Liqiang WAN,Xianglin LI,Dan WANG,Kaiyun XIE,Feng HE,Huigang LU,Weida WU

Institute of Animal Sciences,Chinese Academy of Agricultural Sciences,Beijing 100193,China

Growth Performance of Alfalfa(Medicago sativa) under Different Nitrogen Application Levels

Liqiang WAN,Xianglin LI*,Dan WANG,Kaiyun XIE,Feng HE,Huigang LU,Weida WU

Institute of Animal Sciences,Chinese Academy of Agricultural Sciences,Beijing 100193,China

Nitrogen addition is rather important to the growth of alfalfa.In this study, the effects of different nitrogen application levels on various growth characteristics of alfalfa were investigated.The results showed that nitrogen application exhibited no significant effects on plant height and stem diameter but posed significant effects on branch number of alfalfa.Branch number of alfalfa in each cutting raised with the increasing application level of nitrogen,with significant differences among different treatments(P＜0.05);individual aboveground biomass increased with the increasing application level of nitrogen,but the increasing trend gradually tended to be steady from the first to the fourth cutting.Nitrogen application affected significantly(P＜0.05) individual aboveground biomass of alfalfa in the first,second and fourth cutting,but exhibited no significant effects on individual aboveground biomass of alfalfa in the third cutting.With the increase of nitrogen application level,total surface area,total volume,collar diameter and crossing number of alfalfa roots increased gradually,but total length,average diameter and furcating number of alfalfa roots increased first and then declined.Total length,average diameter and furcating number of alfalfa roots reached the maximum in N60 treatment.

Nitrogen application;Alfalfa;Growth characteristics;Biomass;Roots

A lfalfa(Medicago sativa),known as"king of forage",is a deeprooted perennial high-quality leguminous forage with the widest distribution and the longest history of cultivation in the world.Currently,the cultivation area of alfalfa exceeds 3.3 million hm2.In addition,alfalfa also possesses the largest cultivation area (1 045.3 thousand hm2)in China,accounting for 78.5%of the totalarea of artificial grassland.Nitrogen fertilizer application is of very important significance for forage growth.Studies have shown thatplant height,branch number and individual weight of alfalfa plants are improved with the increase of application level of ammonium nitrate[1].Nitrate nitrogen and ammonium nitrogen are the main forms ofnitrogen absorbed by alfalfa from the soil.However,the absorption amount of different forms of nitrogen by alfalfa varies. To be specific,the absorption amount ofnitrate nitrogen is higher than thatof ammonium nitrogen.In addition,differentforms of nitrogen pose varying effects on the growth characteristics of alfalfa[2].Nitrate nitrogen can promote tillering and contributes to increasing the weight of alfalfa shoots;ammonium nitrogen can not only improve the yield and quality of alfalfa,reduce the contents of acid detergent fiber,neutral detergent fiber and cellulose in alfalfa stems by about 8%,but also improve the in vitro digestibility of dry matter in alfalfa[3].Nevertheless, no consistent conclusions have been drawn on the effects of nitrogen addition on the growth performance of alfalfa.In this study,the effects ofdifferentnitrogen application levels on plant height,stem diameter,branch number,morphological indicators of roots, and biomass of alfalfa were investigated,aiming atproviding a theoretical reference for fertilizer management of alfalfa in production practices.

Materials and Methods

Materials

Experimental alfalfa(Medicago sativa L.)materials were provided by Barenbrug(Beijing)International Co., Ltd.Experimental fertilizers:urea,AR, containing(NH2)2CO≥99.0%;potassium sulfate,AR,containing K2SO4≥99.0%;superphosphate,CP,containing 14%-15%available phosphorus (P2O5).Alfalfa seedlings were grown in plastic pots 28 cm in upper diameter, 24 cm in bottom diameter and 32 cm in height.Experimental soil:0-30 cm topsoilcollected from the experimental field in Institute of Animal Sciences, Chinese Academy of Agricultural Sciences.Soil bulk density:1.43 g/cm3; field capacity:17.3%;pH 7.39;organic matter:21.73 g/kg;total nitrogen: 0.844 g/kg;rapidly available phosphorus:4.85 mg/kg;rapidly available potassium:108.09 mg/kg;nitrate nitrogen:11.88 mg/kg;ammonium nitrogen:5.33 mg/kg.Soil samples were sifted and dried before the experiment.

