Search Results (50)

Optimizing irrigation and nitrogen (N) management in saline soils is critical for sustainable cotton production in arid regions that have been subjected to climate change. In this study, a two-year factorial field experiment (3 salinity levels × 3 N rates × 3 irrigation quotas) is integrated with the RZWQM2 model to (1) identify water–N–salinity thresholds for cotton yield and (2) to project climate change impacts under SSP2.4-5 and SSP5.8-5 scenarios (2031–2090) in Xinjiang, China, a global cotton production hub. The results demonstrated that a moderate salinity (6 dS/m) combined with a reduced irrigation (3600 m³/hm²) and N input (210 kg/hm²) achieved a near-maximum yield (6918 kg/hm²), saving 20% more water and 33% more fertilizer compared to conventional practices. The model exhibited a robust performance (NRMSE: 5.94–12.88% for soil–crop variables) and revealed that warming shortened the cotton growing season by 1.2–9.5 days per decade. However, elevated CO₂ (832 ppm by 2090) levels under SSP5.8-5 increased yields by 22.6–42.1%, offsetting heat-induced declines through enhanced water use efficiency (WUE↑27.5%) and biomass accumulation. Critically, high-salinity soils (9 dS/m) required 25% additional irrigation (4500 m³/hm²) and a full N input (315 kg/hm²) to maintain yield stability. These findings provide actionable strategies for farmers to optimize irrigation schedules and nitrogen application, balancing water conservation with yield stability in saline-affected arid agroecosystems that have been subjected to climate change. Full article

The utilization rate of phosphorus fertilizer is low in Xinjiang, China, due to the fact that phosphorus is easily fixed by the widely distributed lime soil, leading to the limited contribution of phosphorus fertilizer to crop yield and a decline in crop quality. Phosphate-soluble bacteria can convert insoluble phosphates in the soil into soluble phosphates, playing an important role in soil phosphorus circulation and plant growth. In this study, two bacteria with strong phosphate-solubilizing ability, Enterobacter hormaechei (P1) and Bacillus atrophaeus (P2), were selected from severely salinized soils in Xinjiang, China. The taxonomic status of the strains was determined by analyzing the colony morphology and 16S rRNA gene sequence similarity. Then, the content of organic acids and the activity of acid phosphatase and phytase in the P1 and P2 fermentation broths were measured. Finally, field experiments were conducted in 20 April–2 October 2023 in Wulanwusu, Xinjiang, China, to analyze the effects of phosphate-solubilizing bacterial agents (P1, P2, and P3 (P1 + P2)) on soil physicochemical properties, microbial diversity, and cotton yield. The results showed that both P1 and P2 could significantly solubilize phosphates and produce indole-3-acetic acid (IAA), lactic acid, and tartaric acid. In the cotton field under phosphorus fertilization, the cotton yield of P1, P2, and P3 treatments increased by 10.77%, 8.48%, and 14.00%, respectively, compared with no bacterial agent treatment (CK) (p < 0.05). In addition, the application of phosphate-solubilizing bacterial agents also significantly increased the content of available nutrients and the abundances of Acidobacteria, Bacteroidetes, Fusarium, Bacteroidetes, and Verrucobacteria in the soil compared with CK. In summary, inoculating with phosphate-solubilizing bacteria could promote cotton growth and yield formation by increasing soil available nutrients and altering soil microbial communities. This study will provide a basis for the efficient utilization of phosphorus resources and sustainable agricultural development. Full article

