Effects and Mechanisms of Granulated Compost on Soil Nitrogen Supply and Crop Uptake: Preliminary Evidence from a ¹⁵N Tracing Field Experiment in Tobacco

Abstract

Organic fertilizer granulation represents a promising strategy for modifying nitrogen (N) release from compost in soil. Nevertheless, there is a lack of large-scale field trials exploring its impact on tobacco production and soil N supply. This research conducted a preliminary study by employing ¹⁵N tracing technology to investigate the effects of granular compost on soil N transformation and supply; on the yield and quality of tobacco leaves; and on the distribution of granular compost-derived N among the different soil N pools and tobacco plant organs. The results revealed that the 2 cm diameter granule organic fertilizer treatment (G2) significantly increased tobacco leaf yield by 15% compared to conventional fertilization (CK). However, the 4 cm diameter granule organic fertilizer (G4) treatment resulted in a reduction in leaf yield. Notably, the quality of tobacco leaves remained unaffected compared to conventional fertilization treatment; the N content ranged from 15 to 25 g kg⁻¹, which was within the high-quality range. The results also indicated that direct N supply to the tobacco from granular compost was limited. The G2 and G4 treatments provided 2.8% and 2.2% of the N in the fertilizer to the tobacco plants, respectively, with more than 93% of the N in the tobacco plants derived from the soil. Therefore, both of these particle sizes of granular compost facilitated the absorption of soil N by tobacco plants. At the end of the growth period, the N content derived from the G4 granular fertilizer in the soil was significantly higher than that from the G2 fertilizer. This may be due to the slower nutrient release mechanism and longer release period of the G4 fertilizer compared to G2. Our results suggested that granulated compost fertilizer (both G2 and G4) has the potential to enhance soil N supply. Despite the elevated nitrogen levels observed in leaves treated with 4 cm diameter granular fertilizer, an integrated assessment of yield performance demonstrates that the 2 cm diameter granular organic fertilizer delivers superior economic benefits. However, G2 may also have a higher potential for N loss. Further investigations under field conditions are necessary to validate the applicability of granular fertilizer of different particle sizes and its specific mechanisms of impact.

1. Introduction

Nitrogen (N) is an essential nutrient for plant growth and development. The use of N fertilizers is a key strategy in modern agriculture to meet the growing demand for food from an increasing population. However, the excessive use of chemical N fertilizers can potentially cause negative impacts that outweigh benefits, including soil and groundwater pollution, nutrient imbalances in plants, and greenhouse gas emissions [1,2]. The utilization of compost amendments produced from livestock manure and crop straw has the potential to reduce the leaching of chemical N fertilizers and alleviate associated economic and environmental burdens.

Studies have shown that compared to urea application, cow dung increases the content of ammonium nitrogen (NH₄⁺) and nitrate nitrogen (NO₃⁻) in the soil [3,4]. The use of organic fertilizers reduced ammonia (NH₃) emissions from the soil by 47–71% compared to 100% synthetic N fertilizer [5]. Cheng et al. [6] suggested that the application of organic fertilizers promoted the mineralization of labile organic N and overall nitrification, with NH₄⁺ oxidation being the only source of NO₃⁻. Furthermore, organic amendments increased the contents of potentially mineralizable N, readily mineralizable N (Pool I), and intermediate mineralizable N (Pool II) by 140–355% compared to synthetic fertilizer treatments [7]. Studies have shown that organic fertilizers can significantly increase microbial biomass nitrogen (MBN), and microorganisms are key regulators of N transformation. Microbial biomass nitrogen serves as a source (mineralization) and sink (fixation) of N during mineralization and immobilization processes and, to some extent, can regulate the retention and supply of soil N [8,9]. Recalcitrant N pools include hydrochloric acid hydrolysable N (HCl-N) and stable soil N (SSN). The former acts as a transitional N connecting inorganic N (InorgN, NH₄⁺-N, NO₃⁻-N) and MBN with SSN and quantitatively corresponds to the sum of the hydrochloric acid hydrolysable amino acid N and hydrochloric acid hydrolysable unknown N [10]. Some studies have also suggested that it includes amino acids, humic acids, and N-containing heterocyclic compounds bound to minerals [11,12]. Stable soil N is the most stable N pool, formed by the combination of NH₄⁺ with clay minerals and humic acids or the hydrolysis-condensation of amino acids and amino sugars [13]. Limited research is currently available on the phase characteristics of the transformation of various N pools in soil following compost application.

Organic amendments could act as a slow-release fertilizer to enhance the net primary productivity of soil [7], as organic N is the primary form of N in these amendments, which needs to be mineralized into InorgN before it can be taken up and utilized by crops, which is a slow and continuous process. In agricultural production, N in organic amendments (such as manure, green manure, and compost) has a lower recovery rate in crops (27%), although the recovery rate in the years following application (10%) was higher than that of mineral fertilizers [14]. Equilibration among the N mineralization, immobilization, and loss rates in compost reflects the effectiveness and usability of compost for plants [15,16]. A large portion of NH₄⁺ can be absorbed and utilized by crops when the N mineralization period of organic amendments coincides with the peak N demand of crops. However, if the N mineralization rate does not match crop demand, there can be high residual N or loss, especially during periods of heavy rainfall, and there is an increased risk of NO₃⁻ leaching and gaseous losses through N₂O and N₂. Some NH₄⁺ also has the potential to be lost through rapid volatilization via conversion to NH₃ in the soil water phase. Alternatively, microorganisms can assimilate NH₄⁺ and NO₃⁻ into the soil N pools, but their utilization by subsequent crops is slow and depends on remineralization. Furthermore, NH₄⁺ can bind with soil minerals, forming mineral-bound N that creates SSN [17]. The distribution and dynamic changes in soil N from different forms of organic amendments may be influenced by factors such as fertilizer type, application method, cropping history, and environmental and soil factors. For example, Ding et al. [13] explored the influence of straw on the mineralization of powdered manure during tobacco cultivation. Their results showed that straw promoted the conversion of NH₄⁺ from manure into HCl-N, followed by gradual remineralization [13]. There is limited research on the changes in soil N pools that arise from the use of organic amendments [13,18].

