• Soil samples were collected from the following locations of Karachi.

• Nurseries (Glushan-e-Iqbal)

• Garden soil (Safari park)

• Staff town (Karachi University)

• Agriculture fields (Malir and Gaddap)

Nutrient broth and Nutrient agar were used for isolation and purification of the bacterial strains. Tris minimal salt medium (Fasim et al., 2002) was used for the initial screening of metal solubilization activity. NBRIP (Nautiyal, 1999), Pikovskya (1948) and MPVK medium (Son et al., 2006), were also used to check solubilization activity of selected bacterial strains.

Tris minimal medium (Appendix 7) was employed for solid and liquid culture, containing the carbon source, usually D-glucose10g l^-l. All chemicals were either of Oxoid, BDH or Merck.

Soil samples from nurseries, garden soils and agricultural fields (Malir and Gaddap) were brought to the laboratory in sterilized universals. Ten gram soil was aseptically weighed and transferred to Erlenmeyer flasks containing 100 ml nutrient broth and incubated at 37°C for 24 hrs on 100 rpm. After 24 hrs, incubated samples were diluted by serial dilution method. A series of tenfold dilution was made for each sample by pippeting 1 ml aliquots into 9 ml sterile water. Each dilution of (0.1 ml) series was then spread on the nutrient agar plates and incubated at 37°C. Morphologically distinct colonies were picked and purified by streaking on nutrient agar. The selected strains were stored at 4 ˚C as stabs and at -70 ˚C in 40% glycerol.

Sadabahar plants of Karachi university staff town were picked along roots and soil. The plants were immediately brought to the laboratory in sterilized universals. Only the root portion of the plant was used for the isolation of bacteria. For this purpose, soil was removed gently by repeated washing with sterilized distilled water. The roots were then transferred to mortar and pestle and gently crushed. The paste obtained was transferred to Erlenmeyer flasks containing 100 ml nutrient broth and incubated at 37 °C for 24 hrs. A series of tenfold dilutions were made for each sample by pippeting 1ml aliquot into 9 ml sterile NaCl (0.89%). Each dilution of the series was spread (0.1 ml) on the nutrient agar plates and incubated at 37 °C for 24 hrs. Morphologically distinct colonies were picked and purified by streaking on nutrient agar plates.

All the bacterial strains were screened for solubilization activity towards the following insoluble compounds.

• Zinc phosphate

• Zinc oxide

• Calcium phosphate

All the above metal compounds were added to the tris minimal medium to give a final concentration of 5mM except for ZnO which was added to give a final concentration of 14mM. Overnight growth culture in nutrient broth was used as inoculum. A drop of the inoculum (10µl) placed on to the tris glucose agar plates amended with 5mM zinc phosphate in presence of 1% glucose and incubated at 30°C for 3 days. The plates were examined every day for metal solubilization activity by observing halos formation around colonies. The diameter of the zones of solubilization (halo formation) were observed and the efficiency of the solubilization was calculated according to the formula (Nguyen et al., 1992)

Four best Phosphate solubilizing bacterial strains were also identified by 16s ribosomal RNA gene sequence homology in addition to biochemical tests. PCR amplification was carried out using thermocycler (Eppendorf). Colony PCR was carried out and the PCR reaction mixture consisted of 2.5 µl (20 pico mole) of each forward (TAC CGC GGC TGC TGGCAC) and backward primer (TGG AGA GTT TGA TCC TGC CTC AC), MasterAmp Taq DNA polymerase; 0.5µl, MasterAmp Taq 10X PCR Buffer; 10µl, MasterAmp 10X PCR Enhancer; 15µl, 25 mM MgCl₂ Solution; 5µl, dNTP mix.; 8µl and dH2O; 56.5µl. Thermal cycling was done by initially denaturation at 96° C for10 minutes followed by 10 cycles at 94° C for 1minute. Then 52° C for 30 sec and 72° C for 1 min. Annealing temperature was 55° C for 30 sec for 30 cycles. Last elongation step was five min at 72°C. A 10 µl reaction mixture was used to visualize PCR product on 1% Agarose gel. The remaining PCR product was purified by using Qiagen purification kit and sequencing was done commercially by using universal 16sRNA primer on an automated ABI prism 377DNA sequencer. Sequence Data was analyzed by BLAST aligothirm (www.ncbi.nlm.nih.gov/blast/cgi)

The bacterial strains were grown in liquid tris minimal media to study their solubilization activity of insoluble metal compounds in liquid tris minimal medium. Three replicates (each of 250 ml ) samples of liquid tris minimal salt medium un supplemented (control), supplemented with 5mM zinc phosphate and 14mM zinc oxide were inoculated with an overnight grown pre culture of organisms (2.5% inoculum) and incubated in shakibutor (100 rpm) at 20 ˚C for 10 days.

