for cyanobacterial diversity in rice fields of Meghalaya revealed presence of a limited number of species predominantly belonging to heterocystous cyanobacteria such as Nostoc and Anabaena. However, based on morphological study alone, definite identification of the organisms was difficult. Hence, the identities of the organisms were established using partial sequence of 16S rRNA gene. In this work, we report the isolation, molecular identification, and characterization of a cyanobacterial species that could be a potential biofertilizer in acidic rice fields. The organism was identified as an Anabaena sp. and was characterized with respect to its growth, heterocyst frequency, nitrogen fixation, photochemical and respiratory activities, as well as various enzymes related to nitrogen metabolism to establish its potential as a biofertilizer strain under acidic conditions. Further, its rice root colonization ability as well as associative nitrogenase activity in the presence of nitrate was also assessed. This Anabaena strain was isolated from all the rice fields taken as study sites and found to be able to withstand cold environment and acidic condition of the region. It showed adequate growth, heterocyst frequency, and nitrogenase activity in the presence of nitrate in its vicinity. These features could make this organism a preferred biofertilizer strain in rice fields having lower pH as low pH usually restricts growth of most other cyanobacterial strains. In addition, this strain showed compatibility with nitrate and thus could be simultaneously used along with nitrate-based fertilizers to augment nitrogen content of the paddy fields and lower chemical fertilizer load.
Research & Reviews: A Journal of Microbiology & Virology
Volume 1, Issue 3, December 2011, Pages 1-12.
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ISSN 2230 9853 © STM Journals 2011. All Rights Reserved.
1
Molecular Identification and Characterization of a Rice Field
Cyanobacterium for Its Possible Use as Biofertilizer in Acidic Environment
Mayashree B. Syiem1*, Natasha A. Nongrum1, B. Bashisha Nongbri1, Amrita Bhattacharjee1,
David L. Biate2, Arvind K. Misra2
1Department of Biochemistry,North-Eastern Hill University, Shillong-793022, Meghalaya, India
2Department of Botany, CAS,North-Eastern Hill University, Shillong-793022, Meghalaya, India
*Author for Correspondence E-mail: mayashreesyiem@yahoo.co.in, Tel No: +91-364-
2722126, Telefax: +91-364-2550108
1. INTRODUCTION
Cyanobacteria are ubiquitous
photosynthetic prokaryotes with
remarkable degree of morphological and
developmental diversity [1, 2]. They are
found in almost every aquatic and
terrestrial environment [3, 4] and play an
important role in nutrient recycling and
maintenance of organic matter in aquatic
systems including lakes, rivers, and
wetlands. Besides, their nitrogen-fixing
potential is crucial to these ecosystems [5,
6]. They enhance productivity in variety of
environments due to their ability of both
carbon assimilation and nitrogen fixation.
In addition, they secrete a number of
important biologically active substances
that improve soil structure [7, 8].
Cyanobacterial presence in rice field
ecologies is of interest to scientists,
especially from rice-growing countries.
The positive effects of cyanobacteria in
Abstract
Initial screening for cyanobacterial diversity in rice fields of Meghalaya revealed presence
of a limited number of species predominantly belonging to heterocystous cyanobacteria
such as Nostoc and Anabaena. However, based on morphological study alone, definite
identification of the organisms was difficult. Hence, the identities of the organisms were
established using partial sequence of 16S rRNA gene. In this work, we report the isolation,
molecular identification, and characterization of a cyanobacterial species that could be a
potential biofertilizer in acidic rice fields. The organism was identified as an Anabaena sp.
and was characterized with respect to its growth, heterocyst frequency, nitrogen fixation,
photochemical and respiratory activities, as well as various enzymes related to nitrogen
metabolism to establish its potential as a biofertilizer strain under acidic conditions.
Further, its rice root colonization ability as well as associative nitrogenase activity in the
presence of nitrate was also assessed. This Anabaena strain was isolated from all the rice
fields taken as study sites and found to be able to withstand cold environment and acidic
condition of the region. It showed adequate growth, heterocyst frequency, and nitrogenase
activity in the presence of nitrate in its vicinity. These features could make this organism a
preferred biofertilizer strain in rice fields having lower pH as low pH usually restricts
growth of most other cyanobacterial strains. In addition, this strain showed compatibility
with nitrate and thus could be simultaneously used along with nitrate-based fertilizers to
augment nitrogen content of the paddy fields and lower chemical fertilizer load.
