Estimation of Phosphatase activity of Phosphate solubilizing bacteria based on their

characterization and its effectiveness on plant growth

ABSTRACT
Sustainable agriculture aims to minimize the use of hazardous chemical fertilizers, without
affecting the crop production. The use of microorganisms to avail the macronutrients to the plants is the
modern and traditional method used by the farmers in many countries. Phosphorus (P) is one of the
macronutrients responsible for the growth and development as it is central atom in energy currency (ATP)
also. There are some genera of bacteria responsible for the solubilization of ortho-Phosphate to avail the
crop plants in fields. The Phosphate solubilizing bacteria collectively called as PSBs have ability to
produce the phosphatase and phytase enzymes, and the micro inoculum prepared using the PSBs is an
important and environment friendly approach to increase the crop productivity without any kind adverse
effects on the fertility of soil. The present investigation aims to isolates the phosphate solubilizing
microorganisms, to characterized the bacterial isolates on the basis of morphology and biochemical
identification methods and then to estimate the phosphate solubilizing activity of the isolated
microorganisms and preparation of a cost effective, environmentally safe bio-fertilizer to maintain
biological as well as chemical balance of soil and to increase the crop production for the fulfillment of the
fundamental need of food in healthy manner.
KEY WORDS : Phosphate solubilizing bacteria, Solubilization, Characterization, biofertilizer

