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Article

The Responses of Soil Extracellular Enzyme Activities and Microbial Nutrients to the Interaction between Nitrogen and Phosphorus Additions and Apoplastic Litter in Broad-Leaved Korean Pine Forests in Northeast China

by
Liming Chen
1,2,†,
Lixin Chen
1,†,
Meixuan Chen
1,
Yafei Wang
1 and
Wenbiao Duan
1,*
1
College of Forestry, Northeast Forestry University, Harbin 150040, China
2
State Key Laboratory of Tree Genetics and Breeding, College of Forestry, Northeast Forestry University, Harbin 150040, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Forests 2024, 15(10), 1764; https://doi.org/10.3390/f15101764
Submission received: 4 September 2024 / Revised: 27 September 2024 / Accepted: 7 October 2024 / Published: 8 October 2024
(This article belongs to the Section Forest Soil)
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Abstract

:
The impact of nitrogen and phosphorus deposition alternations, as well as apoplastic litter quality and quantity, on soil nutrient cycling and soil carbon pool processes in forest ecosystems is of considerable importance. Soil ecological enzyme chemistry is a powerful tool for elucidating the nutrient limitations of microbial growth and metabolic processes. In order to explore the responding mechanisms of soil ecological enzyme chemistry to the simultaneous changes in apoplast input and nitrogen and phosphorus deposition in temperate coniferous and broad-leaved mixed forests, an outdoor simulating experiment was conducted. The results demonstrate that the treatments involving apoplastic material and nitrogen and phosphorus additions had significantly impacted soil nutrient levels across different forest types. Apoplastic treatments and N-P additions had a significant effect on the soil total organic carbon (TOC), dissolved organic carbon (DOC), soil total soluble nitrogen (TSN), soil available phosphorus (SAP), soil total nitrogen (TN), soil total phosphorus (TP), and microbial biomass carbon (MBC). However, the effects on soil microbial biomass (MBN) and microbial biomass phosphorus (MBP) were insignificant. The apomictic treatments with N and P addition did not result in a statistically significant change in soil C-hydrolase activities (β-1,4-glucosidase BG, β-1,4-xylosidase BX, cellobiohydrolase CBH, phenol oxidase POX, and peroxidase PER), N-hydrolase activities (β-1,4-N-acetylglucosaminidase NAG and L-leucine aminopeptidase LAP), or P-hydrolase activities (Acid phosphatase AP). Although the apomictic treatments did not yield a significant overall impact on carbon hydrolase activity, they influenced the activity of specific enzymes, such as CBH, LAP, and PER, to varying degrees. The effects on BG, BX, CBH, AP, and C-hydrolase activities were significant for different stand types. The impact of apomictic treatments and N-P additions on soil nitrogen hydrolase activities was inconsequential with a minimal interactive effect. The highest correlation between PER, LAP, and N-hydrolase activities was observed in conjunction with elevated levels of nitrogen and phosphorus addition (N3L0, original litter treatment, and high amounts of N and P addition). These findings may provide a theoretical foundation for the management of ecosystem function in broad-leaved Korean pine forests.

1. Introduction

Nutrient cycling in ecosystems represents a pivotal concern in contemporary ecological research. In particular, the extensive utilization of fossil fuels is a concern [1]. The Industrial Revolution has led to an increase in the deposition of nitrogen and phosphorus in ecosystems, thereby altering the nutrient cycle of these ecosystems, especially forest ecosystems. It has been demonstrated that alterations in nitrogen levels considerably influence forest ecosystems’ productivity. Phosphorus is a crucial nutrient element that is indispensable for plant growth and development, providing a fundamental material basis and environmental context for plant survival [2,3]. Alterations in nitrogen levels significantly impact forest ecosystem productivity. Phosphorus is a vital nutrient essential for plant growth and development, providing a critical material foundation and environmental context for plant survival. Since the onset of the Industrial Revolution, the extensive use of fossil fuels and fertilizers has resulted in increased atmospheric deposition of nitrogen and phosphorus. Soil extracellular enzymes are crucial in soil organic matter decomposition and nutrient cycling [4,5]. As per the Resource Allocation Theory (RAT), variations in extracellular enzyme activities (EEAs) can directly influence essential ecosystem functions such as material decomposition, nutrient cycling, and plant–microbe interactions, subsequently impacting ecosystem productivity and carbon balance [6]. Nitrogen and phosphorus additions have been observed to enhance the activities of enzymes involved in carbon and phosphorus acquisition [7,8]. Nevertheless, the current findings are inconclusive; some suggest that applying nitrogen fertilizers has no substantial effect on cellulase activity, while others indicate a neutral or modest impact on soil carbon, nitrogen, and phosphorus-acquiring enzyme activities within grassland ecosystems [9,10]. These conflicting results suggest that other environmental factors may regulate the response of extracellular enzyme activities to nitrogen and phosphorus inputs.
Soil dissolved organic carbon (DOC) was found to increase, while soil microbiome biomass carbon (MBC) was decreased initially following apoplastic addition [11]. Subsequently, the soil MBC content was increased with apoplastic addition [12]. Consequently, the outcomes regarding the impacts of nitrogen and phosphorus deposition and apoplastic addition treatments on soil reactive organic carbon pools remain contentious among the scientific community. The quality and quantity of apoplastic matter significantly influenced soil enzyme activities. Activities of catalase, polyphenol oxidase, and urease in the soil exhibited a notable increase of more than 15% when the proportion of needles in the apoplastic treatment decreased [13]. Another study revealed that the activities of urease and deaminase were considerably higher under mixed apoplastic treatments than single apoplastic treatments. This finding suggests that apoplastic diversity does not directly correlate with heightened microbial activity, and the chemical composition of apoplastic material plays a significant role in influencing microbial activity [14,15]. Additionally, it has been observed that the activities of β-1,4-glucosidase and β-N-acetylaminoglucosidase were markedly reduced in comparison to those in the leaf apoplastic doubling treatment and mixed apoplastic doubling treatment under conditions of apoplastic exclusion. The deposition of nitrogen and phosphorus due to global climate change alters the physicochemical properties of soils in forest ecosystems, affecting the functional status of microorganisms and soil enzyme activities [14,16]. The impact of nitrogen and phosphorus supplementation on soil enzyme activity is complex and variable. Some studies have suggested that nitrogen addition may decrease cellulase activity while increasing urease and sucrase activities [17]. However, the effect of nitrogen addition on β-1,4-glucosidase activity tends to shift from the promotion to inhibition of its activity as the concentration of nitrogen addition increases [18]. Despite this, there is a lack of evidence regarding the mechanism through which simultaneous alterations in apoplastic inputs and nitrogen and phosphorus additions influence soil ecological enzyme chemistry by modulating soil enzyme activities.
This study aimed to explore the following questions and address the research objectives by determining the soil physicochemical properties, microbial biomass, and extracellular enzyme activities through various nitrogen and phosphorus additions and apomictic treatments in northeastern broad-leaved Korean pine forests. This study aims to investigate the limiting effects of nitrogen and phosphorus (N and P) addition and dieback treatments on soil enzyme activities and microbial nutrients in diverse forest ecosystems. In particular, it seeks to elucidate the ecological and enzyme chemical responses of soil extracellular enzymes (EEAs) to nitrogen and phosphorus addition and dieback treatments and to ascertain the key factors affecting soil extracellular enzymes and microbial communities. It was hypothesized that the soil properties and pH in the apomictic and nitrogen and phosphorus addition treatments may be the most significant factors influencing soil EEAs and microbial communities.