In global drought-prone cotton-growing (Gossypium hirsutum L.) areas, saline water irrigation has become a key strategy to alleviate the shortage of freshwater resources. Against this backdrop, the synergistic effect of mineralization degree (MD) and sodium adsorption ratio (SAR) on the rhizosphere microecological regulation mechanism remains unclear. To address this issue, this study constructed an experimental framework of the interaction between MD and SAR, aiming to explore their effects on the bacterial community structure in the rhizosphere of cotton and the soil environment. The soil type in the study area is saline–sodic sandy loam. In the experimental design, three MD levels (3 g/L, 5 g/L, 7 g/L) were set, and under each mineralization condition, three SAR levels (10 (mmol/L)^1/2, 15 (mmol/L)^1/2, 20 (mmol/L)^1/2) were arranged. In addition, local freshwater irrigation was used as the control group (CG), resulting in a total of 10 treatment schemes. The aim of this study was to investigate the effects of varying levels of irrigation water MD and SAR on the structure of bacterial communities in cotton rhizosphere soil and the soil environment. The results indicated that saline water irrigation could enhance the diversity and richness of the bacterial community in the rhizosphere soil of cotton and alter its community structure. Under treatment with the MD of 3 g/L and the SAR of 10 (mmol/L)^1/2, the diversity and richness of the bacterial community in the cotton rhizosphere reached their peak levels. Compared with the CG, the Chao1 index significantly increased by 260 units, while the Shannon index increased by 0.464. When the MD does not exceed 5 g/L, reducing SAR can enhance the diversity and network stability of the rhizosphere bacterial community, thereby synergistically promoting the accumulation of soil nutrients. The key soil environmental factors driving changes in the rhizosphere bacterial community structure mainly include soil moisture content, total nitrogen, nitrate nitrogen, and total organic carbon. The concentrations of total nitrogen, nitrate nitrogen, available phosphorus, and available potassium significantly increased by 19.66%, 26.10%, 89.41%, and 49.76% respectively (p < 0.05). This study provides a theoretical basis for sustainable irrigation and microbial regulation strategies in saline–alkali cotton fields at the theoretical level, and offers a new perspective for revealing the mutual feedback mechanism between bacterial community assembly and soil environment under saline conditions. From a practical perspective, this research offers valuable hands-on experience for optimizing agricultural ecological management in saline–alkali sandy loam soils, thereby contributing to the sustainable development of agriculture on such lands. Full article

Soil salinization and alkalization can cause great losses to agricultural production in arid regions. Cotton, a common crop in arid and semi-arid regions in China, often encounters saline stress and alkaline stress. In this study, NaCl (8 g·kg⁻¹), Na₂CO₃ (8 g·kg⁻¹⁾, and a compound material (an organic polymer compound material) were mixed with field soil before cotton sowing, and the ion content, photosynthetic characteristics, and metabolite levels of the new cotton leaves were analyzed at the flowering and boll-forming stage, aiming to clarify the photosynthetic and metabolic mechanisms by which compound material regulates cotton’s tolerance to saline stress and alkaline stress. The results showed that the application of the compound material led to an increase in the K⁺/Na⁺ ratio, stomatal conductance (Gs), efficiency of PSII photochemistry (ψPSⅡ), potential activity (Fv/Fo), and chlorophyll content (Chla and Chlb), as well as the abundances of D-xylonic acid and DL-phenylalanine in the NaCl treatments. Additionally, there were increases in the K⁺ content, K⁺/Na⁺ ratio, Chla/b ratio, net photosynthetic rate (Pn), transpiration rate (Tr), ψPSⅡ, and D-saccharic acid abundance in the Na₂CO₃ treatments. A correlation analysis and a metabolic pathway analysis revealed that the compound material mainly regulated the photosynthetic characteristics of and the ion balance in the new leaves through regulating the abundance of key metabolites when the cotton was under NaCl stress or Na₂CO₃ stress. Furthermore, the positive impact of the compound material on the cotton’s NaCl stress tolerance was stronger than that on the cotton’s Na₂CO₃ stress tolerance. Full article

To improve cotton yield in salinized arid fields, excess salt is removed and phosphorus content is increased. Adjusting phosphate fertilizer timing with water and fertilizer reduces phosphorus binding with calcium ions. Salt removal precedes phosphate application, enhancing soil phosphorus availability and promoting better growth. However, the optimal time for delaying phosphate fertilizer drip irrigation remains unclear. Therefore, this study evaluated the total salt, soil available phosphorus, and cotton yield under the condition of delayed phosphate fertilizer application. We conducted a field experiment using a completely randomized design to adjust the timing of phosphatic fertilizer application and apply the same amount of pure phosphorus. Specifically, “t” was defined as the total duration of one irrigation cycle, and the starting points for phosphorus application were as follows: T1, 1 h; T2, 1 h + 1/3 t h; T3, 1 h + 2/3 t h; CK, 1/3 t h. These values represent the duration of salt leaching through irrigation in each treatment. Phosphate fertilizer was applied to the soil after salt washing was complete. The results revealed that the T2 treatment exhibited the highest SPAD value (64.53), which was 11.46% and 15.48% higher than that of the T1 and T3 treatments. The 0–20 and 20–40 cm soil layers under the T2 treatment had the highest pH values of 9.12 and 9.37, representing increases of 1.93%, 1.21%, 4.50%, and 1.38% compared with T1 and T3 treatments, respectively (p < 0.05). At the bud stage, the Olsen-P in the T2 treatment was 82.86% and 26.53% higher than that in the T1 and T3 treatments, respectively (p < 0.05). The T2 treatment achieved the highest yield of 6492.09 kg/hm², which was 31.47%, 31.53%, and 2.77% higher than that of T1, T3, and CK. Overall, the T2 treatment increased cotton yield and reduced the adsorption of calcium ions to available phosphorus in salinized soil. This study provides an effective technical approach for the sustainable development of salinized cotton fields in Xinjiang. Full article

