Effect of Long-Term Fertilization Practices on the Stability of Soil Organic Matter in the Northeast Black Soil Region in China

Abstract

Soil organic matter (SOM) is an important carbon pool in terrestrial ecosystems and plays a key role in soil functions. Nevertheless, the effects of fertilization practices on the physical, chemical, biological, and comprehensive stability of SOM are still unclear. We carried out a long-term field experiment in the northeast black soil region in China with four different fertilization practices: no fertilizer (CK), single chemical fertilizer (NPK), chemical fertilizer + straw (NPKJ), and chemical fertilizer + organic manure (NPKM). The content of particulate organic matter (POM) and mineral-associated organic matter (MAOM), compound composition of SOM, carbon mineralization characteristics, active soil organic matter (ASOM), and inert soil organic matter (ISOM) were tested. The results showed that the application of fertilizers significantly increased the contents of POM and MAOM to 2.59–4.65 g kg⁻¹ and 32.69–34.65 g kg⁻¹ (p < 0.05), but decreased the MAOM/POM values by 37.8–42.4%, indicating reduced the physical stability of SOM. Fertilization practices increased the contents of aromatic, nitrogen-containing compounds and decreased the oxygen compounds of SOM, representing enhancement of the chemical stability. The contents of ASOM and ISOM increased in fertilization practices, while the biological stability index (BSI) under the NPKJ and NPKM treatments was lower than the CK treatment, suggesting that the biological stability decreased under the manure and straw application. In addition, the comprehensive stability of SOM increased by 26–116% through a reduction in the physical and biological stability, coupled with an increase in the chemical stability. Collectively, our study demonstrated that the application of manure and straw enhanced both the comprehensive stability and content of SOM and reduced the physical and biological stabilities while increasing the chemical stability, which made the largest contribution to the comprehensive stability.

1. Introduction

Soil organic matter (SOM) has positive effects on many aspects of soil properties. Increasing SOM content can improve soil fertility and health [1], such as by enhancing water retention, improving soil structure, promoting nutrient cycling, and increasing microbial diversity [2]. SOM is one of the largest carbon (C) reservoirs on the earth’s surface, where the C storage is equivalent to twice the atmospheric C storage, and small changes to it will also lead to huge changes in atmospheric C dioxide concentration, thus affecting global warming [3]. Therefore, it is essential to promote the long-term retention of SOM in the soil to increase the transfer of atmospheric C to the soil and prevent its rapid release. The application of organic fertilizers is known to enhance SOM content compared to chemical fertilizers [4]. While different organic fertilizers have been shown to impact the accumulation of SOM diversely [5,6], their effects on the stability of SOM remain poorly understood. Therefore, evaluating the stability of SOM and understanding the underlying soil processes under contrasting fertilization practices, especially over long-term periods, is of great interest for enhancing arable land quality, promoting sustainable agriculture, and achieving consistently high crop yields.

The stability of SOM refers to its ability to resist mineralization or decomposition, as well as its ability to recover to the original level after certain interference [7]. The stability information of SOM is the basis of studying the storage and transformation process of SOM and its function, which directly affects the C fixation and storage capacity, and also determines soil fertility [8]. SOM stability can be divided into physical stability, chemical stability, and biological stability, and is affected by the combined effects of various factors [9]. Particulate organic matter (POM) and mineral-associated organic matter (MAOM) can be used to characterize the physical stability of SOM and are thought to have distinct turnover rates as a consequence of their differences in degrees of physical protection. MAOM is better at spatially separating SOM from microbes and enzymes [10]. Thus, the physical stability of SOM increases with the content of MAOM [3]. The differences in the chemical stability of SOM appear as differences in the chemical structure of SOM compounds [11]. Previous studies have found that the main chemical components of SOM are carbohydrates, lignin, nitrogen-containing compounds, lipids, and other components [12,13,14]. It is generally believed that the carbohydrate substances in the soil (oxy-alkyl carbon) are mostly unstable and easily decomposed C components, while the SOM rich in lignin (aromatic C) is relatively stable and not easily decomposed due to its intrinsic molecular characteristics [15,16]. The resistance of SOM against microbial decomposition serves as an indicator of its biological stability, which is usually impacted by the activity of microorganisms, enzymes, and other environmental factors [17]. Studies have shown that as the content of active soil organic matter (ASOM) that is available for microorganisms increases, the biological stability of SOM decreases [18]. The biological stability of SOM was evaluated by the biological stability index (BSI) [9].