2. Materials and Methods

2.1. Study Area

This study was conducted in the Liangshui National Nature Reserve (E:128°54′30″ N:47°11′30″), a mountainous area in the eastern part of Northeast China, with an altitude ranging from 400 m to 500 m (Figure 1). The predominant soil type is dark brown forest soil, and the forest type is a mixed broad-leaved forest, mainly consisting of Pinus koraiensis, which is widely distributed across the area, with a forest coverage of 98%. The climate is characterized by a temperate continental monsoon, exhibiting relatively mild and humid conditions. The formation of this climate is primarily attributed to a combination of factors, including land and water distribution, altitude, and the influence of the southeast monsoon. These factors contribute to a distinctly contrasting climate in the central plains of the Northeast. The average annual precipitation is 676 mm, with 120–155 days of yearly rainfall. The number of snow days ranges from 130 to 155. The average annual evaporation is 805 mm, the relative humidity is 78% annually, and the number of sunshine hours is approximately 1850 per year, with a sunshine rate of 43.5%. The tree layer is primarily composed of the following species: purple linden (Tilia amurensis), chaff linden (Tilia mandshurica), stinking pine (Abies nephrolepis), mountain poplar (Abies nephrolepis), maple birch (Betula costata), Mongolian oak (Quercus mongolica), Phellodendron amurense, Betula platyphylla, Abies fabri, and Acer mono (Table 1).

2.2. Experimental Design and Sample Collection

The experimental sample plots were selected in September 2017 in undamaged, representative, and adjacent sections of two forest types: a 65-year-old Korean pine plantation (I) and a 230-year-old virgin broadleaf Korean pine forest (F). Three fixed sample plots of 20 m × 30 m in size were established within each forest type, and the data for each sample point are presented in Table 1. The 65-year-old Korean pine plantation pure forest had standard sample plots, designated as No. 1, No. 2, and No. 3, which were situated by the downslope, mid-slope, and up-slope, respectively. The 230-year-old primitive broad-leaved Korean pine forest had sample plots designated as No. 4, No. 5, and No. 6, which were situated according to the up-slope, mid-slope, and downslope, respectively. Additionally, 12 smaller sample squares (2 m × 2 m) were randomly positioned within each standard sample plot and numbered from 1 to 12. Sample squares 1 to 4 were set up to maintain the original apomictic material on the ground surface in its original state. The sample squares numbered 5–8 were set up to remove apomictic material from the ground surface. The apomictic material removed from squares 5–8 was subsequently added to squares 9–12, which were set up as a double apomictic material. The intensity and frequency of nitrogen (N) and phosphorus (P) treatments were determined based on an analysis of local summer rainfall records over an extended period and the identification of natural N and P deposition in the region. This approach was informed by assessing the background values of natural N and P deposition and by reviewing international treatments of similar studies. The intensity and frequency of N and P treatments were established by the background value of natural N and P deposition concerning analogous international research methodologies. The nitrogen and phosphorus fertilizers were applied in N0, N1, N2, and N3 gradients using (NH4)2SO4 (manufactured by Tianjin Zhiyuan Chemical Reagent Co., Ltd., with a nitrogen content of 21.2%) as the nitrogen source. The other plots were the control (0 gN·m−2·a−1, 0 gP·m−2·a−1), a plot with a low concentration of nitrogen and phosphorus treatment (5 gN·m−2·a−1, 5 gP·m−2·a−1), and a plot with a medium concentration of nitrogen and phosphorus treatment (30 gN·m−2·a−1, 20 gP·m−2·a−1), with three replicates for each treatment. There was a total of 72 sample plots. Different levels of (NH4)2SO4 and (NH4)2HPO4 dissolved in 2 L of water were uniformly and systematically sprayed in each sample plot to simulate nitrogen–phosphorus deposition within each sample plot from May 2018 onwards (May, July, and September). Equal amounts of water were sprayed in the control sample plot to avoid the effects of biogeochemical cycling caused by the imposed water. Nitrogen and phosphorus were applied at equal rates in each column, with the rates fully randomized across columns within each block group. The apomictic treatments and nitrogen and phosphorus additions were implemented annually from 2018 to 2019, with the introduction of new apomictic material being sorted and sprayed with nitrogen and phosphorus fertilizer after each sampling event, per the sample plot design.
The apoplastic treatments were as follows: (i) the removal of apoplastic litter (RL), involving the elimination of soil surface apoplastic litter and discernible humus within the sample plots, coupled with the periodic removal of new apoplastic litter; (ii) the preservation of the initial apoplastic litter (CK), henceforth referred to as the apoplastic control treatment; and (iii) the addition of twice the amount of apoplastic litter (AL). The process for adding apoplastic litter was as follows: Twelve distinct locations, each 2 m × 2 m in size and spaced 2 m apart, were randomly selected within each sample plot in May 2018. Sample squares 1–4 were established within these locations to maintain the original apoplastic layer’s integrity. To remove any fallout and preserve the original apoplastic condition, sample squares 5–8 were created. Conversely, sample squares 9–12 were assigned for the even and consistent addition of apoplast sourced from the previously removed apoplastic material.

2.3. Sample Processing and Analysis of Nutrients

2.3.1. Analysis of Soil Properties

Seasonal sampling was conducted at the end of May, early August, and early October 2018. Eight sample points were randomly selected within each plot to obtain soil samples from the 0–10 cm layer with a soil auger. The samples were mixed into a single soil sample, and three replicates of the mixed samples were taken for each treatment. The samples were packed in self-sealing bags and placed in a portable refrigerator, where they were immediately transported back to the laboratory. Some soil samples were passed through a 2 mm sieve (10 mesh) and placed in a refrigerator set at 4 °C for storage. The remaining soil samples were placed in a shaded area and allowed to air-dry, after which they were used for the determination of soil physicochemical properties. Some soil samples were subjected to a 2 mm sieving process (10 mesh) and stored in a refrigerator at 4 °C for the determination of soil enzymes, soil microorganisms, and other indicators. In contrast, some of the soil samples were placed in a shaded area to air-dry and used for the determination of the physical and chemical properties of the soil.
The soil pH was determined by the electrode potential method, while the soil organic carbon (TOC) was determined by the potassium dichromate oxidation–external heating method, and the soil dissolved organic carbon (DOC) was determined by extracting distilled water with a total organic carbon analyzer (soil/water ratio of 1:5). The soil total soluble nitrogen (TSN) was determined by the K2SO4 leaching method. The soil effective phosphorus (SAP) was determined by means of 0.5 mol/L of NaHCO3 extraction, with the phosphorus content of the extract subsequently being determined by molybdenum antimony scandium colorimetry [19]. The total nitrogen content was determined by the semi-micro Kjeldahl method, the total phosphorus content was determined by NaOH melting-molybdenum antimony resist color-ultraviolet spectrophotometry, the total potassium content was determined by NaOH melting–atomic absorption, and the alkaline nitrogen content was determined by diffusion absorption. Furthermore, quick phosphorus was determined by NaHCO3 extraction–molybdenum antimony resist color-ultraviolet spectrophotometry, and the total phosphorus was determined by NaHCO3 extraction–molybdenum antimony resist color-ultraviolet spectrophotometry.

