2.1 Study sites and sample collection
Four forest sites with different ages were selected at the Huaiping forest farm in Yongshou County, Shaanxi Province, China. R. pseudoacacia is the dominant tree species on the farm, which represents a natural forest protection area on the Loess Plateau (108°5’6”–108°5′11” E, 34°12’–34°50’ N). The study area has a temperature semi-arid climate, with a mean annual temperature of 11.30°C and a mean annual precipitation of 569.90 mm. The elevation ranges from 1368.3 to 1393.6 m. The main undergrowth vegetation in the four sites with different forest ages is summarized in Table S1.
Soil sampling was carried out in R. pseudoacacia plantations aged 15, 25, 35, and 45 years (designated RS15, RS25, RS35, and RS45, respectively) with gentle slopes (Table S1). Six 20 × 20-m plots for each forest age treatment were established in July, 2020, and the distance between any two plots was more than 50 m. Rhizosphere soil was defined as the soil tightly adhering to fine roots (<2 mm diameter) of R. pseudoacacia after gentle mechanical disturbance (28). After removing the litter horizon, rhizosphere soil samples were collected from the top 20 cm. Six soil cores were collected in each plot based on an “S”-shaped pattern and mixed to form one composite sample. Subsequently, each composite sample was divided into two parts. One part was immediately frozen at –20°C with dry ice and transported to the laboratory, where it was stored at –80°C until DNA extraction. The other part was air-dried for used in physicochemical analyses after transportation to the laboratory.