Soil extracellular enzyme stoichiometry reveals the increased P limitation of microbial metabolism after the mixed cultivation of Korean pine and Manchurian walnut in Northeast China

Korean pine (Pinus koraiensis) and Manchurian walnut (Juglans mandshurica) are the main tree species used for plantation regeneration in Northeast China, and their mixed cultivation is typically used to address a decline in stand productivity in long-term monocultures. However, little is known about the changes in the C, N and P requirements of soil microbial metabolism between monocultures and mixed plantations. In this study, we examined the effect of plantation type on the soil microbial metabolic limitations by investigating the soil extracellular enzyme activities across three plantation types (P. koraiensis monocultures, J. mandshurica monocultures, P. koraiensis and J. mandshurica mixed plantations) of the same age (32 years old). We used ecoenzymatic stoichiometry methods to reveal the patterns of microbial metabolic C, N and P limitations along a soil depth gradient of 0–10, 10–20, and 20–40 cm by collecting a total of 81 composite soil samples including three plantation types. With the increased soil depth, the activities of C-, N- and P-acquiring enzymes in mixed plantations decreased from 93.60 nmol g−1 soil h−1 to 20.52 nmol g−1 soil h−1, 33.46 nmol g−1 soil h−1 to 22.09 nmol g−1 soil h−1, and 11.80 nmol g−1 soil h−1 to 7.30 nmol g−1 soil h−1, respectively. Enzymatic vector analysis (vector angle >45°) revealed P limitation rather than N limitation of microbial metabolism across all plantation types. Of the plantations, the mixed stands had the greatest vector length (1.15 ± 0.02) and vector angle (56.91 ± 1.47). The interactions of plantation type and soil depth had significant effects on microbial metabolic C and P requirements. Changes in soil available nutrient contents and stoichiometry ratios of C, N and P across different plantation types were the two most important soil variables affecting microbial metabolic C and P requirements.