Effects of Organic and Inorganic Fertilization on Soil Organic Carbon and Enzymatic Activities

Abstract

The overapplication of mineral nitrogen (N) fertilizer has become a scientific problem to be solved in the North China Plain; meanwhile, most organic manure cannot be recycled into the soil. To make full use of organic manure and decrease the applied rate of mineral N fertilizer, a four-year fertilization experiment was carried out to assess the effects of substituting mineral fertilizer with organic manure on soil organic carbon (SOC), labile organic carbon (LOC), stable organic carbon (UOC), microbial biomass carbon (MBC), and soil enzymatic activity. There were six treatments including unfertilized control (CK); mineral fertilizer without organic manure (FR); and mineral nitrogen substituted by organic manure at 20%, 40%, 60%, and 80% (FM1, FM2, FM3, and FM4), respectively. The results showed that SOC, UOC, and LOC were significantly increased by 13.30~40.56%, 41.10~121.92%, and 4.91~15.57% with increasing amounts of organic manure, respectively. Adding organic manure also enhanced urease, catalase, sucrase, and cellulase activities as well as MBC in surface soil (0–20 cm). The present study revealed the activities of urease, catalase, sucrase, and cellulase were closely related to SOC and its components. Following organic manure application, MBC and enzymatic activities had active effects in enhancing LOC and UOC sequestration based on path analyses. Additionally, organic manure increased SOC by direct and indirect enhancement of UOC and LOC, respectively, and improved SOC sequestration as compared to FR treatment. Finally, the study revealed that SOC quantity and quality were both significantly improved by combined application of mineral fertilizer and organic manure, and this effect was enhanced with the increase of organic manure application. Considering the improvements in soil properties and grain yields, substituting mineral fertilizer with organic manure might be a promising alternative in the North China Plain.

1. Introduction

Fertilizer application has increased China’s production several times according to previous estimates [1,2]. This has allowed China to feed 22% of the global population using only 7% of the total arable land area over the past 50 years [2]. However, soil organic carbon (SOC) content is declining in some areas of China, and soil health have been altered by long-term excessive use of fertilizers [3,4,5]. Low efficiency of utilization and high energy consumption are the major problems associated with the chemical fertilizer application in China [3,6]. Long-term single application of chemical fertilizer increases the cost of agriculture and environmental destruction. These unsolved problems have motivated the development of various approaches for optimizing fertilization and increasing crop yield based on soil properties, crop nutrient requirements, and environmental carrying capacity.

As the material basis of soil fertility, SOC is mainly derived from exogenous organic materials as well as crop residues and their exudates, and the changes of SOC affect soil fertility and health directly [7]. Normally, organic fertilizer application accelerates crop growth; meanwhile, the accompanying increase in root area and exudates further promotes SOC accumulation [8]. Because organic fertilizers carry a lot of available nutrients and are very easy to obtain, the application of organic manure is one of the important traditional measures of farmland fertilizer cultivation in China. Similar to natural soil, organic manure typically contains labile organic carbon (LOC), stable organic carbon (UOC), and available nitrogen, which not only promote carbon sequestration, but also stimulate soil biodiversity [9,10,11]. Thus, long-term application of organic manure or combined application of organic and inorganic fertilizers is a more effective way to increase total SOC and its labile components than the sole chemical fertilizer application [9,10,12]. Some studies also proved that partial substitution of fertilizer with organic manure could reverse the negative effects of excess fertilization [9]. In addition, in terms of improving soil fertility and maintaining soil nutrient balance, the combined application of organic and inorganic fertilizers has also shown significant improvement effects [9,13]. In fact, soil carbon sequestration and decomposition occur simultaneously, but the application of organic manure might complicate the process of SOC accumulation and transformation [7]. Most previous studies have only revealed that there is a certain quantitative relationship between soil fertility (such as, SOC, soil enzyme activities, etc.) and organic fertilizer application, but the mechanism of the relationship between organic fertilizer application, SOC, and soil biological activities remains unclear [14,15]. Therefore, more quantitative data on the effects of organic manure on soil biochemical properties are still needed from further research effects of chemical fertilizer reduction combined with organic manure on SOC and enzymatic activities.

