Optimized nitrogen application methods to improve nitrogen use efficiency and nodule nitrogen fixation in a maize-soybean relay intercropping system

YONG Tai-wen, CHEN Ping, DONG Qian, DU Qing, YANG Feng, WANG Xiao-chun, LlU Wei-guo,YANG Wen-yu

1 College of Agronomy, Sichuan Agricultural University/Key Laboratory of Crop Eco-physiology and Farming System in Southwest,Ministry of Agriculture, Chengdu 611130, P.R.China

2 Shehong Farm Bureau, Suining 629200, P.R.China

1.lntroduction

In China, the continuous increase of food production largely depends on chemical nitrogen (N) fertilizer inputs(Zhu and Chen 2002).However, the overuse of N fertilizer and low N use efficiency (NUE) leads to wasted resources and environmental pollution and is contrary to sustainable agricultural production (Kwong et al.2002; Xing and Zhu 2002; Wang et al.2017).Surplus N fertilization results in substantial soil acidification, with N leaching and emissions as direct triggers of water and air pollution (Guo et al.2010;Zhao et al.2012; Zhang et al.2013), which is contrary to environmental-friendly agricultural production.

As the main food and economic crops in China, maize and soybean are widely cultivated.However, during maize cropping seasons from 1980 to 2010 in China, the NUE declined from 30.2 to 29.9 kg grain kg-1N; in contrast, NUE improved from 39.4 to 53.2 kg grain kg-1N in the USA (Yu et al.2015).To achieve both food security and agricultural sustainable development, optimal N management practices are urgently needed.Suitable N nutrient management and cropping systems can improve nutrient use efficiency and utilization of solar energy while being environmentally friendly.

Efficient N management technology has become one of the most urgent requirements for sustainable agriculture in China.Researchers are trying to reduce N input directly by decreasing chemical N fertilization rates (Liu et al.2011;Ruan et al.2013; Yan et al.2013) and combining chemical N with organic fertilizer or new fertilizer types (slow release or controlled release) to meet crop demand (Shoji et al.2001;Fernández-Escobar et al.2004; Meng et al.2005; Noellsch et al.2009).Previous studies have shown that optimal nutrient management strategies can significantly reduce N fertilization rate and increase crop yield, with multiple benefits for agriculture and the environment (Chen et al.2006; Ju et al.2009; Constantin et al.2010; Ruan et al.2013).In wheat-maize rotation cropping system, the total N fertilizer reduced from 430 to 360 kg N ha-1with improved treatments,resulting in a maize yield increase by 7-14%, and a reduction in wheat yield, N2O and NO emissions by 1-2, 7 and 29%,respectively (Liu et al.2011).In addition, reduced N with optimal fertilization practices improve NUE, thereby reducing seasonal cumulative N2O emission (Yan et al.2013).

Global food demand is increasing with economic development and population growth.Especially, China feeds approximately 20% of the world’s population with 7% of the world’s farm land (Godfray et al.2010; Tilman et al.2011; Larson 2013).In China, the average unit grain yield increases from 1.09 tonne ha-1in 1949 to 6.51 tonne ha-1in 2014 (Zhou 2017).However, large amounts of soybeans are imported annually to meet the grain and soybean oil consumption in China.Relay intercropping and intercropping systems can be high-yielding, land-use efficient, and resource-efficient, and can efficiently control weeds, diseases and pests (Li et al.2006; Corre-Hellou et al.2011; Rodríguez-Navarro et al.2011; Zuo et al.2015; Wang et al.2017; Yang et al.2017).In particular, those systems including soybean crops can increase soybean yield with limited arable land.On one hand, legume-based cropping system can reduce N leaching by 50% compared with conventional systems (Drinkwater et al.1998).Soybeans reduce N input requirements by biological nitrogen fixation,which meets 50-60% of N demand (Salvagiotti et al.2008).On the other hand, fertilization methods have a significant impact on intercropped crops, with better performance when heterogeneous N was supplied at interspecific rather than intraspecific rows (Wu et al.2014).Interspecific facilitation can increase resource use efficiency and land productivity without negative impacts on the environment (Oljaca et al.2000; Li et al.2001, 2003; Hochman et al.2011).Therefore,soybean-based relay intercropping and intercropping systems, with improved fertilization methods can increase soybean yield and decrease environment costs.However,environmental factors, e.g., light, heat, and rainfall, can limit cropping systems.Intercropping systems are employed in areas with two (or three) crops a year, such as the Huang-Huai-Hai region in China (Liu et al.2017), whereas relay intercropping systems are used in areas with one crop a year(or three crops in two years), such as in Southwest China(Yang et al.2017).

