Impact of Nitrogen Application Frequency on Cotton Yield and Nitrogen Use Efficiency under Integrated Water-Fertilizer Management

Impact of Nitrogen Application Frequency on Cotton Yield and Nitrogen Use Efficiency under Integrated Water-Fertilizer Management | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Impact of Nitrogen Application Frequency on Cotton Yield and Nitrogen Use Efficiency under Integrated Water-Fertilizer Management Xiaoqian Wu, Jun Zhang, Yuwen Wu, Leru Zhou, Bolang Chen This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7923043/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 12 You are reading this latest preprint version Abstract Water-nitrogen (N) coupling is recognized as a crucial strategy for enhancing crop yield and improving nitrogen use efficiency (NUE).However, the specific mechanisms by which the frequency of split nitrogen fertilizer applications influences cotton yield and NUE under water-nitrogen coupling conditions remain poorly understood. To determine optimal N fertilization strategies,a two-year consecutive field experiment was conducted from 2023 to 2024, comparing three treatments: no nitrogen fertilizer (CK), nitrogen fertilizer applied in eight follow-up applications (N8) and nitrogen fertilizer applied in ten split applications (N10). The results indicated that the N8 treatment maintained the highest levels of soil organic matter and alkali-hydrolyzable nitrogen content in the 0 ~ 20 cm soil layer compared with other treatments. When nitrogen application rates were consistent, adjusting the frequency of nitrogen application could improve the distribution of dry matter and nitrogen accumulation in cotton within the Xinjiang cotton-growing region. Specifically, the N8 treatment achieved a balanced relationship between vegetative and reproductive growth, facilitating greater accumulation of dry matter and nitrogen in reproductive organs. In both 2023 and 2024, the N8 treatment increased net photosynthetic rate by 25.27% and 45.21%, transpiration rate by 55.30% and 46.42%, and stomatal conductance by 38.89% and 55.95%, seed cotton yield by 31.94% and 36.78%, nitrogen fertilizer utilization rates by 56.07% and 49.11%, respectively,compared to the CK treatment, while intercellular CO₂ concentrations decreased by 18.76% and 22.91%, respectively. Moreover, cotton seed yield is highly significantly and positively correlated with dry matter accumulation and nitrogen accumulation.Under irrigation under plastic film irrigation, applying N fertilizer in eight doses effectively ensured soil nutrient availability, promoted dry matter and nitrogen in cotton, promote photosynthesis, and thereby enhanced cotton yield. Biological sciences/Ecology Earth and environmental sciences/Ecology Earth and environmental sciences/Environmental sciences Biological sciences/Plant sciences cotton nitrogen application frequency dry matter accumulation nitrogen accumulation yield nitrogen use efficiency Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Cotton is a vital cash crop, boasting the highest yield among all fiber crops [ 1 ]. Xinjiang is China’s largest commercial cotton-producing region as ample sunshine and optimal accumulated temperature, with its cotton planting area and output accounting for 86.24% and 92.24% of national total, respectively[ 2 ]. However, cotton production faces several constraints, particularly related to fertilizer supply. Commercial fertilizers contribute to at least 40% of the productivity achieved in intensive, high-yield cotton cultivation systems[ 3 ]. Nevertheless, excessive fertilizer application and unscientific fertilization practices have resulted in nutrient fixation, leaching, and losses, thereby reducing fertilizer utilization efficiency, limiting yield improvements, and hindering the advancement of modern agricultural production[ 4 ][ 5 ]. Given the scarcity of arable land resources and the demand for high cotton yields, optimizing fertilizer application strategies is one of the most critical approaches to enhancing cotton productively. Increasing nitrogen is an essential nutrient for crop growth and development[ 6 ][ 7 ]. In cotton production systems, nitrogen fertilizer is the primary yield-limiting factor. Therefore, the application of nitrogen fertilizer has been emphasized as a critical measure to improve cotton yield. According to crop fertilizer requirements, soil fertilizer supply capacity, and fertilizer performance, adjusting the frequency of nitrogen application can provide a sufficient nitrogen supply throughout the cotton growth cycle, reduce nitrogen losses, improve nitrogen utilization efficiency, maintain soil nitrogen balance, and achieve synergistic improvements in yield and nitrogen fertilizer use efficiency [ 8 ][ 9 ]. Increasing nitrogen fertilization generally enhances cotton plant height, fruit branch number, and effective bolls per plant. However, excessive nitrogen application can inhibit fruit branch formation and reduce the number of effective bolls per plant[ 10 ][ 11 ]. Thus, proper nitrogen application during the growth process can enhance cotton yield and fiber quality. Nitrogen is the fundamental element constituting substances such as cotton proteins, chloroplasts, and nucleic acids[ 12 ]. In the early growth stages, it promotes root growth, strengthens seedlings, and preserves buds[ 13 ]. During the later stages, it strengthens bolls and enhances fiber quality[ 14 ]. As the core nutrient in cotton cultivation, nitrogen helps coordinate vegetative and reproductive growth to achieve high yields of premium cotton[ 15 ]. There are significant differences in nutrient requirement characteristics of cotton at various growth seasons. So, Rational nitrogen allocation is a key strategy for improving both cotton yield and nitrogen use efficiency. For example, Liu et al [ 16 ] demonstrated that a single application of nitrogen fertilizer decreased the cotton harvest index and seed cotton yield. Guo et al [ 17 ]reported that splitting nitrogen applications into 40% at pre-planting, 15% at the square grain stage, 23% at the primordial stage, and 22% at the full flower stage resulted in the highest cotton yield, with the lowest abscission rate. Li et al.[ 18 ] also found that 70% fertilization at the flowering stage can promote nitrogen uptake and utilization efficiency of cotton with a nitrogen application rate of 270kg-ha − 1 . However, Yang et al[ 19 ] reported that the highest biomass and yield were achieved with a nitrogen application rate of 225 kg-ha − 1 , split as 0% at pre-planting, 40% at first flowering and 60% at full bloom. Similarly, the study of Raphael et al. [ 20 ] has proved that nitrogen fertilization in the later growth stage of cotton will affect the production of assimilates, thus improving seed cotton yield. Therefore, it is important to determine an appropriate nitrogen application frequency in improving cotton yield and reducing nitrogen loss. Applying fertilizer at the right time and crop growth stage ensures an adequate nutrient supply when crop needs it, while also preventing fertilizer waste and environmental pollution[ 21 ]. Drip irrigation fertilization technology is the best method for achieving real-time, precise nutrient delivery[ 22 ][ 23 ]. In Xinjiang, the mulched drip irrigation technique with integrated water and fertilizer management is the main cultivation method used to boost cotton yield and improve fertilizer use efficiency[ 24 ][ 25 ]. Applying nitrogen through drip irrigation beneath plastic mulch in Xinjiang has been shown to significantly increase cotton yields by approximately 43.38% compared to unfertilized fields, ensuring consistently high productivity[ 26 ][ 27 ]. Uzen et al [ 28 ]found that an appropriate nitrogen application frequency facilitated cotton yield accumulation under a fixed nitrogen rate. However, under drip irrigation conditions, it remains to be thoroughly investigated how to determine both the frequency of nitrogen application and the optimal distribution ratio per application to concurrently enhance cotton yield, improve nitrogen fertilizer utilization efficiency, and reduce labor costs. In this study, we examined soil physico-chemical properties, cotton nitrogen uptake and allocation, and yield under varying nitrogen fertilizer application frequencies. The objectives of this study were to (1) clarify the effects of nitrogen fertilizer frequency on the soil environment, (2) uncover the regulatory mechanisms of nitrogen fertilizer frequency on cotton nitrogen uptake and yield, and (3) offer an empirical foundation for improving cotton production through integrated water-fertilizer technology in arid and semi-arid regions. 2. Materials and Methods 2.1 Experimental field profile The experiment was conducted under field conditions from 2023 to 2024 at the cotton experimental station (44°10′E, 86°58′N) in Hutubi County, northern Xinjiang. The region experiences a temperate continental arid and semi-arid climate, and cotton cultivation follows a continuous cropping and annual ripening system. The field soil was a gray desert loam, with the following properties in the 0–20 cm layer, 11.54 g-kg − 1 organic matter, 22.4 mg-kg − 1 alkaline N, 18.4 mg-kg − 1 Olsen-P, 207.6 mg-kg − 1 exchangeable potassium, and pH 8.26. 2.2 Experimental design and field management Three nitrogen fertilizer application frequency treatments were established: no nitrogen fertilizer (CK), nitrogen fertilizer applied in 8 doses (N8) and nitrogen fertilizer applied in 10 times (N10), with each treatment was replicated 3 times, resulting in a total of 9 monitoring plots, each measuring 10 m×6.9 m = 69 m 2 . Fertilizer and irrigation were applied uniformly to ensure that the total amounts of fertilizer irrigation remained consistent across treatments. The irrigation quota during cotton growing season was 450 mm, with 10 irrigation events in all cases. Nitrogen fertilizer was applied as urea, with 20% used as a basal fertilizer before sowing and the remaining 80% applied via fertilization (see Table 1 ). Phosphate and potash were supplied as heavy calcium superphosphate and potassium sulfate, respectively, all applied as basal fertilizers. The total nutrients inputs were 300 kg N ha − 1 , 150 P 2 O 5 ha − 1 , 90 kg K 2 O ha − 1 . the test crop was Jinken 1441, sown in mid-April and late-April and harvested in early-mid October. The planting method was drip irrigation under the plastic film mulch, with a row spacing of (66 + 10) cm, a plant spacing of 9.2 cm, and a planting density of 19.0 ×10 4 plants ha − 1 . Field management practices were consistent with those used in local cotton production. Table 1 Nitrogen application rate under different treatments. Treatment Nitrogen application rate/kg·hm -2 Base fertilizer Topdressing N8 60 0 16.8 24 24 48 48 36 24 19.2 0 N10 60 16.8 16.8 19.2 19.2 43.2 43.2 28.8 19.2 16.8 16.8 2.3 Determination standard and method 2.3.1 Soil pH、conductivity、organic matter and alkali-hydrolyzable nitrogen [ 29 ] content determination Soil samples were collected from 0–20 cm and 20–40 cm soil layers of each plot during the bud, blooming and boll opening stages of cotton, the samples were air-dried naturally, thoroughly mixed, and sieved for analysis. Soil pH and electrical conductivity were measured using the electrode method; organic matter content was determined by the external heating method with potassium dichromate; Alkali-hydrolyzable nitrogen content was measured using the alkaline hydrolysis diffusion method. 2.3.2 Determination of plant dry matter and nitrogen [ 29 ] content During the bud, blooming, and boll opening stages, 5 representative cotton plants were randomly sampled from each plot, the plants were separated into roots, stems, leaves, and reproductive organs, and were first heated at 105 ℃ for 30 min to halt enzymatic activity and then dried at 80 ℃ until a constant weight was reached. The dried plant tissues were ground using a pulverizer, and total nitrogen content was determined by the semi-micro Kjeldahl method. 