Effects of Organic Fertilizer Substitution for Chemical Fertilizer on Grain Yield and 2-Acetyl-1-pyrroline (2-AP) of Fragrant Rice

Abstract

Organic fertilizer replacing a portion of chemical fertilizers is a key strategy for improving grain qualities and economic benefits. Fragrant rice, favored by consumers for its superior quality and rich aroma, has garnered significant attention. However, there is little information on the effect of organic fertilizer replacing a portion of chemical fertilizers on the grain yield and 2-AP of fragrant rice. Taking Meixiangzhan2 and Qingxiangyou19 as experimental materials, five different experimental treatments were designed: all urea (T1), 33.3% organic fertilizer substitution for urea (T2), 66.7% organic fertilizer substitution for urea (T3), all organic fertilizer (T4), and no fertilizer as a control (CK). The results showed that the T2 treatment could significantly increase the grain yield of Meixiangzhan2 to 62.50 g·pot⁻¹ and Qingxiangyou19 to 67.88 g·pot⁻¹ due to the increase of 27.90% and 26.03% over T1, and 72.18% and 59.45% over CK, respectively. Compared with T1, the T2 treatment could markedly enhance the 2-AP content in Meixiangzhan2 (418.01 μg kg⁻¹, up by 7.70%) and Qingxiangyou19 (378.53 μg kg⁻¹, up by 9.12%). Relative to CK, the aroma content of these two varieties under the T2 treatment rose by 22.05% and 31.04%, respectively. The main reasons were due to the increase in pyrroline-5-carboxylic acid, 1-pyrroline, proline dehydrogenase, and pyrroline-5-carboxylate synthase. The dry matter accumulation, leaf area, and photosynthetic rate of Meixiangzhan2 and Qingxiangyou19 were also significantly increased, and the activities of nitrate reductase and glutamine synthetase were also significantly improved. Moreover, the activities of peroxidase and catalase in rice sword leaves were remarkably improved, and the content of malondialdehyde was significantly decreased. The results showed that 33.3% of organic fertilizer instead of chemical fertilizer had the positive effect of increasing the grain yield and improving the aroma of rice, which was worth further popularization and application.

1. Introduction

Rice (Oryza sativa), a core cereal crop, serves as the primary energy source for approximately half of the global population. Within the Chinese agricultural system, rice holds distinct strategic importance, with its products forming the dietary foundation for over 65% of the population [1]. Alongside transformations in socioeconomic structures and rising consumer demands, the market has placed increasing emphasis on the nutritional quality and flavor profiles of rice. Among these, fragrant rice, enriched with the volatile aromatic compound 2-acetyl-1-pyrroline (2-AP), has emerged as a significant category in the premium rice market [2]. Notably, the escalating sale prices and consumption volumes of fragrant rice reflect a market trend that has spurred systematic research within the scientific community, spanning germplasm innovation, cultivation physiology, and the regulation of aroma metabolism [3,4].

In rice cultivation’s nutrient system, nitrogen metabolism exerts a dual regulatory effect on the yield formation and quality development [5]. Previous studies have shown a significant dose-dependent relationship between nitrogen fertilizer application and the agronomic traits of fragrant rice varieties. Specifically, when nitrogen application rates range from 150 to 220 kg N/ha, both fragrant and non-fragrant varieties exhibit positive responses in yield components, including the number of effective panicles, grains numbers per panicle, and thousand-grain weight [6]. Reasonable nitrogen management not only significantly enhances the rice yield, but also improves dry matter accumulation and nitrogen use efficiency [7]. Furthermore, increased nitrogen input elevates the protein content in rice grains, reduces the chalky grain percentage, and enhances the milling quality, leading to higher brown rice and head rice recovery rates. These changes positively impact the nutritional value and processing characteristics of rice [8]. More critically, nitrogen metabolism is intricately linked to the biosynthetic pathway of 2-acetyl-1-pyrroline (2-AP). For instance, applying 30 kg/ha of nitrogen at the heading stage significantly increases the aroma content in fragrant rice grains [9]. In summary, nitrogen fertilizer application plays a pivotal role in improving rice yield and enhancing the grain aroma.

Within the context of sustainable agriculture, the integrated application of organic and inorganic fertilizers has emerged as a critical strategy to address nutrient supply imbalances caused by single-fertilizer use. Organic fertilizers, characterized as slow-release nutrient sources, offer the advantage of regulating the nutrient release compared to traditional chemical fertilizers, thereby more effectively meeting the nutrient demands of rice throughout its growth cycle [10]. To date, research on the substitution of chemical fertilizers with organic fertilizers in conventional rice systems has been relatively extensive. Multiple studies have demonstrated that the scientifically balanced application of organic fertilizers with nitrogen fertilizers can significantly enhance the rice yield, improve the grain quality, and increase the nutrient use efficiency [11,12]. Previous studies showed that organic fertilizer substitution for chemical could improve grain qualities, increasing the nitrogen use efficiency and reducing the adverse effects of excessive nitrogen application on the environment [13]. However, the impact of varying substitution ratios of organic fertilizers for nitrogen fertilizers on the aroma characteristics of fragrant rice, particularly in early-season indica rice, remains insufficiently explored in existing studies.

