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Mountain Aspect Influences the Genetic Clustering
of Psychrotolerant Phosphate Solubilizing Pseudomonads
in the Uttarakhand Himalayas
Govindan Selvakumar ÆPiyush Joshi Æ
Pankaj K. Mishra ÆJaideep K. Bisht Æ
Hari S. Gupta
Received: 8 May 2009 / Accepted: 2 July 2009 / Published online: 28 July 2009
ÓSpringer Science+Business Media, LLC 2009
Abstract Fourteen cold tolerant phosphate solubilizing
bacteria isolated from high altitude representative locations
of the two major mountain aspects of the Uttarakhand
Himalayas (cooler north and warmer south facing slopes)
were selected for this study. The tricalcium phosphate
(TCP) solubilizing abilities of the isolates were estimated
at three different incubation temperatures viz., 4, 15, and
30°C under in vitro conditions. Irrespective of their geo-
graphical origin, all the isolates recorded maximum P
release values at 30°C. At 4°C, the isolates from the north
facing slope were found to release significantly higher
levels of P, as compared to the isolates from the south
facing slopes. Alternatively at 15°C, the isolates from the
south facing slope were found to release significantly
higher levels of P. Initial confirmation of their genus level
identity as Pseudomonads was arrived by amplification of a
990 bp fragment of the 16S rRNA gene using genus spe-
cific primers. Further putative species level identification
was arrived by sequencing of the 16S rRNA gene. The
diversity among the isolates was determined by rep-PCR
using the primers BOX, ERIC, and ERIC2. A composite
dendrogram constructed using the rep-PCR profiles
revealed that the isolates from the north and south moun-
tain aspects formed separate major clusters. The extent of
diversity was greater among the isolates from the south
mountain aspect. This study reveals the potential of rep-
PCR in determining the genetic diversity among
Pseudomonads selected for a single functional trait, but
varying in their geographical origin.
Introduction
Microbial diversity is the key to the integrated functioning
of nutrient cycles and decomposition in terrestrial ecosys-
tems. These systems range from high altitude areas, trop-
ical forest, savannah, temperate evergreen forests, to
agricultural ecosystems. Inherent to each system is the
unique microbial flora, whose characteristics and diversity
are affected by a variety of anthropogenic and non-
anthropogenic factors. In the mountain eco-system, among
the various non-anthropogenic factors that affect diversity
of living beings, the direction of the mountain slope is most
important. In physical geographical terms, the word
‘‘aspect’’ refers to the direction to which a mountain slope
faces. The directional effect on living beings is highly
pronounced in the Uttarakhand Himalayas (74°500–95°400
East and 26°200–35°400North), which harbours a myriad of
microbial wealth. The mountain slopes of this region are
broadly divided into two categories as north and south
facing. While the southern facing slopes are generally
warmer and receive more sunlight, the northern facing
slopes are characterized by lesser duration and intensity of
sunlight [1].
Among the various tools used for microbial diversity
studies, PCR based tools have become hugely popular due
to the ease in handling and repeatability. The presence of
repetitive elements in the prokaryotic genome has spawned
a new PCR based technique called the rep-PCR technique
which utilizes the oligonucleotide derived repetitive
sequences present in bacterial strains for distinguishing
between closely related members of the same genus. The
G. Selvakumar (&)P. Joshi P. K. Mishra
J. K. Bisht H. S. Gupta
Vivekananda Institute of Hill Agriculture, Indian Council
of Agricultural Research, Almora 263601, India
e-mail: gselva74@rediffmail.com
123
Curr Microbiol (2009) 59:432–438
DOI 10.1007/s00284-009-9456-1
Repetitive Extragenic Palindromic (REP) and Enterobac-
terial Repetitive Intergenic Consensus (ERIC) sequences
were originally described for members of the family
Enterobacteriaceae [2], but were later reported in several
Gram-negative bacteria and close relatives within the same
phyla [3]. Similarly the presence of repeat sequences called
the BOX elements were reported in the Gram positive
bacterium Streptococcus pneumoniae [4]. The conserved
status of these repetitive sequences make them ideal tools
for biodiversity studies. The utility of rep-PCR in differ-
entiating members of several eubacterial genera is well
documented [5,6].
