Chemical and Biological Amendments and Crop Rotation Affect Soil Carbon and Nitrogen Sequestration by Influencing the Carbon and Nitrogen Contents of Soil Aggregates

Abstract

Soil organic carbon (SOC) and total nitrogen (TN) sequestration are vital for maintaining soil fertility and mitigating climate change. This study aimed to evaluate the effects of different amendments (chemical and biological) and crop rotations on SOC, TN sequestration, and soil aggregate distribution. A six-year field study was conducted, involving five different treatments: a monoculture of peanut (PC), a monoculture of maize (MC), a maize-peanut rotation (M-PR), and peanut continuous cropping with chemical (PCCA) and biological (PCBA) amendments. Soil properties, aggregate size distribution, SOC, TN, and enzyme activities were measured. The results show that the bulk density increased, while the field water−holding capacity and porosity decreased with depth. M-PR had the highest macroaggregate (>0.25 mm) proportion, increasing by 21.6–50.8%. SOC and TN increased with aggregate size and were 23.9–103.6% and 7.0–82.9% higher, than PC and MC, respectively, under the treatments. PCCA showed the highest SOC, TN, and enzyme activities. Structural equation modeling indicated that the C and N contents of aggregates directly influenced SOC and TN sequestration. In conclusion, crop rotation and amendments, especially PCCA, effectively improve soil C and N sequestration, and enhance the soil structure, thereby reducing degradation risks, and potentially decreasing on−farm greenhouse gas emissions.

1. Introduction

Continuous cropping involves cultivating the same crop repeatedly on the same field over a long duration to maximize yields [1], and it is widely practiced in China’s agricultural systems. However, this practice can lead to unfavorable growing conditions, increased pest infestations, higher disease occurrence, and reduced crop yields [2]. Additionally, continuous cropping has been shown to decrease organic carbon (C) sequestration and compromise soil aggregate stability [3]. This issue is particularly pronounced in crops with high economic value and intensive cultivation demands, such as peanuts (Arachis hypogaea L.) [4]. Peanut ranks among the leading economic and oilseed crops worldwide [5]. Its economic value surpasses that of many other crops, contributing to the continuous expansion of its production scale [6]. Despite this, peanuts face challenges in sustaining or increasing production in China [7], primarily due to crop succession issues, limited arable land, water scarcity, and the intensification of agricultural practices [8]. Research has shown that extended periods of continuous cropping can cause the degradation of soil microbial communities [9], the suppression of soil enzyme activities [10], a buildup of autotoxic substances, and disruptions in nutrient balance, thereby heightening the vulnerability of peanuts to diseases and ultimately leading to reductions in yield and quality [11]. To address the challenges associated with continuous cropping, various measures have been implemented, which are critical for the sustainable development of peanut production.

Maize (Zea mays L.) monoculture is one of the most significant food production systems in dryland regions [12]. In recent decades, maize yields have accounted for nearly one−third of China’s total feed and food supply [13]. Prolonged and intensive soil utilization has resulted in a series of detrimental effects, including soil erosion, the deterioration of soil physicochemical properties, and a decline in soil fertility [13]. The manifestation of continuous cropping obstacles is closely linked to alterations in soil chemical characteristics [14], the proliferation of soil-borne pathogens and pests [15], and significant shifts in soil microbial community structure and function [16]. To mitigate these challenges, adopting appropriate cropping systems and strengthening soil management practices are essential for improving soil chemical properties and altering microbial community structures [17,18]. Research has confirmed that the appropriate implementation of crop rotation strategies and soil amendment applications can significantly mitigate the adverse impacts associated with continuous cropping [16,19]. Crop rotation helps break the cycle of pests and diseases by introducing crops with varying levels of resistance and tolerance, thereby reducing the accumulation of pests and pathogens [20]. Additionally, crop rotation improves the soil nutrient structure, prevents the excessive depletion of specific nutrients by a single crop, and facilitates the balanced restoration of soil nutrients [21]. The root structures and growth patterns of different crops contribute to improving soil physical properties, enhancing aeration, and increasing water permeability, thus preventing soil compaction [22]. The maize–peanut rotation system has been increasingly implemented across Northeast China [13]. Nevertheless, studies investigating its effectiveness in mitigating continuous cropping obstacles and elucidating the underlying regulatory mechanisms remain scarce. Soil amendments refer to materials applied to enhance the properties of cultivated soils and boost crop productivity [23]. They support plant growth by optimizing the soil structure, increasing nutrient accessibility, improving water-holding capacity, stimulating microbial communities, adjusting soil pH, and minimizing the presence of toxic compounds [16]. Research has indicated that the prolonged application of humic acid enhances peanut resistance to continuous cropping stress, with a particularly pronounced effect on the growth of peanut seedlings [24]. Moreover, applying organic amendments has proven to be an efficient approach to improving soil aggregation and increasing organic carbon levels [25]. While recent research has examined the impacts of different cultivation methods on peanut−related disorders, evidence remains scarce regarding the effects of long−term management practices on the soil’s physical attributes and enzyme activities within continuous peanut cropping systems.

Increasing soil carbon is a potential mechanism to help offset the rising concentration of CO₂ in the atmosphere [26], and reducing its conversion into greenhouse gases is crucial for mitigating subsequent climate change [27]. Soil aggregates are crucial for enhancing the retention of soil organic carbon (SOC) and facilitating carbon sequestration [28]. First, aggregates physically enclose organic carbon, reducing its exposure to microorganisms and slowing the decomposition rate [29]. Second, aggregates provide a habitat for microorganisms, fostering the development of stable microbial communities that promote organic carbon accumulation [30]. Additionally, organic matter within aggregates combines with minerals to form organic−mineral complexes [31], which are more resistant to decomposition, thus enhancing carbon stability [32]. Nevertheless, soil aggregate stability is affected by different cultivation methods and soil amendment applications [33]. Research has demonstrated that rotations involving tobacco and maize markedly enhance SOC accumulation within macro-aggregates and elevate total nitrogen (TN) levels [34]. Nevertheless, studies have also reported that the carbon content of soil aggregates shows no significant variation across different crop rotation systems [35], as aggregates respond differently to various rotations, leading to varying levels of SOC sequestration. The addition of soil amendments such as organic fertilizers [36] and humic acid [37] can directly increase soil organic matter, promote aggregate formation, and enhance aggregate stability. The organic and mineral components in amendments improve the soil structure, facilitating the sequestration of more organic carbon and nitrogen in stable aggregates [38]. These findings suggest that SOC sequestration varies with aggregate size and is influenced by the type of soil, amendment, and crop [39].

