Effects of Tree Species and Soil Enzyme Activities on Soil Nutrients in The Dryland Plantations

Long-term afforestation of different tree species strongly changes the soil physicochemical and biological properties. However, how tree species through litter quality and soil enzyme activities affect the succession of soil nutrients is still unclear in the dryland plantations. In this study, samples of surface soil (0–20 cm) and woody litter were collected from 55 years Caragana korshinskii, and 50 years Armeniaca sibirica, Populus hopeiensis, Platycladus orientalis, and Pinus tabulaeformis, and the natural grassland, and tested for the carbon, nitrogen, phosphorus, and potassium contents, as well as the soil sucrase (SC), urease (UE), and alkaline phosphorus (ALP) activities.


Introduction
The global arti cial forest area has reached 2.78×10 6 km 2  However, in long-term dryland plantations, how soil enzyme activities respond to the effects of different tree species on soil nutrients is unclear due to differences in litter speci city and site conditions. Therefore, understanding the relationship between soil enzyme activities, litter properties, and soil nutrients is the key to exploring the changing mechanism of soil fertility in dryland plantations.
The total area of the Loess Plateau in China is about 6.28×10 5 km 2 , spanning the semi-humid, semi-arid and arid areas, and the expansion of farmland and the reduction of vegetation have caused serious soil erosion problems (Zou and Luo 1997). Since 1950, large-scale afforestation activities have been carried out in the Loess Plateau, especially the "Grain for Green project" in 1999 (Chen et al. 2015). Caragana korshinskii, Armeniaca sibirica, Populus hopeiensis, Platycladus orientalis, Pinus tabulaeformis, etc. are common afforestation tree species in the Loess Plateau because of their nitrogen xation, drought resistance and/or rapid growth characteristics (Zou and Luo 1997). Many studies have focused on the changes of litter decomposition, soil enzyme activities, and nutrient properties and in plantations in semihumid area Zhang et al. 2018b;Zhang et al 2018c). However, in dryland plantations, the effects of different tree species on soil nutrients may be small and slow, and the soil nutrients response to litter properties and soil enzymes may be different compared to that in other regions. Therefore, the objective of this study was to (1) compare the changes in soil nutrients of different tree species plantations after long-term afforestation, (2) analyze the differences in the litter chemical properties and soil enzyme activities of different tree species plantations, and (3) reveal the relationship among the litter chemical properties, soil enzyme activities and soil nutrients in dryland plantations. We hypothesized that long-term afforestation promoted the increase of soil nutrients and enzyme activities, but may be reduced by the in uence of evergreen tree species. Due to the phosphorus limitation in the Loess Plateau , we predicted that the litter phosphorus content and alkaline phosphatase were important factors that determined the succession of soil nutrients in dryland plantations.

Study area
The study site is located at the Gongjing Forest Farm (104.3°E, 35.9°N) in Yuzhong County, Gansu Province, western Loess Plateau, China. The Gongjing Forest Farm was established in 1959, with about 86 km 2 and an average altitude of 2250 m. The forest farm is located in a semi-arid area and has a temperate continental monsoon climate. Average annual precipitation is 381 mm, annual average temperature is 7.2 °C (1985-2019), and more than 50% of annual precipitation is concentrated in May to September. Over the past 35 years, average annual precipitation and temperature showed an increasing trend. The soil type is loessial soil (Calcaric Cambisol, FAO classi cation), with no gravel. The plantations are pure forests, including C. korshinskii, A. sibirica, P. hopeiensis, P. orientalis, and P. tabulaeformis. Before afforestation, plantations plots had the same agricultural cultivation activities.

