Effects of Soil C／N Ratio on Apple Grow th and Nitrogen Utilization，Residue and Loss

Shunfeng GE，Yihua REN，Ling PENG，Haigang XU，Mengmeng JI，Shaochong WEI，Yuanmao JIANG

State Key Laboratory of Crop Biology，College of Horticulture Science and Engineering，Shandong Agricultural University，Tai'an 271018，China

According to statistics，the planting area and output of China's apple in 2011 reached 1.99million hectares and 35million tons，respectively，both accounting for more than 50% of the world apple planting area and output.The fruit industry has gradually become a major pillar industry for increasing farmers'income in the apple producing areas［1］.Nitrogen is the essential macro nutrient for apple's growth and development，and increasing the application of chemical nitrogen fertilizer is one of major measures to increase the apple production［2－3］.At present，the net nitrogen application rate has reached 400－600 kg·hm2in China's apple orchard，but the nitrogen productivity tends to decline，and the caused environmental pollution tends to aggravate［4－8］.Therefore，improving nitrogen use efficiency，reducing its negative impact on the environment，and taking into account the ecological benefits while improving apple production，is the guiding ideology for the study of soil nitrogen in China currently［9］.Soil carbon and nitrogen ratio（C／N）is a sensitive indicator of soil quality，and a indicator for measuring the nutrient balance of soil nitrogen，which has a major impact on the soil nitrogen cycling［10］.Currently the soil nitrogen content in China's apple orchard has reached as high as0.8－1.3 g／kg，while the organic matter content is low，only about 1%［5，11］.Together with the lack of effective management measures in the ground，the soil C／N ratio is out of balance，which is an important reason for orchard soil quality degradation，decline in soil microbial diversity，serious soil-borne diseases，and decline in fruit quality［12］.Soil C／N ratio is usually considered a sign of mineralization capacity of soil nitrogen.Low C／N ratio can accelerate microbial decomposition and nitrogen mineralization rates，and high C／N ratio can play a certain role in limiting the soil microbial activity，so that the decomposition and mineralization rate of organic matter and organic nitrogen is reduced and the ability of soil to fixate the organic carbon is increased［10］.Currently the researches of soil C／N ratio are mainly focused on the tobacco quality and physiological and biochemical indicators and the ways to improve soil C／N ratio by adding organic matter［13－14］，but there are few studies on the effects of C／N ratio on apple plant growth and soil nitrogen cycling.

Therefore，using15N isotope tracer technique，in this study we research the effects of different soil C／N ratios on the nitrogen balance of apple plant－soil system，in order to provide a theoretical basis for the soil improvement and rational nitrogen application in the apple orchard.

1　Materials and methods

1.1Experimental design The experiment was carried out in the Horticultural Experiment Station of Shandong Agricultural University.The place features a temperate semi-humid continental monsoon climate.The average annual temperature is12.9℃，and the annual frost-free period is about 195 d.The average annual rainfall is697 mm and the rainfall is mainly concentrated in June to September.The soil for experiment is brown soil［（organic matter of10.82 g／kg；total nitrogen of1.01 g／kg；available phosphorus of 26.21 mg／kg；available potassium of 130.61 mg／kg；pH（H2O）6.40］.There are 8 treatments set in this experiment as follows：

Each C／N ratio is regulated by adding different amounts of glucose based on the soil for experiment.

The potted plant is used for this experiment.The pot has diameter of 30 cm and height of 40 cm，and 23 kg of soil is placed into each pot.On March 21，2012，two-year old apple trees（Borkh.cv.‘Fuji’／Malus hupehensis）were transplanted in the pots，one pot for one plant.5 pots were prepared for each treatment，and the planting height was 60 cm.After the plants grew steadily，24 pots of robust pest disease-free plants in the similar way of growing were selected.3 pots were for each treatment and each pot was a repetition.On April21，3 g of10.25%15N－urea（produced from Shanghai Research Institute of Petrochemical Technology），4 g of superphosphate and 3 g of potassium sulphate were applied to each pot，and watering was immediately done after fertilization.

In the stopping period of plant growth（September 10），the destructive sampling was carried out on the whole plant，to determine the shoot length and plant fresh weight.Then the whole plant was decomposed into roots，stems，and leaves.After the samples were rinsed in the order of water→detergent→water→1%HCl→3 times deionized water，they underwent 30 min of water-removing under 105℃，and then they were dried to constant weight at 80℃.After being crushed with electric grinder and sifted through a 60-mesh sieve，they were mixed and bagged for use.All soil in the pot was removed while the plant was decomposed，and after it was weighed and mixed evenly，quartering sampling was used to determine the15N abundance of soil samples.ZHT－03 spectrometer（Beijing Analytical Instrument Factory）was used to determine the15N abundance of samples.