Among the various geochemical cycles mediated by
microbes, phosphate solubilization assumes considerable
significance due to the indispensability of phosphorous in
plant nutrition. Though the soil phosphorus levels are quite
sufficient to sustain plant growth, most forms of phos-
phorus are present in fixed forms and thereby require
transformation to be converted to plant utilizable forms.
Bacterial mineral phosphate solubilization is a well estab-
lished phenomenon and has predominantly reported at
mesophilic temperatures [7]. Recent studies have shifted
the focus from P solubilization at the mesophilic range to
the psychrotolerant range by mutant strains [8]. Since the
soils of the Uttarakhand Himalayas are generally acidic in
nature and are prone to fixation of phosphorous [9], there
exists a need for biologically mediated phosphorus nutri-
tion of plants. Some progress has been made in this
direction mainly from the Indian Himalayan Region (IHR)
[10]. These studies have revealed the predominant nature
and role of the genus Pseudomonas, in phosphate solubi-
lization under cold temperature conditions. But the factors
that contribute to the diversity of such cold tolerant strains
are less understood. Since mountain aspect plays a defining
role in the diversity and distribution of living organisms,
this study was undertaken to determine the genetic diver-
sity among a set of elite Pseudomonads selected for a
single plant growth promotional trait, but originating from
two highly variable mountain aspects of the Uttarakhand
Himalayas.
Materials and Methods
Site Characteristics, Strain Isolation, and Maintenance
The rhizospheric soil samples used for isolation of bacteria
were collected from alpine and sub alpine locations of the
state of Uttarakhand located in the N.W. Indian Himalayan
Region. At the time of sample collection various parame-
ters like altitude, atmospheric temperature, soil tempera-
ture, and the direction of slope were recorded. The
altitudinal variation represented by the sampling sites
ranged from 1600 m to 3800 m above mean sea level. The
mean soil and atmospheric temperatures of the north facing
slope at the time of soil collection were 12 and 15.8°C,
respectively, while the south facing slopes recorded war-
mer temperature regimes of 21.2 and 24°C, respectively.
Rhizospheric samples collected from both cultivated and
uncultivated eco-systems were transported in sterile plastic
bags to the laboratory under controlled temperature con-
ditions (5 ±1°C). The processed soil samples were seri-
ally diluted, spread plated on full strength nutrient agar,
and incubated at 4°C. Morphologically distinct bacterial
colonies from each sample were selected, purified and
maintained on nutrient agar slants and 50% glycerol stocks
at -80°C. All the subsequent experiments were conducted
after raising fresh cultures. By following the isolation
strategy mentioned above we were able to isolate five
hundred psychrotolerant bacterial isolates, which were
subject to selection for phosphate solubilization at cold
temperatures.
Selection of Efficient Phosphate Solubilizing Bacterial
Isolates
The tricalcium phosphate (TCP) solubilization by the iso-
lates was estimated at three different incubation tempera-
tures viz., 4, 15, and 30°C, by inoculating 1 ml of bacterial
suspension (3 910
7
cells/ml) in 50 ml of NBRIP broth
[11], containing (per liter) glucose, 10 g; Ca
3
(PO
4
)
2
,5g;
MgCl
2
6H
2
O, 5.0 g; MgSO
4
7H
2
O, 0.25 g; KCl, 0.2 g;
(NH
4
)
2
SO
4
, 0.1 g in Erlenmeyer flasks (150 ml). The pH of
the medium was adjusted to 7.0 before autoclaving. The
flasks were incubated for 7 days. At the end of the incu-
bation period the cell suspension was centrifuged at
10,000 rpm for 10 min and the P content in the supernatant
was spectrophotometrically estimated by the ascorbic acid
method [12]. Based on their P solubilizing potential at 4°C,
14 isolates were designated as elite and used for further
experimental procedures.
Genomic DNA Extraction
The genomic DNA was extracted by inoculating 1 ml of
bacterial suspension (10
7
cells/ml), of the individual iso-
lates in 50 ml of LB broth in Erlenmeyer flasks (150 ml),
and incubating them for 24 h at 30 ±1°C at a uniform
speed of 110 rpm. Cells were harvested by centrifugation
at 8,000 rpm for 20 min, washed with 0.85% normal sal-
ine, and resuspended in 200 ll TE buffer. The cell pellet
was lysed with a combination of 10 ll of SDS (10%) and
5ll of proteinase K (10 mg/ml), followed by extraction
with phenol: chloroform (1:1) and chloroform: isoamyl
alcohol (24:1). The extracted DNA was dissolved in 20 ll
TE buffer and used as the template for the PCR reactions.