A previous study has found that the application of organic fertilizers results in a decrease in MAOM/POM value, indicating that the effects of organic fertilizers reduce the physical stability of SOM [19]. The application of organic fertilizers increased the content of POM and MAOM in the soil, but the POM content responds more rapidly to the application of organic fertilizers, resulting in a decrease in the value of MAOM/POM [20]. Straw return and the combined application of organic–inorganic fertilizers could increase the contents of aliphatic, aromatic, carbohydrate, and silicone compounds in soil, thereby improving the chemical stability of SOM [21]. Changes in active soil organic matter (ASOM) in soil induced by fertilization practices also further affected the biological stability of SOM. A previous study observed that the application of organic fertilizers increased the content of mineralizable organic matter in the soil and reduced the biological stability of SOM [22]. Fertilization practices have different effects on the physical, chemical, biological, and comprehensive stability of SOM, which is helpful to the restoration of SOM. However, the detailed process by which fertilization practices affect comprehensive stability is still unclear.

In northeast China, the average content of SOM in virgin soil is 58.6 g kg⁻¹, which plays a crucial role in safeguarding grain production in China [23]. However, due to long-term high-intensity utilization from continuous cultivation and too little organic matter return, SOM content is gradually declining [20]. It has been documented that the total SOM in topsoil in northeast China has decreased by one-third, and the topsoil SOM has even decreased by 50% in severely degraded soils [21]. Therefore, it is an urgent issue to determine the stability and content of SOM in the northeast black soil region. Current results on the stability of SOM under fertilization practices tend to focus on the single aspect of physical or chemical stability; the effects of fertilization practices on the comprehensive stability of SOM are rarely studied. Therefore, we used pyrolysis–gas chromatography/mass spectrometry (Py GC-MS) technology and incubation experiments to explore the effects of different fertilization practices on the physical, chemical, biological, and comprehensive stability of SOM. We hypothesized that: (1) fertilization practices would change the content of POM, MAOM, and MAOM/POM, decreasing physical stability; (2) fertilization practices would affect the composition of SOM compounds and lead to changes in the chemical stability of SOM; (3) fertilization practices could affect the composition of ASOM and inert soil organic matter (ISOM), thus affecting the biological stability of SOM. The aim of this study was to analyze the stability mechanism of SOM under various fertilization practices, provide theoretical and practical guidance for restoring SOM content in the northeast black soil region, and promote the sustainable development of agriculture in this region.

2. Materials and Methods

2.1. Experimental Site

The long-term experiment was established at the National Field Observation and Research Station of Hailun Agroecosystems, part of the Chinese Academy of Sciences, located in Heilongjiang Province at coordinates (47°27′ N, 126°55′ E) (Figure 1). This region is characterized by a typical temperate continental monsoon climate, featuring hot and rainy summers, and cold and dry winters. The mean annual temperature in this region is approximately 1.5 °C, with monthly temperature averages ranging from −23 °C in January to 21 °C in July. The mean annual precipitation in this region totals 550 mm, with approximately 65% of this precipitation occurring from June to August. The frost-free period in this region lasts for approximately 120 days. The climatic conditions from 1990 to 2022 are shown in Figure 2. The soil in this region is primarily derived from sedimentary materials with a loamy loess composition and is classified as a Mollisol [24].

2.2. Experimental Design and Field Management

The field experiment was initiated in May 1990 with a soybean-corn rotation system, including four treatments: (1) no fertilizer (CK), (2) single chemical fertilizer (NPK), (3) chemical fertilizer and straw added at 4500 kg hm⁻² (NPKJ), and (4) chemical fertilizer and organic manure added at 3750 kg hm⁻² (NPKM). The basic physical and chemical properties are shown in

Table 1. The soil’s basic physical and chemical properties in 1990.

pH	SOC	Sand	Silt	Clay
	g kg−1	g kg−1	g kg−1	g kg−1
6.2	25.7	250	340	410

. The experiment has a complete randomized complete-block design with three replicates. Each plot was 12 × 5.6 m. Chemical fertilizers were applied at the same rate in all fertilized plots. Urea was applied to provide 20.25 kg N hm⁻² and 120 kg N hm⁻² for soybean and corn, respectively. Ammonium hydrogen phosphate was applied to provide 51.75 kg P hm⁻² and 60 kg N hm⁻² for soybean and corn, respectively. Potassium sulfate was applied at a rate of 30 kg K hm⁻². The organic manure was pig manure and collected in the same way every year. We adopted the soybean and corn rotation planting mode; the straw comes from the last seasonal crops. Specifically, when soybean is cultivated, soybean straw is returned to the field, while corn straw is returned when corn is grown.