Experimentaltreatments

Single-factor completely randomized experimental design was performed with four nitrogen gradients (N0:0 kg N/hm2;N30:30 kg N/hm2;N60:60 kg N/hm2;N90:90 kg N/hm2). Each treatment was repeated four times.Each potwas filled with 17 kg of dried soil.According to the determination results of soil fertility,150 kg/hm2P2O5and 100 kg/hm2K2O were applied primarily before sowing as base phosphorus,potassium fertilizers.Five alfalfa seeds were sown in each pot.At three-leafstage,alfalfa seedlings were singled(alfalfa seedlings with robust and uniform growth were selected); three seedlings were preserved.Base nitrogen fertilizer was applied after finalsingling.Topdressing nitrogen fertilizer was applied after each mowing (1/3 of the application amount of base nitrogen fertilizer).

Determination items and methods Observation and determination of biological characteristics

At initial flowering stage,plant height,stem diameter and branch number ofeach alfalfa seedlings were measured.Measurement of plant height:the vertical height of alfalfa seedlings was measured with a ruler;measurement of stem diameter:the diameter of the main stem 2 cm away from the ground was measured with a vernier caliper; branch number:the number of branches on each alfalfa seedling was counted.

Determination of morphological indicators of roots

Alfalfa roots were rinsed clean with water,placed separately into a ziplock bag and brought back to the laboratory,and then scanned using EPSON Perfection V700 Photo scanner.The scanning images of alfalfa roots were analyzed using WinRHIZO analysis software.

Determination of yield

Alfalfa seedlings were mowed at initial flowering stage,and 5 cm tall stubbles were preserved.After mowing,alfalfa samples were weighed immediately, placed into a large envelope,and brought back to the laboratory for drying.Alfalfa samples were fixed at 105℃for 15 min and dried to a constant weight at 65℃,to obtain the yield of alfalfa in each pot.In addition,the biomass of each alfalfa seedling was calculated.Alfalfa seedlings were grown for one year,and harvested four times.

Data processing

Excle 2010 and SAS(9.0)software were used for charting,analysis ofvariance,and significance test;variance analysis and multiple comparison were performed with Duncan method (P＜0.05).

Results and Analysis

Effects ofdifferentnitrogen application levels on plant height of alfalfa

Plant height refers to the vertical height of the main stem,which is one of the major indicators reflecting the growth conditions and productivity of forage.Overall,with the increase of nitrogen application level,plant height of alfalfa increased gradually.To be specific,average plant height of alfalfa from the first to the fourth cutting was 52.3,73.1,72.0 and 56.8 cm,respectively.Compared with the first cutting, plant height of alfalfa in the second, third and fourth cutting increased by 39.77%,37.67%and 8.60%,respectively,which might be due to the slow growth of alfalfa in the first cutting in winter.As shown in Table 1,Table 2, Table 3 and Table 4,plantheight of alfalfa in the first cutting in N30,N60 and N90 treatments increased by 7.37%, 4.49%and 15.63%compared with N0 treatment,respectively;plant height of alfalfa in the second cutting in N30, N60 and N90 treatments increased by 17.06%,25.00%and 21.40%compared with N0 treatment,respectively; plant height of alfalfa in the third cutting in N30,N60 and N90 treatments increased by 2.68%,2.52%and 3.30%compared with N0 treatment, respectively;plant height of alfalfa in the fourth cutting in N30,N60 and N90 treatments increased by 2.23%,1.79% and 2.90%compared with N0 treatment,respectively.Therefore,nitrogen application was conducive to increasing the plantheight of alfalfa in the first and second cutting but exhibited so significanteffects on the plantheightof alfalfa in the third and fourth cutting, which mightbe due to that root nodule of alfalfa atearly growth stage exhibited a weak ability of nitrogen fixation that could not meet the demand for growth of alfalfa,while nitrogen application promoted the growth ofalfalfa.

