## 2 Materials and Methods
### Morphological and Biochemical Characterization of PGPR
#### 2.2.4 Functionality Tests  
For functionality tests, isolates were revived and re-cultured to form a single colony. Several tests were carried out to identify the beneficial bacteria which can produce indole acetic acids (IAA), and fix nitrogen, solubilize phosphorus and potassium. 1. Nitrogen fixation ability  
For nitrogen fixation ability, nitrogen fixation bacteria (NFB) medium was prepared [48]. The composition (g/L) of the medium was: MgSO\({}_{4}\cdot\)7H\({}_{2}\)O (0.20); DL-malic acids (5.00); KOH (4.00); NaCl (0.10); K\({}_{2}\)HPO\({}_{4}\) (0.50); and CaCl\({}_{2}\) (0.02). Another component (ml/L) included was: Trace elements solution (2.00); Fe-EDTA (4.00); 5% alcoholic solution of Bromothymol Blue (2.00); and vitamin solution (1.00). For trace element composition (mg/200 mL distilled water), they included MnSO\({}_{4}\cdot\)H\({}_{2}\)O (235); NaMoO\({}_{4}\) (200); ZnSO\({}_{4}\cdot\)7H\({}_{2}\)O (24); H\({}_{3}\)BO\({}_{3}\) (280); CuSO\({}_{4}\cdot\)5H\({}_{2}\)O (8). For the vitamin solution, they included biotin (10 mg/100 mL distilled water) and pyridoxine (20 mg/100 mL distilled water). Finally, all these components were combined and the pH of the solutions was maintained at 6.8 with NaOH before sterilization. NFB media-containing petri dishes were divided into four segments to simulate the replications. The replications were done by each quarter of the agar plates getting a separate inoculum disc. One loopful of bacterial stock cultures from nutrient agar slants was transferred into 10 mL of sterile nutrient broth in a universal bottle. Later, these bottles were incubated for 18 h at 35-37 \({}^{\circ}\)C in an incubator shaker with a controlled temperature. A sterile plaque disc from filter papers was inserted into the bacterial inoculum for 30 min before being transferred into the NFB medium plate. The plate was incubated for 24 h. The nitrogen contents were measured using micro-Kjeldahl method [48]. 2. Phosphate solubilization ability  
Pikovskaya agar medium was employed to test the ability of the bacteria to solubilize phosphorus [49]. The medium was cooked using standard compositions (g/L) as follows: (NH\({}_{4}\))\({}_{2}\)SO\({}_{4}\) (0.50); glucose (10.0); Ca\({}_{3}\)(PO\({}_{4}\))\({}_{2}\) (5.00); MgSO\({}_{4}\cdot\)7H\({}_{2}\)O (0.10); NaCl (0.20); KCl (0.20); FeSO\({}_{4}\cdot\)7H\({}_{2}\)O (0.002); yeast extract (0.50), MnSO\({}_{4}\cdot\)H\({}_{2}\)O (0.02); and agar (15.0). These components were mixed and sterilized before being poured into a sterilized petri dish. Petri dishes containing Pikovskaya agar medium were segmented into four segments representing the replications. One loopful of bacterial stock cultures from nutrient agar slants was transferred into 10 mL of sterile nutrient broth in a universal bottle. The bottlewas incubated for 18 h at 35-37 \({}^{\circ}\)C in an incubator shaker with a controlled temperature. A sterile plague disc from filter papers was inserted into the bacterial inoculum for 30 min before being transferred into the Pikovskaya agar medium plate. The plate was incubated for 24 to 168 h. The ability to solubilize phosphorus was shown by a clear halo zone formation due to the organic acid production from the bacteria that were solubilizing tricalcium phosphates in the media. The presence of a clear halo zone was determined and measured every 24 h using a caliper by deducting the colony diameter from the total diameter [49]. Potassium solubilization ability  
The chosen isolates were cultured on a modified Aleksandrov agar medium in order to test the potassium solubilizing ability [50]. The composition of Aleksandrov agar medium in (g/L) was: glucose (5.00); FeCl\({}_{3}\cdot\)6H\({}_{2}\)O (0.01); MgSO\({}_{4}\cdot\)7H\({}_{2}\)O (0.50); CaCO\({}_{3}\) (0.10); Ca (H\({}_{2}\)PO\({}_{4}\))\({}_{2}\) (2.00); agar (20.0); and potassium aluminium silicate (muscovite mica) (3.00). This component was mixed and sterilized before being poured into a sterilized petri dish.