Currently, the majority of studies addressing continuous cropping challenges have concentrated mainly on the fundamental physical and chemical characteristics of soils in peanut monoculture systems [40]. Despite the availability of diverse soil amendments, the potential of biochar, as an emerging amendment, to enhance soil quality under continuous peanut cropping systems remains insufficiently explored. Soil enzyme activity directly influences the mineralization of organic matter and the transformation of nutrient forms [41], thereby regulating nutrient availability to plants [42]. However, the mechanisms by which soil amendments and crop rotation systems regulate soil carbon (C) and nitrogen (N) sequestration remain inadequately understood. To address this knowledge gap, a six-year field experiment was conducted to assess the impacts of different cropping systems—continuous peanut cropping, continuous maize cropping, and peanut-maize rotation—as well as the application of chemical and biological amendments within peanut monoculture systems, on soil C and N sequestration, the distribution of C and N across soil aggregates, and the activity of C- and N-cycling enzymes. We hypothesize that peanut-maize rotation and amendments enhance soil organic carbon (SOC) and total nitrogen (TN) sequestration by promoting soil aggregate formation and stability. Our objectives are to: (1) assess the effects of different cropping systems and amendments on aggregate stability; (2) quantify the relationship between aggregate-associated C and N contents and SOC and TN; and (3) determine how crop rotation and amendment application promote soil aggregate stability and enzyme activity, as well as elucidating their potential mechanisms.

2. Materials and Methods

2.1. Site Description

A long-term field trial began in 2011 at the Peanut Scientific Research Center of Shenyang Agricultural University (40°48′ N, 123°33′ E) in Liaoning Province, China. The study area experiences a semihumid climate, characterized by an average annual temperature between 7.0 °C and 8.1 °C, and an average annual precipitation of approximately 547 mm. According to the US Soil Taxonomy, the soil at the experimental site is categorized as a Hapli–Udic Cambisol. The area was mixed with sand before the start of the experiment to reduce the soil fertility, for specific details, refer to Gao et al., [43]. The basic chemical properties of the dry soil at the beginning of the experiment in 2011 are shown in

Table 1. Basic chemical properties of the soil at the beginning of the experiment (2011).

Soil Depth (cm)	pH	Organic Carbon (g/kg)	Total Nitrogen (g/kg)	Total Phosphorus (g/kg)	Total Potassium (g/kg)	Available Nitrogen (mg/kg)	Available Phosphorus (mg/kg)	Available Potassium (mg/kg)
0–20	5.59 ± 0.10	6.95 ± 0.11	0.95 ± 0.01	0.41 ± 0.02	17.64 ± 1.14	78.30 ± 4.52	10.37 ± 1.25	52.56 ± 6.47
20–40	5.84 ± 0.21	8.52 ± 0.14	1.03 ± 0.02	0.49 ± 0.04	18.40 ± 1.24	120.45 ± 9.78	19.72 ± 2.35	71.23 ± 5.45

Note: Data are presented as mean ± standard deviation, n = 3.

.

2.2. Experimental Design and Field Management

Five treatments were established in the experiment: continuous maize cultivation (MC), maize–peanut rotation (M-PR), peanut monoculture (PC), peanut monoculture amended with chemical inputs (PCCA), and peanut monoculture amended with biological agents (PCBA). These five treatments are detailed in

Table 2. Nutrient input and modifier dosage in different treatments (kg/hm²).

Treatment	Nutrient Input (kg/hm2)
	N	P2O5	K2O	Chemical Modifier	Biological Modifier
MC	225	75	75	0	0
M-PR	225 ①/60 ②	75/82.5	75/112.5	0	0
PC	60	82.5	112.5	0	0
PCCA	58.1	79.4	110.3	750	0
PCBA	56.6	82.3	112.3	0	45

MC: maize continuous cropping; M-PR: peanut−maize rotation; PC: peanut continuous cropping; PCCA: peanut continuous cropping + chemical amendments; PCBA: peanut continuous cropping + biological amendments. Starting from 2011, the M-PR treatment was planted to peanuts in odd years with the same fertilizer application as the PC treatment, and it was planted to maize in even years with the same fertilizer application as the MC treatment. ① The amount of fertilizer applied to maize and ② The amount of fertilizer applied to peanuts.

. In 2011, a randomized complete block design (RCBD) was implemented to establish the treatments, with three replicates for each across 15 plots measuring 6 m by 4.5 m. The ridges were spaced 90 cm apart, with two rows of peanuts planted on each ridge. The row spacing was 30 cm, and the spacing between individual plants within the rows was 15 cm. For maize, the planting method was manual hole planting with single−seed sowing, a plant spacing of 37 cm, and a row spacing of 90 cm. The fertilizers tested included urea, diammonium phosphate, potassium sulfate, chemical amendments, and biological amendments. The chemical amendment was prepared by mixing biochar and silica powder in a 1:1 ratio. The biochar (C: 33.32%; N: 0.5%; P₂O₅: 0.84%; K₂O: 0.59%) was produced from corn cobs pyrolyzed at temperatures between 450 and 500 °C. The selected microbial amendment was a Trichoderma-based fertilizer (C: 43.32%; N: 7.5%; P₂O₅: 0.54%; K₂O: 0.47%). In the experiment, equal nutrient fertilization with a single application of base fertilizer was used. The nutrient input and amendment dosage for each treatment are shown in Table 2.

