Effects of Carbon-Based Fertilizer on Soil Physical and Chemical Properties, Soil Enzyme Activity and Soil Microorganism of Maize in Northeast China

Abstract

The soil environment is an important factor that affects the growth of maize. Our study discusses the effect of carbon-based fertilizer on the rhizosphere soil microenvironment. Xianyu 335 and Jingke 968 maize varieties were selected, and six treatments were set up as follows: no fertilizer, chemical fertilizer, or carbon-based fertilizer (3 t/hm², 3.75 t/hm², 4.5 t/hm², and 5.25 t/hm²). The results showed that the carbon-based fertilizer significantly reduced the soil pH value in the late growth stage. Carbon-based fertilizer can significantly improve the conductivity of soil solution. On 8 July (jointing stage), the conductivity of the two varieties of soil was the highest at 3.75 t treatment, 259.38% and 169.26% higher than that of chemical fertilizer, respectively. Carbon-based fertilizer significantly increased the soil carbon flux. The soil carbon flux of Jingke 968 showed a trend of first rising and then falling with the increase in the application amount of carbon-based fertilizer. On 8 July (jointing stage) and 28 July (tasseling stage), the 4.5 t treatment reached the maximum value, and on 16 August (early filling stage) and 4 September (waxy stage), the 3.75 t treatment reached the maximum value. Carbon-based fertilizer significantly increased the content of nitrate nitrogen, ammonium nitrogen, available phosphorus, and available potassium in the topsoil. Carbon-based fertilizer had a significant effect on soil enzyme activity and significantly increased soil neutral phosphatase activity in the late growth stage. On 16 August (early stage of grouting), β-Glucosidase activity was significantly higher in 3 t and 3.75 t treatments than other treatments in Xianyu 335 and Jingke 968. The activity of α-Xylosidase reached the highest at 3.75 t. The activities of polyphenol oxidase and catalase reached their maximum at 5.25 t treatment on 4 September (waxy ripening) and 26 September (full ripening). Carbon-based fertilizer significantly increased the soil bacterial diversity index (Shannon index, ACE index and Chao1 index), but had no significant impact on the fungal diversity index, and significantly increased the abundance of soil bacterial and fungal populations.

1. Introduction

The soil environment directly affects the absorption of water and nutrients by roots, root activity, and root growth distribution, thus affecting the growth and yield formation of aboveground parts. When we review the history of agricultural development in China, the substantial increase of grain yield is closely related to the input of fertilizer [1]. Since the 1980s, China has started using many chemical fertilizers, and the grain yield has increased rapidly. At the same time, due to the blind pursuit of high yield, the wrong concept of “high yield and multiple fertilization” has been deeply rooted in the hearts of the people, which not only wastes a lot of resources, resulting in low resource utilization, but also seriously damages the ecological environment [2]. In the preparation process, biochar has formed a structure with a large specific surface area and multiple pores [3], and has the characteristics of multipolar functional groups, polyaromatic hydrocarbons, high carbon content, and stable physical and chemical properties [4]. Some research results show that carbon-based fertilizer retains the alkalinity and super adsorption of biochar [5]. The extremely high exchange activity of the carbon-based fertilizer surface makes the soil cation exchange capacity significantly improved, and the soil water holding capacity and water supply capacity are improved. Applying carbon-based fertilizer can improve soil ventilation and permeability, and can adjust soil pH value to be close to neutral [6]. Carbon-based fertilizer treatment significantly reduced soil bulk density and increased soil porosity [7]. After the application of carbon-based fertilizer, the soil water content in the topsoil was significantly increased, the soil bulk density decreased with the increase in the amount of carbon-based fertilizer [8], and the soil capillary porosity and field water capacity were significantly increased, which had a great impact on the soil pH value [5]. Carbon-based fertilizer has a significant effect on soil improvement and carbon fixation, which is mainly reflected in the increase in soil organic matter, electrical conductivity, effective nutrient elements, and trace nutrients [9].

Carbon-based fertilizer has high cation exchange capacity and developed pores, so it has strong adsorption characteristics, has retention function for nutrients, can promote the fixation of nutrients in the soil, delay the release and leaching of fertilizer in the soil, play a slow release role for nutrients, reduce the loss of fertilizer and soil nutrients, and improve the fertilizer utilization rate [8,9,10]. The leaching release period of carbon-based fertilizer nutrients in soil solution was significantly delayed [11]. The decomposition of organic matter can also produce a variety of acids and enzymes, accelerate the decomposition and transformation of various complex organic matters, rapidly activate soil nutrients, and increase the effectiveness of nutrients [12]. Carbon-based fertilizer significantly increased the content of organic matter, total potassium, and available potassium in peanut soil [13,14]. The application of carbon-based fertilizer increased the content of soil available phosphorus in different growth stages of silage maize, and the content of organic matter increased by 0.70% and 14.90% in the filling and mature stages of maize, respectively [15]. Compared with organic fertilizer, the soil water-soluble nitrogen and water-soluble phosphorus treated with carbon-based fertilizer increased by 11.92% and 9.67%, respectively [16]. The continuous use of carbon-based fertilizer significantly increased the content of soil organic carbon, free particulate organic carbon, and closed particulate organic carbon, and the effect was significant compared with the same amount of nitrogen, phosphorus, and potassium nutrients [17]. Combined application of nitrogen fertilizer and biochar can promote soil nitrification by increasing soil organic matter abundance and changing soil organic matter composition [18]. The results of using rice hull biochar and ammonium phosphate to prepare ammonium phosphate carbon-based fertilizer showed that, compared with single chemical fertilizer, nitrogen release in sandy soil was uniform and slow, significantly improving soil organic matter and having little negative impact on the environment [19]. The results showed that the water-soluble phosphorus in the soil was significantly reduced, while the high content of phosphorus retained in the fertilizer particles continued to be released in the later stage of corn growth, and was fully utilized, improving the utilization efficiency of phosphorus [20].

The activity of microbial communities is affected by environmental factors such as soil pH, temperature, water, nutrient content, etc. [21]. Carbon-based fertilizer has a good application effect on improving the soil’s ecological environment. After being applied to the soil, carbon-based fertilizer improves the soil porosity, pH, water content, and nutrient level, and the decomposition of organic matter produces a large amount of organic carbon, which provides a suitable microenvironment for the survival of microorganisms and has an impact on different microbial communities [22]. The porous structure on the biochar surface also provides a refuge for bacteria, which adsorb to the surface and are not easy to wash [23]. Previous studies have shown that carbon-based fertilizer can increase the richness and diversity of soil microorganisms and change the composition of bacterial communities in rhizosphere soil. Carbon-based fertilizer can change the structure and diversity of soil fungi and bacteria communities and optimize the soil ecological environment by affecting the chemical changes of soil microorganisms [24]. The application of carbon-based fertilizer is beneficial to improving the ratio of soil fungi/bacteria and the diversity of soil microbial community structure [25]. The diversity index (Simpson and Shannon) and richness index (Chao1) of soil bacteria and fungi in the treatment of carbon-based fertilizer were generally higher than those of chemical fertilizer and biochar alone. There was a significant difference in bacterial and fungal community structure between the treatment of chemical fertilizer and biochar alone [20]. Carbon-based fertilizer significantly increased the abundance of bacteria in red soil, and applying carbon-based fertilizer is a more effective method to improve soil bacterial structure [26]. The soil environment directly affects the physiological activity of microorganisms. Carbon-based fertilizer can improve the characteristics of the soil environment and significantly impact the metabolic activity of microorganisms. It is believed that the study of the utilization rate of microorganisms to nutrients by the Biolog microplate method can reflect the metabolic activity of microorganisms to a certain extent [27]. Carbon-based fertilizer has rich nutrient components, which can meet the metabolic needs of various microorganisms. Adding carbon-based fertilizer is conducive to maintaining the ability of soil microorganisms to use carbon source substrate, thus enhancing the metabolic activity. With the increase in the application amount of carbon-based fertilizer, microbial species’ number, richness, and dominance significantly increase [28].

Soil enzyme is an integral part of the soil system and is mainly composed of enzymes secreted by plant roots and residues, soil animals and their remains, and microorganisms [29]. The soil enzyme system is considered to be the most active part of the soil, which can catalyze the decomposition of substances in the soil, promote the transformation of soil substances, and promote the circulation of soil nutrients [30]. The research shows that the response of soil enzyme activity to the change of fertilization management, land use mode, and planting system is faster than other soil indicators [31]. In the soil environment, factors such as ion concentration, pH value, redox potential, and nutrient element composition directly affect enzyme synthesis and reaction activity [32]. Carbon-based fertilizer has high pH, loose porous structure, and large surface area. After being applied to the soil, it changes the soil’s physical and chemical properties and nutrient structure, thus affecting the activity of soil enzymes. The application of carbon-based fertilizer significantly increased the activities of urease, sucrase, and phosphatase in the 0–40 cm soil layer [33]. Carbon-based fertilizer significantly increased the activities of sucrase and catalase in sandy soil and loam [34]. Carbon-based fertilizer improve the activities of xylosidase, glucosidase, and cellobiose hydrolase to varying degrees [35,36]. This experiment provides a theoretical basis and technical reference for the rational application of carbon-based fertilizer by studying the influence mechanism of soil physical and chemical characteristics, soil enzyme activity, and soil microorganisms after the treatment of carbon-based fertilizer.

