Soil extracellular enzyme stoichiometry (EES) is the essential predictor in nutrient status and resource limitation of soil microorganisms, whose metabolism has a vital role in biogeochemical cycling and ecosystem function. However, little is known about how N₂-fixer tree species with different planting patterns affect soil nutrient resources in terms of extracellular enzyme activity (EEA) or EES within aggregates in degraded karst ecosystems. In this study, we evaluated soil EEA and EES related to carbon (C), nitrogen (N), and phosphorus (P) cycles across two eight-year-old pure plantations of legume species [Dalbergia odorifera T. Chen (PD) and Acrocarpus fraxinifolius Wight ex Arn. (PA)] and a mixed plantation of the two tree species listed above (MP). Meanwhile, a nearby undisturbed shrubland was used as a control (CK). We concluded that the activities of C-, N-, and P-acquiring enzyme increased to different degrees in the N₂-fixer tree species stands (particularly in MP) compared to CK in all aggregates. Compared to CK, MP significantly increased by 39.0%, 54.0%, 39.3%, and 24.8% in total C-acquiring EEA, 41.1%, 60.5%, 47.8%, and 12.5% in total N-acquiring EEA, and 100.4%, 79.7%, 69.2%, and 56.4% in total P-acquiring EEA within >2 mm, 1–2 mm, 0.25–1 mm, and <0.25 mm aggregates, respectively. Furthermore, the logarithmic transformed ratio of C-, N-, and P-acquiring enzyme activities was 1.20:1.08:1, which deviated from the global ratio (1:1:1). Vector analysis of EEA showed that the vector length (VL) within aggregates was significantly lower than that of CK in all stands of N₂-fixer species except PD; while in all treatments, vector angle (VA) was <45° for all aggregate sizes, except in MP, where VA reached 45° for <0.25 mm aggregate. These indicated soil microbes were limited by C and N together. However, MP significantly alleviated microbial C and N limitation than CK (p < 0.05). There were obvious positive relationships between enzyme C:N, C:P, and N:P ratios. VL was markedly negatively linked to VA. EES was markedly related to most soil nutrients and microbial biomass stoichiometry ratios. Changes in soil EEA and EES were primarily driven by available phosphorus (AP), microbial biomass carbon (MBC), soil C:N and MBN:MBP ratios. Together, our results demonstrate the influences after introducing N₂-fixer tree species (particularly MP) for vegetation recovery on soil microbial nutrient limitation and ecological processes in aggregate level and will contribute to the development of ecological restoration practices and fertility management in degraded karst ecosystems of southwest China.

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 (E_C:N), C:P ratio (E_C: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.

The extracellular enzyme activity (EEA) and enzymatic stoichiometry (EES) of soil are useful indicators of shifts in soil nutrition and microbial resource requirements. Nevertheless, it is uncertain how the limitation of soil microbial nutrients is altered by a Eucalyptus plantation mixed with a N₂-fixing tree species. Our study examined the microbial nutrient limitation in two plantations: a pure Eucalyptus plantation (PP) and a mixed plantation (Eucalyptus and Erythrophleum fordii, MP) in rhizosphere and non-rhizosphere soils, beginning with two indicators, soil EEA and EES. In this study, the soil EEA was considerably (p < 0.05) greater in the MP contrasted to the PP, and the enzyme C:N:P ratios of the PP (1.12:1:1.10) and MP (1.07:1:1.08) both diverged from the global average EEA (1:1:1), and the deviation degree of the PP was greater. The results of the vector analysis demonstrated that the vector angle (VA) and vector length (VL) were considerably (p < 0.05) smaller in the MP contrasted to the PP. In comparison to the PP, the MP had a considerably (p < 0.05) poorer carbon quality index (CQI). Additionally, both microbial and soil properties have a considerable impact on soil EEA and EES, according to variance partitioning analysis (VPA) and redundancy analysis (RDA). In summary, our results show that the restriction of microorganisms on C and P in rhizosphere soils is usually weaker than that in non-rhizosphere soils and that the addition of N₂-fixing tree species to Eucalyptus plantations can lessen but not completely remove the restriction of soil microorganisms on C and P. Future management practices involving mixed plantations with N₂-fixing trees species could help decrease microbial nutrient limitation and promote sustainable plantations.