The Response of Soil Organic Nitrogen to the Application of Green Manure Mixed with Phosphate Fertilizer at Manure Microsite on Acidic Soil

Abstract

The application of green manure (GM), particularly leguminous varieties, demonstrates significant benefits for crop cultivation in acidic soils by enhancing the soil organic nitrogen (No) pool. To maximize these agronomic advantages, it is crucial to implement scientifically grounded application strategies. To this end, an incubation experiment was conducted to investigate the content, movement distance, and accumulation of acidic soil organic N (No) at different distances from the GM application microsite. Stylosanthes GM (10 or 40 ton/ha) was applied with or without phosphate fertilizer (monocalcium phosphate, MCP) at 44 kg/ha P, placed on the surface of soil cylinders. The GM/fertilizer and soil were incubated for 14 and 28 d. The results indicated the total organic nitrogen (TNo) content—including both non-acid-hydrolyzable N (NAHNo) and acid-hydrolyzable N (AHNo) fractions—significantly (p < 0.05) increased at the GM microsite after GM application. The influence of GM generally weakened as the distance from the site increased, and the spatial impact range exhibited significant modulation by three key parameters: incubation period, GM rates, and MCP addition. Subsequent analysis revealed a positive correlation between GM rate/incubation period and the movement distance of No fractions at GM microsite, demonstrating rate-dependent temporal dynamics. They were also increased by the addition of MCP after a longer incubation period but inhibited after a shorter period. This information will improve the efficiency of GM use, with or without MCP addition, and decrease the environmental load due to N pollution caused by GM.

1. Introduction

Most of the soils in south China are acidic soils, such as Latosol, and are generally deficient in organic matter, nitrogen (N), and other nutrients, with high acidity and aluminum toxicity. Applying green manure (GM), especially legumes, is an effective way to solve these problems. This is because GM can provide not only N, phosphorus (P), potassium (K), and other macronutrients necessary for plant growth but also iron, manganese, copper, zinc, and other micronutrients. It can effectively increase the organic carbon content and improve physical and chemical properties, improving the quality of the soil [1,2,3,4,5]. N is the most abundant mineral nutrient element in most leguminous GM, and because of this, the effect of leguminous GM on soil N is likely stronger than its effect on other mineral nutrients. Stylosanthes (Stylosanthes sp.) is a leguminous GM commonly used in the tropics; of all the Stylosanthes species, Stylosanthes guianensis is perennial and tolerant of aluminum and manganese toxicity, making it well suited for tropical acidic soils [6]. It is often intercropped in orchards and economic forests in southern China, used to improve soil fertility, suppress weeds, reduce soil erosion, and so on. Despite being one of the three major nutrient elements, with high demand in plant growth, N is one of the most commonly deficient elements in crop growth. In practice, a large amount of N fertilizer is often applied to meet the needs of crop growth. Applying GM, especially organic manure such as leguminous GM, can greatly reduce the application of chemical fertilizers; specifically, GM can replace synthetic N fertilizers, a practice that has been recently advocated by the Chinese government. GM has more beneficial effects on the soil when it is applied scientifically; however, inappropriate applications can lead to N losses in the form of nitrate and nitrogen oxides, which can be detrimental to the environment [7]. GM changes the contents of various forms of organic N (No) in the soil; the soil N pool will be different because of these changes, which can play an important role in soil fertility and ecological protection. Bremner [8] divided soil organic nitrogen into non-acid-hydrolyzable N (NAHNo) and acid-hydrolyzable N (AHNo), including amino acid N (AANo), ammonium N (ANo), amino sugar N (ASNo), and acid hydrolyzable unidentified N (AHUNo). Of the different kinds of No, AHNo is given more attention due to its variety of forms, clear components, and functions. GM may have some effects on soil organic N with different components. Xia et al. [9] found that covering green manure could increase the amount of active No fractions, e.g., AANo, by 32–44%. Yu et al. [10] found that long-term applications of GM of Chinese milk vetch (Astragalus sinicus L.) could improve and increase soil No. Xie et al. [11] also found that applying Chinese milk vetch could significantly increase the AHNo and non-acid-hydrolyzable N (NAHNo) contents in the soil. However, the present research on the effects of GM on soil organic N has mainly focused on bulk soil and has not reported on soil at fertilizer microsites. “Soil fertilizer microsite” refers to a special regional environment [12] in which fertilizer is added to soil with a concentration 10 times greater (or more) than that of the bulk soil, applied intensively (strip application, hole application, band application, etc.) near the fertilizer. At the microsite, the physical, chemical, physio-chemical, and biological properties will be distinctly different from those of the bulk soil, producing a series of special physical, chemical, physio-chemical, and biological reactions. Therefore, to better understand the scientific application of GM, it is important to study the transfer and transformation of organic carbon and N at leguminous green manure microsites. Changes in organic carbon fraction dynamics at the manure microsite on the acidic soil have been researched before [13]; however, there is a lack of documentation on the response of No at manure microsite on acidic soil.

Therefore, this study examines the transfer and transformation of soil No on the acidic soil at the microsite of GM and phosphate fertilizer to provide a basis for the scientific application of GM.

2. Materials and Methods

2.1. Materials

2.1.1. Green Manure

Stylosanthes guianensis cv. Reyan No. 2 was used as the experimental material. This cultivar, developed through selective breeding from CIAT184, demonstrates enhanced nutrient utilization efficiency and exceptional adaptability to challenging tropical acidic soils characterized by low organic matter content and aluminum toxicity. As one of the most extensively utilized Stylosanthes varieties in China’s tropical regions, it serves as a perennial leguminous green manure crop in sustainable agroecosystems. Its primary applications include intercropping with tropical fruit trees and cash crops, where it fulfills multiple ecological functions including soil amelioration (through nitrogen fixation and organic matter accumulation), weed suppression, and erosion control. The aboveground part was cut to maintain a vegetative stage over a 25 cm height; therefore, this part was sampled. The site was located in the experimental station of Tropical Crops Genetic Resources Institute, Chinese Academy of Tropical Agricultural Science, namely in Danzhou City, Hainan Province, P. R. China (19°29.101′ N, 109°29.167′ E). The sample was oven-dried at 70 °C and passed through a 1 mm mesh. The N content was 2.86%; the P content was 0.22%; and the K content was 1.15%, based on the dry material.

