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Int. J. Engg. Res. & Sci. & Tech. 2014 N W Bagalkar, 2014

EVOLUTION OF PHOSPHOBACTER

FROM SALINITY AFFECTED SOIL

IN AMARAVATI DISTRICT

N W Bagalkar1*

The salinity affected soil was investigated for the study of phosphate solubilization by bacteriaand fungi. In the present study 87 soil sample were collected from salinity affected area ofAmravati district (Daryapur, Bhatkuli, and Anjangaon), among these samples, 34 samples showedthe ability to solubilize the inorganic insoluble phosphate. From the study it was observed thatthe fungi (Aspergillus spp., Penicillium spp.) have more solubilizing ability of inorganic insolublephosphate than bacteria, i.e., B. cereus, B. megaterium, Bacillus subtilis, pseudomonas spp.,Enterobacter spp., Hence the application of biofertilizer prepared by above mentioned fungishould be helpful to increase the crop yield by solubilizing large concentration of inorganic insolublephosphate.

Keywords: Phosphate Solubilizing Bacteria (PSB), Pikovaskayas agar, Biofertilizer

*Corresponding Author: N W Bagalkar � [email protected]

INTRODUCTION

Microorganisms have the ability to solubilize the

insoluble phosphates and maintain the nutrient

status of soil (Richardson, 2001). Microorganisms

are central to the soil P cycle and play a significant

role in mediating the transfer of P between

different inorganic and organic soil P fractions,

subsequently releasing available P for plant

acquisition (McLaughlin et al., 1988 and Oberson

et al., 2001). Phosphorus (P) is one of the major

essential macronutrients for plant and is applied

to soil in the form of phosphatic fertilizers. In soil

inorganic and organic forms of phosphorus is

present. The inorganic forms of the element in

1 Department of Microbiology Sant Gadge Baba Amravati University, Amravati (INDIA).

Int. J. Engg. Res. & Sci. & Tech. 2014

ISSN 2319-5991 www.ijerst.com

Vol. 3, No. 2, May 2014

© 2014 IJERST. All Rights Reserved

Research Paper

soil are compound of calcium, iron, aluminum and

fluorine. The organic forms are compounds of

phytins, phospholipids and nucleic acid which

come mainly by way of decaying vegetation.

Therefore, soils containing high organic matter

are also rich in organic forms of phosphorus

(Subbarao, 1982).

Amravati district of Maharashtra state in India

is a part of alluvial valley of Purna basin. The

district lies between latitude 20o, 37’ and 21o, 26’

N and longitudes 76o, 37’ and 78o, 27’ E in

northeastern part of Maharashtra. The district

covers an area of about 12,212 sq/km, out of

which 3053 sq. km (25%) is covered by Purna

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alluvium. The salinity-affected area is 1756 sq.

km. (58% of alluvium) and occurs along central

part of river basin, which is located in

northwestern part of the district. The salinity in

shallow as well as deep groundwater on one hand

and scarcity of surface storage due to low rainfall

on the other makes the situation worse for

management of water resources for both drinking

irrigation and industrial use.

A large portion of soluble inorganic phosphate

applied to the soil as chemical fertilizer is

immobilized rapidly and becomes unavailable to

plant (Gorstein, 1986). Microorganisms are

involved in a range of processes that affect the

transformation of soil phosphorous and are thus

an integral part of the soil phosphorous cycle. In

particular, soil microorganisms are effect in

releasing phosphorous form inorganic and

organic pools of total soil phosphorous through

solubilization and mineralization (Hilda and Fraga,

1999).

The average soil phosphorus concentration is

about 0.05 ppm and varies widely among soil.

The phosphorus concentration required by most

plants varies from 0.003 to 0.3 ppm and depends

on the crop species and level of production

maximum corn grain yield may be obtained with

0.01 ppm phosphorus, if the yield potential is low,

but 0.05 ppm phosphorus is needed under high

yield potential. Organic phosphorus represents

about 50% of total phosphorus in soil and typically

varies between 15 and 80% in most soils. Soil

organic phosphorus decrease with depth and the

distribution with depth also vary among soils. The

phosphorus content of soil ranges from about 1-

3%. Therefore, the quantity increases with

increasing organic C and N. However, the C: P,

N: P ratios are more variable among soils than

the C: N ratio, (Miller and Donahue, 1990).