2. Materials and Methods

2.1. Experimental Design

The tested variety Xianyu 335 was selected by Tieling Xianfeng Seed Research Co., Ltd. (Tieling, China). and Jingke 968 by the Maize Research Center of Beijing Academy of Agricultural and Forestry Sciences (Beijing, China). Carbon-based fertilizer is provided by Heilongjiang Wuchang Runnong Science and Technology Co., Ltd. (Wuchang, China). Its nutrient content is 45.6% organic matter, 4% nitrogen (N), 3% phosphorus (P₂O₅) and 3% potassium (K₂O). We started the preliminary experiment in 2018, and achieved good experimental results. In 2019, we modified and improved the plan based on 2018, and achieved good application results from carbon-based fertilizer, and achieved good application data. The test for this manuscript was conducted in the Xiangyang Demonstration Base of Northeast Agricultural University in 2019. The planting density is 82,500 plants per hectare. The area has a temperate continental climate, rich in light and heat resources, and moderate rainfall. Daily mean values of the weather variables at the experimental site during the six months of the maize growing season in 2019 are listed in

Table 1. Daily mean values of the weather variables at the experimental site during the six months of the maize growing season in 2019.

Month	Average Temperature (°C)	Precipitation (mm)	Sunshine Hours (h)
April	11.6	46.2	200.6
May	18.5	70.5	234.1
June	22.7	115.2	224.5
July	27.4	31.1	275.4
August	25.2	47.4	223.2
September	18.5	52.4	185.9

. The test soil is chernozem, with loose texture and basic soil fertility: organic matter 28.21 g/kg, total nitrogen 1.8 g/kg, alkali-hydrolyzable nitrogen 105.84 mg/kg, available phosphorus 47.32 mg/kg, available potassium 166.15 mg/kg, and pH = 6.82. The experiment adopted a completely random design, and a total of 6 treatments were set up, namely, non-fertilization treatment (CK1), conventional fertilization treatment (CK2): nitrogen (N) 180 kg/hm², phosphorus (P₅O₂) 90 kg/hm², and potassium (K₂O) 90 kg/hm². The four carbon-based fertilizer treatments were: 3 t/hm², 3.75 t/hm², 4.5 t/hm², and 5.25 t/hm². For chemical fertilizer treatment, N 120 kg/hm², P₅O₂ 90 kg/hm², and K₂O 90 kg/hm² were applied as a base fertilizer before sowing, N 60 kg/hm² was applied at the jointing stage, and carbon base fertilizer was applied at one time. After ridging, mechanical furrow was opened on the ridge, carbon base fertilizer was applied, and then the ridge was closed. The plot has 8 ridges, 0.65 m wide, 7 m long, and an area of about 36.4 m². Each treatment is repeated 3 times and arranged randomly. The experiment was sown on 28 April, and sampling analysis was conducted on 8 July (jointing stage), 28 July (tasseling stage), 16 August (early filling stage), 4 September (waxy ripening stage), and 26 September (full ripening stage) [37].

2.2. Data Collection

2.2.1. Determination of Soil pH and Conductivity

We weighed 16 g of air-dried topsoil and put it into a 50 mL beaker; we added 40 mL of distilled water, stirred for 1 min, and placed it for half an hour. We inserted the pH electrode (PHSJ-6L, Shanghai, China) into the upper clarifier, turned it gently, and recorded the data when the reading reached a stable level. At the same time, we measured the conductivity of the clarifier with a conductivity meter (DDSJ-307F, Shanghai, China).

2.2.2. Determination of Soil Carbon Flux

Soilbox-343 portable soil respiration system (Beijing, China) was used to measure the soil carbon flux. The measurement time was 8:30–10:30 a.m. Four positions were randomly selected from each experimental area to measure the soil respiration. Weeds and humus on the surface were picked up during the measurement. The breathing chamber was covered in the area to be tested, and the contact edge between the breathing chamber and the soil was sealed with dry soil. We measured continuously for 15 min each time, and the measurements were recorded automatically every 30 s. The soil respiration rate was calculated according to the change of CO₂ concentration per unit time.

2.2.3. Determination of Soil Available Nutrient Content

The content of ammonium nitrogen and nitrate nitrogen in soil was determined by SAN++flow analyzer (Skalar, Beijing, China), the available phosphorus was determined by molybdenum–antimony colorimetry, and the available potassium was determined by flame photometer (WGH6400, Shanghai, China).

2.2.4. Determination of Soil Enzyme Activity

Soil Xylosidase and Glucosidase was detected by fluorescence microplate detection technology, urease was detected by phenol–sodium hypochlorite colorimetry, neutral phosphatase was detected by sodium diphenyl phosphate colorimetry, and polyphenol oxidase and catalase were detected by microplate absorption light method.

2.2.5. Determination of Soil Microorganism

On 8 July, 16 August, and 26 September, the rhizosphere soil samples of Jingke 968 chemical fertilizer treatment and 4.5 t treatment were collected. The corn rhizosphere was dug out from the soil as a whole. The soil loosely combined with the root was shaken off by shaking the soil, and then the soil closely combined with the root was brushed off with a brush to remove the roots and small stones in the soil. After passing the 2 mm sieve, the soil samples were placed in a sterile sealed bag and stored in a refrigerator at −80 °C for standby.

Soil microbial analysis analyzed the population structure of bacteria and fungi through the variable region of 16S rRNA gene V3–V4 of bacteria and ITS1 of fungi. High-throughput sequencing was conducted in Novogene Co., Ltd. (https://cn.novogene.com/, Beijing, China). Complete gene DNA extraction, PCR amplification, Miseq library construction, and Miseq sequencing were performed. The raw data obtained by sequencing was spliced and filtered to obtain the effective data. Then OTUs (Operational Taxonomic Units) clustering and species classification analyses were carried out based on valid data. According to the OTUs clustering results, the sequence of each OTU was annotated to obtain the corresponding species information and abundance distribution based on species. At the same time, the abundance A Diversity calculation, and other analyses, were performed to obtain the species richness and evenness information within the sample, common and unique OTUs information among different samples or groups, etc. Multiple sequences of OTUs were compared to explore the differences in community structure between different samples or groups.

2.3. Statistical Analysis

Excel 2016 was used to sort out the data, and SPSS 24 statistical software was used for data analysis; all data were tested for homogeneity of variance and then analyzed for one-way ANOVA (p < 0.05 is the significant difference, p < 0.01 is the extremely significant difference). At the same time, we used Origin 9.6 software to map the data.

3. Results

3.1. Soil pH Value and Soil Electrical Conductivity

Soil pH is one of the important factors affecting soil fertility, which is closely related to the transformation of soil nitrogen, phosphorus, and potassium, as well as the decomposition of organic matter. It can be seen from Figure 1 that the pH value of Xianyu 335 rhizosphere soil had no significant difference between the treatments on 8 July and 28 July; The fertilization treatments on 16 August and 4 September were significantly lower than those without fertilization. There was no significant difference between the fertilization treatments on 16 August, and the lowest was 4.5 t on 4 September, which was significantly lower than other carbon-based fertilizer treatments. On 26 September, the chemical fertilizer treatment was the highest, and the carbon-based fertilizer treatment was 3 t > 4.5 t > 5.25 t > 3.75 t. Except for the 3 t and 4.5 t treatments, the differences between the other carbon-based fertilizer treatments were significant. The pH of rhizosphere soil of Jingke 968 was not significantly different among the treatments on 8 July. On 28 July, there was no significant difference among the treatments of no fertilizer, chemical fertilizer, and carbon-based fertilizer, but there was a significant difference between 3 t and 5.25 t. On 16 August, there was no significant difference among the treatments of no fertilizer, chemical fertilizer, and 4.5 t. On 4 September and 26 September, the treatment without fertilization was the highest, and was significantly higher than other treatments, and the treatment of 3 t was significantly higher than that of chemical fertilizer treatment (Figure 1).

The soil conductivity reflects the accumulation of soil salt. It can be seen from Figure 1 that the carbon-based fertilizer significantly improved the soil conductivity. On 8 July, in Xianyu 335, the soil conductivity of the carbon-based fertilizer treatment was significantly higher than that of the non-fertilization and chemical fertilizer treatment. There was a significant difference between the carbon-based fertilizer treatment, which was 3.75 t > 4.5 t > 5.25 t > 3 t. At 28 July and 16 August, the soil conductivity of 3 t, 4.5 t, and 5.25 t treatments were significantly higher than that of non-fertilization and chemical fertilizer treatments, and there were also significant differences between carbon-based fertilizer treatments, with the 4.5 t treatment significantly higher than each treatment. On 4 September and 26 September, the 3 t treatment was significantly higher than each treatment. On 8 July, in Jingke 968, the soil conductivity of the carbon-based fertilizer treatment was significantly higher than that of non-fertilization and chemical fertilizer treatment. There was a significant difference between carbon-based fertilizer treatment, which was 3.75 t > 4.5 t > 5.25 t > 3 t. On 28 July, except for 3.75 t, the other treatments were significantly higher than those without fertilization and chemical fertilizer, and there was a significant difference between carbon-based fertilizer treatments, which was 5.25 t > 4.5 t > 3 t > 3.75 t. On 16 August and 26 September, the 5.25 t treatment was significantly higher than each treatment. On 4 September, the treatment of carbon-based fertilizer was significantly higher than that of chemical fertilizer and non-fertilizer treatment, and the treatment of 4.5 t was significantly higher than that of each treatment (Figure 1).