2.1.2. Soil

Ferralsols are typical acidic soils found in the Chinese tropics. Soil samples from the top 15 cm of cultivated soil layer were collected using the stainless soil sampler in December of 2013 from an experimental station located at the Tropical Crops Genetic Resources Institute, CATAS (19°30.633′ N, 109°30.194′ E). The soils were then air-dried and sieved through a 1 mm mesh.

The soil physio-chemical properties were as follows: soil pH: 5.36; organic matter: 1.35%; alkali-hydrolyzed N content: 56.1 mg/kg; 0.025 M HCl-0.03 M NH₄F extracted P: 7.0 mg/kg; 1 M NH₄OAc extracted K: 87.2 mg/kg; bulk density: 1.32 g/cm³.

2.2. Column Incubation Experiment

The six treatments were as follows: CK, the control (no addition of monocalcium phosphate or GM); MCP, phosphate fertilizer (monocalcium phosphate, P 44 kg/ha); GML, Stylosanthes GM (10 ton/ha); GMH, Stylosanthes GM (40 ton/ha); GML + MCP, Stylosanthes GM (10 ton/ha) + phosphate fertilizer (monocalcium phosphate, P 44 kg/ha); GMH + MCP, Stylosanthes GM (40 ton/ha) + phosphate fertilizer (monocalcium phosphate, P 44 kg/ha). The treatments were incubated for 14 days or 28 days, and each had three replicates.

The soil incubation method was used, and the experiment was conducted using a biological incubation box (Model LRH-550; Shaoguan Taihong Medical Equipment Ltd., Shaoguan, Guangdong Province, China) in a laboratory of Tropical Crops Genetic Resources Institute, Chinese Academy of Tropical Agricultural Science from 2013 to 2018. A paraffin wax container and petroleum jelly in a 2:1 ratio were used [13]. The inner diameter and height of the wax container were 10 cm and 20 cm, respectively. The soil was placed in the wax container, and the final bulk weight of the soil was 1.32 g/cm³. The soils were pre-incubated for 10 d to restore soil microbial activity before the experiment.

Reagent-grade MCP fertilizer (AR, Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) and GM were mixed in a nylon bag on top of the soil cylinder. No MCP or GM was added to the soil cylinder in the control treatment. Each treatment had three replicates, and the 36 soil cylinders containing the different soil treatments for the 14 d and 28 d incubation times were arranged in a completely randomized design. The wax blocks were incubated vertically in the biological incubation box at 25 °C after being sealed with Parafilm. After 14 or 28 d of incubation, Parafilm was removed from the soil cylinder. The soil cylinders were placed on the polyvinylchloride base of the amended sectioning apparatus [13]. These were sectioned from the top of the soil cylinder using a sharp, stainless-steel knife. The soils in the slices were kept for analysis. To make the figures clearer, only the data from the 0–100 mm soil layer for N are included in the data collected from the manure microsite, as these had the most significant influence on the overall results.

2.3. Analysis of the Samples

The soil organic nitrogen fractions were determined using a sequential extraction procedure according to Bremner’s [8] acid hydrolysis–distillation method (Figure 1). A soil sample of 5.00g, sieved through a 0.25 mm mesh, was weighed and placed into a plug.; Twodrops of n-octanol and 20 mL of 6 M HCI were added to the sample. The hydrolysis tube was shaken to fully mix the soil and acid. The bottle was then placed into a constant temperature oven set at 110 °C, where the soil–acid mixture was hydrolyzed for 12 h. After hydrolysis, the sample was filtered using filter paper while it was still hot. The filtrate was collected in a 50 mL volumetric flask and cooled, and the residue was rinsed with a small amount of deionized water several times with a constant volume of 50 mL. Hydrolysate of 20 mL was added to the 50 mL beaker; placed the beaker with filtrate in crushed ice; and with the help of a pH meter, slowly added 5 M of NaOH solution drop by drop, stirring until the pH of the hydrolysate reached about 5. The mixture was then neutralized with 0.5 M NaOH so that the pH of the hydrolysate reached 6.5 ± 0.3. The acid solution was transferred to a 50 mL volumetric bottle with a small funnel and kept the volume to scale. (1) Determination of AHNo: Before sampling, the volumetric flask was inverted several times to make the suspension uniform. Using a pipette with a wide end, 5 mL of acid solution was absorbed and placed in a boiling tube. To this, 0.5 g of nitrogenous mixture catalyst and 2 mL of concentrated sulfuric acid were added. The mixture was then boiled for 1.5 h and allowed to cool. The N content in the extract was analyzed using a Kjeldahl N meter. (2) Determination of ANo: Neutralized acid hydrolysate at 20 mL was placed in a dissolving tube, and 0.14 g MgO was added. After distillation for about 2 min, titration was performed. (3) Determination of ANo and ASNo: The neutralized acid hydrolysate (20 mL) was added to the boiling tube, followed by 20 mL monoboric acid buffer solution (pH = 11.2). The mixture was distilled for 4 min and then titrated. The determination results minus the ASNo content in (2) are the amino sugar nitrogen content. (4) Determination of AANo: 5 mL of neutralized acid hydrolysate was added to a 10 mL scale test tube, followed by l mL of 0.5 M NaOH solution. The mixture was heated in boiling water until the solution was reduced to 2~3 mL, then removed and cooled. In total, 0.5 g of citric acid and 0.1 g of ninhydrin were added, and the test tube was returned to the boiling water bath. After heating for 1 min, it was shaken for 10 s (without removing it from the water bath) and left in the water bath for 9 min. After cooling, 10 mL of monoboric acid phosphate buffer and 1 mL of 5 M NaOH were added and distilled for 4 min. (5) AUNo = AHNo − (Ano + ASNo + AANo): Total organic N (TNo) and organic N fractions were measured via the micro-Kjeldahl method presented by Bremner [8]. The non-hydrolyzable N (acid-insoluble N) was estimated by subtracting the total hydrolyzable N from TNo according to the method presented by Bremner [8]. Soil pH was measured in a 1:2.5 soil–water suspension using a thin, combined glass–calomel electrode pH meter (Model pp-15, Sartorius AG, Göttingen, Germany), following the method outlined by the Soil Science Society of China [14]. The bulk density of the soil was determined using the cutting ring method [14]. Soil organic matter was analyzed using the wet combustion method, as described by the Soil Science Society of China [14]. Soil-available phosphorus (P) was extracted using 0.03 M NH₄F with 0.025 M HCl [14]. The P concentrations in the extracts were determined with a spectrophotometer (Model V-1200, Shanghai Mapada Instrument Co., Ltd., Shanghai, China), using the molybdenum blue method [14]. Soil-available potassium (K) was extracted using 1 M NH₄OAc (pH 7), and K concentrations in the extracts were analyzed using a flame photometer [14]. GM samples were digested with HNO₃ using a microwave digestion system (CEM MARS, Matthews, NC, USA), and the P concentrations in the solution were determined with a spectrophotometer (Model V-1200; Shanghai Mapada Instrument Co., Ltd., Shanghai, China), using the molybdenum blue method [14].The GM N release rate was determined according to the N amount in the dry mass of Stylosanthes GM added to the column at the beginning and end of the incubation experiment.