Effects of different nitrogen application levels on stem diameter of alfalfa

Stem diameter refers to the diameter of the main stem,which is also a comprehensive reflection of other biologicalcharacteristics in addition to robust growth.To be specific,average stem diameter of alfalfa from the first to the fourth cutting was 1.693,2.806, 1.694 and 2.384 mm,respectively. Compared with the first cutting,stem diameter of alfalfa in the third cutting exhibited no significant differences; stem diameter ofalfalfa in the second and fourth cutting increased by 65.74%and 40.82%,respectively. With the increase of nitrogen application level,stem diameter of alfalfa in the first,second and fourth cutting increased gradually,butthatin the third cutting showed an opposite trend.As shown in Table 1,Table 2,Table 3 and Table 4,stem diameter of alfalfa in the first cutting in N30,N60 and N90 treatments increased by 3.57%,5.73%and 9.86%compared with N0 treatment, respectively;stem diameter of alfalfa in the second cutting in N30,N60 and N90 treatments increased by 4.98%, 5.68%and 5.09%compared with N0 treatment,respectively;stem diameterof alfalfa in the third cutting in N30, N60 and N90 treatments was reduced by 0.44%,3.91%and 4.31%compared with N0 treatment,respectively; stem diameter of alfalfa in the fourth cutting in N30,N60 and N90 treatments increased by 0.15%,1.54%and 6.06%compared with N0 treatment, respectively,which might be due to that alfalfa in the third cutting exhibited a strong ability of symbiotic nitrogen fixation that could meet the demand forgrowth ofalfalfa.

Table 1 Effects of different nitrogen application levels on biological characteristics of alfalfa in the first cutting

Table 2 Effects of differentnitrogen application levels on biological characteristics of alfalfa in the second cutting

Effects of different nitrogen application levels on branch number of alfalfa

Branch number is one of the majorindicators reflecting the branching ability and growth status of individuals. Under adequate nutrition conditions, individuals with robust growth had more branches and higher yield.With the progress of growth period,branch number of alfalfa increased gradually. To be specific,average branch number of alfalfa from the first to the fourth cutting was 11.50,14.92,16.86 and 25.50,respectively.Branch number of alfalfa in the second,third and fourth cutting increased by 29.74%,46.61% and 121.74%compared with the first cutting,respectively.With the increase of nitrogen application level,branch number of alfalfa increased gradually. As shown in Table 1,Table 2,Table 3 and Table 4,branch number of alfalfa in the firstcutting in N30,N60 and N90 treatments increased by 3.26%, 18.48%and 28.26%compared with N0 treatment,respectively;branch number of alfalfa in the second cutting in N30,N60 and N90 treatments increased by 7.26%,8.06%and 17.74% compared with N0 treatment,respectively;branch number of alfalfa in the third cutting in N30,N60 and N90 treatments increased by 6.29%,6.29%and 11.89%compared with N0 treatment, respectively;branch number of alfalfa in the fourth cutting in N30,N60 and N90 treatments increased by 0.15%, 1.54%and 6.06%compared with N0 treatment,respectively.With the increase ofmowing times,the promotion effects of nitrogen application on branch number of alfalfa decreased gradually.

Effects of different nitrogen application levels on individual aboveground biomass of alfalfa