The risk of soil salinization is prevalent in arid and semi-arid regions, posing a critical challenge to sustainable agriculture. This study addresses the need for accurate assessment of regional root-zone soil salt content (SSC) and understanding of underlying driving mechanisms, which are essential for developing effective salinization mitigation and water management strategies. A remote sensing inversion technique, initially proposed to estimate root-zone SSC in cotton fields, was adapted and validated more widely to non-cotton farmlands. Validation results (with a coefficient of determination R² > 0.53) were obtained using data from a three-year (2020–2022) regional survey conducted in the arid Manas River Basin (MRB), Xinjiang, China. Based on this adapted technique, we analyzed the spatiotemporal distributions of root-zone SSC across all farmlands in MRB from 2001 to 2022. Findings showed that root-zone SSC decreased significantly from 5.47 to 3.77 g kg⁻¹ over the past 20 years but experienced a slight increase of 0.15 g kg⁻¹ in recent five years (2017–2022), attributed to cultivated area expansion and reduced irrigation quotas due to local water shortages. The driving mechanisms behind root-zone SSC distributions were analyzed using an approach combined with two machine learning algorithms, eXtreme Gradient Boosting (XGBoost) and SHapley Additive exPlanation (SHAP), to identify influential factors and quantify their impacts. The approach demonstrated high predictive accuracy (R² = 0.96 ± 0.01, root mean squared error RMSE = 0.19 ± 0.03 g kg⁻¹, maximum absolute error MAE = 0.14 ± 0.02 g kg⁻¹) in evaluating SSC drivers. Factors such as initial SSC, crop type distribution, duration of film mulched drip irrigation implementation, normalized difference vegetation index (NDVI), irrigation amount, and actual evapotranspiration (ET_a), with mean ($\left|\mathrm{SHAP} \mathrm{value}\right|$) ≥ 0.02 g kg⁻¹, were found to be more closely correlated with root-zone SSC variations than other factors. Decreased irrigation amount appeared as the primary driver for recent increased root-zone SSC, especially in the mid- and down-stream sections of MRB. Recommendations for secondary soil salinization risk reduction include regulation of the planting structure (crop choice and extent of planting area) and maintenance of a sufficient irrigation amount. Full article

The relationship between soil structure and salt accumulation is unclear; thus, experiments on salt accumulation under different soil structures were conducted in cotton fields in arid areas of northwest China. Thirty-nine sets of soil samples were collected from the 0 to 180 cm profile of three experimental areas. The total salt content of the soil extracts and the particle size distribution of the soil samples were determined using a JENCO TDS and a laser particle size analyzer, respectively, and the fractal dimension of the soil structure was obtained using fractal theory. Pearson’s correlation analysis and Tukey’s test (p < 0.01) were used to analyze the correlation between soil salinity, soil particle size distribution, and fractal dimensions in the three profiles. The results showed soil salinity accumulation was affected mutually by soil texture and soil structure, and soil salinity tended to accumulate in fine-grained soil. The soil fractal dimension (D) could indicate soil texture and quantify soil salinity content. When the sand content was more than 50%, there was a significant positive correlation between the soil fractal dimension and soil salinity (correlation coefficient R = 0.943). The results provide valuable insights into cotton production in arid areas. Full article

