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May 1, 2017 - (in press), 2012. [11] Rekha, R. and Sreeramulu, K. R.. (2015). Isolation, characterization and Identification of Efficient. Potash Solubilizing ...
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)

Research Article

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)

Research Article

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)

Matthew T and Tallapragada P*

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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