2.3. Soil Sampling

After harvesting in October 2016, undisturbed soil cores were collected using soil rings (volume: 100 cm³) at depths of 0–20 cm and 20–40 cm in each plot for the determination of soil physical properties (soil bulk density, soil porosity, and field water–holding capacity). The mixed soil samples were subsequently milled to a 0.25 mm particle size in order to determine the total nitrogen (TN) and soil organic carbon (SOC) contents. These analyses were conducted using an Element III elemental analyzer (Bremen, Germany), with the instrument calibrated with three standard samples, following the combustion oxidation method as described in [44]. Additionally, undisturbed soil samples from the 0–20 cm and 20–40 cm depths were uniformly collected with a shovel, while the adjacent soil was manually disrupted. From each sampling plot, three soil cores were obtained and the undisturbed samples were homogenized prior to laboratory analysis. The soil samples were divided into small clods along the natural soil structure, screened through a 10 mm sieve, and air-dried naturally for an analysis of the soil aggregate structure, SOC and TN contents, and soil enzyme activities.

Regarding the soil’s physical properties, the soil bulk density for the 0–20 cm and 20–40 cm horizons was calculated according to Blake and Hartge [45], the soil porosity was calculated according to Yang [46], and the field water holding capacity was calculated using Liu et al.’s equations [47].

$$\mathrm{Soil} \mathrm{bulk} \mathrm{density}(\mathrm{g}/{\mathrm{cm}}^3)={\displaystyle \frac{\mathrm{Soil} \mathrm{dry} \mathrm{weight} \left(\mathrm{g}\right)}{{\mathrm{The} \mathrm{volume} \mathrm{of} \mathrm{cutting} \mathrm{ring} (\mathrm{cm}}^3)}}$$

(1)

$$\mathrm{Soil} \mathrm{porosity} \left(\%\right)=\left(1 - {\displaystyle \frac{\mathrm{Soil} \mathrm{bulk} \mathrm{density}}{\mathrm{Soil} \mathrm{gravity}}}\right)\times 100\%$$

(2)

$$\mathrm{Field} \mathrm{water} \mathrm{holding} \mathrm{capacity} \left(\%\right)={\displaystyle \frac{{(\mathrm{M}}_1 {- \mathrm{M}}_2)}{{(\mathrm{M}}_2{ - \mathrm{M}}_0)}}\times 100\%$$

(3)

M₀—Quality of the empty cutting ring after drying, g;

M₁—Cutting ring before drying with wet soil sample quality, g;

M₂—Dried cutting ring with dry soil sample mass, g.

2.4. Separation of Soil Aggregates and Determination of SOC and TN in Soil Aggregates

Aggregate stability was determined using the wet sieve method, as adapted from the method of Mustafa et al. [27]. Using this technique, the soil samples were separated into five categories: 2 mm, 1 mm, 0.5 mm, 0.25 mm, and 0.053 mm. Briefly, 50 g of air-dried soil was placed in a beaker, slowly wetted with water, and allowed to soak for 30 min. The sieve was then placed into a bucket filled with distilled water. Under constant temperature conditions (25 °C), the soaked soil sample was placed on the top sieve. The water level was adjusted to just reach the edge of the 2 mm sieve, and the soil was sieved through a set of 2 mm, 1 mm, 0.5 mm, 0.25 mm, and 0.053 mm sieves using an automatic sieving device. The soaked samples were shaken in water for 30 min, and the material remaining on top of each sieve was collected. The aggregates, classified by particle sizes, of >1 mm, 1–0.5 mm, 0.5–0.25 mm, 0.25–0.053 mm, and <0.053 mm, were successively transferred into pre-weighed beakers, and then they were dried and weighed. The aggregates were separated according to particle size into macroaggregates (>0.25 mm), microaggregates (0.25–0.053 mm), and fine fractions consisting of silt and clay (<0.053 mm) [27]. Soil particles were analyzed using a particle analyzer [48]. Soil samples were separated by wet sieving and, then ground to pass through a 0.15 mm mesh, and the SOC and TN contents of aggregates of different particle sizes were determined using a Vario EL III Elemental Analyzer (Elementar, Germany).

2.5. Contribution Rate of Aggregates of Different Particle Sizes to SOC and TN

The contribution rates to soil C and N by aggregates of different particle sizes were calculated according to the method described in [48], using the following equation:

$$CR={\displaystyle \frac{C_i\times W_i}{C_T}}$$

(4)

Here, CR represents the contribution rate (%), C_i represents the C or N content within aggregates of a specific particle size (g kg⁻¹), W_i is the weight percentage of water-stable aggregates of that particle size (%), and C_T denotes the total C or N content of the corresponding soil layer (g kg⁻¹).

2.6. Soil Enzyme Activities

Soil urease (S-UE) and soil acid invertase (S-AI) were determined using Comibio kits (Beijing Beiluo Biotechnology Co., Ltd., Beijing, China), according to the manufacturer’s instructions. For S-UE, one unit of enzyme activity (µg/d/g) was defined as the production of 1 µg NH₃-N per g of soil sample per day. For S-AI, one unit of enzyme activity (mg/d/g) was defined as the production of 1 mg glucose per g of soil sample per day [49].

2.7. Statistical Analysis

All treatment means (n = 3) were analyzed for significant differences using a one−way analysis of variance (ANOVA), followed by Duncan’s multiple range post-hoc test at p < 0.05. Data analysis was performed using the Windows-based SPSS 19.0 statistical package. In the structural equation model (SEM), the first component, which accounted for more than 70% of the variance, was used for each new indicator. The SEM was conducted using the R package “piecewiseSEM” to investigate the relationships between soil aggregate size, soil organic carbon (SOC), total nitrogen (TN), aggregate carbon and nitrogen content, and related enzyme activities under crop rotation and amendment treatments [50]. To obtain the optimal SEM, we first ran an a priori model, including all potential pathways, and then sequentially removed insignificant pathways based on modification indices. Model fit was evaluated using R², Fisher’s C statistic, the Akaike Information Criterion (AIC), and p-value (with p > 0.05 indicating a sufficient fit). Linear mixed−effects models were applied using the “nlme” package in R to explore the effects of SOC and TN on urease (S-UE) and soil acid invertase (S-AI) activities [51].

3. Results

3.1. Soil Physical Properties

As the soil depth increased, the bulk density gradually increased, while the field water-holding capacity and porosity decreased, showing an inverse trend with the bulk density (

Table 3. Soil bulk density, field water capacity, and porosity of different treatments in 0–20 cm and 20–40 cm soil layers.