INTRODUCTION
Phosphorus (P) is an essential and significant
macronutrient for food production. It also plays an
important role in photosynthesis, cell division,
energy transfer and storage, respiration and other
plant functions (Dadarwal et al., 1997). Although,
only 25% of the Phosphorus applied to the soil is
available for the crops and the rest become
unavailable due to its binding to soil minerals and
elements such as calcium (Ca), magnesium (Mg),
aluminium (Al), Iron (Fe) present in soil leads to
losses in several crop yields.
This bound form of phosphorus can solubilize by
various types of microorganisms called as
Phosphate Solubilizing Microorganisms (PSM’s).
Strains from the genera Pseudomonas,
Enterobacter, Bacillus and Rhizobium are among
the most powerful phosphate solubilizers
(Rodríguez et al., 1999). It possesses the ability to
bring insoluble phosphates in the soluble forms by
secreting organic acids. These acids lower the pH
and bring about the dissolution of bound forms of
phosphorus.
These PSB’s also produce ubiquitous class of
enzymes such as phosphatase and phytase. These
enzymes help to convert Organic and inorganic
phosphorus compounds that are not readily
available to plants into a soluble form that plants
can absorb. Phosphatases are generally classified
as alkaline or acid phosphatases based on their pH.
Acid phosphatases are plant-derived hydrolases
works better under acidic conditions. Alkaline
phosphatases are hydrolases produced by bacteria
under alkaline and neutral soil conditions (Stephen
et al., 1994). Whereas, Phytases are a type of
enzyme that catalyzes the hydrolysis of phytic acid
(inositol hexakisphosphate) an indigestible,
organic form of phosphorus into a more digestible
form. Apart from this, adding phosphatase to the
soil root interface improves plant efficiency in
utilizing organic P components (Sharpley 1985).
Biofertilizers are usually carrier-based inoculants
containing beneficial microorganisms.
Incorporation of such microorganisms in carrier
material enables long-term storage, easy handling
and high effectiveness of biofertilizers (Malik et
al., 2017). Phosphorus biofertilizers, in the form of
microorganisms, can increase the availability of
phosphates for plant growth by solubilizing them.
Incorporating Phosphate solubilizing
microorganisms as a biofertilizer could reduce the
need of chemical fertilizers in crop production.
This study aims to investigate and characterize
various species of Phosphate solubilizing bacteria
found in soils and their use in plant development
in the form of biofertilizers.
MATERIALS AND METHODS
1) Isolation of Phosphate Solubilizing Bacteria
(PSB)
Rhizospheric soil sample was collected from the
Sant Gadge Baba Amravati University area in
Amravati region and kept in a sterile zip lock
plastic bag. 1 gm of soil sample was serially
diluted from 10-1 to 10-10 and plated on
Pikovskaya’s agar medium and subjected to
incubation. After 48 h of incubation, colonies
showing clear halo zone were picked, sub-cultured
and were restreaked on Nutrient agar slants for
further use.
2) Phosphate solubilization activity
The phosphate solubilizing ability of PSB isolates
were measured by phosphate solubilization index
(PSI) as given by (Akintokun et al., 2007).
PSI = Colony diameter + Halo zone diameter
Colony diameter
These isolates were inoculated on Pikovskaya’s
agar plate and incubated at 37°C for 48h. After
incubation, the diameters of colonies and clear
zone were measured and the PSI were calculated.
3) Cultural and morphological study
The cultural characters of isolates such as size,
shape, colour, margin, elevation etc were recorded.
The gram staining and microscopic study of
isolates were done according to standard method
as per Bergey’s manual of determinative
bacteriology.
4) Biochemical identification
Various biochemical tests such as IMViC, Catalase
test, Oxidase test, Carbohydrate utilization test,
Nitrate reduction test and Triple sugar iron test for
PSB isolates were performed and results were
recorded.
5) Characterization of isolates
5.(a) Organic acid production :
Organic acid production by PSB isolates was
estimated by method of (sperber 1958). PSB
strains were inoculated on Pikovskaya’s broth
followed by incubation at 30°C for 7 days. After
incubation the culture was centrifuged. 2 ml of
supernatant was taken in a flask and few drops of
phenolphthalein indicator were added and titrated
against 0.01N Sodium hydroxide (NaOH). The
volume of NaOH consumed by culture filtrate was
the total titrable acidity of the culture filtrate. The
total titrable acidity was demonstrated by ml of
0.01 N NaOH consumed by culture filtrate.
5.(b) Thin layer chromatography:
Presence of organic acid in media produced by
PSB isolates using Thin layer chromatography
described by Gupta and Sahoo (2017). Isopropanol
: 2N ammonia in 7:3 ratio was used as a solvent
system to developed chromatogram. The bacterial
culture spotted on the chromatographic paper and
allowed to run. The chromatogram was air dried
and dipped in 0.4% bromocresol green prepared in
ethanol. After air dried the Rf values of the yellow
spots of organic acids appeared on blue
background was measured and compared with Rf
values of standard organic acid for identification.
5.(c) Ammonia production:
The Phosphate Solubilizing Bacterial isolates were
tested for the production of ammonia in peptone
broth. The isolates were inoculated in 10 ml
peptone broth and incubated at 30°C for 48 h. After
incubation, 1 ml Nessler’s reagent was added to
each tube. Development of yellow to brown colour
indicates production of ammonia (Cappucino and
Sherman 1992).
5.(d) Indole acetic acid production:
The production of Indole acetic acid was estimated
by the method described by Gutierrez et al. (2009).
The isolated PSB strains were inoculated in 4 ml
LB broth developed with tryptophan (1mg/ml) and
subjected to incubation at 30°C with continuous
shaking for 5 days. After incubation, the culture
was centrifuged at 3000 rpm for 20 min. Then 1 ml
of supernatant taken into test tube in which
Salkowski’s reagent (1.2% FeCl3 in 37% sulphuric
acid) was added and incubated for 30 min at room
temperature. Then optical density was measured at