Given this background, we hypothesized that (1) organic fertilizer substitution for chemical fertilizer can increase the grain yield of fragrant rice and (2) organic fertilizer substitution for chemical fertilizer can improve the 2-AP content of fragrant rice. This study primarily investigated the effects of different ratios of organic and chemical fertilizer combinations on the grain yield, 2-AP formation, and its physiological traits of fragrant rice (Qingxiangyou19 and Meixiangzhan2). Both Qingxiangyou19 and Meixiangzhan2 are widely planted in the area of south China because of their better grain qualities. This was achieved by measuring physiological activities at key growth stages (mid-tillering, panicle initiation, and heading stages), the yield and its components, the 2-acetyl-1-pyrroline (2-AP) content, and substances related to 2-AP biosynthesis. Mid-tillering, panicle initiation, and heading stages are important periods that determine the panicle number per ha, spikelets per panicle, and 1000-grain weight, respectively. The results elucidated the specific impacts of substituting chemical fertilizers with organic fertilizers on the aroma and physiological characteristics of fragrant rice, providing a scientific foundation and practical fertilization guidance for the development of high-quality, efficient cultivation techniques for fragrant rice.

2. Materials and Methods

2.1. Plant Materials and Experimental Design

This study was conducted during the early rice-growing season (March to July) of 2024 in a net-room at the Experimental Research Farm of the College of Agriculture, South China Agricultural University, Guangzhou, China (23°16′ N, 113°23′ E, altitude 11 m). The net-room is mainly composed of stainless steel wires with a diameter of 1 cm to prevent rats and birds from entering. The region is characterized by a typical subtropical monsoon climate, with an average temperature of 23.1 °C and annual precipitation of 2536.7 mm in 2024. The experimental soil was a sandy loam, with 21.44 organic C, 1.70 g kg⁻¹ total N, 1.24 g kg⁻¹ total P, 22.85 g kg⁻¹ total K, 79.03 mg kg⁻¹ available P, 108.30 available K, and pH = 6.50. Two indica-type fragrant rice varieties with distinct jasmine-like aroma traits, Qingxiangyou19 and Meixiangzhan2, were selected as test materials, with seeds provided by the College of Agriculture, South China Agricultural University.

For seed pre-treatment, surface sterilization was performed using a 5% sodium hypochlorite solution, followed by five rinses with ultrapure water. Seeds were then soaked in water for 12 h and germinated in a dark constant-temperature incubator at 38 °C for 12 h. Germinated seeds were uniformly sown in PVC seedling trays. After 22 days of seedling growth, three-leaf-age seedlings were selected and transplanted into plastic pots (bottom diameter 26 cm, height 25 cm) filled with 10 kg of dry sandy loam soil. Five-hill seedlings were transplanted in a cross line, with four rice seedlings in each hill for every pot.

A gradient substitution strategy was employed to establish the following five fertilization treatments:

CK: no fertilizer application (zero-nitrogen control).

T1: full application of urea (100% urea).

T2: 33.3% organic fertilizer substitution for urea (33.3% organic fertilizer + 66.7% urea).

T3: 66.7% organic fertilizer substitution for urea (66.7% organic fertilizer + 33.3% urea).

T4: full application of organic fertilizer (100% organic fertilizer).

Each treatment was planted for 10 pots and arranged in a split plot design. The organic fertilizer used in the experiment was sourced from Xinyangguang Organic Fertilizer Co., Ltd., Changchun Jilin Province, China. This kinds of organic fertilizer belonged to animal manure (43% organic matter, 2.31% nitrogen, 2.98% phosphorus, and 3.82% potassium). Nitrogen application rates for all treatments were standardized at 150 kg N/ha.

2.2. Measurement of Yield and Its Component Factors

During the maturity stage, tree pots were randomly selected from each treatment for harvesting (n = 12 hills). The grains were manually threshed and air-dried to an approximately 14% moisture content for yield determination. The number of panicles per treatment was calculated by averaging the panicle count per plant from two different pots. The total number of grains per panicle was determined by manually threshing each panicle and counting the grains. A seed air-screen cleaner was used to separate the grains, enabling the calculation of the filled grain number and seed-setting rate. The thousand-grain weight was obtained by counting and weighing the filled grains from each treatment and calculating the average.