1.2Data calculation and statistical analysis Ndff（Contribution rate of15N absorbed from fertilizer to be allocated to plant organ to total nitrogen of this organ，%）＝［15N abundance of sample（%）15N natural abundance（%）］／［（15N abundance of fertilizer－15N natural abundance（%）］×100；

Nitrogen use efficiency（%）＝［Ndff×total nitrogen of plant organs（g）］／Fertilizer application rate（g）×100；

Soil15N residue＝Soil15N abundance×Total soil weight×soil layer nitrogen content；

Nitrogen residual rate（%）＝Soil15N residues／fertilizer application rate（g）×100；

Nitrogen loss rate＝100%nitrogen utilization rate nitrogen residual rate.

Microsoft Excel 2003 was used for drawing tables，and the experimental data used DPS 7.05 for one-factor analysis of variance.LSD method was used for difference significance comparison.

2　Results and analysis

2.1Effects of soil C／N ratio on plant grow th The shoot length and plant fresh weight of apple trees reflect the growth status of plants.As can be seen from Table1，shoot length and plant fresh weight both increase first and then decrease with the gradual increase in soil C／N ratio.After the exogenous carbon is added to gradually increase the soil C／N ratio to25，shoot length and plant fresh weight gradually increase to the maximum，then with the further increase in the soil C／N ratio，shoot length and plant fresh weight both gradually decrease.When soil C／N ratio＝40，shoot length and plant fresh weight are both lower than CK.

Statistical analysis results show that the shoot length and plant fresh weight under the three treatments of15，20 and 25 are significantly higher than that under other treatments，but there are no significant differences among the three（P＜0.05）.It shows that adding the exogenous carbon to adjust the soil C／N ratio（15 to 25）is conducive to the growth of apple plants.

Table 1 Effects of soil C／N ratio on apple shoot length and plant fresh weight

2.2Effects of soil C／N ratio on plant absorption of fertilizer nitrogen As can be seen from Fig.1，with the increase in soil C／N ratio，plant's utilization rate of fertilizer15N－urea increases first and then decreases.When soil C／N ratio＝25，the plant's utilization rate of15N is the greatest，reaching 22.87%，and there is no significant difference between the plant's utilization rate of15N when soil C／N ratio＝25 and the plant's utilization rate of15N when soil C／N ratio＝20，but the plant's utilization rate of15N under both is significantly higher than that under other treatments.When soil C／N ratio＝40，the plant's utilization rate of15N is the lowest，only 15.43%，lower than that under CK treatment（16.65%）.

The nitrogen absorbed the plant is on the one hand from the nitrogen provided by the soil，and on the other hand from fertilizer nitrogen application.Fig.2 shows that 29.56%－45.65%of total nitrogen absorbed by the apple plant is from fertilizer nitrogen，and 54.35%－70.44%of total nitrogen absorbed by the apple plant is from the soil nitrogen，indicating that most of the nitrogen absorbed by the plants is from the nitrogen provided by the soil.When soil C／N ratio is gradually increased to25，the proportion of total nitrogen absorbed by the apple plants from the fertilizer nitrogen is gradually increased（from 32.67%to 45.65%），and the proportion of total nitrogen absorbed by the apple plants from the soil nitrogen is gradually decreased（from 67.33%to 54.35%）.

With the further increase in the soil C／N ratio，the proportion of total nitrogen absorbed by the apple plants from the fertilizer nitrogen is gradually decreased（from 45.65%to29.56%，and the proportion of total nitrogen absorbed by the apple plants from the soil nitrogen is gradually increased（from 54.35%to 70.44%）.It indicates that when the soil C／N ratio is too high（＞25）or too low（＜15），the plant will use more nitrogen in the soil，but absorb less fertilizer nitrogen.

2.3Effects of soil C／N ratio on15N residues and losses As can be seen from Table2，with the increase in the soil C／N ratio，the15N residues will increase in the soil.When the soil C／N ratio＝40，the15N residue amount and residue rate are both the highest in the soil，863.89 mg and 28.80%，respectively，1.32 times the15N residue amount and residue rate under CK treatment.There is no significant difference in the soil nitrogen residue amount among the treatments with C／N of25 or more，but significantly higher than the soil nitrogen residue amount under other four treatments.

Compared with CK，the rate of nitrogen losses under all treatments by adding exogenous carbon is reduced.Nitrogen loss rate is gradually reduced and then increased with the increase in the soil C／N ratio.The15N loss amount and15N loss rate under the treatment of C／N＝25 is the lowest（1496.08 mg and 49.87%，respectively），only 81.04%of CK.Statistical analysis shows that there are no significant differences in the nitrogen losses between the treatments of C／N＝20 and C／N＝25，but significantly lower than that under other treatments；there are no significant differences in the nitrogen losses among the three treatments of CK，C／N＝10 and C／N＝40，but significantly higher than that under other treatments.