Overall,individual aboveground biomass of alfalfa was improved with the increase of nitrogen application level;however,from the first to the fourth cutting,the increasing trend tended to be steady gradually(Fig.1), indicating that the dependence of the growth of alfalfa on nitrogen fertilizer was reduced gradually.In the first mowing,individual aboveground biomass of alfalfa in N90 treatment increased by 23.17%compared with N0 treatment,with significant differences (P＜0.05),but individual aboveground biomass of alfalfa in N30 and N60 treatments exhibited no significant differences compared with N0 treatment. In the second cutting,individual aboveground biomass of alfalfa reached the maximum of13.72 g/plant in N90 treatment and the minimum of 10.56 g/plant in N0 treatment.In addition,individual aboveground biomassof alfalfa in N30,N60 and N90 treatments increased by 14.68%,15.32% and 29.95%compared with N0 treatment,respectively,exhibiting significantdifferences(P＜0.05).In the third cutting,individual aboveground biomass of alfalfa exhibited no significantdifferences among different treatments.In the fourth cutting,individual aboveground biomass of alfalfa reached the maximum of 10.30 g/plant in N90 treatment and the minimum of 8.49 g/plantin N0 treatment;individual aboveground biomass ofalfalfa in N90 treatment increased by 21.34%compared with N0 treatment,exhibiting significant differences(P＜0.05),but individual aboveground biomass of alfalfa in N60 and N30 treatments exhibited no significantdifferences compared with N0 treatment.Therefore,in the third cutting,the effects of nitrogen application on individual aboveground biomass of alfalfa became weak.In this study,alfalfa seedlings were harvested four times in one year,individualaboveground biomass showed a descend order offirst cutting＞second cutting＞third cutting＞fourth cutting (Fig.1).Accumulated biomass of alfalfa from the first to the fourth cutting was 41.42-53.69 g/plant.In each cutting,individual aboveground biomass of alfalfa accounted for 31.43%, 26.03%,22.61%and 19.94%of the annual biomass,respectively.Thus, with the progress of growth period,individual aboveground biomass of alfalfa was reduced gradually.

Table 3 Effects of differentnitrogen application levels on biological characteristics of alfalfa in the third cutting

Table 4 Effects of different nitrogen application levels on biological characteristics of alfalfa in the fourth cutting

Effects of different nitrogen application levels on morphological characteristics ofalfalfa roots

Nitrogen application posed significant effects on morphological developmental characteristics of alfalfa roots(Table 5).With the increase of nitrogen application level,totalsurface area,totalvolume,collar diameter and crossing number of alfalfa roots increased correspondingly.In N30,N60 and N90 treatments,totalsurface area of alfalfa roots increased by 3.72%, 23.52%and 15.19 compared with control,respectively;total surface area of alfalfa roots in N60 and N90 treatments was significantly higher compared with control(P＜0.05);totalsurface area of alfalfa roots in N30 treatment exhibited no significant differences compared with control.In N30, N60 and N90 treatments,total volume of alfalfa roots increased by 12.68%, 27.16%and 26.31%compared with control,respectively;total volume of alfalfa roots in N60 and N90 treatments was significantly higher compared with control(P＜0.05);totalvolume of alfalfa roots in N30 treatment exhibited no significant differences compared with control.In N30,N60 and N90 treatments,root collar diameter of alfalfa increased by 19.42%, 17.73%and 24.36%compared with control,respectively,with significant differences(P＜0.05);however,root collar diameter of alfalfa exhibited no significant differences among N30, N60 and N90 treatments.Crossing number of alfalfa roots in N90 treatment was significantly higher than that in other treatments,which increased by 20.19%,17.58%and 6.19%compared with N0,N30 and N60 treatments,respectively;crossing number of alfalfa roots exhibited no significant differences among N0,N30 and N60 treatments.In addition,total length, average diameter and furcating number of alfalfa roots increased first andthen declined with the increase of nitrogen application level.To be specific, totallength,average diameter and furcating number of alfalfa roots in N60 treatmentreached the maximum.Total length of alfalfa roots in N60 treatment increased by 20.19%and 5.89% compared with N0 and N90 treatments,respectively;total length of alfalfa roots in N60 treatment was significantly higher than that in control and N90 treatment but exhibited no significant differences compared with N30 treatment.Average diameter of alfalfa roots in N60 treatment was significantly higher than that in other treatments,which increased by 82.69%,48.62%and 37.23%compared with control,N30 and N90 treatments,respectively;average diameter of alfalfa roots in N30 and N90 treatments was significantly higher than that in control,but average diameter of alfalfa roots exhibited no significant differences between N30 and N90 treatments.Furcating number ofalfalfa roots in N60 treatmentwas significantly higher than that in other treatments, which increased by 8.84%,6.35%and 3.85%compared with N0,N30 and N90 treatments,respectively.Root tip number of alfalfa reached the maximum of70 490.3 in N60 treatmentand the minimum of 63 550.3 in control, exhibiting no significant differences among differenttreatments.

