Binglin Chen，Hongkun Yang，Weichao Song，Chunyu Liu，Jiao Xu，Wenqing Zhao，Zhiguo Zhou*

Key Laboratory of Crop Physiology Ecology and Production Management，Nanjing Agricultural University，Jiangsu Collaborative Innovation Center for Modern Crop Production，Nanjing 210095，China

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Effect of N fertilization rate on soil alkalihydrolyzable N，subtending leaf N concentration，fiber yield，and quality of cotton

Binglin Chen1，Hongkun Yang1，Weichao Song，Chunyu Liu，Jiao Xu，Wenqing Zhao，Zhiguo Zhou*

Key Laboratory of Crop Physiology Ecology and Production Management，Nanjing Agricultural University，Jiangsu Collaborative Innovation Center for Modern Crop Production，Nanjing 210095，China

A R T I C L E I N F O

Article history:

in revised form

30 March 2016

Accepted 6 June 2016

Available online 14 June 2016

Cotton（Gossypium hirsutum L.）

Soil alkali-hydrolyzable nitrogen

Subtending leaf nitrogen

concentration

Fiber yield

Fiber properties

Nitrogen use efficiency

A B S T R A C T

Soil alkali-hydrolyzable nitrogen，which is sensitive to N fertilization rate，is one of the indicators of soil nitrogen supplying capacity.Two field experiments were conducted in Dongtai（120°19″E，32°52″N），Jiangsu，China in 2009 and Dafeng（120°28″E，33°12″N），Jiangsu province，China in 2010.Six nitrogen rates（0，150，300，375，450，and 600 kg ha-1）were used to study the effect of N fertilization rate on soil alkali-hydrolyzable nitrogen content（SAHNC），subtending leaf nitrogen concentration（SLNC），yield，and fiber quality.In both Dongtai and Dafeng experiment station，the highest yield（1709 kg ha-1），best quality（fiber length 30.6 mm，fiber strength 31.6 cN tex-1，micronaire 4.82），and highest N agronomic efficiency（2.03 kg kg-1）were achieved at the nitrogen fertilization rate of 375 kg ha-1.The dynamics of SAHNC and SLNC could be simulated with a cubic and an exponential function，respectively.The changes in SAHNC were consistent with the changes in SLNC.Optimal average rate（0.276 mg day-1）and duration（51.8 days）of SAHNC rapid decline were similar to the values obtained at the nitrogen rate of 375 kg ha-1at which cotton showed highest fiber yield，quality，and N agronomic efficiency.Thus，the levels and strategies of nitrogen fertilization can affect SAHNC dynamics.The N fertilization rate that optimizes soil alkali-hydrolyzablenitrogencontentwouldoptimizethesubtendingleafnitrogen concentration and thereby increase the yield and quality of the cotton fiber.

?2016 Crop Science Society of China and Institute of Crop Science，CAAS.Production and hosting by Elsevier B.V.This is an open access article under the CC BY-NC-ND license（http://creativecommons.org/licenses/by-nc-nd/4.0/）.

1.Introduction

Cotton growth is influenced by several factors including genotype，environmental conditions，and management practices. Among them，nitrogen is one of the limiting factors for yield and quality［1］.As an essential macronutrient，nitrogen is required more than other nutrients for cotton production during its long growth season［2］.Farmers tend to apply excessive nitrogen toensure high yield［3，4］.This practice not only increases environmental pollution and nitrogen loss，but also delays maturity and dramatically reduces fiber quality［5］.A better understanding of the effect of N fertilization rate on soil alkali-hydrolyzable nitrogen content（SAHNC）and subtending leaf nitrogen concentration（SLNC）wouldaidindevelopingimprovedNmanagement practices based on nitrogen requirement.