To further understand the effects of organic manure application on soil carbon sequestration and enzyme activities, we carried out a field experiment in central China. The objectives were to: (1) investigate the influence of chemical fertilizer combined with manure at different ratios on SOC, LOC, UOC, and soil enzymatic activities; (2) evaluate the direct and indirect effects of organic manure on SOC and its components after four years of intensive application; and (3) obtain a quantitative estimate of the effects of combined fertilizer and manure application at different ratios to soil biochemistry properties.

2. Materials and Methods

2.1. Experimental Site

The study was conducted in an experimental fertilization field (latitude 33°33′ N, longitude 114°02′ E) situated in Henan province, China. The experiment was started in June 2012 on a well-drained field representing typical irrigable cropland of the North China plain, with an average annual temperature and precipitation of 14.80 °C and 852 mm, respectively. The average number of sunshine hours and frost-free periods are 2659 h and 121 days, respectively. Soil nutrient concentrations in June 2012 are shown in Table 1. The soil type is low-yielding vertisol and the soil of the experimental site has grown winter wheat (Triticum aestivum L.) and summer maize (Zea mays L.) in rotation for more than 30 years.

2.2. Experimental Design

The experiment included two crops per year—winter wheat (Triticum aestivum L.) and summer maize (Zea mays L.)—and six treatments including unfertilized control (CK), chemical fertilizer at a standard rate (FR), and chemical fertilizer containing 20%, 40%, 60%, 80% organic manure (FM1, FM2, FM3, and FM4, respectively). The amount of fertilizer used during the wheat-growing season is shown in Table 2. No fertilizer was applied to the CK group; the remaining plants were treated with 225 kg N/ha + 90 kg P₂O₅/ha + 90 kg K₂O/ha, except that in FM1, FM2, FM3, and FM4 the nitrogen (N) was replaced with organic manure at a ratio of 20%, 40%, 60%, and 80% respectively. In the maize season, all treatments had the same amount of N fertilizer (240.00 kg N/ha). Thus, the total amount of N, P₂O₅, and K₂O was 465, 180, and 180 kg/ha in all treatments. Organic manure and P₂O₅ and K₂O fertilizers were applied to the soil before sowing, and 40% and 60% of the total amount of N fertilizer was applied before sowing and during the elongation stage, respectively. The organic manure was made of chicken compost. A randomized block design was used to prepare three replicates of each of six treatments. Each plot was 5 × 9 m². The winter wheat (Triticum aestivum L.) and summer maize (Zea mays L.) varieties were Zhengmai 9023 and ZhengDan 958, respectively. Mature wheat and maize were harvested in June and October, respectively. Crop yield was measured manually after crop maturity.

2.3. Soil Sampling and Chemical Analysis

Soil sampling was performed at the time of harvest in June 2016, and five samples were obtained from each plot at depth intervals of 20 cm. Fresh soil samples were bulked and immediately transported to the laboratory for determination of MBC and microbial biomass nitrogen (MBN) contents by chloroform fumigation extraction [16], and of soil water content by oven-drying (105 °C for 24 h) and gravimetric analysis. The remaining soil samples were air-dried and used to determine SOC, LOC, and UOC and enzymatic activity. SOC was measured with the potassium dichromate heating method [16]; LOC and enzymatic activity were measured as previously described [17]; and UOC was calculated by subtracting LOC from total organic carbon [17].

2.4. Calculations and Statistical Analysis

Differences between groups were evaluated by analysis of variance using SPSS software (SPSS 22.0 Inc., Chicago, IL, USA). Statistical significance was determined using the least significant difference method. Path analysis was carried out as previously described [18]. Interaction analysis was performed with the following equations:

COM = (OCF − NOCF)/OCF × 100%

IFM = FMOCF − FOCF

IFMC = IFM/FMOCF × 100%

where COM is the contribution of fertilization; OCF is the SOC of the fertilizer treatment; NOCF is the SOC with no fertilization treatment; IFM is the interaction effect of chemical fertilizer combined with organic manure on SOC and its fractions; FMOCF is the SOC of chemical fertilizer combined with organic manure treatment; FOCF is the SOC of chemical fertilizer treatment; and IFMC is the contribution of IFM to SOC and its fractions.

