PHOSPHATE SOLUBILIING
BACTERIA FROM
RHIZOSPHERE SOIL OF “ZEA
MAYS”.
Yusra Farooq & Asghari Bano
Dept. of Plant sciences
Quaid e Azam University
Islamabad
INTRODUCTION
• In the recent advances of science, it is much more
easier to appreciate
“ the biofertilizer technology as a substitute to
chemical fertilizers|”
• phosphorous solubilizing bacteria are of significant
importance
• It reduces the cost and increases the yield and
quality
CONT…
• The PSBs release phosphorous from insoluble and fixed / adsorbed
forms
• Phosphorous is desiderate nutrient in soil and a major building block
of DNA
• Storage of energy  (ADP) and (ATP)
• Purpose transporatation and mechanical work
CONT…
• The agricultural land that are heavily used are often deficient in
phosphorous. So, the addition of phosphorus fertilizers ensure that
crops will reach their full potential
• Phosphorus (P) is vitally important and major growth-limiting nutrient,
and unlike the case for nitrogen and other atmospheric gases, there is
no large or small atmospheric source that can be made biologically
available (Ezawa et al. 2002).
CONT…
• Phosphorous occurs in two forms in soil, organic and inorganic.
• Microorganisms work on a mechanism to enhance the P availability to
plants by mineralizing organic P in soil and other than it by
solubilizing precipitated phosphates (Chen et al., 2006; Kang et al.,
2002; Pradhan and Sukla, 2005)
CONT…
• Microorganisms synthesize organic acids that are used for the major
mechanism of mineral phosphate solubilization (Halder et al.,1990).
• Microbial cell and its surroundings area is acidified by the production
of organic acids like Gluconic acid, isobutyric, acetic acids ,glycolic,
oxalic, malonic, and succinic acid, have also been reported among
phosphate solubilizers.
CONT…
• The current study was designed to determine the effect of three PSB
isolated from rhizospheric soil of maize from Gilgit region and
innoculated in a Maize variety Islamabad Gold in pot experiment.
MATERIAL
& METHODS
MATERIAL & METHODS
• Rhizospheric soil was collected from the maize (Zea Mays) plant grown
at an altitude of 1600 m.a.s.l. in Gilgit.
• Soil was used for the isolation of microbes that are phosphorous
solubilizing.
• The sample collection was performed at the vegetative stage
CONT…
• The microbial isolation was carried out by taking 10gm of
Rhizospheric soil and dissolved in 90ml of distilled water.
• The full loop of soil suspension from this prepared soil suspension
was inoculated on Pikovskaya’s medium (Miller, 1972) by streak plate
technique.
CONT…
• The plates are placed in incubator for 48 hours and temperature was
adjusted at 30 0C.
• The halozone forming isolates were selected and re-streaked until the
pure cultures are obtained.
• Single colonies appearing on Pikovskaya’s agar plates were
preserved in liquid broth of Pikovskaya’s and on agar slants in cold
and dry place for further analysis.
CONT…
• Fresh cultures after 24 hour incubation on agar plates (Miller, 1972).
were used for identification of bacterial strains on the basis of colony
and cell morphology.
• The colour shine and shape of bacterial isolates was recorded after 24
hours.
CONT…
• Catalase test was also performed to detect the presence of catalase
enzyme in bacterial colonies of Phosphorous solubilizing bacterias.
• Fresh culture that is 24 hour old was used and bacterial colony was
placed on a glass slide and one drop of H2O2 (30%) was dropped on
the colony if gas bubbles appear, it is the indicator of catalase
enzyme. (McFadden, 1980).
CONT…
• Siderphore production was checked by using the chrome azurole S
agar (CAS) ( Clark and Bavoil,1994) .
• The plates of Chrome azurole S agar were prepared and spot
inoculated with bacterial isolate.
• The plates are incubated at 30◦ C for 5 days. Positive indication is the
appearance yellow – orange halo around the colony .
CONT…
• The Colony diameter, halozone and solubilization index of bacterial
isolates was measured.
• Autoclaved Pikovskaya’s media was poured into sterilized petri-plates
under sterilized and hygienic environment, when media is solidified ;
a pin point inoculation was made on agar plates under aseptic
conditions.
• The plates were incubated at 28 oC for 7 days.
CONT…
• Solubilization index indicates the ability and extent of PSM to
solubilize the insoluble phosphate and it is calculated by the formula :
the ratio of the total diameter (colony + halozone) and the colony
diameter (Edi-Premono, 1996)
• SI = colony diameter + halozone diameter
Colony diameter
CONT…
• Bacteriocin activity was detected by using a saturated culture of
indicator strain (VF39) grown in TY medium, it was diluted 10-2 and 1 ml
was mixed with approximately 25 ml of soft TY agar (0.6% wt/vol)
containing 5 mM Ca+2 that is the final concentration.
• The isolated colonies of strain to be verified for bacteriocin activity
were inoculated into soft agar within two hours after agar solidification.
• Halozones are the indicators surrounding the stab inoculated cultures.
The plates were incubated approximately for 48 hours and than result is
recorded (Oresenil et al., 1999).