Most of the Indian soil (98%) is poor in

availability phosphorus to plants (Gaur, 1987).

Agricultural soils generally contain adequate

amount of total phosphorus but the amount of

plant available phosphorous is small (Singh and

Kapoor, 1994). Therefore application of

phosphatic fertilizers is essential for optimum

crop yield. But the utilization efficiency of

phosphate fertilizers by plant is only 20-25%

largely due to its chemical fixation in soil (Dave et

al., 2003).

It is generally accepted that the mechanism

of mineral phosphate solubilization by Phosphate

Solubilizing Bacteria (PSB) strains is associated

with the release of low molecular weight organic

acids (Goldstein, 1995; Kim et al., 1997) like

formic acid, acetic acid, lactic acid, sulphuric acid

and propionic acid, which through their hydroxyl

and carboxyl group chelate the cations bound to

phosphate, thereby converting it into soluble

forms (Kpomblekou and Tabatabai, 1994).

However, P-solubilization is a complex

phenomenon, which depends on many factors

such as nutritional, physiological and growth

conditions of the culture (Reyes et al., 1999).

There is experimental evidence to support the role

of organic acids in minerals phosphate

solubilization (Haldar et al., 1990).

The present investigation is carried out to study

the performance of phosphate solubilizing

microorganism in salinity affected area if Amravati

district (Daryapur, Bhatkuli, and Anjangaon)

Though lot of work have been made on phosphate

solubilizing microorganism from different parts

of India and Maharashtra but not much work has

been done on phosphate solubilizing microorganism

or on report is available on study of phosphate

solubilizing microorganism in salinity affected

area of Amravati district (Figure 1).

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The objective of this study was to isolate and

characterized PSB from the salinity affected area

in Daryapur, Bhatkuli, and Anjangaon.in Amravati

district. As chemical fertilizers and phosphate

solubilizing biofretilizer are used by farmer to

increase the production in present days, there is

necessity to have a comparative study on

potential of native phosphate solubilizing

microorganism, which will help to diagnose the

need of phosphate solubilizing biofertilizer

application in the Amravati district (Daryapur,

Bhatkuli, and Anjangaon).

MATERIALS AND METHODS

Sample Collection

Total 87 soil samples were collected from salinity

affected area of Amravati district (Daryapur,

Bhatkuli, and Anjangaon) in sterilized container.

The soil suspension was prepared by mixing 1 g

of soil sample in 9 mL distilled water then

supernatant was discarded and soil sample was

point inoculated on previously prepared and

sterilized pikovaskaya’s agar plates. Then the

pikovaskaya’s agar plates were incubated at 28±2oC for 24-48 h. And after completion of incubation

time, Zone of phosphate solubilization was

recorded. The colonies showing clear zone of

solubilization were further subculture on

pikovaskaya’s agar plates.

Microscopic Study of Bacteria

Size, shape, arrangement and gram’s nature of

the isolates were studied for gram’s staining.

Smear was prepared from the isolated culture

on clean glass slide, heat fixed and stained. The

stained smear was observed under microscope

(Oil immersion lance-100x). The fungal isolates

were identified up to generic level based on their

colony morphology and microscopic examination

as outlined in the manual of (Gilman, 1957)

Identification of Bacterial IsolatedThrough Biochemical Test

The PSBs isolated from salt affected soils were

identified up to generic level based on

Figure 1: Map of Study Area

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morphological and biochemical tests as specified

in Bergey’s Manual of Determinative Bacteriology

(Holt et al., 1994). Biochemical test were

performed as suggested by (Garrity et al., 2001)

which included following tests Grams, IMViC

reaction, catalase test, starch hydrolysis test

oxidation fermentation test, Phenyl alanine

deamination test, Nitrate reduction, Gelatin

hydrolysis test, Urea hydrolysis test,

Dehydrogenase test, Casein hydrolysis test,

Citrate utilization test, Indol production test, Triple

Sugar Iron (TSI) test, Carbohydrate fermentation

test (Glucose, Fructose, Sucrose, Arabinose,

Mannitol , Lactose, Treholase, Galactose,

Raffinose), Motility test, Endospore staining and

capsule staining.