Experimental design
Under similar soil and climatic conditions, analyzing the changes and interactions of plants and soil is a widely used method in ecological research (Bhojvaid and Timmer 1998;Jaiyeoba 1998). In this study, we selected C. korshinskii (55 years), A. sibirica (50 years), P. hopeiensis (50 years), P. orientalis (50 years), and P. tabulaeformis (50 years) plantations as study plots, and a natural grassland near the plantations as a reference plot. The distance between different plantations and the grassland is less than 10 km, and their abiotic factors are consistent, so it can be considered that plants are the key factors affecting soil nutrient cycling (Delgado-Baquerizo et al. 2013). The dominant species of the grassland were Artemisia ordosica and Festuca ovina, and the community height was about 10 cm. The detailed information of plantations and grassland were shown in Table 1. In each vegetation type, 5 replicate sample squares with the distance of more than 30 m from each other were randomly set up, including plantation squares (10 m×10 m) and grassland squares (1 m×1 m). Vegetation has the greatest impact on the surface 0-20 cm soil (Goebes et al. 2019), so we collected the surface 0-20 cm soil. The soil was collected in early July 2019, and the woody litter was collected in early October 2019. In each sample square, soil and litter samples were collected according to the diagonal method (5 points) and mixed them.

Litter and soil sampling
Before collecting soil samples, rst removed litter, herbs and crusts, and then used a 5 cm soil drill to obtain the 0-20 cm soil. After thoroughly mixing the soil at 5 points, removed the plant residues and divided them equally into 3 parts. The rst part soil sample was packed in an aluminum box to determine soil water content. The second part soil sample was air-dried in a natural environment without light and passed through a 2 mm sieve to determine the chemical properties, pH, and particle composition of the soil. The third part soil sample was stored at 4 ℃ for determination of soil enzyme activities.
Simultaneously, at the center of each sample square, a steel cylinder (ring knife) with a volume of about 100 cm 3 was used to collect a portion of the soil that remained intact and determine the soil bulk density (Zhang et al. 2018c). 120 soil samples were obtained: 6 study plots × 5 replicate squares × 4 parts (determining different properties). In addition, the leaf litter of woody plants was collected form each plantation square, dried and crushed, and a total of 25 litter samples were obtained.

Measurement of litter and soil properties
Using the combustion method determined litter carbon (LC, g·kg -¹) and nitrogen (LN, g·kg -¹) and soil total carbon (TC, g·kg -1 ) and nitrogen (TN, g·kg -¹), tested by the Elementar vario MACRO cube Organic Element Analysis (Germany Elementar) (Zhao et al. 2018). Litter phosphorus (LP, g·kg -¹) and soil total phosphorus (TP, g·kg -¹) and were measured using the molybdenum antimony colorimetric method after litter was digested with the concentrated sulfuric acid and hydrogen peroxide and soil was digested with the concentrated sulfuric acid and perchloric acid (Bao 2000). The LP and TP were test by the Smartchem 140 Automatic discontinuous chemical analyzer (France Alliance). Litter potassium (LK, g·kg -1 ) and soil total potassium (TK, g·kg -1 ) were measured using the ame atomization method after digestion (Cao et al. 2007), and tested by the TRACE AI1200 Atomic Absorption Spectrometer (Canada Aurora).
Using the potassium dichromate external heating method determined soil organic carbon (OC, g·kg -1 ). After soil was leached by potassium chloride, using the indophenol blue colorimetric method and the phenol disulfonic acid colorimetric method determined the soil ammonium nitrogen and nitrate nitrogen.
Soil available nitrogen (AN, mg·kg -1 ) was de ned as the sum of soil ammonium nitrogen and nitrate nitrogen. Soil available phosphorus (AP, mg·kg -1 ) was measured using the molybdenum antimony colorimetric method after sodium bicarbonate leaching (Bao 2000). The AN and AP were tested by the Smartchem 140 Automatic discontinuous chemical analyzer. Soil available potassium (AK, mg·kg -1 ) was measured using the ame atomization method after ammonium acetate leaching (Cao et al. 2007), and tested by the TRACE AI1200 Atomic Absorption Spectrometer.
The soil samples in the steel cylinder and aluminum box were dried and weighed to determine the soil bulk density (BD, g·cm -3 ) and soil water content (SWC, %). BD was equal to the ratio of the dry soil mass to 100 cm 3 , and SWC was equal to the 100 × the ratio of soil water mass to dry soil mass (Zhang et al. 2018c; Zhao et al. 2018). Using a pH meter to determine the soil pH in a 1:5 w/v soil-water ltration solution (Zhang et al. 2018c).