2.3. Soil Sampling

Soil samples were collected in August 2022. After removing the litter and vegetation, three soil samples were collected from 0−20 cm soil layers in each plot and then were mixed. The composite soil samples were carefully placed into sterile bags and transported to the laboratory for further analysis. We divided the soil samples into two subsamples. The first set of subsamples was passed through a 2.00 mm sieve and stored at 4 °C prior to conducting the incubation experiment [25]. The other set of subsamples was air-dried at room temperature and passed through a 2.00 mm sieve to determine the content of POM and MAOM, and passed through a 0.25 mm sieve to determine the content of soil organic carbon (SOC) and the compounds of SOM.

2.4. Laboratory Methods

2.4.1. SOC, POM, and MAOM

A Vario Max elemental analyzer (Elementar, Hanau, Germany) was used to measure SOC [26]; the conversion coefficient between SOC and SOM was 1.724 [27]. The content of POM and MAOM was determined as described by Wu et al. (2024) [28]. Briefly, the <2 mm portion of the 10 g air-dried soil was weighed into a 50 mL centrifuge tube, after which 10 mL of sodium hexametaphosphate solution (0.5%) (Sigma-Aldrich, Shanghai, China) and beads were added. The sample was shaken slowly (90 rpm min⁻¹) for 18 h at room temperature to fully disperse the soil particles. Then, the fully dispersed soils were placed at the top of the 0.053 mm sieve and continuously rinsed with deionized water until the sieved water became clear. The organic substances passing through the sieve were defined as MAOM (< 0.053 mm), while those retained on the sieve were defined as POM (>0.053 mm).

2.4.2. Soil Organic Compounds

The compounds of the SOM were measured using Py–GC/MS which consisted of a single shot PY2020iD pyrolyzer (Frontier Laboratories, Koriyama, Japan) coupled to a GCMS-QP2010 (Shimadzu, Japan) [29]. Briefly, 5 mg of soil sample (passed through a 0.15 mm mesh) was placed in a white gold boat and subsequently dropped into the quartz pyrolysis cracking tube. The pyrolysis temperature was 550 °C, helium was used as a carrier gas, and the pyrolysis products were transferred online to a gas chromatograph (GC). The injection temperature of the GC and the GC/MS interface was 300 °C. The GC oven ran a temperature program of 40 °C for 3 min, 10 °C min⁻¹ to 260 °C, followed by a ramp (15 °C min⁻¹) to a final temperature of 300 °C for 5 min. The GC instrument was equipped with a DB-5 column, with a length of 30 m, diameter of 0.25 mm, and film thickness of 0.25 μm. The MS scanned in the range of m/z 29–500. Pyrolysis products were identified using the National Institute of Standards and Technology compound library database (2011 Edition) according to their dominant mass ions (m/z). Each compound was identified based on the literature and using the NIST library, and classified into one of seven categories comprising: (1) saturated hydrocarbons (e.g., alkanes, cycloalkanes); (2) unsaturated hydrocarbons (e.g., alkenes, alkynes, etc.); (3) aromatic compounds; (4) oxygenated chemicals (e.g., phenols, esters, etc.); (5) nitrogen-containing compounds (e.g., amines, nitrile compounds); (6) siliceous compounds; and (7) other compounds (mainly furans and levoglucosan) [30,31].

2.4.3. Soil Mineralization Rate Constant

The potentially mineralizable organic matter was assessed by incubating intact soil samples (50 g) at a constant 25 °C temperature and 25% moisture with C-CO₂ quantification over 60 days, as described by Semenov et al. (2018) [20]. Soil samples were incubated in a glass 750 mL wide-mouth bottle in triplicate. Subsequently, the sealed wide-mouth bottle was placed in a constant temperature incubator at 25 °C for incubation without light. During the incubation period, the gas in the wide-mouth bottle was collected with a syringe at 1, 3, 5, 7, 14, 21, 30, 45, and 60 days, respectively. After sampling, the sealing cover of the wide-mouth bottle was opened and it was ventilated in the fume hood for 20 min. After taking out the plastic cup, it was weighed and water was replenished to maintain the soil moisture content before quickly being put back into the wide-mouth bottle, which was sealed and transferred to the constant temperature incubator for further incubation. The C-CO₂ concentration was determined in a KristalLux 4000 M gas chromatograph to determine the flow rate (mg 100 g⁻¹ d⁻¹), cumulative C-CO₂ production (mg 100 g⁻¹), and the carbon content of ASOM by the beginning of incubation [9].

