Effects of Controlled Release Nitrogen Fertilizer on Seedling Growth and Nutrient Uptake of Bitter Gourd

Baige ZHANG，Jian CAO，Zhao SONG，Qiang Ll，Xinping CHEN，Fusuo ZHANG

1.Vegetable Research Institute，Guangdong Academy of Agricultural Sciences，Guangzhou 510640，China；

2.Guangdong Provincial Key Laboratory for New Technology for Vegetables，Guangzhou 510640，China；

3.Department of Plant Nutrition，College of Resources and Environmental Sciences，China Agricultural University，Beijing 100097，China；

4.Foshan Institute of Agricultural Sciences，Foshan 528145，China

Effects of Controlled Release Nitrogen Fertilizer on Seedling Growth and Nutrient Uptake of Bitter Gourd

Baige ZHANG1，2，3，Jian CAO1，2，Zhao SONG1，2，Qiang Ll4，Xinping CHEN3*，Fusuo ZHANG3

1.Vegetable Research Institute，Guangdong Academy of Agricultural Sciences，Guangzhou 510640，China；

2.Guangdong Provincial Key Laboratory for New Technology for Vegetables，Guangzhou 510640，China；

3.Department of Plant Nutrition，College of Resources and Environmental Sciences，China Agricultural University，Beijing 100097，China；

4.Foshan Institute of Agricultural Sciences，Foshan 528145，China

Different application rates of controlled release nitrogen fertilizer（CRFN）were designed to evaluate their effects on the growth and root morphology of bitter gourd （Momordica charantia L.）seedlings，and thus determine the optimal nitrogen amount and suitable nitrogen content in substrate at seedling transplanting，in comparison with conventional fertilizer application.CRFN was applied at five levels，0，0.6，1.2，2.4 and 4.8 kg N/m3，and conventional fertilizer was applied at 0.6 kg N/m3as control.Four replicates were included in each treatment.The results showed that 0.6-2.4 kg N/m3CRFN provided sufficient N nutrient for bitter gourd，with higher shoot and root dry weights，root length and root surface area than control treatments.Correspondingly，the total inorganic nitrogen in substrate ranged from 99.3 to 162.5 mg/pot at seedling transplanting in these treatments.1.2 kg N/m3was proven to be the optimal CRFN rate.Compared with conventional nitrogen fertilizer application，1.2 kg N/m3CRFN in substrate increased the dry weight，nitrogen uptake and improved root morphology indices of seedlings，and more than 83.3 mg/pot inorganic nitrogen could be carried with substrate at transplanting，revealing a potential to reduce N-deficient risk after rain and basal N input by continuous release of CRFN.

Controlled release fertilizer；Bitter gourd；Seedling；Root；Nutrient uptake

B itter gourd （Momordica charantia L）is an annual climbing herb in the genus Momordica of the family Cucurbitaceae.Its fruit is rich in proteins and carbohydrates，17 kinds of amino acids，momordicin，insulin，and other beneficial substances. All its root，stem，leaf，flower，fruit and seed can be used as medicine［1］.Bitter gourd is widely planted in tropical and subtropical regions，and its output in southern China has reached 105-225 thousand Yuan［2-5］.However，in recent years，excessive application of nitrogen fertilizer not only failed to improve crop production，but also increased the cost and thus reduced the economic efficiency of farmers.However，no standard for nutrient management for bitter gourd has been developed so far.For example，184 kg/hm2N was recommended for bitter gourd in Tai wan and 243 kg/hm2N in Australia［2，6］. In contrast，a larger amount of nitrogen fertilizer was applied for bitter gourd in southern China，up to 321 kg/hm2on average，with great difference among regions，and 30%-40%of the nitrogen was applied as basal fertilizer［7-12］.The undeveloped roots of the newly transplanted bitter gourd seedlings have poor nutrient absorption.On the other hand，leaching losses may not be totally prevented in southern China where the average annualrainfall sometimes exceeds 2 000 mm.Therefore，reducing the application of basal fertilizers and improving seedling nu-trients are two effective measures to reduce the risk of leaching losses［13-14］. However，traditional nitrogen fertilizers fail to achieve continuous release of nitrogen without damaging crop roots at early growth stage［15］.On the contrary，controlled release nitrogen fertilizer（CRFN）provides the possibility for nitrogen regulation in the rhizosphere of bitter gourd seedlings.How to maximize the nitrogen amount in substrate carried with seedling transplanting and obtain healthy seedlings with developed root system has not been reported［16］.