Treatment	Physical Properties
	Bulk Density (g/cm3)		Field Water-Holding Capacity (%)		Porosity (%)
	0–20 cm	20–40 cm	0–20 cm	20–40 cm	0–20 cm	20–40 cm
MC	1.15 ± 0.02 c	1.28 ± 0.03 c	36.10 ± 0.24 d	32.61 ± 0.42 b	56.54 ± 0.85 a	51.66 ± 1.16 b
M-PR	1.09 ± 0.01 d	1.20 ± 0.02 d	45.14 ± 0.31 a	39.25 ± 0.46 a	58.80 ± 0.22 a	54.88 ± 0.75 a
PC	1.30 ± 0.01 a	1.40 ± 0.02 ab	29.60 ± 0.57 e	27.17 ± 0.43 d	51.11 ± 0.98 b	47.08 ± 0.80 cd
PCCA	1.23 ± 0.02 b	1.36 ± 0.02 b	41.33 ± 0.31 b	32.40 ± 0.46 b	53.57 ± 0.61 b	48.86 ± 0.67 c
PCBA	1.29 ± 0.03 a	1.45 ± 0.02 a	37.77 ± 0.49 c	30.95 ± 0.33 c	51.38 ± 1.06 b	45.29 ± 0.79 d

Different letters indicate significant differences among treatments. (p < 0.05, Duncan test). Data are presented as mean ± standard deviation, n = 3. MC: maize continuous cropping; M-PR: peanut-maize rotation; PC: peanut continuous cropping; PCCA: peanut continuous cropping + chemical amendments; PCBA: peanut continuous cropping + biological amendments. Starting from 2011, the M-PR treatment was planted to peanuts in odd years with the same fertilizer application as the PC treatment, and planted to maize in even years with the same fertilizer application as the MC treatment.

). At the 0–20 cm depth, the bulk density of the M-PR treatment was the lowest at 1.09 g/cm³, which was 19.3% lower than that of the PC treatment and 5.5% lower than that of the MC treatment. The field water-holding capacity and porosity of the M-PR treatment were the highest, at 45.14% and 58.8%, respectively, significantly higher than the 52.5% and 15.0% of the PC treatment. The bulk density of the PCCA treatment was lower than that of the PC and PCBA treatments. Compared to the PC treatment, the field water-holding capacity and porosity of PCCA increased by 39.6% and 4.8%, respectively, and they were 9.4% and 4.3% higher than those of the PCBA treatment. At the 20–40 cm depth, the bulk density of the M-PR treatment was the lowest at 1.20 g/cm³, and its porosity was the highest at 54.9%. No significant difference in soil porosity was observed between the PCCA and PC treatments.

3.2. Soil Aggregate Size Distribution

Under different treatments, the mass fraction of the aggregates increased as the particle size decreased (

Table 4. The mass fraction of the water-stable aggregates of different sizes (%).

Soil Depth	Treatment	Aggregate Size
		>1 mm	1–0.5 mm	0.5–0.25 mm	0.25–0.053 mm	<0.053 mm
0–20 cm	MC	6.49 ± 0.30 a	4.24 ± 0.36 bc	13.13 ± 0.42 b	31.05 ± 0.20 a	45.09 ± 0.26 c
	M-PR	3.58 ± 0.18 b	8.21 ± 0.30 a	17.22 ± 0.26 a	27.19 ± 0.62 b	43.79 ± 0.76 c
	PC	2.25 ± 0.15 cd	3.99 ± 0.30 c	13.01 ± 0.33 b	22.24 ± 0.48 c	58.51 ± 0.68 a
	PCCA	1.93 ± 0.07 d	4.77 ± 0.17 bc	16.98 ± 0.42 a	25.76 ± 0.33 b	50.56 ± 0.92 b
	PCBA	2.61 ± 0.20 c	5.07 ± 0.26 b	13.05 ± 0.36 b	26.42 ± 0.48 b	52.85 ± 1.12 b
20–40 cm	MC	10.71 ± 0.38 b	10.38 ± 0.38 b	12.69 ± 1.61 b	28.74 ± 1.66 bc	37.49 ± 3.11 a
	M-PR	13.48 ± 0.82 a	12.62 ± 0.79 a	12.80 ± 0.59 b	22.47 ± 3.37 c	38.63 ± 5.23 a
	PC	1.76 ± 0.08 c	5.37 ± 0.62 cd	20.55 ± 4.61 a	34.01 ± 1.70 b	38.31 ± 5.23 a
	PCCA	2.67 ± 0.26 c	6.40 ± 0.32 c	10.66 ± 0.27 b	47.31 ± 4.11 a	32.96 ± 3.27 a
	PCBA	2.64 ± 0.02 c	4.19 ± 0.19 d	12.46 ± 0.66 b	36.56 ± 0.92 b	44.15 ± 0.23 a

Different letters indicate significant differences among treatments. (p < 0.05, Duncan test). Data are presented as mean ± standard deviation, n = 3. MC: maize continuous cropping; M-PR: peanut-maize rotation; PC: peanut continuous cropping; PCCA: peanut continuous cropping + chemical amendments; PCBA: peanut continuous cropping + biological amendments. Starting from 2011, the M-PR treatment was planted to peanuts in odd years with the same fertilizer application as the PC treatment, and planted to maize in even years with the same fertilizer application as the MC treatment.

). In the 0–20 cm soil layer, compared to the other treatments, the M-PR treatment had the highest proportion of large aggregates (>0.25 mm), accounting for 29.0%, which was 50.7% and 21.6% higher than that of the PC and MC treatments, respectively. The mass fraction of microaggregates (0.25–0.053 mm) was highest in the MC treatment at 31.1%, which was significantly higher than that in the other treatments, with the PC treatment showing the lowest content. Regarding the silt and clay particles (<0.053 mm), compared to the PC treatment, the mass fractions of silt and clay in the M-PR, PCCA, and PCBA treatments were significantly reduced by 33.6%, 15.7%, and 10.7%, respectively. In the 20–40 cm soil layer, the M-PR treatment had the highest mass fraction of large aggregates (>0.25 mm) at 38.9%, which was 40.5% higher than that in the PC treatment. Compared to the PCCA treatment, the mass fractions of microaggregates (0.25–0.053 mm) in the MC and PC treatments were significantly reduced by 64.6% and 39.1%, respectively. The PCBA treatment had the highest mass fraction of silt and clay particles (<0.053 mm) at 44.2%, while the PCCA treatment had the lowest, although the differences between all treatments were not significant.

