2.3.Soil sampling
On 4 July 2016, at the mid-bloom stage of alfalfa in the first planting year, soil samples were collected from mulberry and alfalfa rhizosphere soils by shaking the soil off the root. One part of the sample was used for soil analyses, while the other part was used for the determination of carbon source use via Biolog plates. For extraction, 10 g of soil were shaken with 100 mL 0.1 M Tris buffer (pH 7.5) for 10 min and centrifuged for 10 min at 2,600 g. The supernatant was diluted, and 150 mL of the 10-3 dilutions were inoculated into Biolog-Ecoplates (Biolog, Hayward, CA, USA). Each of the 96-well plates contained 31 different carbon sources separately and a blank well, each replicated three times. The plates were incubated at 28 °C, and substrate use condition was monitored by measuring absorbance via an automated plate reader (ELX808, Lab systems, Helsinki, Finland) at 590 nm every 24 h for 192 h.