530 nm and the standard curve was created by
serially diluting IAA solution (50 mg mL−1) in LB
medium.
5.(e) Analysis of Zinc solubilization:
The Zinc Solubilization Agar media contains
(Dextrose 10.000g/L, Magnesium sulphate
heptahydrate 0.200 g/L, Potassium chloride
0.200g/L, Dipotassium hydrogen phosphate
0.100g/L, Zinc Oxide 1.000g/L) was used. ZnO
was used as a zinc source. Other than ZnO, ZnCO3
and (ZnPO3)4 are also used as insoluble zinc
sources. The isolates were inoculated into media
and incubated at 30°C for 72 h. After incubation,
the colonies that exhibited clear zone were
considered as zinc solubilizers (Sunithakumari et
al., 2016).
5.(f) Estimation of Phosphatase activity:
Phosphatase activity was estimated by the method
described by Eivazi And Tabatabai (1977). The
selected isolates of PSB based on their phosphate
solubilization efficiency inoculated in
Pikovskaya’s broth in which tri calcium phosphate
was replaced by (p-glycerophosphate) then
subjected to incubation at 28°C for 78h with
continuous shaking. After incubation, 10 ml of the
broth culture was taken and centrifuged at 4°C at
10,000 rpm for 10 minutes. Then after discarding
supernatant pellets were suspended in 5 ml sterile
distilled water. These cells provided a source of
enzymes. Take 1 ml of sample in a 50 ml flask,
add, 4 ml of modified universal buffer, 1 ml of p-
nitrophenyl phosphate solution which was made in
the same buffer and 0.25 ml of toluene and agitate
the flask for a few seconds to mix these
ingredients. Plug the flask and subjected to
incubation at 37°C. After incubation of 1 h, add
4ml of 0.5M sodium hydroxide and 1 ml of 0.5M
calcium chloride. Swirl the flask and and filtered
the content through a filter paper. Measure the
absorbance ( yellow colour intensity) of mixture at
420 nm. Later, the phosphatase activity was
determined by 1 mole of PNP released/ml of
filtrate/h.
6) Preparation and application of biofertilizer
The two strains of PSB selected for biofertilizer
preparation based on their zone of solubilization
and enzyme activity. The selected isolates were
inoculated in Nutrient broth. Peat soil, Coconut
husks and Charcoal were selected as a carrier
materials. The incubated bacterial culture(10ml) of
PSB2 and PSB6 respectively were mixed with 50g
peat soil and 50g Coconut husks(i.e. mixture a
with PSB2 and mixture b with PSB6) separately.
These two mixtures subjected to curing process for
7 days. Then the selected seeds(Mungbean and
Chickpea) were coated with prepared biofertilizer
with jaggery solution and sowed in soil and plant
growth was observed.
RESULTS AND DISCUSSIONS
1)Isolation of Phosphate solubilizing bacteria
Soil sample was collected and subjected to serial
dilution. The diluent was loaded on Pikovskaya’s
agar plate followed by incubation. The PSB with
high phosphate solubilization were isolated and
point inoculated on Pikovskaya’s medium to
obtain individual growth. These isolates were
designated as PSB1, PSB2, PSB3, PSB4, PSB5,
PSB6, PSB7, PSB8, PSB9, and PSB10 (Fig1).
2)Phosphate solubilization activity
The zone of phosphate solubilization by PSB
isolates was measured, its efficiency was
calculated and results were noted down. From the
observation table 1, it was observed that PSB 6,
PSB 2 and PSB 7 have the ability to solubilize
phosphate in large amounts.
3) Cultural and Morphological study
The isolated strains of PSB are subjected to
cultural and morphological characterization. In
which all the PSB isolates were white in colour,
had translucent colonies, most of them had entire
margin except PSB4 and PSB9 which had irregular
margin (Table 2). Again it was observed that all
PSB isolates were gram positive except PSB5 and
PSB6 which were gram negative in nature (Table
2).
4) Biochemical identification
There are various types of biochemical tests were
performed for Isolated PSB strains. As mentioned
in (Table 3), it was observed that all PSB isolates
were showed Indole test negative. Methyl red test
were positive for all PSB isolates. Voges-
prausker’s test showed negative results for all PSB
isolates. Among 10 PSB isolates PSB1, PSB2,
PSB3, PSB6, PSB8, PSB9 utilized citrate as a
carbon source while others showed negative test
for citrate utilization. The catalase test was carried
out to determine the nature of the bacteria involved
in producing the Catalase enzyme. It was observed
that all PSB isolates were catalase positive.
Oxidase test were showed positive by all PSB
isolates which determine the nature of the bacteria
involved in producing the Oxidase enzyme. All
PSB isolates showed Nitrate reduction test
negative which indicated that all isolates failed to
reduce nitrates into nitrites. Carbohydrates such as
Fructose, Sucrose, Mannitol and Lactose were
fermented by all PSB isolates by producing acid
and gas. Triple sugar iron test was negative for all
PSB isolates.
5) Characterization of isolates
5.(a) Organic acid production:
Phosphate solubilizing bacteria solubilize
insoluble phosphate into soluble form by
producing organic acids such as malic acid, acetic
acid, gluconic acid, citrate acid etc. So it is
important to estimate organic acid production by
PSB isolates thus all PSB isolates tested for
organic acid production. The isolates were
inoculated in Pikovskaya’s broth and were
centrifuged. After centrifugation, 2 ml supernatant
was taken in conical flask with Few drops of
phenolphthalein indicator and titrated against 0.01
N NaOH. The total consumption of NaOH by PSB
cultures was recorded (Table 4). The consumption
of NaOH was found higher in PSB10. Obtained
results were compared with (Padmavathi and
Swetha 2016). In their study, they inoculated
culture in Reyes basal medium containing five
phosphate sources in presence of an indicator
bromocresol green. They have obtained results
showing change in colour from blue to yellow in
medium indicated production of organic acid.
5(b) Thin layer chromatography:
Isolated phosphate solubilizing bacteria were
capable to produced organic acid according to
(Table 5) but to identify which organic acid
produced by isolates. Thin layer chromatography
was performed for all isolates. Isopropanol: 2N
ammonia in 7:3 ratio was prepared to developed
chromatogram as a solvent system. All PSB