2.3.3 Photosynthetic parameters and SPAD values From each experimental plot, six plants in the central two rows were randomly selected, and the fourth-leaf-from-the-base of each cotton plant was measured. The photosynthetic parameters ( net photosynthetic rate, intercellular CO₂ concentration, transpiration rate, and stomatal conductance ) were measured using the LCi T/LCpro T photosynthesis analyzer (ADC Bio Scientific, UK) on clear, windless mornings with ample natural light between 11:00 AM and 1:00 PM during the budding stage (July 22, 2023, and August 13, 2023) and the budding stage (July 11, 2024, and August 24, 2024). The SPAD value of cotton inverted quadruple leaves was measured using an SPAD-502 chlorophyll meter. Five leaf sections were sampled from different locations, and the average value was taken to characterize the chlorophyll content of the treated cotton leaves. 2.3.4 Yield determination During the cotton boll opening period, the number of cotton plants and bolls was recorded in a 6.67 m 2 sampling area within each plot. 30 bolls were randomly harvested from the upper, middle, and lower parts of the cotton plants to determine the average boll weight, cotton yield was calculated based on plant density, boll number per plant, and average boll weight, with a yield coefficient of 90% was applied to account for seed cotton conversion. 2.3.5 Nitrogen use efficiency [30] Nitrogen fertilizer utilization rate(%)=(N-N 0 ) /F t (1) (where N is total N uptake by cotton in fertilized plots,N0 is total N uptake by cotton in unfertilized (control) plots, and F t is the amount of N fertilizer applied) 2.4 Data Processing and Statistical Methods Microsoft Excel 2016 was used for data processing. One-way analysis of variance (ANOVA) was performed with SPSS 25.0 software. The significance of differences between treatments was compared with the new complex extreme deviation test (Duncan's method), and plotted with Origin 2021 software. 3. Results 3.1 Effect of frequency of nitrogen application on soil chemical properties 3.1.1 Effect of frequency of nitrogen application on soil pH The impacts of nitrogen application frequency on soil pH across different soil layers during various fertility stages of cotton are depicted in Fig. 1 . At the bud stage: in 2023, no significant differences were observed in the pH levels of the 0–20 cm soil layer among the treatments; the pH of the 20–40 cm soil layer in the N10 treatment was significantly higher than that of the other treatments, and the N8 treatment showed a significantly higher pH compared to the CK treatment. In 2024, there were no significant variations in the pH values of both the 0–20 cm and 20–40 cm soil layers across all treatments. At the blooming stage: in 2023, the pH of the 0-20cm soil layer in the N10 treatment was significantly higher than that in the other treatments, while no significant difference was found between the CK and N8 treatments; for the 20-40cm soil layer, the N8 treatment had a significantly higher pH than the CK and N10 treatments, whereas no significant difference was observed between the CK and N10 treatments. In 2024, no significant differences were detected in the pH levels of the 0-20cm and 20-40cm soil layers among the various treatments. At the boll opening stage: in 2023, the pH of the 0-20cm soil layer did not show significant differences among the treatments; however, the pH of the 20-40cm soil layer in the N8 treatment was significantly lower than that in the other treatments, with no significant difference observed between the CK and N10 treatments. In 2024, no significant differences were found in the pH levels of the 0-20cm and 20-40cm soil layers across all treatments. 3.1.2 Effect of frequency of nitrogen application on soil conductivity The effects of nitrogen application frequency on soil conductivity in different soil layers during cotton fertility stages (Fig. 2 ). At the bud stage: in 2023, in the 0 ~ 20cm soil layer, conductivity was significantly higher under the N10 compared to other treatments, and no significant difference was observed between CK and N8 ; in the 20 ~ 40cm soil layer, there was no significant difference in conductivity was detected among treatments. In 2024, there was no significant difference in conductivity was found in the 0 ~ 20cm soil layer across treatments; in the 20 ~ 40cm soil layer, conductivity was significantly higher under N8 and N10 compared to CK, with no significant difference between N8 and N10. At the blooming stage: in 2023, there was no significant difference in conductivity in either the 0 ~ 20cm or 20 ~ 40cm soil layers among treatments. In 2024, in the 0 ~ 20cm soil layer, conductivity was significantly higher under N10 compared to other treatments, with no significant difference between N8 and CK; in the 20 ~ 40cm soil layer, conductivity was significantly higher under N8 and N10 compared to CK, with no significant difference between N8 and N10. At the boll opening stage: in 2023, in both the 0 ~ 20cm and 20 ~ 40cm soil layers, conductivity was significantly higher under N8 compared to CK, with no significant difference between N8 and N10. In 2024, there was no significant difference in conductivity were found in the 0 ~ 20cm soil layer; in the 20 ~ 40cm soil layer, conductivity was significantly higher under N8 and N10 compared to CK, with no significant difference between N8 and N10. 3.1.3 Effect of frequency of nitrogen application on soil organic matter content The effect of nitrogen application frequency on soil organic matter in different soil layers during various fertility periods of cotton is depicted in Fig. 3 . At the bud stage: in 2023, the organic matter content in the 0–20 cm and 20–40 cm soil layers under the N8 treatment was significantly higher than that under the CK, and no significant differences was observed between the CK and N10 treatments. In 2024, the organic matter content in the 0–20 cm and 20–40 cm soil layers under the N8 treatment was significantly higher than that under the CK treatment, the organic matter content between the N8 and N10 treatments was not significantly different. At the blooming stage: in 2023, the organic matter content in the 0–20 cm soil layer under both the N8 and N10 treatments was significantly higher than that under the CK treatment, with no significant difference between the N8 and N10 treatments; in the 20–40 cm soil layer, there was no significant difference in organic matter content among the treatments. In 2024, the organic matter in the 0–20 cm and 20–40 cm soil layers under both the N8 and N10 treatments was significantly higher than that under the CK treatments. At the boll opening stage: in 2023, the organic matter of 0 ~ 20cmcm soil layer under the N8 treatment was significantly higher than that under the CK treatment, while no significant difference was observed between the CK and N10 treatments; in the 20 ~ 40 cm soil layer, no significant difference in organic matter content was found among the treatments. In 2024, the organic matter content in the 0 ~ 20 cm and 20 ~ 40 cm soil layer under the N8 treatment was significantly higher than that under the CK treatment, but no significant difference was observed between the N8 and N10 treatments. 3.1.4 Effect of frequency of nitrogen application on soil alkali-hydrolyzable nitrogen content The effects of nitrogen application frequency on soil alkali-hydrolyzable nitrogen (AN) in different soil layers during cotton fertility stages (Fig. 4 ). At the bud stage: in 2023, in the 0–20 cm soil layer, AN under the N8 treatment was significantly higher than that under other treatments, and there was no significant difference between CK and N10 treatments; in the 20–40 cm soil layer, there was no significant difference in AN among treatments. In 2024, in both the 0–20 cm and 20–40 cm soil layers,AN under the N8 treatment was significantly higher than that under CK treatment, and there was no significant difference between CK and N10 treatments. At the blooming stage: in 2023, in the 0–20 cm soil layer, the alkali-hydrolyzable nitrogen (AN) under the N8 treatment was significantly higher than that under the CK treatment, and there was no significant difference between the N8 and N10 ; in the 20–40 cm soil layer, there was no significant difference in the AN among treatments. In 2024, in both the 0–20 cm and 20-40cm soil layers, AN under N8 was significantly higher than that under the CK treatment, and there was no significant difference between the CK and N10 treatments At the boll opening stage: in 2023, in the 0–20 cm soil layer, the alkali-hydrolyzable nitrogen (AN) under the N8 treatment was significantly higher than that under other treatments, and there was no significant difference between CK and N10 treatments; in the 20–40 cm soil layer, there was no significant difference in AN among treatments. In 2024, the AN in both the 0 ~ 20 cm and 20 ~ 40 cm soil layers under the N8 treatment was significantly higher than that under the CK treatment, and there was no significant difference between N8 and N10. 3.2 Effect of frequency of nitrogen application on dry matter and nitrogen accumulation in cotton 3.2.1 Effect of frequency of nitrogen application on dry matter quality As shown in Fig. 5 , in 2023, the total dry matter accumulation in cotton organs across different growth stages was recorded in 2023 and 2024. In 2023, the dry matter accumulation ranged from 3902.4 to 6553.1 kg-hm − 2 at bud stage, 5252.4 to 9370.1 kg-hm − 2 at the blooming stage, and 6503.28 to 12415.4 kg-hm − 2 at the boll opening stage. In 2024, these values were 2654.3 to 5699.6 kg-hm − 2 , 4960.3 to 7573.6 kg-hm − 2 and 6086.6 to 10287.2 kg-hm − 2 at bud, blooming, and boll opening stages, respectively. When comparing the N8 treatment to the CK treatment in 2023, dry matter mass of cotton increased by an average of 46.43% at the bud stage, 78.39% at the blooming stage, and 58.14% at the boll opening stage.In 2024, under the N8 treatment, the average increases compared to CK were 80.45% at the bud stage, 52.68% at the blooming stage, and 58.37% at the boll opening stage. Similarly, for the N10 treatment in 2023, the average increase in dry matter compared to CK was 67.92% at bud stage, 37.14% at the blooming stage and 90.91% at the boll opening stage. In 2024, the N10 treatment resulted in average increases of 114.73% at the bud stage, 40.58% at the blooming stage, and 69.01% at the boll opening stage compared to CK. 3.2.3 Effect of frequency of nitrogen application on nitrogen uptake As shown in Fig. 6 , the total nitrogen accumulation in cotton organs at the bud, blooming, and boll opening stages ranged from 154.84 ~ 331.50 kg-hm − 2 , 225.65 ~ 443.51 kg-hm − 2 and 204.64 ~ 420.58 kg-hm − 2 in 2023, respectively; and 152.74 ~ 373.04 kg-hm − 2 , 252.57 ~ 430.61 kg-hm − 2 and 275.84 ~ 489.70 kg-hm − 2 in 2024, respectively; under the N8 treatment compared to CK, nitrogen accumulation increased by an average of 80.05% (bud), 96.54% (blooming) and 82.19% (boll opening) in 2023, and 92.66%, 70.48% and 53.40% in 2024, respectively; under the N10 treatment compared to CK, increases averaged 114.08% (bud), 38.86% (blooming) and 105.52% (boll opening) in 2023, and 144.22%, 53.45% and 77.53% in 2024, respectively. 3.3 Effects of frequency of nitrogen application on photosynthetic characteristics and spad values of cotton 3.3.1 Effects of frequency of nitrogen application on photosynthetic characteristics The frequency of nitrogen application significantly influenced the photosynthetic characteristics of cotton during the bud and boll stages ( Fig. 7 ). In 2023, the net photosynthetic rate, transpiration rate, and stomatal conductance of the N8 treatment were higher than those of other treatments, while no significant differences were observed between the CK and N10 treatments. The intercellular CO₂ concentration in the CK treatment was higher than in other treatments, with no significant difference between the N8 and N10 treatments. In 2024, the net photosynthetic rate, transpiration rate, and stomatal conductance of the N8 treatment were significantly higher than those of other treatments; but the intercellular CO₂ concentration in the CK treatment was significantly higher than in other treatments. 3.3.2 Effects of frequency of nitrogen application on SPAD values The frequency of nitrogen application significantly influenced SPAD values during the budding and boll-setting stages of cotton ( Fig. 8 ). In 2023, SPAD values for the N8 treatment showed no significant difference compared to the N10 treatment, while were significantly higher than the CK treatment, representing increases of 13.71% and 37.21%, respectively. In 2024, the SPAD values of the N8 treatment during the budding and boll-setting stages were significantly higher than those of other treatments, compared with the CK treatment, increased by 21.73% and 6.90%, respectively. 