3.2. Soil Moisture Content and Soil Carbon Flux

It can be seen from Figure 2 that the soil water content in the rhizosphere of Xianyu 335 increased first and then decreased, and there was no significant difference between the treatments on 8 July. On 28 July, 3.75 t, 4.5 t, and 5.25 t were significantly higher than those of chemical fertilizer and non-fertilizer treatment; they were, respectively, 27.32%, 28.02% and 27.04% higher than those of the chemical fertilizer treatment. On 16 August, the soil water content increased at first and then decreased with the increase in the application amount of carbon-based fertilizer; 3.75 t, 4.5 t and 5.25 t were significantly higher in the treatment of fertilizer and no fertilizer, which were 26.75%, 34.62%, and 32.25% higher than the treatment of fertilizer, respectively. On 4 September, 3.75 t and 4.5 t fertilizer treatments were significantly higher; they were 11.95% and 12.07% higher, respectively, while the other treatments and fertilizer treatments had no significant difference. On 26 September, 3.75 t, 4.5 t, and 5.25 t were significantly higher than those of chemical fertilizer treatment; they were 23.34%, 51.99%, and 39.56% higher, respectively, and 4.5 t was significantly higher than those of each treatment. The change trend in soil water content in the rhizosphere of Jingke 968 was similar to Xianyu 335, and there was no significant difference between the treatments on 8 July. On 28 July, 3.75 t, 4.5 t, and 5.25 t were significantly higher than those of chemical fertilizer and non-fertilizer treatment; they were 18.76%, 21.76%, and 24.89% higher than those of chemical fertilizer treatment, respectively. On 16 August, the treatment of carbon-based fertilizer was significantly higher than that of chemical fertilizer and non-fertilizer treatment, and the treatment of 4.5 t and 5.25 t was significantly higher than that of each treatment. On 4 September, the soil water content gradually increased with the increase in the application amount of carbon-based fertilizer; 3.75 t, 4.5 t and 5.25 t were significantly higher in the treatment of chemical fertilizer and no fertilizer; they were 10.61%, 12.45%, and 15.37% higher than the treatment of chemical fertilizer, respectively. On 26 September, there was no significant difference between 3 t, 3.75 t, and 5.25 t and fertilizer treatments (Figure 2).

The soil carbon flux reflects the rate of carbon dioxide released by roots, soil microorganisms, soil animal respiration, and the decomposition of substances in the soil. It can be seen from Figure 2 that the soil carbon flux gradually decreases and tends to be flat with the promotion of the maize growth process. Xianyu 335 on 8 July, 3.75 t, 4.5 t, and 5.25 t were significantly higher than those of chemical fertilizer and no fertilizer treatment, with 5.25 t > 4.5 t > 3.75 t. On 28 July, 4.5 t and 5.25 t were significantly higher than those of chemical fertilizer and non-fertilizer treatment; they were 103.05% and 83.22% higher than those of chemical fertilizer treatment, respectively. On 16 August, 5.25 t was significantly higher than that of non-fertilization treatment, and there was no significant difference with fertilizer treatment. On 4 September and 26 September, 4.5 t was significantly higher than that of chemical fertilizer and non-fertilizer treatment; they were 249.83% and 68.79% higher than that of chemical fertilizer, respectively. The soil carbon flux of Jingke 968 increased at first and then decreased with the increase in the application amount of carbon-based fertilizer at each sampling date. On 8 July, 3.75 t and 4.5 t were significantly higher than those of fertilizer and no fertilizer treatments. On 28 July, 4.5 t was significantly higher than that of all treatments, which was 150.96% higher than that of chemical fertilizer. On 16 August and 4 September, 3.75 t was the highest, significantly higher than that of chemical fertilizer and no fertilizer treatment. There was no significant difference between the treatment of carbon-based fertilizer and chemical fertilizer on 26 September (Figure 2).

3.3. Nitrate Nitrogen Content in Soil

Soil available nitrogen reflects the strength of soil nitrogen supply capacity and is the main indicator of soil fertility. It can be seen from

Table 2. Effects of carbon-based fertilizer on soil nitrate nitrogen content (mg/kg).

Variety	Treatment	8 July	28 July	16 August	4 September	26 September
Xianyu335	CK1	6.54 ± 0.92 e	5.33 ± 0.46 e	5.62 ± 0.20 d	2.28 ± 0.12 d	10.47 ± 1.03 b
	CK2	17.35 ± 0.45 c	12.21 ± 0.66 d	7.32 ± 0.56 c	4.81 ± 0.09 c	10.92 ± 0.46 b
	3 t	15.27 ± 0.47 d	13.20 ± 0.31 cd	10.52 ± 0.76 b	4.94 ± 0.14 c	11.30 ± 0.66 ab
	3.75 t	17.81 ± 0.65 c	13.53 ± 0.69 c	11.17 ± 0.31 b	5.17 ± 0.08 b	11.24 ± 1.58 ab
	4.5 t	19.38 ± 0.76 b	15.55 ± 0.63 b	12.69 ± 0.64 a	5.41 ± 0.08 a	11.79 ± 0.60 ab
	5.25 t	21.27 ± 1.09 a	16.81 ± 0.67 a	10.81 ± 0.91 b	5.26 ± 0.18 ab	12.92 ± 0.40 a
Jingke968	CK1	6.35 ± 0.62 c	6.65 ± 0.83 c	4.18 ± 0.36 f	2.88 ± 0.13 c	9.67 ± 1.09 c
	CK2	14.10 ± 0.72 b	11.06 ± 2.59 b	8.43 ± 0.86 e	2.79 ± 0.08 c	12.26 ± 0.75 b
	3 t	8.54 ± 1.15 c	11.09 ± 1.01 b	10.58 ± 0.57 d	2.88 ± 0.69 c	12.85 ± 0.26 ab
	3.75 t	21.79 ± 2.46 a	11.60 ± 0.96 b	12.20 ± 0.69 b	5.05 ± 0.10 b	12.78 ± 0.48 ab
	4.5 t	23.21 ± 0.69 a	12.53 ± 0.72 ab	12.70 ± 0.50 ab	6.32 ± 0.26 a	12.70 ± 0.40 ab
	5.25 t	20.89 ± 2.85 a	14.78 ± 0.82 a	13.70 ± 0.13 a	6.81 ± 0.01 a	13.51 ± 0.16 a

Notes: Data are expressed as mean ± standard deviation. Different letters within the same column indicate significant difference at the 5% level.

that the content of nitrate nitrogen in soil decreases first and then increases with the progress of growth. Fertilization significantly increased the content of nitrate nitrogen in soil, and the effect of carbon-based fertilizer treatment was significant, showing an upward trend with the increase in application amount. Except for 26 September, the content of nitrate nitrogen in the soil of Xianyu 335 under fertilization treatment was significantly higher than that without fertilization, which was 30.17~225.27% higher. On 8 July, 4.5 t and 5.25 t were significantly higher than those of chemical fertilizer treatment; they were 11.71% and 22.61% higher, respectively. On 28 July, 3.75 t, 4.5 t, and 5.25 t were significantly higher than those of chemical fertilizers; they were 10.80%, 27.36%, and 37.64% higher, respectively. On 16 August, carbon-based fertilizer was significantly higher than chemical fertilizer, which was 43.70~73.37% higher. On 4 September, 3.75 t, 4.5 t, and 5.25 t were significantly higher than those of chemical fertilizers; they were 7.43%, 12.43%, and 9.30% higher, respectively. On 26 September, 5.25 t was significantly higher than that of non-fertilization and chemical fertilizer, which was 23.44% and 18.32% higher, respectively. There was no significant difference between carbon-based fertilizer treatments. On 8 July, in Jingke 968, except for the 3 t treatment, other treatments were significantly higher than those without fertilizer and chemical fertilizer, and 3.75 t, 4.5 t, and 5.25 t were 54.57%, 64.61%, and 48.17% higher than those of chemical fertilizer treatment, respectively. On 28 July, fertilization treatment was significantly higher than non-fertilization treatment, which was 66.27~122.28% higher, and 5.25 t was significantly higher than fertilizer treatment, which was 38.65% higher. On 16 August, fertilization treatment was significantly higher than no fertilization, and carbon-based fertilizer was significantly higher than chemical fertilizer, which was 25.46~62.51% higher. On 4 September, 3.75 t, 4.5 t, and 5.25 t were significantly higher than those of chemical fertilizer and non-fertilizer treatment; they were 81.02%, 126.40%, and 144.12% higher than those of chemical fertilizer treatment, respectively. On 26 September, fertilization treatment was significantly higher than non-fertilization treatment, which was 26.78~39.74% higher, 5.25 t was significantly higher than chemical fertilizer treatment, which was 10.22% higher, and the difference between carbon-based fertilizer treatment was not significant (Table 2).