cultures were inoculated on TLC sheet coated with
silica gel and allowed to run. Then the sheets were
dipped in 0.4% Bromocresol green followed by air
drying. PSB4 and PSB5 showed yellow spot on
blue background on silica gel sheet. The Rf value
of PSB4 and PSB5 were obtained 0.27 and 0.30
respectively which coincided with Gluconic acid
The present results were compared with (Sahoo
and Gupta 2017). In their study, they obtained Rf
value around 0.9 and 0.6 which coincided with
oxalic acid and citric acid respectively..
5.(c) Ammonia production:
Ammonia producing bacteria can mineralize
organic nitrogen in the soil into ammonia, which
can then be used by other microbes including PSB
to produce ammonium phosphate, which is a
soluble form of phosphate. Therefore PSB isolates
were tested for ammonia production in which all
isolates were inoculated in peptone broth at 30°C
for 48 h. After incubation, 1 ml Nessler’s reagent
was added in each tube. The qualitative analysis
was done by observing change in colour from
colourless to yellow and brown. Faint yellow
colour indicated ammonia production in small
amount whereas dark yellow or brown colour
indicated ammonia production in large amount. In
quantitative measurement, colorimetric analysis
was done in which it was observed that PSB 6
produced ammonia in high amount than other
isolates (Graph 2). These results were compared
with (Walpola and Yoon 2013).
5.(d) Indole acetic acid production:
The production of Indole acetic acid(IAA) by
Phosphate solubilizing bacteria can enhance root
growth and development, which in turn increases
the plant’s ability to take up nutrients, including
phosphorus. The PSB isolates were inoculated in
LB broth supplemented with tryptophan and
subjected to incubation at 30° C for 5 days. After
incubation, centrifuged it and Salkowski’s reagent
was added. After 30 min colour change was
observed from yellow to red it was because
Salkowski’s reagent react with IAA produced by
bacteria. The intensity of the color was used to
estimate the concentration of IAA produced by the
isolates by colorimetric analysis at 540 nm. In
which it was observed that PSB9 produced IAA in
high amount(i.e 79 µg/ml) than other isolates. The
present results are in line with the study of
(Walpola and Yoon 2013) who investigated with a
pot experiment conducted under green house
conditions and found IAA production 10 and 7.5
µg/ml respectively for P. agglomerans and B.
anthina. IAA induce a quick response such as Cell
elongation also a long-term response such as cell
division and differentiation within plants (Ahmad
et al., 2008).
5.(e) Analysis of Zinc solubilization:
Zinc is an essential micronutrient in plant growth
and development. Deficiency of zinc in plants can
causes stunted growth, inhibits flowering etc. But
Zinc present in soil in inorganic form which is not
uptake by plants directly therefore there are some
microorganisms which can convert this insoluble
form of Zinc in soluble which is accessible for
plants by secreting organic acids such as gluconic
acid. Adequate zinc levels are essential for proper
seed production and development and it is also an
essential plant growth regulator. Thus, PSB
isolates were tested for Zinc solubilization test in
which isolates point inoculated on Zinc
solubilization media involved ZnO as a Zinc
source and incubated. After incubation, zone of
solubilization was observed (Fig. 2). It was
observed that PSB2, PSB5, PSB6, PSB7 and PSB9
were capable to solubilized Zinc and formed clear
zone of solubilization around the colonies where
other isolates failed to solubilize zinc. These
results were compared with (Sunithakumari et al.,
2016).
5.(f) Estimation of Phosphatase activity:
Phosphatase is an enzyme released by PSB which
contribute to phosphate solubilization in efficient
manner. This enzyme hydrolyzes organic form of
phosphorus into inorganic form i.e. hydrogen
phosphate and dihydrogen phosphate before its
implementation by roots of plants. (Marschner,
1995; He et al., 2004). Among ten PSB isolates,
five were selected for estimation of phosphatase
activity based on their ability to solubilize
phosphate and these isolates were subjected to
Estimation of phosphatase activity which was
done followed by (Eivazi And Tabatabai, 1977).
The obtained results indicated that PSB6 showed
highest activity of phosphatase i.e. 26.49 And least
with PSB i.e. 17.22 (Table 7). These present results
were compared with (Goswami and Damor 2016).
In their study, they estimated phosphatase activity
of four isolates out of which they suggested the
better efficiency of strain P 12 in enzyme
production i.e. 28.50
6) Preparation and application of biofertilizer
Biofertilizers are beneficial microorganisms that
promote plant growth by increasing the
availability of nutrients to the plants. There are
various importance of biofertilizers such as it
reduces the need of chemical fertilizers, it
enhances nutrient uptake, cost effective, reduces
soil degradation, promotes sustainable agriculture
etc. Also biofertlizers are easy to handle and can
store for long duration so it can use by farmers
affordably. So to prepare biofertilizer the isolates
were selected which were capable to solubilize
phosphate in large amount and enzyme activity i.e.
PSB2 and PSB6 (Mixture a and Mixture b). Out of
2 prepared biofertilizers a and b were applied to
plants planted in second row of root trainer and
fourth row of root trainer respectively via seed
coating. It was observed that the plants applied
with biofertilizers showed more growth than
control plant.
The comparative examination was done. In which
it was observed that the growth of plants applied
with phosphatic bioferilizer were maximum than
the control plant which was uninoculated with any
PSB isolates and biofertilizer (Graph 4 and 5).
From overall discussion it was found that the
Phosphate solubilizing bacteria stimulates growth
of the roots and enhances the plant’s ability to take
up nutrients and water from soil. The use of
phosphatic biofertilizers can reduce the need for
chemical fertilizers and for farmers it is cost
effective. Overall, phosphatic biofertilizers play a
significant role in promoting sustainable
agriculture.
 Photo 1: Isolates of PSB