2.3. Total Aboveground Biomass Accumulation (TAB) and Determination of Leaf Area

At the tillering stage (MT), panicle initiation stage (PI), and full heading stage (HS) of rice growth, four representative rice plants were randomly selected from each treatment. The plants were cleaned and separated into their respective organs (stems, leaves, and panicles). Leaf area per plant was calculated by measuring the leaf area of standard leaves using the length–width coefficient method. Subsequently, the separated plant components were oven-dried at 80 °C to a constant weight, after which the aboveground dry matter accumulation (TAB) was determined [9].

2.4. Determination of Photosynthetic Characteristics

The photosynthetic characteristics of rice flag leaves were measured at the full heading stage at 9:00–11:00 on a sunny morning using a portable photosynthesis system (LI-6400, LI-COR, Lincoln, NE, USA). During the measurements, the leaf surface environmental parameters were set as follows: the photosynthetic active radiation intensity was maintained at 1100~1200 μmol·m⁻²·s⁻¹, an ambient CO₂ concentration was held at 400.0 μmol·mol⁻¹, the temperature was controlled at 30.0 ± 0.5 °C, and the relative humidity was kept within the range of 60~80% [9].

2.5. Determination of Anti-Stress Enzyme Activity and MDA Content in Leaves

Samples were collected at key rice growth stages (tillering, panicle initiation, and full heading stages). From each treatment, 25 flag leaves were randomly selected and stored in an ultra-low-temperature freezer at −80 °C for subsequent analysis of the leaf antioxidant response parameters. The activities of peroxidase (POD) and catalase (CAT), as well as the malondialdehyde (MDA) content, were determined following the methods described by Mostofa et al. (2020) [14]. POD and CAT activities were expressed as U·min⁻¹·g⁻¹ FW (FW, fresh weight), while the MDA content was expressed as μmol·g⁻¹ FW.

2.6. Determination of Leaf-Nitrogen-Metabolizing Enzyme Activity

Sampling was conducted at the tillering, panicle initiation, and full heading stages of rice growth. From each treatment, 25 flag leaves were randomly selected and stored at −80 °C for the analysis of key nitrogen metabolism enzyme activities. The extraction and activity levels of nitrate reductase (NR) and glutamine synthetase (GS) were determined following the methods described by Mo et al. (2018) [9]. Absorbance was measured at 540 nm, with NR activity expressed as mg·min⁻¹·g⁻¹ FW and GS activity expressed as U·min⁻¹·g⁻¹ FW.

2.7. Determination of 2-AP Content

At the maturity stage, approximately 10 g of fresh grain samples were randomly collected from each treatment and stored at −80 °C for the determination of the aroma characteristic compound 2-acetyl-1-pyrroline (2-AP) content. The preparation and measurement of the 2-AP content in grain samples were conducted following the method described by Mo et al. (2018) [9]. The 2-AP content was analyzed using a GCMS-QP 2010 Plus system (Shimadzu Corporation, Kyoto, Japan), with the 2-AP content expressed in units of μg·kg⁻¹ FW.

2.8. Determination of P5C, Pyrroline, Methylglyoxal Content, and P5CS Activity in Leaves

At the tillering, panicle initiation, and full heading stages, 20 flag leaves were randomly selected from each treatment and stored at −80 °C for the determination of compounds related to 2-AP biosynthesis. The contents of Δ1-pyrroline-5-carboxylate (P5C), pyrroline, and methylglyoxal, as well as the activity of Δ1-pyrroline-5-carboxylate synthetase (P5CS), were measured according to the methods described by Mo et al. (2018) [9]. The contents of P5C, pyrroline, and methylglyoxal were expressed as μmol·g⁻¹ FW. Absorbance was measured at 535 nm and P5CS activity was expressed as μmol·g⁻¹·h⁻¹ FW.

2.9. Data Analysis

Experimental data were subjected to outlier removal and data standardization using Microsoft Excel 2016 and IBM SPSS Statistics Version 27.0.1 (IBM Crop., Armonk, NK, USA). Multiple comparisons were performed using the least significant difference (LSD) test. Data are presented as means ± standard error (Mean ± SE). All analyses were based on three independent biological replicates.

3. Results

3.1. Yield and Yield Components

Fertilization treatments significantly influenced the rice yield. As shown in

Table 1. Effect of organic fertilizer substitution for chemical fertilizer on the yield and yield-related attributes of fragrant rice.