3.4. Ammonium Nitrogen Content in Soil

It can be seen from

Table 3. Effect of carbon-based fertilizer on soil ammonium nitrogen content (mg/kg).

Variety	Treatment	8 July	28 July	16 August	4 September	26 September
Xianyu335	CK1	25.53 ± 4.36 b	3.25 ± 0.08 e	2.38 ± 0.15 e	1.14 ± 0.12 d	0.05 ± 0.01 e
	CK2	60.09 ± 1.83 a	4.86 ± 0.11 bc	2.76 ± 0.10 d	1.98 ± 0.15 c	0.07 ± 0.01 de
	3 t	60.24 ± 2.04 a	4.45 ± 0.09 d	2.92 ± 0.09 d	1.34 ± 0.09 d	0.09 ± 0.01 d
	3.75 t	60.40 ± 2.70 a	4.75 ± 0.12 c	3.17 ± 0.14 c	2.33 ± 0.11 c	0.13 ± 0.01 c
	4.5 t	63.82 ± 4.09 a	4.97 ± 0.03 b	4.86 ± 0.14 b	3.97 ± 0.59 a	0.25 ± 0.01 b
	5.25 t	62.00 ± 3.54 a	5.40 ± 0.11 a	5.12 ± 0.11 a	2.92 ± 0.19 b	0.390.03 a
Jingke968	CK1	22.24 ± 3.26 c	3.10 ± 0.25 d	2.65 ± 0.20 d	0.89 ± 0.20 e	0.05 ± 0.01 f
	CK2	63.72 ± 2.20 ab	4.20 ± 0.08 bc	3.42 ± 0.08 c	1.54 ± 0.12 d	0.07 ± 0.01 e
	3 t	58.06 ± 3.95 b	3.85 ± 0.17 c	3.29 ± 0.17 c	1.65 ± 0.06 cd	0.12 ± 0.01 d
	3.75 t	63.74 ± 1.36 ab	4.25 ± 0.32 bc	3.58 ± 0.32 bc	1.83 ± 0.12 c	0.15 ± 0.01 c
	4.5 t	64.24 ± 4.03 a	4.44 ± 0.16 b	3.89 ± 0.20 b	2.21 ± 0.11 b	0.22 ± 0.01 b
	5.25 t	64.86 ± 3.31 a	5.35 ± 0.39 a	4.38 ± 0.35 a	3.86 ± 0.17 a	0.27 ± 0.01 a

Notes: Data are expressed as mean ± standard deviation. Different letters within the same column indicate significant difference at the 5% level.

that the content of ammonium nitrogen in soil gradually decreases with the progress of growth. Fertilization significantly increased the content of ammonium nitrogen in the soil, and the effect of carbon-based fertilizer treatment was significant, showing an upward trend with the increase in application amount. Except for the 3 t treatment on 4 September and the chemical fertilizer treatment on 26 September, the soil ammonium nitrogen content of Xianyu 335 in each sampling period was significantly higher in the fertilization treatment than in the non-fertilization treatment, which was 17.81~150.00% higher. On 8 July, there was no significant difference between carbon-based fertilizer and chemical fertilizer treatment. On 28 July, 5.25 t was significantly higher than that of chemical fertilizer treatment, which was 11.15% higher. On 16 August, 3.75 t, 4.5 t, and 5.25 t were significantly higher than those of chemical fertilizers; they were 15.00%, 76.14%, and 85.44% higher, respectively. On 4 September, 4.5 t and 5.25 t were significantly higher than those of chemical fertilizer treatment, which were 100.72% and 47.33% higher, respectively. On 26 September, 3.75 t, 4.5 t, and 5.25 t were significantly higher than those of chemical fertilizers; they were 89.95%, 254.32%, and 463.00% higher, respectively. The soil ammonium nitrogen content of Jingke 968 at each sampling date was significantly higher in fertilization treatment than in non-fertilization treatment, which was 24.19~440.00% higher. On 8 July, there was no significant difference between carbon-based fertilizer and chemical fertilizer. On 28 July, 5.25 t was significantly higher than that of chemical fertilizer, which was 27.42% higher. On 16 August, 4.5 t and 5.25 t were significantly higher than those of chemical fertilizer treatment; they were 13.87% and 28.20% higher, respectively. On 4 September, 3.75 t, 4.5 t, and 5.25 t were significantly higher than those of chemical fertilizers; they were 18.61%, 43.51%, and 150.93% higher, respectively. On 26 September, carbon-based fertilizer treatment was significantly higher than chemical fertilizer treatment, which was 78.04~291.48% higher (Table 3).

3.5. Available Phosphorus Content in Soil

The soil available phosphorus reflects the soil phosphorus supply capacity and is also the direct source of plant phosphorus. It can be seen from

Table 4. Effect of carbon-based fertilizer on available phosphorus content in soil (mg/g).

Variety	Treatment	8 July	28 July	16 August	4 September	26 September
Xianyu335	CK1	31.82 ± 1.53 c	32.02 ± 1.32 c	26.76 ± 2.76 b	30.85 ± 2.64 b	33.03 ± 1.50 bc
	CK2	46.46 ± 4.55 a	42.39 ± 4.58 ab	39.03 ± 3.63 a	34.43 ± 1.79 ab	35.52 ± 2.35 bc
	3 t	36.72 ± 4.02 bc	38.12 ± 2.87 bc	35.20 ± 2.00 a	30.96 ± 3.76 b	32.18 ± 3.77 c
	3.75 t	36.36 ± 3.70 bc	38.62 ± 2.50 bc	35.49 ± 3.62 a	34.41 ± 1.36 ab	37.35 ± 1.81 ab
	4.5 t	43.72 ± 4.60 ab	41.89 ± 2.12 ab	38.48 ± 3.42 a	35.32 ± 2.75 ab	40.72 ± 2.08 a
	5.25 t	41.15 ± 4.87 ab	45.11 ± 5.04 a	40.77 ± 2.80 a	37.82 ± 3.41 a	40.14 ± 1.78 a
Jingke968	CK1	34.47 ± 1.62 d	34.69 ± 0.94 c	20.54 ± 1.04 d	26.05 ± 4.46 d	32.64 ± 4.31 b
	CK2	50.43 ± 4.53 ab	37.74 ± 1.71 c	35.81 ± 2.24 c	32.62 ± 3.77 c	37.90 ± 3.27 ab
	3 t	41.93 ± 2.28 c	42.44 ± 4.97 b	39.16 ± 3.32 bc	35.06 ± 0.73 bc	34.00 ± 3.25 b
	3.75 t	44.71 ± 3.54 bc	44.15 ± 1.86 ab	41.67 ± 3.07 ab	37.22 ± 3.61 abc	36.52 ± 2.89 ab
	4.5 t	50.70 ± 2.83 ab	46.23 ± 2.01 ab	43.56 ± 1.81 ab	40.27 ± 2.92 ab	37.32 ± 2.28 ab
	5.25 t	54.71 ± 5.62 a	48.22 ± 2.12 a	44.13 ± 2.51 a	42.50 ± 2.30 a	42.44 ± 2.73 a

Notes: Data are expressed as mean ± standard deviation. Different letters within the same column indicate significant difference at the 5% level.

that fertilization significantly increased the content of soil available phosphorus. The content of available phosphorus in Xianyu 335 soil on 8 July was significantly higher than that without fertilization, which was 15.40~46.00% higher, and the difference between 4.5 t, 5.25 t, and fertilizer was not significant. On 28 July, 4.5 t, 5.25 t, and fertilizer treatments were significantly higher than those without fertilizer, and the difference between fertilizer treatments was not significant. On 16 August, fertilization treatment was significantly higher than no fertilization, and there was no significant difference between carbon-based fertilizer and chemical fertilizer treatment. On 4 September, the 5.25 t treatment was significantly higher than that without fertilization, and the difference between other treatments and fertilizer was not significant. On 26 September, 4.5 t and 5.25 t were significantly higher than those without fertilizer and chemical fertilizer; they were 14.64% and 13.00% higher, respectively, than those of chemical fertilizer. The content of available phosphorus in the soil of Jingke 968 showed a gradual increase with the increase in the application amount of carbon-based fertilizer at each sampling date. On 8 July, fertilization treatment was significantly higher than non-fertilization treatment, which was 21.64~58.71% higher. There was no significant difference between the 4.5 t and 5.25 t treatments and chemical fertilizer. On 28 July, the treatment of carbon-based fertilizer was significantly higher than that of non-fertilization and chemical fertilizer, which was 12.47~27.77% higher than that of chemical fertilizer. On 16 August, fertilization treatment was significantly higher than non-fertilization treatment; 3.75 t, 4.5 t, and 5.25 t were significantly higher than chemical fertilizer treatment, which were 9.35%, 16.36%, and 23.25% higher, respectively. On 4 September, 4.5 t and 5.25 t were significantly higher than those of chemical fertilizers; they were 23.45% and 30.30% higher, respectively. On 26 September, 5.25 t was significantly higher than that without fertilization, and there was no significant difference between carbon-based fertilizer and chemical fertilizer treatment (Table 4).