Measurement of soil enzyme activities
The soil enzyme activities were measured using fresh soil samples stored at low temperature and converted into dry soil enzyme activities through SWC. The 3,5-dinitrosalicylic acid colorimetry method, indophenol blue colorimetry method and phenol colorimetric method by a spectrophotometer was used to determine soil sucrase (SC, mg·d -¹·g -¹), urease (UE, mg·d -¹·g -¹) and alkaline phosphatase (ALP, mg·d -¹·g -¹), respectively (Guan et al. 1986).

Statistical analysis
A one-way ANOVA and a least signi cant difference (LSD) multiple comparison test (P<0.05) were used to compare the differences in soil physicochemical properties, enzyme activities, and litter chemical properties of different tree species plantations. A redundancy analysis (RDA) was used to estimate the contribution of effect variables (litter chemical properties and soil enzyme activities) on response variables (soil nutrients). A bivariate correlation analysis was used to explore the relationship among litter chemical properties, soil enzyme activities and soil nutrients. In this study, SPSS 17.0, OriginPro 2021 and CANOCO 4.5 were used for data analysis and charting.

Soil physicochemical properties
The soil BD, pH, and SWC were signi cantly different between ve plantations and grassland (Table 2). Compared with grassland, plantations reduced soil BD and SWC by 3.36%-17.65% and 3.81%-61.7%, and soil pH varied from 8.05 in grassland to 7.87 in P. tabuliformis plantations. The soil BD of P. orientalis, A. sibirica, and P. tabuliformis was signi cantly greater than that of C. korshinskii and P. hopeiensis (P<0.05). The highest SWC (13.89%) was observed in the P. hopeiensis plantation, which was higher than that of C. korshinskii, A. sibirica, P. orientalis, and P. tabuliformis by 14.54%, 56.52%, 60.19%, and 37.01%, respectively. The lowest SWC (5.53%) was observed in the P. orientalis plantation, which was signi cantly lower than that (11.87%) of C. korshinskii plantation by about 50% (P<0.05). The soil pH of C. korshinskii, P. hopeiensis and P. orientalis was signi cantly greater than that of P. tabuliformis (7.87).
tabuliformis plantation was the lowest (2.04 g·kg -1 ). The changing trends of LN and LP in different tree species plantations were similar.
Relationship among litter chemical properties, soil enzyme activities and soil nutrients RDA showed that the litter chemical properties, soil enzyme activities and nutrients of different tree species plantations were quite different ( Figure 4). The ve plantations were clustered into four groups, and C. korshinskii, P. hopeiensis and A. sibirica were each grouped into one group, while P. orientalis and P. tabuliformis were clustered into one group, with similar soil and litter properties. The 62.2% of soil nutrient variation could be explained by litter chemical properties and soil enzymes, of which the contribution rate of the rst and second axes was 56.5%. The soil SC, UE, and ALP positively affected soil TK, AK, TP, AP, TN, AN, and OC, and three soil enzyme activities were positively related with each other. In addition to soil TK, The LC negatively affected soil nutrients, and the LK had less positive or negative effects on soil nutrients. But the LN and LP positively affected the soil TC, OC, TN, AN, TP, AP, and AK, and the positive relationship was observed between LN and LP. The positive in uences of the LN, LP, and ALP on soil nutrient variability reached a signi cant level (P<0.05).
Bivariate correlation analysis illustrated the correlation between litter chemical properties, soil enzyme activities and nutrients ( Table 3)