2.3.2. Analysis of Microbial Biomass and Enzyme Activity

The microbial biomass carbon (MBC), nitrogen (MBN), and phosphorus (MBP) were determined by the chloroform fumigation and leaching method in accordance with the protocol previously outlined in the literature [20,21,22]. Eight soil extracellular enzyme activities (six hydrolases and two oxidases) were assayed using a fluorescence assay about soil C (β-1,4-glucosidase (βG), β-1,4-xylosidase (βX), and cellobiose hydrolase. The cycle-related enzymes include CBH, phenol oxidase, POX, peroxidase (PER), N (β-1,4-acetamido-glucosidase and NAG), and L-leucine aminopeptidase (LAP), as well as the phosphorus acquisition cycle-related enzyme, acid phosphatase (AP) [23]. Hydrolase activity was prepared by adding 1 g of soil (converted to a dry weight basis) to a solution of 25 mL of acetate buffer (50 mmol L−1; pH 5). The soil suspension was prepared by mixing 1 g of soil (converted to dry weight), 25 mL of acetate buffer (50 mmol L−1; pH 5.0), and vortexing. The suspension was then centrifuged at 3500 rpm for 5 min, and the process was repeated three times. The resulting soil suspension was added to a 96-well microtiter plate, with 200 μL of the suspension and 50 μL of the substrate (200 μmol L−1, 4-methylumbelliferyl β-D-cellobiose glycoside, prepared in ultrapure water) added to each well. The microtiter plates were then incubated in the dark at 20 °C for 4 h. At the conclusion of the incubation period, 10 μL of a 1 mol L−1 NaOH solution was added to each well. Following a 1 min incubation period, the soil suspension was extracted and analyzed with a multifunctional enzyme marker (Spectra Max M5, Sunnyvale City, CA, USA). After the incubation was terminated for 1 min, the fluorescence was determined with a multifunctional enzyme labeler (Spectra Max M5, USA) with excitation at 365 nm and fluorescence at 460 nm. The oxidase activity was determined by adding 50 μL of the substrate (200 μmol L−1 L-DOPA, prepared in ultrapure water) and 10 μL of 0.3% hydrogen peroxide to the prepared soil suspension. All microtiter plates were incubated at 20 °C for 20 h in the dark, and following the termination of the incubation period, the absorbance values were determined using a multifunctional enzyme labeling instrument (Spectra Max M5, USA) with fluorescence at 465 nm. Each sample was subjected to four replicates. The enzyme–substrate was procured from Sigma, and the other reagents were of analytical grade. Ultimately, the enzyme activity was calculated from the dry weight of the soil and the reaction time and expressed as nmol h−1·g−1 [24].

2.3.3. Statistical Analysis

The effects of nitrogen (N) and phosphorus (P) addition and apoplastic treatments on soil chemical properties, microbial biomass, and enzyme activities were determined by an analysis of variance (ANOVA) using the IBM SPSS Statistics software version 26.0 (IBM Corp., Armonk, NY, USA). The relative contributions of N addition and apoplastic treatments in explaining changes in soil properties, microbial biomass, and ecological enzyme activities were quantified using a two-way ANOVA. Stepwise regression analyses were employed to identify the soil chemical indicators that most effectively explained the changes in soil enzyme activities and microbial community characteristics. A principal component analysis (PCA) was conducted using Canoco software (Canoco for Windows 4.5) to illustrate the effects of N P addition, litter treatments, and their interactions on soil enzyme activities in broad-leaved Korean pine forests.

3. Results

3.1. The Litter Treatment Regulates Nitrogen and Phosphorus Additions to Soil Nutrients and Microbial Activities

A comparison showed that the litter treatment (L) had a significant effect on the soil TOC in the broad-leaved Korean pine forest (F) and Korean pine plantation (I) (Table S1). Nitrogen and phosphorus application had no significant effect on the soil TOC in the planted pine forest but had a significant effect on the broad-leaved Korean pine forest, where nitrogen and phosphorus application and litter treatment reduced the soil TOC content. Specifically, N and P application (N1, N2, N3) reduced the soil TOC content by 5.18%–33.85%, and apomictic treatments reduced the TOC content by 5.82%–12.76% (Table S1; Figure 2A). The results of the ANOVA show that apoplastic treatments and N P amendments and their interactions had significant effects on the DOC in soils of broad-leaved Korean pine forest and Korean pine plantation (Table S1). The apoplastic treatments and N P amendments significantly increased the soil DOC in the Korean pine plantation except the N3 treatment (Table S1; Figure 2B), whereas the apoplastic treatments and N P amendments significantly decreased the soil DOC in the broad-leaved Korean pine forest. The apoplastic treatments and N P amendments did not affect the TSN in soils of the broad-leaved Korean pine forest and Korean pine plantation (Table S1; Figure 2C). N P addition and the interaction between the litter treatment and N P addition had no significant effect on the SAP in the Korean pine plantation and broad-leaved Korean pine forest (Table S1). The litter treatment resulted in a significant reduction in the SAP in the Korean pine plantation, while no significant effect was observed in the broad-leaved Korean pine forest (Table S1; Figure 2D). The results of the variance analysis indicate that the litter treatment, N P addition, and their interaction had a significant impact on the TN in the Korean pine plantation and broad-leaved Korean pine forest (Table S1). Nitrogen and phosphorus significantly reduced the nitrogen (TN) content of the broad-leaved Korean pine forest by 11.09% to 36.87%. Conversely, the effect of nitrogen and phosphorus addition on the TN content of the Korean pine plantation was insignificant. However, the TN content increased by 6.49% under the N2 treatment (Table S1; Figure 2E). The interaction between litter treatment and nitrogen (N) and phosphorus (P) addition, as well as the interaction between litter treatment and N P addition, had a significant impact on the total phosphorus (TP) in the broad-leaved Korean pine forest. The application of the litter treatment and N P addition resulted in an increase in the TP in the Korean pine plantation, although this was not statistically significant (Table S1; Figure 2F). The application of the litter treatment and the interaction between the litter treatment and N P addition had a significant impact on the soil pH in the Korean pine plantation. The addition of N P reduced the soil pH. The litter treatment resulted in an increase in the soil pH in the Korean pine plantations and a decrease in the soil pH in the broad-leaved Korean pine forest; however, this was not statistically significant (Table S1; Figure 2G).
The litter treatment and nitrogen and phosphorus addition and their interaction significantly affected the MBC in the Korean pine plantation and broad-leaved Korean pine forest (Table S2). The litter treatment significantly increased the MBC of the Korean pine plantation and broad-leaved Korean pine forest. The RL treatment increased the MBC of Korean pine plantation and broad-leaved Korean pine forest by 42.75% and 5.39%, respectively, and the AL treatment increased these values by 69.57% and 7.61%, respectively (Table S2; Figure 3A). The addition of N P significantly reduced the MBC of the Korean pine plantation and broad-leaved Korean pine forest except for the N1 treatment in the Korean pine plantation. The litter treatment and N P addition had no significant effect on the MBN and MBP in the Korean pine plantation and broad-leaved Korean pine forest (Table S2; Figure 3B,C). In addition to the MBP of the Korean pine plantation under the N1 treatment, N P addition increased the MBP and MBN of the Korean pine plantation. Furthermore, under the N3 treatment, the addition of NP reduced the broad-leaved Korean pine forest’s MBN and MBP. Under the litter treatment, except for the MBN of the Korean pine plantation and broad-leaved Korean pine forest treated with AL, the MBN and MBP of the Korean pine plantation were increased. However, this treatment decreased the MBN and MBP of the broad-leaved Korean pine forest (Table S2).