3.6. Soil Available Potassium Content

Soil available potassium is an important indicator to judge the abundance and deficiency of soil potassium. According to

Table 5. Effects of carbon-based fertilizer on soil available potassium content (mg/g).

Variety	Treatment	8 July	28 July	16 August	4 September	26 September
Xianyu335	CK1	181.47 ± 2.35 c	146.16 ± 4.71 d	144.80 ± 6.22 b	143.44 ± 0.00 c	120.35 ± 2.35 d
	CK2	248.03 ± 2.35 b	155.66 ± 7.07 d	140.72 ± 2.35 b	150.23 ± 2.35 c	135.29 ± 4.07 c
	3 t	184.19 ± 4.07 c	181.47 ± 10.26 c	131.21 ± 4.07 b	151.59 ± 0.00 c	133.93 ± 2.35 c
	3.75 t	239.88 ± 2.35 b	208.64 ± 7.06 b	147.51 ± 4.07 b	152.95 ± 2.35 c	138.01 ± 2.35 c
	4.5 t	291.50 ± 18.82 a	222.22 ± 6.22 a	195.06 ± 2.35 a	176.04 ± 4.07 b	152.95 ± 2.35 b
	5.25 t	292.86 ± 6.22 a	223.58 ± 8.48 a	180.11 ± 24.79 a	201.85 ± 26.20 a	262.97 ± 2.35 a
Jingke968	CK1	181.47 ± 2.35 d	146.16 ± 2.35 d	139.36 ± 7.06 b	161.10 ± 6.22 cd	114.91 ± 4.07 d
	CK2	195.06 ± 2.35 d	169.25 ± 13.10 c	120.35 ± 11.76 c	161.10 ± 2.35 cd	140.72 ± 2.35 c
	3 t	211.36 ± 2.35 c	205.92 ± 9.41 b	161.10 ± 9.41 a	157.02 ± 2.35 d	139.36 ± 4.07 c
	3.75 t	261.61 ± 4.07 b	214.07 ± 2.35 ab	167.89 ± 0.00 a	173.32 ± 10.26 bc	150.23 ± 13.10 c
	4.5 t	262.97 ± 12.45 b	219.51 ± 8.48 ab	176.04 ± 4.07 a	180.11 ± 10.78 ab	176.04 ± 4.07 b
	5.25 t	284.71 ± 16.97 a	222.22 ± 8.48 a	166.53 ± 13.10 a	186.91 ± 6.22 a	216.79 ± 4.07 a

Notes: Data are expressed as mean ± standard deviation. Different letters within the same column indicate significant difference at the 5% level.

, fertilization significantly increased the content of soil available potassium. Except for the 5.25 t treatment on 16 August, the content of soil available potassium at each sampling date showed a trend of increasing with the increase in the amount of carbon-based fertilizer. On 8 July, Xianyu 335, 4.5 t and 5.25 t were significantly higher than those of chemical fertilizer and non-fertilizer treatments; they were 17.52% and 18.07% higher than those of chemical fertilizer, respectively. On 28 July, carbon-based fertilizer treatment was significantly higher than chemical fertilizer and non-fertilizer treatment, which was 16.58~43.63% higher than chemical fertilizer treatment. On 16 August and 4 September, 4.5 t and 5.25 t were significantly higher than those of chemical fertilizer and non-fertilizer treatments, and there was no significant difference among other treatments. On 26 September, fertilization treatment was significantly higher than that without fertilization, 4.5 t and 5.25 t were significantly higher than that of chemical fertilizer, which were 13.05% and 94.38% higher, respectively. On 8 July, 28 July and 16 August, the treatment of carbon-based fertilizer was significantly higher than that of chemical fertilizer and non-fertilizer treatment, which was 8.35~45.96% higher than that of chemical fertilizer treatment on 8 July, 15.80~31.30% higher on 28 July, and 33.86~38.37% higher on 16 August. On 4 September and 26 September, 4.5 t and 5.25 t were significantly higher than those of chemical fertilizer and non-fertilizer treatment; they were 11.80% and 16.02% higher than those of chemical fertilizer treatment on 4 September and 25.10% and 54.06% higher on 26 September, respectively (Table 5).

3.7. Soil Neutral Phosphatase Activity, Soil Urease Activity and Soil Β-Glucosidase Activity

Soil phosphatase can hydrolyze phosphate ester to produce orthophosphate, and its activity directly affects the decomposition and transformation of soil organic phosphorus. It can be seen from Figure 3 that Xianyu 335 had no significant difference between carbon-based fertilizer and chemical fertilizer treatment on 8 July, 28 July, and 16 August. On 4 September, 5.25 t was significantly higher than other treatments, and there was no significant difference among other treatments. On 26 September, 4.5 t and 5.25 t were significantly higher than those of chemical fertilizer and non-fertilizer treatments; they were 28.65% and 78.53% higher than those of chemical fertilizer, respectively. On 8 July, Jingke 968 had no significant difference between other treatments and chemical fertilizers except 4.5 t. On 28 July, there was no significant difference between carbon-based fertilizer treatment and chemical fertilizer treatment. On 16 August, 4.5 t and 5.25 t were significantly higher than those of chemical fertilizer treatment; they were 19.49% and 39.83% higher, respectively. There was no significant difference between other treatments. On 4 September, 4.5 t was significantly higher than that of chemical fertilizer and no fertilizer; they were 15.44% and 24.60% higher, respectively, and 3.75 t, 5.25 t and chemical fertilizer were not significantly different. On 26 September, carbon-based fertilizer treatment was higher than chemical fertilizer treatment, with no significant difference (Figure 3).

Soil urease can catalyze urea to produce ammonia, which can reflect the soil fertility level to a certain extent. It can be seen from Figure 3, Figure 4, Figure 5 and Figure 6 that on 8 July, the treatment of Xianyu 335 with 3.75 t was significantly higher than that with chemical fertilizer and no fertilizer, and the difference between the treatment of 3 t and chemical fertilizer was not significant. On 28 July, there was no significant difference between 3 t treatment and chemical fertilizer. On 16 August, the treatment of carbon-based fertilizer was significantly higher than that of chemical fertilizer; they were 13.67%, 34.36%, 37.83%, and 38.07% higher, respectively. On 4 September, there was no significant difference among the treatments. On 26 September, there was no significant difference between 3 t and fertilizer treatment. On 8 July, Jingke 968, 3 t was significantly higher than chemical fertilizer treatment, which was 12.29% higher, and 4.5 t and 5.25 t were significantly lower than chemical fertilizer treatment. On 28 July, there was no significant difference between other treatments and fertilizer except 3.75 tons. On 16 August and 4 September, the soil urease activity gradually increased with the increase in the application amount of carbon-based fertilizer. On 16 August, 4.5 t and 5.25 t were significantly higher than chemical fertilizer; they were 23.65% and 29.44% higher, respectively. On 4 September, 4.5 t and 5.25 t were significantly higher than chemical fertilizer; they were 12.29% and 14.59% higher, respectively. On 26 September, urease activity gradually decreased with the increase in the application amount of carbon-based fertilizer, and there was no significant difference between 3 t, 3.75 t, and chemical fertilizer (Figure 3).

β-Glucosidase participates in soil carbon cycle, and its hydrolysate is the main nutrient source of soil microorganisms. It can be seen from Figure 3 that the soil β-Glucosidase activity increased first and then decreased with the advancement of the growth process. In the 4.5 t and 5.25 t treated soil of Xianyu 335, β-Glucosidase activity reached its maximum on 28 July, and other treatments reached its maximum on 16 August. On 8 July, the enzyme activity increased first and then decreased with the increase in the application amount of carbon-based fertilizer. The activity of 3.75 t was the highest, which was 398.15% higher than that of chemical fertilizer. On 28 July, the carbon-based fertilizer treatment was significantly higher than the chemical fertilizer treatment, with the highest activity of 4.5 t, which was 165.98% higher than the chemical fertilizer treatment. On 16 August, the enzyme activity gradually decreased with the increase in the amount of carbon-based fertilizer application, and 3 t and 3.75 t were significantly higher than that of chemical fertilizer treatment, which were 100.45% and 33.82% higher, respectively. On 4 September, there was no significant difference between carbon-based fertilizer and chemical fertilizer treatment. The change trend on 26 September was similar to that on 8 July, and 3.75 t was significantly higher than that of chemical fertilizer by 45.85%. On 8 July, 3.75 t of Jingke 968 was significantly higher than that of chemical fertilizer and non-fertilizer treatment, which was 156.04% higher than that of chemical fertilizer. On 28 July, fertilization treatment was significantly higher than non-fertilization treatment, and there was no significant difference between carbon-based fertilizer and chemical fertilizer treatment. On 16 August, 3 t and 3.75 t were significantly higher than those of chemical fertilizer and no fertilizer treatment; they were 126.58% and 293.11% higher than those of chemical fertilizer, respectively. On 4 September, 3.75 was 33.78% higher than chemical fertilizer, and there was no significant difference between other carbon-based fertilizer treatments and chemical fertilizer. On 26 September, 3.75 t and 5.25 t treatments were significantly higher than other treatments; they were 54.48% and 33.86% higher than chemical fertilizers, respectively (Figure 3).