Keywords: Acidic pH, Anabaena sp., phylogenetic analysis, enzyme assays,
associative nitrogen fixation
Research & Reviews: A Journal of Microbiology & Virology
Volume 1, Issue 3, December 2011, Pages 1-12.
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ISSN 2230 9853 © STM Journals 2011. All Rights Reserved.
2
rice fields are well established.
Cyanobacteria do not compete with rice
plants as they are self-sufficient in
generating their carbon and energy needs.
Instead, they are known to increase soil
fertility, regulate plant growth and improve
soil health because of their ability to fix
both atmospheric CO2 and N2 [6, 915].
Over the years, there have been several
reports of widespread occurrence of
different cyanobacterial species such as
Nostoc, Anabaena, Gloeotrichia,
Gloeocapsa, Camptylonema,
Cylindrospermum, Scytonema,
Tolypothrix, Aulosira, Westiellopsis, and
many other genera in Indian rice fields [9,
1619]. Among these, Nostoc and
Anabaena are most prevalent. During
1970s, use of cyanobacteria as biofertilizer
in rice fields was popularized in an attempt
to reduce the chemical fertilizer load on
the rice fields [6]. However, research in
recent times revealed that this biofertilizer
program is challenged with a number of
problems including low survival rate of
inoculums, incompatibility with chemical
fertilizers, low nitrogen release, as well as
their inefficient adaptability to
environmental conditions and to
competition with naturally existing soil
microflora [20]. In addition, diverse soil
ecologies dictate cyanobacterial
distribution and abundance. There have
been reports of low soil pH, among other
factors, adversely affecting cyanobacterial
diversity, existence, and abundance [19,
2123].
Soil pH of rice fields of Meghalaya ranges
from 5.2 to 7.1. During the course of
collection of cyanobacterial strains for
diversity studies, we found it difficult to
identify many strains belonging to the
family Nostocaceae by simple microscopy
as these isolates showed close similarity in
their morphology. Their identification thus
was established by molecular studies using
16S rRNA gene-specific primer. Among
the isolates, an Anabaena strain was found
to be ubiquitous in all rice fields that were
undertaken for the study. The sites were
selected to include a wide range of acidic
pH. The presence and abundance of this
Anabaena strain indicated that it could
tolerate and thrive in a wide variation of
field pH, particularly acidic in nature. As
most cyanobacteria prefer neutral to
alkaline pH, we decided to characterize
this strain for its possible application as
biofertilizer in acidic rice fields.
2. MATERIALS AND METHODS
2.1. Sample Collection
Soil and water samples were collected
from eight different rice fields, i.e.,
Umkhen, Mairang, Umiam, Nongstoin,
Mawphlang, Mawlai Mawroh, Syntu
Ksiar, and Sung Valley in the state of
Meghalaya during the month of December,
2009, when there was no rice plantation
and fields were dry as well as in the month
of August of 2010 when the rice planted
into the fields was about forty-five days
old. There was continuous rain throughout
the months of June, July, and August
2010, due to which rice fields were
waterlogged.
2.2. Measurement of pH
The pH of samples collected was measured
by using method described by Black [24].
2.3. Isolation and Purification of
Cyanobacteria
Cyanobacterial strains were isolated and
purified after inoculating the samples in a
nitrogen-free (BG-110) medium and
subsequently planting on 1.2% nutrient
agar for many successive generations.
Axenic isolates thus achieved were grown
and maintained in batch cultures in the
above medium at 25 ± 2 ° C and at a
photon fluence rate of 50 µmol m2s-1 [25].
Research & Reviews: A Journal of Microbiology & Virology
Volume 1, Issue 3, December 2011, Pages 1-12.
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Morphological identification was done
under an Olympus BX 53 light microscope
using description given by Desikachary
[26].
2.4. Molecular Techniques for
Identification Study
Light microscopic study showed presence
of cyanobacterial isolate No. NEHU7 in all
collection sites. To attain pure and axenic
culture of this cyanobacterium, single-
spore colonies were raised. For this
purpose, sporulation was induced in the
cyanobacterium by inoculating 0.5 µg ml1
of sample in sulphate deficient BG-110
medium [27]. Single-spore colonies were
raised in 1.2% nutrient agar plates using
1000-fold diluted spore suspension. The
isolated colonies from the plates were
transferred to liquid media and maintained
in axenic state in batch cultures.