Calculation of the movement distance and accumulation amount: It is assumed that the N content difference between the treatments where GM and/or MCP was applied (including the five GML, GML + MCP, GMH, GMH + MCP, and MCP treatments) and the CK treatment is the N supplied by the GM and/or MCP [13]. Based on this, the movement distance in this research is the distance from the manure/fertilizer site to the nearest site where the N content between the treatments with the added GM and/or MCP and CK was not significantly different (p < 0.05), and the No acculturation amount is the total difference in content between the treatment and CK within the movement distance multiplied by the corresponding soil mass.

2.4. Statistical Analysis

The data calculation was performed by the use of the Microsoft office Excel 2021. The analysis of variance (ANOVA) and correlations were calculated using SPSS 13.0 after the data were computed using Microsoft Office Excel 2016. For the ANOVA, the least significant difference (LSD) test was used to compare the difference between the means of the fertilizer treatments at the same distance from the manure site for the same incubation time at the p < 0.05 significance level. Duncan’s test was used to compare the movement distance and the accumulation amount means of the treatments for the same incubation time and the same after a different incubation time; the significance level was p < 0.05. The effects of incubation time, movement distance, fertilizer treatments, and their interaction on the soil No fractions were tested via three-way ANOVA, and the significance level was p < 0.05. The figures were produced using Sigmaplot 10.0.

3. Results

3.1. Dynamics of the Nitrogen Release Rate

After its application to soil, GM decomposes and is transformed by soil microbes. The decomposition changes over time. As a leguminous GM, Stylosanthes contains a higher amount of N, so N in the GM is released as it continuously decomposes; therefore, the N release rate is probably the main factor in N movement and transformation at the manure microsite on the acid soil. Figure 2 shows the temporal dynamics of the N release rate of Stylosanthes GM in the four treatments. The analytical results indicate that the N release rate increased rapidly after 14 d of incubation; however, the rate of increase in the decomposition rate decreased between 14 and 28 d. The results also show that the N release rate of the GML + MCP treatment was significantly lower than the single application of GM after 14 d; however, for the high-GM treatment, the N release rate was only slightly decreased by the MCP addition after 14 d of incubation. After 14 d of incubation, the N release rate in the single-application GM treatment increased slowly. The N release rate in the treatment mixed with MCP increased more than that of the single-application GM treatment; after 28 d incubation, the N release rate in the treatment mixed with MCP was higher than the single-application GM treatment, especially for the low-rate treatment. These results indicate that the GM application mixed with MCP restricted the N release between 0 and 14 d of incubation, and then the restriction gradually disappeared. Figure 1 indicates that the GM N release rate in the GMH treatment (the high-GM application: 40 t/ha of Stylosanthes GM) was significantly higher than that of the GML treatment (the low-GM application: 10 t/ha Stylosanthes GM) during the 28 d incubation, indicating high rates of GM could significantly increase the N release.

3.2. Changes in Soil Organic Nitrogen

3.2.1. Total Organic Nitrogen

Total organic nitrogen (THo) is the major soil nitrogen pool; the content is high and commonly difficult to change via fertilization. After the GM application, THo content at the manure microsite was likely influenced by much of the N released after GM decomposition. The effect is shown in Figure 3; the results indicate that the GM application, with or without MCP, could increase the AHNo content at the manure microsite. This effect was mainly produced close to the manure site, and the influence generally weakened as the distance increased from the site. The influencing scale depended on the incubation time, the GM treatments, and the MCP addition.

The analytical results for the changes in movement distance (Figure 3) indicate that the TNo movement distances in the GMH and GMH + MCP treatments were 6 mm and 4 mm longer (p < 0.05), respectively, after 14 d of incubation, and both were 6 mm longer (p < 0.05) than in the GML and GML + MCP treatments after 28 d of incubation. These results indicate that the GM rate increase facilitated TNo movement regardless of MCP addition. The analytical results for the MCP addition effect indicate that the GML + MCP and GMH + MCP treatments were 2 mm and 4 mm shorter (p < 0.05), respectively, after 14 d of incubation, and 10 mm and 12 mm longer (p < 0.05), respectively, after 28 d of incubation compared with the GML and GMH treatment. This indicates that MCP can inhibit TNo movement after a shorter incubation and facilitate TNo movement after a longer incubation. The analytical results in Figure 3 also indicate that the TNo movement distances in the GM treatments after 28 d of incubation were significantly (p < 0.05) longer than those of corresponding GM treatments after 14 d of incubation. These results indicate that incubation time can significantly positively affect TNo movement.

The GM application not only changed the TNo movement distance but also changed the TNo accumulation amount. The analytical results in Figure 3 indicate that the TNo accumulation amounts in the GMH and GMH + MCP treatments increased by 24.1% and 22.1% (p < 0.05), respectively, after 14 d of incubation. They also increased by 30.1% and 47.0% (p < 0.05), respectively, more than in GML and GML + MCP treatments after 28 d of incubation. These results indicate that GM rate increase could facilitate TNo accumulation regardless of MCP addition. The analytical results for the effect of MCP on the TNo accumulation amounts indicate that in the GML + MCP and GMH + MCP treatments, they decreased by 25.9.6% and 27.1% (p < 0.05), respectively, compared with the amounts in the GML and GMH treatments after 14 d of incubation, and by 35.8% and 53.4% (p < 0.05), respectively, compared with the GML and GMH treatments after 28 d of incubation. This indicates that MCP can inhibit TNo accumulation after a shorter incubation and facilitate TNo accumulation after a longer incubation. The analytical results in Figure 3 also show that the TNo accumulation amounts in the GM treatments after 28 d of incubation were significantly (p < 0.05) higher than those of corresponding GM treatments after 14 d of incubation. These results indicate that the incubation time can significantly positively affect TNo accumulation.