Maize-soybean relay intercropping system can increase NUE, light use efficiency, and land productivity, which has become a major planting pattern in Southwest China (Gao et al.2010; Yan et al.2010; Xiang et al.2012; Yang et al.2014, 2017; Yong et al.2015; Wang et al.2017).Despite these benefits, few studies are focused on the lower N application rate and its application methods to maizesoybean relay intercropping system.The early study showed that crops performed better at lower N application rate (270 kg N ha-1), and the fertilizer placement can increase grain yield (Dong et al.2014).However, the lower N applied rate and fertilization methods on NUE and soybean biological nitrogen fixation are still unclear in the maize-soybean relay intercropping system.Therefore, this study was performed to test the optimized N management strategy for maize-soybean relay intercropping system in Southwest China.It was hypothesized that lower N input with optimized fertilization locations for maize-soybean relay intercropping system would increase NUE, N agronomy efficiency (NAE), and total yield compared with conventional N application rate and location.The objectives of this study were to: (1) investigate NUE, NAE and yield responses of crops to lower N and fertilization locations and to (2)determine ureide, leghemoglobin content, and nitrogenase activity responses of soybean to lower N and fertilization locations.

2.Materials and methods

2.1.Experimental site

The experiment was performed in Renshou County (29°40′-30°16′N(xiāo), 104°00′-104°30′E), Sichuan Province, Southwest China, during the 2012 and 2013 cropping seasons(April-November).The climate of the experimental site is subtropical monsoon humid, with a mean annual temperature of 17.4°C and mean annual rainfall of 1 009.4 mm.The frost-free period lasts approximately 312 days, and the average annual sunshine is 1 196.6 h.The soil is anthrosol with a clay loam texture, and the soil (0-20 cm) nutrient contents are shown in Table 1.

A semi-compact maize variety (Zea mays L.Chuandan 418)and shade-tolerant soybean variety (Glycine max L.Nandou 12) were chosen as the experimental crops.In the maize-soybean relay intercropping system, a wide-narrow row planting was adopted, with wide rows of 160 cm and narrow rows of 40 cm.The total ratio of maize to soybean rows was 2:2.Maize plants were in the narrow rows with a row spacing of 40 cm, soybean plants in the wide rows with a row spacing of 40 cm.The distance between maize and soybean rows was 60 cm.The plant density was 52 500 plants per ha for maize and 105 000 plants per ha for soybean.The plant spacing was 19 cm for all treatments,with a post-emergence density of one maize plant and two soybean plants per hole (Fig.1).

The total number of plots was 15, and the area of each plot was 6 m×6 m.To avoid edge effects, border rows were planted along the edges of plots.Maize was sown on 6 April 2012 and 2 April 2013 and harvested on 3 August 2012 and 29 July 2013.Soybean was sown on 16 June 2012 and 14 June 2013, with simultaneous application of maize topdressing and soybean base fertilizer, and harvested on 27 October 2012 and 2 November 2013.

2.2.Experimental design and treatments

Five fertilizer treatments were investigated in a singlefactor randomized block design with three replications in the maize-soybean relay intercropping system.Three total nitrogen application rates included no nitrogen application(NN: 0 kg N ha-1), conventional nitrogen application rate employed by the local farmer (CN: 330 kg N ha-1), and lower nitrogen (LN: 270 kg N ha-1) with three topdressing distances(LND), e.g., 15 cm (LND1), 30 cm (LND2) and 45 cm (LND3)from maize rows.The NN and CN treatments were set as the control group (CK).Urea (N content: 46%), CaP2H4O8(P2O5content: 12%) and KCl (K2O content: 60%) were used as N, P and K fertilizer.All maize treatments received 105 kg P2O5ha-1and 112.5 kg K2O ha-1as base fertilizer,and all soybean treatments received 63 kg P2O5ha-1and 52.5 kg K2O ha-1as base fertilizer.