Table 5 Effects of different nitrogen application levels on morphological characteristics of alfalfa roots

Discussions

Alfalfa requires various nutrients in the growth and development process,which can notbe satisfied simply from the soil but also need external materials.As an important component of proteins,nucleic acids,chlorophyll, and some hormones in plants,nitrogen is the primary factor for crop growth and yield formation[4-7].Previous studies have shown that nitrogen application could significant improve the yield ofcerealcrops[8-10].As a leguminous forage,alfalfa has a strong ability of symbiotic nitrogen fixation. Thus,no consistent conclusion has been drawn on whether nitrogen application is necessary in alfalfa production[11].Some studies suggest that nitrogen application has no improving effects on alfalfa yield[12];large-scale application of nitrogen fertilizer will inhibit the development of root nodules, reduce the infection of root hairs and nitrogenase activity[13];several studies even show that nitrogen application poses negative effects on alfalfa. These views suggest that the amount of nitrogen fixed by alfalfa can meet their basic needs,requiring no further application of nitrogen fertilizer.However,other studies show that nitrogen application under certain conditions exhibited significant promotion effects on alfalfa yield.Applying nitrogen fertilizer can significantly promote the yield of alfalfa in the planting year.A large number ofstudies in North America[14]report that,when nitrate nitrogen contentin soilwas lowerthan 15 mg/kg, organic matter content was lower than 15 g/kg,ambienttemperature was below 15℃,applying a small amount of nitrogen fertilizer in sowing can improve the yield;otherwise,nitrogen application has no significant effects. Studies have shown thatalfalfa can utilize nitrogen in deep soil,resulting in nitrogen deficiency and soilfertility reduction,which requires application of nitrogen fertilizer.

Nitrogen application can improve the yield ofalfalfa in the planting year. Specifically,nitrogen application improves significantly the yield of alfalfa in the first and second cutting,but exhibits no significant effects in the third cutting[15].Zeng et al.[3]reported thatsimple application ofnitrogen fertilizer improved the yield ofalfalfa;to be specific,the yield of alfalfa applied with 60 and 30 kg N/hm2in the first cutting was improved by 10.7%and 5.5% compared with CK,respectively;the yield of alfalfa applied with 60 and 30 kg N/hm2in the second cutting was improved by 3.1%and 3.4%compared with CK,respectively;thus,yield increment in the first cutting was higherthan that in the second cutting.In this study,experimentalresults indicated that individual aboveground biomass of alfalfa increased with the increasing nitrogen application level, which varied significantly among differenttreatments in the first,second and fourth cutting(P＜0.05),but exhibited no significantdifferences among different treatments in the third cutting, which may be due to that alfalfa is capable of symbiotic nitrogen fixation. Therefore,the improving effects of nitrogen fertilizer on alfalfa yield were relatively complex.Before the formation of effective root nodules,applying a small amount of nitrogen fertilizer can promote the growth and improve the yield of alfalfa;after the formation of effective root nodules,alfalfa can meet the needs via symbiotic nitrogen fixation,and nitrogen application exhibits no significant effects on alfalfa yield.Large-scale application of nitrogen fertilizer will inhibit the development of root nodules.Under nitrogen deficiency conditions,rhizobia can not effectively fix nitrogen,which can not fully meet the demands for nitrogen nutrition ofalfalfa[16].