G. Selvakumar et al.: Influence of Mountain Aspect on Pseudomonad Clustering 433
123
Amplification of the 16S rRNA Gene Using
Pseudomonas Specific Primers
Initial genus level confirmation of the isolates was carried
out in our laboratory. The primers Ps-for (50GGTCTGA-
GAGGATGATCAGT30) and Ps-rev (50TTAGCTCCACC
TCGCGC30) as described by [13], were used to amplify a
990 bp fragment of the 16S rRNA gene of the selected
isolates. The reaction was carried out in a 20 ll reaction
mixture containing 50 ng template DNA, 1X reaction
buffer, 2 mM MgCl
2
, 200nM dNTP mixture, and 200 nM
each of primer and one unit of Taq Polymerase (Bangalore
Genei, India), in a thermal cycler (BioRad, USA). The
thermal profile used was initial denaturation at 94°C for
3 min, followed by 34 cycles of denaturation step (92°C
for 15 s), annealing (68°C, 1 min), and extension (72°C for
1 min), and a single final extension step (72°C for 6 min).
Gel electrophoresis was performed on a 1.2% agarose gel
to visualize the amplified product. The gel was stained with
ethidium bromide, visualized and documented using
a documentation system (Alpha Imager TM 1200). The
sequencing of the 16S rRNA gene was carried out at
the Institute of Microbial Technology, Chandigarh and
Bangalore Genei, Bangalore using universal eubacterial
primers. The putative stain assignment was based on results
of a query using the BLAST programme [14].
Repetitive Element PCR
The primers evaluated in this study were ERIC1R (50-ATG
TAA GCT CCT GGG GAT TCA C-30) and ERIC2 (50-
AAG TAA GTG ACT GGG GTG AGC G-30) for ERIC-
PCR; ERIC2 (50-AAG TAA GTG ACT GGG GTG AGC
G-30) for ERIC2-PCR and BOX (50-GAT CGG CAA GGC
GAC GCT GAC G-30) for BOX-PCR [15]. All the reac-
tions were carried out in a 20 ll reaction mixture con-
taining 50 ng template DNA, 2 mM MgCl
2
, 0.25 mM
dNTP mixture, and 0.25 lM each of primer and one unit of
Taq Polymerase (Bangalore Genei, India), in a thermal
cycler (BioRad, USA). The thermal profile used was initial
denaturation at 95°C for 2 min, followed by 30 cycles of
denaturation step (92°C for 30 s), annealing (50°C, 1 min),
and extension (65°C for 8 min), and a single final extension
step (65°C for 8 min). Gel electrophoresis was performed
on a 1.5% agarose gel to visualize the amplified products
along with a pair of ladders (1 kb and 500 bp, Bangalore
Genei, India). The gel was stained with ethidium
bromide and documented using a documentation system
(Alpha Imager TM 1200). The presence or absence of
distinct and reproducible bands in each of the individual
PCR profiles was converted into binary data, and the
pooled binary data were used to construct a composite
dendrogram. The software NTSYSpc, version 2.02i [16],
was used to calculate the Jaccard distance index [17] and
construct the dendrogram using the Unweighted Pair-
Group Method with Arithmetic Average (UPGMA). Sta-
tistical analysis was performed with the Statistical Package
for Social Sciences (SPSS) software, and treatment means
were compared at 5% level of significance, by a two tailed
ttest.
Results
Strain Isolation and Characteristics of the Isolates
The nature and properties of the selected isolates are pro-
vided in Table 1. Differential staining and microscopic
examination revealed that all the isolates were Gram neg-
ative rods. When incubated on nutrient agar at 15°C they
produced whitish, translucent circular colonies of 2–3 mm
diameter, after 48–72 h incubation. Most isolates grew at a
wide temperature range of 4–30°C, but maximum growth
was found to occur at 30°C. All the isolates recorded
higher P release values from Tri Calcium Phosphate at
30°C. At 15 and 30°C, the inter quartile range which
denotes the spread of the values, was higher among the
isolates originating from the warmer south facing slopes,
while at 4°C the spread was greater amongst the isolates
originating from the cooler north facing slope (Fig. 1).