2.5. SOM Stability Indexes

The physical stability of SOM was characterized by the MAOM/POM value; the chemical stability was characterized by the sum of the contents of aromatic compounds, nitrogen compounds, silicon-containing compounds, and hydrocarbons; and the biological stability was characterized by BSI.

The values of the BSI correspond to the ratio between ASOM (C₀) and ISOM (C_total-C₀). The C₀ content was determined by the cumulative amount of C-CO₂ released during the incubation of soil samples and subsequent approximation of the obtained curve with the use of the first-order kinetic equation. These were calculated using the following equation of the first-order kinetics [13]:

$${\mathrm{C}}_{\mathrm{t}}={\mathrm{C}}_0\times \left(1-{\mathrm{e}}^{-\mathrm{k}\mathrm{t}}\right)$$

(1)

where C_t is the cumulative C-CO₂ amount (mg 100 g⁻¹ soil) over time t; C₀ is the content of active (potentially mineralizable) carbon (mg 100 g⁻¹); and k is mineralization rate constant.

According to the accumulation curve of C-CO₂ production, k was fitted by nonlinear estimation and the biological stability of SOM was assessed by the corresponding BSI as [13]

$$\mathrm{B}\mathrm{S}\mathrm{I}={\displaystyle \frac{{\mathrm{C}}_{\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}}-{\mathrm{C}}_0}{{\mathrm{C}}_0}}$$

(2)

The comprehensive stability of SOM is calculated from the physical, chemical, and biological stability, and the formula is as follows:

$${\mathrm{S}\mathrm{O}\mathrm{M}}_{\mathrm{s}\mathrm{t}\mathrm{a}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}}=0.5\times {\sum }_{\mathrm{n}}^{\mathrm{i}}\mathrm{s}\mathrm{t}{\mathrm{P}}_{\mathrm{i}}^2\times \sin \left(2\times {\displaystyle \frac{\mathsf{\pi }}{\mathrm{n}}}\right)$$

(3)

$$\mathrm{s}\mathrm{t}{\mathrm{P}}_{\mathrm{i}}={\displaystyle \frac{{\mathrm{P}}_{\mathrm{i}}}{\mathrm{P}}}$$

(4)

where P_i and P represent the index values of different stability indicators and their maximum values, respectively. n represents the number of indicators and π is 3.14 [32].

2.6. Statistical Analysis

SPSS v17.0 (IBM, Armonk, NY, USA) was used for statistical analysis. One-way identification analysis of variance (ANOVA) and Duncan’s multiple range test for all data were used between treatments (p < 0.05). The partial least squares path modeling (PLS-PM) in R 4.2.3. Origin 2021 was used to draw the figures.

3. Results

3.1. Physical Stability of SOM

Fertilization practices increased the contents of POM and MAOM, while decreasing the MAOM/POM value, thus reducing the physical stability of SOM (Figure 3). The NPKJ and NPKM treatments increased the amount of POM to 2.59–4.65 g kg⁻¹ and increased the content of MAOM to 32.69–34.65 g kg⁻¹ compared with the CK treatment. The NPKJ and NPKM treatments significantly increased the contents of POM and MAOM by 36.8% and 47.4% and 4.3% and 6.9%, respectively (p < 0.05), but NPK treatment significantly decreased the content of MAOM by 3.2% (p < 0.05) compared with CK treatment. Fertilization practices significantly decreased the MAOM/POM compared with CK treatment (p < 0.05), and the lowest POM/MAOM value was observed in the NPKM treatment, indicating that fertilization practices reduced the physical stability of SOM.