Cultivar	Fertilization Treatment	Effective Panicle (Panicle per Hill)	Grain Number per Panicle (Grain per Panicle)	Grain-Filling Percentage (%)	1000-Grain Weight (g)	Yield (g/pot)
Meixiang zhan2	CK	8.00 ± 0.58 c	90.00 ± 2.88 b	77.4 ± 0.31 a	16.35 ± 0.11 c	36.28 ± 1.48 d
	T1	10.00 ± 0.58 b	95.29 ± 4.21 b	74.68 ± 0.40 b	17.23 ± 0.06 b	48.86 ± 2.09 bc
	T2	12.67 ± 0.33 a	112.25 ± 2.26 a	78.00 ± 0.97 a	17.65 ± 0.09 a	62.50 ± 0.37 a
	T3	10.67 ± 0.67 b	97.56 ± 4.93 b	69.79 ± 1.00 c	17.35 ± 0.15 ab	50.11 ± 1.56 b
	T4	9.33 ± 0.88 bc	95.47 ± 4.36 b	73.02 ± 0.91 b	17.26 ± 0.07 b	44.54 ± 2.23 c
Qingxiang you19	CK	9.33 ± 0.33 d	71.29 ± 3.63 c	75.89 ± 1.39 ab	21.12 ± 0.05 a	42.49 ± 0.88 c
	T1	10.67 ± 0.33 c	96.83 ± 3.12 ab	76.70 ± 0.74 a	21.29 ± 0.22 a	53.86 ± 0.59 b
	T2	14.00 ± 0.58 a	105.44 ± 1.53 a	71.18 ± 2.22 abc	21.51 ± 0.23 a	67.88 ± 4.26 a
	T3	12.33 ± 0.33 b	98.21 ± 3.09 ab	68.07 ± 2.22 c	21.21 ± 0.15 a	55.85 ± 1.32 b
	T4	10.33 ± 0.33 cd	93.98 ± 4.85 b	70.39 ± 1.92 bc	21.39 ± 0.17 a	46.62 ± 2.15 b
ANOVA	Variety (V)	12.960 **	4.596 *	5.964 *	2060.677 **	85.928 **
	Fertilization Treatments (F)	22.660 **	15.201 **	10.488 **	9.155 **	93.850 **
	V×F	0.260 ns	2.614 ns	2.629 ns	3.350 *	2.502 ns

Within the same column, different lowercase letters indicate significant differences between treatments (p < 0.05), while identical letters denote no statistical difference (p ≥ 0.05). Data are presented as means ± standard error (Mean ± SE). CK, no fertilization; T1, 100% urea application; T2, 2/3 urea and 1/3 organic manure; T3, 1/3 urea and 2/3 organic manure; T4, 100% organic fertilizer application. V: Variety; F: Fertilization Treatments; V×F: Interaction between variety and fertilization treatments; ** represent a significant difference at p < 0.01, * represent a significant difference at p < 0.05, respectively; ns represents a non-significant difference.

, the grain yields of the two fragrant rice varieties across different treatments followed the trend: T2 > T3 > T1 > T4 > CK. Specifically, compared to the sole urea application (T1), the yield of Meixiangzhan2 under the T2 treatment increased significantly by 27.9%, reaching approximately 62.5 g per pot. Similarly, Qingxiangyou19 exhibited the highest yield under the T2 treatment, approximately 67.88 g per pot, representing a 26.03% increase over T1. For Meixiangzhan2, the T2 treatment resulted in significant increases of 26.7% in effective panicles and 18.06% in grains per panicle compared to T1. A similar pattern was observed in Qingxiangyou19, where the T2 treatment increased effective panicles and grains per panicle by 31.2% and 8.89%, respectively, relative to T1. Notably, significant differences in the rice yield were also observed among the varying ratios of organic and inorganic fertilizer combinations. Compared to T3, the T2 treatment increased the yield, effective panicles, and grains per panicle of Meixiangzhan2 by 24.72%, 18.74%, and 15.06%, respectively. In Qingxiangyou19, the T2 treatment improved the yield, effective panicles, and grains per panicle by 21.54%, 13.54%, and 7.36%, respectively, compared to T3.

3.2. Average Growth Rate (AGR)

The average growth rate (AGR) of the two fragrant rice varieties, Meixiangzhan2 and Qingxiangyou19, under different treatment conditions is presented in Figure 1. The substitution of nitrogen fertilizer with organic fertilizer significantly affected the average growth rate of both varieties, with the T2 treatment exhibiting the most pronounced effect. Compared to the T1 treatment, the T2 treatment increased the AGR of Meixiangzhan2 by 16.84% and 90.65% during the MT-PI and PI-HS stages, respectively, with the most substantial increase observed during the PI-HS stage. During the PI-HS stage, the T3 treatment resulted in a significant increase of 74.97% in the AGR compared to T1. Similarly, for Qingxiangyou19, the T2 treatment elevated the AGR by 33.85% and 15.67% during the MT-PI and PI-HS stages, respectively, relative to T1, although these increases did not reach statistical significance. Among the treatments involving organic fertilizer substitution for nitrogen fertilizer, the T2 treatment exerted a stronger positive effect on the AGR than the T3 treatment, though the difference between the two was not statistically significant.