Further statistical scrutiny revealed that at 4°C the isolates
from the north facing slope released significantly higher
levels of P, compared to the isolates from the south facing
slopes. Alternatively at 15°C, the isolates from the south
facing slope released significantly higher levels of P
(Table 2). Though the isolates from the warmer south
facing slopes released higher levels of P at 30°C, the results
were non-significant.
Confirmation of Genus Level Identity and Putative
Species Level Identity
Using primers that specifically amplified the 16S rRNA
gene of the genus Pseudomonas, we obtained an amplicon
size of approximately 990 bp in all the isolates which
confirmed the identity of the isolates at the genus level
(Fig. 2a). Based on the results of the BLAST query con-
ducted with the 16S rRNA gene sequences, three isolates
were putatively identified as Pseudomonas poae and one
each as P. lurida and P. fragi, respectively. While three
isolates shared a high degree of identity with both P. poae
and P. trivialis one isolate shared 99% identity with
P. lurida and P. tolaasi. Species level assignment was not
possible for three isolates, due to the lack of suitable
matches in the public domain (Table 1). In general it could
be observed that most isolates fell in the tightly linked
434 G. Selvakumar et al.: Influence of Mountain Aspect on Pseudomonad Clustering
123
P. poae/lurida cluster, which makes the species level
identification difficult.
Diversity Analysis by rep-PCR
The PCR profiles obtained using the different primers
revealed that the resolving power of the ERIC primers was
superior to the BOX primer. The BOX primer generated
amplified products varying from 400 bp to 2500 bp
(Fig. 2b), while the ERIC and ERIC-2 primers generated
amplified bands ranging from 100 bp to 5 Kb in size
(Fig. 2c and d). The composite dendrogram constructed
using the rep-PCR profiles revealed the presence of two
major clusters among the isolates (Fig. 3). A careful perusal
of the dendrogram revealed that the six isolates that formed
Table 1 Nature of the selected psychrotolerant phosphate solubilizing pseudomonads
Isolate
designation
Altitude
(amsl)
Mountain
aspect
Origin of
the isolates
Putative 16S rRNA
identification
Identity
(%)
Gen Bank
Accession number
Phosphate solubilization (lg/ml)
4°C15°C30°C
CS11RH1 1800 m South CL Pseudomonas fragi 99.0 EU 255303 76.86 118.40 229.10
CS11RP1 1800 m South CL Pseudomonas sp. 95.0 GQ 132058 89.28 126.30 176.00
CS11RH4 3000 m South CL NS – – 77.52 139.32 269.70
CT4RH2(2) 1990 m South CL Pseudomonas poae/trivialis 99.0 FJ 643472 71.47 92.50 202.30
RT5RP(2) 3100 m North FL Pseudomonas poae 99.0 EU 600372 114.14 77.11 171.40
RT6RP 3800 m North FL Pseudomonas poae/trivialis 99.0 EU 255305 102.53 72.77 182.30
PB2RP2 2000 m North CL Pseudomonas sp. 95.0 FJ 643473 95.26 100.19 193.42
PB2RP1(1) 2000 m North CL NS – – 87.70 91.90 173.10
PB1RP3 2000 m North CL Pseudomonas sp. 97.0 FJ 643474 70.53 108.55 258.80
PC7RP(2) 1990 m North CL Pseudomonas lurida/tolaassii 99.0 EU 255304 107.57 91.34 180.60
PB2RP1(2) 2000 m North CL Pseudomonas poae/trivialis 99.0 FJ 643471 81.30 83.23 175.40
NM4RP1 2290 m North FL Pseudomonas poae 99.0 FJ 643470 84.49 92.91 201.70
NS12RH2(1) 1780 m South CL Pseudomonas poae 99.0 EU 600373 73.30 116.42 195.40
M2RH3 2560 m South CL Pseudomonas lurida 99.0 EU 600374 72.74 88.58 130.14
amsl above mean sea level, CL cultivated land, FL forest land, NS not sequenced
668688
N =
ASPECT
SouthNorth
Soluble P (µg/ml)
300
200
100
0
4°C
15°C
30°C
9.00
2.00
Fig. 1 Boxplot of TCP solubilization by the isolates from north and
south aspects at various incubation temperatures. The vertical
whiskers represent the maximum and minimum values; the box area
represents the inter-quartile range; the dark line within boxes