3.2. Chemical Stability of SOM

The chemical stability of SOM is related to the content of chemical compounds and increases with the higher content of aromatic compounds, nitrogen-containing compounds, and silicon-containing compounds, which are known to be stable in SOM. In comparison to CK treatment, the hydrocarbon compounds that are not stable decreased by 7.8% and 14.2% under NPKJ and NPKM treatments (

Table 2. Relative contents of organic compounds (%) in soil under different fertilization practices.

Treatments	CK	NPK	NPKJ	NPKM
AI	22.79 ± 1.52 a	21.65 ± 2.76 b	18.30 ± 1.31 c	17.66 ± 1.04 c
UH	49.00 ± 2.32 b	52.26 ± 5.17 a	47.91 ± 4.70 c	43.97 ± 3.21 d
OC	21.43 ± 3.44 a	15.45 ± 2.77 c	18.57 ± 4.51 b	18.04 ± 3.90 b
AR	2.15 ± 0.61 d	3.15 ± 0.27 c	5.59 ± 0.79 b	9.25 ± 1.13 a
NC	3.02 ± 0.27 d	5.66 ± 0.46 c	8.08 ± 0.52 b	9.45 ± 0.81 a
SI	0.14 ± 0.04 d	0.26 ± 0.07 c	0.32 ± 0.06 b	0.47 ± 0.08 a
OT	1.47 ± 0.12 b	1.57 ± 0.22 a	1.23 ± 0.53 c	1.16 ± 0.17 c

Notes: CK, no fertilizer; NPK, single chemical fertilizer; NPKJ, chemical fertilizer and straw; NPKM, chemical fertilizer and organic manure. AI, saturated hydrocarbons; UH, unsaturated hydrocarbons; AR, aromatic compound; OC, oxygenated chemicals; NC, nitrogen-containing compound; SI, siliceous compound; OT, other compounds. Different lowercase letters in the same row indicate significant differences among different fertilization treatments, p < 0.05.

), while there was little change in hydrocarbon content under NPK treatment. Moreover, compared to CK treatment, the aromatic compound content increased by 46.5%, 160%, and 330% under NPK, NPKJ, and NPKM treatments. Additionally, compared to CK treatment, NPK, NPKJ, and NPKM treatments decreased the oxygen content by 27.9%, 13.3%, and 15.8%. Furthermore, nitrogen-containing compounds increased by 87.4%, 167.5%, and 212.9% under NPK, NPKJ, and NPKM treatments, respectively, compared with CK treatment. Finally, the silicon-containing compounds increased by 85.7%, 128.6%, and 235.7% under NPK, NPKJ, and NPKM treatments, respectively, compared with CK treatment. In NPK, NPKJ, and NPKM treatments, the content of aromatic compounds, nitrogen compounds, and silicon compounds, which are stable, increased, indicating all fertilization practices improved the chemical stability of SOM.

3.3. Biological Stability of SOM

Fertilization practices had a significant impact on the contents of ISOM and ASOM, as well as the BSI value, which serves as an indicator for the biological stability of SOM (p < 0.05). Large BSI values indicate a high biological stability of SOM. The application of fertilizers increased the amount of ASOM to 19.86–27.67 g kg⁻¹ and increased the content of ISOM to 20.00–22.55 g kg⁻¹. In comparison to CK treatment, NPK, NPKJ, and NPKM treatments resulted in a 2%, 7%, and 13% increase in ISOM contents (Figure 4). Similarly, ASOM contents also increased by 18%, 30%, and 39% under NPK, NPKJ, and NPKM treatments. NPK, NPKJ, and NPKM treatments reduced the value of BSI; NPKM treatment has the smallest BSI value. In addition, fertilization practices increased the mineralization rate constant (k) by 9.3%, 31.4%, and 50% in NPK, NPKJ, and NPKM treatments. The results showed that all fertilization practices reduced the biological stability of SOM.

3.4. The Content and Comprehensive Stability of SOM

Fertilization practices significantly increased the contents of SOM to 39.86–50.22 g kg⁻¹ (Figure 5, p < 0.05). Compared with CK treatment, SOM contents in NPK, NPKJ, and NPKM treatments were significantly increased by 8.4%, 15.6%, and 21.4% (p < 0.05). Fertilization practices increased the comprehensive stability of SOM by 26%, 101%, and 117% under NPK, NPKJ, and NPKM treatments (p < 0.05) compared with CK treatment. Fertilization practices increased the comprehensive stability of SOM by affecting the physical, chemical, and biological stability of SOM (Figure 6). Fertilization practices had the most significant impact on the chemical stability of SOM, with a path coefficient of 0.909, and the least significant effect on the physical stability of SOM, with a path coefficient of 0.645 (p < 0.05). The chemical stability of SOM contributed the most to its comprehensive stability, with a path coefficient of 0.993. Consequently, fertilization practices primarily enhanced the comprehensive stability of SOM by improving its chemical stability. Additionally, the comprehensive stability of SOM directly influences its content.