Table 2 Effects of soil C／N ratio on 15 N residues and losses in apple－soil system

3　Discussions and conclusions

Adding the glucose to improve soil C／N ratio can significantly affect apple plant growth and nitrogen utilization.The studies of Li Xueli et al［14］on tobacco suggest that the soil C／N ratio in 24－28 is most conducive to the carbon and nitrogen metabolism during the tobacco plant growth，improving the coordination of internal chemical components of tobacco and improving quality of tobacco，but when the soil C／N ratio is too high，then it is the other way around.The studies of Liu Shiliang et al［15］also show that suitable soil C／N ratio will help improve the content of chlorophyll and nitrogen content and nitrate reductase activity，but with the further increase in C／N ratio，these indicators show a decreasing trend.In this experiment，when the soil C／N ratio is15－25，the plant growth is in the best conditions，and its shoot length and plant fresh weight are significantly higher than that under other treatments，but when C／N ratio is increased to40，plant's shoot length and fresh weight are even lower than that under the control，which is consistent with the above conclusions.The uptake and utilization efficiency of fertilizer nitrogen also confirms this point.

When the soil C／N ratio is 15－25，the plant's utilization rate of15N is the highest，but with the further increase in C／N ratio，the utilization rate of fertilizer nitrogen is gradually decreased，and when C／N ratio is40，the fertilizer nitrogen utilization rate is even lower than that under the control treatment without the addition of exogenous carbon.This may be due to the fact that when C／N ratio is high，there is more added exogenous glucose and adding a lot of soluble exogenous carbon promotes the microorganisms in the soil propagate in a large number；the propagation of microorganism need to consume part of nitrogen，and microorganism and apple plant will compete for the nitrogen in the soil，thus affecting the growth of apple plants and roots'absorption of nutrients［16－17］；when the soil C／N ratio is low，there will be less carbon sources for microbes and microbial activity is decreased，thus affecting the availability of nutrients［10，18］，making the nutrients difficult to be absorbed by the apple plant roots，thereby inhibiting plant growth and development.From the perspective of apple plant growth and nitrogen uptake，the soil C／N ratio in 15－25 is the best.

The nitrogen absorbed by the crops is mainly from the nitrogen provided by soil and nitrogen provided by fertilizer，and soil nitrogen supply is main based on mineralization and release of organic matter.Studies have shown that soil mineralized nitrogen amount is significantly positively correlated with amount of nitrogen absorbed by crops［19］，while soil mineralized nitrogen amount is significantly correlated with the organic carbon level［20］.Even if in the case of considerable application of nitrogen fertilizer，there is more than about 50%of nitrogen absorbed by crops from the soil［21］.

This study shows that with the increase in the soil C／N ratio，the proportion of nitrogen absorbed by crops from the soil nitrogen supply first decreases and then increases，while the proportion of nitrogen absorbed by crops from the fertilizer nitrogen supply first increases and then decreases.It suggests that low C／N ratio can accelerate microorganisms'decomposition of organic nitrogen in the soil and nitrogen mineralization rate，thereby improving soil's nitrogen supply potential and nitrogen supply capacity［22－23］.When the C／N ratio is at a relatively high level，it will increase soil microorganisms'＂retaining＂on the applied fertilizer nitrogen，so that the availability of exogenous nitrogen is reduced，resulting in less absorption of nitrogen fertilizers by the plant.Suitable C／N ratio plays a certain role in limiting soil microbial activity，decelerating the decomposition and mineralization of organic matter and organic nitrogen，which is conducive to improving the soil organic carbon level.

At the same time，it will inhibit the too high intensity of＂retaining＂of soil microorganisms on the applied nitrogen，which is conducive to giving full play to the effectiveness of nitrogen fertilizer，and promoting the plant's uptake and utilization of nitrogen fertilizers［10，18］.

The study results in this experiment show that with the increase in the soil C／N ratio，the soil residual nitrogen will increase.Yao Huaiying et al［24］pointed out promoting the microbial fixation of inorganic nitrogen was an effective way to reduce nitrogen losses.Li Shiqing et al［25］confirmed that the application of organic fertilizer can promote microbial nitrogen fixation，and effectively reduce nitrogen and ammonia volatilization losses.The high soil organic carbon content can provide adequate carbon for the microorganisms，helping to increase the number and improve the activity；the excess fertilizer nitrogen that can not be absorbed by the roots，can be assimilated into microbial body to transform into stable organic nitrogenous matter，thereby effectively reducing nitrogen gaseous losses［26－28］.If the soil C／N ratio is higher，the amount of soil nitrogen fertilizer residues will be greater，but the nitrogen losses first decrease and then increase.

It is mainly due to the fact that when the soil C／N ratio is at a very high level，the proportion of fertilizer nitrogen absorbed the plants is reduced，and the excessive exogenous carbon makes the soil structure loose，increasing the risk of deep nitrogen leaching losses.In this experiment，the fertilizer nitrogen loss rate under each treatment is in 49.87%－61.54%，higher than the nitrogen loss rate in the field experiment，which may be related to serious nitrogen leaching losses caused by frequent watering in the summer pot experiment.

From the effects of soil C／N ratio on apple plant growth and nitrogen balance situation，when the soil C／N ratio is in 15－25，it can promote plant growth and development，reduce nitrogen losses and improve fertilizer use efficiency.Therefore，when applying biological straw or exogenous carbon to improve the orchard soil C／N ratio in the apple production，it is necessary to adjust the addition of exogenous carbon and control the soil C／N ratio within the appropriate range，according to the level of soil nitrogen，so as to achieve high yield，high quality and high efficiency.

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