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Article

Effects of Litter Input on Soil Enzyme Activities and Their Stoichiometric Ratios in Sandy Soil

by
Haiyan Gao
1,2,†,
Shengnan Zhang
1,2,†,
Zhiguo Yang
1,3,
Hongbin Xu
1,
Haiguang Huang
1,3,
Chunying Wang
1,2 and
Lei Zhang
1,*
1
Inner Mongolia Academy of Forestry Sciences, Hohhot 010010, China
2
Key Laboratory of State Forestry and Grassland Administration for Sandy Land Biological Resources Conservation and Cultivation, Hohhot 010010, China
3
Inner Mongolia Duolun Hunshandake Sandland Ecosystem Observation and Research Station, Xilingol League 027300, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2025, 15(5), 1152; https://doi.org/10.3390/agronomy15051152
Submission received: 2 April 2025 / Revised: 29 April 2025 / Accepted: 1 May 2025 / Published: 8 May 2025
(This article belongs to the Section Soil and Plant Nutrition)
Browse Figures
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Abstract

:
Litter serves as a crucial source of soil nutrients in sandy land ecosystems. Soil enzyme activities and their stoichiometric ratios act as essential “bridges” linking microbial metabolism with nutrient cycling, thereby reflecting the availability of soil nutrients and the sensitivity to microbial substrate limitations. To investigate the effects of litter quality changes on soil nutrients, enzyme activities, and stoichiometric ratios in sandy land, leaf litter and surface soil were collected from four sand-fixing forests in the Mu Us Sandy Land, including YC (Corethrodendron fruticosum), NT (Caragana korshinskii), ZSH (Amorpha fruticose), and SL (Salix cheilophila). These samples were then used for indoor cultivation. Experiments with these four leaf litter types were carried out; one treatment with no litter added served as the control. Our aim was to systematically study the changing characteristics of enzyme activities related to soil carbon, nitrogen, and phosphorus with different litter inputs. The results indicate the following: (1) Compared to the control treatment with no litter added (CK), the addition of all four types of litter significantly increased soil organic carbon, total nitrogen, and alkaline nitrogen contents. The addition of NT and YC litter significantly increased dissolved organic carbon, microbial biomass carbon (MBC), and microbial biomass nitrogen (MBN). (2) The addition of the four types of litter had different effects on the soil enzyme activity, showing increasing trends overall. A chemical analysis of the enzyme activity revealed that the soil was limited in nitrogen and phosphorus. After the addition of the ZSH, NT, and YC litter, the enzymatic C/P acquisition ratio (EC/P) and enzymatic N/P acquisition ratio (EN/P) decreased significantly, alleviating the limitation of phosphorus. After the addition of the NT litter, the enzymatic C/N acquisition ratio (EC/N) increased significantly, alleviating the limitation of soil nitrogen. (3) A correlation analysis showed that the soil nutrients had varying degrees of correlation with enzyme activity and their stoichiometric ratio. The redundancy analysis results show that MBN, TN, MBC/MBN, organic carbon, and available nitrogen were key factors influencing soil enzyme activity and stoichiometric ratios. These results provide a reference for nutrient cycling during sandy soil restoration, and they provide essential data support for the development of fragile ecosystem models in the context of global change.