3.2.2. Acid-Hydrolyzable Nitrogen

Acid-hydrolyzable nitrogen (AHNo) is No extracted using heated 3N HCl or 6 N HCl for 12 to 24 h, including amino acid N (AANo), ammonium N (ANo), amino sugar N (ASNo), and acid-hydrolyzable unidentified N (AHUNo) [8]. Of the different kinds of No, AHNo is given more attention due to its variety of forms, clear components, and functions. After GM application, AHNo is likely influenced by much of the N released after GM decomposition. This effect is shown in Figure 4. The results indicate that GM application with or without MCP can increase AHNo content. This effect was mainly produced close to the manure site, and the influence gradually weakened with increasing distance from the site. The influencing scale depended on the incubation time, GM treatments, and MCP addition.

The analytical results for the change in movement distances (Figure 4) indicate that the AHNo movement distances in the GMH and GMH + MCP treatments were 14 mm and 6 mm longer (p < 0.05), respectively, after 14 d of incubation and 6 mm and 12 mm longer (p < 0.05), respectively, than in the GML and GML + MCP treatments after 28 d of incubation. These results indicate that a GM rate increase can facilitate movement regardless of MCP addition. The analytical results for the effect of MCP addition indicate that the distances in the GML + MCP and GMH + MCP treatments were 6 mm and 14 mm shorter (p < 0.05), respectively, after 14 d of incubation and 16 mm and 22 mm longer (p < 0.05), respectively, after 28 d of incubation compared with the GML and GMH treatments. This indicates that MCP can inhibit AHNo movement after a shorter incubation and facilitate AHNo movement after a longer incubation. The analytical results in Figure 4 also show that the AHNo movement distances in the GM treatments after 28 d of incubation were significantly longer (p < 0.05) than those of corresponding GM treatments after 14 d of incubation. These results indicate that incubation time can significantly positively affect the AHNo movement.

The GM application not only changed the AHNo movement distance but also changed the AHNo accumulation amount. The analytical results in Figure 4 indicate that the AHNo accumulation amounts in the GMH and GMH + MCP treatments increased by 77.9% and 30.0% (p < 0.05), respectively, after 14 d of incubation and by 21.8% and 46.0% (p < 0.05), respectively, compared with those in the GML and GML + MCP treatments after 28 d of incubation. These results indicate that a GM rate increase could facilitate AHNo accumulation regardless of MCP addition. The analytical result for the MCP addition effect on the AHNo accumulation amount indicated that in GML + MCP and GMH + MCP treatment, they decreased by 25.6% and 45.7% (p < 0.05), respectively, compared with in the GML and GMH treatments after 14 d of incubation and by 35.6% and 62.5% (p < 0.05), respectively, compared with in GML and GMH treatment after 28 d of incubation. This indicates that MCP can inhibit AHNo accumulation after a shorter incubation and facilitate AHNo accumulation after a longer incubation. The analytical results in Figure 4 also show that the AHNo accumulation amounts in the GM treatments after 28 d of incubation were significantly longer (p < 0.05) than those in the corresponding GM treatments after 14 d of incubation. These results indicate that incubation time can significantly positively affect AHNo accumulation.

Amino Acid Nitrogen

Amino acid nitrogen (AANo) is one of the most important AHNo components and one of the most readily oxidizable organic nitrogen compounds in soil nitrogen [8]. Therefore, AANo plays a significant role in soil N transformation. GM contains a large amount of N, and its application likely has a significant effect on the content and distribution of AANo, as shown in Figure 5. The results indicate that GM application with or without MCP can increase the AANo content. This effect was mainly produced close to the manure site, and the influence gradually weakened with increasing distance from the site. The influencing scale depended on the incubation time, GM treatments, and MCP addition.

The analytical results for the change in the movement distance (Figure 5) indicated that the AANo movement distance in the GMH treatment was 4 mm longer (p < 0.05) than in the GML treatment after 14 d of incubation, and there was no significant (p > 0.05) difference between the GML and GMH treatments with respect to AANo movement distances after 28 d of incubation. There was no significant difference between the GMH + MCP and GML + MCP treatments with respect to AANo movement distances after 14 d incubation; compared with the GML + MCP treatment, the AANo movement distance in the GMH + MCP treatment was 10 mm longer (p < 0.05) after 28 d of incubation. These results indicate that a GM rate increase could not significantly change AANo movement after single-GM application with a longer incubation or after MCP addition with a shorter incubation; however, it did facilitate AANo movement after a single-GM application with a shorter incubation and after MCP addition with a longer incubation. The analytical results for the effect of MCP addition on AANo movement distance indicate that there was no significant difference (p > 0.05) between the GML and GML + MCP treatments or between the GMH and GMH + MCP treatments with respect to AANo movement distance after 14 d of incubation; however, the AANo movement distances in the GML + MCP and GMH + MCP treatments were 8 mm and 18 mm longer (p < 0.05), respectively, than in the GML and GMH treatments after 28 d of incubation. This indicates that MCP can facilitate AANo movement after a longer incubation but cannot significantly affect AANo movement after a shorter incubation. The analytical results in Figure 5 also show that the AHNo movement distances in the GM treatments after 28 d of incubation were significantly longer (p < 0.05) than those of the corresponding GM treatments after 14 d of incubation. These results indicate that incubation time can significantly positively affect AANo movement.