Fertilizer granulation can lead to the formation of complexes containing organic C and N (with diameters of up to 4 cm), which may have significant impacts on the distribution patterns of pores, C and N nutrients, oxygen, and water resources within the granules, as well as its interactions with the surrounding soil matrix [19]. A single organic fertilizer granule can be regarded as an independent microecosystem with abundant nutrient sources. The microbial community and structure in the granular matrix may be different from those in the fertilizer sphere and nonfertilizer sphere soil, so the mineralization–immobilization–turnover (MIT) of N can occur independently of the microbial ecosystem in the soil. This means that the MIT of fertilizer determines the proportion of MBN that is released to the soil matrix. Unlike powdery organic fertilizer, the unique ecological hot spot inside granular fertilizer results in a significantly higher MBN content than that in the surrounding soil. As a major factor regulating organic N remineralization, MBN has the potential to mineralize more inorganic nitrogen. Microorganisms then determined the amount of net N mineralization and dissolved organic N (DON) by regulating related enzyme activities in the fertilizer substrate [19]. Studies have shown that granular organic fertilizers (≤10 mm in diameter) delay N release by approximately one month, reducing the risk of N loss during crop seedling stages [18]. Similarly, granular organic-inorganic fertilizers (3–4 mm in diameter) appear to slow nutrient release rates and offer advantages as a plant nutrient source compared to organic or chemical fertilizers alone [20]. However, research on larger particle sizes (>10 mm in diameter) is limited.

The objective of this research was to examine and analyze the following: (a) The impact of granular fertilizer on soil N pools during the growth period of tobacco. (b) The dynamic transformation of N derived from granular fertilizer sources in the soil. (c) The effects of granular fertilizer on yield, N uptake, and accumulation in tobacco.

2. Materials and Methods

2.1. Overview of the Trial Site

The field application trial of large granular manure was conducted from 26 April 2020 to 1 September 2020, in the Tobacco Planting Technology Demonstration Park, Linquan Town, Qianxi County, Bijie City, Guizhou Province, China (E 105.90°, N 27.02°). Linqian is located at an altitude of 1200 m, with an annual average temperature of 14.2 °C. The region falls under a subtropical monsoonal humid climate zone, with an average annual precipitation of 926 mm and a yellow loam soil type. The air-dried soil was mixed with sterile water at a ratio of 1:5 (w/v) for 2 h and centrifuged at 3500 rpm for 20 min. The pH of the suspension was determined using a pH meter (multi 340i, WTW GmbH, Ihlow, Germany). Total organic carbon (TOC) and total nitrogen (TN) combustion analysis were performed using the trace element analyzer UNICUBE^® (element, Hesse, Germany) equipped with a thermal conductivity detector (TCD) [13]. Total phosphorus (TP) was determined using the molybdenum antimony anti-colorimetric method after digestion in HClO₄-H₂SO₄, while available phosphorus (Olsen-P) was determined using the same method after extraction with 0.5 M NaHCO₃. Available potassium (AK) was extracted using a solution of 1 M NH₄OAc (pH 7.0), and its concentration was measured by flame photometer. Inorganic nitrogen compounds (NH₄⁺-N, NO₃⁻-N) were extracted with 2 M KCl and analyzed by continuous flow analyzer (AutoAnalyzer 3, Bran & Luebbe, Hamburg, Germany) [13]. Soil pH was 5.30, total organic carbon (TOC) was 13.53 g kg⁻¹, total nitrogen (TN) was 1.53 g kg⁻¹, total phosphorus (TP) was 0.96 g kg⁻¹, available phosphorus (Olsen-P) was 5.68 mg kg⁻¹, available potassium (AK) was 223.8 mg kg⁻¹ and InorgN (NH₄⁺-N, NO₃⁻-N) was 32.4 mg kg⁻¹.

2.2. Preparation of Test Material

2.2.1. Preparation of ¹⁵N-Labeled Compost

Based on the MIT theory of N in soil, the input of an available C source, such as glucose, could encourage the immobilization and dispersion of exogenous N to promote a more even distribution of ¹⁵N throughout the N pool in compost [6,21]. The particulars are outlined below:

Compost was obtained from Organic Biotechnology Ltd., located in Beijing, China. It is primarily formed by mixing cow manure and vegetable residues, followed by aerobic composting. We added (¹⁵NH₄)₂SO₄ to the compost, which had a N abundance of 50%, and the added amount was equivalent to 30% of the fertilizer’s total N (on a dry basis). Glucose was also added to the compost at a rate of 2000 mg C kg⁻¹ every 20 days to improve homogeneity among the various N components in the compost. This method was successfully implemented in our previous research, which showed that a pulse addition of glucose could promote the uniformity of ¹⁵N enrichment in different N pools in compost, including InorgN, hot water-soluble organic N (HWON), Microbial Biomass Nitrogen (MBN), hydrochloric acid-hydrolysable N (HCl-N) and stable soil N (SSN), in which the APE (atom percent excess) of ¹⁵N was consistent at 2.40 ± 0.1% [22]. The relatively small error among the different active N pools (meaning that the ¹⁵N labeling was uniform) guaranteed the accuracy of the calculated percentage of N derived from the soil and the fertilizer. The compost labeled with ¹⁵N had a TN content of 8.1 g kg⁻¹, P₂O₅ content of 9.7 g kg⁻¹, and K₂O content of 16.9 g kg⁻¹.

2.2.2. Preparation of Granular Compost

On 26 April 2020, modified starch was added to the compost at a rate of 10% as a binder to enhance granulation. After the water content was adjusted to 36.7%, the mixture was manually granulated into particles of diameters 2 cm and 4 cm. Both types of granules had the same density (2.39 kg m⁻³), which implied that the weight of an individual 4 cm diameter granular fertilizer was approximately 8 times that of a 2 cm diameter granular fertilizer. Due to the purely manual production process of the granular fertilizer, no additional heat is generated and no nutrient loss occurs during its fabrication.

2.3. Experimental Design

The tobacco used was Yunnan 87 (Nicotiana tabacum L. Yunnan No. 87), which had 3 real leaves at transplant. The entire process from tobacco seed germination to seedling formation was accomplished using a floating seedling tray within the incubator and lasts about 60 days. This tray, typically made of molded polystyrene, was filled with a specialized substrate for optimal growth and floats in nutrient-rich water within the seedling pond. During the seedling bed stage, various techniques such as leaf cutting and refinement are employed to cultivate robust and vigorous seedlings.