3.4 Effect of frequency of nitrogen application on cotton yield, yield components and nitrogen fertilizer utilization rate In both 2023 and 2024, no significant differences in cotton harvest density were observed among treatments (Table 2 ). In 2023, the number of cotton bolls per plant under N8 and N10 was significantly higher than that under the CK treatment, and with no significant difference between N8 and N10 treatments. In 2024, the N8 treatment exhibited a significantly higher number of bolls per plant compared to other treatments, while no significant differences were found between the CK and N10 treatments. Regarding cotton boll weight, no significant differences among treatments were observed in 2023, however, in 2024, the N8 treatment showed significantly higher boll weights than other treatments, with no significant difference between CK and N10. In 2023, in terms of seed cotton yield, the N8 treatment significantly outperformed CK, and there was no significant difference between N8 and N10. In 2024, the N8 treatment yielded significantly more than CK. Compared to CK, the N8 treatment increased seed cotton yield by 31.94% in 2023 and 36.78% in 2024. There was no significant difference in nitrogen fertilizer use efficiency between the N8 and N10 treatments in either year. Table 2 Effects of nitrogen application times on cotton yield traits.Data are mean ± standard deviation. Different lowercase letters in the same column indicated significant difference between different treatments in the same year (P < 0.05). Year Treatment Number of plants harvested /×10 4 ·hm -2 Number of bells per plant Boll count/g Seed cotton yield/kg·hm -2 Nitrogen fertilizer utilization rate/% 2023 CK 17.93 ± 0.76a 4.51 ± 0.20b 4.38 ± 0.30a 3183 ± 274b N8 19.34 ± 1.01a 4.94 ± 0.23a 4.90 ± 0.44a 4200 ± 299a 56.07 ± 17.37a N10 18.03 ± 0.92a 5.07 ± 0.11a 4.66 ± 0.37a 3831 ± 360a 71.98 ± 11.87a 2024 CK 15.99 ± 0.83a 4.98 ± 0.47b 4.49 ± 0.09b 3205 ± 165c N8 17.29 ± 0.57a 5.68 ± 0.13a 4.92 ± 0.29a 4384 ± 242a 49.11 ± 22.34a N10 17.19 ± 1.36a 5.22 ± 0.18ab 4.67 ± 0.08ab 3770 ± 234b 71.29 ± 7.74a 3.5 Correlation of cotton seed cotton yield with various indicators The correlation analysis results between cotton yield, soil physicochemical properties, cotton growth indicators, and cotton photosynthetic characteristics are shown in Fig. 9 . Statistical analysis indicates that cotton yield shows no statistically significant correlation with pH or alkali-hydrolyzable nitrogen. Conversely, cotton yield exhibits a highly significant positive correlation with organic matter content; it also shows a highly significant positive correlation with dry matter accumulation and nitrogen accumulation. Furthermore, cotton yield demonstrates a highly significant positive correlation with net photosynthetic rate, transpiration rate, stomatal conductance, and SPAD values, while exhibiting a highly significant negative correlation with intercellular CO₂ concentration. 4. Discussion 4.1 Effect of frequency of nitrogen application on soil chemical properties The nutrient content of soil significantly influences its fertility status, directly affecting cotton yield and quality[ 31 ][ 32 ]. In this experiment, we observed that the varying N application frequencies had a relatively minor impact on soil pH. This may be attributed to the fact that urea, a water-soluble fertilizer, is typically applied in multiple split doses as a follow-up fertilizer. It is rapidly absorbed by plants and becomes diluted with water, diminishing its inherent acid-base properties, and thus having a limited impact on soil pH. Cai et al[ 33 ] demonstrated that, compared to a single follow-up fertilizer application, three to four follow-up applications led to a greater concentration of soil NO 3 − N in the upper soil layer (0–40 cm) during the flowering and sizing stages of wheat. Qiu et al. [ 34 ] showed that, with an N application rate of 300 kg-hm − 2 , a fertilization schedule of nine times (20% basal application, 20% in three applications at the bud stage, 30% in three applications at anthesis, and 30% in three applications at full bloom) significantly increased the levels of soil nitrate, ammonium, and alkali-hydrolyzable nitrogen (AN) compared to a no-N fertilization treatment. Zhang et al.[ 35 ] found that excessive water inflow and prolonged high-frequency irrigation were primary factors contributing to carbon leaching under certain conditions. In contrast, this study revealed that under drip irrigation beneath a membrane, the surface soil organic matter and alkali-hydrolyzable nitrogen in the N8 treatment were significantly higher than those in the control (CK) treatment. This environment was conducive to the crop's absorption of soil nutrients, thus promoting the growth and development of cotton and enhancing yield formation. 4.2 Effect of nitrogen application frequency on dry matter and nitrogen accumulation in cotton Appropriate dry matter accumulation and its coordinated dynamics are crucial for establishing an optimal population structure and achieving high cotton yields[ 36 ][ 37 ][ 38 ]. Lu et al[ 39 ] demonstrated that regulating fertilizer application timing and nitrogen dosage could maintain balanced nitrogen uptake and utilization by reproductive organs, thereby increasing biomass and ultimately enhancing yields. Ren et al[ 40 ] reported that, during later growth stages, dry matter accumulation increased by 54.6% and 73.3% in Ningjin and Changyi experimental areas, respectively, under a nitrogen allocation ratio of 3:5:2 compared to a 0:10:0 treatment. Abdelraouf et al[ 41 ] found that increasing fertilization frequency and shortening intervals in 2014/2015 and 2015/2016 improved nitrogen uptake efficiency, cumulative N uptake and grain N content in both observed and simulated values. This study showed that both N8 and N10 treatments enhanced dry matter accumulation in cotton roots, stems and leaves and reproductive organs. At the bud stage, the N10 treatment exhibited higher total dry matter than N8, during the blooming stage, the N8 treatment increased overall plant dry matter by promoting accumulation in reproductive organs; and at the boll open stage, the N10 treatment boosted total plant dry matter by enhancing accumulation in vegetative organs. These differences may stem from varying nutrient demands across cotton growth stages[ 42 ], and the flowering stage is a critical phase for reproductive growth, the N8 treatment supplied high nitrogen during this period to meet nutrient demand, ensuring sufficient nitrogen availability, accelerating the transition to reproductive growth, and facilitating nitrogen uptake and redistribution from vegetative to productive organs. The N10 treatment applied nitrogen later in the flowering stage, sustaining vigorous vegetative growth and reducing nitrogen transfer efficiency to seeds, which hindered nutrient translocation from leaves and stems[ 43 ]. Therefore, appropriate nitrogen application rates and allocation ratio are essential for optimizing plant dry matter accumulation. Nitrogen uptake and utilization are prerequisites for dry matter accumulation, which forms the basis for high cotton yields[ 44 ][ 45 ][ 46 ].Uzen et al [ 28 ] showed that moderate fertilization frequency significantly enhanced total nitrogen uptake compared to high or low frequencies. Feng et al[ 47 ] reported that apparent nitrogen loss was 48.3% under single nitrogen application but reduced it to 38.5% with split applications. This study showed that both N8 and N10 treatments increased nitrogen accumulation in cotton roots, stems, leaves, and reproductive organs, the N8 treatment effectively balanced vegetative and reproductive growth, promoting nitrogen accumulation in reproductive organs and facilitating photosynthate translocation. Wang et al[ 48 ] noted that while nitrogen content increased with fertilizer dosage, excessive nitrogen slowed or reduced nitrogen accumulation, highlighting the importance of optimal nitrogen application for maximizing accumulation rates. The present study showed that the N10 treatment at the bud stage exhibited higher N accumulation than N8; the N8 treatment at the blooming stage enhanced total plant nitrogen accumulation by increasing uptake in both vegetative and reproductive organs; the N10 treatment at the boll open stage increased the N accumulation of the whole cotton plant by promoting uptake in vegetative organs. 4.3 Effect of nitrogen application frequency on photosynthetic characteristics and SPAD values in cotton Cotton yield exhibits a strong correlation with plant growth and development indicators, and enhancing leaf photosynthetic properties can promote plant growth, development, and dry matter accumulation. Tian et al.[ 9 ] demonstrated that during the late growth stage of cotton, adjusting nitrogen application frequency and proportion can increase net photosynthetic rate, transpiration rate, and stomatal conductance while reducing intercellular CO₂ concentration.The results of this study indicate that under the N8 treatment, cotton exhibited higher net photosynthetic rates, transpiration rates, and stomatal conductance compared to the CK and N10 treatments, while intercellular CO₂ concentrations were lower than those in the CK treatment. This suggests that varying nitrogen application frequencies exert distinct effects on cotton photosynthesis, with the application of nitrogen fertilizer in eight installments more effectively promoting photosynthetic activity. Nitrogen fertilization increased net photosynthetic rate, transpiration rate, and stomatal conductance while decreasing intercellular CO₂ concentration, consistent with studies by Dai et al.[ 49 ] and Li et al.[ 50 ]. This may occur because under nutrient-sufficient conditions, stomata open to enhance crop respiration, allowing CO₂ to enter plant cells. This reduces intercellular CO₂ concentration, increases photosynthetic rate, and ultimately boosts cotton photosynthetic capacity. Under the experimental conditions, the SPAD values of the N8 treatment were significantly higher than those of the CK treatment, while the N10 treatment showed no significant difference from the CK treatment. This indicates that an appropriate nitrogen application frequency can promote the vegetative growth of cotton, increase chlorophyll content in cotton leaves, and lay a material foundation for high cotton yields [ 51 ]. 4.4 Effect of different frequency of nitrogen application on cotton yield, yield components and nitrogen fertilizer utilization rate Nitrogen nutrition plays a critical role in regulating the cotton yield formation, improper fertilizers leads to excessive vegetative growth or nutrient deficiency, reducing boll number, boll weight and yield. Conversely, rational nitrogen management coordinates vegetative and reproductive growth, ensuring high-yield and high-quality cotton production[ 52 ][ 53 ][ 54 ].Yang et al[ 55 ] found that in the Yangtze River Basin cotton region, one-time fertilization yielded similar results to traditional three-time fertilization but outperformed two-time fertilization. Luo et al [ 56 ] in the cotton region of the Yangtze River Basin, showed that among all one-time fertilization schedules, the one-time application of fertilizer to the first flower stage maximized yield, comparable to traditional three-time applications. In this study, N8 and N10 treatments increased seed cotton yield by raising boll number per plant and boll weight, with N8 being the most effective compared to the CK treatment under a total pure N rate of 300 kg-hm − 2 . Nitrogen fertilizer utilization efficiency is a key metric reflecting crop nitrogen uptake and utilization[ 57 ][ 58 ]. Cai et al[ 33 ] showed that splitting follow-up nitrogen applications 3–4 times, with about 16.7% applied at the flowering and the irrigating stages, significantly improved yield, WUE (Water Use Efficiency), PFPN (Partial Productivity of Fertilizer with Nitrogen), and NHI (Nitrogen Harvest Index). In this study, the N10 treatments slightly improved N fertilizer utilization efficiency compared to N8, though the difference was not statistically significant. 