The GM application not only changed the AANo movement distance but also changed its accumulation amount. The analytical results in Figure 5 indicate that after 14 d of incubation, the AANo accumulation amount in the GMH treatment was the highest, significantly (p < 0.05) higher than in the GML, GML + MCP, and GMH + MCP treatments. There was no significant difference (p > 0.05) between the other three treatments. After 14 d of incubation, AANo continuously accumulated, and the amounts in the treatments after 28 d of incubation were significantly higher (p < 0.05) than those after 14 d of incubation. The analytical results also indicate that after 28 d of incubation, the AANo accumulation amount in the treatments changed significantly; the GMH + MCP treatment accumulated most of the AANo, and there was a significant difference (p < 0.05) from the other three treatments. The GML + MCP treatment accumulated less (p < 0.05) AANo than the GMH + MCP treatment and more (p < 0.05) than the GML and GMH treatments. There was no significant difference (p > 0.05) between the GML and GMH treatments with respect to the AANo accumulation amount. These results indicate that after 28 d of incubation, the increased GM rate facilitated AANo accumulation after a single-GM application with a shorter incubation and MCP addition with a longer incubation; however, it could change the AANo accumulation after MCP addition with a shorter incubation or a single-GM application after a longer incubation. These results also indicate that MCP can inhibit AANo accumulation after a shorter incubation and facilitate AANo accumulation after a longer incubation compared with the corresponding single-GM application. The analytical results in Figure 5 also show that the AANo accumulation amounts in the GM treatments after 28 d of incubation were higher (p < 0.05) than those of the corresponding GM treatments after 14 d of incubation. These results indicate that the incubation time can significantly positively affect AANo accumulation.

Ammonium Nitrogen

Ammonium nitrogen (ANo) is the ammonia product of acid hydrolysis in soil [8]. Its sources are complex, including inorganic N from soil and organic manure, so it is likely changed by the application of leguminous GM, which contains a significant amount of N. The effect of GM application on ANo is shown in Figure 6. The results indicate that GM application with or without MCP can increase AANo content. This effect was mainly produced close to the manure site, and the influence gradually weakened with increasing distance from the site. The influencing scale depended on the incubation time, GM treatments, and MCP addition.

The analytical results for the change in ANo movement distance (Figure 6) indicated that, compared with the GML + MCP treatment, the ANo movement distances in the GMH + MCP treatment were 8 mm (p < 0.05) and 4 mm (p > 0.05) longer, respectively, after 14 d and 28 d of incubation. Compared with the GML + MCP treatment, the ANo movement distances in the GMH + MCP treatment were 2 mm (p > 0.05) and 8 mm (p < 0.05) longer, respectively, after 14 d and 28 d of incubation. These results indicate that a GM rate increase can facilitate ANo movement regardless of MCP addition. The analytical results for the effect of MCP addition on ANo movement indicate that in the GML + MCP and GMH + MCP treatments, the distances were 8 mm and 12 mm shorter (p < 0.05), respectively, after 14 d of incubation, and 6 mm and 10 mm longer (p < 0.05), respectively, after 28 d of incubation compared with the GML and GMH treatments. This indicates that MCP can inhibit ANo movement after a shorter incubation and facilitate ANo movement after a longer incubation. The analytical results in Figure 6 also show that the AHNo movement distances in the GM treatments after 28 d of incubation were significantly longer (p < 0.05) than those in the corresponding GM treatments after 14 d of incubation. These results indicate that incubation time can significantly positively affect ANo movement.

The GM application not only changed the ANo movement distance but also changed the ANo accumulation amount. The analytical results in Figure 6 indicate that the ANo accumulation amounts in the GMH treatment increased by 54.6% (p < 0.05) and 17.8% (p < 0.05) compared with those in the GML treatment after 14 d and 28 d of incubation, respectively. Compared with the GML + MCP treatment, the ANo accumulation amount in the GMH + MCP treatment increased by 9.5% and 52.2% (p < 0.05), respectively, after 14 d and 28 d of incubation. These results indicate that a greater GM rate can increase the ANo accumulation amount regardless of MCP addition. The analytical results for the effect of MCP addition on ANo accumulation indicate that the ANo accumulation amounts in the GML + MCP and GMH + MCP treatments decreased by 22.3% and 45.0%, respectively, compared with the GML and GMH treatments after 14 d of incubation and increased by 3.2% (p > 0.05) and 33.3% (p < 0.05), respectively, compared with the GML and GMH treatments after 28 d of incubation. This indicates that the MCP can inhibit ANo movement after a shorter incubation and facilitate ANo movement after a longer incubation. The analytical results in Figure 6 also show that the AHNo accumulation amounts in the GM treatments after 28 d of incubation were significantly (p < 0.05) greater than those of the corresponding GM treatments after 14 d of incubation. These results indicate that incubation time can significantly positively affect ANo accumulation.

Amino Sugar Nitrogen

There are two main forms of amino sugar nitrogen (ASNo) in soil: one is a macromolecular compound that is dispersed and heterogeneous, the other is a combination form closely bound to inorganic colloids [8]. The main components of ASNo, determined via gc-ms, are glucosamine, galactosamine, and cell wall acid. It is mainly derived from the microbial cell wall of soil microbial biosynthesis and can reflect the N assimilation and utilization of soil microorganisms [15]; therefore, the ASNo content in soil is closely related to soil microbial activity, quantity, and community structure. This indicates that the effect of fertilization on ASNo is likely to be small. This effect is shown in Figure 7. The results indicate that this effect was mainly produced close to the manure site, and the influence gradually weakened with increasing distance from the site. The influencing scale depended on the incubation time and the treatments.

The analytical results for the change in movement distance (Figure 7) indicate that the ASNo movement distances in the GMH and GMH + MCP treatments were 14 mm and 18 mm longer (p < 0.05), respectively, after 14 d of incubation and 10 mm and 14 mm longer (p < 0.05), respectively, than in GML and GML + MCP after 28 d of incubation. These results indicate that the increased GM rate facilitated the movement regardless of MCP addition. The analytical result for the effect of MCP addition on ASNo movement indicated that the distances in the GML + MCP and GMH + MCP treatments were 8 mm and 14 mm shorter (p < 0.05), respectively, after 14 d of incubation and 30 mm and 34 mm longer (p < 0.05), respectively, after 28 d of incubation compared with the GML and GMH treatments. This indicates that MCP can inhibit ASNo movement after a shorter incubation and facilitate ASNo movement after a longer incubation. The analytical results in Figure 7 also show that the ASNo movement distances in the GM treatments without MCP addition after 28 d of incubation were significantly (p < 0.05) shorter than those in the corresponding GM treatments after 14 d of incubation; however, the ASNo movement distances in the GM treatments with MCP addition after 28 d of incubation were significantly (p < 0.05) longer than those in the corresponding GM treatments after 14 d of incubation. These results indicate that incubation time can significantly inhibit the ASNo movement for the treatments without MCP addition and facilitate the ASNo movement for the treatments with the addition of MCP.