All fertilizers for crops were closely placed to the plant stems in NN and CN treatments, respectively.In CN treatment, the total N fertilizer (330 kg N ha-1) was applied at 270 kg N ha-1for maize and 60 kg N ha-1for soybean.The N fertilizer for maize was applied at 120 kg N ha-1for base fertilizer and 150 kg N ha-1for topdressing, and N fertilizer for soybean was applied as base fertilizer.In optimized fertilization for LN, all base fertilizers for maize were closely placed to the plant’s stems.The total N fertilizer (270 kg N ha-1) was applied at 120 kg N ha-1for maize base fertilizer,and 150 kg N ha-1for maize topdressing.Topdressing for maize was integrated with soybean base fertilizer, and strips placed at distances of LND1, LND2 and LND3 from maize rows (Fig.2).

2.3.Determination of crop shoot biomass, grain yield,and N content

Four representative plants from each plot were separately sampled at five fully developed trifoliate leaf stage (V5),full bloom stage (R2), beginning seed stage (R5), and physiological maturity stage (R8) of soybeans (Fehr et al.1971), respectively.Crops were harvested from 12 m2areas of each plot to calculate grain yield and shoot biomass.Samples were divided into different organs and exposed to 105°C for 30 min to destroy tissues; they were oven-dried at 80°C to a constant weight before weighing.Nitrogen concentration in plant tissues was determined using BUCHI Auto Kjeldahl Unit K-370 (Buchi Labortechnik AG, Switzerland) according to the Kjeldahl method (Hou et al.2006).

2.4.Determination of symbiotic nitrogen fixation by soybean

Soybean samples were collected at the V5, R2 and R5 stages (Fehr et al.1971).Shoots were cut at ground level and divided into different organs.Root systems attached to the soil block (20 cm long×40 cm width×30 cm deep) were dug out and gently shaken to remove bulk soil.The entire root system was placed in nylon nets, soaked in water,and carefully washed and separated for counting nodules and lateral roots and for weighing nodules.The roots and nodules of four representative plants from each plot were stored for morphology and physiology determination.Root volume was measured with WinRHIZO System (WinRHIZO Pro LA2400; Regent Instruments Inc., Quebec, Canada);the main root length was manually determined with a ruler.Roots were then exposed to 105°C for 30 min and ovendried at 80°C to a constant weight before weighing.

Table 1 Properties of the experimental soil in 2012 and 2013

Fig.1 Maize-soybean relay intercropping in Renshou County,Sichuan Province, Southwest China, in August 2013.

The acetylene (C2H2) reduction assay (ARA) was used to assess nitrogen fixation.The nodule samples were collected in 10-mL penicillin bottles in the presence of 20% (v/v)acetylene and then incubated for 30 min in a 30°C constant temperature water bath.Next, 0.1 mL of gas sample from each bottle was analyzed for ethylene content by a Shimadzu GC-2010 Plus Gas Chromatograph with Flame Ionization Detector (FID) (Siczek and Lipiec 2011).To measure the content of leghemoglobin, 2 g fresh soybean root nodules were ground into homogenate with approximately 5 volumes of phosphate buffer solution (5°C; pH 6.8;6.8 mol L-1).The samples were centrifuged at 100 r min-1for 15 min at 15°C, the sediment was discarded, and the supernatant was centrifuged at 39 000 r min-1for another 20 min at 5°C.The final supernatant was measured using a spectrophotometer (Mapada Instruments, Shanghai,China) at 540 nm (Riley and Dilworth 1985).Ureide content was analyzed in dry leaves, stems, pods, shells,and grains.The steps were modified according to Young and Conway (1942): 0.4 g tissue samples were added to 8 mL 0.1 mol L-1phosphate buffer (pH 7.0) containing 50%alcohol (v/v), and added to a water bath (80°C) for 5 min.The samples were centrifuged at 5 000 r min-1for 10 min,and clarified supernatant was collected and stored at-20°C.Then, 1.5 mL extracted samples mixed with 0.5 mL of 0.5 mol L-1NaOH were boiled for 6 min.After chilling to room temperature, 0.5 mL of 0.65 mol L-1HCl was added to the mixture and boiled for 6 min.After chilling, 0.5 mL of 0.4 mol L-1phosphate buffer (pH 7.0) and 0.5 mL of 0.5%C6H8N2·HCl were added and samples were kept for 6 min at room temperature.Samples were chilled to 0°C and then 2.5 mL concentrated HCl and 0.5 mL of 2% K3[Fe(CN)6] were added; samples were prechilled to 0°C and kept at room temperature for 15 min.Finally, the ureide concentration was measured by a spectrophotometer at 535 nm.