1. Introduction

For humans, sandy land serves not only as a focal point for ecological governance but also as a crucial barrier to sustaining regional development. Within sandy ecosystems, water is scarce and soil is poor. These ecosystems are widely distributed across arid and semi-arid regions globally and continue to face the threat of expansion. Nevertheless, China has successfully achieved an ecological reversal characterized by greening and sand retreat through measures such as the restoration of appropriate vegetation and the reconstruction of soil functions [1,2]. Litter, as a product of plant metabolism, is an important “bridge” connecting soil and plants. It is also a core part of the biogeochemical cycling of key elements, such as carbon, nitrogen, and phosphorus, in terrestrial ecosystems and crucial for the soil nutrient cycle [3,4]. Litter decomposition research has revealed the mechanisms of nutrient cycling, providing key scientific support to reverse sandy ecosystems and improve soil function and ecological stability. Conducting indoor litter addition cultivation experiments allows for the uniform regulation of the soil temperature, moisture, and pH conditions. The results have certain guiding significance and can act as reference values for future field litter decomposition experiments [5].
Soil enzymes are biocatalysts produced by plant root secretion, residue decomposition, microbial metabolism, animal activities, etc. They participate in soil organic matter decomposition, nutrient cycling, and energy metabolism and have certain ecological indicators [6,7]. The enhancement of soil enzyme activity can improve the effectiveness of soil nutrients, such as C, N, and P, by accelerating the degradation and conversion rates of organic and inorganic substances [8]. Soil enzyme activity and its stoichiometric characteristics are key biological indicators for characterizing microbial metabolic requirements and nutrient limitations. They can profoundly reflect the coupling process and equilibrium state of the carbon, nitrogen, and phosphorus cycles in ecosystems [9,10]. Litter input directly or indirectly regulates soil enzyme synthesis and activity expression by changing the substrate supply and microbial community structure, thereby affecting the stoichiometric ratio [11,12]. Soil enzyme activity stoichiometry is the ratio of soil enzyme activity related to nutrient acquisition, which describes the relationship between soil nutrient availability and microbial nutrient demand [13]. Sinsabaugh et al. (2008) [14] believed that soil enzyme activity stoichiometry can be used as an effective tool to assess the biogeochemical balance between microbial community metabolism and nutrient demand and environmental nutrient availability at a global scale. Soil enzyme activity stoichiometry is an effective indicator of microbial nutrient demand and is closely related to soil nutrient stoichiometry [15,16]. Commonly used litter quality indicators, such as carbon, nitrogen, and phosphorus, are related to soil enzyme activity [17,18]. Large changes in litter N and P content may lead to changes in N and P acquisition enzyme activity during decomposition [19] and changes in soil enzyme activity stoichiometry. When litters with different quality or stoichiometric characteristics decompose, soil microorganisms adapt to these different input nutrients by adjusting the enzyme stoichiometry of C/N, C/P, or N/P [20,21]. Existing studies have shown that in forest ecosystems, increases or decreases in litter input can significantly change the activity ratio of hydrolases (such as β-glucosidase and N-acetylglucosaminidase) and oxidases (such as peroxidase), thereby affecting the enzyme stoichiometric characteristics. For example, a field experiment in a subtropical Castanopsis carlesii forest showed that litter input could significantly increase the activities of cellulose hydrolases and leucine aminopeptidase, while also increasing the carbon and nitrogen contents of microbial biomass [11]. Similarly, litter addition in Chinese fir plantations promoted the growth and reproduction of soil microorganisms by increasing the input of organic carbon, ultimately driving an increase in soil enzyme activity [22]. In a degraded grassland ecosystem, soil extracellular enzyme activity and stoichiometry methods were used to study the effects of different litter types on soil extracellular enzyme activity and nutrient limitation. Litter type affects soil extracellular enzyme activity. Compared with Leymus chinensis, the input of Sansevieria trifasciata stimulated microorganisms to produce more soil extracellular enzymes to obtain nutrients [23].
Current research is focusing more on forest and farmland systems with rich organic matter, but insufficient attention has been paid to fragile ecosystems with high heterogeneity and a low nutrient matrix, such as sandy land. In sandy ecosystems, due to their unique physical structure and poor nutrient conditions, soil enzyme activity is more susceptible to the input of exogenous organic matter. As a result, the regulatory mechanism of litter input on soil enzyme activity may follow unique patterns [24,25]. Compared to other ecosystems, litter decomposition in sandy soils may face more significant microbial metabolic trade-offs. However, the response mechanisms of enzyme stoichiometry characteristics (such as C/N enzymes and C/P enzymes) to litter input have yet to be clarified [9]. Studies have shown that soluble organic components released during litter decomposition can accelerate the turnover of soil organic matter by activating oxidase systems (such as peroxidase) [26,27], while changes in the carbon/nitrogen stoichiometric ratio will reconstruct the microbial community structure and extracellular enzyme secretion strategy [10,28]. For example, an increase in the litter carbon/nitrogen ratio reduces the energy flux and metabolic efficiency of soil microbial food webs but increases the uniformity of energy distribution [10]. Recent research on sandy ecosystems has focused on the improvement effects of litter on soil physical and chemical properties, but there is still a gap in understanding the response pathways of enzyme activity and its stoichiometric ratio to litter input [26,28]. This study selected the leaf litter of four common sand-fixing plant species, namely, Corethrodendron fruticosum, Caragana korshinskii, Amorpha fruticose, and Salix cheilophila. We hypothesized that (1) litter input in sandy ecosystems may promote the, alkaline phosphatase, leucine aminopeptidase, and polyphenol oxidase activities in sandy soils, and (2) different types of litter input can enhance soil enzyme activity and improve soil chemical properties, thereby causing the stoichiometric ratio of soil enzyme activity to shift in a direction that is favorable for litter decomposition. We systematically analyze the response of soil enzyme activity and its stoichiometric ratio to different litter inputs in sandy land. This will not only help improve the carbon and nitrogen cycle theory of arid ecosystems but will also provide a theoretical basis for litter management after vegetation restoration in desertified land, which is of great significance for improving the carbon sink function.

2. Materials and Methods

2.1. Sampling Location

The soil and litter used in this study were collected from the Ulan Tologai Desert Control Station (37°28′~39°21′ N, 107°23′~110°30′ E) in Ordos, Inner Mongolia, China (Table 1). Located on the southern edge of the Mu Us Desert, the soil type is aeolian sandy soil, with fixed dunes, semi-fixed dunes, mobile dunes, and inter-dune lowlands mosaic distribution. The average altitude is about 1200 m, with a temperate continental semi-arid climate; an average annual temperature of 6.0~8.5 °C; an annual precipitation of 250~440 mm, mostly concentrated in July–September; an annual evaporation of 1800~2500 mm; an average annual wind speed of 4.8 m·s−1; and vegetation types mainly composed of artificial shrubs and trees.

2.2. Experimental Design

In early July 2024, four plots were selected for restoration, each consisting of 10-year-old plants of the following species: Corethrodendron fruticosum (YC), Caragana korshinskii (NT), Amorpha fruticose (ZSH), and Salix cheilophila (SL). Leaves of the four types of plants were collected and brought back to the laboratory for drying. To eliminate the influence of the physical size of the leaves on the research results, they were cut to 2 mm and used as litter. In late July, soil samples from the 0–10 cm soil layer were collected from four plots. To ensure the consistency of the soil in each treatment for indoor cultivation, the samples were fully mixed and placed in a 4 °C car refrigerator. Then, they were brought back to the laboratory and sieved through a 2 mm sieve to remove large plant, animal, and litter residues. Next, 500 g of fresh soil samples was weighed and put into 1000 mL culture bottles. The water content was adjusted to 60% of the field water holding capacity, and the bottles were placed in a 25 °C incubator for pre-cultivation for 7 days. During this period, the missing water was supplemented using the weighing method every 3 days. Litter was added after the microorganisms had recovered and stabilized. The addition rate was 2 g/100 g dry soil. The plant samples were added and mixed thoroughly with the soil, with no litter added in the control treatment, for a total of 5 treatments. Each treatment was repeated 3 times, for a total of 15 culture bottles. The soil chemical properties and enzyme activity were measured after the 108th day of cultivation.