Soil available nitrogen is derived from mineralization of soil organic matter and the available ingredients of fertilizers［6］.It can be directly absorbed and used by crop roots and is an important indicator for evaluating soil nutrients［7-9］.However，soil available nitrogen is easily leached out during rainfall，resulting in dramatic reduction in a short time［10］. Soil alkali-hydrolyzable N is not readily leached and also can be directly absorbed and used by crop roots.Thus，soil alkali-hydrolyzable N is an alternative indicator of soil nitrogen-supplying capacity［11］.In developing bolls，more than 80%of nutrients are provided by subtending leaves［12］. These leaves have a close“source and sink”relationship with bolls［13］，and are the major nutrient provider for fiber growth［12］.The source-to-sink transportation of nutrients is influenced by the activity of the source［14］.Nitrogen is an important factor in photosynthesis and carbon and nitrogen metabolism in cotton leaves［15］.The influence of nitrogen on boll development is derived primarily from its effect on the source［16］.Plant nitrogen content is a key factor in photosynthesis，growth，and light utilization efficiency［17-20］. Photosynthesis decreases with decreasing leaf nitrogen concentration［21，22］.For this reason，leaf nitrogen content is considered to be an indicator of a plant's environmental adaptability［23，24］，and can be used in real-time quantification of the effect of nitrogen fertilization rate on yield and quality［25，26］.Sufficient nitrogen supply to subtending leaves is important for securing a high cotton yield［27］.

Crops absorb nitrogen from the soil.High soil nitrogen content is necessary to maintain high leaf nitrogen［28］.Yield and fiber quality are dependent on the distribution of nitrogen in the plant［29，30］.Despite many studies of soil and leaf nitrogen，the effects of changes in SAHNC after nitrogen fertilization on SLNC have not been reported.In the present study，we varied nitrogen fertilization rates in cotton-growing areas in the lower reaches of the Yangtze River to investigate the relationship between SAHNC and SLNC，in order to generate information for better N management practice.

2.Materials and methods

2.1.Experimental design

Two experiments were conducted in sandy loam fields at Dongtai（120°19″E，32°52″N）in 2009 and Dafeng（120°28″E，33°12″N）in2010，Jiangsuprovince.Thecultivarwas Xiangzamian 8，a high-yielding commercial BT-transgenic upland cotton （Gossypium hirsutum L.）.Soils at the two sitescontained26.10and11.70 g kg-1organicmatter，52.95 and 63.78 mg kg-1alkaline-hydrolyzable N，21.21 and 21.65 mg kg-1available P，and 131.54 and 230.00 mg kg-1available K，respectively.Soil organic matter，available P，and available K were determined following Sui et al.［31］.

A complete randomized block design was employed with 4 replications and 6 treatments.Each plot was 10 m long and 4 m wide with a plant density of 24，900 plants ha-1.Nitrogen fertilization rates were 0，150，300，375，450，and 600 kg ha-1and are referred to as N0，N1，N2，N3，N4，and N5，respectively. Of the fertilization，30%was base nitrogen，40%initial flowering nitrogen，and 30%flowering nitrogen.As basal fertilizer，150 kg P2O5ha-1and 225 kg K2O ha-1as single superphosphate（17%of P2O5）and potassium chloride（55% of K2O）were applied.For flower fertilization，urea（46%of N）was spot-applied near the roots.Cotton plants were seeded on April 5 in 2009 and 2010，transplanted on May 20，and topped on August 10.All plots were managed in accordance with local high-yield farming practices.

2.2.Sampling and processing

Soil samples were collected at 0-20 cm depth once every 20 days in 2009 and once every 15 days in 2010 from the initial flowering stage until opening of the boll on the 13th fruiting branch.After air-drying and screening through a 100-mesh sieve，soil samples were used for determination of soil alkali-hydrolyzable nitrogen.

White flowers at the first node of fruiting branches between numbers 1-6，7-12，and 13 and above（referred to as FB1-6，F(xiàn)B7-12，and FB13+，respectively）were tagged with plastic tags used for noting the flowering date.Tagged bolls and their subtending leaves were collected every 7 days from 10 DPA until opening at 9:00-10:00 a.m.（local time）.The subtending leaves were washed with distilled water and the main vein was removed.The samples were inactivated at 105°C for 30 min，dried at 80°C to constant weight，and ground to powder for determination of leaf nitrogen.

