IJBPAS, May, 2017, 6(5): 931-940
ISSN: 2277–4998
ISOLATION, CHARACTERIZATION AND IDENTIFICATION OF POTASSIUM SOLUBILIZING FUNGI FROM RHIZOSPHERE SOIL IN BANGALORE MATTHEW T AND TALLAPRAGADA P* Department of Microbiology, Centre for Postgraduate Studies, Jain University, 18/3, 9th Main, 3rd block, Jayanagar, Bangalore- 560011, India *
Corresponding Author: E Mail: -
[email protected], Phone number: +919448533337 th
th
Received 7 Dec. 2016; Revised 8 Feb. 2016; Accepted 11th March 2017; Available online 1st May 2017
ABSTRACT In this research, an attempt was made to find fungi that can solubilize the insoluble potassium from rhizosphere soil collected from Bangalore. Soil samples were collected and mixed with insoluble potassium (Feldspar) and incubated for 1 week at room temperature. After adaptation 1 gm of soil was inoculated in 100 ml liquid medium containing 1% glucose, 0.05%yeast extract and 0.5% feldspar and incubated at 370 C on 120 rpm for 1 week. Enriched samples were inoculated after serial dilution up to 10-6 on Aleksandrov agar. Colonies exhibiting clear zone of potassium solubilization were selected as potassium solubilizers from the 10-4, 10-5 and 10-6 dilutions containing plates. Secondary screening was carried out on the basis of study of zone of activity of the different isolates by using Khandeparkar’s selection ratio. Optimization experiment showed that solubilisation activity was more in glucose supplemented medium for (carbon source), urea (for nitrogen source), potassium chloride (for potassium source) and at the temperature of 30oC. One fungal isolate (Aspergillus terreus) showed good solubilizing activity. Keywords: Isolation, Identification, Potassium, Solubilizing, Fungi INTRODUCTION Land (soil) remains a limited resource
exhausting it of the minerals. This has
which cannot be expanded. Overtime,
resulted in increasing demand for chemical
farmers have continued to cultivate this
fertilizers for cultivation of crops in
limited resource thereby depleting and
developing
countries.
These
chemical 931
IJBPAS, May, 2017, 6(5)
Research Article
Matthew T and Tallapragada P*
fertilizers have in turn continued to degrade
involved with over 60 different enzyme
the soil in particular and the general
systems in plants. Besides its potential to
environment subsequently endangering the
resist drought and disease [2, 3], it helps in
plant, animal, microbial and human health
the production of starch, controls root
[1].
growth
Fixation of added nutrients/fertilizers in
movement
soil reduces the efficiency of applied
contributes to quality.
potassium fertilizers and thus a large
Among Nitrogen (N), Phosphorus (P) and
quantity
become
Potassium (K), Potassium is the third
Rhizosphere
important plant nutrient. Potassium is
microorganisms contribute significantly in
essential macronutrient for plant growth
solubilization of bound form of soil
and plays significant roles in activation of
minerals in the soil [15]. However,
several
potassium nutrients are released slowly
protein synthesis, photosynthesis, enzymes,
from the rock materials and their use as
as well as in resistance to diseases and
fertilizer
insects etc. [4].
of
added
unavailable
to
fertilizers
plants.
often
causes
insignificant
and in
regulates plant
metabolic
the
cells
processes
stomata and
also
including
increases in the yield of crops [10].
India totally depends on import of potassic
It has therefore become expedient to
fertilizers and farmers use very little or not
explore better ways and methods to meet
of potassium in crop production [5].
the nutritional needs of the crops in ways
Therefore, more research is needful to
that reduce or eliminate the adverse effects.
unearth more efficient and native strains of
Ways that are eco-friendly and promote
these
sustainable agriculture.
microorganisms for use.
Potassium
(K)
macronutrient
is for
a
major
plant
essential
growth.
The
potassium
solubilizing
MATERIALS AND METHODS Sample Collection
concentrations of soluble potassium in the
Rhizosphere soil samples were randomly
soil are usually very low and more than
collected from different locations in and
90% of potassium in the soil exists in the
around Bangalore. From each location,
form of insoluble rocks and silicate
samples were collected from six different
minerals. Potassium (K), one of the
sites. The collected samples were pooled
seventeen chemical elements required for
together to make the composite sample [6].
plant growth and reproduction, is often
Adaptation and Enrichment
referred to as “the regulator” since it is 932 IJBPAS, May, 2017, 6(5)
Research Article
Matthew T and Tallapragada P*
The composite soil samples collected were
under
mixed with insoluble potassium (Feldspar)
lactophenol cotton blue as stain. Molecular
and incubated for 1 week at room
characterization of the isolate was done
temperature. After adaptation 1 gm of soil
with help of 18S rRNA sequence analysis.
was inoculated in 100 ml liquid medium
Steps followed were: Genomic DNA was
containing 1% glucose, 0.05% yeast extract
isolated from the fungal sample. The 600-
0
compound
microscope
using
and 0.5% feldspar and incubated at 37 C
650 bp ITS region was amplified using
on 120 rpm for 1 week [7].
high–fidelity PCR polymerase. The PCR the
product was sequenced bidirectionally. The
Rhizosphere Soil and Screening for
sequence data was analyzed to identify the
potassium Solubilization
culture and its closest neighbours [8].