Table 1:- Efficiency of Phosphate Solubilization

Isolates Colony diameter Halo zone Phosphate

(mm) (mm) solubilization
efficiency
PSB 1 2 17 9.5
PSB 2 1 19 20
PSB 3 1 12 13
PSB 4 2 20 11
PSB 5 2 20 11.5
PSB 6 1 21 22
PSB 7 1 19 20
PSB 8 2 10 6
PSB 9 2 9 5.5
PSB 10 2 8 5
 Table 2:- cultural and morphological characters

Isolates Colour Size(mm) Shape Margin Elevation Opacity Gram Shape Arrangement
nature
PSB 1 White 2 Circular Entire Convex Opaque Gram Small Single
positive rods
PSB 2 White 1 Circular Entire Convex Opaque Gram Small Single
positive rods
PSB 3 White 1 Circular Entire Convex Opaque Gram Cocci Single
positive
PSB 4 White 2 Irregular Irregular Convex Opaque Gram Small Single
negative rods
PSB 5 White 2 Circular Entire Convex Opaque Gram Small Single
negative rods
PSB 6 White 1 Circular Entire Convex Opaque Gram Small Single
negative rods
PSB 7 White 1 Circular Entire Convex Opaque Gram Small Single
positive rods
PSB 8 White 2 Circular Entire Convex Opaque Gram Small Single
positive rods
PSB 9 White 2 Irregular Irregular Convex Opaque Gram Small Single
positive rods
PSB 10 White 2 Circular Entire Convex Opaque Gram Small Single
positive rods

Table 3:- Biochemical tests for phosphate solubilizing bacterial isolates

Tests PSB PSB PSB PSB PSB PSB PSB PSB PSB PSB
1 2 3 4 5 6 7 8 9 10
Indole - - - - - - - - - -
Methyl red + + + + + + + + + +
Voges - - - - - - - - - -
prausker’s
Citrate + + + - - + - + + -
Catalase + + + + + + + + + +
Oxidase + + + + + + + + + +
Nitrate - - - - - - - - - -
reduction
H2S H2S - - - - - - - - - -
production

Acid + + + + + + + + + +
Gas + + + + + + + + + +
Sucrose Acid + + + + + + + + + +
Gas + + + + + + + + + +
Fructose Acid + + + + + + + + + +
Gas + + + + + + + + + +
Mannitol Acid + + + + + + + + + +
Gas + + + + + + + + + +
Lactose Acid + + + + + + + + + +
Gas + + + + + + + + + +
Table 4: - Titrimetric analysis of organic acid production

Isolates Total NaOH

consumed(ml)
PSB 1 5.5
PSB 2 7.4
PSB 3 4.1
PSB 4 8.3
PSB 5 4.5
PSB 6 8.0
PSB 7 5.4
PSB 8 4.3
Graph 1: Production of organic acid by PSB isolates
PSB 9 4.4
PSB 10 8.9