Discussion
Effects of tree species on soil nutrients Long-term afforestation of different tree species signi cantly changed soil BD, pH, and SWC. The decrease of soil BD was caused by the active activities of more abundant soil animals and microorganisms after afforestation (Frouz et al. 2001). Except for P. hopeiensis, other four tree species plantations signi cantly decreased SWC (Table 2), which was consistent with the ndings of Cao et al. (2007), who reported that large-scale afforestation resulted in soil drying and water depletion. Compared with grassland, woody plants consumed more soil water, and canopy and litter layers of plantations intercepted precipitation (Sato et al. 2004). And we also found the pH of alkaline soils decreased after long-term afforestation, because woody plants could produce more organic acids or anions to enter the soil (Wang et al. 2021b). A meta-analysis showed that afforestation promoted soil pH neutralization (Hong et al. 2018). In this study, SWC and pH of broadleaf tree species (C. korshinskii and P. hopeiensis) were higher than those of coniferous tree species (P. orientalis and P. tabuliformis) by 35.66%-151.18% and 0.50%-2.29%, suggesting that the impact of tree species on soil properties was species-speci c (Hong et al. 2018), which may be due to differences in water requirements, plant traits, litter Our results also showed that deciduous plantations (C. korshinskii and P. hopeiensis) had higher soil TC, OC, TN, AN, and AK contents than those in evergreen plantations (P. orientalis and P. tabuliformis) by 1.34%-166.87% (Figure 1). In addition, C. korshinskii was not only a deciduous tree species, but also a leguminous tree species. The leguminous tree species had higher nitrogen and carbon input from xation (Wang et al. 2019). Gei and Powers (2013) compared the effects of legumes and non-legumes tree species on soil properties in Costa Rican dry plantations, who con rmed that legumes had higher soil TC, TN, and nitrate nitrogen contents. These ndings indicated that deciduous tree species, especially legumes, played a more important role than evergreen tree species in dryland vegetation restoration ( 2020) illustrated that Robinia pseudoacacia, C. korshinskii, A. sibirica and P. tabuliformis had signi cant differences in nitrogen and phosphorus resorption e ciency, green leaf nutrients, and growth rates, which may lead to the production of different quality litters (Bai et al. 2019). In our study, high-quality litters (higher LN, LP, and LK with lower LC) were observed in C. korshinskii, A. sibirica and P. hopeiensis, while low-quality litters (higher LC with lower LN, LP, and LK) in P. orientalis and P. tabuliformis. We also found that the LC, LN, and LP under P. tabuliformis were signi cantly higher than those under P. orientalis by 3.32%, 34 ). In addition, P. orientalis presented lowest soil SC, UE, and ALP than C. korshinskii, A. sibirica, P. hopeiensis, P. tabuliformis, and grassland. However, in semi-humid plantations, soil enzyme activities in P. orientalis were higher than that in Sophora davidii (a leguminous shrub) (Li et al. 2020), suggesting that P. orientalis may not be suitable for afforestation in arid areas (Zhang and Chen 2007), which was also supported by the lowest quality litter, SWC and soil nutrients of P. orientalis in this study.
Response of soil nutrients to litter chemical properties and soil enzyme activities Vegetation played a major role in the improvement of soil nutrients, and the in uence of different vegetation types on soil nutrients was controlled by litter (Sariyildiz et al. 2005;Wang et al. 2021a). Nutrients return to the soil through the litter decomposition was the main link in the material cycle of the ecosystem (Wang et al. 2008). The high-quality litter and high soil enzyme activity could accelerate the nutrient release of litter and improve the supply capacity of soil available nutrients for plant growth (Wang et al. 2008;Noll et al. 2016). Therefore, the litter properties and soil enzyme activity determined the level of soil fertility (Pan et al. 2013;Bohara et al. 2020). Our results showed that the variation in soil nutrients following long-term afforestation with different tree species were caused by soil TC, OC, TN, AN, AP, and AK, which were signi cantly positively affected by the LN, LP, UE, and ALP (table 3). Firstly, the LN and LP represented the litter quality, which decided the decomposition rate and microbial metabolism, thereby affecting the nutrient cycle (Prescott 1996 Secondly, the differences in soil carbon, nitrogen and phosphorus contents were similar to that in soil enzyme activities in different tree species plantations, especially C. korshinskii and P. orientalis, indicating that there was a strong interaction between soil nutrients and enzymes (Table 3). Some studies have reported that soil nutrients were closely related to soil enzyme activities (Zhang et (Table 3).
In addition, the signi cant positive correlation between LN, UE, TN, AN and LP, ALP, AP demonstrated that nitrogen and phosphorus existed an obvious coupling relationship, which was con rmed by Zhao and Zeng (2019), who noted that phosphorus addition strongly altered the impact of nitrogen addition on soil nitrogen and phosphorus transformations. Another nitrogen addition test revealed that soil speci c acid phosphatase, N-acetyl glucosaminidase, and oxidative enzyme activities signi cantly increased with nitrogen levels increasing (Li et al. 2019), which suggested that improved soil nitrogen availability could promote the cycling of the phosphorus and other elements. Zhang et al. (2018b) and Zhang et al. (2021) pointed out that phosphorus limitation existed in the Loess Plateau and might become more severe in the future. Soil microbes promoted the extraction of restricted elements by regulating the production of soil enzymes and the e ciency of nutrient utilization (Bai et al. 2021). The signi cant positive correlation between TC, OC, TN, AN, LN and LP, OC, TN, AN, LN and ALP, and LN, SC, UE, and AP illustrated that soil microbes might tend to consume more carbon and nitrogen to increase the soil phosphorus availability, alleviate the limitation of phosphorus on the growth of plants and microorganisms, and meanwhile, improve other nutrients in the soil (Yan et al. 2020;Bai et al. 2021). It was also proved that in the C. korshinskii plantation with the highest LN, TC, OC, TN and AN, the LP, soil ALP and AP in whose was higher than that in other plantations and grasslands (Figure 1, 2&3). And the LP and ALP had a larger correlation coe cient with soil nutrients than LN and UE (Table 3). Therefore, these results revealed that the LP and ALP were the key factors driving soil nutrient changes in dryland plantations limited by phosphorus, which supported our prediction that the LP and ALP were important for soil nutrient dynamics.