2.3. Research Methods

2.3.1. Determination of Litter Chemical Properties

The total carbon (PTC) content of the plants was determined using the potassium dichromate method, the total nitrogen (PTN) content was determined using the salicylic acid–zinc powder reduction method, and the phosphorus (PTP) content was determined using the vanadium–molybdenum yellow colorimetric method. The specific experimental operation steps were based on the research method of Bao Shidan [29].
The initial chemical contents of the leaves of the four litters are shown in Table 2. There was no significant difference in the total carbon content of the leaves of the four litters. The total nitrogen content of NT and ZSH was significantly higher than that of SL. The total phosphorus content of ZSH was significantly higher than that of other plants.

2.3.2. Soil Chemical Properties Determination

The soil organic carbon (OC) content was determined using the potassium dichromate volumetric method-external heating method. Total nitrogen (TN) content was determined by the Kjeldahl method. The total phosphorus (TP) content was determined by the HClO4-H2SO4 method. The determination of the available nitrogen (AN) content was carried out with the sampling alkaline diffusion method. The specific experimental operation steps refer to the research method of Bao Shidan [29]. The chloroform fumigation-extraction method was used to determine soil microbial biomass carbon (MBC) and microbial biomass nitrogen (MBN). The soluble organic carbon (AC) content was determined according to the method of Jones et al. [30], using a TOC (Wuhan, China) total organic carbon analyzer.

2.3.3. Soil Enzyme Activity Determination and Stoichiometric Ratio Calculation

Soil cellulose (CBH), β-1,4-xylosidase (BX), β-1,4-glucosidase (BG), leucine aminopeptidase (LAP), β-1,4-acetylglucosaminidase (NAG), alkaline phosphatase (ALP), catalase (CAT), and polyphenol oxidase (PPO) were determined with the microplate method using a multifunctional microplate reader. For specific operation steps, refer to the kit instructions (Suzhou Gres Biotechnology Co., Ltd., Suzhou, China). Cellulose, β-1,4-xylosidase, and β-1,4-glucosidase were used as the C-acquisition enzymes; leucine aminopeptidase and β-1,4-acetylglucosaminidase were used as the N-acquisition enzymes; and alkaline phosphatase was used as the P-acquisition enzyme.
The soil enzyme stoichiometric ratios (E) were calculated as follows [31]:
EC/N = ln(CBH + BX + BG)/ln(LAP + NAG)
EC/P = ln(CBH + BX + BG)/lnALP
EN/P = ln(LAP + NAG))/lnALP
where CBH is soil cellulose, BX is β-1,4-xylosidase, BG is β-1,4-glucosidase, LAP is leucine aminopeptidase, NAG is β-1,4-acetylglucosaminidase, and ALP is alkaline phosphatase.
A vector analysis of the soil enzyme activity was used to predict the microbial nutrient limiting factors. The following formulas were used [31]:
V L = X 2 + Y 2
V A = D e g r e e s [ a t a n ⁡ ( 2 X , Y ) ]
where
X = C B H + B X + B G ( C B H + B X + B G ) + L A P
Y = C B H + B X + B G ( C B H + B X + B G ) + ( N A G + L A P )
where VL represents the vector length, VA represents the vector angle, and VL represents the microbial C limit. The longer the length, the greater the limit. A VA less than 45° indicates a relative nitrogen limitation, while a VA greater than 45° indicates a relative phosphorus limitation. X represents the activity of the C-acquisition enzyme relative to the P-acquisition enzyme, and Y represents the activity of the C-acquisition enzyme relative to the N-acquisition enzyme.

2.4. Statistical Analysis

The Excel 2010 software was used for data collation, and the SAS 9.1 software was used for variance analysis (ANOVA procedure). The Origin 2021 software was used for mapping, and the Canoco 5 software was used for redundancy analysis of the correspondence between soil enzyme activity and chemical properties.

3. Results

3.1. Effects of Litter Input on Soil Physical and Chemical Properties

As shown in Figure 1, the litter type significantly affected the soil chemical properties. TN and AN increased significantly to varying degrees compared to the CK with the addition of the four types of litter. The OC content in the SL, ZSH, and NT treatments was significantly higher than that in the CK and YC treatments. The AC content in the ZSH, NT, and YC treatments was significantly higher than that in the SL and CK treatments. The TP content in the SL treatment was significantly higher than that in the YC treatment. The C/N and C/P in the SL and ZSH treatments were significantly higher than those in the CK, NT, and YC treatments. The N/P in the ZSH and YC treatments was significantly higher than that in the other treatments.