Fig.2 Diagrammatic sketch of maize-soybean relay intercropping system and fertilizer application locations in 2012 and 2013.In the maize-soybean relay intercropping system, strip width is 2 m.Maize and soybean were sown in two separate rows with 40 cm width, and 60 cm between maize and soybean rows.Nitrogen application rates were 330 kg ha-1 (CN: 270 kg N ha-1 for maize and 60 kg N ha-1 for soybean); 270 kg N ha-1 (LN, shared by maize and soybean).Fertilization locations under LN were 15 cm(LND1), 30 cm (LND2), and 45 cm (LND3) from maize rows, CN and NN were applied between two plants in the same planting row.Post-emergence densities were 1 maize plant and 2 soybean plants per hole.Maize planting density was 52 500 plants ha-1,and soybean planting density was 105 000 plants ha-1.indicates fertilizer application locations.

2.5.Calculation

Plant organ N accumulation (NA, kg ha-1), total N accumulation (TNA, kg ha-1), NUE (%), NAE (kg kg-1) and nitrogen fixation (ARA, mL h-1plant-1) were calculated using the following formulas (Bellaloui et al.2014; Duan et al.2014).

Total grain yield and shoot N uptake of maize-soybean relay intercropping system were the sum of component crops grain yield and shoot N uptake, respectively.The NUE and NAE of the maize-soybean relay intercropping system were determined by the total N uptake and total yield relative to the amount of N supplied.

2.6.Statistical analysis

The study was performed using one-factor design, namely,lower N application with three different topdressing positions.Conventional N application and no N application were set as the control.Therefore, data were analyzed with oneway ANOVA of SPSS (ver.15, SPSS, Chicago, IL, USA)in several steps: treatments were considered the fixed factor, and data were the dependent variable.Significant differences among treatments were determined by the least significant difference (LSD) (P≤0.05).

3.Results

3.1.Crop yield and nitrogen uptake

Yield data of all five treatments showed that different N fertilization treatments had remarkable effects on crop grain yield (P＜0.05) (Table 2).Compared with CN, LN increased the system grain yield by 5.7% in 2012 and by 3.3% in 2013.In addition, the system grain yield of LN was increased by 34.8% in 2012 and by 25.6% in 2013 compared with NN.Under different fertilization locations, crops performed better in LND1 and LND2 than that in LND3.The highest grain yield of maize and soybean was in LND1 and LND2,at 23.6 and 16.2% higher than LND3, respectively.In addition, the system grain yields of LND1 and LND2 were higher than LND3.Maize and soybean shoot N uptakes increased by 2.9 and 24.5% in LN compared with CN,respectively.Compared with LND3, shoot N uptake of maize and soybean in LND1 increased by 24.3 and 25.1%, and in LND2 increased by 14.9 and 13.6%, respectively (Table 3).

3.2.Nitrogen utilization

NAE and NUE were significantly affected by N fertilization treatments (Table 4).Compared with CN, the NAE and NUE of maize, soybean, and the relay intercropping system increased by 35.2, 98.3 and 51.6%, and 32.2, 160.4 and 72.5% in LN, respectively.The optimization of fertilization location increased the NAE and NUE of maize, soybean and the relay intercropping system by 242.6, 40.1 and 155.8%in LND1, and 77.0, 85.2 and 81.2% in LND2 compared with LND3, respectively.

3.3.Soybean nodule nitrogen fixation

The number and dry weight of soybean nodulesNodulenumber and dry weight gradually increased from the V5 to R5 stage (Fehr et al.1971) (Fig.3).Across the growth stages of soybeans, lower N fertilization promoted nodule growth.Compared with CN, the nodule number and dry weight in LND1 increased by 32.0 and 28.5% at the V5 stage, by 41.4 and 42.3% at the R2 stage, and by 47.5 and 35.8% at the R5 stage, respectively.In addition,compared with CN, the nodule number and dry weight in LND2 increased by 56.0 and 44.2% at the V5 stage, by 63.2 and 49.4% at the R2 stage, and by 40.4 and 29.1% at the R5 stage, respectively.Optimization of fertilization location promoted soybean nodule growth, and the nodule number and dry weight of LND1 and LND2 were higher compared with LND3, respectively.