Enriched samples were inoculated after
Primers used were: ITS-Forward Primer 5’
Isolation
of
Rhizofungi
serial dilution up to 10
-6
from
on Aleksandrov
– GRAAGNAHADGTVGKAAYAWSG –
agar medium constituted as 1% glucose,
3’.
0.05%
FeCl3,
TCCTNCGYTKATKGVTADGH – 3’. The
0.01% CaCO3, 0.2% CaPO4 and 0.5%
crude sequence was submitted and aligned
potassium aluminium silicate, agar 3 % at
to/by
MgSO4.7H2O,
0.0005%
0
ITS-Reverse
the
Primer
National
5’
Centre
for
pH 6.5 [16] and incubated at 37 C for 1
Biotechnology
week. Fungal Colony exhibiting clear zone
database. The accession number from
of potassium solubilization on Aleksandrov
NCBI is KX775949.
agar was selected as potassium solubilizer.
Solubilization Activity and Optimization
Secondary Screening was carried out on the
Conditions for Efficient K Solubilization
basis of study of zone of activity of the
The isolated K solubilizing fungus was
isolate by using Khandeparkar’s selection
tested for optimization its K solubilizing
ratio. Ratio = D/d = Diameter of zone of
activity under varying conditions of carbon,
clearance / Diameter of growth [7].
nitrogen, potassium, temperature and pH
Characterization of the isolate
sources used.
Morphological and molecular identification
A loopful of 48 hour old grown fungi
of the isolate was done to ascertain what
culture
fungi it is. The isolate was grown in on
Aleksandrov
czapeck dextrose agar medium and its
capacity flask containing either of different
colony characteristics were studied. The
sugars: fructose, galactose, glucose and
morphology of the isolate was studied
xylose with added flask for control which
was
Information
–
inoculated medium
(NCBI)
into
broth
in
25ml 50ml
933 IJBPAS, May, 2017, 6(5)
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Matthew T and Tallapragada P*
was not inoculated. All the inoculated
standard curve generated.
flasks plus the control were incubated at
each parameter was carried out thrice to
28±2oC for 10 days. Same was done for
obtain
nitrogen sources (beef extract, NaNO3,
referenced to the standard curve to obtain
peptone and urea), potassium sources (KCl
the estimate of solubilization.
and K2SO4), varying temperatures (25oC,
RESULTS AND DISCUSSION
o
o
o
the
average
Estimation for
which
was
now
30 C, 35 C and 40 C) and varying pH (6.5,
Isolation and Solubilization Activity of
7.0, 7.5and 8.0) [6].
Fungi
Quantitative Estimation of Potassium
Of the colonies that were able to grow on
Release
Aleksandrov agar one of these isolated
Different concentrations of KCl solution,
colonies was found to make a clearance
ranging from 0 – 100 ppm, were used for
zone indicating k-solubilization fig. 4
preparation of standard curve. Sodium
giving a Khandeparker’s ratio of 2.33 Table
cobaltinitrite solution (5ml) was added
1.
slowly to each test tube containing varied
Morphological
concentrations of potassium and volume
Characters of Isolated Strain
made up to 10ml by adding distilled water.
The fungal isolate was circular, whitish,
o
and
The reaction mixture was incubated at 37 C
cotton-like
for 45 minutes to precipitate the potassium
pigmentation under fig. 1. It was identified
and centrifuged at 13,000 rpm for 5
to be Aspergillus terreus fig. 3. The isolate
minutes to permit the precipitate to settle
was found to be closest to Aspergillus sp. 7
down in the tube. The supernatant was
BRO-2013 18S ribosomal RNA gene,
decanted, precipitate collected and washed
partial sequence; internal transcribed spacer
twice with distilled water and once with
1, 5.8S ribosomal RNA gene, and internal
absolute ethanol. After washing, 10ml of
transcribed spacer 2, complete sequence;
conc. HCl was added to the precipitate and
and 28S ribosomal RNA gene, Sequence
incubated at 37OC for 15 - 20 minutes to
ID: gb|KF367546.1 fig. 2. The next closest
develop the green colour and absorbance
homologue was found to be Uncultured
was
fungus clone CMH583 18S ribosomal RNA
measured
at
600nm
using
the
colorimeter [9]. Following
the
colonies
Biochemical
showing
yellow
gene, partial sequence; internal transcribed same
procedure
and
spacer 1, 5.8S ribosomal RNA gene, and
conditions, potassium was estimated in 5ml
internal transcribed spacer 2, complete
of culture supernatant, with reference to the
sequence; and 28S ribosomal RNA gene, 934
IJBPAS, May, 2017, 6(5)
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Matthew T and Tallapragada P*
Sequence ID: gb|KF800672.1|. This agrees
K2SO4 (372.31mg/l). This is in line with
with the finding of Rekha and Sreeramulu
the fact that potassium nutrients are
[11] who stated that most of the fungi
released slowly from the rock materials and
noticed
their
that
were
efficient
potash
solubilizers were Aspergillus sp.
use
as
fertilizer
often
causes
insignificant increases in the yield of crops [10].