2.3.Soil alkali-hydrolyzable nitrogen content and subtending leaf nitrogen concentration

Soil alkali-hydrolyzable nitrogen content was quantified by the method of Roberts et al.［11］.Briefly，5-g samples of soil were distilled with 2 mol L-1NaOH for 5 h and then with 10 mol L-1NaOH for 7 min.Boric acid（40 g L-1）was used to absorb the liberated NH3using the method of direct steam distillation.Soil alkali-hydrolyzable nitrogen content was quantified by conductometric titration.Subtending leaf nitrogen concentration was measured by the Kjeldahl method with a continuous flow analyzer（Bran and Luebbe TRAACS Model 2000 Analyzer）［32］.

2.4.Fiber yield and quality

Open bolls were hand-collected in each plot with three replications.Seed cotton was ginned when the water content was less than 11%.Total boll numbers and boll weight were calculated as the average of 20 consecutive plants in the central row of each plot.Fiber yield was determined from ginned seed cotton in a sampling area of 2.5 m×2.2 m（row length×row width）.Cotton fiber length of the harvested bolls was measured using a Y-146 cotton fiber photodometer（Taicang Elec-tron Apparatus Co.，Ltd.，Suzhou，China）and measurements were repeated six times for each air-dried

2.5.Statistical analysis

Analysis of variance（ANOVA）was performed using the least significant difference（LSD，P=0.05）test.Letters in the tables indicate significant differences at P＜0.05.All statistical analyseswereperformedwithSPSSstatisticalpackage version 17.0［33］.The graphs were constructed using OriginPro 8.0 software［33］.In order to assess the effects of treatments on cotton yield and fiber quality，ANOVA with a randomized complete block design was used.Regression analyses were performed with SPSS.

3.Results

sample.The fiber strength was determined by HVI-1000（Uster，Switzerland）.Fiber micronaire was measured with a Y145C Airometer（Ningbo Textile Instrument Factory，Ningbo，China）.

Fig.1-Soil alkali-hydrolyzable nitrogen content dynamics in cotton field at different rates of nitrogen application.SAHNC，soil alkali-hydrolyzable nitrogen content.T，days after flowering.

3.1.Effect of N fertilization rate on SAHNC

SAHNC after initial flowering stage following fertilization with nitrogen at different rates is shown in Fig.1.SAHNC at N0 showed a consistent decline that could be fitted with a quadratic function.Between N1-N5，SAHNC increased initially and then declined dramatically followed by an increase.The dynamics could be fitted with a cubic function.

By simulation of SAHNC variation at different nitrogen fertilization rates，the highest（Y1）and lowest（Y2）SAHNC，the start（T1）and end（T2）days of SAHNC rapid decline，duration of SAHNC rapid decline（ΔT=T2-T1），and the average rate of SAHNC rapid decline（ΔV=（Y2-Y1）/ΔT）were calculated and are shown in Table 1.T1 fell and then rose，ΔV and ΔT rose and then fell with the same inflection points at N3，and Y2 increased with nitrogen rate.These findings indicated that at N3（375 kg N ha-1），the highest SAHNC appeared earlier with faster decline in the rapid decline period and shorter duration than other nitrogen rates.

Table 1-Characteristics of alkali-hydrolyzable content in cotton fields at different rates of nitrogen application.

3.2.Effect of N fertilization rate on characterization of SLNC

SLNC declined with days after flowering and was significantly higher in FB1-6than in FB7-12and FB13+（Fig.2）.The dynamics of SLNC has been proposed to be describe by YN=At-B［34］，where YN（%）is the subtending leaf nitrogen concentration，t（days）is days after flowering，A is the subtending leaf nitrogen concentration on the flowering day（t=1），and b is the rate of decline of subtending leaf nitrogen concentration. Table 2 shows that at the same fruit branch positions，A tended to increase and B tended to decrease，indicating that the nitrogen concentration increased and the rate of declinedecreased with increasing nitrogen rate.At the same nitrogen rates，A tended to increase and B tended to decrease with increase in fruit branch position，indicating that the higher the fruit branch position，the lower was the nitrogen concentration and the higher the decline rate.