In April 2020, a field that was flat and had uniform fertility was selected for the microplot experiment. Three adjacent microareas were selected for the three treatments, namely, conventional fertilization (CK), application of granular compost with a diameter of 2 cm (G2) and application of granular compost with a diameter of 4 cm (G4). A single ridge film was used to cover the holes, and the row spacing × plant spacing was 1.1 m × 0.55 m. The microareas were characterized by a length of 8 m and a width of 4.4 m. A total of 5 ridges were created within each microarea. The protected rows consisted of two ridges on each side, while the treated rows had three ridge rows in the middle. To maintain consistency and ensure accurate data collection, each individual ridge was treated as a repetition. Within each ridge, a total of 14 tobacco plants were transplanted. In terms of the cost of our trials, this setup allowed for systematic observation and analysis of the experimental variables. The entire process, from transplanting the seedlings on April 25, 2020, to harvesting the upper leaves of tobacco plants in mid-August 2020, lasts approximately 110 days.

The procedures for transplanting and fertilization were as follows: ridges were made one week before transplanting according to the local tobacco planting methods. The dimensions of the transplanting holes and ridges are shown in Figure 1. The transplanting hole was similar to a frustum of a cone, with upper and lower base radii of R = 0.25 m and r = 0.05 m, respectively, and a height h = 0.20 m. The three treatments were as follows: ① CK: the base fertilizer for tobacco (N-P₂O₅-K₂O, 10-15-24, ≥49%, recommended amount of fertilizer was 525 kg ha⁻¹) was applied and fully mixed with the soil on the ridge. Then, soil holes were dug, tobacco seedlings were placed in the holes, and their root areas were covered with soil and film. Then, to bacco water-soluble root fertilizer (N-P₂O₅-K₂O: 22-14-10, ≥46%, the recommended amount was 37.5 kg ha⁻¹) was applied. Additionally, 150 kg ha⁻¹ (N-P₂O₅-K₂O: 13-15-25, ≥53%, the recommended amount was 150 kg ha⁻¹) was applied at 10 days and 30 days after transplanting. The fertilizer applied by the CK treatment corresponded to a total N amount of approximately 100 kg ha⁻¹, a P₂O₅ amount of 129 kg ha⁻¹, and a K₂O amount of 204 kg ha⁻¹. ② G2: Forty fertilizer pellets with diameters of 2 cm were evenly placed in each hole, and the spaces between the granules were filled with soil. The tobacco seedlings were placed in the holes, and their root areas were covered with soil and film. ③ G4 treatment: 5 fertilizer pellets with diameters of 4 cm were evenly dispersed in each hole, some soil was sprinkled over the fertilizer to fill in the gaps between the granules. Then, tobacco seedlings were placed in the holes, and their root areas were covered with soil and film. The plants in the G2 and G4 treatments did not receive any additional fertilizer. The amount of granular fertilizer applied in the G2 and G4 treatments was 7560 kg ha⁻¹, equivalent to 58 kg ha⁻1 of total N, 70 kg ha⁻¹ of P₂O₅ and 122 kg ha⁻¹ of K₂O. All granular of fertilizer are applied on the same day the seedlings are transplanted.

Field management involved routine manual weeding as needed based on weed growth and topping when the central inflorescence of the tobacco plants had opened more than 50%. The tobacco plants had an average of 18 leaves, and other management practices were consistent with the conventional agricultural production methods employed locally.

2.4. Sample Collection and Processing

2.4.1. Soil Sampling

Six plants were chosen on days 45, 75, and 105 after transplanting, and soil samples were collected using a steel soil auger with a diameter of 30 mm from the 0–20 cm depth at a horizontal distance of 15 cm from the tobacco stems [13]. After visible roots and gravel were removed, some soil samples were passed through a 2 mm sieve and stored in a refrigerator at 4 °C for determination of soil N pools and ¹⁵N abundance. Another part was air-dried for further processing [13]. Most importantly, we rigorously ensured that there were no obvious residues of granular compost in the collected soil samples.

2.4.2. Plant Sampling

Mature tobacco leaves were collected when they turned “fully yellow.” The upper, middle, and lower leaves were harvested separately. The terms “upper leaves,” “middle leaves,” and “lower leaves” refer to the 1st leaf to the 6th leaf, the 7th to the 12th, and the 13th to the 18th leaf of the tobacco plant from the top to the root [23], respectively. On July 13th, when the lower leaves of the plants had fully “yellowed,” six plants were randomly selected. The middle leaves, upper leaves, stems, and roots of other plants were harvested according to the degree of “yellowing”. The stages were as follows: the middle leaves were harvested twice on July 18th and July 25th, while the upper leaves were collected in two batches on August 2nd and 8th. The stems and roots were collected on August 15th. The soil around the roots was thoroughly washed with deionized water to obtain the roots. All samples were then roasted according to the “442” (with ten key temperature stable points) flue-cured tobacco process, and the weight of the biomass was recorded.

2.5. Measurements

2.5.1. Soil N Pools and N Content of Plants

The method described by Ding et al. [13] and Stevenson [24] was adopted for the sequential extraction of N pools. At a ratio of v: w = 1:4, 20 g soil (dry base) samples were extracted sequentially by 2 M KCl, 0.5 M K₂SO₄, 80 °C hot deionized water and 6 M HCl successively to separate InorgN, MBN, HWON, and HCl-N. Specifically, 20 g fresh soil samples were passed through a 2 mm sieve and were extracted by 80 mL 2 M KCl at 180 rpm/25 °C, and all the fluids were filtered by quantitative filter paper via a continuous flow analyzer (SEAL-AA3, Bran & Luebbe, Hamburg, Germany) to measure the InorgN content. The residue remaining after the extraction of InorgN and filter paper were dried at 50 °C and fumigated to extract MBN, and the leachate was filtered through a 0.45 μm filter and stored at −20 °C. InorgN and MBN were defined as labile N components. The residue remaining after the extraction of MBN was dried at 50 °C and extracted in an 80 °C water bath for 2 h and then shaken at 150 rpm/25 °C for 1 h [22]. The extract was filtered through a 0.45 μm filtration membrane and stored at −20 °C for further analysis [23]. This part was defined as HWON. The residue remaining after the extraction of HWON was placed in an oven and dried at 50 °C. Then, 100 mL 6 M HCl was added, and the sample was kept in a 100 °C water bath for 12 h. The extract was frozen at −20 °C after it was passed through a 0.45 μm filtration membrane, and this component was defined as HCl-N [22]. HWON and HCl-N were defined as moderately liable N pools. Finally, the residue remaining after HCl-N extraction was defined as stable soil N (SSN) [22]. Soil TN was the sum of the five N components. The MBN, HWON and HCl-N contents were determined using a total organic carbon analyzer (Vario TOC Cube, Elementar, Hanau, Germany).