In this experiment，CRFN was applied at five different rates to determine their effects on the dry matter accumulation，root morphology and nutrient uptake of bitter gourd seedlings，as well as the optimal CRFN amount and suitable nitrogen content in substrate at seedling transplanting.The results will provide a theoretical basis for nutrient regulation for bitter gourd at early seedling stage.

Materials and Methods

Experimental materials and design

The controlled release nitrogen fertilizer（44%），coated with polyester，enabled the nutrients to be available to plants within 60 d，which was proven using the methods described by GB/T 23348-2009.The bitter gourd variety Fenglu and the substrate for seedling establishment，which contained 55.7% organic matters，22.2 g/kg total nitrogen，8.46 g/kg total phosphorus and 14.3 g/kg total potassium，were provided by the Vegetable Research Institute，Guangdong Academy of Agricultural Sciences.

The field trials were conducted in the plastic greenhouses of the Vegetable Research Institute，Guangdong Academy ofAgriculturalSciences. Seeds were disinfected and germinated by soaking in 10%hydrogen peroxide for 12 h，wrapped with gauze and cultured in dark in an incubator at a temperature of 27℃ and a relative humidity of 70%for 3 d，before the germinated seeds were sown in field. Six treatments were set for fertilizer application:treatments T1 to T5 for CRFN and treatment Conv for conventionalfertilizer application.Twenty seedlings were included in each treatment.The experiment was ended on May 25（four-leaf seedling stage）.The NPK doses in each treatment were listed in Table 1.

Measurement items and methods

Seedling growth was evaluated at four-leaf stage，15 d after seed sowing. The SPAD value of the newest，fullyexpanded leaves was read using a portable chlorophyll meter.The shoots and roots of five similar sized seedlings were sampled from each repetition，fixed at 100℃ for 30 min，dried at 75℃ to constant weight before their dry weight was measured. After digestion with boiling H2SO4and H2O2，the contents of total nitrogen，phosphorus and potassium in shoot samples were respectively determined using Kjeldahl method，vanadate colorimetric method and flame photometry，as described in Agrochemical Analysis of Soil.The CRFN granules in substrate were picked out，washed，ground，filtered through nylon mesh of 0.1 mm pore size into a 100 ml volumetric flask，diluted to the mark with water to measure the amount of nitrogen residual with Kjeldahl method.The substrate was sieved through 3 mm mesh，then 5 g of the sample was weighed and dissolved in 100 ml of 0.01 mol/L CaCl2solution，oscillated for 1 h to measure the content of inorganic nitrogen（Nmin，including NO3-N and NH4+-N）with TRAACS 2000 Continuous-Flow Analyzer（three-channel） and water content in matrix.The roots of seedlings were washed and scanned using EPSON PERFECTION 4990 PHOTO to analyze the total root length，surface area and mean of root diameter using Win RHIZO software. The data were analyzed in Excel，and analysis of variance was performed with SAS 11.5 statistical software.

Table 1 Experimental design kg/m3

Table 2 Dry weight and nitrogen content of shoot and root in different treatments（n=5）

Results and Analysis

Effects of controlled release fertilizer amount on seedling growth of bitter gourd

Shoot growth and leaf chlorophyll SPAD value of bitter gourd seedlings Fifteen days after seed sowing，the seedlings of bitter gourd entered to four-leaf stage.As shown in Fig.1A，the seedlings in the treatment with 1.2 kg N/m3were 11.4 cm high on average and grew faster than those in all other treatments.The seedlings in treatments with 0.6 and 2.4 kg N/m3were very close in average heights（12.5 and 12.1 cm）.The treatment with 4.8 kg N/m3significantly reduced the average height of seedlings to only 8.3 cm.In general，seedling height and CRFN rate shared a significant correlation，fitting the equation y=-0.46x2+ 1.48x+11.7（R2=0.90）.

Leaf chlorophyll is one of the indicators characterizing nitrogen supply in plants.We found that they fit the equation of y=-1.74x2+8.37x+21.1（R2=0.84）in this experiment.Asshown in Fig.1B，the SPAD value in the newest fully-expanded leaves in the treatment with 1.2 kg N/m3was the highest，and it had no significant difference between the control treatment without any additional N supply and the treatment with 0.6 kg N/m3，the treatment with 2.4 kg N/m3and the treatment with 4.8 kg N/m3（P＞0.05）. Overall，the leaf SPAD value showed an increase before a decrease with the increase of nitrogen level in substrate，indicating that the nitrogen level in substrate had certain influence on plant chlorophyll content.Excessive nitrogen resulted in dark green or burnt looking leaves and reduced leaf chlorophyll due to nitrogen toxicity.