Roots are not only the material basis for growth and regeneration of alfalfa,but also the major organs of nutrient absorption and nodule formation[17-18].Currently,most researches are focused on alfalfa rhizobia[19-21].As a recognized deep-rooted plant,root traits ofalfalfa are closely related to its biomass;biomass directly affects aboveground yield.Some studies have found that applying nitrogen under potted conditions can improve root biomass of alfalfa.Based on pot experiment,Saindon et al.[22]found that rootbiomass of17-week-old Medicago sativa L.cv.Algonguin and other seven alfalfa varieties in nitrogen application treatment(400 mgN/L)was 2.94-3.36 g/plant,which was significantly higher than that in nitrogen fixation treatment(artificial rhizobium inoculation)of2.29-2.78 g/plant.Khan etal.[23]conducted a pot experiment and found that root biomass of 80-day-old Gilboa alfalfa was 0.34,0.54 and 0.93 g/plant after application of 0,3 and 6 mmol/Lrespectively;root biomass in nitrogen application treatment was improved by 58.8%and 173.5%compared with control.Esechie et al.[24]conducted a potexperimentand found that root biomass of 8-week-old Batini alfalfa was 0.36,0.61 and 0.83 g/plant after application of 0,3 and 6 mmol/L,respectively;root biomass in nitrogen application treatment was improved by 69.4%and 130.6%compared with control;similar results were also found in Trifecta alfalfa,which indicates that after application of either nitrate nitrogen or ammonium nitrogenunder potted conditions,root biomass of alfalfa increases with the increasing nitrogen application level.In the present study,experimental results indicate that,with the increase of nitrogen application level,total surface area, total volume,collar diameter and crossing number of alfalfa roots increased gradually,buttotallength,average diameter and furcating number of alfalfa roots increased first and then declined;totallength,average diameter and furcating number of alfalfa roots reached the maximum in N60 treatment.The corresponding mechanism between root growth indicators and biomass of alfalfa requires further investigation.

Conclusions

Different nitrogen application levels pose varying effects on the growth performance of alfalfa.Application of nitrogen fertilizer exhibits no significant effects on plantheightand stem diameter of alfalfa,but significantly affects the branch number of alfalfa.Thus, branch number of alfalfa raises with the increase of nitrogen application level;aboveground biomass of alfalfa is improved with the increase of nitrogen application level.However,with the increase of cutting times,the biomass of alfalfa increases slowly, suggesting that nitrogen application affects significantly the biomass ofalfalfa.Growth indicators of alfalfa are improved with the increase of nitrogen application level.To be specific,total surface area,total volume,collar diameter and crossing number of alfalfa roots increase gradually,but total length,average diameter and furcating number of alfalfa roots exhibit no significant variations.In this study,total length,average diameter and furcating number of alfalfa roots reached the maximum in N60 treatment among various nitrogen application treatments.

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不同氮素添加水平對(duì)紫花苜蓿生長性能的影響

萬里強(qiáng)，李向林*，王 丹，謝開云，何 峰，呂會(huì)剛，吳維達(dá)
（中國農(nóng)業(yè)科學(xué)院北京畜牧獸醫(yī)研究所，北京100193）

氮素添加對(duì)于紫花苜蓿生長具有重要作⒚。該試驗(yàn)研究了不同氮素添加水平對(duì)于紫花苜蓿生長性能各個(gè)指標(biāo)的影響機(jī)制。結(jié)果表明，施氮對(duì)不同茬次苜蓿的株高、莖粗影響效果不明顯，而對(duì)苜蓿的分枝數(shù)影響效果顯著。每一茬次的苜蓿分枝數(shù)都隨施氮量的增加而增加，并且處理之間差異顯著（P＜0.05）；苜蓿單株地上生物量隨施氮量的增加而增加，但從第一茬到第四茬，增加的趨勢逐漸平緩。施氮對(duì)第一茬、第二茬和第四茬的苜蓿單株生物量有顯著影響（P＜0.05），而對(duì)第三茬苜蓿單株生物量影響不顯著；隨著施氮量的增加，紫花苜蓿根系的總表面積、總體積、根頸粗和交叉數(shù)呈逐漸增加的趨勢，而紫花苜蓿的總根長、平均直徑和分叉數(shù)隨施氮量的增加并未呈現(xiàn)逐漸增加的趨勢，而是先增加后降低，N60處理的紫花苜?？偢L、平均直徑和分叉數(shù)最大。

氮素添加；紫花苜蓿；生長性狀；生物量；根系

國家自然科學(xué)基金項(xiàng)目（31372370）資助。

萬里強(qiáng)（1973-），男，甘肅禮縣人，博士，研究員，主要從事牧草生理生態(tài)學(xué)研究，E-mail：wanliqiang@caas.cn。*通訊作者。

2014-10-13

Supported by NationalNaturalScience Foundation of China(Grant No.31372370).

*Corresponding author.E-mail:lxl@caas.cn

Received:October 13,2014 Accepted:January 5,2015

修回日期2015-01-05