The GM application not only changed the ASNo movement distance but also changed the ASNo accumulation amount. The analytical results in Figure 7 indicate that the ASNo accumulation amounts in the GMH and GMH + MCP treatments increased by 123.0% and 129.9% (p < 0.05), respectively, after 14 d of incubation and by 201.6% and 66.6% (p < 0.05), respectively, compared with the GML and GML + MCP treatments after 28 d of incubation. These results indicate that an increased GM rate can facilitate ASNo accumulation regardless of MCP addition. The analytical results for the effect of MCP addition on the ASNo accumulation amounts indicated that in the GML + MCP and GMH + MCP treatments, they decreased by 27.9% and 25.7% (p < 0.05), respectively, compared with the GML and GMH treatments after 14 d of incubation and by 362.2% and 155.2% (p < 0.05), respectively, compared with the GML and GMH treatments after 28 d of incubation. This indicates that MCP addition can inhibit ASNo movement after a shorter incubation and facilitate ASNo movement after a longer incubation. The analytical results in Figure 7 also show that the ASNo accumulation amounts in the GM treatments after 14 d of incubation were significantly (p < 0.05) shorter than those in the corresponding GM treatments after 28 d of incubation. These results indicate that incubation time can significantly positively affect ASNo accumulation.

Acid-Hydrolyzable Unknown Nitrogen

Acid-hydrolyzable unknown nitrogens (AHUNos) are N compounds cannot be identified in the acidolysis process [8]. As AHUNo is the major contributor to soil reactive N [15], its changes are important for N transformation in acidic soil. The effect is shown in Figure 8. The results indicate that this effect was mainly produced close to the manure site, and the influence gradually weakened with increasing distance from the site. The influencing scale depended on the incubation time and treatments.

The analytical results for the change in movement distance (Figure 8) indicate that the AHUNo movement distances in the GMH and GMH + MCP treatments were 14 mm (p < 0.05) and 4 mm (p > 0.05) longer, respectively, than those in the GML and GML + MCP treatments after 14 d of incubation; the AHUNo movement distances in the GMH and GMH + MCP treatments were 12 mm and 22 mm longer (p < 0.05), respectively, than in the GML and GML + MCP treatments after 28 d of incubation. These results indicate that an increased GM rate can facilitate AHUNo movement regardless of MCP addition. The analytical results for the effect of MCP addition on AHUNo movement indicate that AHUNo movement distances in the GML + MCP and GMH + MCP treatments were 8 mm and 18 mm shorter (p < 0.05), respectively, than those in the GML and GMH treatments after 14 d of incubation and 16 mm and 26 mm longer (p < 0.05), respectively, than those in the GML and GMH treatments after 28 d of incubation. This indicates that MCP can inhibit AHUNo movement after a shorter incubation and facilitate AHUNo movement after a longer incubation. The analytical results in Figure 8 also show that there was no significant difference for the AHUNo movement distance between GM treatments without MCP addition after 28 d of incubation with the corresponding GM treatments after 14 d of incubation, and AHUNo movement distance in the GMH and GMH + MCP treatments after 28 d of incubation were significantly longer (p < 0.05) than those in the corresponding GMH and GMH + MCP treatments after 14 d of incubation. These results indicate that incubation time can significantly positively affect AHUNo movement.

The GM application not only changed the AHUNo movement distance but also changed the AHUNo accumulation amount. The analytical results in Figure 8 indicate that the AHUNo accumulation amounts in the GMH and GMH + MCP treatments increased by 104.4% and 54.0% (p < 0.05), respectively, after 14 d of incubation and by 37.7% and 35.5% (p < 0.05), respectively, compared with the GML and GML + MCP treatments after 28 d of incubation. These results indicate that an increased GM rate can facilitate AHUNo accumulation regardless of MCP addition. The analytical results for the effect of MCP addition on AHUNo accumulation amounts indicate that in the GML + MCP and GMH + MCP treatments they decreased by 41.2% and 55.7% (p < 0.05), respectively, compared with those in the GML and GMH treatments after 14 d of incubation and by 85.5% and 82.6% (p < 0.05), respectively, compared with those in the GML and GMH treatments after 28 d of incubation. This indicates that MCP can inhibit AHUNo accumulation after a shorter incubation and facilitate AHUNo accumulation after a longer incubation. The analytical results in Figure 8 also show that the AHNo accumulation amounts in the GM treatments after 28 d of incubation were significantly (p < 0.05) higher than those in the corresponding GM treatments after 14 d of incubation. These results indicate that incubation time can significantly positively affect AHUNo accumulation.

3.2.3. Non-Acid-Hydrolyzable N

Non-acid-hydrolyzable nitrogen (NAHNo) is a complex compound formed by the condensation of amino acids and amino sugars during soil acidification, accounting for approximately 10% to 20% of total soil nitrogen [15]. This effect is shown in Figure 9. The results indicate that this effect was mainly produced close to the manure site, and the influence gradually weakened with increasing distance from the site. The influencing scale was independent of the incubation time and treatments.

The analytical results for the change in movement distance (Figure 9) indicate that the NAHNo movement distance in the GMH treatment was 6 mm and 8 mm longer (p < 0.05) than in the GML treatment after 14 d and 28 d of incubation, respectively. Furthermore, the NAHNo movement distance in the GMH + MCP treatment was 2 mm (p > 0.05) and 20 mm longer (p < 0.05) than in the GML + MCP treatment after 14 d and 28 d of incubation, respectively. These results indicate that an increased GM rate can facilitate NAHNo movement regardless of MCP addition. The analytical results for the effect of MCP addition on NAHNo movement indicate that NAHNo movement distances in the GML + MCP and GMH + MCP treatments were 2 mm and 6 mm shorter (p < 0.05), respectively, after 14 d of incubation and 4 mm and 16 mm longer (p < 0.05) than in the GMH treatment after 28 d of incubation. This indicates that MCP can inhibit NAHNo movement after a shorter incubation and facilitate NAHNo movement after a longer incubation. The analytical results in Figure 9 also show that the NAHNo movement distances in the GMH and GMH + MCP treatments after 28 d of incubation were significantly (p < 0.05) longer than those in the corresponding GMH and GMH + MCP treatments after 14 d of incubation. These results indicate that incubation time can significantly positively affect NAHNo movement.