3.2. The Litter Treatment Regulates Nitrogen and Phosphorus Additions to Soil Hydrolase Activities

The litter treatment and nitrogen and phosphorus addition had no significant effect on the activities of soil carbon hydrolases (BG, BX, CBH, POX, and PER) and nitrogen hydrolases (NAG and LAP) and phosphorus hydrolases (AP) in broad-leaved Korean pine forest (Table S3; Figure 4). The activities of the enzymes BG, BX, CBH, AP, and the C-hydrolase (BG + BX + CBH + PEROX + PHOX) were found to be significantly affected by the different forest types, namely the Korean pine plantation and the broad-leaved Korean pine forest (Table 2). The interactions between the litter treatment, N and P applications, and different forest types were found to be relatively minor. In general, the soil hydrolase activity of the Korean pine plantation (F) was observed to be higher than that of the broad-leaved Korean pine forest (I). The application of the litter treatment reduced the activities of BG, BX, NAG, AP, PHOX, and C-hydrolase. However, CBH, LAP, PEROX, and N-hydrolase activities were higher under the litter removal treatment (RL) than the other litter treatments (CK and AL). As the NP addition (NO, N1, N2, N3) increased, the activities of BG, BX, CBH, LAP, AP, and PEROX initially decreased and then increased. Conversely, the activities of PHOX, C-hydrolase, and N-hydrolase increased with the NP addition (Table S3). The results of the principal component analysis demonstrated that the first and second principal components collectively explained 53.4% and 13.1% of the variance, respectively (Figure 5). The correlation between the treatment combinations and PEROX, LAP, and N-hydrolase activity was the most significant, while the correlation with PHOX was the least pronounced. In general, the combination of the original litter treatment (CK) and high N P (N3) (N3L0) had the most pronounced effect on enzyme activity.

3.3. Soil Enzyme Activities Correlated with Chemical Indices and Microbial Biomass Properties

Table 3 indicates the significance and strength of the correlation between the soil microbial biomass, enzyme activity, and soil properties. The soil TOC, PH, and MBC were negatively correlated with all soil enzymes and hydrolase activities, the TOC was significantly negatively correlated with BG, CBH, and LAP activities, and the soil PH was significantly negatively correlated with CBH. The DOC was significantly negatively correlated with BG, BX, and C-hydrolase activities. The SAP was significantly negatively correlated with PEROX. The soil TN and CBH were significantly negatively correlated. There was a significant positive correlation between the soil MBN and LAP activity.

