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.