Effect of media on the efficiency of phosphate solubilization was studied by using following media

• Pikovskaya (Pikovskaya, 1948)

• Tris minimal salt medium (Fasim et al., 2002)

• NBRIP (National Botanical Research Institute’s Phosphate growth medium) (Nautiyal, 1999)

• MPYK [(modified Pikovskya media) (Son2006)]

Effect of various/individual components of the Tris minimal media was further checked as described by Nautiyal (1999) on the efficiency of phosphate solubilization. Effect of each component was studied individually by deleting; each component one by one to note the minimum requirements of the strains for phosphate solubilization as described by Nautiyal (1999). Efficiency of solubilization was calculated as described in section 2.5.1. Experiment was done in 3 replicates and plates were incubated at 20 °C for 10 consecutive days.

The bacterial strains which showed efficient phosphate solubilization activity in plate assay were selected to study phosphate release (PO₄) in liquid media by using colorimetric method (slight modification of EPA 424 F ascorbic acid method). Briefly the bacterial strains were grown in liquid tris minimal medium amended with 5 mM zinc phosphate and incubated at 20°C at 100rpm. An aliquot of 10 ml was aseptically removed from each flask at particular intervals of time (0, 1, 2, 3, 4, 5, 6, 8, 10, 13, 15, 17, 20, 22 and 25 days) and centrifuged at 5,000g for 15 min. The supernatant was filtered through a sterilized 0.45µm millipore filter and assessed for free phosphate contents in the filtrate. Phosphate in the solution was visualized at 880 nm (UV visible thermo nicholet evolution-300). Working standards were prepared in the range of 0.01 to 6ppm from stock of 200ppm.

The primary selection criteria for the phosphate solubilizing bacterial strains, which were picked for further studies, were as follows.

• Producing clear and sharp halos of more than 20mm

• Producing halos formation with in 48 hrs.

• Continually showing the solubilization trait up to 10 generations

• Continuously showing the gradual increase in the diameter of halos formation

• Showing the solubilization of both zinc phosphate and calcium phosphate

Only ten bacterial strains (7%) were fulfilled the above mentioned criteria and they were selected for future studies

Qualitative screening of phosphate solubilization was carried out by using tris glucose agar plates amended with either zinc phosphate or calcium phosphate. All the ten bacterial strains showed solubilization of zinc phosphate up to 5mM except CMG860 which showed solubilization of zinc phosphate up to 14mM .

Ten efficient bacterial strains which showed solubilization of all tested compounds in plate assay were then grown on liquid tris minimal media to study their solubilization trait in liquid medium. Solubilization of zinc phosphate and calcium phosphate were inspected daily and clearance/transparency of the media against control was noted visually and consider as a positive sign of solubilization (Figure 64). Transparency of the media is a primary indicator of solubilization to be visualized as halos formation is considered a primary indicator of solubilization in plate assay. Although a plate represents a closer environment to the soil because of the contrary results in solid and liquid media, it is necessary to check the solubilization in both media. In plate bacteria come in contact with a limited patrticles at a time while in liquid media their tolerance as well as more thorough study was achieved.

16srRNA

Quantification of Free Phosphate Via Calorimertic Assay

Release of soluble phosphate in liquid media as a result of solubilization of zinc phosphate was determined at different time intervals. Results are shown in Figure 1 CMG860 released maximum amount of soluble phosphate in the culture broth with 4709 mg/L concentration after 6days of incubation (Figure 1). CMG851 released maximum amount of soluble phosphate in the culture broth with 2580mg/L after 4 days of incubation (Figure 1). CMG854 released maximum amount of soluble phosphate in the culture broth after five days of incubation with concentration of 1132mg/L (Figure 1). while CMG57 released maximum amount of soluble phosphate in the culture broth after 8th days of incubation with concentration of 1646mg/l (Figure 1).

Phosphate solubilization is a phenotype exhibited by many soil borne microorganisms known as PSM (phosphate solubilizing microorganisms). In the natural environment, rhizophere of different plant species interact with phosphate solubilizing bacteria (PSBs) which play an important ecophysiological role (Perez et al., 2007). In fact, they mobilize insoluble inorganic phosphates from their mineral matrix to the bulk soil where they can be absorbed by plant roots, consecutively plants supply root borne carbon compounds, mainly sugars, which can be metabolized for bacterial growth (Deubel et al., 2000; Goldstein, 1995).

Figure 1

Figure1: Release of free phosphate in the media

Figure 2

Figure 2: Auxins Production Abilities of PSBs via Standard Bioassay Method

Keys: A = CMG851 B = CMG852 C = CMG854 D = CMG855 E = CMG857 F = Control G = CMG860

Figure 3

Figure3: Effect of CMG860 on Rye Seedling in Flask (Pilot Scale Experiment)

Keys: A = Only garden soil B = Garden soil and zinc phosphate C = CMG860 + zinc phosphate + garden soil

Figure 4

Figure4: Effect of CMG860 on Rye Seedling (Pilot Scale Experiment)

Keys: A = Only garden soil B = Garden soil and zinc phosphate C = CMG860 + zinc phosphate + garden soil

Figure 5

Table1: Identification of Selected Bacterial strains via 16srRNA

Figure 6

Table2: Effect Of PSBs On Plant Growth Promotion