The carbon pool management index (CPMI) for each treatment was calculated based on a published method [19]. CK was used as the reference when calculating the carbon pool index (CPI) and lability index (LI). Lability (L) reflected changes in the proportion of LOC in the soil; and LI accounted for changes in the proportion of labile carbon in the soil between a reference and treatment site. Based on changes in SOC between reference and sample soils, CPI, LI, and CPMI were calculated as follows:

$\mathrm{CPI} =\frac{{\mathrm{SOC}}_{sample}}{{\mathrm{SOC}}_{reference}}$

$\mathrm{L} =\frac{\mathrm{LOC}}{\mathrm{UOC}}$

$\mathrm{LI} =\frac{{\mathrm{L}}_{sample}}{{\mathrm{L}}_{reference}}$

CPMI = CPI × LI × 100

where ${\mathrm{SOC}}_{sample}$ and ${\mathrm{SOC}}_{reference}$ are the SOCs of sample and reference soils; and ${\mathrm{L}}_{sample}$ and ${\mathrm{L}}_{reference}$. are the L of sample and reference soils.

3. Results

3.1. Effects of Different Fertilization Treatments on Soil Enzymatic Activity

The relationship between the applied amount of organic manure and urease, catalase, sucrase, and cellulase activities was evaluated in the present study. Urease activity was higher in FR than in CK, and increased linearly with the proportion of organic manure (p < 0.05; Figure 1). Thus, the increase in urease activity was approximately equal between FR and FM1. Similar trends were observed for catalase, sucrase, and cellulase activities; the latter two, which are closely related to organic carbon transformation and carbon cycling, were also significantly improved by combined application of organic and inorganic fertilizer (p < 0.05). These results demonstrate that soil enzymatic activity was enhanced by application of organic manure significantly compared to CK. In addition, both FM3 and FM4 treatments significantly enhanced urease (increased by 18.43~28.77%), catalase (increased by 54.07~69.66%), sucrase (increased by 42.38~126.11%), and cellulase (increased by 27.50~35.67%) activities when compared to FR treatment. Meanwhile, only urease (higher by 13.09%) and catalase (higher by 29.21%) activities in FM2 treatment and catalase (higher by 4.53%) activity in FM1 were significantly higher than those in FR.

3.2. Effects of Different Fertilization Treatments on SOC Fractions and CPMI

SOC and CPMI varied according to the fertilizer treatment (Table 3). LOC was about two to four times higher in the treatment groups than that in CK depending on the amount of organic manure applied, whereas SOC (higher by 13.30~40.56%), UOC (higher by 41.10~121.92%) and LOC (higher by 4.91~15.57%) in FM2, FM3, and FM4 treatments were significantly higher than that in FR. However, FM1 had little effect on SOC sequestration when compared to FR. CPI, L, and LI were increased relative to CK, especially when organic manure content was high (i.e., FM2, FM3, and FM4). LOC and SOC were increased after 4 years of organic manure treatment. What is more, the increment of CPMI values of FM2, FM3, and FM4 treatments reached a significant level compared with the FR treatment. Thus, the practice of combining inorganic and organic fertilizer significantly improved CPMI.

MBC also varied across treatments (Table 4), and was higher in FM1, FM2, FM3, and FM4 than in FR, and increased with the applied amount of organic manure. MBN was similar between the FM3 and FM4 treatment groups, and MBC/MBN and qMB showed the same trend as MBC.

3.3. Effects of Interaction of Chemical Fertilizer and Organic Manure on SOC Fractions

SOC was enhanced by 7.62% in FR and by 10.39%, 18.49%, 29.15%, and 34.25% in FM1, FM2, FM3 and FM4, respectively, relative to CK (Table 5). FR treatment caused a 4.35% decline in UOC but increased LOC by 46.59%. Compared to CK, COM of LOC and UOC was markedly increased in FM1, FM2, FM3, and FM4. IFM values also indicated that combined application of chemical fertilizer and organic manure increased SOC, LOC, and UOC by 3.01~28.83%, 10.00~54.19%, and 1.84~13.45%, respectively.