Conclusions
This study emphasized the importance of litter quality and soil enzyme activities in the succession of soil nutrients in dryland plantations. Long-term afforestation led to signi cant changes in soil nutrients, litter chemical properties and soil enzyme activities. The soil TC in different plantations increased, and soil TP and TK signi cantly decreased. The C. korshinskii signi cantly increased soil nutrients (TC, OC, TN, AN, and AK), and its soil AP also increased. While the P. orientalis signi cantly decreased soil nutrients (TN, TP, AP, TK, and AK). Therefore, soil nutrients in dryland plantations were signi cantly affected by tree species. The 62.2% of the total variation of soil nutrients could be explained by the litter quality and soil enzyme activities, and the LP and soil ALP had greater effect on soil nutrients than the LN and soil UE.
And the signi cant positive correlations between the LP, soil ALP and AP and the LN, soil UE, TN and AN further indicated that there was an important coupling relationship between nitrogen and phosphorus, which may promote the phosphorus cycle and alleviate the phosphorus limitation. In short, tree species, LP, and soil ALP determined the changes in soil nutrients in phosphorus-de cient dryland plantations. These ndings will help guide the establishment and management of dryland plantations, and it is recommended to choose leguminous tree species for afforestation, such as C. korshinskii, in the phosphorus-limited dryland.

Declarations
Yage Li wrote the manuscript, Chun Han and Yage Li revised the manuscript, Changming Zhao and Shan Sun guided the experimental design of the study.

Availability of data and materials
The datasets used and/or analyzed in this study are available from the corresponding author on reasonable request.   Table 3 Correlation coe cients of litter chemical properties, soil enzyme activities and soil nutrients in plantations.