3.3. Soil Aggregates C and N Distribution

In different soil layers, the SOC content increased as the aggregate particle size increased (Figure 1). In the 0–20 cm soil layer, for the same aggregate particle size class, the PCCA treatment had the highest SOC content, while the MC treatment had the lowest. Compared to MC and PC, the SOC content of aggregates >1 mm in the M-PR, PCCA, and PCBA treatments increased significantly by 71.1%, 146.8%, and 99.9%; and by 24.3%, 79.3%, and 45.2%, respectively. For aggregates sized 1–0.5 mm, the SOC content increased significantly by 46.2%, 139.0%, and 113.7%; and by 43.7%, 134.8%, and 110.0%, respectively. For aggregates sized 0.5–0.25 mm, the SOC content increased by 18.8%, 103.9%, and 57.2%; and by 20.2%, 106.4%, and 59.1%, respectively. In the microaggregates (0.25–0.053 mm), the SOC content increased by 13.6%, 51.6%, and 46.3%; and by 16.5%, 55.4%, and 50.0%, respectively. For silt and clay particles (<0.053 mm), SOC content increased by 11.7%, 53.1%, and 27.1%; and by 9.4%, 49.9%, and 24.5%, respectively (Figure 1a). In the 20–40 cm soil layer, the SOC content in the PCCA and PCBA treatments was significantly higher than that in the other treatments for the same aggregate components. Compared to MC and PC, the SOC content in the PCCA and PCBA treatments increased significantly by 9.3–81.8% and 21.6–47.0%, respectively (Figure 1b). In both soil layers, the contribution of different aggregate sizes to SOC followed the order of: large aggregates (>0.25 mm) > microaggregates (0.25–0.053 mm) > silt and clay particles (<0.053 mm).

The variation in the soil TN content mirrored that in SOC (Figure 1). With an increasing soil depth, the TN levels progressively declined, and a reduction in the TN content was also observed as the aggregate particle size decreased. Within the 0–20 cm soil depth, aggregates under the PCCA treatment exhibited the highest TN content, with PCBA ranking second; both treatments exhibited significantly greater values than the other treatments. Compared to MC and PC, the TN content of aggregates larger than 1 mm in the PCCA treatment increased significantly by 116.2% and 102.2%, while in PCBA, it increased significantly by 80.8% and 69.1%. For aggregates sized 1–0.5 mm, the TN content increased by 99.1% and 94.2%, and by 87.2% and 82.5%, respectively. For aggregates sized 0.5–0.25 mm, the TN content increased by 84.1% and 90.8%, and by 76.1% and 82.6%, respectively. In the microaggregates (0.25–0.053 mm), the TN content increased by 60.6% and 52.9%, and by 50.5% and 43.3%, respectively. For silt and clay particles (<0.053 mm), the TN content increased by 38.1%, 32.7%, 24.7%, and 19.8% (Figure 1c). In the 20–40 cm soil layer, the TN content in the PCCA and PCBA treatments was significantly higher than that in the other treatments for the same aggregate particle sizes. Compared to MC and PC, the TN content in the PCCA and PCBA treatments increased significantly by 13.3–67.5% and 10.4–48.7%, respectively (Figure 1d). In both soil layers, the contribution of different aggregate sizes to TN followed the order of: large aggregates (>0.25 mm) > microaggregates (0.25–0.053 mm) > silt and clay particles (<0.053 mm).

The treatments exhibited varying impacts on soil SOC and TN levels, with PCCA and PCBA demonstrating superior performance to the other treatments. At the 0–20 cm depth, of SOC content of PCCA was significantly higher than that of MC and PC, increasing by 54.4% and 44.6%, respectively. The TN content also significantly increased by 24.8% and 23.6%. In contrast, the SOC content of PCBA increased by 26.6% and 17.5% compared to that of MC and PC, respectively, and its TN content increased by 15.2% and 14.2%. At the 20–40 cm depth, PCCA and PCBA also performed well in terms of the SOC and TN content (

Table 5. SOC and TN contents of bulk soil at different soil depths.

Treatments	0–20 cm		20–40 cm
	SOC (g/kg)	TN (g/kg)	SOC (g/kg)	TN (g/kg)
MC	9.11 ± 0.08 d	1.05 ± 0.03 d	8.75 ± 0.06 c	0.98 ± 0.03 d
M-PR	10.32 ± 0.21 c	1.15 ± 0.02 c	8.86 ± 0.06 c	1.05 ± 001 c
PC	9.77 ± 0.12 cd	1.06 ± 0.01 d	9.39 ± 0.15 b	1.03 ± 0.01 cd
PCCA	14.04 ± 0.42 a	131 ± 0.02 a	11.64 ± 0.15 a	1.27 ± 0.02 a
PCBA	11.48 ± 0.15 b	1.21 ± 0.01 b	9.64 ± 0.05 b	1.18 ± 0.02 b

Different letters indicate significant differences among treatments. (p < 0.05, Duncan test). Data are presented as mean ± standard deviation, n = 3. MC: maize continuous cropping; M-PR: peanut-maize rotation; PC: peanut continuous cropping; PCCA: peanut continuous cropping + chemical amendments; PCBA: peanut continuous cropping + biological amendments. Starting from 2011, the M-PR treatment was planted to peanuts in odd years with the same fertilizer application as the PC treatment, and planted to maize in even years with the same fertilizer application as the MC treatment.

).

By analyzing the contribution rates of aggregates of different sizes to C and N, we found that, in all treatments, the silt and clay particles contributed the most to SOC and TN, ranging from 30.9% to 69.1% for SOC and from 32.0% to 67.6% for TN (

Table 6. Contribution rates of aggregates to soil C and N under different treatments.