3.4. Linear Relationship between Organic Manure Application and SOC Increase

The linear regression analysis revealed that SOC, LOC, and UOC were positively correlated with amount of nitrogen in the applied organic manure (Figure 2). The increases in SOC and LOC were positive whereas the increase in UOC was negative when the amount of nitrogen in the applied organic manure was zero based on the fitted curves, suggesting that SOC and LOC but not UOC could be increased by applying no organic manure or FR only. The regression analyses between SOC, LOC, and UOC and amount of nitrogen in the applied organic manure all reached a significant level (p < 0.01), respectively. The intersection point of the solid and dotted lines was x = 75.57, y = 1.52. This indicated that the applied organic manure was more effective in increasing LOC than SOC when the applied nitrogen amount was <75.57 kg/ha; however, when the applied nitrogen amount was >75.57 kg/ha, organic manure had a greater influence on SOC than on LOC. SOC, LOC, and UOC were significantly increased with an increase in the amount of applied nitrogen as organic manure based on the linear regression analysis in Figure 2. There was a linear relationship between the increase in SOC and changes in the activities of urease, catalase, sucrase, and cellulase at the significant level (p < 0.01) (Figure 3).

3.5. Crop Yields of Different Treatments

The results of this study show that CK treatment had the lowest grain yield, and the grain yields of wheat and maize under FM2 treatment were significantly higher than those of FR treatment (Figure 4). Although the yields of soil with FM1, FM3, and FM4 treatments were higher than those of soil with a single fertilizer application (FR treatment), they did not reach the level of significant difference (p < 0.05).

4. Discussion

In the present study, organic manure greatly improved soil organic matter and active components according to analysis results (Table 3 and Table 4). This change might play a positive role in improving vertisol structure because some previous studies have confirmed the close relationship between soil structure and SOC content. For example, Hidalgo et al. [20] and Wang et al. [21] revealed that vertisol exhibits the features of heavy clay, including drying shrinkage and bulking, waterlogging, and low SOC. Fortunately, organic matter contained in organic manure can not only reduce the shear strength of montmorillonite and restrict its expansion and shrinkage, but can also supply active organic colloids for the formation of organo-mineral composites, which in turn form macro-aggregate bridges that physically protect SOC [21,22]. Such aggregates play a critical role in balancing the water, fertilizer, air contents, and temperature of soil by influencing soil microbial enzyme activities, which can affect mass and energy flow in ecological systems and improve soil fertility [23]. Thus, combined application of mineral fertilizers and organic manure enhanced soil fertility by increasing SOC, LOC, UOC, MBC, and soil enzymatic activities, and the applied amount of fertilizer was positively correlated with nutrient content (Table 3). Others have reported that SOC and soil microbial biomass and enzymatic activities were higher in soil treated with compost as compared to chemical fertilizer [24], and that SOC and MBC were higher in plants subjected to long-term treatment with chemical fertilizer combined with farmyard manure as compared to fertilizer alone [9]. In the study, organic manure application had variable effects on SOC and LOC, which may be due to differences in nutrient availability, microbial activity, and microstructural change [24].

SOC, which reflects the balance between organic carbon accumulation and decomposition, is an important index for evaluating soil productivity and fertility of cropland. Changes in SOC can reveal the effect of fertilization on SOC turnover and sequestration, given that it is mainly influenced by fertilization practices and is independent of natural factors [19,25]. On the other hand, soil microbial biomass—the living component of SOC—can increase SOC; indeed, the turnover rate of soil microorganisms is often used to predict changes in SOC [26,27]. Therefore, in order to clarify the factors contributing to the increase in SOC, it is necessary to evaluate the relationships between SOC, LOC, UOC, MBC, and soil enzymatic activity. In this study, we evaluated the direct and indirect effects of applied manure on SOC, LOC, and SOC by path analysis (Figure 5). UOC and LOC were affected to a greater degree than SOC by organic manure treatment; the direct coefficients of determination of manure on UOC, LOC, and SOC were 1093.18, 277.40, and 60.47, respectively. However, manure application had greater indirect effects on LOC and SOC through MBC than on SOC through LOC and UOC, suggesting that organic manure used in farming injects large amounts of UOC into soil.