This study assesses soil productivity in a 15-hectare cotton field using an integrated approach combining field data, laboratory analysis, and remote sensing techniques. Soil samples were collected and analyzed for key parameters including nitrogen (N), humus, phosphorus (P₂O₅), potassium (K₂O), carbonates, pH, and electrical conductivity (EC). In addition to low salinity, these analyses showed low results for humus and nutrient parameters. A Pearson correlation analysis showed that low organic matter and high salinity had a strong negative correlation with crop productivity, explaining 37% of the variation in NDVI values. Remote sensing indices (NDVI, SAVI, NDMI, and NDSI) confirmed these findings by highlighting the relationship between soil properties and spectral reflectance. This research demonstrates the effectiveness of remote sensing in soil assessment, emphasizing its critical role in sustainable agricultural planning. By integrating traditional methods with advanced remote sensing technologies, this study provides actionable insights for policymakers and practitioners to improve soil productivity and ensure food security. Full article

The assessment of soil health relies on key parameters such as soil aggregates and organic matter content. Therefore, examining the impact of irrigation water ion composition and variations in salinity on soil aggregates and organic matter is imperative, which is key to developing a theoretical basis for the sustainable utilization of saline water resources, particularly in extremely arid regions. This experiment was conducted to investigate the impact of different irrigation water salinity treatments (T3: 3 g/L, T5: 5 g/L, and T7: 7 g/L) on the root zone soil of cotton fields. Each salinity treatment included three variations of the sodium adsorption ratio (SAR) at S10: 10 (mmol/L)^1/2, S15: 15 (mmol/L)^1/2, and S20: 20 (mmol/L)^1/2. Local freshwater irrigation served as the control, resulting in a total of 10 treatments. Our findings show that the soil Ca²⁺ and Mg²⁺ content increased with higher irrigation water salinity but decreased with increasing irrigation water SAR. The relative macroaggregate stability and the content of water-stable macroaggregates and soil organic matter (SOM) decreased as the irrigation water salinity and SAR increased. In comparison to T3S20, T5S10 did not improve the soil Na⁺ content but significantly increased the soil Ca²⁺ content by 147.76%, while the water-stable aggregate and SOM saw a notable increase of 7.66% and 9.86%, respectively. Reducing the SAR in brackish water lessens its negative impact on soil aggregates in cotton fields. This is primarily because Ca²⁺ counteracts the dispersive effect of high Na⁺ concentrations and promotes aggregate formation. Irrigation water with a salinity of 3 g/L and an SAR of 10 (mmol/L)^1/2 positively affected the stabilization of soil aggregates and organic matter. Full article

Soil salinization, a significant global challenge, threatens sustainable development. This study explores the potential of magnetized ionized water irrigation and Bacillus subtilis application to mitigate this issue. The former method is hypothesized to enhance soil salt leaching, while the latter is expected to improve soil nutrient availability, thereby increasing microbial diversity. To address the unclear impact of these interventions on soil quality and cotton productivity, this study employs four different experimental methods: magnetized ionized water irrigation (M), application of 45 kg ha⁻¹ B. subtilis (B), a combination of 45 kg ha⁻¹ B. subtilis with magnetized ionized water irrigation (MB), and a control treatment with no intervention (CK). This study aims to clarify the effects of these treatments on soil bulk density (BD), field capacity (FC), salinity and alkalinity, nutrient content, microbial activity, and cotton crop yield and quality. Additionally, it aims to evaluate the efficacy of these methods in improving saline soil conditions by developing a soil quality index. The results showed that using magnetized ionized water for irrigation and applying B. subtilis, either alone or together, can effectively lower soil pH and salt levels, enhance microbial diversity and abundance, and improve the yield and quality of cotton. Notably, B. subtilis application significantly decreased BD and enhanced FC and nutrient content (p < 0.05). A correlation was found where soil nutrient content decreased as pH and salt content increased. Furthermore, a strong correlation was observed between the major soil bacteria and fungi with BD, FC, and salt content. Comparatively, M, B, and MB significantly boosted (p < 0.01) the soil quality index by 0.21, 0.52, and 0.69 units, respectively, and increased (p < 0.05) cotton yield by 5.7%, 14.8%, and 20.1% compared to CK. Therefore, this research offers eco-friendly and efficient methods to enhance cotton production capacity in saline soil. Full article