represents the median value. The hollow circles represent outliers
which exceed the remaining observations by at least thrice the
standard deviation of the entire data series. The numerals adjoining
the hollow circles represent the case numbers of the outliers. The
outliers were included for the ttest
Table 2 Effect of in vitro incubation temperature on TCP solubili-
zation by the selected isolates
Temperature
(8C)
P solubilized (lg/ml) tvalue
North origin
(n=8)
South origin
(n=6)
4 92.94 76.86 2.49*
15 89.75 113.58 2.84*
30 192.09 195.29 0.15
* The difference between the observed mean values is significant at a
level 0.05 by a two tailed ttest
G. Selvakumar et al.: Influence of Mountain Aspect on Pseudomonad Clustering 435
123
the first major cluster had their origins from rhizospheric
soil samples collected from the south aspect, while the eight
isolates that formed the second cluster originated from
samples collected from the north aspect. The genetic clus-
tering of the isolates found support in the putative species
level identification based on 16S rRNA gene sequences.
The highly diverse south facing cluster branched into two
sub-clusters, which further gave rise to three distinct minor
clusters. The first minor cluster was formed by P. fragi and
Pseudomonas sp., the lone P. lurida isolate formed the
second cluster while the third cluster was formed by the
P. poae/trivialis group of isolates. The less diverse north
Fig. 2 a Amplification of Pseudomonas specific 16S rRNA gene
(size approx 990 bp); bBOX PCR amplification profile of the
psychrotolerant Pseudomonads; cERIC PCR amplification profile of
the psychrotolerant Pseudomonads; dERIC-2 PCR amplification
profile of the psychrotolerant Pseudomonads. M1-100 bp ladder,
M2-500 bp ladder, 1-CS11RH1, 2-CS11RP1, 3-CS11RH4, 4-
CT4RH2(2), 5-RT5RP(2), 6-RT6RP, 7-PB2RP2, 8-PB2RP1(1), 9-
PB1RP3, 10-PC7RP(2), 11-PB2RP1(2), 12-NM4RP1, 13-
NS12RH2(1), 14-M2RH3
Fig. 3 Composite dendrogram
showing the genetic relatedness
between the isolates originating
from different mountain aspects
436 G. Selvakumar et al.: Influence of Mountain Aspect on Pseudomonad Clustering
123
facing cluster was predominated by the P. poae/lurida/
tolaassii group of isolates, which is evident from the almost
identical band pattern of the amplification products that can
be visualized in lanes 5–12 of Fig. 2b, c, and d. Another
interesting feature was the extent of similarity among the
isolates that formed the individual clusters. While the south
cluster gave rise to a sub cluster that branched into minor
clusters with less than 70% similarity among them, the
north cluster gave rise to a sub cluster that branched into
two minor clusters were almost 90% similar. Hence it could
be concluded that the isolates originating from the south
aspect shared only 46% similarity with the isolates from the
north aspect, and were genetically more diverse than the
isolates from the northern slopes.
Discussion
In recent years, considerable attention has been devoted to
cold-adapted microorganisms that successfully colonize
cold habitats that comprise more than 80% of the earth’s
biosphere. The renewed interest in these microorganisms
could be attributed to the major role they play in nutrient
turnover processes at low temperatures [18]. While a
considerable portion of earth remains frozen throughout the
year, the alpine soil environments are characterized by
dramatic seasonal shift in physical and biochemical prop-
erties, due to intermittent snow cover and fluctuating sub
freezing temperatures in winter and intense, desiccating
sunshine punctuated by infrequent rains during summer
[19]. The atmospheric and edaphic selection processes in
the alpine regions have helped in the evolution of a variety
of living organisms that have adapted to cold stress.