5. Conclusions In summary, nitrogen frequency significantly influenced soil nutrient content, dry matter and nitrogen accumulation, photosynthetic characteristics,yield traits and nitrogen fertilizer utilization efficiency in Xinjiang cotton fields. The N8 treatment maintained high soil organic matter and alkali-hydrolyzable nitrogen levels throughout the reproductive period. Adjusting nitrogen application frequency under consistent nitrogen rates optimized dry matter and nitrogen distribution, promote photosynthesis,enhancing the nitrogen fertilizer utilization efficiency and yield. Under the experimental conditions, drip fertilization with water in 8 split applications (N8) yielded the best results. Declarations Author Contributions: Conceptualization, X.W. and B.C., methodology, X.W., J.Z. and L.Z., software,Y.W. and X.W., validation, J.Z. and X.W., formal analysis, X.W. and L.Z., investigation, X.W. and J.Z., resources, B.C., data curation, X.W. and L.Z., writing-original draft preparation, X.W.; writing review and editing, X.W. and B.C., funding acquisition, B.C. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the Science Foundation for Outstanding Young Scholars of Xinjiang Uygur Autonomous Region (Grant No. 2024D01E06), the Key Laboratory Project of "Soil and Plant Ecological Processes in Xinjiang"(Grant No. 24XJTRZWY02); the Project for Xinjiang Agricultural University Graduate Research and Innovation(Grant No. XJAUGRI2025068); the Project for Young Top-Notch Talents in Science and Technology of Xinjiang Uygur Autonomous Region (Grant No. 2022TSYCCX0085), the National Natural Science Foundation of China (Grant No. 32360793 and 31960629), the Key Research and Development Project in Xinjiang Uygur Autonomous Region (Grant No. 2022B02033-1), and Special Topics of Major Science and Technology in Xinjiang Uygur Autonomous Region (Grant No.2022A02007-2). We thank David for editing the English text of a draft of this manuscript. Data Availability Statement: The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author. Acknowledgments: We appreciate and thank the anonymous reviewers for helpful comments that led to an overall improvement of the manuscript. Conflicts of Interest: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. References Dai, F.; Chen, J.; Zhang, Z.; Liu, F.; Li, J.; Zhao, T.; Hu, Y.; Zhang, T. & Fang, L. Cottonomics: a comprehensive cotton multi-omics database. 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1","display":"","copyAsset":false,"role":"figure","size":57782,"visible":true,"origin":"","legend":"\u003cp\u003eSoil pH changes at different growth stages of cotton under different nitrogen application frequencies from 2023 to 2024. Figure a, b and c represent cotton bud stage, blooming stage and boll opening stage in 2023 respectively; d, e and f stand for cotton bud stage, blooming stage and boll opening stage in 2024, respectively. The different lowercase letters in the same column indicate significant differences for the different nitrogen application frequencies (p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7923043/v1/176077d4c4096e989585cf31.png"},{"id":95526376,"identity":"ea207a00-32be-4641-af12-0d21580be230","added_by":"auto","created_at":"2025-11-10 10:06:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":75577,"visible":true,"origin":"","legend":"\u003cp\u003eChanges of soil conductivity at different growth stages of cotton under different nitrogen application frequencies from 2023 to 2024.Figure a, b and c represent cotton bud stage, blooming stage and boll opening stage in 2023 respectively; d, e and f stand for cotton bud stage, blooming stage and boll opening stage in 2024, respectively.The different lowercase letters in the same column indicate significant differences for the different nitrogen application frequencies (p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7923043/v1/3dd6c505204586015ee8dc28.png"},{"id":95381390,"identity":"7c3edcef-0738-4769-aea8-dd76740cda0c","added_by":"auto","created_at":"2025-11-07 12:05:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":81254,"visible":true,"origin":"","legend":"\u003cp\u003eChanges of soil organic matter at different growth stages of cotton under different nitrogen application frequencies from 2023 to 2024.Figure a, b and c represent cotton bud stage, blooming stage and boll opening stage in 2023 respectively; d, e and f stand for cotton bud stage, blooming stage and boll opening stage in 2024, respectively.The different lowercase letters in the same column indicate significant differences for the different nitrogen application frequencies (p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7923043/v1/2279b506709fa125b7914613.png"},{"id":95525362,"identity":"5c93a43c-c8d0-4e75-aabc-f320639e7ca9","added_by":"auto","created_at":"2025-11-10 10:04:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":90035,"visible":true,"origin":"","legend":"\u003cp\u003eChanges of soil alkali-hydrolyzed nitrogen in different growth periods of cotton under different nitrogen application frequencies from 2023 to 2024.Figure a, b and c represent cotton bud stage, blooming stage and boll opening stage in 2023 respectively; d, e and f stand for cotton bud stage, blooming stage and boll opening stage in 2024, respectively.The different lowercase letters in the same column indicate significant differences for the different nitrogen application frequencies (p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7923043/v1/162448b49fa41ece21d67e6c.png"},{"id":95525813,"identity":"11747ad5-6e69-442b-b015-d205a66331c1","added_by":"auto","created_at":"2025-11-10 10:05:42","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":110065,"visible":true,"origin":"","legend":"\u003cp\u003eChanges of dry matter accumulation in cotton at different growth stages under different nitrogen application frequencies from 2023 to 2024.Figure a, b and c represent cotton bud stage, blooming stage and boll opening stage in 2023 respectively; d, e and f stand for cotton bud stage, blooming stage and boll opening stage in 2024, respectively.The different lowercase letters in the same column indicate significant differences for the different nitrogen application frequencies (p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7923043/v1/a46d95f38945193d3ed17ea0.png"},{"id":95381396,"identity":"5d475299-b7d4-4e4b-ae4f-c43c36984639","added_by":"auto","created_at":"2025-11-07 12:05:29","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":121346,"visible":true,"origin":"","legend":"\u003cp\u003eChanges of nitrogen accumulation in cotton at different growth stages under different nitrogen application frequencies from 2023 to 2024.Figure a, b and c represent cotton bud stage, blooming stage and boll opening stage in 2023 respectively; d, e and f stand for cotton bud stage, blooming stage and boll opening stage in 2024, respectively. The different lowercase letters in the same column indicate significant differences for the different nitrogen application frequencies (p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7923043/v1/44492f55c677c2da072cf0df.png"},{"id":95531256,"identity":"796c7d18-b93e-4067-b74d-b7c86f557776","added_by":"auto","created_at":"2025-11-10 10:22:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1898090,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7923043/v1/c9257cc9-94cd-4c80-9f82-42baeb68fa27.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Impact of Nitrogen Application Frequency on Cotton Yield and Nitrogen Use Efficiency under Integrated Water-Fertilizer Management","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eCotton is a vital cash crop, boasting the highest yield among all fiber crops [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Xinjiang is China\u0026rsquo;s largest commercial cotton-producing region as ample sunshine and optimal accumulated temperature, with its cotton planting area and output accounting for 86.24% and 92.24% of national total, respectively[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. However, cotton production faces several constraints, particularly related to fertilizer supply. Commercial fertilizers contribute to at least 40% of the productivity achieved in intensive, high-yield cotton cultivation systems[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Nevertheless, excessive fertilizer application and unscientific fertilization practices have resulted in nutrient fixation, leaching, and losses, thereby reducing fertilizer utilization efficiency, limiting yield improvements, and hindering the advancement of modern agricultural production[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e][\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Given the scarcity of arable land resources and the demand for high cotton yields, optimizing fertilizer application strategies is one of the most critical approaches to enhancing cotton productively.\u003c/p\u003e\u003cp\u003eIncreasing nitrogen is an essential nutrient for crop growth and development[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e][\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In cotton production systems, nitrogen fertilizer is the primary yield-limiting factor. Therefore, the application of nitrogen fertilizer has been emphasized as a critical measure to improve cotton yield. According to crop fertilizer requirements, soil fertilizer supply capacity, and fertilizer performance, adjusting the frequency of nitrogen application can provide a sufficient nitrogen supply throughout the cotton growth cycle, reduce nitrogen losses, improve nitrogen utilization efficiency, maintain soil nitrogen balance, and achieve synergistic improvements in yield and nitrogen fertilizer use efficiency [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e][\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Increasing nitrogen fertilization generally enhances cotton plant height, fruit branch number, and effective bolls per plant. However, excessive nitrogen application can inhibit fruit branch formation and reduce the number of effective bolls per plant[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e][\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Thus, proper nitrogen application during the growth process can enhance cotton yield and fiber quality.\u003c/p\u003e\u003cp\u003eNitrogen is the fundamental element constituting substances such as cotton proteins, chloroplasts, and nucleic acids[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In the early growth stages, it promotes root growth, strengthens seedlings, and preserves buds[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. During the later stages, it strengthens bolls and enhances fiber quality[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. As the core nutrient in cotton cultivation, nitrogen helps coordinate vegetative and reproductive growth to achieve high yields of premium cotton[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. There are significant differences in nutrient requirement characteristics of cotton at various growth seasons. So, Rational nitrogen allocation is a key strategy for improving both cotton yield and nitrogen use efficiency. For example, Liu et al [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] demonstrated that a single application of nitrogen fertilizer decreased the cotton harvest index and seed cotton yield. Guo et al [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]reported that splitting nitrogen applications into 40% at pre-planting, 15% at the square grain stage, 23% at the primordial stage, and 22% at the full flower stage resulted in the highest cotton yield, with the lowest abscission rate. Li et al.