1. K SOLUBILIZATION
Solubilization was best (49.23mg/l) at the
ACTIVITY the
temperature of 30oC followed by 41.54mg/l
solubilization was best in glucose broth
at 25oC, then the least were 41.54mg/l at
26.15mg/l
galactose
35oC and at 40oC fig. 8. Same temperature
(22.31mg/l), then fructose (14.62mg/l) and
of 30oC was found to be the temperature at
the least was xylose (10.77mg/l) fig. 5. This
which Aspergillus performed best by
agrees with the study by Barasso, C. B.
Maharani et al., [14].
et.al., [12], who stated that Aspergillus
At pH 7.5 solubilization was best yielding
produces citric acid in medium supplement
87.69mg/l
with carbon sources like glucose, galactose
45.39mg/l at pH 6.5, next was 26.15mg/l at
and the citric acid production enhances the
pH 7 and the least was 22.31mg/l at pH 8
solubilization of the insoluble nutrients.
fig. 9. This differs from findings of other
As seen on fig. 6, the solubilization was
researches that show best solubilization at
most in urea (106.92mg/l), followed by
lower pH of 3 to 4. The difference could be
NaNO3 (103.08mg/l), then beef extract
attributed to low quantity of solubilization
(91.54mg/l) and lastly peptone (64.62mg/l).
as it has been reported by Trivedi, M. et.
Solubilization as observed on fig. 7 was
al., [13] that pH lowers with more quantity
more in KCl broth (476.15mg/l) than
of potassium solubilized.
From
(fig.
5)
it
is
followed
seen by
that
at
pH
7.5,
followed
Table 1: K solubilization zone formation by isolates by Khandeparkar’s ratio: D/d Diameter of Zone of clearance (D) Diameter of growth of Colony (d) in in mm mm Aspergillus terreus 14 06 D = Diameter of zone of clearance, d = Diameter of growth of isolate Isolate
by
D/d Ratio 2.33
Fig.1: Aspergillus terreus plates
935 IJBPAS, May, 2017, 6(5)
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Matthew T and Tallapragada P*
Aligned sequence data: 2B (632bp) TGGAAGTAAAAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTACCGAGTGCGGGTC TTTATGGCCCAACCTCCCACCCGTGACTATTGTACCTTGTTGCTTCGGCGGGCCCGCCAGCGTTGCTGG CCGCCGGGGGGCGACTCGCCCCCGGGCCCGTGCCCGCCGGAGACCCCAACATGAACCCTGTTCTGAAA GCTTGCAGTCTGAGTGTGATTCTTTGCAATCAGTTAAAACTTTCAAAATGGATCTCTTGGTTCCGGCATC GATGAAGAACGCAGCGAAATGCGATAACTAATGTGAATTGCAGAATTCAGTGAATCATCGAGTCTTTGA ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTCCGAGCGTCATTGCTGCCCTCAAGCCC GGCTTGTGTGTTGGGCCCTCGTCCCCCGGCTCCCGGGGGACGGGCCCGAAAGGCAGCGGCGGCACCG CGTCCGGTCCTCGAGCGTATGGGGCTTCGTCTTCCGCTCCGTAGGCCCGGCCGGCGCCCGCCGACGCA TTTATTTGCAACTTGTTTTTTTCCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATA TCAATAAGCGGAG G Fig. 2: Gene sequence of the isolate (Aspergillus terreus)
Fig. 3: Phylogenetic tree of Aspergillus terreus
Fig. 4: Aspergillus terreus colony showing solubilisation zone on Aleksandrov medium
936 IJBPAS, May, 2017, 6(5)
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Research Article
Fig. 5: K solubilization by the culture using different sugars (carbon sources)
Fig. 6: K solubilization by the culture using different proteins (nitrogen sources)
Fig.7: K solubilization by the culture using different potassium sources
Fig. 8: K solubilization by the culture at different temperatures
937 IJBPAS, May, 2017, 6(5)
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Matthew T and Tallapragada P*
Fig. 9: K solubilization by the culture at different pH
CONCLUSION
are hereby most sincerely acknowledged
This study attempted to find such fungal
for their different roles played for the
microorganisms that so contribute to the
success of this research.
solubilization to the bound insoluble form
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