4. Discussion

4.1. Responses of Soil Chemical Properties and Soil Microbial Biomass to N and P Addition and Litter Treatments

Nitrogen and phosphorus in soil are essential nutrients that regulate plant growth and development [25,26]. They also serve as the primary material basis and environmental conditions that support plant survival. The effects of the litter treatment and N P addition on the soil organic carbon (TOC) were inconsistent. The findings indicate that incorporating nitrogen and phosphorus had a negligible impact on the TOC concentration of the Korean pine plantation. However, this addition in the broad-leaved Korean pine forest resulted in a pronounced negative effect, leading to a notable decline in the TOC levels. The most pronounced reduction was observed, reaching 33.85%. Implementing low and medium nitrogen management measures resulted in an enhancement in the soil’s effective nitrogen content, accompanied by a reduction in the soil’s carbon/nitrogen ratio (C/N). This alteration established auspicious circumstances for the liberation of nutrients by microorganisms during the decomposition of organic matter. Concurrently, it stimulates microbial activity and expedites the release of nutrients during the decomposition of organic matter [27]. In contrast, the litter treatment typically resulted in a reduction in the total organic carbon (TOC) content of the soil. This result indicates that the impact of nitrogen and phosphorus addition on the total organic carbon (TOC) was more intricate and dependent on the specific soil type. At the same time, the litter treatment demonstrated more pronounced adverse effects across all soil types. As the fresh litter input increases, the potential for inhibiting the soil organic carbon loss also increases [28]. Conversely, nitrogen and phosphorus additions alter the microbial enzymes and metabolic activities during litter decomposition, inhibiting decomposition and increasing soil carbon pools [29,30]. It has been demonstrated that litter treatment exerts a considerable influence on the soil-dissolved organic carbon (DOC) content in both Korean pine plantations and broad-leaved Korean pine forests. However, in the broad-leaved Korean pine forest, adding nitrogen and phosphorus resulted in a notable reduction in the DOC content. The addition of nitrogen and phosphorus had a negligible impact on the DOC of the Korean pine plantation, suggesting that the direct addition of these nutrients may facilitate the sequestration of soil organic carbon within a specific range while simultaneously reducing the mineralization and decomposition of organic carbon [31,32]. The observed increase in the DOC content in the Korean pine plantation under the litter treatment may indicate enhanced biomass circulation and replenishment, which could contribute to an improvement in the soil carbon storage capacity. The increased DOC may be attributed to the enhanced fixation of carbon released by litter decomposition in the broad-leaved Korean pine forest. Prior research indicates that soil nitrogen availability rises with nitrogen deposition, exhibiting a robust positive correlation with DOC decomposition. This increase in soil nitrogen availability may stimulate microbial activity, leading to an upsurge in carbon-degrading enzymes and accelerating the decomposition of DOC [33]. However, an elevated nitrogen input may also precipitate soil acidification and reduce soil carbon pools [34]. While the impact of nitrogen and phosphorus addition on dissolved organic carbon (DOC) in Korean pine plantations is relatively limited, its detrimental effect on broad-leaved Korean pine forests cannot be overlooked. As an active soil management strategy, litter treatment has been demonstrated to significantly increase the soil’s soluble organic carbon content in both Korean pine plantations and broad-leaved Korean pine forests. This result has shown to positively affect maintaining and enhancing the carbon sink function of forest ecosystems.
The present study demonstrates that nitrogen (N) and phosphorus (P) addition had no significant impact on the SAP. In contrast, the litter treatment resulted in a notable reduction in the SAP in the context of the Korean pine plantation. However, the interaction between the litter treatment and N addition significantly affected the TP in the broad-leaved Korean pine forest, resulting in an increase in the TP content. A recent study on the response of soil phosphorus fractions to the litter treatment demonstrated that the content of soil phosphorus fractions in primary and secondary forests underwent significant changes [35]. The input of nitrogen can increase the demand or limitation of phosphorus in the soil, thereby exacerbating the limitation of phosphorus [36]. This is a determining factor in microbial nutrient uptake [37,38]. Additionally, it was determined that phosphorus availability represents a limiting factor for microbial growth. Furthermore, the addition of nitrogen and phosphorus could significantly reduce the total nitrogen content of the broad-leaved Korean pine forest with a maximum decrease of 36.87%. However, in the case of the Korean pine plantation, except for the N2 treatment, the addition of N and P had no significant effect on the TN (Table S1; Figure 2E). The alterations to soil nutrients resulting from the introduction of nitrogen and phosphorus may influence the nutrient cycle by affecting EEAs and enhancing the absorption of nitrogen by plants [39,40].
Our results demonstrate that the litter treatment had markedly disparate effects on the soil microbial biomass carbon (MBC) in the Korean pine plantation and broad-leaved Korean pine forest. The impact of NP supplementation on the MBN and MBP exhibited notable discrepancies. The N1 treatment resulted in a significant increase in the MBP, while the N3 treatment led to a significant decrease in MBN (Table S2; Figure 2). It can be demonstrated that litter treatment increases the soil microbial biomass carbon (MBC). However, this effect manifests differently in the context of Korean pine plantations and broad-leaved Korean pine forests. Specifically, the increase in MBC in the Korean pine plantation is more significant under the presence of NP addition, vegetation, and litter, contributing to the input of soil C through material circulation, leading to an increase in the MBC content and, consequently, an enhancement in ecosystem productivity. This process also results in soil microorganisms intensifying their consumption of P [41,42,43]. Therefore, soil microorganisms’ utilization efficiency of soil organic carbon was enhanced [44,45]. This point suggests that the microbial response to carbon accumulation may be slower or less sensitive to litter treatment in the broad-leaved Korean pine forest than in the Korean pine plantation. Nitrogen enrichment has been demonstrated to inhibit fungal activity by reducing the soil pH and inhibiting the degradation rate of lignin. However, it has also been shown to enhance soil enzyme activity and the ability of soil fungi to decompose litter, increasing the soil microbial biomass C/N ratio [46]. This finding underscores the diversity of forest types in response to soil management measures, thereby underscoring the necessity of considering the specific needs of forest types when implementing forest management and soil protection measures. In the Korean pine plantation, adding nitrogen led to a tendency towards soil acidification, with a particularly pronounced decrease in pH under the N2 treatment. The application of nitrogen (N) and phosphorus (P) fertilizer also reduced the soil’s pH level in the broad-leaved Korean pine forest. However, the impact was less pronounced when compared to the results of the Korean pine plantation. In the broad-leaved Korean pine forest, except for the N1 treatment, the majority of the N P addition treatments resulted in a reduction in the soil pH, although this did not reach statistical significance. In both the Korean pine plantation and the broad-leaved Korean pine forest, adding nitrogen and phosphorus reduced the pH value of the soil, resulting in a certain degree of soil acidification. This phenomenon may have impacted the growth environment of plant roots in the soil, thereby indirectly influencing the overall health and productivity of the forest. Furthermore, soil acidification may also impede the activity of certain soil microorganisms, influencing soil nutrient cycling and biodiversity. It is therefore imperative to gain an understanding of and to be able to control the effects of N P addition on soil pH if we are to achieve sustainable forest management and soil conservation.

4.2. Responses of C-, N-, and P-Degrading Enzymes to N P Addition and Litter Treatments

Soil enzymes serve as a significant indicator for assessing soil fertility and microbial activity, which directly influence the efficacy of soil nutrient cycling. Carbon, nitrogen, and phosphorus hydrolases are significant enzymes in soil, facilitating the decomposition of organic matter and nutrient cycling [47,48]. In this study, the increase in N and P addition (NO, N1, N2, and N3) resulted in declines in BG, BX, CBH, LAP, AP, and PEROX activities, which subsequently increased. Conversely, PHOX, C-hydrolase, and N-hydrolase activities demonstrated an upward trend with the rise in N and P addition. The litter treatment decreased the activities of BG, BX, NAG, AP, PHOX, and C-hydrolase, but the activities of CBH, LAP, PEROX, and N-hydrolase under the litter removal treatment (RL) were higher than those under the other litter treatments (Table 2; Figure 3). The findings indicate that while the litter treatment had no discernible impact on the overall carbon hydrolase activity, it exerted varying effects on the activity of specific soil enzymes, including CBH, LAP, and PEROX. Adding nitrogen can stimulate the activity of carbon hydrolase, thereby promoting the decomposition of organic matter and the release of carbon. The addition of phosphorus has been demonstrated to exert a promoting effect on the activity of phosphorus hydrolase, thereby facilitating the release and circulation of phosphorus [31,32]. Furthermore, nitrogen addition has been demonstrated to influence litter decomposition and the soil environment. Different levels of nitrogen addition can elicit disparate reactions in soil enzyme activity and nutrient cycling [49,50,51]. Additionally, nitrogen and phosphorus addition may also impact the activity of nitrogen hydrolase, though the precise effects may fluctuate depending on the environmental conditions. The present study demonstrates that different forest types (Korean pine plantations and broad-leaved Korean pine forests) exerted notable influences on the activities of several enzymes, including BG, BX, CBH, AP, and C-hydrolase (BG + BX + CBH + PEROX + PHOX). The findings indicate that the forest type was a significant determinant of the soil hydrolase activity, with the soil hydrolase activity of the Korean pine plantation (F) exhibiting a higher mean value than that of the broad-leaved Korean pine forest (I). The addition of litter can provide organic matter and nutrients, thereby promoting the activity of carbon, nitrogen, and phosphorus hydrolases. The carbon present in the litter can be utilized as a substrate to stimulate the activity of carbon hydrolase. Additionally, the nitrogen and phosphorus in the litter can serve as nutrients, stimulating the activity of the corresponding hydrolases. Consequently, incorporating litter can augment the activities of carbon, nitrogen, and phosphorus hydrolases within the soil. In conclusion, the responses of carbon, nitrogen, and phosphorus-degrading enzymes to nitrogen and phosphorus addition and litter treatment are complex and affected by many factors, including long-term fertilization, nutrient limitation, and microbial community dynamics [52,53]. It has been reported that the co-addition of N and P significantly affect the soil extracellular enzymes involved in C, N, and P cycling in subtropical forests, indicating that soil microorganisms have different needs for N and P under different concentrations of N and P addition [54]. In our study, nitrogen and phosphorus addition had disparate effects on the soil extracellular enzyme activities involved in carbon, nitrogen, and phosphorus cycling in the broad-leaved Korean pine forest soil. However, the overall effect was not statistically significant. This may be true because the addition of nitrogen and phosphorus provides exogenous resources for soil microorganisms, affecting the availability of soil nitrogen and phosphorus [55] and leading to the mutual transformation of N limitation and C limitation. An understanding of these responses is crucial for the elucidation of the fundamental mechanisms governing nutrient cycling in terrestrial ecosystems and can provide invaluable insights into the impact of human activities, such as fertilization and nutrient deposition, on forest soil health and the functioning of broad-leaved Korean pine forests.