Phosphate Solubilization by Plate Assay

Solubilization of tricalcium phosphate was

detected in Pikovskaya’s Agar medium (Sundara-

Rao and Sinha., 1963) each isolate was point

inoculated in at the center of Pikovskaya’s Agar

plate and inoculated for 24 – 48 h the development

of clear around the colony indicated phosphate

solubilizing activity.

Observe the zone of solubilization and maser

diameter around the colony

RESULTS

On the basis of cultural character, Morphological

character and biochemical character phosphate

solubilizing bacteria was identifying. Following

character was compare with ‘BERGEY’S

MANUAL’ and all phosphate solubilizing bacteria

and fungi were identified. That is Bacillus cereus,

Bacillus megaterium, Bacillus subtilis,

pseudomonas spp., Micrococcus spp.,

Enterobacter spp., fungi (Aspergillus spp.,

Penicillium spp.)

Out of this isolate fungi (Aspergillus spp.,

Penicillium spp.) having efficiency of Phosphate

solubilization was more as compare to other

isolated phosphate solubilizing bacteria that is

(285). But Enterobacter spp. having efficiency of

Phosphate solubilization was less as compare

to other isolated phosphate solubilizing bacteria

that is (127). Efficiency of Phosphate solubilization

was determined by plate assay using

Pikovaskaya’s Agar Medium (Figure 2).

To Isolate Phosphate SolubilizingBacteria and Fungi from Amravati District(Daryapur, Bhatkuli, and Anjangaon)

• Colonies showing zone of clearance were

observed on Pikovaskaya‘s agar plates.

• The ability to solubilize precipitated phosphate

was positively exhibited by Pseudomonas spp.

Bacillus cereus, Bacillus megaterium, Bacillus

subtilis, Micrococcus spp., Enterobacter spp.,

fungi (Aspergillus spp., Penicillium spp.).

• All phosphate solubilizing bacteria and fungi

was selected and subculture on Pikovaskaya‘s

agar plates for further studies.

Determination of Efficiency of Phosphate

solubilization, solubilize by. Bacillus cereus,

Bacillus megaterium, Bacillus subtilis

pseudomonas spp., Micrococcus spp.


Penicillium spp.)

% of Efficiency of PSB was calculated by using

following formula

100lub ×

ColonyofDiameter

DiameterilizaionSo

Table 1 represents the percentage efficiency

of different PSB using Pseudomonas spp.

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Table 1: Efficiency of Phosphate Solubilization

S. No. PSB Strain Colony Diameter Solubilization Diameter / % Efficiency 48 Hr

1 Pseudomonas spp. 0.9 1.6 177

2 Bacillus cereus 1 1.6 160

3 Pseudomonas spp. 0.8 1.4 175

4 Bacillus megaterium 1.1 1.8 163

5 Aspergillus spp 0.7 2 285

6 Penicillium spp 0.8 1.6 200

7 Enterobacter spp. 1.1 1.4 127

8 Bacillus subtilis 0.9 1.4 155

9 Micrococcus spp. 0.7 1.4 200

10 Fungi (N. I.) 0.8 1.8 225

11 Fungi (N. I.) 1.1 2 180

Figure 2: Result of Efficiency of Phosphate Solubilizer

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Bacillus cereus, Bacillus megaterium, Bacillus

subtilis, pseudomonas spp., Micrococcus spp.,


Penicillium spp.)

DISCUSSION

Phosphate soluibililizing bacteria survive well in

the soils which provide organic substrates,

nutrient, minerals, adequate moisture and

tolerable environmental conditions. Efficient

phosphate solubilizer always prefers soils which

have good carbon for their survival.