CMI can be used to describe soil quality by comparing the ability of different management practices to predict the long-term effectiveness of soil nutrient supply, productivity, and carbon pools [28]. In the study, CMI was enhanced by both chemical fertilizer and organic manure application (Table 3). However, a more potent effect was observed with larger amounts of manure (e.g., FM3 or FM4 treatments), possibly due to an increase in annual carbon input and changes in organic matter quality. Previous studies reported that manure without or with inorganic fertilizer increased CMI as compared to single chemical fertilizer treatment upon long-term application [28,29]. Thus, CMI may be a better indicator of SOC sensitivity to agricultural practices, with higher values reflecting a more stable SOC [28,30]. The analysis results indicate that combining chemical fertilizers and organic manure is effective for maintaining or improving SOC. Substituting fertilizer with manure can enhance both the quantity and quality of SOC.

On the other hand, the addition of organic manure significantly increased soil enzyme activities in this study (Figure 1), which was an indirect reference basis for revealing the effect of organic matter application on soil fertility. Soil enzymatic activity, which reflects the efficiency of nutrient transformation, can serve as an indicator for cropland fertility. For instance, urease has high affinity for N fertilizers and plays an important role in N transformation by hydrolyzing organic amide compounds into inorganic N compounds [31]. Thus, urease activity reflects soil nitrogen-supplying capacity and may be significantly affected by the amount of organic manure that is applied. As a redox enzyme, catalase contributes to the transformation of pedological mass; moreover, the oxidation strength can be indirectly determined by measuring catalase activity [32]. The degree of mineralization of SOC is closely related to cellulase activity, because SOC is a major substrate of the enzyme [33]. Thus, according to the characteristics of simultaneous growth of soil enzyme activities and SOC components in the study (Table 3, Figure 1), it could be inferred that the application of organic manure could promote soil fertility to some extent.

In addition, soil enzymatic activity is closely associated with SOC [34]. Application of chemical fertilizer containing organic manure can complicate the catalytic reaction because the latter comprises a large amount of organic–inorganic material. However, the study showed that not only were urease, catalase, sucrase, and cellulase activities increased by combined application of chemical fertilizers and organic manure (Figure 1), but that the enzymes played distinct roles in the sequestration of SOC, LOC, and UOC (Figure 6). The manure had the most potent effect on catalase, followed by sucrase, cellulase, and urease; it also indirectly influenced UOC to a greater extent than LOC and MBC through soil enzymatic activity. Compared to LOC and UOC, MBC may be more closely associated with the activities of the four enzymes, and the indirect effect of MBC on LOC through enzyme activity was greater than that of LOC and UOC on SOC. Thus, organic manure has variable effects on the activities of soil enzymes, which play an important role in the accumulation and transformation of MBC and LOC.

The above-described results may be explained by the fact that applied manure can enhance loosely combined humus, which when absorbed by soil microorganisms intensified their enzymatic activities, leading to acceleration of SOC cycling. However, applying mineral fertilizers alone was more beneficial for improving the amount of firmly combined humus while avoiding the consumption of a large number of organic substances, which is important for preserving soil structure and nutrient levels [35,36]. Under combined application of mineral fertilizers and organic manure, increased SOC sequestration was mainly due to an improvement in UOC, with LOC playing a secondary role in SOC fixation. Additionally, MBC had a synergistic effect on LOC, whereas the presence of organic manure stimulated the activity of catalase, which had a greater effect on the SOC fraction than the other three enzymes. More importantly, the application of organic manure could significantly increase grain yield, which is the ultimate goal of agricultural practice (Figure 4). Thus, combined application of mineral fertilizers and organic manure can improve SOC, LOC, and UOC and overcome the problems of nutrient deficiency and short effective period. Additionally, applying the proper amount of organic manure has a positive effect on improving crop productivity.

5. Conclusions

Combined application of mineral fertilizer and organic manure improved SOC and enzymatic activity of soil. A positive linear relation was observed between SOC fractions (SOC, LOC, and UOC) and the amount of nitrogen in the applied organic manure. Following organic manure application, MBC and the activities of urease, catalase, sucrase, and cellulase indirectly and directly enhanced sequestration of LOC and UOC, respectively. These findings provide insight into the quantitative relationships between the amount of organic manure applied to soils and SOC, LOC, and UOC, and suggest that combined application of mineral fertilizer and organic manure is a sustainable management option for improving SOC quantity and quality and consequently.