For molecular identification, genomic DN
A was isolated from the cyanobacterium
(isolate No. NEHU7) using MiniPrep of
Bacterial Genomic DNA Method [28]. For
securing amplification of 16S rDNA
homologous sequence, primer CF16S-7
was designed using Mac Vector software®
based on cyanobacterial 16S rDNA
sequences retrieved from GenBank. PCR
amplification was carried out using
primers CF16S-7 (forward, 5-
GGCTCAGGATGAACGCTG-3′) and
FGPS 1490 (reverse, 5' TGGAAAGCTTG
ATCCTGGCT-3') following the
methoddescribed by Normand et al. [29] in
a GeneAmp 9700 Gold thermal cycler
(Applied Biosystems). Amplification was
carried out following 35 cycles of
denaturation at 94 oC for 1 min, annealing
for 1 min at 61 oC, elongation at 72 o C for
1 min. Final extension was allowed for
10 min at 72 o C. Each reaction mixture
contained 1 μl of template DNA, 2.5 μl of
10X PCR buffer and 2.5 μl of each primer.
Total reaction volume was made up to
25 μl by adding requisite quantity of
ultrapure water. Target amplicon was
extracted from the cut portion of the gel
using phenol extraction followed by
chloroform: isoamyl alcohol purification.
Purified amplicons were sequenced with
the cycle sequencing method using
BigDye Terminator v3.1 Cycle
Sequencing Kit (Applied Biosystems) on a
3130 Genetic Analyzer (Applied
Biosystems). Related sequences were
retrieved from GenBank using BLAST
search. Sequenced amplicons were aligned
with retrieved sequences using ClustalW
(http://www.ebi.ac.uk/Tools/clustalw2/ind
ex.html) (Table I). Phylogenetic analysis
was performed with the PHYLIP version
3.69 [30]. The neighbor-joining algorithm
based on a matrix of pair-wise distances
corrected for multiple base substitutions by
the method of Kimura [31] was used to
generate the phylogenetic tree with a
bootstrap confidence value of 1000.
2.5. Measurement of Growth
Growth was measured as increase in
chlorophyll a content. 3 ml cyanobacterial
culture was centrifuged at 2000 rpm. To
the pellet, 3 ml of methanol was added and
the tube was vortexed to mix the added
methanol with the pelleted cells. This was
then incubated for 5 min in boiling water
bath for extraction of chlorophyll. The
tube was centrifuged and absorbance was
read for the supernatant at 663 nm [32].
2.6. Heterocyst Frequency and
Nitrogenase Assay
Heterocyst percentage was calculated
using an Olympus BX 53 light
microscope. Acetylene reduction activity
by nitrogenase enzyme was estimated in
vivo by gas chromatography using a Tracor
540 Gas Chromatograph fitted with a
Porapak “T” column and a flame
ionization detector [33].
Research & Reviews: A Journal of Microbiology & Virology
Volume 1, Issue 3, December 2011, Pages 1-12.
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2.7.Assay of Electron Transport
Activities
Photochemical and respiratory activities
were estimated by measuring O2 evolution
and consumption at 25 ± 2 °C and at a
photon fluence rate of 50 µmol m2s1
using a Clark-type polarographic O2
electrode installed in a 3-ml Plexi glass
container with magnetic stirrer [34].
2.8. Nitrate Uptake, Nitrate Reductase,
and Glutamine Synthetase
(Transferase) Activities
Nitrate uptake experiments were initiated
by adding NaNO3 (100 μM) to the ten-
day-old cell suspension. Uptake of nitrate
was measured by the rate of its depletion
from the medium. Samples were
withdrawn after 3 h of incubation,
subjected to rapid centrifugation at 5000
X g and the cell-free supernatants were
analyzed for residual nitrate. Nitrate
concentration was measured by the method
of Cawse [35]. Ferredoxin-dependent
nitrate reductase activity was measured
using dithionite reduced methyl viologen
as reductant [36, 37]. Glutamine
synthetase (transferase) activity was
measured as described by Sampio et al.
[38]. Protein was measured according to
Lowry et al. [39].
