Soil enzymes

Soil samples were taken from each plot in the beginning of May 2011 and 2014 as a mixed sample from 14 soil cores of the top horizon (0–10 cm). Denitrification enzyme activity (DEA) was measured in 2011 only according to refs. ^242,243. Urease activity was determined in 2011 only by incubating 1 g of fresh soil with 1.5 ml of 0.08 M substrate (urea) solution at 37 °C for 2 h²⁴⁴. Released ammonium was extracted with 12 ml of a 1 M potassium chloride/0.01 M hydrochloric solution and determined by a modified Berthelot reaction. The abundance of ammonia oxidation gene of archaea, bacteria and nitrogen fixation gene in soil bacteria different functional genes (nifH, amoA) is quantified via real-time qPCR analysis in both 2011 and 2014 and averaged across years. The abundance of ammonia nitrite-oxidizing Nitrobacter bacteria and archaea was estimated in 2014 using respective amoA gene, while NS-like and NB-like NOBs were targeted by primer sets for 16S rRNA genes for NS and nxrA primers genes specific for NB²⁴⁵. The abundance of nitrite-oxidizing Nitrospira was estimated using 16S rRNA gene primer specific for Nitrospira²⁴⁵. For potential nitrification, ammonium and nitrate concentrations were measured after CaCl₂ extraction, potential nitrification was determined according to ref. ²⁴⁶ and averaged across years. Activities of the soil enzymes soil enzyme ß-glucosidase, N-acetyl-ß-glucosaminidase, and ß-xylosidase were determined according to ref. ²⁴⁷ as described in detail in ref. ²⁴⁸, using fluorescent 4-methylumbelliferone substrates (4-MUF; Sigma-Aldrich, St. Louis, USA) and a buffered solution (pH 6.1).

Open datasets from Biodiversity Exploratories: refs. ^249–252.