Author Contributions

Conceptualization, C.Z. and J.Z.; methodology, C.Z., Z.Z. and J.Z.; software, C.Z., Z.Z. and F.L.; validation, C.Z. and J.Z.; formal analysis, Z.Z. and F.L.; investigation, C.Z. and Z.Z.; resources, C.Z., F.L. and J.Z.; data curation, Z.Z.; writing—original draft preparation, C.Z. and Z.Z.; writing—review and editing, Z.Z.; visualization, Z.Z. and F.L.; supervision, C.Z. and J.Z.; project administration, C.Z. and J.Z. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The dataset for this article is also available by contacting the corresponding author.

Conflicts of Interest

The authors declare no conflict of interest.

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures and Tables

Figure 1. Soil enzyme activities under different organic manure treatment conditions. Different lowercase letters in the same box denote significant differences at p < 0.05, respectively; short horizontal lines at the top of each column denote standard error.

Figure 2. Linear regression analysis between increases in SOC, LOC, and UOC and amount of applied N. Values on the horizontal axis denote the amount of applied N in organic manure, and those on the vertical axis denote increases in SOC, LOC, and UOC vs. CK. The solid line and upper and lower dotted lines represents the regression lines for SOC, LOC, and UOC, respectively. ** p < 0.05.

Figure 3. Linear regression analysis between increases in SOC and urease (a), catalase (b), sucrase (c), and cellulase (d) activities. Values on the horizontal axis denote increases in urease, catalase, sucrase, and cellulase activities, respectively, whereas those on the vertical axis denote the increase in SOC vs. CK. ** p < 0.05.

Figure 4. The oven-dried grain yields of wheat and maize under different experimental treatments after four years. Different lowercase letters in the same box denote significant differences at p < 0.05, respectively.

Figure 5. Path analyses between organic manure amount, SOC, and the various stable fractions. Solid and dotted lines denoted direct and indirect effects, respectively. Values indicated within lines are path coefficients reflecting the relative influence of manure on the dependent variables (SOC, LOC, UOC, and MBC) in the same path; those in brackets are coefficients of determination representing the degree of influence of manure on dependent variables in the same path.

Figure 6. Path analyses between organic manure and activities of four enzymes affecting SOC and the various stable fractions. Solid and dotted lines and values are as described in the legend for Figure 5.

Table 1

Physical and chemical properties of soil in field under study.

Total Nitrogen(g/kg)	Total Phosphorus(g/kg)	Total Potassium(g/kg)	Available Phosphorus (mg/kg)	Available Potassium(mg/kg)	Sand(%)	Silt (%)	Clay(%)	SOC(g/kg)
0.57	0.76	21.20	33.06	125.60	45.60	40.70	13.70	5.92

SOC, soil organic carbon.

Table 2

Composition of various treatments.

Treatment	Organic Manure a(t/ha)	Chemical Fertilizer b(kg/ha)			Chemical Fertilizer c(kg/ha)
		N	P2O5	K2O	N	P2O5	K2O
CK	0	0	0	0	0	0	0
FR	0	225	90	90	240.00	90.00	90.00
FM1	2.11	180	47.55	51.45	240.00	90.00	90.00
FM2	4.21	135	5.25	12.9	240.00	90.00	90.00
FM3	6.32	90	0	0	240.00	52.80	64.35
FM4	8.42	45	0	0	240.00	10.50	25.80

^a Total N, P₂O₅, and K₂O contents of organic manure were 2.14%, 2.02%, and 1.83% respectively. ^b Wheat-growing season. ^c Maize-growing season. CK, unfertilized control; FM1/2/3/4, chemical fertilizer containing 20%, 40%, 60%, 80% organic manure, respectively; FR, conventional fertilization.

Table 3

Change in soil organic carbon fraction and carbon pool management index under different treatments.