Dry matter accumulation and nitrogen absorption in bittergourd seedlings As shown in Table 2，T3 had the highest shoot dry weight among all the treatments with different N rates，while the shoot dry weight in T1，T2 and T4 had no significant difference，indicating that 1.2 kg N/m3was the most suitable dose for dry matter accumulation in shoot of bitter gourd seedlings.The root dry matter in T2 was the highest，followed by that in T3，T5，T4 and T1，but the difference among all the treatments was insignificant.With the increase of CRFN in substrate，the nitrogen concentration in seedling shoots was also increased. Among all the treatments，shoot nitrogen concentration in T5 was the highest，up to 50.7 g/kg，while the seedling growth was significantly inhibited，indicating that 4.8 kg/m3CRFN could result in luxury uptake of nitrogen.Root nitrogen concentration in T4 and T5 was the highest，while that in T3 and T2 had no significant difference，but was still significantly higher than that in T1.The five treatments with different nitrogen rate were listed here in a decreasing order of shoot nitrogen uptake as T3≥T5＞T2＞T4＞T1，in a decreasing order of root nitrogen uptake as T5≥T2＞T3=T4＞T1.It could be concluded from Table 2 that more nitrogen was stored in roots than in shoots to prevent the shoots from nitrogen toxicity when the nitrogen rate in substrate was 2.4 kg/m3，slightly exceeding plantdemand.However，when the nitrogen rate in matrix was 4.8 kg/m3，excessive nitrogen was transported to shoots，so that the shoot growth was inhibited and the leaves appeared burnt looking due to nitrogen toxicity.

Root morphology of bitter gourd seedlings The data proved that the amount of CRFN affected root morphology ofbittergourd seedlings（Table 3 and Fig.2）.Among all the treatments with differentrates of CRFN，T3 had the largest root length，surface area and projected area，followed by T2 and T4.The root length and projected area in T5 was smaller than in T1，indicating that the root growth and developmentofbitter gourd seedlings were inhibited in the substrate supplemented with 4.8 kg N/m3.On the contrary，the average root diameter of bitter gourd seedlings was increased with the increase of CRFN rate.

lnorganic nitrogen content in substrate carried with transplanting

As shown in Table 4，the total nitrogen amount can be carried with transplanting in each pot included two parts，the inorganic nitrogen in substrate and the controlled release nitrogen residual. The nitrogen residual in CRFN granules and the inorganic nitrogen content in substrate were both increased with the increase of CRFN rate from 0 to 4.8 kg/m3we applied.According to seedling growth status it was found that 59.8-66.0 mg/plantwas the suitable range for nitrogen residual in CRFN granules at transplanting，39.5-96.5 mg/plant for inorganic nitrogen，and 99.3-162.5 mg/plant for the total nitrogen content in substratecarried with transplanting.

Table 3 Root morphology indices of bitter gourd transplants（n=5）

Table 4 N in different treatments（n=5） mg/plant

In summary，the treatment T3 with 1.2 kg/m3nitrogen rate was most suitable for the growth of bitter gourd seedlings，and the total nitrogen content carried with transplanting was also the most suitable，indicating it was considered as the optimal CRFN treatment（Opt-N）.

Comparison between optimal controlled release nitrogen fertilizer treatment（Opt-N）and conventional fertilizer application（Conv-N）Seedling growth and nitrogen uptake The seedling growth was compared between Opt-N treatment and Conv-N treatment（Table 5）.The resultsrevealed thatcompared with Conv-N，the Opt-N treatmentincreased the dry weight，nitrogen uptake，SPAD value in the newest fullyexpanded leaves and seedling height by 12.5%，14.0%，20.1%and 17.0%，respectively.But the root cap in Conv-N was 12.1%higher than that in Opt-N.Compared with the treatment without any additional N application（No-N），the treatments of Opt-N and Conv-N both increased shoot dry weight，nitrogen concentration，SPAD and seedling height by 15.2% ，16.9% ，20.4%，9.6%and 2.8%，2.6%，0.3%，-6.3%.