Soil Layer (cm)	Treatment	>1 mm		1–0.5 mm		0.5–0.25 mm		0.25–0.053 mm		<0.053 mm
		C-CR%	N-CR%	C-CR%	N-CR%	C-CR%	N-CR%	C-CR%	N-CR%	C-CR%	N-CR%
0–20 cm	MC	7.56 ± 1.23 a	7.42 ± 0.53 a	9.95 ± 1.09 ab	9.40 ± 0.54 ab	15.46 ± 1.03 b	14.16 ± 1.50 b	37.21 ± 6.94 ab	35.72 ± 5.31 b	30.87 ± 6.50 c	32.01 ± 7.29 c
	M–PR	6.71 ± 1.46 ab	4.82 ± 0.94 b	5.18 ± 0.93 c	3.86 ± 0.70 c	8.30 ± 0.15 c	6.86 ± 0.06 c	16.70 ± 1.48 d	15.70 ± 0.77 c	61.20 ± 2.04 a	62.80 ± 2.85 a
	PC	3.62 ± 0.42 c	2.97 ± 0.38 c	4.45 ± 0.52 c	4.52 ± 0.43 c	15.48 ± 1.91 b	14.77 ± 2.3 b	32.36 ± 3.14 bc	35.52 ± 2.90 b	36.80 ± 4.85 bc	41.13 ± 4.43 b
	PCCA	3.88 ± 0.32 c	4.13 ± 022 bc	8.74 ± 0.98 b	8.46 ± 0.63 b	24.70 ± 4.50 a	24.92 ± 3.06 a	30.12 ± 1.03 c	37.75 ± 0.63 b	41.16 ± 1.91 b	47.47 ± 3.90 b
	PCBA	5.17 ± 0.69 bc	5.08 ± 0.84 b	11.88 ± 2.43 a	10.77 ± 2.25 a	21.45 ± 4.25 a	23.96 ± 4.10 a	43.36 ± 1.82 a	46.84 ± 2.38 a	35.04 ± 2.21 bc	38.88 ± 3.26 bc
20–40 cm	MC	4.85 ± 0.19 a	4.77 ± 0.19 a	5.91 ± 0.61 c	6.45 ± 0.20 b	8.75 ± 0.10 c	13.58 ± 0.21 c	12.67 ± 0.63 d	14.21 ± 1.04 b	59.77 ± 1.10 b	67.58 ± 5.41 a
	M-PR	3.42 ± 0.16 b	3.44 ± 0.07 c	4.34 ± 0.10 d	4.27 ± 0.11 c	8.86 ± 0.10 c	15.52 ± 0.87 b	15.65 ± 0.25 b	17.14 ± 0.66 a	51.77 ± 0.63 c	61.03 ± 0.73 b
	PC	2.84 ± 0.28 b	2.39 ± 0.20 d	6.59 ± 0.58 c	5.77 ± 0.62 b	9.39 ± 0.26 b	11.62 ± 0.36 d	11.65 ± 0.38 e	12.96 ± 0.42 c	59.90 ± 2.07 b	66.49 ± 1.45 a
	PCCA	5.05 ± 0.54 a	4.30 ± 0.38 b	10.40 ± 1.38 a	9.70 ± 1.37 a	11.64 ± 0.27 a	14.55 ± 0.77 bc	14.28 ± 0.64 c	13.91 ± 0.23 bc	47.95 ± 2.68 d	57.84 ± 2.57 b
	PCBA	4.80 ± 0.39 a	3.57 ± 0.19 c	8.71 ± 0.36 b	6.85 ± 0.20 b	9.64 ± 0.09 b	17.24 ± 1.41 a	16.73 ± 0.71 a	15.02 ± 0.45 b	69.11 ± 1.74 a	66.60 ± 1.44 a

Different letters indicate significant differences among treatments. (p < 0.05, Duncan test). Data are presented as mean ± standard deviation, n = 3. MC: maize continuous cropping; M-PR: peanut-maize rotation; PC: peanut continuous cropping; PCCA: peanut continuous cropping + chemical amendments; PCBA: peanut continuous cropping + biological amendments. Starting from 2011, the M-PR treatment was planted to peanuts in odd years with the same fertilizer application as the PC treatment, and planted to maize in even years with the same fertilizer application as the MC treatment.

). In the 0–20 cm soil layer, compared to the other treatments, the PCCA and PCBA treatments increased the contribution rates of the 0.5–0.25 mm aggregates to SOC and TN by 38.6–197.6% and 63.0–263.3%, respectively. In the M-PR treatment, the silt and clay particles (<0.053 mm) were the main contributors to SOC and TN, accounting for 61.2% and 62.8%, respectively. In the 20–40 cm soil layer, the main contributors to SOC and TN in the PCCA and PCBA treatments were microaggregates (0.25–0.053 mm) and silt and clay particles (<0.053 mm). Moreover, compared to the MC and PC treatments, the PCCA and PCBA treatments showed a greater advantage in the contribution rates of macroaggregates (>0.25 mm) to SOC and TN. Overall, the contribution rates of different aggregate size classes to the total SOC and TN generally decreased with increasing aggregate size.

3.4. Soil Aggregate Enzyme Activities

Across various soil depths, the S–UE activity of aggregates under the PCCA treatment was consistently the highest across all particle size classes, significantly surpassing that observed in the other treatments (Figure 2a). In the 0–20 cm soil layer, compared to MC and PC, urease activity in the M-PR, PCCA, and PCBA treatments increased by 0.35–9.5%, 13.7–45.4%, and 2.6–21.9%, respectively. The S–UE activity was the highest in large soil aggregates (>0.25 mm), followed by silt and clay particles (<0.053 mm), and then microaggregates (0.25–0.053 mm). Within the 20–40 cm soil depth, the S-UE activity across aggregate size classes under the M-PR treatment did not significantly differ from that under the PC treatment, but it remained significantly higher than that observed under MC. Compared to the M–PR treatment, PCCA significantly increased S-UE activity by 10.3–30.2% in the 0–20 cm layer and by 8.8–41.7% in the 20–40 cm layer. In the 0–20 cm soil layer, the S-UE activity followed the order of: large aggregates (>0.25 mm) > silt and clay particles (<0.053 mm) > microaggregates (0.25–0.053 mm).