2.9. Co-cultivation Experiment with Rice
Rice seeds of local variety “Synteng” were
collected from ICAR Complex (Umiam,
Shillong, India). The seeds were washed,
surface sterilized using 1% sodium
hypochlorite solution, and grown on
autoclaved Perlite in glass beakers. The
seedlings were irrigated with 10 times
diluted BG-11o media supplemented with
2 mM NaNO3 from time to time. 10day-
old five rice seedlings were transferred
together to 15 ml capacity tubes. 5 ml BG-
11o medium (“ symbol indicates a
nitrogen-free medium, i.e., BG-11 medium
without a nitrogen supplement) was
poured into the tubes carefully dipping the
roots in the medium. Exponentially
growing cultures of the test organism were
introduced into the tubes (initial
chlorophyll a content of 1 µg ml1 per
tube). Association was confirmed under
microscope as well as by estimating
chlorophyll a concentration in the rice
roots after removing loosely associated
filaments using a sonic bath.
2.10. Associative Nitrogen Fixing Ability
Associative nitrogenase activity of the
cyanobacterium was estimated [40] in the
rice roots after removing loosely
associated filaments using a sonic bath.
Roots were excised and chlorophyll a was
extracted from the cyanobacterial
filaments associated with the roots using
3 ml methanol.
3. RESULTS
Seventy-three cyanobacterial strains were
isolated (Table I). Thirty-four were found
to be Nostoc while 16 belonged to
Anabaena species. Of these, isolate No.
NEHU7 was found in all the rice fields.
This strain was purified and single spore
colonies of this cyanobacterium were
raised to obtain genetically identical and
pure culture for further studies
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Volume 1, Issue 3, December 2011, Pages 1-12.
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Table I Cyanobacterial Isolates from Rice Fields within the State of Meghalaya.
*Anabaena sp. reported in this study was isolated from these sites.
Values in the parentheses indicate pH of
the collected water samples. Under light
microscope, long filaments with spherical
intercalary heterocysts were observed.
Heterocysts of different filaments showed
tendency to align together forming a
cluster of heterocysts. Akinetes were not
apparent in the field samples.
To establish the identity of the isolate, pure
culture was raised from spores. Spore
differentiation in the cyanobacterium was
initiated by inoculating 0.5 µg ml1 of
sample in sulphate deficient BG-110
medium [27]. Kyndiah and Rai in 2007
have already shown the adverse effect of
phosphate and sulphate restriction in
growth medium on cyanobacteria.
Sulphate restriction seems to have more
severe effect. As a protective measure,
when cyanobacteria are inoculated in a
medium, deficient in any form of sulphate,
the cells differentiate into spores and
remain dormant as long as the adverse
condition persists. Since each cell has the
potential to differentiate into spore, a
dilute solution of spore suspension, when
spread onto agar made in appropriate
medium, supplemented with required
sulphate, developed into a pure colony.
Purity of such colonies is guaranteed as it
was raised from a single spore. Such
colonies are therefore very useful for
molecular studies as this eliminates
chances of contamination.
Partial sequence of 16S rRNA gene was
used to identify the isolate at molecular
level. 16S rRNA sequence is referred here
as “partial” because only a part (the initial
5' end) sequence was successfully
sequenced out of the approximate 1.5 kb
(being the approximate total size), which
was further used for analysis in the present
study. The word approximate has been
used for the genome size because the
generally accepted size of the 16S rRNA is
1500 bp. However, variability in size
exists among different organisms, e.g., in
Anacystis nidulans, this gene is sequenced
to be 1487 bp, whereas in Anabaena sp.
strain PCC 7120, it is 1489 bp, in E. Coli,
it is 1541 bp, and in Bacillus, it is 1554 bp.
The variability is due to the presence of
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Volume 1, Issue 3, December 2011, Pages 1-12.
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6
functional and structural regions in the 16S
rRNA gene, whereby such differences can
be tolerated at the structural regions.
Amplicon of approximately 1.5 kb was
obtained with the primer pair used.
However, additional bands were also
observed, which persisted even when the
stringency conditions were altered and the
annealing temperature increased to 61 o C.
There was no amplification when the
annealing temperature was raised above
61 o C. Consequently, the band of interest
was eluted by excising part of the gel.
Sequencing yielded a partial sequence of
249 bases. Multiple alignments with the
BLAST-retrieved sequences showed
homology of 94 and 92% with Anabaena
sp. XPORK15F (Acc. No. EF568905.1)
and Nostoc sp. (Acc. No. AB187508.1)
respectively (Table II).