The alpine and sub alpine regions of the N.W. Indian
Himalayan region are known to be a rich source of
microbial diversity. During our earlier forays in the region
we were able to describe diverse cold tolerant strains of
plant growth promoting bacteria including Serratia mar-
cescens, Pantoea dispersa, and Exiguobacterium acetyli-
cum [20–22]. An interesting observation from our
laboratory reveals that intense screening of psychrotolerant
bacterial isolates from the region for phosphate solubili-
zation at 4°C resulted in recovery of Pseudomonads alone.
This finding is in concurrence with the earlier observation
[23], which attributed the predominance of Pseudomonads
in alpine environments to their metabolic diversity. A
similar observation was made by Gulati et al. [24], who
isolated several phosphate solubilizing strains of P. poae
from the rhizosphere of the Himalayan sea buckthorn, but
the P solubilizing ability of this bacterium under cold
temperature conditions has not been reported so far. By
sequencing the 16S rRNA gene we were able to infer that a
majority of our isolates fell in the tightly linked P. poae/
P. trivialis cluster, thereby making the species assignment
difficult. The predominance of Pseudomonads in cold
environments is well established, the causes for the
diversity within a group of functionally similar Pseudo-
monads are a researchable issue. While several factors have
been attributed to this diversity [25], very little information
is available on the physico-geographical factors that
influence the diversity of microorganisms. An exception to
this is the spatial orientation based on distance alone which
receives wide attention from microbial ecologists. But
some of the other finer factors such as the mountain aspect
remain to be relatively lesser explored. A recent study
carried out in the Evolution Canyon of Israel has revealed
differences in the cell wall fatty acid profile of 130 B.
simplex stains originating from the cool north facing slope
and the warmer south facing slope. Greater unsaturation
was observed in the fatty acids of the isolates from the
north facing slopes, which probably accounts for their cold
tolerance [26]. An interesting observation of the present
study was that isolates originating from the cooler north
facing slopes were superior in solubilizing TCP at 4°C,
while isolates from the warmer south facing slopes solu-
bilized greater levels of TCP at 15°C. While the observed
phenomena can be partly attributed to the thermal accli-
matization of the isolates, the multitude of factors that
contribute to the observed phenomena are a researchable
issue. The absence of a definitive trend at 30°C could be
attributed to their psychrotolerant nature.
Earlier studies involving rep-PCR profiles were used to
differentiate between closely related strains of the same
Pseudomonad species [27]. The unique feature of this study
is the construction of a dendrogram with rep-PCR profiles,
which reveals that mountain aspect has a definite influence
on the genetic clustering of cold tolerant Pseudomonads
that were selected for a single functional plant growth
promotional trait. It is a well established fact that the micro
climatic factors of a location are largely determined by its
geographical orientation [28]. The relevance of this is
largely evident in the hills where locations separated even
by a few meters experience entirely different microcli-
mates. In the context of this paradigm, the aspect or the
direction of the mountain slope has a major influence of the
soil characteristics including the microclimatic conditions
since slopes on either side of a mountain receive varying
levels of sunlight, that in turn influence the temperature,
moisture level, soil characteristics, and the biological
activities of the soil.
Though a myriad of factors are known to influence the
metabolic activity of microbes, temperature is the single
most important factor that has a profound influence on the
cell morphology and physiology [29]. Likewise the edaphic
characteristics including soil formation processes, moisture
profiles, organic matter contents, are also influenced by
G. Selvakumar et al.: Influence of Mountain Aspect on Pseudomonad Clustering 437
123
temperature [30]. The greater diversity in the south facing
slopes could be attributed to copious amounts of sunlight
which leads to elevated soil temperatures and hence greater
microbial activity. The north facing slopes receive lesser
sunlight and hence lower temperatures and lower metabolic
rates. But the intriguing fact in this study remains the
influence of aspect on the clustering of the isolates. It is
quite possible that the aspect induced micro-climatic fac-
tors select a particular genotype of bacteria that are highly
adapted to the prevailing conditions. This is a researchable
issue since many factors are responsible for this selection
process. But never the less this study is a novel attempt to
derive meaningful conclusions on the ecological aspects of
Pseudomonas species diversity.
Acknowledgments Part of this research work was supported by
ICAR, under the project ‘‘Application of Microorganisms in Agri-
culture and Allied Sectors’’. Mr. Sanjay Kumar is acknowledged for
technical assistance.
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