3.2. Effects of Litter Input on Soil Microbial Biomass and Its Stoichiometric Ratio

As can be seen in Figure 2, after litter input, soil microbial biomass carbon showed an increasing trend to varying degrees, with significantly higher values in the SL, NT, and YC treatments than in the CK treatment. The MBN in the SL and ZSH treatments was significantly lower than that in the CK treatment and significantly higher in the NT and YC treatments than in the CK treatment. The carbon/nitrogen ratio of microbial biomass in the SL, ZSH, and YC treatments was significantly higher than that in the CK treatment.

3.3. Effects of Litter Input on Soil Enzyme Activities and Their Stoichiometric Ratios

Figure 3 presents the changes in soil enzyme activity with different litter inputs. Six hydrolases and two oxidases related to the C, N, and P cycles were measured. The CBH activity showed an increasing trend with litter input and was significantly higher in the SL, NT, and YC treatments than in the CK and ZSH treatments. The BX activity increased significantly in the ZSH and YC treatments compared to the other treatments and decreased significantly in the SL and NT treatments compared to the CK treatment. The BG activity increased significantly after the input of the four litter types compared to the CK. The activity of LAP increased significantly under the SL, ZSH, and YC treatments compared to the CK and NT treatments, and it decreased significantly under the NT treatment compared to the CK treatment. The NAG activity and alkaline phosphatase activity under the ZSH, NL, and YC treatments were significantly higher than those under the CK and SL treatments. The CAT activity in the SL and ZSH treatments was significantly higher than that in the other treatments. The PPO activity was significantly higher with the four litter additions than in the CK treatment, but there were no significant differences among them.
In addition, the calculations of the stoichiometric ratios of enzyme activities related to the C, N, and P cycles (Figure 4) show that the litter input had a significant effect on the stoichiometric ratios of soil enzymes. Compared to the CK, the soil enzyme C/N ratio decreased significantly by 3.15%, and the soil enzyme N/P ratio increased significantly by 7.93% under the SL treatment. Under the ZSH treatment, the soil enzyme C/N and soil enzyme C/P ratios were significantly reduced by 1.04% and 1.63%, respectively. The C/N ratio of soil enzymes in the NT treatment increased significantly by 5.64%, and the N/P ratio of soil enzymes decreased significantly by 8.54%. Under the YC treatment, the soil enzyme C/P and soil enzyme N/P ratios were significantly reduced by 7.91% and 9.36%, respectively. The stoichiometric carrier characteristics of the soil enzymes (Figure 5) show that the VL of YC was significantly reduced, and the VA was significantly increased, indicating that the addition of YC leaf litter significantly reduced the limitation of C but increased the relative limitation of P. The VA of SL was significantly lower than that of the CK, while the VA of NT was significantly higher than that of the CK, indicating that SL was relatively more restricted by N, while NT was relatively more restricted by P.

3.4. Effects of Soil Factors on Enzyme Activity and Enzyme Stoichiometric Ratio

From the redundancy analysis (RDA) of the soil chemical properties, soil enzyme activity, and stoichiometric ratios (Figure 6), it was found that the CBH and MBN were extremely significantly positively correlated. BX was significantly positively correlated with OC, TN, C/P, and N/P and extremely significantly negatively correlated with MBN. BG was extremely significantly positively correlated with TN, significantly positively correlated with AN and AC, and significantly negatively correlated with TP. LAP had a very significant positive correlation with OC, TN, C/N, C/P, and MBC/MBN and a very significant negative correlation with MBN. NAG was significantly positively correlated with AC, AN, MBN, and MBC; significantly negatively correlated with OC and C/N; and significantly negatively correlated with MBC/MBN. ALP was extremely significantly positively correlated with AC, AN, and MBC; significantly positively correlated with the N/P and MBN; and significantly negatively correlated with TP. CAT had an extremely significant positive correlation with OC, C/N, and C/P; a significant positive correlation with MBC/MBN; and an extremely significant positive correlation with MBN and MBC. PPO had a very significant positive correlation with AN and MBC. VL was significantly positively correlated with TP; significantly negatively correlated with TN; and extremely significantly negatively correlated with the N/P. VA had a very significant positive correlation with AC, AN, MBN, and MBC and a very significant negative correlation with MBC/MBN. The EC/N had a very significant positive correlation with MBN; a significant positive correlation with AC and AN; and a very significant negative correlation with MBC/MBN. The EC/P and EN/P showed a very significant negative correlation with AC, AN, MBN, and MBC; the EC/P showed a significant positive correlation with TP and MBC/MBN; and the EN/P showed a very significant positive correlation with MBC/MBN.
Figure 6 shows that the explanations for the soil enzyme activity and stoichiometric ratios along the I and II sorting axes are 51.95% and 29.46%, respectively. Therefore, the cumulative explanation of the correlation between the soil chemical properties and the soil enzyme activity and stoichiometric ratios reaches 81.41%. This indicates that the first two axes cover most of the information regarding the soil chemical properties, soil enzyme activities, and their stoichiometric ratios, suggesting that the first axis plays an important role. To determine the importance of the soil chemical properties on soil enzyme activity, the Monte Carlo test was performed to rank the soil chemical properties (Table 3). The results show that the influence of the soil chemical properties on the soil enzyme activity and stoichiometric ratio was as follows: MBN > TN > MBC/MBN > OC > MBC > AN > C/N > AC > N/P > TP > C/P. The effects of MBN, TN, MBC/MBN, and OC content on soil enzyme activity reached an extremely significant level (p < 0.01), and the effect of AN content on soil enzyme activity reached a significant level (p < 0.05). This shows that MBN, TN, MBC/MBN, OC, and AN contents are key factors explaining the differences in soil enzyme activity and stoichiometric ratios.