3.8. Soil α-Xylosidase Activity, Soil Polyphenol Oxidase Activity and Soil Catalase Activity

α-Xylosidase is also an important enzyme involved in the hydrolysis of carbon-containing organic matter, and plays a very important role in the transformation of soil carbon. It can be seen from Figure 4 that, in the two varieties of soil, α-Xylosidase activity was consistent, and the change trend of xylosidase activity increased first and then decreased with the advancement of the growth process, reaching the maximum value on 16 August. In Xianyu 335, on 8 July, the treatment of carbon-based fertilizer was significantly higher than that of chemical fertilizer and no fertilizer, with 3.75 t being the highest, which was 1058.87% higher than that of chemical fertilizer. On 28 July, 3.75 t, 4.5 t, and 5.25 t were significantly higher than those of chemical fertilizer and non-fertilizer treatments They were119.96%, 172.23%, and 140.59% higher than those of chemical fertilizer, respectively. On 16 August, 3 t and 3.75 t were the highest, significantly higher than other treatments; they were 113.21% and 193.02% higher than chemical fertilizer, respectively. On 4 September, 3.75 t was significantly higher than that of chemical fertilizer and non-fertilizer treatment, which was 82.69% higher than that of chemical fertilizer. There was no significant difference among the treatments on 26 September. On 8 July, in Jingke 968, 3.75 t was significantly higher than other treatments, which was 138.29% higher than chemical fertilizer. On 28 July, 3 t was significantly higher than that of chemical fertilizer treatment. On 16 August, the enzyme activity increased at first and then decreased with the increase in the application amount of carbon-based fertilizer. The 3.75 t treatment reached the maximum value, which was significantly higher than other treatments and 193.02% higher than chemical fertilizer. On 4 September, 3.75 t and 4.5 t were significantly higher than those of chemical fertilizer and non-fertilizer treatments; they were 60.91% and 32.59% higher than those of chemical fertilizer, respectively. There was no significant difference between the treatments on 26 September (Figure 4).

Soil polyphenol oxidase can promote the oxidation and decomposition of phenols in soil to produce quinones, which is an important oxidase in the process of humic acid synthesis. It can be seen from Figure 4 that the change trend of polyphenol oxidase activity of the two varieties was similar. From 8 July to 16 August, the change range was small. The activity increased rapidly on 4 September and decreased slightly on 26 September. Xianyu 335 had no significant difference between the treatment of 3 t, 5.25 t, and chemical fertilizer on 8 July. On 28 July, there was no significant difference between 4.5 t, 5.25 t, and chemical fertilizer treatments. On 16 August, 3 t, 4.5 t, and 5.25 t were significantly higher than that of chemical fertilizer; they were 36.81%, 28.28%, and 43.51% higher, respectively. On 4 September and 26 September, the enzyme activity gradually increased with the increase in the application amount of carbon-based fertilizer. On 4 September, 4.5 t and 5.25 t were significantly higher than that of chemical fertilizer; they were 15.12% and 20.85% higher, respectively. On 26 September, the enzyme activity of carbon-based fertilizer treatment was significantly higher than that of chemical fertilizer treatment. On 8 July, the activity of soil polyphenol oxidase in Jingke 968 increased gradually with the increase in the amount of carbon-based fertilizer; 3.75 t, 4.5 t, and 5.25 t were significantly higher than that of chemical fertilizer treatment, which were 24.75%, 24.20%, and 29.12% higher, respectively. On 28 July, there was no significant difference between other treatments and fertilizer except 4.5 t treatment. On 16 August, the 3 t treatment was significantly higher than chemical fertilizer, which was 30.27% higher. On 4 September and 26 September, 5.25 t was significantly higher than that of each treatment; they were 45.16% and 106.77% higher than that of chemical fertilizer, respectively. There was no significant difference between other treatment and chemical fertilizer (Figure 4).

Soil catalase, which can catalyze hydrogen peroxide to produce oxygen and water, is one of the enzymes involved in humic acid synthesis. It can be seen from Figure 4 that the soil catalase activity increases first and then decreases with the advancement of the growth process. On 8 July and 28 July, the catalase activity of Xianyu 335 decreased first and then increased with the increase in the application amount of carbon-based fertilizer. On 8 July, 5.25 t treatment was the highest, which was 24.99% higher than that of chemical fertilizer. On 28 July, the enzyme activity of carbon-based fertilizer and chemical fertilizer treatment was not significantly different except 4.5 t treatment. On 16 August, the treatment of 3 t and 5.25 t was significantly higher than that of chemical fertilizer; they were 26.69% and 11.10% higher respectively. On 4 September, the enzyme activity decreased slightly at first and then increased gradually with the increase in the application amount of carbon-based fertilizer. There was no significant difference between the treatment of carbon-based fertilizer and chemical fertilizer. On 26 September, the enzyme activity gradually increased with the increase in the application amount of carbon-based fertilizer, and 5.25 t was significantly higher than that of chemical fertilizer, which was 32.97% higher. The catalase activity of Jingke 968 showed a gradual upward trend with the increase in the application amount of carbon-based fertilizer on 8 July, 4 September, and 26 September, and a first downward and then upward trend with the increase in the application amount of carbon-based fertilizer on 28 July and 16 August. On 28 July, 5.25 t was significantly higher than that of chemical fertilizer by 16.91%. On 16 August, 3 t and 5.25 t were significantly higher than those of chemical fertilizer and non-fertilizer treatments; they were 19.48% and 29.37% higher than those of chemical fertilizer, respectively; The enzyme activity of 5.25 t treatment on 26 September was significantly higher than that of other treatments, which was 29.88% higher than that of chemical fertilizer (Figure 4).

3.9. Soil Bacteria

The higher the Shannon index, Simpson index, ACE index, and chao1 index, the higher the species diversity. According to the sequencing results of bacteria,

Table 6. α-diversity of soil bacteria treated with carbon-based fertilizer and chemical fertilizer.

Time	Treatment	Coverage Rate (%)	Shannon Index	Simpson Index	ACE Index	Chao1 Index
8 July	chemical fertilizer	98.87 ± 0.15 a	9.29 ± 0.06 b	0.99 ± 0.007 a	2561.14 ± 51.63 c	2503.36 ± 103.72 c
	carbon-based fertilizer	98.57 ± 0.32 ab	9.67 ± 0.15 a	0.99 ± 0.001 a	3040.47 ± 241.41 b	2835.87 ± 13.5 b
16 August	chemical fertilizer	98.47 ± 0.47 ab	9.44 ± 0.06 b	0.99 ± 0.004 a	3236.90 ± 81.05 ab	3330.97 ± 108.11 a
	carbon-based fertilizer	98.20 ± 0.10 b	9.57 ± 0.21 ab	0.99 ± 0.004 a	3168.98 ± 214.67 b	3223.84 ± 219.93 a
26 August	chemical fertilizer	98.30 ± 0.17 ab	9.50 ± 0.29 b	0.99 ± 0.002 a	3205.54 ± 57.34 b	3193.91 ± 211.57 a
	carbon-based fertilizer	98.43 ± 0.35 ab	9.73 ± 0.01 a	0.99 ± 0.004 a	3364.56 ± 93.67 a	3325.49 ± 184.80 a

Notes: Data are expressed as mean ± standard deviation. Different letters within the same column indicate significant difference at the 5% level.

shows the various indicators of bacterial community diversity. The result analysis shows that the coverage rate of the established bacterial library is above 98%, indicating that the established library can truly reflect the bacterial diversity of the sample environment. The aromatic index of carbon-based fertilizer treatment was higher than that of chemical fertilizer treatment on three sampling dates, indicating that the level of bacterial community diversity of carbon-based fertilizer treatment was higher; it was 4.42% higher than that of chemical fertilizer treatment on 8 July, and the difference was not significant on 16 August and 26 September. There was no significant difference between Simpson index treatments. The ACE index of carbon-based fertilizer treatment was significantly higher than that of chemical fertilizer treatment on 8 July and 26 September; it was 18.71% and 4.96% higher respectively. The chao1 index was significantly higher than that of chemical fertilizer treatment on 8 July, which was 13.28% higher, and there was no significant difference between 16 August and 26 September (Table 6).

