Microbial community succession drives soil delta15N dynamics through reorganization of nitrogen-cycling functional gene architecture

Soil samples were collected from the Dongting Lake watershed, a representative erosion-prone region in the red soil hilly area of southern China. The region is characterized by a subtropical monsoon humid climate with mean annual temperature of 16-18 degrees C and annual precipitation of 1300-1800 mm.Surface soil samples (0-20 cm depth) were collected from two land-use types: cropland (Chengtoushan area, Lixian County, Changde City, Hunan Province) and forestland (Mangshan National Nature Reserve, Chenzhou City, Hunan Province). For each land-use type, three sampling plots (20 m x 20 m) were established with five-point sampling method. Fresh cropland and forestland soils were mixed at a 1:1 mass ratio to prepare microbial inoculum.A microcosm incubation experiment was conducted with gamma-irradiated sterile soil (35 kGy, two times) in 150 mL glass culture flasks containing 30 g air-dried soil. Two treatments were established: (1) soil microbial suspension inoculation and (2) sterile deionized water control. Flasks were incubated at 25 degrees C under dark conditions. Destructive sampling was performed at 0, 7, 15, 30, 60, and 120 days with three replicates per treatment per time point, yielding a total of 36 samples.Total genomic DNA was extracted from soil samples stored at -80 degrees C. Bacterial communities were characterized by amplifying the V3-V4 region of 16S rRNA gene using primers 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3'). Fungal communities were characterized by amplifying the ITS1 region using primers ITS1F (5'-CTTGGTCATTTAGAGGAAGTAA-3') and ITS2R (5'-GCTGCGTTCTTCATCGATGC-3'). Sequencing was performed on the Illumina MiSeq PE300 platform at Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China).