Optimal drip irrigation management in shallow groundwater areas needs to clarify the effects of flow rate and layout designs on the soil moisture, salt distribution, cotton root length density, plant height, leaf area, and yield. In this study, a one-year field experiment was conducted from April to October 2018 in the fifth company of the 16th Regiment in Alar City, Xinjiang, to investigate the effects of various drip flow rates and layout designs of cotton growth. Two drip flow rates (2.8 and 5.6 L·h⁻¹) and two layout designs (one film, two drip tapes, and six rows; one film, three drip tapes, and six rows) were applied to explore the optimal combination, resulting in a total of four treatments that were irrigated three times in the whole growth period. Soil moisture, salt distribution, cotton root length density, plant height, and leaf area were measured. The main results were as follows: (1) Under the same layout designs, the soil moisture content was higher and the soil salinity was lower when the drip flow rate was 5.6 L·h⁻¹, and the cotton root length density, plant height, leaf area, and yield were significantly higher than that of 2.8 L·h⁻¹. (2) Under the same drip flow rate, the soil desalination rate, cotton growth indexes, and yield under the three-tapes treatment were significantly higher than the values of the two-tapes treatment. The actual yield of treatment D was 21.56%, 19.23%, and 11.71% higher than that of treatments A, B, and C, respectively. (3) The crop evapotranspiration of cotton during the two irrigation cycles showed an increasing trend, and the groundwater contribution showed a smaller and then increasing trend. Overall, the combination of three tapes and a drip flow rate of 5.6 L·h⁻¹ had the highest cotton yield and net income, which were 6211.36 kg·hm⁻² and 4820.21 kg·hm⁻² for the theoretical and actual yields. The results of this study can provide a reference for the management of limited irrigation leaching soil salinity and cotton cultivation in shallow groundwater areas. Full article

Capillary water, serving as a crucial intermediary between groundwater and crop root layer moisture, is important for both soil retention and crop utilization. To investigate the effect of mulched drip irrigation (MDI) on upward capillary water in cotton fields with different application years (0, 10, 14, 18, 20, and 24 years) in the saline–sodic region of Northwest China, an indoor soil column test (one-dimensional capillary water rise experiment) was conducted. The results showed that the wetting front transport law, capillary water recharge, and wetting front transport rate over time exhibited an increasing trend in the early stages of MDI application (10 and 14 years), peaking at 18 years of application, followed by a decreasing trend. The relationship between the capillary water recharge and rising height was fitted based on the Green–Ampt model, and their slopes reveal that 14 and 18 years of MDI application required the largest amount of water per unit distance, indicating an excellent water-holding capacity beneficial for plant growth. Conversely, 0 years required the smallest amount of water per unit distance. Based on the movement characteristics of upper capillary water, we confirmed that the MDI application years (0–18 years) improves soil infiltration capacity, while the long-term application years (18–24 years) reduces groundwater replenishment to the soil. Furthermore, the HYDRUS-1D model was employed to simulate the capillary water rise process and soil moisture distribution under different MDl application years. The results showed an excellent consistency with the soil column experiments, confirming the accuracy of HYDRUS-1D in simulating the capillary water dynamics in saline–sodic areas. The results would provide suggestions to achieve the sustainable development of long-term drip-irrigated cotton fields. Full article

Saline soils limit plant growth due to high salinity. Straw returning has proven effective in enhancing soil adaptability and agricultural stability on saline lands. This study evaluates the effects of different straw-returning methods—straw mulching (SM), straw incorporation (SI), and straw biochar (BC)—on soil nutrients, water dynamics, and salinity in a barley–cotton rotation system using field box experiments. SM improved soil water retention during barley’s jointing and heading stages, while SI was more effective in its filling and maturation stages. BC showed lesser water storage capacity. During cotton’s growth, SI enhanced early-stage water retention, and SM benefited the flowering and boll opening stages. Grey relational analysis pinpointed significant water relationships at 10 cm and 20 cm soil depths, with SM regulating water across layers. SM and BC notably reduced soil conductivity, primarily within the top 20 cm, and their effectiveness decreased with depth. SI significantly lowered soil conductivity at barley’s jointing stage. SM effectively reduced salinity at 10 cm and 20 cm soil depths, whereas BC decreased soil conductivity throughout barley’s jointing, filling, and heading stages. For cotton, SI lowered soil conductivity at the seedling and boll opening stages. SM consistently reduced salinity across all stages, and BC decreased conductivity in the top 30 cm of soil during all growth stages. Both SM and BC significantly enhanced the total nutrient availability for barley and cotton, especially improving soil organic carbon and available potassium, with BC showing notable improvements. At barley’s heading stage, SI maximized dry matter accumulation, while SM boosted accumulation in leaves, stems, and spikes during the filling and maturation stages. Straw returning increased barley yield, particularly with SM and BC, and improved water use efficiency by 11.60% and 5.74%, respectively. For cotton, straw returning significantly boosted yield and water use efficiency, especially with SI and SM treatments, enhancing the total bolls and yield. In conclusion, straw returning effectively improves saline soils, enhances fertility, boosts crop yields, and supports sustainable agriculture. These results provide a robust scientific foundation for adopting efficient soil improvement strategies on saline lands, with significant theoretical and practical implications for increasing agricultural productivity and crop resilience to salt stress. Full article