Seedling root development Controlled release nitrogen fertilizer and conventional composite nitrogen fertilizer had different effects on root morphology ofbittergourd seedlings（Table 6 and Fig.3）.The total root length，root projected area and surface area in treatment Opt-N （156.7 cm，16.3 cm2，47.2 cm2）were all larger than those in treatment Conv-N（110.5 cm，13.8 cm2and 47.2 cm2），but the root diameter in treatment Conv-N was larger than that in treatment Opt-N. The seedlings in all the three treatments （No-N，Opt-N and Conv-N）grew normally.According to seedling growth status，Opt-N was the best treatment，followed by Conv-N and No-N.

lnorganic nitrogen in substrate carried with transplanting As shown in Table 7，the inorganic nitrogen in substrate had no significant difference between the two treatments Opt-N and Conv-N.However，the total nitrogen which can be carried with transplanting in treatment Opt-N was over two times as high as that in treatment Conv-N，because 80 mg/plant of nitrogen was remained in CRFN granules，which accounted for 56%of the total nitrogen carried with transplanting，and could provide sufficient nutrient for seedling growth after transplanting to enhance seedling resistance to stresses.

Table 5 Growth condition，dry weight and N content of plant in different treatments（n=5）

Table 6 Root length，surface area and diameter of bitter gourd transplants（n=5）

The same lowercase letters within the columns mean insignificant difference at 0·05 level.

Table 7 Comparison of nitrogen residuals in different treatments（n=5） mg/plant

Discussion

The nutrients in substrate directly affect seedling root and shoot growth，dry matter accumulation and crop yield after transplanting，so improving the nutrients in substrate is the main measure to obtain high-quality seedlings［17］. Currently，nutrient solution is a major way to provide nutrients to seedlings in plugs，but there are not too many nutrient solution products available on market［18］.Mixing a certain ratio of fertilizers to substrate before seed sowing is another way to provide nutrients to seedlings.In earlyperiod ofplug seedling promotion，0.3 kg N/m3compound fertilizer was applied into substrate for all vegetables to provide enough nutrients throughout the whole seedling stage which is not scientific and reasonable［19］.Then，researchers began to conduct experiments to determine the optimal nitrogen fertilizer in the substrate for plug seedlings of vegetables.For instance，Zhao et al.［20］proposed that 0.2，0.8 and 0.4 kg N/m3were the optimal nitrogen application for tomato，cucumber and pepper.However，by this method the highnitrogen concentration at early period was toxic to seedlings and thus inhibited seedling growth，even led to wilting and death.On the other hand，the inorganic fertilizers released too fast，which also resulted in the lack of nutrients at later period［21］.In this study，we found that during 0.6-2.4 kg N/m3CRFN，the seedlings all grew normally，and the shoot and root dry weights，root length and root surface area were all increased，compared with the control.The results proved that the controlled release nitrogen slowly released nutrients during seedling growth，and seedling could still grow normally even the controlled release nitrogen was applied 6 times as much as conventional nitrogen fertilizer.

Previous survey has shown that 0.6 kg N/m3of common compound fertilizer was supplemented to substrate for bitter gourd seedling planting by most farmers.Compared with such conventional fertilizer application，1.2 kg N/m3controlled release fertilizer increased nitrogen uptake，dry matter accumulation，seedling height，leaf SPAD value，root length，root projected area and root surface area.In addition，the nitrogen content carried with transplanting （44%was the inorganic nitrogen in substrate and 56%was the nitrogen residual remained in CRFN granules）in this treatment with 1.2 kg N/m3controlled release fertilizer was 147.4%more than that in the treatment with conventional fertilizer application.Originated from tropical and subtropical regions，bitter gourd is planted during January-March and July-August in Guangdong when the rainfall is 160.2 and 415.4 mm，accounting for 6.4%and 16.5%of the annual total precipitation［2，22］.The lack of fertilizer after rain has a certain influence on vegetable yield and quality［23］. As controlled release fertilizer enables that a certain of nitrogen nutrient is remained in substrate and can be carried with transplanting，so that it can provide nutrients to seedlings after transplanting to improve seedling resistance to stresses.By studying the effect of controlled release nitrogen fertilizer on cucumber seedling growth in mixed peat and vermiculite，Si et al.［14］found that 85.9 mg/plant inorganic nitrogen can be carried with transplanting by applying 3.42-6.84 kg/m3controlled release fertilizer，and thus reduced basal nitrogen fertilizer by 80%. By conventional fertilizer management for bitter gourd，30%-40%of fertilizer is applied as basal fertilizer，but there is still a risk of leaching loss，as the roots of pre-transplant bitter gourd seedlings are undeveloped［7-8，24-25］.Controlled release fertilizer greatly reduces the leaching loss risk at early seedling growth period of bitter gourd. In this study，the seedlings of bitter gourd can be transplanted carrying 140 mg N each.Supposing bitter gourd is planted at a density of 450 seedlings per 0.066 7 hm2and conventional NPK fertilizer is used as the basal fertilizer，the application of controlled release fertilizer can reduce fertilizer investment by 6 300 g/hm2directly.No more nitrogen fertilizer should be supplemented immediately after rain，which also saves manpower.