The SSN and plant samples (roots; upper, middle, and lower stems; upper, middle, and lower leaves) were ground to a <300 mesh size using a Retsch^® Mixer Mill MM 200 and analyzed using an elemental analyzer (Vario PYRO cube, Elementar Analysensysteme GmbH, Langenselbold, Germany).

2.5.2. Preparation of Samples to Measure δ¹⁵N

The δ¹⁵N isotopic abundance of InorgN was determined by the microdiffusion method [25]. First, two approximately 9 mm diameter Whatman #42 filter papers were wetted with 20 µL of 2.0 M oxalic acid, and then they were hung at the bottom of a clip attached to the center of a culture bottle cap. The filter paper plate was approximately 2.5 cm away from the bottom of the bottle. Then, 30 mL 2.0 M KCl extract was added to a 150 mL blue cap culture flask that contained three glass beads, and 0.3 g MgO and 0.3 g Dai alloy were added (MgO was burned at 575 °C to remove the impurities nitrogen, and the Dai alloy was ground with a mortar until it could pass through a 300 mesh sieve). The culture bottle was capped and sealed and incubated at 25 °C and 140 r min⁻¹ for 7 days. At the end of incubation, the filter paper tray was removed, wrapped with a tin boat and placed in a 96-well plate for vacuum drying [23].

The liquid samples of MBN, HWON, and HCl-N were freeze-dried and ground using a Retsch^® mixer mill MM 200 to a <300 mesh size. The ¹⁵N abundances of each N pool and plant samples were analyzed using an isotope ratio mass spectrometer (Isoprime 100, Elementar Analysensysteme GmbH, Germany) [23].

2.6. Calculation

Nitrogen accumulation in individual plant organs was calculated by multiplying the N concentration by the dry biomass, and the total N accumulation was the sum of the N accumulation in all organs collected.

NA (kg ha⁻¹) = NP × Weight/1000

(1)

where NA refers to the N accumulation (kg ha⁻¹) and NP (g kg⁻¹) and Weight (kg ha⁻¹) refer to the N content and dry biomass of each organ of the tobacco plant, respectively.

The amount of N uptake (%) from the soil (N_{uptake from soil}) in the different organs was calculated using the following equation [13,23]:

N_{uptake from soil} (%) = 1 − ¹⁵N_{uptake from fertilizer} = (1 − ¹⁵N_P-APE/¹⁵N_F-APE) × 100

(2)

The amount of N absorption (kg ha⁻¹) from the soil (N_{uptake from soil}) in the different organs was calculated using the following equation [13,23]:

N_{uptake from soil} (kg ha⁻¹) = NA × (1 − ¹⁵N_P-APE/¹⁵N_F-APE)

(3)

The amount of N absorption (kg ha⁻¹) from the fertilizer (N_{absorbed from fertilizer}) in the different organs was calculated using the following equation [13,23]:

N_{uptake from fertilizer} (kg ha⁻¹) = NA × ¹⁵N_P-APE/¹⁵N_F-APE

(4)

where ¹⁵N _P-APE (%) is the ¹⁵N atomic percentage excess (i.e., atom% the individual organs of the labeled tobacco plant-natural abundance of ¹⁵N) in the labeled plant, and ¹⁵N_F-APE is the ¹⁵N atomic percentage excess (i.e., atom% of fertilizer -natural abundance of ¹⁵N) in the labeled granular fertilizer. The natural abundance of ¹⁵N was 0.3663%.

The residual in the soil (¹⁵N_{granular fertilizer residue}, kg ha⁻¹) was calculated as follows [13,23]:

¹⁵N_{granular fertilizer residue} = N_soil × ¹⁵N_soil-APE/¹⁵N_F-APE

(5)

where N_soil and ¹⁵N_soil-APE are the total soil N content and ¹⁵N atomic percentage excess in the labeled soil pool (i.e., the atom% of the individual soil nitrogen pool-natural abundance of ¹⁵N), respectively.

The promotion efficiency of nitrogen (PEN) from granulated organic fertilizer in soil N supply can be defined as the N absorption from soil by tobacco plants that is stimulated by a unit mass of fertilizer N, i.e., the ratio of the total soil N absorption by tobacco (kg ha⁻¹) to the total amount of granular fertilizer N absorbed (kg ha⁻¹).

2.7. Statistical Analyses

Significant differences (p < 0.05) were assessed by one-way ANOVA among the different N pools in the soil, the N content of each organ of tobacco plants, N accumulation, and the biomass of each organ of tobacco plants during the sampling period for the three treatments. Differences in ¹⁵N recovery efficiency and ¹⁵N recovery of tobacco plants and soils from the granular fertilizer treatments were assessed by independent sample tests. All data were processed using SPSS (ver. 21.0, IBM, Armonk, NY, USA) and Origin Pro 2023.

3. Results

3.1. Soil N Pools

Figure 2 illustrates the dynamic changes in soil available N components during the growth period for each treatment. The InorgN content of all treatments reached its peak 45 days after transplant and gradually decreased thereafter. Notably, there was no significant difference in the soil InorgN content between the 75th and 105th days after transplant, except for the G4 treatment (Figure 2a). On the 45th day, the InorgN content in the CK treatment was 156.3 mg kg⁻¹, representing a 12.1% increase compared to the G2 treatment and a 51.5% increase compared to the G4 treatment (p < 0.05). On the 75th day, the highest InorgN content was observed in the G4 treatment (42.9 mg kg⁻¹, p < 0.05), followed by the CK and G2 treatments at 36.1 and 21.1 mg kg⁻¹, respectively, as shown in Figure 2a.

In terms of MBN, the soil MBN content of G2 and G4 increased gradually throughout the growth period, while the MBN level of CK decreased gradually with the growth of plants, as depicted in Figure 2b. On the 45th day, the MBN content of CK was 30.0% higher than that of G4 and not significantly different from that of G2. On the 75th day, the MBN content of G4 was 30.7% and 87.2% higher than that of G2 and CK, respectively. On the 105th day, the MBN content of G4 was 30.2% and 96.5% higher than that of G2 and CK, respectively. The MBN of each treatment showed no significant difference on the 75th and 105th days.