Treatment	SOC(g/kg)	LOC(g/kg)	UOC(g/kg)	CPI	L	LI	CPMI
CK	8.61 ± 0.23 d	1.17 ± 0.65 e	7.44 ± 0.65 a,b	1.00 ± 0.03 e	0.17 ± 0.10 d	1.00 ± 0.00 e	100.00 ± 0.00 e
FR	9.32 ± 0.29 c	2.19 ± 0.14 d	7.13 ± 0.29 b	1.08 ± 0.03 d	0.31 ± 0.03 c	1.86 ± 0.20 d	200.55 ± 14.81 d
FM1	9.67 ± 0.35 c	2.41 ± 0.02 d	7.26 ± 0.67 b	1.12 ± 0.04 d	0.34 ± 0.02 c	2.04 ± 0.16 c,d	244.47 ± 53.69 d
FM2	10.56 ± 0.19 b	3.09 ± 0.33 c	7.48 ± 0.33 a,b	1.23 ± 0.02 c	0.42 ± 0.06 b,c	2.39 ± 0.42 c	308.87 ± 43.94 c
FM3	12.15 ± 0.46 a	4.17 ± 0.23 b	7.98 ± 0.27 a,b	1.41 ± 0.05 b	0.52 ± 0.01 b	3.11 ± 0.04 b	444.32 ± 11.74 b
FM4	13.10 ± 0.35 a	4.86 ± 0.28 a	8.24 ± 0.63 a	1.52 ± 0.04 a	0.64 ± 0.10 a	3.86 ± 0.19 a	587.12 ± 30.69 a

Values represent mean ± standard error. Different lowercase letters in the same column denote significant differences between treatments at p < 0.05, respectively. CK, unfertilized control; CPI, carbon pool index; CPMI, carbon pool management index; FM1/2/3/4, chemical fertilizer containing 20%, 40%, 60%, 80% organic manure, respectively; FR, conventional fertilization; L, lability; LI, lability index; LOC, labile organic carbon; SOC, soil organic carbon; UOC, untreated organic carbon.

Table 4

Soil MBC and MBN contents under different fertilization treatments.

Treatment	MBC (mg/kg)	MBN (mg/kg)	MBC/MBN	qMB (%)
CK	33.86 ± 4.1 d	23.96 ± 1.05 e	1.41 ± 0.11 d	0.39 ± 0.07 c
FR	75.51 ± 4.57 c	28.79 ± 1.16 d	2.62 ± 0.11 c	0.81 ± 0.03 b
FM1	83.96 ± 2.67 c	31.89 ± 0.44 c	2.63 ± 0.12 c	0.87 ± 0.04 a,b
FM2	93.23 ± 1.67 b	32.63 ± 1.48 b	2.86 ± 0.08 b	0.88 ± 0.02 a
FM3	98.62 ± 5.65 a,b	35.1 ± 1.25 a	2.81 ± 0.03 b	0.82 ± 0.03 b
FM4	103.69 ± 7.35 a	34.25 ± 0.76 a	3.03 ± 0.05 a	0.77 ± 0.02 b

Values represent mean ± standard error. Different lowercase letters in the same column denote significant differences between treatments at p < 0.05, respectively. CK, unfertilized control; FM1/2/3/4, chemical fertilizer containing 20%, 40%, 60%, 80% organic manure, respectively; FR, conventional fertilization; MBC, microbial biomass carbon content; MBC/MBN, MBC to MBN ratio; MBN, microbial biomass nitrogen content; qMB, soil microbial quotient.

Table 5

Contribution of different fertilization treatments to SOC fraction and effects of interaction of chemical fertilizer and organic manure on the SOC fraction.

Treatment	COM (%)			IFM (g/kg)			IFMC (%)
	SOC	LOC	UOC	SOC	LOC	UOC	SOC	LOC	UOC
CK	/	/	/	/	/	/	/	/	/
FR	7.62	46.59	−4.35	/	/	/	/	/	/
FM1	10.39	51.93	−2.43	0.29	0.24	0.13	3.01	10.00	1.84
FM2	18.49	62.08	0.51	1.24	0.89	0.35	11.77	29.00	4.65
FM3	29.15	71.95	6.79	2.83	1.98	0.85	23.31	47.48	10.68
FM4	34.25	75.91	9.68	3.78	2.67	1.11	28.83	54.91	13.45

CK, unfertilized control; COM, contribution of fertilization; FM1/2/3/4, chemical fertilizer containing 20%, 40%, 60%, 80% organic manure, respectively; FR, conventional fertilization; IFM, interaction effect of chemical fertilization containing manure on SOC and its fractions; IFMC, contribution of IFM to SOC and its fractions; LOC, labile organic carbon; SOC, soil organic carbon; UOC, untreated organic carbon.

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