4. Discussion

4.1. Effects of Litter Input Changes on Soil Chemical Properties

Litter decomposition is an important part of nutrient cycling and energy flow and is one of the main processes that maintain ecosystem functions [32]. This study found that the contents of soil organic carbon, soluble organic carbon, total nitrogen, and alkaline nitrogen showed overall increasing trends under the addition of different litter types (Figure 1), providing a better nutrient matrix for microbial growth and promoting the accumulation of microbial biomass carbon content, thereby optimizing the metabolic environment of the microbial community and stimulating its biomass growth [33,34]. Li et al. [32] used Picea asperata leaf litter input to promote soil nutrient improvement. The study by Shen et al. [35] showed that adding any type of litter would significantly increase the soil organic carbon and total nitrogen, but the effect varied depending on the type of litter used. Lu et al. [36] studied Castanopsis carlesii plantations and found that litter input significantly increased soil total nitrogen, available nitrogen, effective phosphorus, and microbial biomass carbon and nitrogen. However, a study by Wei et al. [37] showed that the addition of litter reduced the contents of total carbon, total nitrogen, and soluble organic nitrogen in the soil. The study by Lu et al. [36] showed that the addition of litter showed a significant increasing trend of carbon and a significant decreasing trend of nitrogen content in microbial biomass. This may be related to the soil environment. (1) Sandy soil microorganisms are in a metabolically inhibited state. Exogenous carbon input activates the starvation-limited microbial community, prompting it to accelerate the secretion of extracellular enzymes, such as cellulose hydrolase and β-1,4-glucosidase, thereby accelerating the decomposition of stubborn soil organic matter and promoting the mineralization of soil organic carbon [32]. (2) Litter input releases soluble carbon and nitrogen compounds, which triggers a surge in the metabolic activity of soil microorganisms and triggers a positive stimulation effect [38].
Soil carbon, nitrogen, and phosphorus maintain functional balance through steady-state interactions. Their stoichiometric ratios can reflect the nutrient relationships and balance among the three elements [39,40]. C/N is closely related to soil organic matter decomposition and nutrient cycling and positively correlated with nitrogen fixation efficiency [41]. When the soil C/N is lower than 25, the conversion rate of organic matter is optimal, and nitrogen in excess of that required for biological growth will be released into the soil [42]. In this study, after the addition of ZSH and SL leaves, the C/N ratio increased significantly compared to the CK. The soil C/N in the ZSH treatment was greater than 25; in the SL treatment, it was less than 25; and in the NT treatment, it increased to 12.94 (Figure 1), indicating the greater limitation of total nitrogen after the addition of litter. The differences in content between the different treatments indicate that it was affected by the C and N contents of the litter itself. The soil C/P ratio reflects the availability of phosphorus in the soil. A high C/P ratio indicates that the availability of soil phosphorus is low [43]. This study showed that the C/P ratio increased to varying degrees after litter addition. With the SL and ZSH treatments, it increased significantly (Figure 1), indicating that the addition of litter increased the soil carbon content, indirectly reducing the effectiveness of phosphorus. The soil N/P ratio can be used as a basis for determining whether nitrogen or phosphorus is insufficient in the soil habitat and is one of the indicators for measuring nutrient limitation [44,45]. In this study, the soil N/P after adding litter leaves was between 0.65 and 1.04. Although it was improved to varying degrees compared to the CK, it was lower than the national soil C/N ratio (5.20) [46]. Related studies have found that when the soil N/P is less than 14 or greater than 16, the ecosystem has limited nitrogen or phosphorus, respectively [40,47]. The results of this study also show that the soil N/P is improved to a certain extent after litter addition, with the ZSH showing significant improvement, though the nitrogen was still limited.