The GM application not only changed the NAHNo movement distance but also changed the NAHNo accumulation amount. The analytical results in Figure 9 indicate that the NAHNo accumulation amounts in the GMH and GMH + MCP treatments increased by 58.9% and 41.4% (p < 0.05), respectively, after 14 d of incubation and by 53.0% and 167.4% (p < 0.05), respectively, compared with the GML and GML + MCP treatments after 28 d of incubation. These results indicate that an increased GM rate can facilitate NAHNo accumulation regardless of MCP addition. The analytical results for the effect of MCP addition on NAHNo accumulation indicate that the NAHNo accumulation amounts in the GML + MCP and GMH + MCP treatments decreased by 8.6% (p > 0.05) and 18.7% (p < 0.05), respectively, after 14 d of incubation and by 30.3% and 127.8% (p < 0.05), respectively, compared with those in the GML and GMH treatments after 28 d of incubation. This indicates that MCP can inhibit NAHNo accumulation after a shorter incubation time and facilitate NAHNo accumulation after a longer incubation time. The analytical results in Figure 9 show that the NAHNo accumulation amounts in the GM treatments after 28 d of incubation were significantly (p < 0.05) greater than those in the corresponding GM treatments after 14 d of incubation. These results indicate that incubation time can significantly positively affect NAHNo accumulation.

4. Discussion

N is an essential nutrient for crop growth; however, it is often deficient in acidic soils due to poor soil conservation and high crop requirements. Applying N fertilizer and organic manure, such as GM, is an effective way to solve this problem. Of all the kinds of GM, leguminous GM is one of the best and most widely used for increasing soil N. As one of the major tropical leguminous GMs, Stylosanthes is widely used in the tropics. After the application of Stylosanthes GM, soil nitrogen, especially No, is likely to increase, as Stylosanthes contains a high amount of N as a leguminous crop. This study performed a GM microsite column incubation experiment in a laboratory under controlled conditions to determine changes in No fractions, including NAHNo and AHNo. We used the sequential N fractionation schemes developed by Bremner [8] over different incubation periods. As a chemical fractionation method, Bremner’s scheme represents one of the most widely accepted approaches for soil No fractionation. Among various soil No fractionation methodologies, this technique coexists with several other analytical frameworks, including physical protection-based fractionation, bioavailability assessment protocols, stability-oriented fractionation approaches, isotopic labeling techniques, spectroscopic analyses, and molecular labeling methods. Notably, Bremner’s chemical fractionation scheme has become particularly significant in soil science research, serving as a foundational protocol for nitrogen speciation studies due to its systematic characterization of different nitrogen pools through sequential chemical extraction procedures. Using Bremner’s fractionation schemes, this study clearly confirmed that GM application can increase TNo content, including NAHNo and AHNo (AANo, ANo, ASNo, and AHUNo) at manure microsites. GM application can increase No fraction content and accumulation, mainly because GM, especially leguminous GM, contains large amounts of N in various forms, which are continuously released into the soil through the decomposition of GM. The decomposition of green manure is the process of returning plant nutrients, including of N, to the soil. GM enhances nutrient cycling and utilization efficiency, and soil microbes have positive effects on this process [16,17]. For example, Acinetobacter, Pseudomonas, and Clostridium accelerate the degradation of lignin into bacterial aggregates [18], and several authors have noted that members of Nitrospirae participate in the soil N cycle [19,20]. The analytical results from this study indicate that an increased GM rate can facilitate the movement and accumulation of TNo, including NAHNo and AHNo, regardless of MCP addition; these results also indicate that a greater GM rate can increase the TNo movement distance and accumulation amount, including NAHNo and AHNo. All of these results illustrate that GM application is the main reason for increases in soil TNo, including NAHNo and AHNo, ultimately improving soil quality. Our results are similar to those of Huang et al. [21], who studied the intercropping of Stylosanthes GM in coconut plantations. In their study, GM was used as mulch and green manure, and both GM utilization patterns increased TNo content, including NAHNo and AHNo (AANo, ANo, ASNo, and AHUNo). Related to this research, Xia et al. [9] found that covering fields with Chinese milk vetch (Astragalus sinicus L.) and rapeseed.(Lolium perenne L.) could increase the amount of AANo by 32–44%. Yu et al. [10] showed that organic manure can significantly increase soil total N, including No and inorganic N, and microbial biomass N. Wu et al. [22] found that compared with CK (without corn residue application), a 33% corn residue application treatment showed a significantly higher concentration of soil hydrolyzable NH₄⁺-N, AANo, and ASNo in both the 0–10 and 10–20 cm soil layers; conversely, 67% and 100% corn residue application treatments significantly increased the concentration and proportion of soil HUNo but decreased the hydrolyzable NH₄⁺-N, AANO, and ASNo proportions in the 10–20 cm soil layer. Huang et al. [23] reported that the long-term application of Chinese milk vetch as GM, with or without chemical fertilizer, increased all soil No fractions, including ANo, AANo, ASNo, HUNo, non-hydrolyzable N, and even total N. These results indicate that organic manure—including GM applications mixed with or without phosphate fertilizer—can increase soil No with different fractions. However, the amount of increase depends on the kinds of organic manure fractions, the GM, and the organic manure application rate, as well as time, the type of manure, and whether it is mixed with chemical fertilizer.

