Soil sampling and analysis

On 14^th August 2010, ten soil cores (1.5 cm in diameter and 10 cm in depth) were randomly taken from each plot. Six of the ten cores were randomly selected to be left intact and the other four cores were first sieved through a 2 mm mesh and then through a 1 mm mesh and roots were removed at each step. All roots were collected, washed and dried at 65°C to a constant weight to measure root biomass in each plot. The six intact cores from each plot were stored in a desk refrigerator at -20°C until the incubation experiment was started to simulate the cold season in the region. Sub-samples of sieved soils were used to analyze soil moisture, total carbon and nitrogen, microbial carbon and nitrogen. Soil gravimetric moisture was obtained after oven-drying samples at 105°C for 24 hours.

Total carbon and total nitrogen were determined using an isotope ratio mass spectrometer with a Eurovector Elemental Analyzer (Isoprime-EuroEA 3000, Milan, Italy). Microbial carbon and microbial nitrogen were measured using the fumigation-extraction method with chloroform described by Vance et al. [30] and Brookes et al. [31]. In brief, fumigated and non-fumigated soils (4-g dry weight equivalent) were extracted with 20 ml of 0.5 M K₂SO₄ (soil/extractant ratio 1:5). The fumigation lasted for 16 h using chloroform. Samples were shaken for 1 h and filtered through a Whatman 42 filter paper. Soluble organic C and N in the fumigated and non-fumigated samples were determined using a Shimadzu TOC-VCPH/CPN Analyzer. Microbial carbon and microbial nitrogen were calculated using conversion factors of 2.64 for carbon [30] and 2.22 for nitrogen [31].