Table 5: Optical densities of ammonia production

Isolates Optical density

at 540 nm 70
Production of IAA (ug/ml)

60
PSB 1 56 62
50 57 57 59
56 54
53 53
PSB 2 57 40 46 47
30
PSB 3 57
20
PSB 4 53 10
0
PSB 5 46
PSB PSB PSB PSB PSB PSB PSB PSB PSB PSB
1 2 3 4 5 6 7 8 9 10
PSB 6 62
PSB Isolates
PSB 7 53

PSB 8 47

PSB 9 59 Graph 2: Ammonia Production by PSB isolates

PSB 10 54
 Table 6: Optical density of IAA at 540 nm

Isolated Optical Production of

density IAA by PSB
(nm) isolates(µg/ml) 90
80

Production of IAA by PSB

PSB 1 0.18 61 70 77 77 79
74 75

isolates(µg/ml)
60 70
PSB 2 0.2 77 63 66
50 61 62
40
PSB 3 0.181 63
30
20
PSB 4 0.186 66
10
0
PSB 5 0.197 74
PSB 1PSB 2PSB 3PSB 4PSB 5PSB 6PSB 7PSB 8PSB 9 PSB
PSB 6 0.2 77 10
PSB Isolates
PSB 7 0.198 75

PSB 8 0.187 70
Graph 3: IAA Production by PSB isolates
PSB 9 0.21 79

PSB 10 0.18 62

Table 7: Estimation of phosphatase activity

Isolates Phosphate Phosphatase activity (Mg of

solubilization index P-Nitrophenol/ gm soil)
PSB6 22 26.49
PSB5 11.5 19.45
PSB7 20 22.83
PSB2 20 23.09
PSB3 13 17.22

Photo 2: Zinc Solubilization by PSB isolates Photo 3: Plant growth of germinated seeds
Table 7: Comparative analysis of plant growth of Mugbeans inoculated with the PSB2 and PSB6 isolate with control

Sr.No. Parameters Control plant Plant with Plant with

PSB 2 PSB 6
1 Plant length (cm) 18.9 27.9 32
2 Total fresh weight of plant (g) 0.149 0.580 0.572
3 Total dry weight of plant(g) 0.033 0.045 0.081
4 Shoot length (cm) 11 19.3 23.5
5 Fresh weight of shoot (g) 0.082 0.231 0.233
6 Dry weight of shoot (g) 0.014 0.039 0.026
7 No. of leaves 3 5 4
8 Fresh weight of leaves (g) 0.045 0.155 0.152
9 Dry weight of leaves (g) 0.012 0.010 0.029
10 Root length (cm) 4 3.9 4.1
11 Fresh weight of root (g) 0.006 0.009 0.020
12 Dry weight of root (g) 0.001 0.014 0.007

Graph 4: Comparative analysis of plant growth of Mugbeans inoculated with the PSB2 and PSB6 isolate with control
 Table 8: Comparative analysis of plant growth of Chickpeas inoculated with the PSB2 and PSB6 isolate with control

Sr.No. Parameters Control Plant with Plant with

plant PSB 2 PSB 6
1 Plant length (cm) 24.9 29 31.1
2 Total fresh weight of plant (g) 0.351 0.462 0.456
3 Total dry weight of plant(g) 0.030 0.052 0.05
4 Shoot length (cm) 14 27.9 30
5 Fresh weight of shoot (g) 0.079 0.242 0.255
6 Dry weight of shoot (g) 0.005 0.011 0.027
7 No. of leaves 19 25 32
8 Fresh weight of leaves (g) 0.045 0.069 0.075
9 Dry weight of leaves (g) 0.002 0.010 0.011
10 Root length (cm) 3.2 3.5 3.3
11 Fresh weight of root (g) 0.003 0.007 0.012
12 Dry weight of root (g) 0.002 0.008 0.006

Graph 5: Comparative analysis of plant growth of Chickpeas inoculated with the PSB2 and PSB6 isolate with control
CONCLUTIONS
The isolation and characterization of phosphate
solubilizing bacteria (PSB) has demonstrated their
significant potential for improving plant growth
and development which leads to involvement for
sustainable agricultural practices. Application of
Phosphatic biofertilizer on plants shows
productive effect. The use of biofertilizers can be
more cost-effective, as they can improve soil
fertility and structure of soil. Using Phosphatic
biofertilizers helps reduce dependence on
synthetic fertiliizers.
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