Both HWON and HCl-N are types of moderately active N. As depicted in Figure 2c, the content of HWON in the CK treatment first showed an increasing trend and then decreased, and there was no significant difference between the 45th and 105th days. The HWON content in the G2 and G4 treatments showed a gradually increasing trend. Second, on the 45th day after transplant, there was no difference in HWON between the three treated soils, and on the 75th day, the HWON content in G4 was 12.5% and 22.6% higher than that in CK and G2, respectively. On the 105th day, the HWON contents of G4 and G2 were 30.7 and 17.5% higher than those of the CK treatments, respectively (p < 0.05).

The HCl-N content of CK did not change throughout the growth period (Figure 2d), while the HCl-N content of G2 and G4 showed a trend of initial decrease followed by an increase (p < 0.05). Additionally, on the 45th day, the HCl-N content of G4 was 15.6% and 7.14% higher than that of CK and G2, respectively. On the 75th day, the HCl-N content of CK was 19.11% and 11.4% higher than that of G2 and G4, respectively (p < 0.05). On the 105th day, the HCl-N content of G2 and G4 significantly increased compared to that on the 75th day, but there was no significant difference among the three treatments.

3.2. Fertilizer-Derived N Among Soil N Pools

The N component from granular fertilizer in soil varied over time and reflected the nitrogen supply pattern of the fertilizer. On the 45th day, the TN content derived from fertilizer was 10.83 in G2 soils. The primary N component derived from the granular fertilizer was SSN, accounting for 52%, followed by moderately liable N at 26% and liable N at 22% (Figure 3). From the 45th day to 75th day, the TN content derived from fertilizer decreased to 4.83 mg kg⁻¹, the proportion of SSN and labile N decreased, and the proportion of moderately labile N increased. A comparison of the soil samples on the 75th and 105th days shows that there were no significant differences in the proportions of each N component derived from fertilizer in the soil. However, on the 105th day, the soil showed a significant increase in the content of TN derived from the fertilizer (p < 0.05) (Figure 3).

For G4, from the 45th to 105th day, the TN content derived from fertilizer in the soil decreased from 11.55 mg kg⁻¹ to 7.69 mg kg⁻¹ and then increased to 20.18 mg kg⁻¹. The N components derived from fertilizer were consistently dominated by the moderately liable N pool (60–68%) throughout the growth period (Figure 3). Although the proportion of InorgN derived from fertilizer in the soil of the G4 treatment decreased gradually throughout the growth period, contrary to G2, the proportion of InorgN derived from fertilizer in the soil of the G4 treatment was higher than SSN on the 45th day after transplant, and the SSN proportion in G4 gradually increased while it decreased in G2.

3.3. N in Tobacco Organs

As the tobacco plants grew, the distribution of N in different organs varied among the treatments (

Table 1. The N content (g kg⁻¹) in different organs and the proportion of N derived from soil sources (%).

	Treatment	Upper-Leaf	Mid-Leaf	Lower-Leaf	Upper-Shoot	Mid-Shoot	Lower-Shoot	Root
Yellowing of the lower leaf	CK	30.7 ± 0.89 Aa (n)	17.5 ± 0.51 Aa (n)	13.5 ± 0.39 a (n)	8.6 ± 0.25 Ac (n)	8.4 ± 0.24 Ab (n)	8.2 ± 0.23 Ac (n)	12.0 ± 0.35 Ac (n)
	G2	6.2 ± 0.18 Bc (96.8)	9.4 ± 0.27 Bc (96.6)	8.8 ± 0.25 b (96.8)	21.9 ± 0.63 Aa (96.8)	22 ± 0.64 Aa (97.0)	17.6 ± 0.51 Aa (97.5)	24.1 ± 0.70 Aa (97.1)
	G4	9.9 ± 0.29 Bb (97.7)	14.3 ± 0.41 Bb (97.6)	7.2 ± 0.20 c (99.0)	17.3 ± 0.50 Ab (97.4)	23.7 ± 0.68 Aa (97.3)	12.3 ± 0.35 Ab (98.3)	17.6 ± 0.51 Ab (97.7)
Yellowing of the upper leaf	CK	18.4 ± 0.53 Ba (n)	17.4 ± 0.52 Ab (n)	13.5 ± 0.39 a (n)	8.9 ± 0.26 Ab (n)	4.7 ± 0.14 Bc (n)	4.8 ± 0.14 Bb (n)	10.1 ± 0.29 Ac (n)
	G2	14.4 ± 0.42 Ab (97.5)	13.1 ± 0.38 Ac (97.8)	8.8 ± 0.25 b (96.8)	17.4 ± 0.50 Ba (97.7)	20.6 ± 1.17 Aa (97.5)	12.0 ± 0.35 Ba (96.5)	20.2 ± 0.58 Ba (97.2)
	G4	15.2 ± 0.44 Ab (96.7)	20.2 ± 0.58 Aa (96.7)	7.2 ± 0.20 c (99.0)	8.3 ± 0.24 Bb (97.8)	13.3 ± 0.38 Bb (97.0)	3.9 ± 0.11 Bb (96.2)	16.2 ± 0.47 Ab (96.5)

Note: Uppercase letters indicate significant differences between the same treatment at different time periods; lowercase letters indicate significant differences between different treatments in the same time period (p < 0.05); the upper, middle, and lower leaves are the 1st-6th, 7th-12th, and 13th-18th leaves from the top, respectively.

). Throughout the growth period, the leaf N content of the CK treatment remained consistently higher than that of the stems and roots. However, when the lower leaves matured, the N content of the stem and root in the G2 and G4 treatments was significantly higher than that of the leaves. This was in contrast to observations during the maturation of the upper leaves, when the N content of the leaves was higher than that of the stems and roots in the G2 and G4 treatments (p < 0.05). In addition, when the lower leaves matured, the leaf N content of G2 and G4 was significantly lower than that of CK (p < 0.05). However, the N content of stems and roots of the G2 and G4 treatments was significantly higher than that of CK (p < 0.05).

When the upper leaves matured, the N content of the upper leaves in the CK treatment decreased from 30.7 to 18.4 g kg⁻¹, and the N content of the middle and lower stems decreased by 44.1% and 41.5%, respectively. However, the N content of the middle leaves, upper stems, and roots did not significantly change. In addition, the N content of the upper leaves in the G2 and G4 treatments increased from 6.2 and 9.9 g kg⁻¹ to 14.4 and 15.2 g kg⁻¹, respectively, while the N content of the middle leaves increased from 9.4 and 14.3 g kg⁻¹ to 13.1 and 20.2 g kg⁻¹, respectively. Typically, flue-cured tobacco leaves with a total nitrogen content ranging from 1.5% to 2.5% (15–25 g kg⁻¹⁾ can be classified as high-quality tobacco [13,22]. This range effectively balances other chemical components in the leaves, thereby positively influencing their aroma, taste, and combustibility. Consequently, the middle and upper leaves from the G2 and G4 treatments have reached or approached the standard for high-quality flue-cured tobacco. In addition, the N content of the stems and roots in the G2 and G4 treatments decreased.