Fig.2-Subtending leaf nitrogen concentration at different rates of nitrogen application.FB1-6，F(xiàn)B7-12，and FB13+denote fruiting branches 1-6，7-12，and 13 and above，respectively.

3.3.Correlation between SLNC and SAHNC

The correlations between nitrogen concentration eigenvalue in the SLNC and SAHNC are shown in Table 3.T1，ΔT，and ΔV were found not to be correlated with A and B，whereas Y1 showed significant and positive correlations with A and B（P＜0.01），indicating that the higher the SAHNC，the higher was the A and the lower the B in SLNC.

Table 2-Effect of nitrogen rate on subtending leaf nitrogen concentration.

3.4.Fiber yield，fiber quality，and N use efficiency

Fiber yield and quality were significantly affected by N rate in both years（Table 4）.Fiber yields increased and then decreased with increasing nitrogen rate，with highest yield and qualityachieved at the rate of 375 kg ha-1（Fig.3）.Highest boll number，boll weight，fiber percentage，fiber length，strength，micronaire，and N agronomic efficiency were also achieved at 375 kg ha-1（Tables 4，5）.In contrast，fiber length was unaffected by N rate.Fertilizer partial factor productivity decreased with increasing nitrogen fertilization rate.

Table 3-Correlation between subtending leaf nitrogen concentration and soil alkali-hydrolyzable nitrogen content.

4.Discussion

Fertilizers，especiallynitrogennutrients，promoteplant growth and yield［35，36］.The amount of nitrogen nutrients applied could affect the growth of cotton plants，because nitrogen can influence cotton leaf photosynthesis［37］.Nitrogen deficiency in the leaf reduces the photosynthetic rate［20-22］.Thus，sufficient nitrogen supply，particularly in subtending leaves，is important for achieving high yield and quality.To investigate the relationship between SLNC and SAHNC，we divided cotton plants into three parts:FB1-6，F(xiàn)B7-12，and FB13+.

In these three parts of the cotton plant，SAHNC could be simulated with a cubic function.It increased with nitrogen rate，in agreement with results reported previously［38］.At the nitrogen rate of 375 kg ha-1，the start and end day of SAHNC rapid decline were earlier，with shorter duration of SAHNC fast decrease，than those at other nitrogen rates（Table 1）. SLNC increased with nitrogen rate.The rate of SLNC decline increased with nitrogen rate（Fig.2）.These results indicated that a too-high or too-low N rate is not benefit for SLNC，probably because low SAHNC led to low SLNC，limiting nitrogen transport to bolls；high SAHNC led to high SLNC，but resulted in excessive growth of the SLNC and a lower nitrogen decline rate in the SLNC，hindering the transportation of nitrogen to the bolls.In previous studies［39］，optimal subtending leaf N concentrations were 3.04%，3.28%，and 3.18%in the lower，middle，and upper fruiting branches，respectively.Optimal N concentrations coincide with optimal N fertilization rate，and N fertilization rate also influenced SAHNC（Fig.1）.Thus，appropriate ΔV and ΔT in SAHNC were necessary for higher subtending leaf nitrogen concentration.

Table 4-Effects of nitrogen rate on fiber yield and fiber qualities.

Analysis of the relationship between SAHNC and SLNC（Table 3）indicated that the SLNC was affected by SAHNC:the higher the SAHNC，the higher was the nitrogen concentration and the lower the decline rate of subtending leaf nitrogenconcentration.However，it is not clear whether there is a threshold at which the nitrogen concentration ceases to increase with further SAHNC increase.

Fig.3-Relationships between fiber yield，strength，micronaire，and soil alkali-hydrolyzable nitrogen content on different days after flowering in 2009 and 2010.

Table 5-Effect of nitrogen rate on nitrogen use efficiency.

