Phosphate Solubiliing Bacteria from Rhizosphere Soil of “ Zea mays ”.

1. Phosphate Solubiliing Bacteria from Rhizosphere Soil of “Zeamays”. YusraFarooq & AsghariBano Dept. of Plant sciences Quaid e Azam University Islamabad

2. 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

3. 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

4. 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 (Ezawaet al. 2002).

5. 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)

6. 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.

7. 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.

8. MATERIAL & METHODS

9. 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

10. 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.

11. 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.

12. 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.

13. 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).

14. 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 .

15. 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.

16. 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

17. 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 (Oresenilet al., 1999).

18. 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.

19. 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.

20. 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.

21. RESULTS

22. Table1. Morphological Characteristics of Phosphate solubilizing bacterial strains

23. photographs P1 P3 P2 P1 P3 P2

24. Table 2.Catalase and Sidrophore test for Phosphate solubilizing bacterial strains

25. Table 3. Solubilization Index of Phosphorous Solubilizing strains

26. Table 4. Bacteriocin production of Phosphate solubilizing bacterial strains

27. Table5. Effect of Inoculation of Phosphate solubilizing bacterial strains on leaf area.

28. Table 6. Effect of inoculation of Phosphate solubilizing bacterial strains on Root length, Shoot length, Root/Shoot length and Shoot/Root-length .

29. 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

30. 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 • leaf area by 42% • Root Length 37% • Shoot length 46% • Fresh root weight 80% • Fresh shoot weight 38% • Change in dry weight of root and shoot is observed by 70% and 30% respectively.

31. 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.

32. Future Prospects • There is a need for identification of isolated strains molecular basis by applying the techniques like • PCR (polymerase chain reaction), • 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.

33. QUESTIONS????????????????????????????