4.2. Effects of Litter Input Changes on Enzyme Activities and Stoichiometric Ratios

Soil enzymes are a key component of maintaining soil ecological functions and energy flow. They characterize the microbial metabolic status, respond to environmental disturbances, regulate material transformation, and drive carbon, nitrogen, and phosphorus cycles [48]. Changes in their activity can comprehensively reflect the effectiveness of soil nutrients, energy flow efficiency, and the health of ecological functions [49]. CBH, BX, and BG are enzymes that characterize soil carbon transformation. These three enzymes synergistically accelerate the transformation of plant residues into humus, achieving step-by-step decomposition from macromolecular polysaccharides to soluble carbon sources. By releasing carbon sources and energy, they regulate the composition and function of soil microbial communities, increase soil organic matter content and structural stability, and maintain the dynamic balance of soil nutrient cycles [50,51]. This study found that after litter addition, the CBH and BG increased to varying degrees, while the BX of the SL and NT treatments decreased significantly (Figure 3). A possible reason for this is that litter addition changed the carbon source structure and microbial community function. Microorganisms may prioritize the decomposition of easily degradable cellulose, while the decomposition demand of xylan lags behind. In addition, the glucose produced by cellulose decomposition may reduce the activity of xylosidase through feedback inhibition [52]. LAP and NAG are enzymes that characterize the soil nitrogen cycle and provide more nitrogen sources for microorganisms to decompose organic matter. When litter was added, the LAP content with the SL and ZSH treatments increased, while the NAG content decreased. The NAG content with the NT treatment increased, while the LAP content decreased. The LAP and NAG contents both increased significantly with the YC treatment (Figure 3). A possible reason for this is that the leaves of SL and ZSH litter are rich in easily decomposable sugars and proteins, which preferentially stimulate bacterial proliferation. In turn, this promotes the secretion of LAP, which quickly decomposes the proteins to obtain nitrogen sources, leading to the “high LAP activity” characteristic. At the same time, the antibacterial substances (such as bacteriocins) produced by bacteria inhibit the activity of fungal communities that rely on NAG, resulting in a decrease in NAG content. The leaves of NT litter contain more cellulose and chitin, which are more difficult to decompose. This type of carbon source is more conducive to fungal colonization, stimulating fungi to secrete NAG, which decomposes chitin to obtain nitrogen sources, leading to a “high NAG activity” response [53,54,55]. Polyphenol oxidase decomposes phenolic organic compounds (such as lignin, tannin, etc.), which releases carbon dioxide, water, and small soluble molecules (such as ammonium nitrogen), thereby improving the availability of soil nutrients and accelerating the mineralization process of organic carbon. The activity of polyphenol oxidase increased significantly after litter was added. A possible reason for this is that litter (such as leaves of SL, ZSH, and other plants) contains high concentrations of phenolic compounds. These substances are secondary metabolites of plants and have potential toxicity or inhibitory effects on microorganisms. Polyphenol oxidase decomposes phenolic substances through oxidation, reducing their toxicity and providing available carbon sources for microorganisms. After the litter was added, the accumulation of phenolic substances directly stimulated the microorganisms to secrete polyphenol oxidase in response to environmental pressure. Additionally, sandy soils are low in nitrogen. The carbon source provided by litterfall may intensify the competition between microorganisms for nitrogen in organic matter. Polyphenol oxidase decomposes nitrogen-containing phenolic substances (such as tannins) and releases soluble nitrogen compounds (such as ammonium nitrogen), thus alleviating nitrogen limitation pressure [56].
The stoichiometric ratios of soil enzymes in global ecosystems are typically a C/N ratio of 1.41, a C/P ratio of 0.62, and a N/P ratio of 0.44 [57]. This study showed that an EC/N lower than 1.41 indicated that the sandy soils were nitrogen-limited; an EN/P higher than 0.44 indicated that the soil nitrogen was less efficient than the phosphorus; and an EC/P higher than 0.62 indicated that the phosphorus availability in the soil was insufficient (Figure 4). In addition, the optimum angles of soil enzyme activity in the ZSH, NT, and YC treatments were all greater than 45° (Figure 5), which also indicates that the soil was mainly limited by the P element. The corresponding microorganisms needed to obtain more ALP to obtain the available phosphorus. After the input of the ZSH, NT, and YC litter, the ALP increased significantly (Figure 2), and the EC/P and EN/P decreased significantly, alleviating phosphorus limitation in the soil. This is consistent with the results of most studies [11].

4.3. Main Factors Affecting Microbial Enzyme Activity and Its Stoichiometric Ratio

The growth of soil microorganisms is affected by soil nutrients and indirectly affects soil enzyme activity, resulting in different correlations between soil nutrients and enzyme activity [58,59]. Liu et al. [60] found that the organic matter content was closely related to soil enzyme activity, while Han et al. [61] found that soil nitrogen availability was a key factor in changes in microbial activity. The results of the redundancy analysis showed that MBN, TN, MBC/MBN, OC, and AN significantly affected the soil enzyme activity and stoichiometric ratios, explaining 47.7%, 21.1%, 15.8%, 6.4%, and 1.6% of the changes in the soil enzyme activity and stoichiometric ratios, respectively (Table 3). This indicates that soil carbon- and nitrogen-related indicators can explain most of the changes in the enzyme activity and stoichiometric ratios, which also indicates that the soil is limited in nitrogen and carbon. In addition, MBN was significantly positively correlated with CBH, NAG, ALP, VA, EC/N; TN with BX, BG, LAP; MBC/MBN with LAP, CAT, EC/P, EN/P; OC with BX, LAP, CAT; AN with BG, NAG, ALP, PPO, VA, EC/N (Figure 6), indicating that MBN, TN, MBC/MBN, OC, and AN can regulate soil enzyme activity and different litter inputs affect the soil carbon and nitrogen contents. Luo et al. [62] found that soil chemical properties have a strong influence on enzyme activity. In addition, enzyme activity is also affected by vegetation community activity. Soil enzymes are key mediators of soil organic matter and microbial metabolic activities. Dynamic changes in the activity of carbon and nitrogen acquisition enzymes may be related to the storage changes and availability of carbon and nitrogen in the soil carbon pool [63]. The accumulation of soil organic carbon provides microorganisms with sufficient carbon and nitrogen substrates for absorption and conversion, thereby promoting increases in soil enzyme activity. This also shows that changes in soil enzyme activity and stoichiometry are affected by the availability of soil nutrients [64].