As can be seen from Figure 5, in the rhizosphere soil, the main bacterial phyla (abundance >1%) were Proteobacteria, Actinobacteria, Acidobacteria, Bacteroides, Germatimonadetes, Chloroflexi, Verrucomicrobia, Thaumarchaeota, and Nitrospirae. The total abundance of the above rhizosphere bacteria was more than 70%. On 8 July, the relative abundance of Proteobacteria, Actinobacteria, Chloroflexi, and Thaumarchaeota treated with carbon-based fertilizer was lower than that of chemical fertilizer; they were 10.34%, 59.14%, 10.19% and 37.62%, respectively. However, the relative abundance of Acidobacterium, Bacteroidetes, Gemmatimonadetes, Verrucomicrosbia, and Nitrospirae was higher than that of chemical fertilizers. On 16 August, the relative abundance of Proteobacteria, Actinobacteria, and Bacteroidetes treated with carbon-based fertilizer was lower than that of chemical fertilizer; they were 9.79%, 12.06% and 8.47%, respectively; However, the relative abundance of Acidobacterium, Gemmatimonadetes, Chloroflexi, Verrucomicrobia, Thaumarchaeota, and Nitrospirae were higher than that of chemical fertilizer treatment; they were 18.90%, 27.81%, 14.50%, 27.61%, 292.64%, and 38.26%, respectively. On 26 September, the relative abundance of Actinobacia, Acidobacterium, Chloroflexi, Thaumarchaeota, and Nitrospirae treated with carbon-based fertilizer were lower than that of chemical fertilizer; they were 40.20%, 0.26%, 21.77%, 205.80%, and 42.17% respectively. However, the relative abundance of Proteobacteria, Bacteroides, Gemmatimonadetes, and Verrucomicrosbia was higher than that of chemical fertilizer treatment; they were 8.75%, 1.27%, 15.55%, and 40.97% respectively (Figure 5).

3.10. Soil Fungi

According to the sequencing results of fungi,

Table 7. α diversity of soil fungi treated with carbon-based fertilizer and chemical fertilizer.

Time	Treatment	Coverage Rate (%)	Shannon Index	Simpson Index	ACE Index	Chao1 Index
8 July	chemical fertilizer	99.57 ± 0.12 a	7.59 ± 0.12 a	0.99 ± 0.002 a	1320.73 ± 145.65 a	1465.83 ± 360.16 a
	carbon-based fertilizer	99.63 ± 0.12 a	7.60 ± 0.57 a	0.98 ± 0.010 a	1390.12 ± 23.67 a	1381.76 ± 214.98 a
16 August	chemical fertilizer	99.63 ± 0.06 a	7.01 ± 0.70 a	0.95 ± 0.340 a	1355.14 ± 95.21 a	1330.47 ± 67.41 a
	carbon-based fertilizer	99.63 ± 0.06 a	7.36 ± 0.44 a	0.98 ± 0.007 a	1348.64 ± 93.45 a	1318.02 ± 108.32 a
26 September	chemical fertilizer	99.67 ± 0.06 a	6.75 ± 1.25 a	0.96 ± 0.030 a	1227.89 ± 304.54 a	1194.38 ± 320.00 a
	carbon-based fertilizer	99.60 ± 0.10 a	7.01 ± 0.70 a	0.97 ± 0.014 a	1300.02 ± 31.88 a	1259.88 ± 59.06 a

Notes: Data are expressed as mean ± standard deviation. Different letters within the same column indicate significant difference at the 5% level.

shows various indicators of fungal community diversity. It can be seen from the table that the coverage rate of the established fungal library is above 99.5%, which can reflect the fungal diversity of the sample environment. Among them, there was no significant difference in the aromatic index, Simpson index, ACE index, and chao1 index of carbon-based fertilizer and chemical fertilizer treatment on 8 July, 16 August, and 26 September. It shows that carbon-based fertilizer has no significant effect on the diversity of fungi in rhizosphere soil (Table 7).

As can be seen from Figure 6, in the rhizosphere soil and the species richness of fungi in the top 10 at the gate level gradually increased with the progress of the growth process, and the total abundance of dominant fungi in the top 10 of carbon-based fertilizer treatment on 8 July and 26 September was higher than that of chemical fertilizer treatment. The main mycophyla (abundance >1%) were Ascomycota, Mortierellomycota, Basidiomycota, Chytridiomycota, Aphelidiomycota, Glomeromycota, Olpidiomycota, Mucoromycota, Blastocladiomycota, and Rozellomycota. On 8 July, the abundance of Basidiomycotta, Olpidiomycota, Mucor mycotta, and Blastocladiomycotta treated with carbon-based fertilizer was 49.02%, 241.39%, 30.86%, and 933.33% lower than that of chemical fertilizer, respectively. However, the abundance of Ascomycota, Mortierellomycota, Chytridiomycota, Aphelidiomycota, Glomeromycota and Rozellomycota was higher than that of chemical fertilizer; they were 18.00%, 22.42%, 65.51%, 36.77%, 169.75%, and 2.16%, respectively. On 16 August, the abundance of Ascomycota, Chytridiomycota and Olpidiomycota treated with carbon-based fertilizer was lower than that of chemical fertilizer; they were 7.86%, 64.19%, and 7.33%, respectively. However, the abundance of Mortierellomycota, Basidiomycotta, Aphelidiomycotta, Glomeromycota, Mucoromycota, Blastocladiomycotta, and Rozellomycota was higher than that of chemical fertilizers; they were 23.53%, 21.61%, 184.76%, 72.60%, 219.23%, 7935.71%, and 35.87%, respectively. On 26 September, the abundance of Aphelidiomycota, Glomeromycota, Mucoromycota, and Rozellomycota treated with carbon-based fertilizer was 10,507.59%, 477.90%, 239.31%, and 158.53% lower than that of chemical fertilizer. However, the abundance of Ascomycota, Mortierellomycota, Basidiomycota, Chyridiomycota, Olpidiomycota and Blastocladiomycota was higher than that of chemical fertilizers, which are 13.23%, 63.30%, 30.66%, 1.67%, 19.33%, and 208.65% higher, respectively (Figure 6).

4. Discussion

Many studies have shown that biochar is a good soil conditioner [38,39,40], and carbon-based fertilizer has the essential characteristics and functions of biochar. Therefore, the application of carbon-based fertilizer can effectively improve soil’s physical and chemical properties. Soil pH reflects the active acidity of soil solution and is a critical strength index to measure the soil acidity and alkalinity. There are different research results on the effect of carbon-based fertilizer on soil pH. Some studies show that the soil pH increased by 0.03~0.14 when applying carbon-based fertilizer, and some studies also believe that the application of carbon-based fertilizer significantly reduces the soil pH [41,42]. This study showed that from 8 July to 16 August, there was no significant difference in the soil pH value between carbon-based fertilizer and chemical fertilizer treatment. After 4 September, the soil pH value of carbon-based fertilizer treatment was significantly reduced. The main reason for the decrease in pH value may be the effect of plant root and soil mineralized ion exchange, root exudates, and soil microbial metabolism. Some studies also believe that the change in soil pH value caused by carbon-based fertilizer may be related to the production conditions, such as material and temperature during the processing of carbon-based fertilizer.

There is a close relationship between soil conductivity, soil salt, and water content. Research shows that carbon-based fertilizer can significantly improve soil conductivity [43,44]. Consistent with the results of this study, carbon-based fertilizer treatment significantly increased soil conductivity, on the one hand, because carbon-based fertilizer contains a large number of soluble K, Ga, Mg, and other elements, increasing the concentration of soil mineral ions. On the other hand, because biochar has rich functional groups and an aromatic ring structure, it increases the ion exchange sites, thus improving the surface activity, and thus increasing the soil conductivity. The soil conductivity is higher in the early stage of growth and lower in the later stage, mainly due to soil mineralization, runoff, and infiltration with rainfall. There is a large difference in soil conductivity between the two varieties. Xianyu 335 has a high conductivity when the application rate is low, while Jingke 968 has a high conductivity, which may be due to the different absorption capacity and amount of soil ions between the two varieties. It is also possible that the root exudates of Xianyu 335 can enrich some ions when the application rate is high, limiting ions’ diffusion and migration, thus reducing the soil conductivity. In this study, carbon-based fertilizer significantly increased the soil water content on 28 July, 16 August, and 4 September, which may be due to the water storage capacity of carbon-based fertilizer, which can retain a part of soil water and reduce evaporation, to improve the soil water content.