[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] also found that 70% fertilization at the flowering stage can promote nitrogen uptake and utilization efficiency of cotton with a nitrogen application rate of 270kg-ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. However, Yang et al[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] reported that the highest biomass and yield were achieved with a nitrogen application rate of 225 kg-ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, split as 0% at pre-planting, 40% at first flowering and 60% at full bloom. Similarly, the study of Raphael et al. [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] has proved that nitrogen fertilization in the later growth stage of cotton will affect the production of assimilates, thus improving seed cotton yield. Therefore, it is important to determine an appropriate nitrogen application frequency in improving cotton yield and reducing nitrogen loss.\u003c/p\u003e\u003cp\u003eApplying fertilizer at the right time and crop growth stage ensures an adequate nutrient supply when crop needs it, while also preventing fertilizer waste and environmental pollution[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Drip irrigation fertilization technology is the best method for achieving real-time, precise nutrient delivery[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e][\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In Xinjiang, the mulched drip irrigation technique with integrated water and fertilizer management is the main cultivation method used to boost cotton yield and improve fertilizer use efficiency[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e][\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Applying nitrogen through drip irrigation beneath plastic mulch in Xinjiang has been shown to significantly increase cotton yields by approximately 43.38% compared to unfertilized fields, ensuring consistently high productivity[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e][\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Uzen et al [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]found that an appropriate nitrogen application frequency facilitated cotton yield accumulation under a fixed nitrogen rate. However, under drip irrigation conditions, it remains to be thoroughly investigated how to determine both the frequency of nitrogen application and the optimal distribution ratio per application to concurrently enhance cotton yield, improve nitrogen fertilizer utilization efficiency, and reduce labor costs.\u003c/p\u003e\u003cp\u003eIn this study, we examined soil physico-chemical properties, cotton nitrogen uptake and allocation, and yield under varying nitrogen fertilizer application frequencies. The objectives of this study were to (1) clarify the effects of nitrogen fertilizer frequency on the soil environment, (2) uncover the regulatory mechanisms of nitrogen fertilizer frequency on cotton nitrogen uptake and yield, and (3) offer an empirical foundation for improving cotton production through integrated water-fertilizer technology in arid and semi-arid regions.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1 Experimental field profile\u003c/h2\u003e\n \u003cp\u003eThe experiment was conducted under field conditions from 2023 to 2024 at the cotton experimental station (44\u0026deg;10\u0026prime;E, 86\u0026deg;58\u0026prime;N) in Hutubi County, northern Xinjiang. The region experiences a temperate continental arid and semi-arid climate, and cotton cultivation follows a continuous cropping and annual ripening system. The field soil was a gray desert loam, with the following properties in the 0\u0026ndash;20 cm layer, 11.54 g-kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e organic matter, 22.4 mg-kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e alkaline N, 18.4 mg-kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Olsen-P, 207.6 mg-kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e exchangeable potassium, and pH 8.26.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2 Experimental design and field management\u003c/h2\u003e\n \u003cp\u003eThree nitrogen fertilizer application frequency treatments were established: no nitrogen fertilizer (CK), nitrogen fertilizer applied in 8 doses (N8) and nitrogen fertilizer applied in 10 times (N10), with each treatment was replicated 3 times, resulting in a total of 9 monitoring plots, each measuring 10 m\u0026times;6.9 m\u0026thinsp;=\u0026thinsp;69 m\u003csup\u003e2\u003c/sup\u003e. Fertilizer and irrigation were applied uniformly to ensure that the total amounts of fertilizer irrigation remained consistent across treatments. The irrigation quota during cotton growing season was 450 mm, with 10 irrigation events in all cases. Nitrogen fertilizer was applied as urea, with 20% used as a basal fertilizer before sowing and the remaining 80% applied via fertilization (see Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Phosphate and potash were supplied as heavy calcium superphosphate and potassium sulfate, respectively, all applied as basal fertilizers. The total nutrients inputs were 300 kg N ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 150 P\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 90 kg K\u003csub\u003e2\u003c/sub\u003eO ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. the test crop was Jinken 1441, sown in mid-April and late-April and harvested in early-mid October. The planting method was drip irrigation under the plastic film mulch, with a row spacing of (66\u0026thinsp;+\u0026thinsp;10) cm, a plant spacing of 9.2 cm, and a planting density of 19.0 \u0026times;10\u003csup\u003e4\u003c/sup\u003e plants ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Field management practices were consistent with those used in local cotton production.\u003c/p\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eNitrogen application rate under different treatments.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eTreatment\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"12\"\u003e\n \u003cp\u003eNitrogen application rate/kg\u0026middot;hm\u003csup\u003e-2\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eBase fertilizer\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"11\"\u003e\n \u003cp\u003eTopdressing\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e19.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"1\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e43.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e43.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e28.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"1\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3 Determination standard and method\u003c/h2\u003e\n \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\n \u003ch2\u003e\u003cem\u003e2.3.1 Soil pH、conductivity、organic matter and alkali-hydrolyzable nitrogen\u003c/em\u003e[\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e] \u003cem\u003econtent determination\u003c/em\u003e\u003c/h2\u003e\n \u003cp\u003eSoil samples were collected from 0\u0026ndash;20 cm and 20\u0026ndash;40 cm soil layers of each plot during the bud, blooming and boll opening stages of cotton, the samples were air-dried naturally, thoroughly mixed, and sieved for analysis. Soil pH and electrical conductivity were measured using the electrode method; organic matter content was determined by the external heating method with potassium dichromate; Alkali-hydrolyzable nitrogen content was measured using the alkaline hydrolysis diffusion method.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\n \u003ch2\u003e\u003cem\u003e2.3.2 Determination of plant dry matter and nitrogen\u003c/em\u003e[\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e] \u003cem\u003econtent\u003c/em\u003e\u003c/h2\u003e\n \u003cp\u003eDuring the bud, blooming, and boll opening stages, 5 representative cotton plants were randomly sampled from each plot, the plants were separated into roots, stems, leaves, and reproductive organs, and were first heated at 105 ℃ for 30 min to halt enzymatic activity and then dried at 80 ℃ until a constant weight was reached. The dried plant tissues were ground using a pulverizer, and total nitrogen content was determined by the semi-micro Kjeldahl method.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\n \u003ch2\u003e2.3.3 Photosynthetic parameters and SPAD values\u003c/h2\u003e\n \u003cp\u003eFrom each experimental plot, six plants in the central two rows were randomly selected, and the fourth-leaf-from-the-base of each cotton plant was measured. The photosynthetic parameters ( net photosynthetic rate, intercellular CO₂ concentration, transpiration rate, and stomatal conductance ) were measured using the LCi T/LCpro T photosynthesis analyzer (ADC Bio Scientific, UK) on clear, windless mornings with ample natural light between 11:00 AM and 1:00 PM during the budding stage (July 22, 2023, and August 13, 2023) and the budding stage (July 11, 2024, and August 24, 2024). The SPAD value of cotton inverted quadruple leaves was measured using an SPAD-502 chlorophyll meter. Five leaf sections were sampled from different locations, and the average value was taken to characterize the chlorophyll content of the treated cotton leaves.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\n \u003ch2\u003e2.3.4 Yield determination\u003c/h2\u003e\n \u003cp\u003eDuring the cotton boll opening period, the number of cotton plants and bolls was recorded in a 6.67 m\u003csup\u003e2\u003c/sup\u003e sampling area within each plot. 30 bolls were randomly harvested from the upper, middle, and lower parts of the cotton plants to determine the average boll weight, cotton yield was calculated based on plant density, boll number per plant, and average boll weight, with a yield coefficient of 90% was applied to account for seed cotton conversion.\u003c/p\u003e\n \u003cdiv\u003e\n \u003ch2\u003e\u003cem\u003e2.3.5 Nitrogen use efficiency\u003c/em\u003e[30]\u003c/h2\u003e\n \u003c/div\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Taba\" border=\"1\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNitrogen fertilizer utilization rate(%)=(N-N\u003csub\u003e0\u003c/sub\u003e) /F\u003csub\u003et\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(1)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003e(where N is total N uptake by cotton in fertilized plots,N0 is total N uptake by cotton in unfertilized (control) plots, and F\u003csub\u003et\u003c/sub\u003e is the amount of N fertilizer applied)\u003c/p\u003e\n \u003c/div\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4 Data Processing and Statistical Methods\u003c/h2\u003e\n \u003cp\u003eMicrosoft Excel 2016 was used for data processing. One-way analysis of variance (ANOVA) was performed with SPSS 25.0 software. The significance of differences between treatments was compared with the new complex extreme deviation test (Duncan\u0026apos;s method), and plotted with Origin 2021 software.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Effect of frequency of nitrogen application on soil chemical properties\u003c/h2\u003e\u003cdiv id=\"Sec14\" class=\"Section3\"\u003e\u003ch2\u003e3.1.1 Effect of frequency of nitrogen application on soil pH\u003c/h2\u003e\u003cp\u003eThe impacts of nitrogen application frequency on soil pH across different soil layers during various fertility stages of cotton are depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. At the bud stage: in 2023, no significant differences were observed in the pH levels of the 0\u0026ndash;20 cm soil layer among the treatments; the pH of the 20\u0026ndash;40 cm soil layer in the N10 treatment was significantly higher than that of the other treatments, and the N8 treatment showed a significantly higher pH compared to the CK treatment. In 2024, there were no significant variations in the pH values of both the 0\u0026ndash;20 cm and 20\u0026ndash;40 cm soil layers across all treatments.\u003c/p\u003e\u003cp\u003eAt the blooming stage: in 2023, the pH of the 0-20cm soil layer in the N10 treatment was significantly higher than that in the other treatments, while no significant difference was found between the CK and N8 treatments; for the 20-40cm soil layer, the N8 treatment had a significantly higher pH than the CK and N10 treatments, whereas no significant difference was observed between the CK and N10 treatments. In 2024, no significant differences were detected in the pH levels of the 0-20cm and 20-40cm soil layers among the various treatments.