3.3. Leaf Area and Photosynthetic Characteristics

As shown in Figure 2, the leaf area per plant was significantly influenced by the fertilizer type. Compared to the CK treatment, the T1, T2, T3, and T4 treatments significantly increased the leaf area in both varieties during the PI and HS stages. Among these treatments, T2 exhibited the most pronounced effect. At the HS stage, the leaf area per plant under T2 was 20.11% and 18.18% higher than that under T1 for Meixiangzhan2 and Qingxiangyou19, respectively. However, no significant differences were observed between the T3 and T1 treatments during the PI and HS stages for either variety.

The analysis of the effects of the fertilizer type on the photosynthetic physiological parameters is presented in

Table 2. Effect of organic fertilizer substitution for chemical fertilizer on photosynthetic properties of fragrant rice.

Cultivar	Fertilization Treatment	Transpiration Rate (mmol m−2 s−1)	Net Photosynthetic Rate (µmol m−2 s−1)	Intercellular CO2 Concentration (µmol mol−1)	Stomatal Conductance (mol m−2 s−1)
Meixiang zhan2	CK	6.37 ± 0.47 b	12.88 ± 0.65 b	304.42 ± 1.98 b	0.30 ± 0.02 a
	T1	7.09 ± 0.31 b	14.54 ± 0.80 ab	302.63 ± 5.17 b	0.34 ± 0.05 a
	T2	8.88 ± 0.36 a	15.26 ± 0.64 a	285.82 ± 5.72 c	0.37 ± 0.02 a
	T3	7.41 ± 0.52 b	14.08 ± 0.36 ab	308.51 ± 2.69 b	0.35 ± 0.03 a
	T4	7.14 ± 0.26 b	15.07 ± 0.17 a	322.90 ± 1.92 a	0.39 ± 0.02 a
Qingxiang you19	CK	7.12 ± 0.67 b	13.77 ± 1.57 b	315.52 ± 7.38 a	0.34 ± 0.04 a
	T1	8.85 ± 0.31 ab	14.71 ± 0.50 ab	311.14 ± 0.58 ab	0.38 ± 0.01 a
	T2	9.54 ± 0.67 a	17.25 ± 1.37 a	301.44 ± 1.80 b	0.45 ± 0.05 a
	T3	8.67 ± 0.41 ab	16.36 ± 0.75 ab	309.21 ± 2.42 ab	0.36 ± 0.08 a
	T4	8.44 ± 0.73 ab	15.84 ± 0.31 ab	311.63 ± 3.61 ab	0.39 ± 0.05 a
ANOVA	Variety (V)	13.250 **	5.438 *	4.022 ns	1.948 ns
	Fertilization Treatments (F)	6.238 **	3.483 *	9.784 **	1.285 ns
	V×F	0.411 ns	0.574 ns	3.683 *	0.194 ns

Within the same column, different lowercase letters indicate significant differences between treatments (p < 0.05), while identical letters denote no statistical difference (p ≥ 0.05). Data are presented as means ± standard error (Mean ± SE). CK, no fertilization; T1, 100% urea application; T2, 2/3 urea and 1/3 organic manure; T3, 1/3 urea and 2/3 organic manure; T4, 100% organic fertilizer application. V: Variety; F: Fertilization Treatments; V×F: Interaction between variety and fertilization treatments; ** represents a significant difference at p < 0.01, * represents a significant difference at p < 0.05, respectively; ns represents a non-significant difference.

. The T2 treatment significantly enhanced the carbon assimilation capacity. For Meixiangzhan2, the net photosynthetic rate (Pn) and transpiration rate (Tr) under T2 increased by 4.97% and 25.34%, respectively, compared to T1. For Qingxiangyou19, the corresponding increases in Pn and Tr were 7.8% and 17.32%, respectively, relative to T1. Compared to the T3 treatment (another organic–inorganic fertilizer combination), the T2 treatment increased Pn and Tr in Meixiangzhan2 by 8.38% and 19.83%, respectively. In Qingxiangyou19, Pn and Tr under T2 were elevated by 5.44% and 10.03%, respectively, compared to T3. Notably, as the proportion of organic fertilizer substituting nitrogen fertilizer increased, the intercellular CO₂ concentration exhibited a trend of initially decreasing and then increasing. However, except for the T2 treatment, differences among the other treatments did not reach statistical significance. Stomatal conductance in fragrant rice was less affected by the fertilizer type, with differences observed between treatments, but not at a statistically significant level.

