Response of enzyme stoichiometric ratios and nutrient limitation to tillage depth and manure application amounts in saline-alkali farmland of Ningxia

Objectives This study investigated the effects of tillage practice and organic manure application rate on nutrient status, enzyme activities, and ecoenzymatic stoichiometry in the rhizosphere soil of saline-alkali farmland in Ningxia, China. Changes in soil microbial nutrient limitation were further quantified to provide a theoretical basis for developing high-yield, efficient, and sustainable maize management strategies in saline-alkali soils.

Methods A split-plot field experiment was conducted from 2021 to 2022 in Xidatan, Ningxia. Two tillage depths were assigned to the main plots: conventional tillage at 20−30 cm (TF) and vertical deep rotary tillage at 40−50 cm (DF). Four organic manure application rates were assigned to the subplots: O (0 kg/hm²), O1 (7500 kg/hm²), O2 (15000 kg/hm²), and O3 (22500 kg/hm²). Conventional tillage without organic manure was used as the control (CK). Rhizosphere soil organic matter (SOM), total nitrogen (TN), available phosphorus (AP), available potassium (AK), and the activities of sucrase (SUC), urease (URE), and alkaline phosphatase (ALP) were measured at key maize growth stages. Vector analysis, random forest modeling, and Mantel tests were used to evaluate the responses of soil physicochemical properties, enzyme activities, and ecoenzymatic stoichiometric ratios to different management practices, and to identify the key constraints on nutrient cycling from the perspective of microbial energy-nutrient allocation strategies.

Results Compared with conventional tillage, vertical deep rotary tillage significantly reduced soil pH, improved aggregate stability, and increased SOM, TN, AP, and AK contents. Under DF, organic fertilizers (O1, O2, O3) treatments increased SUC, URE, and ALP activities by 7.7%−31.2%, 4.2%−30.7%, and 2.7%−17.5%, respectively, compared with the O treatment. Relative to the O treatment, DF combined with O1, O2, and O3 increased the stoichiometric ratio of sucrase to urease (E_C∶N) ratio by 1.8%, 6.0%, and 4.2%, respectively on average across the two years at maturity, while the stoichiometric ratio of sucrase to alkaline phosphatase (E_C∶P) increased by 2.1%–8.0% and 2.2%–7.7% in 2021 and 2022, respectively, whereas stoichiometric ratio of urease to alkaline phosphatase (E_N∶P) varied with the variation of organic manure application rate and year. Vector analysis showed that DF combined with organic manure application alleviated microbial C∶N stoichiometric limitation over two consecutive years, whereas phosphorus limitation varied with manure application rate and year. Under DF, both vector length (VL) and vector angle (VA) increased with increasing organic manure input in 2021, indicating simultaneous microbial carbon and phosphorus limitation. In 2022, VL continued to increase whereas VA decreased, suggesting that phosphorus limitation was partially alleviated, although nitrogen limitation persisted. Random forest analysis revealed that mean weight diameter (MWD) of aggregates was the dominant factor driving shifts in nitrogen versus phosphorus limitation, as reflected by VA, whereas soil pH and ecoenzymatic stoichiometric ratios precisely regulated the intensity of carbon limitation, as indicated by VL. These results confirm that ecoenzymatic stoichiometric signals can serve as direct biological indicators of microbial nutrient limitation.

Conclusions Vertical deep rotary tillage can improve soil structure, expand the root zone of maize, and alleviate salinization and activate soil nutrients. Combining vertical deep rotary tillage with an appropriate amount of organic fertilizer can more effectively reduce rhizosphere soil pH, increase organic matter and available nutrient contents, enhance soil aggregate stability and enzyme activity, drive the enzymatic stoichiometric ratios such as soil E_C∶N, E_C∶P, and E_N∶P to balance throughout the entire maize growth period, optimize the microbial acquisition of carbon, nitrogen and phosphorus resources, and achieve precise matching between nutrient supply and crop demand. An excessively low organic fertilizer application rate causes N and P limitation during the maize growth period; while an excessively high organic fertilizer application rate leads to microbial immobilization of nitrogen and phosphorus, aggravates the imbalance of enzymatic stoichiometric ratios related to carbon, nitrogen and phosphorus cycles, and results in phosphorus limitation at the mid-growth stage of maize.