Table II Source Organisms and Accession Number of the Organisms Used for the Study.
The phylogenetic analysis was performed
with the Phylogeny Inference Package,
version 3.69 [30]. The neighbor-joining
algorithm was used to generate the
phylogenetic tree. The neighbor-joining
tree constructed using the 16S rRNA gene
revealed the cyanobacterial isolate to be
forming a tight cluster with Anabaena sp.
as supported by the bootstrap confidence
value of 993.0 (1000 replicates). Further,
Nostoc sp. also formed a sub-cluster within
this group in congruence with reports by
different workers. Studies of 16S rRNA
sequences have shown that genera Nostoc
and Anabaena are too closely related to be
clearly separated into two different genera
[4143]. Similar observations were made
using partial (359 bp) sequences of Nostoc
and Anabaena [41, 4446]. Tamas et al.
(2000) further suggested that the two
genus could be merged into one based on
the similarity of their nifH gene sequences.
This view was also shared by other
researchers [45, 47]. The phylogenetic
analysis evidently showed the
cyanobacterial isolate to be a member of
the genus Anabaena of the order
Nostocales.
Fig. 1 Neighbor-Joining Phylogenetic Tree Constructed with 1000 Bootstrap Replicates.
Values at the Nodes Represent the Bootstrap Values.
Sl. No.
Source organisms
Accession number
1.
Isolate No.NEHU7
Present study
2.
Anabaena sp. XPORK15F
EF568905
3.
Nostoc sp.
AB187508
4.
Tolypothrix sp. TOL328
AM230706
5.
Tolypothrix sp. PCC 7504
AM230669
6.
Nodularia harveyana
AF268020
7.
Cylindrospermum stagnale
AF132789
8.
Anabaenopsis elenkinii
AM773309
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Volume 1, Issue 3, December 2011, Pages 1-12.
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When grown in different nitrogen-
supplemented media, the cyanobacterium
exhibited best growth in the presence of
nitrate indicating its better nitrate-utilizing
ability than ammonia (Figure 2).
Fig. 2 Growth in the Anabaena sp. Expressed as Increase in Chlorophyll a Content in BG110,
and in 1 mM Nitrate and Ammonium-Supplemented Media.
Heterocyst frequency of the cyanobacteria
in nitrogen-free medium was ~7.8 ± 0.4%
during its exponential phase with
corresponding nitrogenase activity of
13.2 ± 1.2 nmol C2H4 produced µg1 chl
ah1. The photosynthetic oxygen evolution
and respiratory oxygen consumption rates
were 579.3 ± 0.9 nmol O2 evolved µg1
Chl a h1 and 295.6 ± 0.5 nmol O2
consumed µg1 Chl a h1, respectively
(Table III). However, heterocyst frequency
was less than half (~44%) of the control
culture when cyanobacterium was grown
in nitrate medium. No heterocyst was
noticed in ammonia-supplemented
medium. Again, nitrogenase activity was
only ~33% of the control in nitrate
medium while no nitrogenase activity was
recorded in the presence of ammonia.
However, in associated state in nitrate
medium, the nitrogenase activity was 1.2
times higher than the cells in free-living
state in the same medium.
Table III Evaluation of Physiological and Biochemical Characters of the Anabaena sp. on
Eighth Day from Inoculation (The Results are Mean of Three Individual Experiments) at pH
6.0.