5. Conclusions

Litter is a key component of soil nutrient cycles in sandy land. Our aim was to investigate the effects of litter quality changes on soil nutrients, enzyme activities, and stoichiometric ratios in sandy land. This study showed that the input of leaf litter of four plant species, YC, NT, ZSH, and SL, increased the contents of carbon- and nitrogen-related chemical indicators in sandy land soil. The addition of NT and YC litter significantly increased the contents of MBC and MBN. This shows that litter has a key ecological function in enhancing soil microbial activity and nutrient fixation. The addition of litter plays an important role in the activities of enzymes related to soil carbon, nitrogen, and phosphorus cycles. The addition of ZSH, NT, and YC litter alleviated phosphorus limitation, and the addition of NT litter alleviated soil nitrogen limitation. This indicates that sandy soil is subject to nitrogen limitation and nitrogen–phosphorus coordinated limitation, providing a new strategy for the coordinated regulation of nutrients in sandy soil. The results of the redundancy analysis show that the soil nutrients had different degrees of correlation with enzyme activity and its stoichiometric ratio. MBN, TN, MBC/MBN, OC, and AN were the core factors influencing soil enzyme activity and the stoichiometric ratio. Their synergistic effects predominantly regulated the coupling process of the carbon/nitrogen/phosphorus cycles in sandy soils by regulating microbial metabolic substrates and energy allocation. In addition, the brief duration of litter addition introduces certain uncertainties. The introduction of litter may influence the microbial community and induce alterations in soil enzyme activity, necessitating comprehensive experimental research for validation. In summary, this study provides a theoretical basis for the optimization of litter management in sandy ecological restoration. The priority selection of efficient litter types, such as ZSH, NT, and YC, can simultaneously alleviate nitrogen and phosphorus limitations and enhance microbially mediated nutrient cycles, thereby improving the ecological function of sandy soils.

Author Contributions

Conceptualization, H.G., S.Z. and L.Z.; formal analysis, H.H., S.Z. and Z.Y.; writing—original draft, H.G., S.Z., H.X. and L.Z.; resources, H.G., S.Z., H.H. and L.Z.; writing—review and editing, S.Z., C.W. and L.Z.; validation, Z.Y.; funding acquisition, H.G., H.H. and L.Z. All authors have read and agreed to the published version of the manuscript.

Funding

The study was funded by “The Inner Mongolia Forestry Science Research Institute’s Research Capacity Enhancement ‘Unveiling and Leading’ project” (2024NLTS03) and “the Natural Science Foundation project of Inner Mongolia Autonomous Region” (2024LHMS03017).

Data Availability Statement

Data will be made available upon request.