In different soil layers, the S-AI activity of aggregates in the PCCA treatment was the highest across all particle sizes, as well as being significantly higher than in the M-PR and PCBA treatments (Figure 2b). In the 0–20 cm soil layer, PCCA demonstrated the highest S-AI activity (42.88 mg/d/g) in the 1–0.5 mm aggregates. Compared to PCBA, the S-AI activity in PCCA significantly increased by 9.7–13.9% in the 0–20 cm layer and by 5.1–45.3% in the 20–40 cm layer. Compared to M-PR, the S-AI activity in PCCA was 1.4–1.9 times higher in the 0–20 cm layer and increased by 8.8–41.7% in the 20–40 cm layer. In the 0–20 cm layer, S-AI activity was higher in large aggregates (>0.25 mm) than in microaggregates (0.25–0.053 mm) and silt and clay particles (<0.053 mm). The S-AI activity of aggregates in the M-PR treatment was significantly lower than that in PC in the 0–20 cm layer but significantly higher than that in MC in both layers. Overall, the S-AI activity in the 0–20 cm layer was higher than that in the 20–40 cm layer.

3.5. Relationship Between Soil Aggregate Size, Soil Aggregate C and N Contents, and Soil Aggregate Enzyme Activities

A structural equation model (SEM) was employed to investigate the interactions among amendments, crop rotation, soil physical attributes, SOC, TN, and aggregate size distributions. The SEM results revealed that amendments had a significantly positive direct impact on soil physical properties, macroaggregates (>0.25 mm), and SOC, with path coefficients of 0.610, 1.376, and 0.844, respectively. However, amendments had a significantly negative direct impact on microaggregates (0.25–0.053 mm) and silt and clay particles (<0.053 mm), with path coefficients of −0.250 and −1.050, respectively (Figure 3a). Crop rotation had a highly significant positive impact on SOC, while it negatively affected microaggregates (0.25–0.053 mm) and silt and clay particles (<0.053 mm), with path coefficients of 1.004, −0.660, and −0.882, respectively. Soil physical properties, microaggregates (0.25–0.053 mm), and silt and clay particles (<0.053 mm) had significantly positive direct effects on SOC, with path coefficients of 0.552, 1.158, and 1.686, respectively. Additionally, SOC had a strong positive influence on TN, with a path coefficient of 1.218 (Figure 3a).

Regarding the SOC content, five pathways (C1, C2, C3, C4, and C5) drove the changes in SOC, with direct action path coefficients (DAPCs) to the SOC content of 0.463, 1.291, 0.702, and 0.965 for C1, C2, C3, and C4, respectively (Figure 3b). Regarding the TN content, the N content of soil aggregates was the primary factor affecting its variation, with direct path coefficients to the TN content of 1.901, −1.339, and 0.462 for N2, N3, and N5, respectively (Figure 3c). The direct effect coefficient between SOC and TN was 0.867. Regarding soil enzyme activities (S-AI and S-UE), SOC was the primary factor influencing S-AI, while TN was the key factor influencing S-UE (Figure 3d). Furthermore, there were significant positive correlations between SOC and TN with both S-AI and S-UE (Figure 3e,f).

4. Discussion

4.1. Effect of Amendments and Crop Rotation on Soil Physical Properties, SOC, and TN

As the soil depth increased, the bulk density progressively rose, whereas field water−holding capacity and porosity declined, displaying trends opposite to those of the bulk density. Crop rotation and the application of chemical amendments significantly reduced the soil bulk density [52,53] and increased the field water−holding capacity and porosity [54]. This effect is attributed to the incorporation of biochar within chemical amendments, which promotes the formation of new soil aggregates, enhances their stability, and increases soil porosity. The microporosity inside biochar is high, which increases the number of soil micropores. These small pores retain more soil water, thus improving the field water-holding capacity [55]. While the application of biological amendments had less effect on the soil bulk density, biological amendments increased soil field water holding capacity due to the microbial agents that they contain, which improve the soil microhabitat and promote plant root growth. The activity of plant roots and the release of large amounts of root exudates contribute to improved soil texture [56].

Nearly 90% of the SOC in topsoil is located within soil aggregates [57]. The composition of aggregates at all levels can control the dynamics of SOC [58]. The circulation of large aggregates determines the stability of SOC within microaggregates [59]. This study found that the adoption of M-PR and, the application of chemical amendments, and biological amendments significantly increased the organic C and TN contents of macroaggregates (>0.25 mm). The treatment that showed the greatest increase was PCCA. This was mainly attributed to the fact that biochar itself carries certain C and N sources, which are not easily decomposed and remain relatively stable after application in soil [60]. The input of biochar increased the content of SOC, which can act as a cementing substance in the formation of crop aggregates. Biochar significantly increased the SOC content of large aggregates, thus promoting the formation of large aggregates [61]. In this experiment, the organic C in aggregates was mainly distributed in large aggregates (>0.25 mm). On the one hand, organic matter is cemented in large aggregates through microaggregates. On the other hand, this may be due to the physical isolation of available carbon sources from microorganisms becoming stronger as the size of large aggregates (>250 μm) increases [62]. In addition to the N contained in biochar, biochar itself can inhibit soil denitrification by altering the ventilation status and moisture content of farmland soil, reducing the emission of N oxides in soil. Moreover, biochar has a strong adsorption effect on N, which effectively reduces the leaching loss of N in soil, thus increasing TN storage in soil [63].

4.2. Effect of Crop Rotation and Amendments on Soil Aggregates

The formation of soil aggregates is influenced by many factors [64]. Crop rotation and amendment application can significantly increase the proportion of large soil aggregates (>0.25 mm) and microaggregates (0.25–0.053 mm); while decreasing the proportion of silt and clay particles (<0.053 mm). Therefore, the use of crop rotation and amendments can promote the agglomeration of <0.053 mm soil aggregates into larger−particle−size aggregates [65]. Crop rotation treatments are more effective than chemical amendments for tilled soils. The main reason why chemical amendments can promote the formation of large soil aggregates is that the biochar in chemical amendments directly or indirectly promotes the production of colloidal material [66]. Biochar binds to soil particles through bonding to form soil aggregates [67]. Numerous studies have demonstrated that biochar application enhances the proportion of soil macroaggregates and strengthens aggregate stability [68,69]. Nevertheless, experiments on amended mortar black soils indicated that biochar application did not markedly alter soil aggregate content, which may be attributed to the intrinsic properties of biochar [70].