Overall, the N content of different organs showed the following trend: CK > G4 > G2 in the upper leaves, G4 > CK > G2 in the middle leaves, CK > G2 > G4 in the lower leaves, and the N content of the stems and roots was consistently G2 > G4 > CK.

3.4. Soil N and Fertilizer N in Tobacco Plants

When all the tobacco leaves were fully mature, the cumulative N amounts in CK, G2 and G4 were 43.34 kg ha⁻¹, 63.27 kg ha⁻¹, and 42.17 kg ha⁻¹, respectively (

Table 2. Agronomically significant plant N utilization characteristics.

Index	Position	Treatment
		CK	G2	G4
N input (kg ha−1)		95	58	58
Recovery of nitrogen (kg ha−1)	Upper leaf	10.86 ± 1.34 ab	12.83 ± 0.70 a	9.86 ± 0.62 b
	Middle leaf	9.74 ± 0.67 a	7.42 ± 0.54 b	9.22 ± 0.27 a
	Lower leaf	7.97 ± 1.02 a	5.43 ± 0.33 b	5.85 ± 0.43 b
	Total for leaf	28.57 ± 2.06 a	25.67 ± 1.05 a	24.93 ± 1.11 a
	Shoot	8.09 ± 1.15 b	25.45 ± 1.81 a	8.60 ± 0.77 b
	Root	6.67 ± 0.63 c	12.13 ± 0.24 a	8.63 ± 0.34 b
	Total	43.34 ± 3.47 b	63.27 ± 2.19 a	42.17 ± 1.97 b
Stimulatory effect (%)	Upper leaf	n	38.67	29.28
	Middle leaf	n	44.10	29.03
	Lower leaf	n	30.25	28.40
	The all leaves	n	38.42 ± 2.82 a	28.98 ± 1.26 b
	Stem	n	35.27 ± 1.00 a	32.13 ± 0.45 b
	Root	n	34.51	27.18
	Total tobacco	n	36.07 ± 0.40 a	29.17 ± 0.17 b

Note: Lowercase letters indicate significant differences between different treatments (p < 0.05). The upper, middle, and lower leaves are the 1st-6th, 7th-12th, and 13th-18th leaves from the top, respectively.

). The amount of N uptake in the G2 treatment was found to be 46% and 48% higher than that in the CK and G4 treatments, respectively, which was statistically significant (p < 0.05). The cumulative N amounts in tobacco leaves in CK, G2, and G4 were 28.57 kg ha⁻¹, 25.67 kg ha⁻¹, and 24.93 kg ha⁻¹, respectively. The N uptake of the upper leaves in the G2 treatment was significantly higher than that in the other two treatments (p < 0.05). Conversely, the N uptake of middle and lower leaves in the CK treatment surpassed that of both G2 and G4 (p < 0.05) (Table 2).

Table 1 provides a simultaneous reflection of the soil-derived N in tobacco treated with granular fertilizer at different stages. Throughout the growth period, the proportion of N derived from the soil in tobacco plants treated with granular fertilizer remained consistently above 93% for the G2 and G4 treatments. During the transition from the maturation of the lower leaf to the maturation of the upper leaf, there was an increase in N content derived from the soil in the upper and middle leaves for the G2 treatment, while a decrease was observed for the G4 treatment. A similar trend was also observed in the roots.

We calculated the promotion efficiency of nitrogen (PEN) for granular fertilizer on soil N release, which was the ratio of N assimilated from the soil in tobacco plants to N absorbed from the granular fertilizer. The results indicated that the G4 treatment significantly reduced the promotion of soil N release by 19.2% compared to G2 (Table 2). This result contradicted the higher absorption of N by tobacco from G2 fertilizer compared to G4 (Figure 4), but it was consistent with the lower proportion of N obtained from the soil by tobacco under G4 treatment compared to G2 (Table 1). This could be related to the decrease in tobacco yield under the G4 treatment.

Figure 4 displays the data for the G2 and G4 granular fertilizers on N supply to crops and N retention in soil, as calculated using the ¹⁵N tracing technique in this study. After the tobacco leaves had fully matured, the soil N amounts derived from compost for the G2 and G4 treatments were 11.04 and 45.0 kg ha⁻¹, respectively. The N not released or lost from the granular fertilizer was 44.98 and 11.44 kg ha⁻¹, respectively. The amounts of N absorbed by the tobacco plants from the granular fertilizer in the G4 and G2 treatments were only 1.26 and 1.64 kg ha⁻¹, respectively, accounting for only 2.17% and 2.85% of the total N in the granular fertilizer. These values were significantly lower than the N absorbed by the tobacco plants from the soil, as the aboveground parts of tobacco absorbed 49.8 and 31.9 kg ha⁻¹ N from the soil, respectively.

3.5. Yield

Table 3. Yield of tobacco plant organs (kg ha⁻¹).

Month	Treatment	Upper-Leaf	Mid-Leaf	Lower-Leaf	Upper-Shoot	Mid-Shoot	Lower-Shoot	Root
Yellowing of the lower leaf	CK	288.42 ± 25.86 b	361.22 ± 44.90 c	406.56 ± 63.90 a	274.98 ± 22.35 c	403.52 ± 65.08 b	604.13 ± 84.50 b	791.58 ± 115.89 b
	G2	336.20 ± 62.53 ab	439.38 ± 40.38 bc	333.24 ± 24.99 b	346.21 ± 27.38 bc	490.72 ± 44.64 ab	719.32 ± 74.42 ab	930.89 ± 46.10 a
	G4	392.08 ± 33.96 a	582.08 ± 35.21 a	381.25 ± 35.21 a	512.53 ± 33.20 a	617.87 ± 31.62 a	887.51 ± 31.99 a	877.10 ± 69.48 ab
Yellowing of the upper leaf	CK	629.54 ± 77.72 b	596.86 ± 41.36 a	406.56 ± 63.90 a	369.09 ± 59.97 a	493.79 ± 69.20 a	630.14 ± 89.83 a	705.44 ± 66.21 a
	G2	891.15 ± 48.38 a	566.16 ± 41.02 a	333.24 ± 24.99 a	300.36 ± 13.44 a	320.76 ± 47.55 b	515.83 ± 23.12 bc	600.72 ± 12.10 bc
	G4	648.84 ± 48.60 b	456.36 ± 16.06 b	381.25 ± 35.21 a	265.32 ± 38.33 a	334.08 ± 41.50 ab	502.35 ± 22.22 b	532.89 ± 25.12 b