4.3. Major Factors Affecting Soil EEAs and Microbial Communities

Our findings reveal that the soil properties under the litter treatment and the nitrogen and phosphorus addition treatment were the primary factors influencing the soil EEAs’ microbial community. The soil nutrient content can alter soil EEAs and ecological enzyme stoichiometry by modifying the availability of soil matrices and influencing soil stoichiometry [46,48]. Furthermore, soil carbon and nitrogen concentrations are the most crucial elements that influence soil EEAs [56]. Furthermore, the soil nutrient content significantly influences the activity and composition of EEAs and microbial communities [57]. The availability and concentration of nutrients can impact the growth and metabolic processes of microorganisms, affecting the expression of EEAs and the composition of microbial communities. The soil total organic carbon (TOC) demonstrated a significantly negative correlation with β-glucosidase (BG), cellobiohydrolase (CBH), and leucine aminopeptidase (LAP) activities. The findings indicate that under elevated TOC circumstances, the organic matter may be decomposed by a more intricate microbial community, which may rely less on particular enzymes. Furthermore, the elevated TOC milieu may facilitate the formation of more resilient organic matter, thereby reducing the necessity for enzymes. Our data indicate a statistically significant negative correlation between dissolved organic carbon (DOC) and both β-xylosidase (BX) and cellulase (CBH) activities. This result may be possible because in an environment with a high DOC, microorganisms may be more inclined to utilize the dissolved organic carbon directly rather than obtain carbon sources through enzymatic decomposition. Furthermore, a negative correlation was observed between the soil available phosphorus (SAP) and peroxidase (PEROX) activity. There was a significant negative correlation between the soil total nitrogen and cellulose-binding hemoglobin (CBH) activity and a significant positive correlation between microbial biomass nitrogen (MBN) and LAP activity. The application of nitrogen (N) can result in an increased demand for or limitation of phosphorus (P) in the soil, thereby exacerbating the existing limitations of P in the soil [36,48]. It is responsible for regulating the absorption of microbial nutrients [37]. Furthermore, it has been documented that phosphorus availability represents a significant limiting factor for microbial growth [6]. The inverse relationship between soil pH and soil enzyme activity demonstrates the optimal pH range for soil enzyme activity. Beyond this range, the enzyme activity will decline. A change in the soil pH may directly impact the structure and function of the enzyme, consequently influencing its activity. The present study revealed that the litter treatment, N addition, and other types of treatments had significant or highly significant effects on the TOC, DOC, TSN, SAP, TN, TP, and MBC in soil. However, no significant effect was observed for the MBN or MBP (Table 4). These effects are not merely reflected in a single factor but also the interaction between them. The contents of the soil total organic carbon (TOC) and dissolved organic carbon (DOC) were found to be significantly affected by the forest type, N P addition, litter treatment, and their interactions. This result suggests that disparate environmental conditions and management strategies influence the accumulation and decomposition of organic matter in soil, which, in turn, impacts the soil carbon cycle. The concentration of soil soluble nitrogen (TSN) and total nitrogen (TN) exhibited a significant response to the forest type, N P addition, litter treatment, and their interaction. This may indicate the inherent complexity of the nitrogen cycle, which is regulated by numerous factors under diverse environmental conditions. The responses of the total phosphorus (TP) and soil pH to the forest type, nitrogen addition, litter treatment, and their interactions indicated that these factors affected the phosphorus availability and soil acid–base balance, which are critical for plant nutrition and microbial activity. The microbial biomass carbon (MBC) was found to be significantly affected by the forest type, N P addition, litter treatment, and their interaction. In contrast, the responses of microbial biomass nitrogen (MBN) and microbial biomass phosphorus (MBP) were not significant. This may reflect the direct dependence of the carbon cycle on the microbial activity, while other factors may limit the nitrogen and phosphorus cycles.

5. Conclusions

Our results show that the litter treatment and nitrogen and phosphorus additions significantly affected the soil nutrients in different forest types. It can be reasonably argued that the litter treatment and N P addition represent efficacious methods for regulating forest soil properties. The litter treatment, N addition treatment, and other treatment types exerted considerable effects on the TOC, DOC, TSN, SAP, TN, TP, and MBC in soil. However, no significant impact was observed for the MBN nor MBP. Soil hydrolase activity was found to be unaffected by both the litter treatment and nitrogen and phosphorus addition. The litter treatment was observed to result in a decrease in the activities of BG, BX, NAG, AP, PHOX, and C-hydrolase (BG + BX + CBH + PEROX + PHOX). In contrast, the litter removal treatment (RL) resulted in an observed increase in the activities of CBH, LAP, PEROX, and N-hydrolase. Additionally, the investigation revealed that distinct forest types, namely Korean pine plantations and broad-leaved Korean pine forests, exerted notable influences on soil hydrolase activity. The soil hydrolase activity of the Korean pine plantation exhibited a general tendency to surpass that of the broad-leaved Korean pine forest. As the levels of nitrogen and phosphorus increased, there was initially a decrease in the activities of the BG, BX, CBH, LAP, AP, and PEROX enzymes before a gradual rise. A strong positive correlation was observed between PEROX, LAP, and N-hydrolase activities under high amounts of N P addition (N3L0), indicating a significant effect on the activity of these enzymes.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/f15101764/s1, Table S1 Mean values (±SE) of the soil chemical properties for the litter and N P addition treatments and two-way ANOVAs results (F and p values) for litter treatments (L), N P addition (N) and their interaction (L × N). Table S2 Mean values (±SE) of the soil microbial properties for the litter and N P addition treatments and two-way ANOVAs results (F and p values) for litter treatments (L), N P addition (N) and their interaction (L × N). Table S3 Mean values (±SE) of the soil enzyme activities for the litter and N P addition treatments and two-way ANOVAs results (F and p values) for litter treatments (L), N P addition (N) and their interaction (L × N).

Author Contributions

L.C. (Liming Chen): data curation, writing—original draft preparation, visualization, investigation, software, validation, and writing—reviewing and editing; L.C. (Lixin Chen): conceptualization, methodology, writing—reviewing and editing, and software; M.C.: visualization and investigation; Y.W.: software, validation, visualization, and investigation; W.D.: supervision and writing—reviewing and editing. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Fundamental Research Funds for the Central Universities (2572021DT04) and National Forestry and Grassland Bureau promotion project (2023133124).

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Acknowledgments

We thank Xiongwen Chen (Department of Biological & Environmental Sciences Alabama A & M University, Normal, Al 35762) for refining and revising the article.

Conflicts of Interest

The authors declare no conflicts of interest.

