Soil samples for isolation of phosphate solubilizing bacterial strains were taken from various localities of Karachi. PSBs were isolated by plating serial dilutions of this soil in the tris minimal medium as described by Shahab and Ahmed 2008. Bacterial strains producing clear and large halos were selected for future studies and were preserved in 20% glycerol.

The bacterial strains which showed efficient solubilization of zinc phosphate via plate assay method were selected for study of phosphate release (PO₄) in liquid media. The bacterial strains were grown in liquid tris minimal medium amended with insoluble phosphate compound (5 mM zinc phosphate) and incubated at 30C at 100rpm. A 10 ml aliquot was aseptically removed from each flask at each intervals of time (0, 1,2,3,4,5,6,7,8,9,10,12 days) and centrifuged at 5,000g for 15 min. the supernatant was filtered through a sterilized 0.45µm Millipore filters and assessed for free phosphate contents in the filtrate using spectrophotometry method of Murphy and Riley (1962). Distilled water 0.95ml and 0.67ml sodium molybdate was added into test tube containing 0.05ml of samples .The phosphate in the solution was visualized by adding 4ml of SnCl2 with the estimation of blue complex at 720 nm(Pharmacia LKB novaspec.11 spectrophotometer) with reference to a standard curve similarly prepared for a period of 10 days.

IAA was determined in vitro by the method of Salkowski (13). All the test strains were screened for IAA production (15). Briefly, test bacterial culture was inoculated in the nutrient broth with tryptophan.(0.1g/l) or without tryptophan incubated at 30°C. Cultures were centrifuged at 3000 rpm for 30 min. Two milliliters of the supernatant was mixed with 2 drops of orthophosphoric acid and 4 ml of reagent Salkowski (50 ml, 35% perchloric acid; 1 ml 0.5 FeCl3).

HPLC chromatograms were produced by injecting 10μl of the filtered extracts onto a – (C18, 5 μm 25x0.46 cm) in a chromatograph equipped with a differential ultraviolet detector absorbing at 280 nm. Mobile phase was methanol and water (80:20 [vol/vol]), flow rate was 1.5 ml/min, Retention times for peaks were compared to those of authentic standards added to the medium and extracted by the same procedures used with bacterial cultures. Quantification was done by comparison of peak heights.

Seeds of chick pea were surface sterilized with 95% ethanol and 2.5% sodium hypochlorite. Then washed with distilled water repeatedly up to 10 minutes. Similar sized seeds of chick pea were selected and Seeds dressing were done via O/N cultures then seeds were put onto Petri plate having underlined soaked filter paper and incubated for 4hrs at 37° C. After 4 hrs seeds, ten seeds per pot were sown at equal depth 1-cm2 sections in to plastic pots having 200g autoclaved soil. Soil in each treatment was moistened with an equal volume of autoclaved distilled water for daily watering. Lateral root and root hair formation was examined. After 22 days root and shoot length of the plants and controls were measured by using centimeter scale.

A total of 70 metal solubilizing bacterial strains were isolated from soil and tentatively identified on the basis of API kit. We selected the strain CMG851, CMG857 and CMG860 because they released maximum amount of free phosphate in the liquid media and among all the other PSBs isolated, they produced the largest halos, of approximately 20-40 mm within 3-4 days of incubation According to de Freitas et al. (1997), good solubilizers produce halos around their colonies with diameters higher than 15 mm. Since it has been reported that some strains loose their solubilizing capability after several cycles of inoculation, we corroborated the persistence of this trait in all three bacterial strains by successive subcultures.

CMG851, CMG857 and CMG860 were finally identified by 16S ribosomal RNA gene sequence homology. Genomic DNA was obtained from bacteria grown overnight at 37C in 10 ml Luria -Bertani medium .Genomic DNA was isolated by Qiagen-kit according to vendor recommendation. PCR amplification was performed with a Epicentre thermocycler (Epicentre ). Colony PCR was performed which consisted of repective bacterial colony, 2.5 µl (20 pico mole concentration) of each forward and backward primer, MasterAmp Taq DNA polymerase, 0.5µlMasterAmp Taq 10X PCR Buffer ,10µlMasterAmp 10X PCR Enhancer 15µl, 25 mM MgCl2 Solution 5µl, dNTP mix. 8µl and dH₂O 56.5µl. Thermal cycling was done by initially denaturation at 95 for 5 minutes followed by 10 cycles at 94 for 1minute. Then 52C for 30 sec and 72 for 1 min, annealing temperature was 55 for 30 sec for 30 cycles. Last elongation step was five min at 72°C. 10 µl reaction mixture was used to visualize PCR product on 1% Agarose gel .PCR product in remaining mixture was purified by Qiagen kit and sequenced by 16s primer using the ABI prism 377 automated DNA sequencer. Sequence Data obtained were analyzed by blastaligothirm (www.ncbi.nlm.nih.gov/blast/cgi)

Determination of phosphate solubilization via plate assay method was performed for 20 days. It was noted that CMG851, CMG854, CMG857 and CMG860 showed best solubilization efficiency and they continue their solubilization trait up to 20 days. CMG851 and CMG854 showed 221% and 250% phosphate solubilization efficiency after 20 days of incubation in plate assay while CMG860 showed gradual increase in the efficiency of phosphate solubilization and it showed maximum (375%) phosphate solubilization efficiency after 20 days of incubation in plate assay (Table 3). Nguen (1992) and Nautiyal (1999) also reported the solubilization of phosphate in plate assay Seshadre, 2000 reported the solubilization of phosphate up to 150 % by pseudomonas species.