4.3. Effect of Crop Rotation and Amendments on Soil Enzyme Activity

Soil enzymes play a critical role in regulating the soil carbon (C) and nitrogen (N) cycles, and their activity is commonly employed as an indicator of soil quality and nutrient dynamics [71]. Soil invertase enzymes are associated with organic matter metabolism in soil and can catalyze the hydrolysis of sucrose into fructose and glucose, thus providing soil organisms (e.g., microorganisms and plants) with nutrients that they can use [72]. Therefore, soil invertase enzyme activity can characterize the hydrolytic conversion and accumulation pattern of organic C in soil, playing a role in the C cycle within the soil [73]. Soil urease is involved in the soil N cycle and is indispensable for converting organic N in the soil to active N, which is then taken up by plants [74]. Variations in the formation environment and cementation types across different aggregate sizes contribute to differences in aggregate stability and internal composition, ultimately influencing soil enzyme activity [75].

Crop rotation and the application of amendments increased S-UE and S-AI activities, with the most significant effect exerted by chemical amendments (Figure 2), corroborating previous studies [76]. Chemical amendments promoted soil enzyme activity, likely because biochar has strong adsorption properties and can adsorb reaction substrates for enzymatic reactions, which, in turn, promotes enzymatic activity and increases soil enzyme levels [77]. However, biochar, when bound to the enzymatic reaction site through adsorption, may reduce soil enzyme activity [78]. The results of the study showed that bio-amendments containing microbial agents can increase soil enzyme activity, likely due to an increase in the number of living microorganisms, which improves the soil microbiota, inhibits the proliferation of harmful bacteria, and enhances the vascular and biochemical activities of the soil. Additionally, various enzymes secreted by soil microorganisms during growth and reproduction can also increase soil enzyme activity [79]. However, some studies have found that biochar application increased the soil enzyme activities involved in nitrogen conversion but decreased those involved in C conversion [80] or had no significant effect [81]. In general, the enzyme activity of macroaggregates (>0.25 mm) was significantly higher than that of microaggregates (0.25–0.053 mm) and powdered silt and clay particles (<0.053 mm). This is because soil enzyme activity is primarily derived from the metabolic activity of microorganisms in the soil, and microorganisms are more abundant in large aggregates than in microaggregates, leading to higher enzyme activity in large aggregates [82].

4.4. Mechanisms of SOC, TN Content, and Enzyme Activity in Relation to Aggregate C, N, and Stability

Soil aggregate composition and stability are strongly interconnected, exerting a major influence on soil fertility and physical properties [27]. Research has revealed a strong positive association between SOC and TN stocks and the stability indices of water-stable aggregates, emphasizing the essential role of aggregate stability in facilitating soil carbon and nitrogen sequestration [83]. Our findings demonstrate that the organic C and N contents of aggregates directly impact SOC and TN (Figure 3b,c), aligning with the results of Wang et al. [84]. Specifically, the SOC and TN contents of aggregates followed the order of: PCCB > PCBA > M-PR > PC > MC. Large aggregates (>0.25 mm) served as the primary carriers of soil SOC and TN (Figure 1), a result consistent with that of other studies. However, our study also revealed that microaggregates (0.25–0.053 mm) and silt and clay particles (<0.053 mm) are major contributors to soil SOC and TN (Table 6). Furthermore, microaggregates (0.25–0.053 mm) and silt and clay particles (<0.053 mm) have a significantly positive influence on soil SOC, which, in turn, has a strong positive effect on TN (Figure 3a). These findings suggest that the addition of amendments increases SOC primarily by enhancing the organic C concentration in free microaggregates (0.25–0.053 mm) and silt and clay particles (<0.053 mm), which subsequently promotes the formation of microaggregates or large aggregates. Aggregates play an essential role in the physical protection of SOC. With the addition of amendments, large aggregates tend to break down into smaller aggregates, significantly increasing the proportion of microaggregates [85]. Compared to the particulate organic matter in large aggregates, the organic matter in microaggregates is more stable, leading to better protection of organic C [86]. Soil enzymes play an essential role in organic matter decomposition and nutrient cycling processes [87]. This research targets enzymes associated with the carbon (C) and nitrogen (N) cycles, specifically S-AI and S-UE. A regression analysis indicates a significant positive correlation between SOC, the TN content, and S-UE and S-AI activities (Figure 3e,f), suggesting that SOC is a primary determinant. SOC not only provides a substrate for enzymes but also plays a crucial role in protecting soil enzymes through the formation of complexes with clay and humic substances. The stability and persistence of soil enzymes are fundamental to maintaining long−term soil fertility, which, in turn, is intrinsically linked to increased crop productivity. This study underscores the role of soil amendments in enhancing aggregate stability, safeguarding organic matter, and sustaining soil enzymatic functions, thereby promoting long-term improvements in crop yields.

5. Conclusions

After 6 years of continuous fertilization, the combination of peanut−maize rotation and continuous peanut cultivation with the application of amendments reduced the bulk density, increased the field water-holding capacity, and enhanced porosity, with the best effects observed in rotation. The use of chemical amendments promoted soil particle aggregation, facilitating the aggregation of fine silt and clay particles (<0.053 mm) into larger aggregates (>0.25 mm), effectively fixing more carbon and nitrogen in these larger aggregates, and outperforming the effects of biological amendments. The formation of larger aggregates improved soil physical properties, enhanced enzyme activity, and promoted the retention of more carbon and nitrogen. For topsoil, changes in the aggregate structure had a significant impact on the field water capacity. For deep soil, the soil’s physical properties and carbon−nitrogen distribution were primarily influenced by larger aggregates. Rotation and the application of amendments enhanced the enzymatic activity of urease and transformation enzymes in different−sized aggregates, with the most pronounced effect observed with chemical amendments.