Note: Lowercase letters indicate significant differences between different treatments at the same time period (p < 0.05); The upper, middle, and lower leaves are the 1st-6th, 7th-12th, and 13th-18th leaves from the top, respectively.

illustrates the tobacco leaf yields of the three treatments. Upon the maturation of the lower leaves, the yield of lower leaves in CK and G4 was 22.00% and 14.41% higher than that in G2, respectively. Additionally, the yields of the middle and upper leaves in G2 and G4 were 21.64% and 61.14% and 35.94% and 16.62% higher than those in CK, respectively. Upon the maturation of the middle and upper leaves, the yield of the upper and middle leaves in CK and G2 increased by 118.27% and 65.23% and 165.06% and 28.85%, respectively, compared to that during the maturation of the lower leaves. While the yield of the upper leaves in G4 increased by 65.49%, the yield of the middle leaves declined by 21.60%. Finally, the tobacco leaf yields were 1631 kg ha⁻¹, 1790 kg ha⁻¹, and 1485 kg ha⁻¹ for CK, G2, and G4, respectively. The yield of the G2 treatment exceeded that of CK and G4 by 9.75% and 20.54%, respectively.

4. Discussion

4.1. N Supply in the Soil-Fertilizer System with Granular Fertilizer Amendment

Recent research has suggested that when organic fertilizers replace synthetic N fertilizers at a proportion of 50% or higher, a potential decrease in grain yield might result [26]. This can be attributed to a possible lack of N supply during early crop growth stages, as organic N requires a longer period of mineralization than synthetic N fertilizer [27,28]. To increase N uptake and crop yield, the slow-release N (organic N) provided by organic fertilizers must undergo mineralization before becoming available to crops [29,30]. Our findings indicated that while the N mineralization rate in granular fertilizers was lower relative to crop uptake, it did not have a negative impact on the overall N accumulation and yield in crops (Table 2 and Table 3). Additionally, the accumulation of N in G2-treated tobacco plants was significantly higher than that in the CK plants (Table 2).

After the tobacco leaves had fully matured, the soil N amounts derived from compost for the G2 was lower than that of G4 treatments. This may be related to the different N release mechanisms of these two fertilizers and the N demand of tobacco at different stages. The amounts of N absorbed by the tobacco plants from the granular fertilizer in the G4 and G2 treatments were significantly lower than the N absorbed by the tobacco plants from the soil. This was consistent with the findings of Yan et al. [14], who suggested that the main nonfertilizer N sources for seasonal crops are the N residues of previous crops and the continuous turnover of soil organic N. Gardner and Drinkwater [31] also proposed that plants take up more than half of their N from sources other than the fertilizers applied in the current year, with a significant portion coming from the mineralization of soil organic N. Furthermore, Scheer et al. [32] conducted an extensive review of studies utilizing ¹⁵N tracers to explore nitrogen (N) cycling and fluxes in agricultural ecosystems. Their findings indicated that the majority (60–90%) of N absorbed by plants originates from preexisting soil N reserves. This clearly demonstrates the reliance of certain cropping systems on the mineralization of soil organic N and residual fertilizers as the primary sources of N. The recent decline in the availability of added organic N sources seems to be associated with carbon limitations, as the carbon source in compost may affect the fixation of biotic N, altering its N transformation and bioavailability [33,34]. Additionally, external inputs of N may promote soil N mineralization and turnover [14,35], particularly under low N input rates [36].

In addition, the InorgN contents derived from the granular fertilizer in soil were approximately 0.10–2.55 mg kg⁻¹ throughout the growth period (Figure 2) and only accounted for 0.4–2.7% of the total InorgN in soil, while the remaining InorgN came from the soil (Figure 3, Figure 4), which was consistent with the findings of Wei et al. [37]. This indicates a lower N supply efficiency in the granular fertilizer in this study. However, it still met the N needs of crops. Therefore, we speculate that the granular fertilizer increases soil N supply by releasing some labile C and N substrates that stimulate microbial activity, i.e., by stimulating N mineralization and promoting the release of soil N. Furthermore, as plants have the advantage of competing with microorganisms for mineral N at the peak of N demand [27,30], a part of the InorgN in the fertilizer matrix is directly transferred to the rhizosphere soil for the uptake and utilization of plants, while the other part of the active N gradually diffuses into the soil matrix in the form of dissolved organic nitrogen (DON). By affecting soil MBN and C/N, DON participates in the MIT process of N in the soil matrix, affecting the release of InorgN in the soil matrix. Further investigation is needed to explore the specific mechanism.

There may be an alternative explanation. Based on the conventional fertilization strategy in the area and in accordance with the approach employed in this study, the recommended amount of conventional N application (in the form of water-soluble fertilizer) for crops was 90 kg N ha⁻¹ (the data were sourced from China Tobacco Corporation, Bijie Tobacco Company). However, due to long-term fertilizer management practices prior to the experiment, a substantial baseline storage of mineral nitrogen in the soil was observed at the beginning of the trial, amounting to 84 kg N ha⁻¹ in the plow layer (0.20 m). This stored mineral N may also be preferentially utilized by crops before the released effective N from the granular fertilizer can be absorbed into the soil.

4.2. Nitrogen Release from Granular Fertilizer to Soil

This study explored the field application effects of large-sized organic fertilizer granules (diameter > 10 mm). It was speculated in Section 4.1 that the release of nutrients in granular fertilizer promotes N release and supply in the soil. Yang et al. [19] determined the content of DON and InorgN in granular organic fertilizer (diameter of 10 mm) through indoor culture experiments, and the results showed that DON decreased sharply within 15 days, while InorgN gradually increased. This indicates that N in granular fertilizer was first converted to DON, and a significant portion of it was mineralized into InorgN within the fertilizer and diffused out from the granules. A small portion of the N diffused into the soil and mineralized. Therefore, for G2 and G4, the formation and diffusion efficiency of InorgN and DON within the fertilizer matrix may affect the content of InorgN (derived from granular compost) in the soil.