Characters
Value
Heterocyst frequency (%)
7.81 ± 0.4
Heterocyst frequency (%) in 1 mM nitrate-supplemented medium
3.42 ± 0.6
Heterocyst frequency (%) in 1 mM ammonia-supplemented medium
0.0
Nitrogenase activity (nmol C2H4 produced µg1 Chl a h1)
13.2 ± 1.2
Nitrogenase activity (nmol C2H4 produced µg1 Chl a h1) in 1 mM
Nitrate-supplemented medium
4.39 ± 0.9
Nitrogenase activity (nmol C2H4 produced µg1 Chl a h1) in
1 mM ammonia supplemented medium
0.0
Associative nitrogenase activity (nmol C2H4 produced µg1 Chl a h1)
in 1 mM nitrate supplemented medium
5.28 ± 1.2
Photosynthesis (nmol O2 evolved µg1 Chl a h1)
579.3 ± 0.9
Respiration (nmol O2 consumed µg1 Chl a h1)
295.6 ± 0.5
Glutamine Synthetase activity (nmol γ-glutamyl hydroxamate formed min1 mg1
protein)
1264 ± 6
Nitrate uptake (µmol nitrate taken up min1 mg1 protein)
4.20 ± 0.5
Nitrate Reductase activity (nmol nitrite formed min1 mg1 protein)
5.38 ± 0.04
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The rate of nitrate uptake in the
cyanobacterium was 4.2 ± 0.5 µmol nitrate
taken up min1 mg1 protein and the nitrate
reductase activity was found to be
5.38 ± 0.04 nmol NO2 formed min1 mg1
protein. A look at glutamine synthetase
activity yielded a value of 1264 ± 6 nmol
γ-glutamyl hydroxamate formed min1 mg1
protein (Table III).
Visual and microscopic observation
established cyanobacterial association with
the rice roots. Even after vigorous sonic
bath there were residual filaments visible
on the roots confirming tight association
between isolate NEHU7 with roots of rice
plantlets. Associative nitrogenase activity
in these root specimens was found to be
~40% of its free-living counterparts in
nitrate medium (Table III).
4. DISCUSSION
Rice field environment presents an ideal
situation for cyanobacterial growth in
terms of optimum amounts of light,
temperature, water and available nutrition.
On the other hand, presence of
cyanobacteria is beneficial to soil as they
contribute to the overall nitrogen economy,
help in phosphate solubilization and in
removal of toxic compounds. This way,
application of cyanobacteria can bring
about an increase (525%) in crop
productivity, especially in case of rice
cultivation [10, 12, 14, 48]. Although
cyanobacterial presence and their
beneficial effects in crop fields are well-
accepted facts, studies relating to factors
affecting their association with the crop
plants have been a less explored area [4,
49]. There is competition among
cyanobacterial strains to form artificial
association with rice plants [48]. This
information has opened up the possibilities
of using selective strains of cyanobacteria
as biofertilizer inoculants in crop fields.
Besides, Nilsson et al. [49] have shown
that temperature and availability of fixed
nitrogen played a determining role in
establishing such artificial association.
However, other factors such as soil and
water pH of the agricultural fields need
further investigation to establish their role
in influencing such associations. It is
known that existing pH of a location is
certainly the most determining factor for
its biodiversity composition. The naturally
existing cyanobacteria prefer neutral to
alkaline pH [50, 51]. Low pH adversely
affects cyanobacterial growth and
performance. The soil and water pH of
Meghalaya was found to be generally
acidic in nature (Table I) and it is known
that soil in Meghalaya is more weathered
in comparison to other north-eastern states
of India. Acidic soil of Meghalaya
developed on sedimentary and
metamorphic rocks under climatic
predominance of humid tropic. Meghalaya
soils are highly leached, having poor base
saturation and low exchange capacity.
They are characterized by high KCl-
extractable aluminium. Presence of low-
activity clay along with subsoil aluminium
toxicity rendered them less fertile. In
addition, being at a higher altitude the
temperature of the region is lower than
mainland India. Any potential biofertilizer
strain(s) of cyanobacteria for this region
must thus be able to function at low pH
and cooler temperature. The importance of
isolating and projecting a cyanobacterium
as biofertilizer strain for acidic pH from
the region has its own benefits. Since soil
and water of Meghalaya are acidic in
nature, isolates from this region are
already used to lower pH and hence
probably have developed strategies to
counter such adverse situations. Therefore,
there is a need to assess biofertilizer
potential of various cyanobacteria from the
region so that specific strains can be sorted
and utilized to enhance soil fertility and
reduce the necessity of chemical fertilizer
input.
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9
The combined effect of low pH and cooler
temperature can be seen on the diversity
and distribution of cyanobacteria in rice
fields across Meghalaya (Table I). Most
sites represented only strains of either
Nostoc or Anabaena. Thus, these strains
are more versatile and adaptive among the
ones that were isolated from these rice
fields. An analysis of cyanobacterial
diversity in the eight rice fields yielded
only eight genera of cyanobacteria (Table
I). Of the total of 73 isolates, 90%
belonged to heterocystous form. Of these,
73% belonged to Nostoc and Anabaena sp.