Acknowledgments

We thank the anonymous reviewers for reviewing our manuscript.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Change characteristics of soil chemical properties with different litter inputs. Different lowercase letters indicate significant differences among the treatments. OC: organic carbon; AC: soluble organic carbon; TN: total nitrogen; AN: available nitrogen; TP: total phosphorus; C/N: organic carbon/total nitrogen; C/P: organic carbon/total phosphorus; and N/P: total nitrogen/total phosphorus.
Figure 1. Change characteristics of soil chemical properties with different litter inputs. Different lowercase letters indicate significant differences among the treatments. OC: organic carbon; AC: soluble organic carbon; TN: total nitrogen; AN: available nitrogen; TP: total phosphorus; C/N: organic carbon/total nitrogen; C/P: organic carbon/total phosphorus; and N/P: total nitrogen/total phosphorus.
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Figure 2. Characteristics of soil microbial biomass and its stoichiometric ratio with different litter inputs. MBN: microbial biomass nitrogen; MBC: microbial biomass carbon. Different lowercase letters indicate significant differences among the treatments.
Figure 2. Characteristics of soil microbial biomass and its stoichiometric ratio with different litter inputs. MBN: microbial biomass nitrogen; MBC: microbial biomass carbon. Different lowercase letters indicate significant differences among the treatments.
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Figure 3. Variation characteristics of soil enzyme activities with different litter inputs. Different lowercase letters indicate significant differences among the treatments. CBH: cellulose; BG: β-1,4-glucosidase; LAP: leucine aminopeptidase; ALP: alkaline phosphatase; BX: β-1,4-xylosidase; NAG: β-1,4-acetylglucosaminidase; CAT: catalase; and PPO: polyphenol oxidase.
Figure 3. Variation characteristics of soil enzyme activities with different litter inputs. Different lowercase letters indicate significant differences among the treatments. CBH: cellulose; BG: β-1,4-glucosidase; LAP: leucine aminopeptidase; ALP: alkaline phosphatase; BX: β-1,4-xylosidase; NAG: β-1,4-acetylglucosaminidase; CAT: catalase; and PPO: polyphenol oxidase.
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Figure 4. Characteristics of stoichiometric ratios of soil enzyme activities under different litter inputs. Different lowercase letters indicate significant differences among the treatments. EC/N: enzymatic C/N acquisition ratio; EC/P: enzymatic C/P acquisition ratio; EN/P: enzymatic N/P acquisition ratio.
Figure 4. Characteristics of stoichiometric ratios of soil enzyme activities under different litter inputs. Different lowercase letters indicate significant differences among the treatments. EC/N: enzymatic C/N acquisition ratio; EC/P: enzymatic C/P acquisition ratio; EN/P: enzymatic N/P acquisition ratio.
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Figure 5. Chemometric carrier characteristics of soil enzymes. Different lowercase letters indicate significant differences among the treatments. VL: vector length; VA: vector angle.
Figure 5. Chemometric carrier characteristics of soil enzymes. Different lowercase letters indicate significant differences among the treatments. VL: vector length; VA: vector angle.
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Figure 6. Redundancy analysis (RDA) of soil enzyme activity, stoichiometry, and soil physiochemical properties. The blue arrows are the response variables and the red arrows are the explanatory variables; MBN: microbial biomass nitrogen; MBC: microbial biomass carbon; MBC/MBN: microbial biomass carbon/microbial biomass nitrogen; TN: total nitrogen; OC: organic carbon; TP: total phosphorus; AC: soluble organic carbon; AN: available nitrogen; C/N: organic carbon/total nitrogen; N/P: total nitrogen/total phosphorus; C/P: organic carbon/total phosphorus; CBH: cellulose; BG: β-1,4-glucosidase; LAP: leucine aminopeptidase; ALP: alkaline phosphatase; BX: β-1,4-xylosidase; NAG: β-1,4-acetylglucosaminidase; CAT: catalase; PPO: polyphenol oxidase; EC/N: enzymatic C/N acquisition ratio; EC/P: enzymatic C/P acquisition ratio; EN/P: enzymatic N/P acquisition ratio; VL: vector length; and VA: vector angle.
Figure 6. Redundancy analysis (RDA) of soil enzyme activity, stoichiometry, and soil physiochemical properties. The blue arrows are the response variables and the red arrows are the explanatory variables; MBN: microbial biomass nitrogen; MBC: microbial biomass carbon; MBC/MBN: microbial biomass carbon/microbial biomass nitrogen; TN: total nitrogen; OC: organic carbon; TP: total phosphorus; AC: soluble organic carbon; AN: available nitrogen; C/N: organic carbon/total nitrogen; N/P: total nitrogen/total phosphorus; C/P: organic carbon/total phosphorus; CBH: cellulose; BG: β-1,4-glucosidase; LAP: leucine aminopeptidase; ALP: alkaline phosphatase; BX: β-1,4-xylosidase; NAG: β-1,4-acetylglucosaminidase; CAT: catalase; PPO: polyphenol oxidase; EC/N: enzymatic C/N acquisition ratio; EC/P: enzymatic C/P acquisition ratio; EN/P: enzymatic N/P acquisition ratio; VL: vector length; and VA: vector angle.
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Table 1. Basic information of the sampling points.
Table 1. Basic information of the sampling points.
Longitude and LatitudeAltitude/mBasic Situation
YC38°51′58.55″ N, 109°15′31.04″ E1266.78Average height 0.7 m, canopy width 0.25 m × 0.30 m, total coverage 90%
NT38°57′21.04″ N, 109°07′44.00″ E1308.67Average height 1.5 m, canopy width 2.65 m × 2.30 m, total coverage 80%
ZSH38°52′19.30″ N, 109°14′06.68″ E1281.18Average height 1.2 m, crown width 1.52 m × 1.02 m, total coverage 85%
SL38°51′58.57″ N, 109°15′31.04″ E1247.77Average height 1.8 m, canopy width 1.30 m × 1.40 m, total coverage 88%
Notes: YC: Corethrodendron fruticosum; NT: Caragana korshinskii; ZSH: Amorpha fruticose; and SL: Salix cheilophila. The same applies below.
Table 2. Initial chemical contents of leaves of the four litter types. Different lowercase letters indicate significant differences among the treatments.
Table 2. Initial chemical contents of leaves of the four litter types. Different lowercase letters indicate significant differences among the treatments.
PTC/g·kg−1PTN/g·kg−1PTP/g·kg−1
YC409.90 ± 16.19 a39.74 ± 5.23 ab1.70 ± 0.02 b
NT436.57 ± 17.30 a44.69 ± 2.49 a1.73 ± 0.04 b
ZSH426.06 ± 21.04 a44.96 ± 5.03 a1.91 ± 0.02 a
SL425.31 ± 29.92 a29.34 ± 6.52 b1.45 ± 0.04 c
Table 3. Importance ranking and significance level of soil chemical properties.
Table 3. Importance ranking and significance level of soil chemical properties.
Soil Chemical PropertiesExplanation/%Contribution Rate%Fp
MBN47.748.611.90.002 **
TN21.121.58.10.002 **
MBC/MBN15.816.111.30.002 **
OC6.46.57.00.002 **
MBC1.91.92.40.066
AN1.61.72.40.040 *
C/N0.70.71.00.390
AC0.40.40.50.756
N/P0.20.20.20.912
TP1.51.52.10.130
C/P0.90.91.40.298
Notes: * and ** indicate significant effects at the 0.05 and 0.01 levels, respectively.
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Gao, H.; Zhang, S.; Yang, Z.; Xu, H.; Huang, H.; Wang, C.; Zhang, L. Effects of Litter Input on Soil Enzyme Activities and Their Stoichiometric Ratios in Sandy Soil. Agronomy 2025, 15, 1152. https://doi.org/10.3390/agronomy15051152

AMA Style

Gao H, Zhang S, Yang Z, Xu H, Huang H, Wang C, Zhang L. Effects of Litter Input on Soil Enzyme Activities and Their Stoichiometric Ratios in Sandy Soil. Agronomy. 2025; 15(5):1152. https://doi.org/10.3390/agronomy15051152

Chicago/Turabian Style

Gao, Haiyan, Shengnan Zhang, Zhiguo Yang, Hongbin Xu, Haiguang Huang, Chunying Wang, and Lei Zhang. 2025. "Effects of Litter Input on Soil Enzyme Activities and Their Stoichiometric Ratios in Sandy Soil" Agronomy 15, no. 5: 1152. https://doi.org/10.3390/agronomy15051152

APA Style

Gao, H., Zhang, S., Yang, Z., Xu, H., Huang, H., Wang, C., & Zhang, L. (2025). Effects of Litter Input on Soil Enzyme Activities and Their Stoichiometric Ratios in Sandy Soil. Agronomy, 15(5), 1152. https://doi.org/10.3390/agronomy15051152

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