\u003c/p\u003e\u003cp\u003eAt the boll opening stage: in 2023, the pH of the 0-20cm soil layer did not show significant differences among the treatments; however, the pH of the 20-40cm soil layer in the N8 treatment was significantly lower than that in the other treatments, with no significant difference observed between the CK and N10 treatments. In 2024, no significant differences were found in the pH levels of the 0-20cm and 20-40cm soil layers across all treatments.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\u003ch2\u003e3.1.2 Effect of frequency of nitrogen application on soil conductivity\u003c/h2\u003e\u003cp\u003eThe effects of nitrogen application frequency on soil conductivity in different soil layers during cotton fertility stages (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). At the bud stage: in 2023, in the 0\u0026thinsp;~\u0026thinsp;20cm soil layer, conductivity was significantly higher under the N10 compared to other treatments, and no significant difference was observed between CK and N8 ; in the 20\u0026thinsp;~\u0026thinsp;40cm soil layer, there was no significant difference in conductivity was detected among treatments. In 2024, there was no significant difference in conductivity was found in the 0\u0026thinsp;~\u0026thinsp;20cm soil layer across treatments; in the 20\u0026thinsp;~\u0026thinsp;40cm soil layer, conductivity was significantly higher under N8 and N10 compared to CK, with no significant difference between N8 and N10.\u003c/p\u003e\u003cp\u003eAt the blooming stage: in 2023, there was no significant difference in conductivity in either the 0\u0026thinsp;~\u0026thinsp;20cm or 20\u0026thinsp;~\u0026thinsp;40cm soil layers among treatments. In 2024, in the 0\u0026thinsp;~\u0026thinsp;20cm soil layer, conductivity was significantly higher under N10 compared to other treatments, with no significant difference between N8 and CK; in the 20\u0026thinsp;~\u0026thinsp;40cm soil layer, conductivity was significantly higher under N8 and N10 compared to CK, with no significant difference between N8 and N10.\u003c/p\u003e\u003cp\u003eAt the boll opening stage: in 2023, in both the 0\u0026thinsp;~\u0026thinsp;20cm and 20\u0026thinsp;~\u0026thinsp;40cm soil layers, conductivity was significantly higher under N8 compared to CK, with no significant difference between N8 and N10. In 2024, there was no significant difference in conductivity were found in the 0\u0026thinsp;~\u0026thinsp;20cm soil layer; in the 20\u0026thinsp;~\u0026thinsp;40cm soil layer, conductivity was significantly higher under N8 and N10 compared to CK, with no significant difference between N8 and N10.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section3\"\u003e\u003ch2\u003e3.1.3 Effect of frequency of nitrogen application on soil organic matter content\u003c/h2\u003e\u003cp\u003eThe effect of nitrogen application frequency on soil organic matter in different soil layers during various fertility periods of cotton is depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. At the bud stage: in 2023, the organic matter content in the 0\u0026ndash;20 cm and 20\u0026ndash;40 cm soil layers under the N8 treatment was significantly higher than that under the CK, and no significant differences was observed between the CK and N10 treatments. In 2024, the organic matter content in the 0\u0026ndash;20 cm and 20\u0026ndash;40 cm soil layers under the N8 treatment was significantly higher than that under the CK treatment, the organic matter content between the N8 and N10 treatments was not significantly different.\u003c/p\u003e\u003cp\u003eAt the blooming stage: in 2023, the organic matter content in the 0\u0026ndash;20 cm soil layer under both the N8 and N10 treatments was significantly higher than that under the CK treatment, with no significant difference between the N8 and N10 treatments; in the 20\u0026ndash;40 cm soil layer, there was no significant difference in organic matter content among the treatments. In 2024, the organic matter in the 0\u0026ndash;20 cm and 20\u0026ndash;40 cm soil layers under both the N8 and N10 treatments was significantly higher than that under the CK treatments.\u003c/p\u003e\u003cp\u003eAt the boll opening stage: in 2023, the organic matter of 0\u0026thinsp;~\u0026thinsp;20cmcm soil layer under the N8 treatment was significantly higher than that under the CK treatment, while no significant difference was observed between the CK and N10 treatments; in the 20\u0026thinsp;~\u0026thinsp;40 cm soil layer, no significant difference in organic matter content was found among the treatments. In 2024, the organic matter content in the 0\u0026thinsp;~\u0026thinsp;20 cm and 20\u0026thinsp;~\u0026thinsp;40 cm soil layer under the N8 treatment was significantly higher than that under the CK treatment, but no significant difference was observed between the N8 and N10 treatments.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section3\"\u003e\u003ch2\u003e3.1.4 Effect of frequency of nitrogen application on soil alkali-hydrolyzable nitrogen content\u003c/h2\u003e\u003cp\u003eThe effects of nitrogen application frequency on soil alkali-hydrolyzable nitrogen (AN) in different soil layers during cotton fertility stages (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). At the bud stage: in 2023, in the 0\u0026ndash;20 cm soil layer, AN under the N8 treatment was significantly higher than that under other treatments, and there was no significant difference between CK and N10 treatments; in the 20\u0026ndash;40 cm soil layer, there was no significant difference in AN among treatments. In 2024, in both the 0\u0026ndash;20 cm and 20\u0026ndash;40 cm soil layers,AN under the N8 treatment was significantly higher than that under CK treatment, and there was no significant difference between CK and N10 treatments.\u003c/p\u003e\u003cp\u003eAt the blooming stage: in 2023, in the 0\u0026ndash;20 cm soil layer, the alkali-hydrolyzable nitrogen (AN) under the N8 treatment was significantly higher than that under the CK treatment, and there was no significant difference between the N8 and N10 ; in the 20\u0026ndash;40 cm soil layer, there was no significant difference in the AN among treatments. In 2024, in both the 0\u0026ndash;20 cm and 20-40cm soil layers, AN under N8 was significantly higher than that under the CK treatment, and there was no significant difference between the CK and N10 treatments\u003c/p\u003e\u003cp\u003eAt the boll opening stage: in 2023, in the 0\u0026ndash;20 cm soil layer, the alkali-hydrolyzable nitrogen (AN) under the N8 treatment was significantly higher than that under other treatments, and there was no significant difference between CK and N10 treatments; in the 20\u0026ndash;40 cm soil layer, there was no significant difference in AN among treatments. In 2024, the AN in both the 0\u0026thinsp;~\u0026thinsp;20 cm and 20\u0026thinsp;~\u0026thinsp;40 cm soil layers under the N8 treatment was significantly higher than that under the CK treatment, and there was no significant difference between N8 and N10.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Effect of frequency of nitrogen application on dry matter and nitrogen accumulation in cotton\u003c/h2\u003e\u003cdiv id=\"Sec19\" class=\"Section3\"\u003e\u003ch2\u003e3.2.1 Effect of frequency of nitrogen application on dry matter quality\u003c/h2\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, in 2023, the total dry matter accumulation in cotton organs across different growth stages was recorded in 2023 and 2024. In 2023, the dry matter accumulation ranged from 3902.4 to 6553.1 kg-hm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003eat bud stage, 5252.4 to 9370.1 kg-hm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e at the blooming stage, and 6503.28 to 12415.4 kg-hm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003eat the boll opening stage. In 2024, these values were 2654.3 to 5699.6 kg-hm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e, 4960.3 to 7573.6 kg-hm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e and 6086.6 to 10287.2 kg-hm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e at bud, blooming, and boll opening stages, respectively. When comparing the N8 treatment to the CK treatment in 2023, dry matter mass of cotton increased by an average of 46.43% at the bud stage, 78.39% at the blooming stage, and 58.14% at the boll opening stage.In 2024, under the N8 treatment, the average increases compared to CK were 80.45% at the bud stage, 52.68% at the blooming stage, and 58.37% at the boll opening stage. Similarly, for the N10 treatment in 2023, the average increase in dry matter compared to CK was 67.92% at bud stage, 37.14% at the blooming stage and 90.91% at the boll opening stage. In 2024, the N10 treatment resulted in average increases of 114.73% at the bud stage, 40.58% at the blooming stage, and 69.01% at the boll opening stage compared to CK.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section3\"\u003e\u003ch2\u003e3.2.3 Effect of frequency of nitrogen application on nitrogen uptake\u003c/h2\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, the total nitrogen accumulation in cotton organs at the bud, blooming, and boll opening stages ranged from 154.84\u0026thinsp;~\u0026thinsp;331.50 kg-hm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e, 225.65\u0026thinsp;~\u0026thinsp;443.51 kg-hm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e and 204.64\u0026thinsp;~\u0026thinsp;420.58 kg-hm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e in 2023, respectively; and 152.74\u0026thinsp;~\u0026thinsp;373.04 kg-hm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e, 252.57\u0026thinsp;~\u0026thinsp;430.61 kg-hm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e and 275.84\u0026thinsp;~\u0026thinsp;489.70 kg-hm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e in 2024, respectively; under the N8 treatment compared to CK, nitrogen accumulation increased by an average of 80.05% (bud), 96.54% (blooming) and 82.19% (boll opening) in 2023, and 92.66%, 70.48% and 53.40% in 2024, respectively; under the N10 treatment compared to CK, increases averaged 114.08% (bud), 38.86% (blooming) and 105.52% (boll opening) in 2023, and 144.22%, 53.45% and 77.53% in 2024, respectively.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Effects of frequency of nitrogen application on photosynthetic characteristics and spad values of cotton\u003c/h2\u003e\u003cdiv id=\"Sec22\" class=\"Section3\"\u003e\u003ch2\u003e3.3.1 Effects of frequency of nitrogen application on photosynthetic characteristics\u003c/h2\u003e\u003cp\u003eThe frequency of nitrogen application significantly influenced the photosynthetic characteristics of cotton during the bud and boll stages ( Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). In 2023, the net photosynthetic rate, transpiration rate, and stomatal conductance of the N8 treatment were higher than those of other treatments, while no significant differences were observed between the CK and N10 treatments. The intercellular CO₂ concentration in the CK treatment was higher than in other treatments, with no significant difference between the N8 and N10 treatments. In 2024, the net photosynthetic rate, transpiration rate, and stomatal conductance of the N8 treatment were significantly higher than those of other treatments; but the intercellular CO₂ concentration in the CK treatment was significantly higher than in other treatments.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003e3.3.2 Effects of frequency of nitrogen application on SPAD values\u003c/h2\u003e\u003cp\u003eThe frequency of nitrogen application significantly influenced SPAD values during the budding and boll-setting stages of cotton ( Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). In 2023, SPAD values for the N8 treatment showed no significant difference compared to the N10 treatment, while were significantly higher than the CK treatment, representing increases of 13.71% and 37.21%, respectively. In 2024, the SPAD values of the N8 treatment during the budding and boll-setting stages were significantly higher than those of other treatments, compared with the CK treatment, increased by 21.73% and 6.90%, respectively.