Various authors reported the solubilization of zinc phosphate by bacterial isolates (Fasim et al 2002., Uzair et al 2006, Gadd et al, 1999). Presence of soluble phosphate content in liquid media was determined at different time intervals. Results are shown in figure 1. CMG851 released maximum amount of phosphate after four days of incubation with concentration of 4709 ppm which drastically decline after 5 ^th day without any further revival while after 25 days incubation it reached minimum level of soluble phosphate i.e. 432ppm.This result is in agreement with Nautiyal 1999. CMG860 released maximum amount of phosphate after six days of incubation with concentration of 2580 ppm which gradually decline and after 25 days incubation it reached minimum level of soluble phosphate i.e. 482ppm.This result is in agreement with Nautiyal1999, Tripura 2007 and chen et al 2006. CMG857 released maximum amount of phosphate after eight days of incubation with concentration of 1646ppm which gradually decline and after 25 days incubation it reached minimum level of soluble phosphate i.e. 132 ppm. This result is in agreement with Nautiyal 1999 who had reported the similar pattern of phosphate solubilization in liquid media. Lin, 2006 also reported solubilization of phosphate by bacterial filtrate in the range of 160-200ug/ml while Katiyar and Goel 2003 reported the solubilization of calcium phosphate by pseudomonas strain up to 180ug/ml.

These bacterial isolates were screened for their ability to produce plant growth regulator, IAA. Varying levels of IAA production were recorded. The range of IAA production in PSBs isolates with tryptophan was 57 μg/ml-288μg/ml. while indole butyric acid was in range of 22-34μg/ml. Similar high level of IAA production is also recorded by other researchers (Glick 1995). However, reports demonstrating production of IAA by gram positive free living soil bacteria are still lacking. Synthesis of significant amounts of IAA by the gram positive phytopathogen Rhodococcus fascians and the gram positive bacterium Bacillus amyloliquefaciens was reported by Vandeputte et al. 2005 and Idris et al 2007 respectively. These bacterial strains varied greatly in their intrinsic ability to produce IAA.

Production of IAA and phosphate solubilization by the PSBs were examined as possible contributing factors of chick pea growth promotion. Tests for production of the auxin IAA were positive for all test stimulant strains, suggesting a potential mechanism whereby these bacteria may regulate plant growth. This interpretation is in line with the well-known characteristic of certain phytohormones (e.g., auxin, ethylene) to elicit stimulatory effects on plant growth promotion. Induction of longer roots with increased number of root hairs and root laterals is a growth response attributed to IAA production by other rhizobacteria, which improves their nutrient uptake efficiency.

The symbiotic performance of three isolates (CMG851, CMG857 and CMG 860) with the chickpea shoot and root was evaluated in greenhouse experiments. At harvest, 22 days after inoculation, shoot length of plants inoculated with strain CMG860 ranked the highest and it was greater than that of the un inoculated plants. CMG860 showed significant root and shoot elongation. (Shahab and Ahmed unpublished data) Almost all lateral roots were densely covered by root hairs whereas very few or none developed on un inoculated control plants(Fig 2).

The findings of the present investigation highlighted that IAA producing bacteria from local soil could be easily isolated and may be exploited as a cheap mean of biofertilizer. However, further studies using IAA mutant strains of these bacterial strains are needed to explore the exact contribution of IAA production in the promotion of plant growth as well as the contribution of other PGP traits. There are numerous soil microflora involved in the synthesis of auxins in pure culture and soil (Barazani and Friedman 1999). Different plant seedlings respond differently to variable auxin concentrations (Sarwar and Frankenberger1994) and type of microorganisms. Substances produced by bacteria are released continuously, and, especially when they are produced on the surfaces or within the plant tissue since the bacteria grow there. It seems probable that plant growth substances produced by PSBs improve plant growth by their direct effects on metabolic processes. However, since they induce proliferation of lateral roots thus increase nutrient absorbing surfaces, this may lead to greater rates of nutrient absorption. This in turn would be expected to significantly increased the shoot and root length of the plants.

The root length increased in chik pea due to the inoculation with plant growth promoting bacteria was in the range of 50-175% as compared with un-inoculated control while increase in shoot length was in the range of 142%-180% as compared with un-inoculated control (Table 24; Figure 61, 62-63).

All three PGPRs, improves plant growth by various mechanisms which could not be mutually exclusive. It is likely that phosphate solubilizing strains might have helped in plant root proliferation and production of plant growth regulators by the bacteria at root interface which resulted in better water absorption and nutrients such as P by host plant (Barea et al., 2005; Gupta et al., 2002; Lambrecht et al., 2000).

Figure 1

Table 1: General characteristics of PSBs used in the study

Figure 2

Table 2: Indole Acetic Acid Production via HPLC by Phosphate Solubilizing Strain

Figure 3

Table 3: Effect on Root and Shoot Length by Phosphate Solubilizing Auxin-Producing Strains (After 10 days)

Figure 4

Figure1: Effect of Plant Growth Promoting Abilities of CMG860 on chick pea Seedlings (after10 days)

Keys: A = Garden soil with zinc phosphate B = Garden soil only C = CMG860 alone without zinc phosphate D = CMG860 with zinc phosphate