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Responsible editor：Qingqing YlN

Responsible proofreader：Xiaoyan WU

Discussion

Planting is a standard method for identifying the purity of rice hybrids，but the accuracy is more dependent on the experience of researchers，but also related to plant variety，environmental conditions，etc.SSR markers can recognize certain DNA fragments in hybrids，so this method is highly accurate，stable and repeatable.In addition，SSR markers can effectively distinguish the sterile line，restorer and maintainer from the true hybrid.All the SSR primers can distinguish the sterile lines from F1 populations of the hybrids［3-5］.

Both the sterile plants and crosspollinated plants in F1 populations of Gangyou 158，II You 88，Wuyou 308 and Tianfengyou 316 can be detected usingRM341，RM297，RM21and RM110，as the genotype of sterile plants and cross-pollinated plants was the same as female parent’s at these sites.On the contrary，primers RM208，RM264，RM242 and RM164 cannot distinguish the cross-pollinated plants of Gangyou 158，II You 88，Wuyou 308 and Tianfengyou 316，because the genotype of the cross-pollinated plants was the same as the restorers’at these sites.

Theoretically，the error can be controlled below 3‰ using SSR primers to distinguish the sterile lines，restorers and other non-hybrid seeds，if the parents are stable and no crosspollinated plants are produced.In the present study，we found that the existence of cross-pollinated plants of the four hybrids is the main reason causing the difference in purity identification with different primers.Therefore，multiple pairs of primers can increase the chance to distinguish cross-pollinated plants，and thus improve the accuracy of purity identification.In addition，the lower purity result should be adopted if the there is great difference in purity identification results using different primers.

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Responsible proofreader：Xiaoyan WU

控释氮肥对苦瓜幼苗生长及养分吸收的影响

张白鸽1，2，3，曹健1，2，宋钊1，2，李强4，陈新平3*，张福锁3（1.广东省农科院蔬菜研究所，广东广州510640；2.广东省蔬菜新技术重点实验室，广东广州510640；3.中国农业大学资源与环境学院，北京 100097；4.佛山市农业科学研究所，广东佛山 528145）

为探讨包膜控释氮肥对苦瓜幼苗生长及养分吸收的影响，确定育苗阶段合理的控释氮肥用量及移栽时适宜的氮素携带量，该试验共设置5个控释肥料用量处理和1个传统育苗施肥处理，控释氮用量分别为0、0.6、1.2、2.4、4.8 kg N/m3，传统育苗氮处理为0.6 kg N/m3，4次重复，随机排列。结果表明，控释氮肥用量在0.6-2.4 kg N/m3范围内时，苦瓜幼苗都可以正常生长，植株地上部和根系干物质的累积量、根长及根系表面积参数都优于对照处理，与此对应的移栽期基质适宜无机氮含量为每穴盘99.3-162.5 mg。控释氮素用量为1.2 kg N/m3时，育苗效果最优。相比于传统的施氮处理，基质中添加1.2 kg N/m3控释氮素促进苦瓜幼苗累积干物质，增加吸氮量改善了根系生长状况，移栽时单株幼苗可实现139.8 mg的带肥移栽，比传统方法培育的幼苗多携带了83.3 mg氮素，可持续为定植后的苦瓜幼苗及时提供养分，降低前期遇雨脱肥风险并减少基肥投入和损失。

控释氮肥；苦瓜；育苗；根系；养分吸收

农业部公益性农业行业项目（201503106，201103003）；农业部现代产业体系建设专项资金（CARS-25-G-36）。

张白鸽（1983-），女，内蒙古巴彦淖尔人，硕士，助理研究员，主要从事蔬菜营养生理方面研究，E-mail：plantgroup@126.com。*通讯作者，博士，教授，主要从事养分资源综合管理等研究工作，E-mail：chenxp@cau.edu.cn。

2015-10-09

Supported by Special Fund for Agro-scientific Research in the Public Interest of China（201503106，201103003）；Earmarked Fund for China Agriculture Research System（CARS-25-G-36）.

*Corresponding author.E-mail:chenxp@cau.edu.cn

Received:October 9，2015 Accepted:November 10，2015

修回日期 2015-11-10