Species like Haphalosiphon, Westiellopsis,
Aulosira, Tolypothrix that have been
reported from various rice fields of
different parts of India were not isolated
from these sites revealing their reduced
adaptability to combined effects of low pH
and cooler temperature. Most common
genus was Nostoc. However, ample data is
already available on rice field Nostoc
isolates from previous researches [18, 40].
On the other hand, the cyanobacterium
Anabaena over the centuries has found its
use in many rice cultivations in the form of
its symbiotic association with the water
fern Azolla in most rice-growing countries.
Therefore, we have focussed our attention
on the most ubiquitous Anabaena strain,
which showed flexibility in adapting to
acidic conditions prevalent in these fields.
Rice root colonization ability and
associative nitrogenase activity of this
strain are indicative of its potential as
biofertilizer.
Field samples of Anabaena and Nostoc
showed confusing morphological
similarity, making it difficult to establish
their identity in the initial stages of
isolation. On subsequent transfer to fresh
liquid medium, the morphology stabilized
as the stress encountered in field
conditions was removed. A heterotrophic
mode of nutrition in form of 10 mM
glucose in dark failed to sustain growth in
some isolates pointing towards the
possibility of them being Anabaena sp.
Presence of a morphologically similar
Anabaena isolate in all collection sites
with diverse pH range prompted us to use
molecular identification studies that
eventually confirmed its identity as
Anabaena (Fig. 1). In this paper, we have
presented phylogenetic tree of the isolate
No. NEHU7 that was purified from
Umiam (pH 5.9) rice field water sample.
We used 16S rRNA gene for identification
study as it is universally present in
prokaryotes and is functionally conserved
across the kingdom. It has been most
reliable and widely used molecule for
identification of bacteria. The primer pair
used in the present study to amplify the
16S rRNA gene was designed to include
both conserved and variable regions which
aided in the precise identification of the
isolate. The identity established was based
both on the results of BLAST similarity
search from NCBI database as well as the
phylogenetic analysis. Multiple alignment
of the amplified sequence with the
sequences retrieved using BLAST search
revealed that differences between these
sequences were most apparent at the
variable regions. It can therefore be
concluded that the sequence contained
enough conserved and variable bases for
resolving its identity and did not require
calculation for BLOSUM scores as this is
used for sequence alignment of proteins.
On day eighth of its transfer to fresh
medium, the organism showed ~83% and
~72% growth in comparison to the
organism growing at pH of 7.6 when the
pH of the culture media were maintained
at 6 and 5 respectively (data not shown).
The lower pH threshold for this strain was
5.0 for its unrestricted survival. At pH 4.0
the growth expressed in terms of
chlorophyll a drops drastically.
Additionally, the isolate could use both
exogenous nitrate and ammonia for its
growth. The growth pattern of the
organism showed its ability to utilize
Research & Reviews: A Journal of Microbiology & Virology
Volume 1, Issue 3, December 2011, Pages 1-12.
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ISSN 2230 9853 © STM Journals 2011. All Rights Reserved.
10
nitrate better than ammonia (Fig. 3). The
growth was sustained for longer period in
the supplemented media. Its heterocyst
frequency and nitrogenase activity were
comparable to many Nostoc species [40,
52] suggesting its definite role in fixed
nitrogen contribution to the rice field
ecosystems on its mass turn over (Table
III). In ammonia-supplemented media,
there was no heterocyst seen in the
filaments and no nitrogenase activity
recorded. However, in nitrate media, there
was retention of ~44% heterocyst
frequency and nitrogenase activity was
retained at ~33% of the control culture
(experiment conducted at pH 6). It had
optimum photosynthetic and respiratory
activities required for carbon dioxide
fixation and generation of energy for
important cellular activities such as
nitrogen fixation. Nitrate uptake and
reductase activities indicated towards
proper ability to utilize available nitrate in
the medium. A value of 1264 nmol γ-
glutamyl hydroxamate formed min1 mg1
protein for glutamine synthetase activity
suggested its efficiency in incorporating
available ammonia into cellular
metabolism. The associative nitrogenase
activity of the Anabaena strain in 1 mM
nitrate medium was higher than the
nitrogenase activity of the free-living
counterpart under similar conditions
(5.28 ± 1.2 and 4.39 ± 0.9 respectively).