CONT…
• Inoculation of selected strains on bases of solubilization index was
performed on maize plant to check the effect on growth.
• Maize seeds of variety Islamabad Gold were collected from National
Agriculture Research Centre Islamabad
• Fresh cultures were inoculated in 100ml broth media of Pikovskaya’s
media and kept on shaker for 48 hours and then centrifuged for 20
minutes at 3000rpm.
CONT…
• Supernatant was discarded and pellet was diluted with distilled water
up to the level of 100ml and the optical density was measured to be 1.
seeds are soaked in it for 2 to 4 hours.
• Prior to soaking in this bacterial diluted medium seeds were sterilized
with 10% chlorox
• After soaking seeds mere sown in pots containing sterilized soil.
CONT…
• Pots were placed in the green house of Quaid e Azam university.
Sterilized water was provided to plants. Plants are harvested after 15
days.
RESULTS
TABLE1. MORPHOLOGICAL CHARACTERISTICS OF PHOSPHATE
SOLUBILIZING
BACTERIAL
STRAINS
Bacterial
isloate
P3
Cell
morphology
spherical
Colony color
Colony shape
Colony shine
transparent
round
Light yellow
P2
spherical
transparent
round
Bluish Green
P1
spherical
transparent
round
Deep orange
PHOTOGRAPHS
P1
P3
P2
P1
P3
P2
TABLE 2.CATALASE AND SIDROPHORE TEST FOR PHOSPHATE
SOLUBILIZING BACTERIAL STRAINS
Bacterial isloate
Siderophore
Catalase
P1
-ve
-ve
P2
-ve
-ve
P3
-ve
+ve
TABLE 3. SOLUBILIZATION INDEX OF PHOSPHOROUS
SOLUBILIZING STRAINS
Bacterial isolate
Diameter of
Colony(cm)
Diameter of
Halozone(cm)
Solubilization
Index
P1
0.56
2.44
5.33a
P2
1.53
4.79
4.12b
P3
0.61
2.28
4.74c
TABLE 4. BACTERIOCIN PRODUCTION OF PHOSPHATE
SOLUBILIZING BACTERIAL STRAINS
Bacterial Isolate
Potency
Inhibition Zone
P2
+ve
9a
P1
+ve
8ab
P3
+ve
7b
TABLE5. EFFECT OF INOCULATION OF PHOSPHATE SOLUBILIZING
BACTERIAL STRAINS ON LEAF AREA.
Treatment
Leaf area
T0
7.71
T1
13.01a
T2
12.07a
T3
11.78a
TABLE 6. EFFECT OF INOCULATION OF PHOSPHATE SOLUBILIZING
BACTERIAL STRAINS ON ROOT LENGTH, SHOOT LENGTH,
ROOT/SHOOT
LENGTH
AND
SHOOT/ROOT-LENGTH
.
Treatment
Root length
Shoot length
11.1c
Root
length/Shoot
length
2.16a
Shoot
length/Root
length
0.46b
T0
24d
T1
38.2a
20.5a
1.87ab
0.54ab
T2
32b
16.5b
1.70b
0.59a
T3
28c
19a
1.69b
0.59a
TABLE 7. EFFECT OF INOCULATION ON FRESH ROOT WEIGHT, FRESH
SHOOT WEIGHT, FRESH ROOT WEIGHT/FRESH SHOOT WEIGHT RATIO AND
FRESH
SHOOT
WEIGHT/FRESH
ROOT
WEIGHT
RATIO
Treatment
Fresh Root
weight
Fresh Shoot
weight
0.75b
Fresh Root
weight/ Fresh
Shoot weight
1.32c
Fresh Shoot
weight/ Fresh
Root weight
0.76a
T0
0.99c
T1
5.16a
1.21a
4.28a
0.23c
T2
1.9b
1.1a
1.72b
0.58b
T3
1.78b
1.18a
1.52bc
0.66ab
CONT…
• all the innoculated plants have shown better growth than the control.
Among the three isolates, plants innoculated with P1 has shown
significantly better performance in
1.
leaf area by 42%
2.
Root Length 37%
3.
Shoot length 46%
4.
Fresh root weight 80%
5.
Fresh shoot weight 38%
• Change in dry weight of root and shoot is observed by 70% and 30%
respectively.
CONCLUSION
• In the present study, it is inferred that all the innoculated plants have
shown better growth than the control but the isolate P1 has shown
efficiency in tri-calcium phosphate solubilization, plants innoculated
with P1 has shown significantly better performance in root length,
shoot length, fresh root weight, fresh shoot weight, leaf area and
chage in dry weight of root and shoot.
• It is concluded that P1 may be applied in the production of
biofertilizer in areas limiting in P or P is fixed and unavailable.
FUTURE PROSPECTS
• There is a need for identification of isolated strains molecular basis
by applying the techniques like
1. PCR (polymerase chain reaction),
2. plasmid profiling and sequencing of 16S-rRNA gene
• RAPD( random amplification of polymorphic DNA), for finding the
diversity among the isolates.
• As well as bioinformatics tools can be applied for the phylogentic
analysis of these three isolates and locating cis acting elements in the
sequence.
QUESTIONS????????????????????????????