*2.3.1 Assay for siderophore production*

Siderophore productions by antagonistic bacterial isolates were tested qualitatively as described by Alexander and Zuberer [56]. 5 μl of antagonistic bacterial cell suspension (5 � 108 CFU/mL) was spot inoculated on Chrome azurol S (CAS) agar plate. The plates were then incubated at 30°C for 5 days. Development of yelloworange halo zone around the bacterial growth was considered as positive for siderophore production. Experiment was performed with a completely randomized design with 3 replications. CAS agar was prepared from 4 solutions. Solution 1 (Fe-CAS indicator solution) was prepared by mixing 10 mL of 1 mmol L�1 FeCl3.6H2O *Potential Role of Rice Plant Growth Promoting Phylloplane and Rhizospheric Bacteria… DOI: http://dx.doi.org/10.5772/intechopen.99854*

(in 10 mmol L1 HCl) with 50 mL of an aqueous solution of CAS (1.21 g L1). The resulting dark purple mixture was added slowly with constant stirring to 40 mL of aqueous solution of hexadecyl trimethyl ammonium bromide (1.821 g L1). The yielded of dark blue solution which was autoclaved, then cooled to 50°C. The entire reagent was freshly prepared for each batch CAS agar. Solution 2 (buffer solution) was prepared by dissolving 30.24 g of piperazine-N, N-bis (2-ethane sufonic acid) (PIPES) in 750 mL of salt solution containing 0.3 g K2PO4, 0.5 g NaCl and 1.0 g NH4Cl. The pH was adjusted to 6.8 with 50% (w/v) KOH, and water was added to bring the volume 800 mL. The solution was autoclaved after adding 15 g of agar then cooled to 50°C. Solution 3 contained 2 g glucose, 2 g mannitol, 493 mg MgSO4.7H2O, 11 mg CaCl2, 1.17 mg MnSO4.2H2O, 1.4 mg H3BO3, 0.04 mg CuSO4.5H2O, 1.2 mg ZnSO4.7H2O, 1.0 mg NaMoO4.2H2O in 70 mL water, autoclaved, cooled to 50°C. Solution 4 was 30 mL filter sterilized 10% (w/v) casamino acid. Finally, solution 3 added to solution 2 along with solution 4, solution 1 was added last, with sufficient.

#### *2.3.2 Assay for indole acetic acid (IAA) production*

IAA production of antagonistic bacterial isolates were carried out as per the procedure described by Patten and Glick [57]. Every isolate was grown in LB media supplemented with (0.005%) L-tryptophan and incubated in shaker at 30°C with 160 rpm for 48 h. Then bacterial culture was centrifuged at 8000 rpm for 15 min and 1 mL culture filtrate was mixed with 4 mL salkowski's reagent (1.5 mL FeCl3.6H2O 0.5 M solution in 80 mL 60% H2SO4) and the mixture was incubated at room temperature for 30 min, presence of pink color indicate qualitatively that isolate produced IAA. Formation of pink color indicated the presence of indoles [58].

#### *2.3.3 Phosphate solubilization assay by antagonistic bacterial isolates*

Phosphate solubilization was determined according to the method of Azman et al. [59]. Sterile filter papers (5.0 mm) were soaked in antagonistic bacterial cell suspension (5 108 CFU/mL) was dispensed using pipette onto sterile filter paper (6.0 mm) that was placed on National Botanical Research Institute's phosphate (NBRIP) agar plate (Glucose (10 g/L), Ca3 (PO4)2 (5 g/L), MgCl2.6H2O (5 g/L), MgSO4.H2O (0.25 g/L), KCl (0.2 g/L), (NH4)2SO4 (0.1 g/L), Bacteriological Agar (15 g/L) [60]. The plates were then incubated at 28°C for 7 days. Phosphate solubilization was assessed by observing the clear halo zone. The experiment was performed with a completely randomized design (CRD) with 3 replications.