4. Discussion

4.1. Effects of Fertilization Practices on the Physical Stability of SOM

The content of POM, MAOM, and MAOM/POM are important indicators evaluating the physical stability of SOM [3]. POM is more sensitive to agricultural management practices than MAOM and has a faster turnover rate and lower stability [28]. POM is predominantly unbound in the soil matrix and is protected from decomposition. The average residence time of POM in agricultural soils is comparatively short. MAOM is adsorbed to the mineral phase and can have very long residence times [19]. The physical stability of SOM is enhanced with an increasing MAOM/POM value [28]. In our study, the contents of POM and MAOM under NPKJ and NPKM treatments were significantly higher than those in the NPK and CK treatments, which is consistent with the results of Karin’s study (p < 0.05) [33]. Compared with the CK and NPK treatments, where plant residues are the single source of organic matter, the increased contents of MAOM and POM in NKPM and NPKJ treatments are attributed to the higher input of organic matter to the soil through manure and straw application [20]. In addition, manure and straw added to the soil decompose to release nutrients that enhance crop growth, leading to increased litter and root exudates, which promote the formation of POM and MAOM [34]. Our results found that the MAOM/POM value reduced in all fertilization treatments compared with CK treatment, and the lowest value was observed in the NPKM treatment, which showed that chemical fertilizer, manure, and straw application could reduce the physical stability of SOM, and the SOM physical stability of NPKM treatment was the lowest, while CK treatment was the highest. POM is an active part of SOM, which is obviously affected by fertilization practices [28]. Manure and straw contain a large amount of active organic matter, which can be rapidly transformed with POM after being added to the soil, resulting in a rapid increase in the content of POM [19]. MAOM has distinct biogeochemical characteristics, including sources with POM, is relatively stable, and is less affected by exogenous input, with little change in content [6]. The increase in POM content was larger than that of MAOM content, resulting in a decline in the MAOM/POM value, which reveals the physical stability of SOM [33].

4.2. Effects of Fertilization Practices on the Chemical Stability of SOM

The chemical stability of SOM is affected by its compound composition, so the analysis of various organic components of SOM is a common method to evaluate the chemical stability of SOM [35]. The chemical stability of SOM increases with a higher content of stable compounds [36]. The stable compounds of SOM in our study mainly include aromatic compounds, nitrogen compounds, silicon compounds, and other compounds, and saturated hydrocarbons, unsaturated hydrocarbons, and oxygen compounds are unstable compounds [13]. Py GC-MS analysis revealed significant changes in the chemical composition of SOM due to the application of chemical fertilizer, manure, and straw, showing an increase in stable components and a decrease in unstable components compared to CK treatment (p < 0.05). The complex chemical structure and high content of stable compounds in organic fertilizers led to a more substantial increase in the stable components [13]. Furthermore, compared with straw, manure contains a greater variety and higher content of stable compounds [37], resulting in higher levels of stable compounds in soil treated with manure than straw. In addition, the decomposition of manure and straw in the soil releases substantial amounts of nutrients that are available for microbial use [38], enhancing microbial activity and increasing microbial biomass, which in turn contributes to a higher content of stable compounds [39]. However, compared to the CK treatment, the NPK treatment also enhanced the chemical stability of SOM; this result was also found in the previous study [19]. Despite having the lowest content of stable compounds, it directly provides N, P, and K nutrients to microorganisms and increases the aromatic compounds derived from microbial sources [29]. Moreover, compared with CK treatment, fertilization practices could promote crop growth and increase the content of plant residues and root exudates, thus increasing the stable compounds of nitrogen compounds and aromatic compounds of plant origin [40]. Therefore, manure and straw application can increase the content of stable compounds of SOM, and thus improve the chemical stability of SOM.