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Forests 15 01764 g001
Figure 1. Location of study area.
Figure 1. Location of study area.
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Figure 2. Soil chemical properties under different levels of nitrogen and amounts of phosphorus addition (N0, N1, N2, and N3) and litter treatments (CK, RL, and AL) in broad-leaved Korean pine (Pinus koraiensis) forests. The values are shown as the means of three replicates (±SE). Different uppercase letters show significant differences among different litter treatments with the same N and P addition application rate, and different lowercase letters indicate significant differences among different N and P additions under the same litter treatment (p < 0.05). No letter indicates that the difference is not significant. F = Korean pine plantation; I = broad-leaved Korean pine forest; (A) TOC = soil total organic carbon; (B) DOC = soil dissolve organic carbon; (C) TSN = soil total soluble nitrogen; (D) SAP = soil available phosphorus; (E) TN = soil total nitrogen; (F) TP = soil total phosphorus; (G) pH = soil PH.
Figure 2. Soil chemical properties under different levels of nitrogen and amounts of phosphorus addition (N0, N1, N2, and N3) and litter treatments (CK, RL, and AL) in broad-leaved Korean pine (Pinus koraiensis) forests. The values are shown as the means of three replicates (±SE). Different uppercase letters show significant differences among different litter treatments with the same N and P addition application rate, and different lowercase letters indicate significant differences among different N and P additions under the same litter treatment (p < 0.05). No letter indicates that the difference is not significant. F = Korean pine plantation; I = broad-leaved Korean pine forest; (A) TOC = soil total organic carbon; (B) DOC = soil dissolve organic carbon; (C) TSN = soil total soluble nitrogen; (D) SAP = soil available phosphorus; (E) TN = soil total nitrogen; (F) TP = soil total phosphorus; (G) pH = soil PH.
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Figure 3. Soil microbial properties under different levels of nitrogen and phosphorus additions (N0, N1, N2, and N3) and litter treatments (CK, RL, and AL) in broad-leaved Korean pine (Pinus koraiensis) forests. The values are shown as the means of three replicates (±SE). Different uppercase letters show significant differences among different litter treatments with the same N and P addition application rate, and different lowercase letters indicate significant differences among different N and P additions under the same litter treatment (p < 0.05). No letter indicates that the difference is not significant. I = broad-leaved Korean pine forest; F = Korean pine plantation; (A) MBC = soil microbial biomass carbon; (B) MBN = soil microbial biomass nitrogen; (C) MBP = soil microbial biomass phosphorus.
Figure 3. Soil microbial properties under different levels of nitrogen and phosphorus additions (N0, N1, N2, and N3) and litter treatments (CK, RL, and AL) in broad-leaved Korean pine (Pinus koraiensis) forests. The values are shown as the means of three replicates (±SE). Different uppercase letters show significant differences among different litter treatments with the same N and P addition application rate, and different lowercase letters indicate significant differences among different N and P additions under the same litter treatment (p < 0.05). No letter indicates that the difference is not significant. I = broad-leaved Korean pine forest; F = Korean pine plantation; (A) MBC = soil microbial biomass carbon; (B) MBN = soil microbial biomass nitrogen; (C) MBP = soil microbial biomass phosphorus.
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Figure 4. Soil enzyme activities under different levels of nitrogen and phosphorus additions (N0, N1, N2, and N3) and litter treatments (CK, RL, and AL) in broad-leaved Korean pine (Pinus koraiensis) forests. The values are shown as the means of three replicates (±SE). Different uppercase letters show significant differences among different litter treatments with the same N and P addition application rates, and different lowercase letters indicate significant differences among different N and P additions under the same litter treatment (p < 0.05). No letter indicates that the difference is not significant. I = broad-leaved Korean pine forest; F = Korean pine plantation; (A) BG = β-1,4-glucosidase; (B) BX = β-1,4-xylosidase; (C) CBH = cellobiohydrolase; (D) LAP = L-leucine aminopeptidase; (E) NAG = β-1,4-N-acetylglucosaminidase; (F) AP = acid phosphatase; (G) PEROX = peroxidase; (H) PHOX = phenol oxidase; (I) C-degrading enzymes; (J) N-degrading enzymes.
Figure 4. Soil enzyme activities under different levels of nitrogen and phosphorus additions (N0, N1, N2, and N3) and litter treatments (CK, RL, and AL) in broad-leaved Korean pine (Pinus koraiensis) forests. The values are shown as the means of three replicates (±SE). Different uppercase letters show significant differences among different litter treatments with the same N and P addition application rates, and different lowercase letters indicate significant differences among different N and P additions under the same litter treatment (p < 0.05). No letter indicates that the difference is not significant. I = broad-leaved Korean pine forest; F = Korean pine plantation; (A) BG = β-1,4-glucosidase; (B) BX = β-1,4-xylosidase; (C) CBH = cellobiohydrolase; (D) LAP = L-leucine aminopeptidase; (E) NAG = β-1,4-N-acetylglucosaminidase; (F) AP = acid phosphatase; (G) PEROX = peroxidase; (H) PHOX = phenol oxidase; (I) C-degrading enzymes; (J) N-degrading enzymes.
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Figure 5. The results of the principal component analysis (PCA) based on soil extracellular enzyme activities under different levels of nitrogen and phosphorus (N0, N1, N2, and N3) and litter treatments (CK, RL, and AL) (L0, L1, and L2) in the broad-leaved Korean pine forest. Different colors and capital letters represent different treatment combinations. BG = β-1,4-glucosidase; BX = β-1,4-xylosidase; CBH = cellobiohydrolase; NAG = β-1,4-N-acetylglucosaminidase; LAP = L-leucine aminopeptidase; PEROX = peroxidase; PHOX = phenol oxidase; AP = acid phosphatase; C- = degrading enzymes (BG + BX + CBH + PEROX + PHOX); N- = degrading enzymes (NAG + LAP).
