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

Cell morphology

Colony color Colony shape Colony shine

P3 spherical transparent round Light yellow

P2 spherical transparent round Bluish Green

P1 spherical transparent round Deep orange

PHOTOGRAPHSP1

P2

P3

P1

P2

P3

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 ofColony(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 Root length/Shoot length

Shoot length/Root length

T0 24d 11.1c 2.16a 0.46b

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

Fresh Root weight/ Fresh Shoot weight

Fresh Shoot weight/ Fresh Root weight

T0 0.99c 0.75b 1.32c 0.76a

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????????????????????????????