Soil Sample

.1 Soil sampling and bacterial isolation

A total of 17 soil samples were collected from the rhizospheres of various crop plants at two sites located in Yangzhou city, Jiangsu province, China (See Table 1). The soil samples were collected from a depth of 1–15 cm, put in polythene bags, and transferred to the laboratory. One gram was taken from each soil sample and added to 10 ml of sterilized distilled water. Serial dilutions of this mixture, up to 10-7 were then prepared. Nutrient agar medium (peptone 10.0 g, beef extract 3.0 g, sodium chloride 5.0 g, agar 20.0 g per liter and final pH 7.0) was prepared and autoclaved at 121°C for 30 min. Isolation was made using pour plate method and the Plates were incubated at 37°C for 48 h. (Three plates

This test is based on removing of iron from chrome azurol S (CAS) by producing of sidrophore. Five microliters of each fresh culture was inoculated onto a plate, which was then inocubated at 28°C for 72 h. The presence of an orange halo (A change in a color of CAS from blue to orange) around a colony indicated a positive result (Schwyn and Neilands, 1987).

3.1.3 Indole-3-acetic acid production

IAA production was assessed in tryptone broth medium ( tryptone 10.0 g, sodium chloride 5.0 g in 1000 ml distilled water and final pH 7.2). The medium was dispensed into tubes and autoclaved at121°C for 15 min. Bacterial isolates cultured for 24h were inoculated into tubes, which containing 5 ml tryptone broth and incubated at 37°C for 7days. Kovac’s reagent (0.5ml) was added, and the formation of a red color in the alcohol layer was considered a positive result.

3.1.4 Phosphate

A single colony from each culture was carefully picked up with sterilized loop. Each colony was emulisified in a drop of sterilized distilled water placed on a glass slide, then mixed thoroughly to make a thin smear. The smear was air-dried, fixed by a flame, and then stained with crystal violet solution for 1-2 min. Subsequently, flooded with iodine for 1 min. and rinsed with alcohol 95% followed with tap water. Directly, the smear was stained with safranin solution for 20 sec., rinsed with tap water, and left to dry. Cells shape and Gram reaction were