Figure 5. The results of the principal component analysis (PCA) based on soil extracellular enzyme activities under different levels of nitrogen and phosphorus (N0, N1, N2, and N3) and litter treatments (CK, RL, and AL) (L0, L1, and L2) in the broad-leaved Korean pine forest. Different colors and capital letters represent different treatment combinations. BG = β-1,4-glucosidase; BX = β-1,4-xylosidase; CBH = cellobiohydrolase; NAG = β-1,4-N-acetylglucosaminidase; LAP = L-leucine aminopeptidase; PEROX = peroxidase; PHOX = phenol oxidase; AP = acid phosphatase; C- = degrading enzymes (BG + BX + CBH + PEROX + PHOX); N- = degrading enzymes (NAG + LAP).
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Table 1. Basic Survey of Broad-leaved Pinus koraiensis Forest and Pinus koraiensis Plantation in the sample plots.
Table 1. Basic Survey of Broad-leaved Pinus koraiensis Forest and Pinus koraiensis Plantation in the sample plots.
Forest TypesSample PlotTopographical FactorsForest Stand Survey Factors
Slope
Aspect		Slope PositionSlope (°)Elevation /mAge
(a)		Average
DBH/cm		Average Tree
Height/m		Stand Density
(Plant/hm2)		Species
Composition		Canopy
Closure
FF1semi-shady slopedownhill8°4156519.9117.7198310Pk+Am0.70
F2semi-shady slopemidslope9°4356522.9119.5595010Pk0.80
F3semi-shady slopeuphill7°4156520.9518.5386710Pk+Bc0.75
II1semi-shady slopeuphill15°48523018.2610.487837Pk+2An+1Bc+Ta-Am0.71
I2semi-shady slopemidslope15°49923013.578.227508Pk+1An+1Ta-Am0.65
I3semi-shady slopedownhill15°47123016.059.9855010Pk+Ta+Bp-Bc0.50
F = Pinus koraiensis Plantation; I = Broad-leaved Pinus koraiensis Forest; Pk = Pinus koraiensis; Ta = Tilia amurensis; Am = Acer mono; An = Abies nephrolepis; Bc = Betula costata; Bp = Betula platyphylla.
Table 2. A summary of the linear mixed-effects models for the effects of the site (forest type), treatment (N P addition), litter, and their interactions on soil enzyme activities. BG = β-1,4-glucosidase; BX = β-1,4-xylosidase; CBH = cellobiohydrolase; NAG = β-1,4-N-acetylglucosaminidase; LAP = L-leucine aminopeptidase; PEROX = peroxidase; PHOX = phenol oxidase; AP = acid phosphatase; C-degrading enzymes = BG + BX + CBH + PEROX + PHOX; N-degrading enzymes = NAG + LAP. The F values are shown in the table. ** p < 0.01.
Table 2. A summary of the linear mixed-effects models for the effects of the site (forest type), treatment (N P addition), litter, and their interactions on soil enzyme activities. BG = β-1,4-glucosidase; BX = β-1,4-xylosidase; CBH = cellobiohydrolase; NAG = β-1,4-N-acetylglucosaminidase; LAP = L-leucine aminopeptidase; PEROX = peroxidase; PHOX = phenol oxidase; AP = acid phosphatase; C-degrading enzymes = BG + BX + CBH + PEROX + PHOX; N-degrading enzymes = NAG + LAP. The F values are shown in the table. ** p < 0.01.
Site (S)Treatment (T)Litter (L)S × TS × LT × LS × T × L
BG10.637 **0.2771.7940.4440.5240.5730.469
BX7.909 **0.1851.6820.2910.8150.1950.422
CBH7.446 **0.4140.1150.3970.4630.130.582
NAG0.2651.381.2280.7430.5991.0290.494
LAP2.8661.0111.5570.1591.3710.4470.537
PEROX2.0860.2920.1870.4770.6980.631.288
PHOX0.0030.8121.1640.1550.4880.540.377
AP11.182 **0.5792.7410.330.321.0851.255
C-degrading enzymes7.980 **0.2922.2680.2811.050.4060.472
N-degrading enzymes0.7120.4372.8920.2811.5790.1040.999
Table 3. Pearson correlation between soil properties, soil enzyme activities, and soil microbial biomass. Values are correlation coefficients. ** p < 0.01.
Table 3. Pearson correlation between soil properties, soil enzyme activities, and soil microbial biomass. Values are correlation coefficients. ** p < 0.01.
TOCDOCTSNSAPTNTPPHMBCMBNMBP
BG−0.242 **−0.236 **−0.1000.104−0.093−0.088−0.056−0.2160.1980.042
BX−0.211−0.243 **−0.0220.095−0.222−0.003−0.045−0.2090.172−0.085
CBH−0.262 **−0.190−0.0780.022−0.057−0.099−0.239 **−0.083−0.0030.136
NAG−0.021−0.1670.080−0.0500.073−0.191−0.032−0.1120.1360.012
LAP−0.245 **−0.1130.0900.092−0.297 **0.007−0.166−0.0320.308 **−0.118
PEROX−0.1910.067−0.009−0.236 **−0.100−0.028−0.129−0.1410.1750.017
PHOX−0.0600.0900.045−0.0830.0600.141−0.138−0.089−0.008−0.010
AP−0.159−0.124−0.0190.2110.1020.083−0.066−0.187−0.008−0.127
C-−0.203−0.268 **−0.1250.114−0.174−0.048−0.036−0.1810.1520.001
N-−0.105−0.200−0.0050.073−0.161−0.089−0.059−0.0380.212−0.047
Table 4. A summary of the linear mixed-effects models for the effects of site (forest type), treatment (N P addition), litter, and their interactions on the soil chemical properties and soil microbial properties. The F values are shown in the table. ** p < 0.01, and *** p < 0.001.
Table 4. A summary of the linear mixed-effects models for the effects of site (forest type), treatment (N P addition), litter, and their interactions on the soil chemical properties and soil microbial properties. The F values are shown in the table. ** p < 0.01, and *** p < 0.001.
Site (S)Treatment (T)Litter (L)S × TS × LT × LS × T × L
TOC381.173 ***58.550 ***26.202 ***38.751 ***5.160 **14.763 ***48.320 ***
DOC25.496 ***10.727 ***56.203 ***9.697 ***102.910 ***2.538 **21.764 ***
TSN0.2910.3090.1780.5652.2930.4671.322
SAP0.0080.1834.979 **0.5682.0350.1831.020
TN27.143 ***26.597 ***14.665 ***11.645 ***0.64918.862 ***16.246 ***
TP0.9932.740 **1.1106.931 **5.038 **5.015 ***5.293 ***
PH10.087 **0.8740.7512.1481.5750.7642.625 **
MBC863.943 ***181.412 ***227.335 ***150.589 ***18.362 ***224.040 ***227.640 ***
MBN3.6470.4850.7720.7480.3780.7730.402
MBP0.9160.1651.1990.2490.1680.7960.207
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Chen, L.; Chen, L.; Chen, M.; Wang, Y.; Duan, W. The Responses of Soil Extracellular Enzyme Activities and Microbial Nutrients to the Interaction between Nitrogen and Phosphorus Additions and Apoplastic Litter in Broad-Leaved Korean Pine Forests in Northeast China. Forests 2024, 15, 1764. https://doi.org/10.3390/f15101764

AMA Style

Chen L, Chen L, Chen M, Wang Y, Duan W. The Responses of Soil Extracellular Enzyme Activities and Microbial Nutrients to the Interaction between Nitrogen and Phosphorus Additions and Apoplastic Litter in Broad-Leaved Korean Pine Forests in Northeast China. Forests. 2024; 15(10):1764. https://doi.org/10.3390/f15101764

Chicago/Turabian Style

Chen, Liming, Lixin Chen, Meixuan Chen, Yafei Wang, and Wenbiao Duan. 2024. "The Responses of Soil Extracellular Enzyme Activities and Microbial Nutrients to the Interaction between Nitrogen and Phosphorus Additions and Apoplastic Litter in Broad-Leaved Korean Pine Forests in Northeast China" Forests 15, no. 10: 1764. https://doi.org/10.3390/f15101764

APA Style

Chen, L., Chen, L., Chen, M., Wang, Y., & Duan, W. (2024). The Responses of Soil Extracellular Enzyme Activities and Microbial Nutrients to the Interaction between Nitrogen and Phosphorus Additions and Apoplastic Litter in Broad-Leaved Korean Pine Forests in Northeast China. Forests, 15(10), 1764. https://doi.org/10.3390/f15101764

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