2.1. Soil samples collection and isolation of rhizobacteria

The study sites were located in Jimma Zone, Oromia regional state, in the southwestern part of Ethiopia. Jimma town is the capital and administrative center of the zone, located 346 km away from the capital city of Ethiopia. The town's geographical coordinates are approximately 7°41′ N latitude and 36° 50′E longitude with annual maximum and minimum temperature of 30 °C and 14 °C respectively (Alemu et al., 2011). The soil used for bacterial isolation was excavated from the 15–20 cm depth of roots of coffee and collected in sterilized plastic bags. The samples were stored at 4 °C in a refrigerator until the time of analysis.

Isolation of rhizosphere bacteria was done based on (Somasegaren and Hoben, 1994) method. In this method, 1 g of soil sample was transferred in 9 ml of sterilized distilled water and mixed by vortex shaker for 20 s. The suspension then diluted up to 10⁻⁹ dilutions. From each dilution, 0.1 ml of the diluted sample was poured onto a sterilized surface plate of nutrient agar (NA) medium supplemented with 100 μg/ml of cycloheximide to defeat fungal growth. Finally, the plate was incubated at 30 °C for 5days (Midekssa et al., 2015). Individual bacterial isolates on nutrient agar were further sub-cultured on the PVK agar medium (containing (g/l) glucose (10), Ca₃(PO₄)₂ (5), (NH₄)₂SO₄ (0.5), NaCl (0.2), MgSO4_7H₂O (0.1), KCl (0.2), yeast extract (0.2), MnSO₄.7H₂O and FeSO₄.7H₂O (0.0003 each), and agar (15)) to obtain a pure culture by streak plate method. Highly grown bacteria culture on PVK agar media was maintained at 4 °C in the refrigerator for further analysis and studies. All of the experiments were done aseptically to disinfect unnecessary microbes.