This implied that even with nitrate in the
vicinity, the cyanobacterium could form
positive association with rice plants that
led to enhanced nitrogenase activity in the
organism. This is a crucial attribute of a
potential biofertilizer that was exhibited by
the Anabaena strain under study. Also,
that the strain could do so in the presence
of nitrate led us to put it forward as a
potential biofertilizer in acidic rice fields
of colder regions along with nitrate-based
chemical fertilizers.
That cyanobacterial diversity and
abundance is low in Meghalaya was earlier
reported by Prasanna and Nayak [18]. Our
collection done on a larger scale, including
eight rice fields across the state, confirmed
their observation. The pH of each site
recorded was found to be acidic in nature,
which also agreed with previous reports
that low soil pH adversely affects
cyanobacterial diversity [19, 22]. Our
finding that most dominant forms of
cyanobacteria in rice fields are Nostoc and
Anabaena substantiated the report [4] of
these two genera being highly versatile.
Enhanced associative nitrogenase activity
exhibited by the Anabaena sp. under study
correlated with the findings of Nilsson et
al. [40] where they have elaborately shown
increase in nitrogenase activity in
cyanobacteria when associated with rice
roots.
5. CONCLUSIONS
We observed from our isolation
experiments that acidic rice fields are poor
in cyanobacterial diversity due to low
existing pH. This study provides evidence
for possibility of selectively using an
Anabaena isolate as biofertilizer along
with nitrate-based chemical enhancers to
boost crop productivity in acidic
conditions. As evident from the
experiments, it would still be able to fix
atmospheric nitrogen, reducing the
required chemical load in the crop fields.
Such isolates from locations exhibiting
low pH may also be useful in other parts of
the country with similar pH conditions in
enhancing soil quality and fertility. This is
because these isolates are preconditioned
to grow under acidic pH and, therefore,
will be able to populate the fields where it
is being used as biofertilizer. However,
further research is needed to assess its
performance during actual field
applications. Research involving tagging
of the inoculants could immensely help in
Research & Reviews: A Journal of Microbiology & Virology
Volume 1, Issue 3, December 2011, Pages 1-12.
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ISSN 2230 9853 © STM Journals 2011. All Rights Reserved.
11
monitoring their survival and population in
the fields.
ACKNOWLEDGEMENTS
We would like to thank UPE scheme of
UGC, Life Sciences, NEHU, Shillong and
Rajiv Gandhi National Fellowship for
ST/SC students for financial support.
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... Extensive studies of N2-fixing cyanobacteria had been carried out in many countries, particularly in India (Choudhary 2011;Syiem et al. 2011;Vijayan and Rai 2015) and a few in Philippines (Roger et al. 1992), Spain (Fernandez-Valiente and Quesada 2004), China (Song et al. 2005), and Iran (Saadatnia and Riahi 2009). Research from conventional rice fields showed that N2-fixing cyanobacteria such as Aulosira, Anabaena, Nostoc, and Scytonema were found abundant, while Cylindrospermum and Rivularia inhabited deep-water type of rice fields (Sinha and Hader 1996). ...
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Cyanobacteria make a major contribution to world photosynthesis and nitrogen fixation, but are also notorious for causing nuisances such as dense and often toxic `blooms' in lakes and the ocean. The Ecology of Cyanobacteria: Their Diversity in Time and Space is the first book to focus solely on ecological aspects of these organisms. Its twenty-two chapters are written by some thirty authors, who are leading experts in their particular subject. The book begins with an overview of the cyanobacteria - or blue-green algae, for those who are not specialists - then looks at their diversity in the geological record and goes on to describe their ecology in present environments where they play important roles. Why is one of the key groups of organisms in the Precambrian still one of the most important groups of phototrophs today? The importance of ecological information for rational management and exploitation of these organisms for commercial and other practical purposes is also assessed. Accounts are provided of nuisances as well as the ecology of the commercially successful Spirulina and the role of cyanobacteria in ecosystem recovery from oil pollution. Many chapters include aspects of physiology, biochemistry, geochemistry and molecular biology where these help general understanding of the subject. In addition there are three chapters dealing specifically with molecular ecology. Thirty-two pages of colour photos incorporate about seventy views and light micrographs. These features make the book valuable to a wide readership, including biologists, microbiologists, geologists, water managers and environmental consultants. The book complements the highly successful The Molecular Biology of Cyanobacteria already published by Kluwer.