Soil carbon flux is an important indicator of soil fertility and quality, closely related to soil nutrient supply and ecosystem productivity [45]. Soil carbon flux is composed of autotrophic respiration and heterotrophic respiration, mainly from plant underground roots, soil microorganisms, and soil animals, and is affected by biological and abiotic factors. Fertilization has a significant effect on soil carbon flux. Previous studies have shown that long-term use of organic fertilizer can improve soil organic carbon content, increase soil carbon flux, improve farmland productivity and promote sustainable development [46]. This study showed that the soil carbon flux of carbon-based fertilizer treatment was significantly higher than that of chemical fertilizer and non-fertilizer treatment on 8 July, mainly because a large amount of organic matter in the carbon-based fertilizer produced a large amount of carbon dioxide during the decomposition process, followed by the decomposition of carbon-based fertilizer, which promoted the growth and reproduction of microorganisms, increased the number of soil microorganisms, and increased metabolic activities, thus strengthening the respiration and increasing the number of roots and biomass. On 8 July and 28 July, the carbon flux was high, and after 16 August, the carbon flux was low and changed little, mainly because the organic materials in the carbon-based fertilizer were gradually decomposed, and the decomposition capacity was gradually reduced and kept at a certain level. In addition, it may be due to active soil organisms and strong root activity in the early stage, which gradually weakened in the later stage. Therefore, the carbon dioxide generated is relatively high in the early stage and gradually decreased in the later stage.

Biochar has stable physical and chemical properties, rich pore structure, and good adsorbability. It can promote carbon fixation in soil, improve soil cation exchange capacity, and absorb more nutrient ions. Biochar mainly produces exchangeable compounds to absorb nutrients [47,48]. Therefore, carbon-based fertilizer can use its super adsorption capacity to adsorb the nutrients crops need in the soil around the fertilizer particles. In addition, its porous structure can play the role of water-retaining fertilizer and slow-release nutrient, and can still release nutrient elements at the later stage of plant growth, reducing soil nutrient leaching, thus improving soil fertility and fertilizer utilization efficiency [49]. Some studies have shown that applying carbon-based fertilizer into the soil can still play the role of soil conditioner after the release of nutrients, and can effectively reduce the loss of nitrogen, phosphorus, and other nutrients in farmland soil [50]. Carbon-based urea has significantly improved the utilization rate of nitrogen fertilizer, reduced nitrate nitrogen leakage into the deep soil, and reduced the risk of nitrogen leaching and groundwater pollution [51]. The results of this study showed that the application of carbon-based fertilizer significantly increased the content of nitrate nitrogen, ammonium nitrogen, available phosphorus, and available potassium in the soil. At the later stage of growth, the treatment of carbon-based fertilizer was still significantly higher than that of chemical fertilizer, indicating that carbon-based fertilizer had a particular slow-release effect on nutrients.

Soil enzyme is an important indicator reflecting soil biological activity and soil fertility, which is conducive to promoting the activation and renewal of soil nutrients [52]. Catalytic reactions occur on the surface of microbial cells, plant roots, and soil particles, participate in the decomposition and transformation of soil nutrients, and catalyze the soil’s complex organic matter and minerals. Some studies believe that carbon-based fertilizer affects soil enzyme activity by promoting microbial activity, improving soil physical and chemical properties and soil nutrients [53,54]. Carbon-based fertilizer provides a sufficient nitrogen source for microorganisms, improves the soil microenvironment, is conducive to the growth of soil animals and microorganisms, accelerates the decomposition of soil organic matter, and provides a sufficient substrate for soil enzymatic reaction [55].

Urease can catalyze the hydrolysis of urea, which is of great significance in improving the nitrogen utilization rate [56]. Phosphatase can accelerate the dephosphorization rate of organic phosphorus, which plays a vital role in soil phosphorus availability [57]. The results of this study showed that the carbon-based fertilizer had no significant effect on urease and neutral phosphatase in the early stage, and the urease neutral phosphatase activity of the carbon-based fertilizer treatment increased significantly in the late stage, possibly because the carbon-based fertilizer had little effect on the mineralization of soil nitrogen and phosphorus in the early stage, and the biological activity increased and the decomposition rate accelerated in the late stage, and thus the activity increased. α- Xylosidase and β- Glucosidase activity is related to organic carbon decomposition and participates in the hydrolysis of carbon-containing organic matter such as cellulose. Its hydrolysate is the primary nutrient source of soil microorganisms [58]. In this study, carbon-based fertilizer treatment significantly improved soil α- Xylosidase and β- Glucosidase activity, mainly because carbon-based fertilizer provides rich soil enzyme substrates [59]. In this study, carbon-based fertilizer significantly increased the activity of soil polyphenol oxidase and catalase. The activity of polyphenol oxidase was lower in the early stage and increased in the later stage, which may be due to the more organic matter provided by carbon-based fertilizer in the early stage, the higher soil carbon content, the fewer extracellular enzymes released by microorganisms, and the gradual increase in enzymes released in the later stage. On the other hand, carbon-based fertilizer promotes the metabolic activity of soil microorganisms and secretes a large number of extracellular enzymes. The different change trend of soil enzymes in the whole growth period may be related to soil microorganisms. The abundance and metabolic activity of soil microorganisms secreting extracellular enzymes are the main factors affecting the activity of soil enzymes. In addition, the release of extracellular enzymes will also be affected by the influence of temperature, water, and other field conditions at different growth periods of microorganisms.

Soil microorganisms are an essential component of the soil ecosystem and a key factor in maintaining soil material cycle and nutrient balance [60]. The functional diversity of soil microorganisms is closely related to the soil environment. Therefore, the functional diversity of microbial communities is also the evaluation standard of the soil environmental system [61]. The distribution of nutrients such as carbon and nitrogen in the soil determines the type and distribution of microorganisms, and the active products of microorganisms also affect the soil’s nutrient structure and fertility level [62]. The influence of carbon-based fertilizer on microorganisms is mainly in two aspects. One is that carbon-based fertilizer provides nutrients for microorganisms, creates a good living and breeding environment, stimulates the activities of microorganisms, and indirectly affects microbial communities through adsorption, retention of nutrients, and improvement of soil properties. The other is that the porous microstructure and large specific surface area of carbon-based fertilizer itself provide a suitable growth environment for microorganisms [63]. Bacteria and fungi are the most abundant microorganisms in the soil. Many studies have shown that soil nutrient conditions have a noticeable effect on bacteria and fungi [64,65,66,67]. Previous studies have shown that, under different fertilization treatments, the metabolic activity of soil microorganisms varies greatly, and the diversity, evenness, and richness of bacteria are significantly correlated with fertilizer types [68]. Both organic fertilizer and compound microbial fertilizer improved the diversity of soil fungi and bacteria population [69].

The results of this study showed that the soil bacterial Shannon index, ACE index, and Chao index of the carbon-based fertilizer treatment were significantly higher than those of the chemical fertilizer treatment on 8 July, while the soil fungal diversity index had no significant difference, indicating that the carbon-based fertilizer treatment significantly improved the soil bacterial structure diversity, and had no significant impact on the fungal structure diversity. At the gate level, the relative abundance of bacteria is slightly different between the carbon-based fertilizer and the chemical fertilizer treatments, among which Proteus is the dominant strain, and the relative abundance is the highest in the three periods, consistent with other research results. In the whole growth period, the blastomonas and verruca microflora in the carbon-based fertilizer treatment were higher than those in the chemical fertilizer treatment, and the nitrifying spirochetes were significantly higher than those in the chemical fertilizer treatment on 8 July and 16 August. At the gate level, the relative abundance of the dominant species of fungi in the carbon-based fertilizer treatment was higher than that in the chemical fertilizer treatment on 8 July and 26 September, the flagella and rhodiola in the whole growth period were higher than that in the chemical fertilizer treatment, and the ascomycetes and ampullaria were higher than that in the chemical fertilizer treatment on 8 July and 26 September. Soil microorganisms and soil enzymes are independent and interact with each other. Microbes are one of the main sources of soil enzymes. Soil enzymes promote the transformation of soil nutrients and provide nutrients and breeding environments for microorganisms. Fertilizers, microorganisms, and soil enzymes promote and interact with each other in the soil, providing good conditions for plant growth. The effect of the carbon-based fertilizer on the species abundance of bacteria and fungi was mainly due to the fact that the carbon-based fertilizer can improve the soil nutrient status and the living environment of microorganisms. It may be because carbon-based fertilizer has a high carbon content and contains a large amount of trace elements.

5. Conclusions

Carbon-based fertilizer treatment reduced the soil pH value in the later stage and significantly impacted soil carbon flux, and showed a gradual decline trend with the advancement of the growth process. Carbon-based fertilizer treatment significantly increased the content of nitrate nitrogen, ammonium nitrogen, available phosphorus, and available potassium in soil. Carbon-based fertilizer significantly increased the activities of soil urease and neutral phosphatase in the late growth stage, and significantly increased the activities of soil urease and neutral phosphatase in the middle growth stage. β-Xylosidase and α-Glucosidase activity was significantly higher than that of chemical fertilizer treatment at 3.75 t treatment. Carbon-based fertilizer increased the activities of phenoloxidase and catalase in late growth stage. Carbon-based fertilizer significantly improved the diversity and abundance of soil bacteria on 8 July. The effect of carbon-based fertilizer on the diversity of fungi was not significant, but it increased the abundance of fungi. Our study explored the characteristics of rhizosphere microenvironment changes after carbon-based fertilizer treatment, revealed the mechanism of the effect of carbon-based fertilizer on maize growth and the soil environment, and provided a theoretical basis and a technical reference for the rational application of carbon-based fertilizer in maize production in the future.