3.4. Antioxidant Enzyme Activities and MDA Content in Leaves

As shown in Figure 3, the substitution of chemical fertilizers with organic fertilizers significantly modulated the antioxidant system of fragrant rice, exerting notable influences on CAT and POD activities, as well as the MDA content. For Meixiangzhan2, compared to the T1 treatment, the T2 treatment increased the CAT activity by 26.91%, 23.66%, and 27.55% at MI, PI, and HS stages, respectively, demonstrating a significant enhancement effect. Similarly, in Qingxiangyou19, the T2 treatment elevated the CAT activity by 18.68%, 20.8%, and 24.56% at the MI, PI, and HS stages, respectively, compared to T1, also indicating a significant positive effect. A similar trend was observed in other treatments, with the CAT activity under T2 being significantly higher than that under T1, T3, and CK, with the lowest CAT activity recorded in the CK treatment. As the proportion of organic fertilizer substituting nitrogen fertilizer increased, the POD activity in both fragrant rice varieties exhibited a trend of initially increasing and then decreasing across the MI, PI, and HS stages. For Meixiangzhan2, compared to T1, the T2 treatment reduced the MDA content by 17.4%, 12.7%, and 14.22% at the MI, PI, and HS stages, respectively. Likewise, in Qingxiangyou19, the MDA content under T2 was reduced by 13.59%, 11.62%, and 16.05% at the same stages, respectively, compared to T1. Notably, during the MI, PI, and HS stages of both varieties, the T3 treatment also reduced the MDA content compared to T1, though the reduction did not reach a statistically significant level.

3.5. Nitrogen Metabolism-Related Parameters in Leaves

As shown in Figure 4, the substitution of chemical fertilizers with organic fertilizers significantly influenced the activities of key nitrogen metabolism enzymes—NR and GS. Across MT, PI, and HS stages of both fragrant rice varieties, the T2 treatment exhibited the highest NR and GS activities among all treatments. For Meixiangzhan2, compared to T1, the T2 treatment increased the NR activity by 21.7%, 28.17%, and 20.68% at the MT, PI, and HS stages, respectively, while the GS activity was enhanced by 23.76%, 20.68%, and 19.67% at the same stages, respectively, demonstrating a significant enhancement effect. Although the NR activity under the T3 treatment was elevated during the PI and HS stages compared to T1, the differences did not reach statistical significance. For Qingxiangyou19, the effect of substituting chemical fertilizers with organic fertilizers was even more pronounced. Compared to T1, the T2 treatment significantly increased NR activity by 47.69% and 41.45% at the PI and HS stages, respectively, while the T3 treatment also significantly elevated NR activity at the PI stage compared to T1. Similarly, GS activity under the T2 treatment increased by 22.29%, 26.63%, and 17.7% at the MT, PI, and HS stages, respectively, compared to T1.

3.6. 2-AP Content of Grain in Fragrant Rice

As shown in Figure 5, varying ratios of organic fertilizer substitution for nitrogen fertilizer exerted a significant regulatory effect on the 2-acetyl-1-pyrroline (2-AP) content in rice grains. For Meixiangzhan2, compared to T1, the T2, T3, and T4 treatments increased the 2-AP content by 7.7%, 4.26%, and 2.85%, respectively, with the T2 treatment showing the most significant enhancement. The increases in T3 and T4, however, did not reach statistical significance. All treatments resulted in a significantly higher 2-AP content compared to the CK treatment. For Qingxiangyou19, the T2 treatment significantly elevated the 2-AP content by 9.12% compared to T1, whereas the difference in the 2-AP content between T1 and T3 was minimal and not statistically significant. These results indicate that the effect of different organic fertilizer substitution ratios on the 2-AP content varies between rice varieties.

3.7. Indicators Related to 2-AP Biosynthesis

As shown in Figure 6, the substitution of chemical fertilizers with organic fertilizers significantly increased the contents of Δ1-pyrroline-5-carboxylic acid (P5C), Δ1-pyrroline-5-carboxylic acid synthetase (P5CS) activity, 1-pyrroline, and methylglyoxal in the leaves of the two fragrant rice varieties, Meixiangzhan2 and Qingxiangyou19. At the HS, for Meixiangzhan2, the T2 treatment resulted in a significantly higher P5C content and P5CS activity in leaves compared to T1, with increases of 11.03% and 11.18%, respectively. In Qingxiangyou19, the T2 treatment elevated the P5C content and P5CS activity by 16.23% and 17.72%, respectively, compared to T1.

A similar trend was observed in the T3 treatment, where the P5C content and P5CS activity were also significantly higher than those in T1. However, during the same period, the differences in the 1-pyrroline content between T1 and T3 were not significant for either variety. Furthermore, the results indicated that, across all treatments, the T2 treatment consistently achieved the highest or near-highest levels of P5C, P5CS, 1-pyrroline, and methylglyoxal in the leaves of both varieties, demonstrating the most favorable metabolic effect.