There appeared to be differences between the two years in the optimal ΔV and ΔT of SAHNC as calculated by cubic function（Table 1）.The ΔV and ΔT values in 2010 were greater than in 2009.However，the soil nitrogen content before transplanting was higher in 2010 than in 2009.Thus，ΔV and ΔT were also affected by initial soil nitrogen concentration（Fig.1，Table 1）as the soil fertility level.High fertility will reduce ΔV and increase ΔT.The calculated optimal ΔV and ΔT are similar to their values obtained at the nitrogen rate of 375 kg ha-1，and the cotton had the highest fiber yield and quality at the nitrogen rate of 375 kg ha-1.Furthermore，at this nitrogen rate，cotton showed the highest N agronomic efficiency.Thus，the highest yield and quality obtained at the nitrogen rate of 375 kg ha-1at the two experiment stations was due to the optimum soil alkali-hydrolyzable nitrogen content and optimal subtending leaf nitrogen concentration.

Although the highest yield，quality，and N agronomic efficiency were obtained at the nitrogen rate of 375 kg ha-1，the low N partial factor productivity cannot be ignored（Table 5，F(xiàn)ig.3）.In the present study，the optimum N rate was nearly four times that of other experiment stations［32］，possibly owing to extremely low plant density and site-specific N management practices in the Yangtze River cotton-producing region.In previous studies，both plant density and nitrogen fertilization rate showed effects on biomass formation of cotton plants and thereby influenced nitrogen uptake and N efficiency［40，41］.Low N use efficiency was the result of differences in cumulative N utilization efficiency［42］.An adequate increase in plant density can also increase seed cotton yield and N efficiency［43，44］.In addition，because of N rateandfiberqualitywerequadraticformswhichwerefittedon significant level（Fig.3），the effect of N fertilizer on fiber quality was consistent with previous findings［45］，suggesting that highest quality was obtained at an N rate of 300-375 kg ha-1. This rate was higher than that used at other experiment stations，owing to the saline-alkaline soil type.Saltstress limits plant growth and fiber development［46］and thereby raises the optimal N fertilization rate.

5.Conclusions

The N fertilization rate could dramatically affect SAHNC.The dynamics of SAHNC from initial flowering to mature stages could be simulated with a cubic function.It consisted of a period of SAHNC rapid decline.SLNC could be described with an exponential function with a period of fast nitrogen increase.The eigenvalue of the period of SAHNC rapid decline was affected by the nitrogen fertilization rate.The nitrogen concentration eigenvalue of SLNC was significantly correlated with the eigenvalue of the SAHNC rapid decline. At both Dongtai and Dafeng experiment stations，the optimal duration and optimal average rate during SAHNC rapid decline，and optimally rapid nitrogen accumulation，could be achieved at the nitrogen rate of 375 kg ha-1.Maximum N agronomic efficiency was also achieved at 375 kg ha-1，indicating this to be the optimal nitrogen rate for cotton grown in the lower reaches of the Yangtze River.At this nitrogen rate，cotton will have an optimal subtending leaf nitrogen concentration to optimize fiber yield and quality.This method can also be used for predicting final fiber quality and yield by measurement of SLNC or SAHNC.Further research is needed to balance yield and N efficiency using integrated management strategies.

Acknowledgments

This research was funded by the National Key Technology R&D Program of China（No.2014BAD11B02），the Special Fund for Agro-scientific Research in the Public Interest（No.201203096），the National Natural Science Foundation of China（Nos. 31401327，30971735），and the China Agriculture Research System（No.CARS-18-20）.

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2 October 2015

Abbreviations:SAHNC，soil alkali-hydrolyzable nitrogen content；SLNC，subtending leaf nitrogen concentration.

*Corresponding author.Tel.:+86 25 84396813.

E-mail address:giscott@163.com（Z.Zhou）.

Peer review under responsibility of Crop Science Society of China and Institute of Crop Science，CAAS.

1Binglin Chen and Hongkun Yang contributed equally to the work.

http://dx.doi.org/10.1016/j.cj.2016.03.006

2214-5141/?2016 Crop Science Society of China and Institute of Crop Science，CAAS.Production and hosting by Elsevier B.V.This is an open access article under the CC BY-NC-ND license（http://creativecommons.org/licenses/by-nc-nd/4.0/）.