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003e3.4 Effect of frequency of nitrogen application on cotton yield, yield components and nitrogen fertilizer utilization rate\u003c/em\u003e\u003c/p\u003e\u003cp\u003eIn both 2023 and 2024, no significant differences in cotton harvest density were observed among treatments (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In 2023, the number of cotton bolls per plant under N8 and N10 was significantly higher than that under the CK treatment, and with no significant difference between N8 and N10 treatments. In 2024, the N8 treatment exhibited a significantly higher number of bolls per plant compared to other treatments, while no significant differences were found between the CK and N10 treatments. Regarding cotton boll weight, no significant differences among treatments were observed in 2023, however, in 2024, the N8 treatment showed significantly higher boll weights than other treatments, with no significant difference between CK and N10. In 2023, in terms of seed cotton yield, the N8 treatment significantly outperformed CK, and there was no significant difference between N8 and N10. In 2024, the N8 treatment yielded significantly more than CK. Compared to CK, the N8 treatment increased seed cotton yield by 31.94% in 2023 and 36.78% in 2024. There was no significant difference in nitrogen fertilizer use efficiency between the N8 and N10 treatments in either year.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eEffects of nitrogen application times on cotton yield traits.Data are mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Different lowercase letters in the same column indicated significant difference between different treatments in the same year (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eYear\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTreatment\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNumber of plants harvested\u003c/p\u003e\u003cp\u003e/\u0026times;10\u003csup\u003e4\u003c/sup\u003e\u0026middot;hm\u003csup\u003e-2\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNumber of bells per plant\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eBoll count/g\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eSeed cotton yield/kg\u0026middot;hm\u003csup\u003e-2\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNitrogen fertilizer utilization rate/%\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e2023\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCK\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e17.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.76a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3183\u0026thinsp;\u0026plusmn;\u0026thinsp;274b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eN8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e19.34\u0026thinsp;\u0026plusmn;\u0026thinsp;1.01a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e4200\u0026thinsp;\u0026plusmn;\u0026thinsp;299a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e56.07\u0026thinsp;\u0026plusmn;\u0026thinsp;17.37a\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eN10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e18.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.92a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3831\u0026thinsp;\u0026plusmn;\u0026thinsp;360a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e71.98\u0026thinsp;\u0026plusmn;\u0026thinsp;11.87a\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e2024\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCK\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e15.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.83a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3205\u0026thinsp;\u0026plusmn;\u0026thinsp;165c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eN8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e17.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e4384\u0026thinsp;\u0026plusmn;\u0026thinsp;242a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e49.11\u0026thinsp;\u0026plusmn;\u0026thinsp;22.34a\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eN10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e17.19\u0026thinsp;\u0026plusmn;\u0026thinsp;1.36a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3770\u0026thinsp;\u0026plusmn;\u0026thinsp;234b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e71.29\u0026thinsp;\u0026plusmn;\u0026thinsp;7.74a\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Correlation of cotton seed cotton yield with various indicators\u003c/h2\u003e\u003cp\u003eThe correlation analysis results between cotton yield, soil physicochemical properties, cotton growth indicators, and cotton photosynthetic characteristics are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e. Statistical analysis indicates that cotton yield shows no statistically significant correlation with pH or alkali-hydrolyzable nitrogen. Conversely, cotton yield exhibits a highly significant positive correlation with organic matter content; it also shows a highly significant positive correlation with dry matter accumulation and nitrogen accumulation. Furthermore, cotton yield demonstrates a highly significant positive correlation with net photosynthetic rate, transpiration rate, stomatal conductance, and SPAD values, while exhibiting a highly significant negative correlation with intercellular CO₂ concentration.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cdiv id=\"Sec26\" class=\"Section2\"\u003e\u003ch2\u003e4.1 Effect of frequency of nitrogen application on soil chemical properties\u003c/h2\u003e\u003cp\u003eThe nutrient content of soil significantly influences its fertility status, directly affecting cotton yield and quality[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e][\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In this experiment, we observed that the varying N application frequencies had a relatively minor impact on soil pH. This may be attributed to the fact that urea, a water-soluble fertilizer, is typically applied in multiple split doses as a follow-up fertilizer. It is rapidly absorbed by plants and becomes diluted with water, diminishing its inherent acid-base properties, and thus having a limited impact on soil pH. Cai et al[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] demonstrated that, compared to a single follow-up fertilizer application, three to four follow-up applications led to a greater concentration of soil NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003eN in the upper soil layer (0\u0026ndash;40 cm) during the flowering and sizing stages of wheat. Qiu et al. [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] showed that, with an N application rate of 300 kg-hm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e, a fertilization schedule of nine times (20% basal application, 20% in three applications at the bud stage, 30% in three applications at anthesis, and 30% in three applications at full bloom) significantly increased the levels of soil nitrate, ammonium, and alkali-hydrolyzable nitrogen (AN) compared to a no-N fertilization treatment. Zhang et al.[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] found that excessive water inflow and prolonged high-frequency irrigation were primary factors contributing to carbon leaching under certain conditions. In contrast, this study revealed that under drip irrigation beneath a membrane, the surface soil organic matter and alkali-hydrolyzable nitrogen in the N8 treatment were significantly higher than those in the control (CK) treatment. This environment was conducive to the crop's absorption of soil nutrients, thus promoting the growth and development of cotton and enhancing yield formation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section2\"\u003e\u003ch2\u003e4.2 Effect of nitrogen application frequency on dry matter and nitrogen accumulation in cotton\u003c/h2\u003e\u003cp\u003eAppropriate dry matter accumulation and its coordinated dynamics are crucial for establishing an optimal population structure and achieving high cotton yields[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e][\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e][\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Lu et al[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] demonstrated that regulating fertilizer application timing and nitrogen dosage could maintain balanced nitrogen uptake and utilization by reproductive organs, thereby increasing biomass and ultimately enhancing yields. Ren et al[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] reported that, during later growth stages, dry matter accumulation increased by 54.6% and 73.3% in Ningjin and Changyi experimental areas, respectively, under a nitrogen allocation ratio of 3:5:2 compared to a 0:10:0 treatment. Abdelraouf et al[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] found that increasing fertilization frequency and shortening intervals in 2014/2015 and 2015/2016 improved nitrogen uptake efficiency, cumulative N uptake and grain N content in both observed and simulated values. This study showed that both N8 and N10 treatments enhanced dry matter accumulation in cotton roots, stems and leaves and reproductive organs. At the bud stage, the N10 treatment exhibited higher total dry matter than N8, during the blooming stage, the N8 treatment increased overall plant dry matter by promoting accumulation in reproductive organs; and at the boll open stage, the N10 treatment boosted total plant dry matter by enhancing accumulation in vegetative organs. These differences may stem from varying nutrient demands across cotton growth stages[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], and the flowering stage is a critical phase for reproductive growth, the N8 treatment supplied high nitrogen during this period to meet nutrient demand, ensuring sufficient nitrogen availability, accelerating the transition to reproductive growth, and facilitating nitrogen uptake and redistribution from vegetative to productive organs. The N10 treatment applied nitrogen later in the flowering stage, sustaining vigorous vegetative growth and reducing nitrogen transfer efficiency to seeds, which hindered nutrient translocation from leaves and stems[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Therefore, appropriate nitrogen application rates and allocation ratio are essential for optimizing plant dry matter accumulation.\u003c/p\u003e\u003cp\u003eNitrogen uptake and utilization are prerequisites for dry matter accumulation, which forms the basis for high cotton yields[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e][\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e][\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].Uzen et al [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] showed that moderate fertilization frequency significantly enhanced total nitrogen uptake compared to high or low frequencies. Feng et al[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] reported that apparent nitrogen loss was 48.3% under single nitrogen application but reduced it to 38.5% with split applications. This study showed that both N8 and N10 treatments increased nitrogen accumulation in cotton roots, stems, leaves, and reproductive organs, the N8 treatment effectively balanced vegetative and reproductive growth, promoting nitrogen accumulation in reproductive organs and facilitating photosynthate translocation. Wang et al[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e] noted that while nitrogen content increased with fertilizer dosage, excessive nitrogen slowed or reduced nitrogen accumulation, highlighting the importance of optimal nitrogen application for maximizing accumulation rates. The present study showed that the N10 treatment at the bud stage exhibited higher N accumulation than N8; the N8 treatment at the blooming stage enhanced total plant nitrogen accumulation by increasing uptake in both vegetative and reproductive organs; the N10 treatment at the boll open stage increased the N accumulation of the whole cotton plant by promoting uptake in vegetative organs.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\u003ch2\u003e4.3 Effect of nitrogen application frequency on photosynthetic characteristics and SPAD values in cotton\u003c/h2\u003e\u003cp\u003eCotton yield exhibits a strong correlation with plant growth and development indicators, and enhancing leaf photosynthetic properties can promote plant growth, development, and dry matter accumulation. Tian et al.