3.8. Correlation Analysis

As shown in Figure 7, the experimental results revealed a positive and significant correlation between the yield and both the number of effective panicles and grains per panicle. The yield was also positively correlated with the transpiration rate, net photosynthetic rate, and stomatal conductance, exhibiting a significant positive correlation with nitrogen metabolism enzymes and antioxidant enzymes. The 2-acetyl-1-pyrroline (2-AP) content showed a significant positive correlation with nitrogen metabolism enzyme activities, as well as with the contents of Δ1-pyrroline-5-carboxylic acid (P5C), pyrroline, and methylglyoxal, and Δ1-pyrroline-5-carboxylic acid synthetase (P5CS) activity. Notably, the 2-AP content was significantly negatively correlated with the malondialdehyde (MDA) content, suggesting a potential link between aroma synthesis in fragrant rice and its stress resistance capacity.

4. Discussion

4.1. Grain Yield and Yield Components

Environmental factors, nutrient management, and cultivation practices significantly influence the rice yield formation [15]. Previous studies have demonstrated that different fertilization strategies can effectively regulate the yield of fragrant rice [16]. In this experiment, substituting chemical fertilizers with organic fertilizers significantly increased the yield of both fragrant rice varieties, with the T2 treatment achieving the best value. The increase in yield was primarily attributed to the synergistic enhancement of effective panicles and grains per panicle [17]. The rational combination of organic and inorganic fertilizers provided sustained nutrient support for grain development during the reproductive growth stage of fragrant rice [18]. Existing studies have indicated that improvements in biomass accumulation, the leaf area, and photosynthetic efficiency can enhance the formation of effective panicles per unit area and the development of grains per panicle [19]. In this study, the T2 treatment, involving organic fertilizer substitution for nitrogen fertilizer, significantly increased biomass accumulation, the leaf area, and net photosynthetic rate in fragrant rice, aligning with the findings of Moe et al. [15]. Changes in the physiological characteristics of fragrant rice further supported its yield benefits. These findings suggest that the ratio of an organic to inorganic fertilizer application may exhibit a threshold effect on the yield formation.

Based on these results, we speculate that organic fertilizer substitution promoted the formation of effective tillers, subsequently increasing the number of effective panicles at the full heading stage. This inference is supported by the theoretical framework of Kakar et al. [20]. Maybe the nutrient release cycle of organic fertilizer is long, which can increase the leaf area and photosynthetic capacity in fragrant rice, finally enhancing aboveground biomass accumulation. This effect strengthened the transport of assimilates from stems to panicles during the reproductive growth stage, ultimately manifesting as a significant yield increase. However, compared to T1, the yield enhancement under T3 did not reach statistical significance. This may be attributed to an excessively high organic fertilizer substitution ratio in T3, which likely led to an insufficient nitrogen supply during the vegetative growth stage, limiting effective tiller formation and, consequently, failing to significantly increase the number of effective panicles, thus constraining yield gains. In summary, organic fertilizer substitution for chemical fertilizers demonstrates substantial potential for yield enhancement in fragrant rice cultivation, though its effectiveness may exhibit a threshold effect. Based on these findings, we recommend adopting a 33.3% organic fertilizer substitution for chemical fertilizer method to meet the rhythm of the nitrogen supply, ensuring a balance between vegetative and reproductive growth, thereby maximizing the fragrant rice yield. This strategy provides a reference scientific basis for the efficient cultivation of fragrant rice. However, since this study was conducted under pot conditions, future research is recommended to further investigate the effects of 33.3% organic fertilizer substitution for urea and other substitution ratios under field conditions to confirm their applicability and effectiveness in practical agricultural production. Future studies should focus on optimizing fertilization ratios and evaluating the performance under different environmental and management conditions to provide more comprehensive fertilization guidance, thereby promoting the cultivation of fragrant rice and the sustainable development of agriculture.

4.2. 2-AP Content, Aroma-Related Precursors, and Aroma Enzymes

It is well established that 2-acetyl-1-pyrroline (2-AP) is the key volatile compound influencing the aroma of fragrant rice [21]. Previous studies have demonstrated that optimizing crop management practices can significantly enhance 2-AP accumulation in fragrant rice [9]. The results of this experiment align with these earlier findings, showing that a fertilization strategy involving the substitution of nitrogen fertilizer with organic fertilizer effectively increased the 2-AP content in fragrant rice grains. Compared to the unfertilized control (CK) and sole nitrogen fertilizer treatment (T1), treatments with organic fertilizer substitution (T2 and T3) elevated the 2-AP content in fragrant rice grains. Notably, the T2 treatment (2/3 urea + 1/3 organic fertilizer) resulted in the highest 2-AP content in the grains of both fragrant rice varieties, with significant differences compared to other treatments (p < 0.05). These findings suggest that an appropriate ratio of organic fertilizer substitution for chemical fertilizer contributes to an increased 2-AP content in fragrant rice grains.