In our study, adding MCP inhibited the movement and accumulation of TNo, including NAHNo, and most AHNo components (AANo, ANo, ASNo, and AHUNo) at the manure microsites after 14 d of incubation and facilitated the movement and accumulation of these No fractions after 28 d of incubation. This is because, at the early stages of GM decomposition, MCP inhibits decomposition so that less N is released into the soil at the manure microsite (Figure 1); as a result, the movement and accumulation of TNo, including NAHNo and AHNo, is inhibited. The inhibition caused by MCP addition at the early stage is likely due to excessive MCP, which may have inhibited the activity of decomposition microbes such as Acinetobacter, Pseudomonas, and Clostridium [20]. After 14 days, the MCP amount decreased as a result of a large amount of P moved into the soil, a phenomenon demonstrated in our previous research [24]. However, at the late stages of GM decomposition, MCP inhibition continuously weakens (Figure 1) such that more N is released into the soil. At this point, MCP addition makes the proportion of GM N and P more suitable for GM decomposition, proving that our treatment design is correct. The N movement in the soil is mainly via mass flow and diffusion [25]. In this research, only diffusion distributes N; as a result, the distance from the manure site is the main factor in the distribution of N, apart from the treatment factor. The GM decomposition change influenced by MCP was similar to our previous research [13]. However, there is still no research on the effect of phosphate fertilizer addition on GM microsites regarding No changes. Xie et al. [11] reported the effect of N fertilizer with GM on soil No, showing that a mixed application of N fertilizer with Chinese milk vetch GM had significantly greater N values in all forms, with the exception of AHUN. The rate affected the content and proportion of soil No fractions. In our study, N movement was faster in the soil, with No fractions moving more than 30 mm in some cases and more than 80 mm in others after 28 d of incubation in most of the GM treatments. This was further than P in the soil, which moved less than 30 mm [25,26]. This means that excessive N can produce non-point pollution faster and more easily than P. At present, non-point pollution from excessive N use is gaining more and more attention [27,28]. Our research confirms the movement distances and accumulation amounts of No fractions after different treatments and incubation times. This will improve our understanding of the spatial–temporal availability of N. Therefore, these results will help farmers decide when and where to apply GM based on an understanding of crop N requirements in tropical acidic soils. N is fully used by crops, and this leads to a decrease in N entering the environment around the soil. Furthermore, non-point-source pollution also decreases. Therefore, this study will be of economic and environmental benefit, especially given that the price of N fertilizers, such as urea, has skyrocketed.

Our controlled incubation study provides clear mechanistic insights into the effects of GM rate, incubation period, distance from manure microsites, and MCP addition on No dynamics. However, certain limitations inherent to laboratory conditions should be acknowledged when extrapolating these findings to field scenarios. Specifically, our experimental design did not account for natural environmental fluctuations in temperature, microbial community dynamics, or hydrological processes—all critical factors influencing nutrient cycling in agricultural systems. The observed discrepancies between our results and field experiment data [21] primarily stem from environmental optimization in controlled settings. Maintaining ideal moisture, aeration, and temperature conditions likely enhanced microbial activity, thereby accelerating GM decomposition rates and consequent N release compared to long-term field observations. Notably, our manure microsite-focused approach demonstrated that No fractions, including of HANo and NHANo, reached high level within weeks, mirroring levels typically requiring years to achieve in bulk soil under field conditions [21]. While both experimental approaches validate the nitrogen-enhancing capacity of GM amendments, this study specifically examined Stylosanthes (leguminous GM), precluding direct comparisons with gramineous GM systems. The existing literature suggests divergent impacts between these GM types: gramineous species (characterized by higher C:N ratios) tend to enhance soil carbon sequestration more effectively, whereas leguminous GMs (with lower C:N ratios) demonstrate superior nitrogen cycling benefits [20]. This functional complementarity warrants further investigation through comparative studies. As a slow-release nutrient source, GM amendments offer distinct advantages over synthetic fertilizers by mitigating nitrogen loss pathways. Our findings support previous reports of reduced leaching potential and decreased greenhouse gas emissions associated with organic amendments [29,30]. Nevertheless, optimizing GM utilization requires species-specific management strategies that account for their unique biochemical composition and decomposition patterns. The growing consensus in sustainable agriculture advocates for GM substitution to reduce synthetic nitrogen fertilizer dependency. Empirical studies demonstrate significant potential for GM integration in fertilization regimes: Kumar and Bordoloi [29] established that Sesbania aculeata GM application with 50% nitrogen reduction (75 kg N/ha vs. conventional 150 kg N/ha) effectively mitigates N₂O emissions while maintaining crop productivity. Similarly, Daba et al. [30] reported that Chinese milk vetch GM substitution for 20% chemical nitrogen fertilizer substantially decreases greenhouse gas intensity in cropping systems. While GM application represents a promising strategy for enhancing tropical crop productivity and soil health, improper management may paradoxically exacerbate environmental risks. The dichotomy lies in application protocols—optimized use improves soil organic matter and nutrient use efficiency, whereas excessive or poorly timed applications can induce non-point source pollution through nitrogen leaching and elevate greenhouse gas emissions. Our manure microsite experiments systematically quantified the spatiotemporal patterns of No fractions (including HANo and NHANo), elucidating their distribution dynamics and movement distances under varying application durations and MCP amendment conditions. These findings enable precision management through three operational guidelines: (1) Synchronizing GM decomposition phases with crop nitrogen demand curves using phenological models; (2) Implementing spatial placement strategies that match nitrogen movement distances with root architecture; (3) Employing MCP-based retardation techniques to modulate nitrogen release kinetics. Such targeted approaches maximize nitrogen utilization efficiency (NUE) while minimizing environmental losses, effectively bridging the gap between agroecological benefits and production requirements.

5. Conclusions

N is an essential element for crop growth and development, but it is often deficient in acidic soils and sometimes becomes a potential source of environmental pollution. Therefore, it is very important to fully use N from fertilizer/manure applications and soil sources. This study clearly demonstrated the content, movement, and accumulation changes in soil No fractions at microsites after using different GM rates, with or without MCP addition. Based on this research, we conclude that increases in the incubation time and GM rate can increase the movement distance and accumulation of TNo, including NAHNo and AHNo (AANo, ANo, ASNo, and AHUNo). Adding MCP inhibited the movement and accumulation of TNo, including NAHNo, and most AHNo components (AANo, ANo, ASNo, and AHUNo) at manure microsites after 14 d of incubation but facilitated their movement and accumulation after 28 d of incubation. These results will guide the appropriate application of GM to improve GM N use efficiency and decrease the environmental N load by reducing rain-induced N leaching loss from the soil.