Temperate grassland shifted from nitrogen to phosphorus limitation induced by degradation and nitrogen deposition: Evidence from soil extracellular enzyme stoichiometry

Soil extracellular enzyme activity (EEA) and enzymatic stoichiometry (ES) can provide a good indication of changes in soil nutrient availability and microbial demands for energy and nutrients. However, across different levels of degraded grasslands, whether the responses of soil EEAs to increasing nitrogen (N) deposition are consistent remains unclear. To explore the effects of N addition on soil EEAs in natural and degraded grasslands, we conducted a 3-year N addition experiment (0, 10, 20, 30, 40 and 50 g N m(-2) yr(-1)) at four sites in temperate grassland of northern China with different levels of degradation. We measured seven soil EEAs, including carbon (C)-acquiring enzyme (beta-1,4-glucosidase (BG)), N-acquiring enzymes (beta-N-acetyl-glucosaminidase (NAG) and leucine aminopeptidase (LAP)) and phosphorus (P)-acquiring enzyme (acid phosphatase (AP)) activities. The length and angle of vectors defined by ratios of enzyme activities (BG/(NAG + LAP) vs. BG/AP) were used to indicate relative microbial investments in C- (length), and N- and P- (angle) acquiring enzymes. Our results showed that N addition significantly increased AP activity by 176%, 116%, 220% and 268%, decreased LAP activity by 39%, 70%, 67% and 99%, but increased NAG activity by 185%, 137%, 164% and 118% in non-degraded, moderately degraded, severely degraded and extremely degraded grassland, respectively. Decreasing enzyme N: P and C: P ratios and increasing vector angle with the increasing levels of degradation suggested that temperate grassland could transform into P-limitation (angles > 45 degrees) from N-limitation (angles< 45 degrees). N addition significantly decreased the enzyme N: P ratio and increased the vector angle (angles > 45 degrees), indicating that N addition further aggravated P limitation. Our findings highlight that grassland degradation would affect the responses of soil EEAs to N addition and cause nutrient limitation transformation. Soil ES might be a sensitive indicator mediated by soil microorganisms to the relative resource limitation in temperate grasslands.