Introducing N2-Fixing Tree Species into Eucalyptus Plantation in Subtropical China Alleviated Carbon and Nitrogen Constraints within Soil Aggregates

Soil extracellular enzymatic activity (EEA) and extracellular enzymatic stoichiometry (EES) within aggregates indicate variations in soil-nutrient effectiveness and the nutrient requirements of microorganisms. However, the responses of soil EEA and EES after introducing N2-fixing tree species into Eucalyptus plantations are poorly understood. Therefore, we examined soils from a 15-year-old pure Eucalyptus urophylla plantation (PP) and mixed E. urophylla and Acacia mangium plantation (MP) based on the theory of EEA and EES at the aggregate scale. Aggregates were separated into four fractions using a dry-sieving procedure: >2, 1–2, 0.25–1, and <0.25 mm. We measured the EEA of soil carbon (C)-, nitrogen (N)-, and phosphorus (P)-acquiring enzymes, and examined potential factors (soil physicochemical properties, microbial biomass, and litterfall [LF]) that may influence EEA and EES. Significantly higher (p < 0.05) EEA levels in all aggregates were found in MP than in PP. The average natural logarithmic ratio of C-, N-, and P-acquiring enzyme activities in our study was 1.44:1.21:1, which deviated from the global mean ratio of 1:1:1 and implied that soil microbes were limited by C and N. Moreover, the enzyme C:N ratio (EC:N), C:P ratio (EC:P), and vector length (VL) were markedly lower (p < 0.05) in bulk soil and most aggregates in MP compared to PP, suggesting that C limitation was more serious in PP than in MP. Furthermore, while the vector angle (VA) of bulk soil and four aggregate sizes were all <45° in both the PP and the MP, they were markedly higher (p < 0.05) in bulk soil and >2 mm aggregate in MP than in PP. This indicated that mixing N2-fixing species with Eucalyptus alleviated but did not eliminate N limitation. Our study also found that nitrate nitrogen (NO3−-N), total nitrogen (TN), and microbial biomass C:P ratio (MBC:MBP) were the main factors driving changes in EEA, while LF was a key factor controlling EES (p < 0.05). Overall, introducing N2-fixing species into the Eucalyptus plantation alleviated but did not eliminate C and N limitation. The results provide specific recommendations for soil-nutrient management in Eucalyptus plantations in subtropical China.