Previous research has confirmed that 2-AP biosynthesis is closely associated with precursor compounds (P5C, pyrroline, and methylglyoxal) and the key enzyme Δ1-pyrroline-5-carboxylic acid synthetase (P5CS) [22]. This experiment validated the positive regulatory effect of organic fertilizer substitution for nitrogen fertilizer on these critical aroma-related factors. The results indicated that the T2 treatment significantly increased the contents of P5C, 1-pyrroline, and methylglyoxal, while also enhancing P5CS activity. Correlation analysis revealed a significant positive relationship between the 2-AP content and both aroma precursors and enzyme activity, consistent with the conclusions of Li et al. [9]. This suggests that organic fertilizer substitution indirectly drives 2-AP biosynthesis by promoting the accumulation of precursor compounds and elevating key enzyme activity. Additionally, differences were observed among treatments with varying organic fertilizer substitution ratios. For instance, compared to T3, the T2 treatment significantly increased the P5C content and P5CS activity, though the difference in the methylglyoxal content was not significant. These results indicate that the effects of different fertilization ratios on parameters related to 2-AP biosynthesis are specific, with the T2 treatment (1/3 organic fertilizer substitution) exhibiting a superior overall effect in promoting aroma compound synthesis.

4.3. Physiological Traits

Nitrate reductase (NR) and glutamine synthetase (GS), key enzymes in nitrogen metabolism, directly regulate the nitrogen uptake and assimilation efficiency [23]. This study revealed that substituting chemical fertilizers with organic fertilizers enhanced NR and GS enzyme activities in flag leaves during PI and HS stages. This enhancement may stem from the sustained nutrient supply provided by organic fertilizers, which offers energy support to maintain high enzyme activity. Elevated NR and GS activities serve as a critical foundation for improving the nitrogen use efficiency. Accordingly, we infer that organic fertilizer substitution optimized the nitrogen supply, providing sufficient nutrients during the vegetative growth stage to promote effective tiller formation, thereby increasing the number of effective panicles at the heading stage. This result is consistent with Moe et al. [24]. Additionally, peroxidase (POD) and catalase (CAT), as key antioxidant enzymes, scavenge reactive oxygen species (ROS) and protect cellular integrity [25], while malondialdehyde (MDA) reflects oxidative stress levels [26]. The results showed that organic fertilizer substitution increased POD and CAT activities. At the same time, it reduced the MDA content. This suggests that organic fertilizers alleviated oxidative stress in fragrant rice, reducing cell membrane damage and enhancing the antioxidant capacity. This phenomenon indicates that organic fertilizer application effectively mitigated oxidative stress in rice, decreasing lipid peroxidation damage to cell membranes and thereby bolstering plant stress resistance, a finding consistent with previous studies [27]. Previous studies have confirmed that increased nitrogen levels positively promote the enhancement of antioxidant enzyme activities [28]. Thus, we hypothesize that the slow-release properties of organic fertilizers prevented drastic fluctuations in the nutrient supply, reducing oxidative damage to plants caused by excessive ROS accumulation. Concurrently, the enhanced activity of key nitrogen metabolism enzymes further supported the high expression of antioxidant enzymes, ultimately manifested as increased POD and CAT activities and a reduced MDA content.

In summary, the substitution of chemical fertilizers with organic fertilizers enhanced the nitrogen use efficiency by increasing the activities of NR and GS, while simultaneously mitigating oxidative stress through an optimized antioxidant system (elevated POD and CAT activities and a reduced MDA content). This dual regulatory mechanism supported the performance of fragrant rice during vegetative growth and stress adaptation. The experimental results ultimately manifested as improvements in both the yield and aroma content.

5. Conclusions

This study systematically evaluated the effects of varying ratios of organic fertilizer substitution for chemical fertilizers on the yield, aroma, and physiological characteristics of the fragrant rice varieties Meixiangzhan2 and Qingxiangyou19. The results demonstrated that substituting 33.3% of urea with organic fertilizer significantly increased the leaf area and photosynthetic characteristics, promoting biomass accumulation, while enhancing the levels of nitrogen metabolism enzymes, stress-resistant enzymes, and aroma synthesis-related compounds. Consequently, this approach improved both the yield and 2-acetyl-1-pyrroline (2-AP) content of fragrant rice. The results of this study indicate that the strategy of substituting chemical fertilizers with organic fertilizers, particularly under the treatment with 33.3% organic fertilizer substitution for urea (T2), significantly improved the yield and aroma quality of fragrant rice. These findings provide a reference scientific basis for the effective enhancement of the yield and aroma in fragrant rice.