From the salinity affected area of Amravati

district. Total 87 soil sample from three different

tahshil (Daryapur, Bhatkuli, and Anjangaon) were

collected and the different species of phosphate

solubilizing bacteria were isolated using

pikovaskaya’s media. Out of 87 soil samples, 34

containing the different species of Phosphate

solubilizing bacteria and fungi. Among these

isolated Phosphate solubilizing bacteria, only

Phosphate solubilizing bacteria were identified on

the basis of following tests Grams, IMViC reaction,

catalase test, starch hydrolysis test, oxidase test,

Phenyl alanine deamination test, Nitrate

reduction, Gelatin hydrolysis test, Urea hydrolysis

test, Dehydrogenase test, Casein hydrolysis test,

Citrate utilization test, Indol production test, Triple

sugar iron (TSI) test, Carbohydrate fermentation

test ( Glucose, Fructose, Sucrose, Arabinose,

Mannitol, Lactose, Treholase, Galactose,

Raffinose), Motility test, Endospore staining and

capsule staining.

Out of isolates 34 are bacteria and fungi in

which 23 are of genus Bacillus, 4 are of

pseudomonas spp. and remaining 7 are of fungi

which are fungi (Aspergillus spp., Penicillium

spp.) and other are not identified. Different species

of bacillus like Ba.cereus are 11, Ba.megaterium

are 7 and Ba.subtilis are 5 in number.

Among the bacteria the genus bacillus was

dominating than pseudomonas spp., among the

Ba.cereus is found to be the most dominating

followed by Ba.megaterium and Ba.subtilis. In

Daryapur tashil Ba.subtilis and fungi Aspergillus

spp. were most dominating than Ba.Megaterium

and Penicillium spp. whereas pseudomonas spp.

completely absent. In Anjangaon tehsil Penicillium

spp. and Ba.Megaterium is dominating followed

by pseudomonas spp. Ba.subtilis and Ba.cereus.

In Bhatkuli tehsil pseudomonas spp. is dominating

followed by Ba.Megaterium, Penicillium spp,

Aspergillus spp., Ba.subtilis and Ba.cereus.

Bilolkar et.al. (1996) reported that Bacillus spp.

was mostly dominating in the soil of Latur,

Osmanabad and Parbhani whereas

pseudomonas spp. was dominating in the soil

from Aurangabad to Nanded district.

Bhattacharya et.al. (1997) reported that

bacteria were found as the predominant

phosphate solubilizing microorganism in all

Vidarbha soil followed by fungi.

Production of enzyme like phosphates is other

mechanism of phosphate solubilization

(Rodriguez and Fraga, 1999). Aspergillus spp

isolates, showed high activity of AP at 11 M of P;

however, the production of this enzyme was under

detection limit in excess of phosphate compared

to limiting condition, which could explained that

the synthesis of alkaline phosphatase by these

bacteria was inducible in low Pi, while it was

repressed in high concentration. These results

are in concordance with solubilization activity

Pikovaskaya’s Agar, where Aspergillus spp.

isolates were strong P solubilizer. Interestingly,

Enterobacter spp. strain produced a smaller drop

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in pH value compared to others isolates. This

might suggest that this strain is capable to

solubilize phosphate by other ways than the

production of organic acid. Therefore, we found

a positive correlation between phosphate-

solubilizing capacity and phosphatase enzyme

activity.

CONCLUSION

It is concluded from the present study that all

isolated phosphate solubilizing bacteria and fungi

from salinity affected soil in Amravati district

(Daryapur, Bhatkuli, and Anjangaon) are very

useful for increasing solubilization of inorganic

insoluble phosphate. This isolate are very

important for increasing crop yield which is taken

in salinity affected soil in productivity of, cotton,

jawar, bazri, soybean (Glycine Max) etc. From

the study it was observed that the fungi

(Aspergillus spp, Penicillium spp.) have the more

solubilizing ability of inorganic insoluble phosphate

than bacteria, i.e., B.cereus, B.megaterium,

pseudomonas spp, Enterobacter spp., Hence the

application of biofertilizer prepared by above

mentioned fungi should be helpful to increase the

crop yield in salinity affected soil by solubilizing

large concentration of inorganic insoluble

phosphate.

ACKNOWLEDGMENT

Author is very much thankful to Principal of J D P

S College Daryapur for allowing working in

laboratory and available all the facilities, Dr. N V

Phirke and Dr. Nikhil Bajaj for their useful guidance

throughout the period of work.

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