4.3. Effects of Fertilization Practices on Biological Stability of SOM

Soil mineralization rate constant and BSI, which are importantly correlated with the contents of ASOM and ISOM, are important indicators to characterize the biological stability of SOM and are affected by fertilization practices [9]. Our study found that the application of manure and straw could increase the contents of ASOM and ISOM and the highest values were observed in NPKM treatment, which provided more organic matter [41]. Fertilization practices can promote crop growth and then increase the amount of litter and crop stubble which are the sources of ASOM [17]. Manure and straw contain a lot of active organic matter, and their addition can directly increase the content of ASOM in soil [34]. The increase in ISOM may be due to organic fertilizers directly supplying organic matter similar in composition to ISOM, thereby boosting the content of ISOM [9]. Alternatively, the manure and straw added to the soil could decompose into stable compounds that then combine with soil minerals to form ISOM [37]. At the same time, the NPK treatment could also increase the content of ISOM compared to CK treatment, as the additional nutrients boost the production of root exudates and microbial products that can be converted into ISOM [17]. Fertilization practices increased the soil mineralization rate constant (k), which is negatively correlated with the biological stability of SOM [25]. One reason is that the application of manure, straw, and chemical fertilizer brought more nutrient sources to microorganisms in the soil [42] and promoted the metabolism of microorganisms, and thus improved the activity of microorganisms [43]. In addition, fertilization practices also promoted crop growth [44], resulting in the release of root exudates, creating a suitable living environment for microorganisms, and improving the activity of microorganisms [41]. However, fertilization practices led to a reduction in the BSI; D.A. Sokolov et al. found this result [9]. ASOM, being more active and more affected by fertilization than ISOM, increased significantly more than ISOM, resulting in a decrease in BSI, which represents the ratio of ISOM to ASOM [9]. Therefore, manure and straw application increased the contents of ASOM and ISOM, decreased the BSI and soil mineralization rate constant, and thus decreased the biological stability of SOM.

4.4. Effects of Fertilization Practices on the Content and Comprehensive Stability of SOM

Fertilization practices increased the content and comprehensive stability of SOM, which are important indicators of soil quality [45]. Our study found that SOM content was increased by the application of straw and manure (Figure 5). Incorporating manure and straw into the soil leads to their decomposition, which releases organic carbon, thereby enhancing the content of SOM [44]. Simultaneously, the copious organic matter and nutrients stimulate root growth, and the root system might modify the chemical and biological processes of the soil by discharging root exudates, which could have an influence on the content of SOM [46]. Furthermore, this study found that the contents of SOM increased more after manure application than straw application, which is because the decomposition rate of manure was faster than that of straw [38]. The application of chemical fertilizer, manure, and straw increased the comprehensive stability of SOM (Figure 5). PLS-PM showed that fertilization practices affected the comprehensive stability of SOM by increasing chemical stability and decreasing physical and biological stability, and finally increasing SOM content. The increase in chemical stability is the main reason for the increase in comprehensive stability (path coefficient is 0.993). Previous studies have shown that there was a strong positive correlation between chemical stability and comprehensive stability, indicating that chemical stability directly affects comprehensive stability, and simultaneously, chemical stability can indirectly affect physical and biological stability [14,25]. Our study collectively showed that applying manure and straw improved the comprehensive stability and content of SOM and significantly decreased its physical and biological stabilities (p < 0.05), while enhancing its chemical stability, which made the most vital contribution to the overall stability.

5. Conclusions

Compared with previous studies that focused on the single stability of SOM, this study considered the physical, chemical, and biological stability of SOM and studied the comprehensive stability of SOM. The final results show that fertilization practices increased the contents of POM and MAOM and decreased the MAOM/POM value, reducing the physical stability of SOM and confirming our hypothesis. In addition, straw and manure application increased the contents of stable aromatic compounds and nitrogen compounds of SOM, thus improving the chemical stability of SOM. Simultaneously, the increased contents of ASOM, ISOM, and the mineralization rate constant, along with the decreased BSI, indicated that microbial activity was enhanced, which in turn accelerated SOM mineralization, ultimately leading to a reduction in its biological stability. In conclusion, the fertilization practices finally improved the content and comprehensive stability of SOM and decreased the physical and biological stability, increasing the chemical stability, which made the most contributions to the comprehensive stability of SOM. Although we have studied the comprehensive stability of SOM, the relationship between SOM comprehensive stability and crop yield is still unclear. In the future, we will further explore the relationship between SOM comprehensive stability and crop yield.