[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] demonstrated that during the late growth stage of cotton, adjusting nitrogen application frequency and proportion can increase net photosynthetic rate, transpiration rate, and stomatal conductance while reducing intercellular CO₂ concentration.The results of this study indicate that under the N8 treatment, cotton exhibited higher net photosynthetic rates, transpiration rates, and stomatal conductance compared to the CK and N10 treatments, while intercellular CO₂ concentrations were lower than those in the CK treatment. This suggests that varying nitrogen application frequencies exert distinct effects on cotton photosynthesis, with the application of nitrogen fertilizer in eight installments more effectively promoting photosynthetic activity. Nitrogen fertilization increased net photosynthetic rate, transpiration rate, and stomatal conductance while decreasing intercellular CO₂ concentration, consistent with studies by Dai et al.[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] and Li et al.[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. This may occur because under nutrient-sufficient conditions, stomata open to enhance crop respiration, allowing CO₂ to enter plant cells. This reduces intercellular CO₂ concentration, increases photosynthetic rate, and ultimately boosts cotton photosynthetic capacity. Under the experimental conditions, the SPAD values of the N8 treatment were significantly higher than those of the CK treatment, while the N10 treatment showed no significant difference from the CK treatment. This indicates that an appropriate nitrogen application frequency can promote the vegetative growth of cotton, increase chlorophyll content in cotton leaves, and lay a material foundation for high cotton yields [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cem\u003e4.4 Effect of different frequency of nitrogen application on cotton yield, yield components and nitrogen fertilizer utilization rate\u003c/em\u003e\u003c/p\u003e\u003cp\u003eNitrogen nutrition plays a critical role in regulating the cotton yield formation, improper fertilizers leads to excessive vegetative growth or nutrient deficiency, reducing boll number, boll weight and yield. Conversely, rational nitrogen management coordinates vegetative and reproductive growth, ensuring high-yield and high-quality cotton production[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e][\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e][\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e].Yang et al[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e] found that in the Yangtze River Basin cotton region, one-time fertilization yielded similar results to traditional three-time fertilization but outperformed two-time fertilization. Luo et al [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e] in the cotton region of the Yangtze River Basin, showed that among all one-time fertilization schedules, the one-time application of fertilizer to the first flower stage maximized yield, comparable to traditional three-time applications. In this study, N8 and N10 treatments increased seed cotton yield by raising boll number per plant and boll weight, with N8 being the most effective compared to the CK treatment under a total pure N rate of 300 kg-hm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eNitrogen fertilizer utilization efficiency is a key metric reflecting crop nitrogen uptake and utilization[\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e][\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Cai et al[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] showed that splitting follow-up nitrogen applications 3\u0026ndash;4 times, with about 16.7% applied at the flowering and the irrigating stages, significantly improved yield, WUE (Water Use Efficiency), PFPN (Partial Productivity of Fertilizer with Nitrogen), and NHI (Nitrogen Harvest Index). In this study, the N10 treatments slightly improved N fertilizer utilization efficiency compared to N8, though the difference was not statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eIn summary, nitrogen frequency significantly influenced soil nutrient content, dry matter and nitrogen accumulation, photosynthetic characteristics,yield traits and nitrogen fertilizer utilization efficiency in Xinjiang cotton fields. The N8 treatment maintained high soil organic matter and alkali-hydrolyzable nitrogen levels throughout the reproductive period. Adjusting nitrogen application frequency under consistent nitrogen rates optimized dry matter and nitrogen distribution, promote photosynthesis,enhancing the nitrogen fertilizer utilization efficiency and yield. Under the experimental conditions, drip fertilization with water in 8 split applications (N8) yielded the best results.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e Conceptualization, X.W. and B.C., methodology, X.W., J.Z. and L.Z., software,Y.W. and X.W., validation, J.Z. and X.W., formal analysis, X.W. and L.Z., investigation, X.W. and J.Z., resources, B.C., data curation, X.W. and L.Z., writing-original draft preparation, X.W.; writing review and editing, X.W. and B.C., funding acquisition, B.C. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This work was supported by the Science Foundation for Outstanding Young Scholars of Xinjiang Uygur Autonomous Region (Grant No. 2024D01E06), the Key Laboratory Project of \u0026quot;Soil and Plant Ecological Processes in Xinjiang\u0026quot;(Grant No. 24XJTRZWY02); the Project for Xinjiang Agricultural University Graduate Research and Innovation(Grant No. XJAUGRI2025068); the Project for Young Top-Notch Talents in Science and Technology of Xinjiang Uygur Autonomous Region (Grant No. 2022TSYCCX0085), the National Natural Science Foundation of China (Grant No. 32360793 and 31960629), the Key Research and Development Project in Xinjiang Uygur Autonomous Region (Grant No. 2022B02033-1), and Special Topics of Major Science and Technology in Xinjiang Uygur Autonomous Region (Grant No.2022A02007-2). We thank David for editing the English text of a draft of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u003c/strong\u003e The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e We appreciate and thank the anonymous reviewers for helpful comments that led to an overall improvement of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u003c/strong\u003e The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\u003cp dir=\"LTR\"\u003e\u003cstrong\u003eDisclaimer/Publisher\u0026rsquo;s Note:\u003c/strong\u003e The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eDai, F.; Chen, J.; Zhang, Z.; Liu, F.; Li, J.; Zhao, T.; Hu, Y.; Zhang, T. \u0026amp; Fang, L. Cottonomics: a comprehensive cotton multi-omics database. \u003cem\u003eDatabase.\u003c/em\u003e 2022, baac080. https://doi.org/10.1093/database/baac080.（2022）\u003c/li\u003e\n\u003cli\u003eChina Statistical Yearbook; China Statistics Press: Beijing, China, 2024. (In Chinese)（2024）\u003c/li\u003e\n\u003cli\u003eZhou, Y.Q.; Li, F.; Xin, Q.C.; Li, Y.M. \u0026amp; Lin, Z.D. Historical variability of cotton yield and response to climate and agronomic management in Xinjiang, China. \u003cem\u003eSci. 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Wheat genetic improvement affects the fate of 15N fertilizer, improving nitrogen uptake and utilization. \u003cem\u003eField Crops Res.\u003c/em\u003e \u003cstrong\u003e333\u003c/strong\u003e, 110078.https://doi.org/10.1016/j.fcr.2025.110078.（2025）\u003c/li\u003e\n\u003cli\u003eXia, H.; Wang, J.; Riaz, M.; Babar, S.; Wang, X.; Xia, X.; Yan, B.; Liu, B. \u0026amp; Jiang, C. Interaction between biochar and nitrogen fertilizer improves nitrogen utilization efficiency, closely connected with rhizosphere microbes involved in nitrogen-cycling. \u003cem\u003eSoil Tillage Res.\u003c/em\u003e \u003cstrong\u003e254, \u003c/strong\u003e106732.https://doi.org/10.1016/j.still.2025.106732.（2025）\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"cotton, nitrogen application frequency, dry matter accumulation, nitrogen accumulation, yield, nitrogen use efficiency","lastPublishedDoi":"10.21203/rs.3.rs-7923043/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7923043/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWater-nitrogen (N) coupling is recognized as a crucial strategy for enhancing crop yield and improving nitrogen use efficiency (NUE).However, the specific mechanisms by which the frequency of split nitrogen fertilizer applications influences cotton yield and NUE under water-nitrogen coupling conditions remain poorly understood. To determine optimal N fertilization strategies,a two-year consecutive field experiment was conducted from 2023 to 2024, comparing three treatments: no nitrogen fertilizer (CK), nitrogen fertilizer applied in eight follow-up applications (N8) and nitrogen fertilizer applied in ten split applications (N10). The results indicated that the N8 treatment maintained the highest levels of soil organic matter and alkali-hydrolyzable nitrogen content in the 0\u0026thinsp;~\u0026thinsp;20 cm soil layer compared with other treatments. When nitrogen application rates were consistent, adjusting the frequency of nitrogen application could improve the distribution of dry matter and nitrogen accumulation in cotton within the Xinjiang cotton-growing region. Specifically, the N8 treatment achieved a balanced relationship between vegetative and reproductive growth, facilitating greater accumulation of dry matter and nitrogen in reproductive organs. In both 2023 and 2024, the N8 treatment increased net photosynthetic rate by 25.27% and 45.21%, transpiration rate by 55.30% and 46.42%, and stomatal conductance by 38.89% and 55.95%, seed cotton yield by 31.94% and 36.78%, nitrogen fertilizer utilization rates by 56.07% and 49.11%, respectively,compared to the CK treatment, while intercellular CO₂ concentrations decreased by 18.76% and 22.91%, respectively. Moreover, cotton seed yield is highly significantly and positively correlated with dry matter accumulation and nitrogen accumulation.Under irrigation under plastic film irrigation, applying N fertilizer in eight doses effectively ensured soil nutrient availability, promoted dry matter and nitrogen in cotton, promote photosynthesis, and thereby enhanced cotton yield.\u003c/p\u003e","manuscriptTitle":"Impact of Nitrogen Application Frequency on Cotton Yield and Nitrogen Use Efficiency under Integrated Water-Fertilizer Management","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-07 12:05:24","doi":"10.21203/rs.3.rs-7923043/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-17T09:20:54+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-07T10:13:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"257282531168643951623141750624369944889","date":"2026-01-04T11:27:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"16115456718162371770870849679069753461","date":"2026-01-04T08:49:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"48163325879921119616635644874375296096","date":"2026-01-02T16:59:10+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-01T08:09:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"307787845368126072391826075083952107597","date":"2025-10-30T03:54:55+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-28T01:41:52+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-10-27T12:45:55+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-23T14:07:05+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-23T14:05:02+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-10-22T11:30:22+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7624e67a-05ca-476b-9c27-a87522ad936e","owner":[],"postedDate":"November 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":57264653,"name":"Biological sciences/Ecology"},{"id":57264654,"name":"Earth and environmental sciences/Ecology"},{"id":57264655,"name":"Earth and environmental sciences/Environmental sciences"},{"id":57264656,"name":"Biological sciences/Plant sciences"}],"tags":[],"updatedAt":"2026-04-24T08:09:43+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-07 12:05:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7923043","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7923043","identity":"rs-7923043","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}