Soil amendments such as humic acid (HA), a biochar-based microbial agent (M), and vermicompost (V) can improve soil quality and promote crop growth. However, it remains unclear whether the co-application of the three soil amendments (HMV) has a synergistic effect on alleviating soil quality deterioration obstacles caused by dry sowing and wet emergence technology in Xinjiang cotton fields. A three-year field experiment was conducted in saline–alkali soils using plastic-film-mulched drip irrigation in Xinjiang, China. Through the orthogonal experiment method, the application amounts of HA, M and V were 75 kg ha⁻¹, 75 kg ha⁻¹ and 225 kg ha⁻¹ respectively in 2021. In 2022, three application amount gradients were used for HA, M and V: 60 kg ha⁻¹, 90 kg ha⁻¹ and 120 kg ha⁻¹ respectively. In 2023, the application amounts of HA, M, and V were 60 kg ha⁻¹, 120 kg ha⁻¹, and 120 kg ha⁻¹. It should be pointed out that V contains HA in the range of 20–35%. This study aimed to explore the improvement effect of a single or combined application of HA, M, and V on soil quality and cotton emergence rate using dry sowing and wet emergence technology in Xinjiang cotton fields. The results showed that the single and combined applications of HA, M, and V improved the soil quality and water–heat–salt environment of the cultivated layer. In the combined application, the cotton seedling emergence rate and yield increased by 1.9–22.8% and 7.0–54.1%. Therefore, it is recommended to jointly apply HA, M, and V to promote cotton seedling emergence and increase yield using dry sowing and wet emergence technology in Xinjiang cotton fields. Full article

Soil salinity affects nutrient uptake by cotton. The cotton bud stage is a very important period in the process of cotton planting and directly affects the yield of cotton. The nutritional status of the bud stage directly affects the reflectance spectra of cotton canopy leaves. Therefore, it is of great significance to nondestructively monitor the nutritional status of the cotton bud stage on salinized soil via spectroscopic techniques and perform corresponding management measures to improve cotton yield. In this study, potted plants with different nitrogen application rates were set up to obtain the reflection spectral curves of cotton bud stage leaves, analyze their spectral characteristics under different nitrogen application rates, and establish spectral estimation models of chlorophyll density. The results are as follows: in the continuum removal spectrum of the cotton bud stage, the lowest point of the absorption valley near 500 nm shifted to the shortwave direction with an increasing nitrogen application rate. The mean reflectance between 765 and 880 nm was significantly different between nitrogen-stressed and nitrogen-unstressed cotton. The average reflectance of the near-infrared band, the absorption valley depths near 500 nm and 675 nm, the first derivative of the 710 nm reflectance, and the second derivatives of the 690 nm and 730 nm reflectance increased with increasing nitrogen application and chlorophyll density, and significant correlations were observed with the chlorophyll density. These parameters were modeled using support vector regression (SVR) and artificial neural network (ANN) methods, two commonly used algorithms in the field of machine learning. The determination coefficients of the three chlorophyll samples via the ANN models were 0.92, 0.77, and 0.94 for the modeling set and 0.77, 0.69, and 0.77 for the verification set. The ratio of quartile to root-mean-square error (RPIQ) of the ANN model was greater than 2.2, and the ratio of the standard error of the measured value to the standard error of the predicted (SEL/SEP) was close to 1, indicating that the chlorophyll density estimation models built based on the ANN algorithm had robust prediction ability. Our model could accurately estimate the leaf chlorophyll density in the cotton bud stage. Full article