From Day 45 to Day 75 after transplant, the content of InorgN from fertilizer in the soil significantly decreased in the G2 and G4 treatments and then remained stable (Figure 2), which was consistent with the trend in N accumulation in tobacco plants (Table S1). Table S1 shows that when the lower leaves of tobacco plants matured (approximately the 75th day after transplant), the accumulation of N was already complete for the most part. Therefore, the dynamics of soil InorgN (including fertilizer-derived InorgN) can reflect the nitrogen demand of tobacco plants. The sharp decline from the early stage (day 45) to the mid-stage (day 75) suggests that tobacco was at its peak demand for nitrogen during this period [38,39].The subsequent stabilization indicates that nitrogen uptake by tobacco plants was essentially complete. The InorgN contributions from G2 and G4 granular fertilizers further corroborate this pattern, demonstrating that the nitrogen supply trend from these granular fertilizers aligns with the soil InorgN dynamics. On Day 45 after transplant, there was no significant difference in the content of soil InorgN and TN between the G2 and G4 treatments (Figure 2a and Figure 3). However, the SSN content and percent in G2 were significantly higher than those in G4 (Figure S1). This may be attributed to the fact that, under the condition of equal TN input, the 2 cm diameter granular organic fertilizer (G2) possesses a larger specific surface area compared to the 4 cm diameter granules (G4), leading to a more rapid release of nitrogen. In addition to the released nitrogen being absorbed by crops or existing in inorganic forms [40], a greater proportion was likely assimilated and immobilized by microorganisms or participated in the formation of more stable organo-mineral complexes [41]. This process consequently increased the content of stable nitrogen pools in the soil. On Day 75, the percent of SSN from fertilizer sources in the soil decreased in the G2 treatment but increased in the G4 treatment (Figure 3), indicating that the nitrogen pools underwent dynamic changes in response to the growth demands of tobacco plants. The only minor fluctuations observed in the proportions of various nitrogen pools between G2 and G4 on both Day 75 and Day 105 further corroborate this interpretation. In addition, the other N components from fertilizer sources were significantly higher in G4 than in G2, except on Day 45 when the same InorgN content was observed (Figure 3), as the tobacco plants grew, resulting in a higher TN content in the final fertilizer source in G4 (Figure 3). In our opinion, up to the 45th day after transplant, the rate at which the G2 fertilizer released InorgN into the soil and retained N was faster than that of G4, but the N fixation efficiency after this period was lower.

The moisture content of the granules after granulation was approximately 36.7%, which may not be conducive to the growth of microorganisms in organic fertilizers. However, G2 granules have a larger contact area with the soil, and soil water (the soil moisture content was approximately 65% at the time of transplanting) can interact with the G2 granules at a faster rate compared to G4. This, in turn, may promote the growth of microorganisms in G2 granules and accelerate the release of InorgN. The InorgN released from the fertilizer may be rapidly absorbed and transformed into organic N, which is then fixed as SSN. SSN is a persistent organic N component similar to humus, and its formation is primarily linked to its combination with clay minerals. Previous studies have demonstrated that as the N content in the soil increases, the relative importance of abiotic N fixation also increases [42,43]. This was mainly attributed to the lattice oxidation of NH₄⁺ [44] and was consistent with our findings in this study.

Additionally, the N not released or lost from the G2 fertilizer was much higher than that of G4 (Figure 4), and the retained G2 fertilizer-sourced N in the soil was significantly lower than that of G4 fertilizer-sourced N. We speculate that the G2 treatment had higher N losses compared to G4. The main reasons were as follows: the availability of large amounts of InorgN may have promoted nitrification in G2 soil. This was due to the larger specific surface area of G2 relative to G4, which theoretically indicated a more pronounced interaction with soil water, leading to better water movement toward the G2 granular fertilizer and the promotion of microbial growth and activity. This could, in turn, enhance N-containing gas loss mediated by microbes. These findings were in accordance with the findings of Yang et al. [19], who observed that a larger contact area between fertilizer and soil may create “hot spots” around the fertilizer and enhance denitrifying microbial activity, thereby accelerating N₂O emissions. Meanwhile, Zhou et al. [45] also found that during rice cultivation, the application of granular fertilizer could reduce ammonia volatilization by 7.6% to 11.0% compared to powdered fertilizer. This reduction is primarily because granular fertilizer has a smaller specific surface area. Large granular fertilizer act as a package., when fertilizer is applied, the film can hinder the loss of N. Furthermore, the slower release of NH₄⁺ from larger granules may reduce the abundance of ammonia-oxidizing bacteria and archaea, which have more affinity for NH₄⁺. This would result in lower N₂O emissions. Consequently, the TN content (derived from fertilizer) resulting from G4 in the soil was ultimately higher than that of the G2 granular fertilizer.

5. Conclusions

Granulation could be a potential way to improve plant N utilization efficiency from fertilizer. Granule size has different effects on the release of N. In this study, a ¹⁵N isotope tracer technique was used to understand the effect of granular organic fertilizer application in field tobacco cultivation. The results clearly showed that the effects of the size of granular organic fertilizer on the yield and quality of flue-cured tobacco were inconsistent. Although the N content of tobacco leaves treated with granular fertilizer was slightly lower than that treated with conventional synthetic nitrogen fertilizer, the yield of flue-cured tobacco was significantly increased with the 2 cm diameter granular fertilizer, while the yield of flue-cured tobacco in the 4 cm treatment slightly decreased. Therefore, despite the higher nitrogen content in leaves from the 4 cm diameter granular fertilizer, a comprehensive consideration of yield factors suggests that the 2 cm diameter granular organic fertilizer can generate better economic returns. From the perspective of production, granular organic fertilizer appears to promote the absorption and utilization of soil N by tobacco plants. Unlike synthetic nitrogen fertilizer, the N within granular fertilizer was gradually released into the soil in the form of easily available organic N and supplied to tobacco plants through soil microbial mineralization. This may impact the absorption and metabolic mechanisms of N by tobacco plants, altering the distribution of N between the soil and tobacco plants. However, since the field verification experiment in this study was only conducted for one year and was influenced by factors such as tobacco variety, annual rainfall, accumulated temperature, and soil conditions, further investigations are needed to understand the importance of granular organic fertilizer for the N MIT processes in soil under complex environmental factors and its potential application in the field.