Table 2 Grain yield under different N application treatments (kg ha-1)

Table 3 Shoot nitrogen uptake under different N application treatments (kg ha-1)

Nitrogen fixation and leghemoglobin contentNitrogen fixation was assessed using ARA (Fig.3).With the advancement of growth stages, ARA first increased and subsequently decreased, peaking at the R2 stage.Under various N levels, the ARA of LN was higher than CN and NN,respectively.Fertilization location significantly affected N fixation ability.Compared with LND3, the ARA of LND1 and LND2 was respectively increased by 51.6 and 16.7% at the V5 stage, by 6.2 and 15.4% at the R2 stage, and by 24.0 and 32.9% at the R5 stage.Leghemoglobin content increased with the advancement of growth stages, peaking at the R5 stage (Table 5).Under LN treatments, the leghemoglobin content of LND1 and LND2 was significantly higher than LND3, respectively.Compared with CN, leghemoglobin content in LND1 and LND2 was significantly increased by 101.1 and 55.8% at the V5 stage, and by 73.6 and 91.7%at the R2 stage, and by 63.3 and 32.3% at the R5 stage,respectively.

Ureide content and N accumulation of soybeanUreides are the dominant long-distance transport forms of N that are fixed in soybean nodules (Collier and Tegeder 2012).At different growth stages of soybean, ureide storage in soybean organs differed (Table 6).During vegetative stages, most ureides are stored in stems, while during reproductive stages, ureide was transferred from stems to pods and stored in grain.At all sampling stages, ureide content was higher in LN compared with CN and NN.Compared with LND3, ureide content of LND1 and LND2 increased by 23.1 and 22.6% in leaves at the V5 stage,by 22.2 and 11.6% in stems at the V5 stage, and by 17.1 and 23.9% in grain at the R8 stage, respectively.Across the growth stages of soybean, N accumulation of leaf was higher than in stems (Table 7).N fertilization influenced the N accumulation of soybean organs, and LN had greater N accumulation than CN and NN.Compared with LND3,N accumulation of LND1 and LND2 increased by 3.9 and 2.3% in leaves at the V5 stage, by 3.1 and 2.8% in stems at the V5 stage, and by 20.7 and 11.9% in pods at the R8 stage, respectively.

Morphological characteristics of soybean rootsHigh levels of N application suppress soybean root system growth(Table 8).The root volume, main root length, and lateral root number were higher in LN compared with CN.At the V5,R2, and R5 stages of soybean, the root volume of CN was decreased by 43.73, 29.19, and 26.35% compared with LN,respectively.Additionally, at the V5, R2, and R5 stages of soybean, the main root length and lateral root number of LN were increased by 29.29, 9.77 and11.16%, and 24.63, 20.99 and 8.43%, respectively.Optimized fertilizer application methods promoted soybean root system growth.At V5,R2 and R5 stages, the root volume, main root length and lateral root number of LND1 and LND2 were greater than those of LND3, respectively.

4.Discussion

4.1.Optimizing N management to improve soybean biological nitrogen fixation

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Fig.3 Effects of different N application treatments on nodulation and biological nitrogen fixation at different growth stages.Nitrogen application rates were 0 (NN),270 kg N ha-1 (LN; shared by maize and soybean), and 330 kg ha-1 (CN; 270 kg N ha-1 for maize and 60 kg N ha-1 for soybean).Fertilization locations under LN were 15 cm (LND1), 30 cm (LND2), and 45 cm (LND3) from maize rows, CN and NN were applied between two plants in the same planting row.V5, five fully developed trifoliate leaf stage; R2, the full bloom stage; R5, the beginning seed stage.Nodules were separately sampled at V5, R2 and R5 stages of soybean.The data are means±SE.Different letters showed significant differences between different treatments under different growth stages by least significant difference (LSD) at P＜0.05.

The present study clearly demonstrated that the maizesoybean relay intercropping system with LN treatments presented an advantage over CN treatment.Moreover,optimization of fertilization methods further contributes to this advantage.The biological nitrogen fixation of soybean can meet almost half of N demand in the lifespan (Salvagiotti et al.2008).However, the grain yield of soybean with full-N is average 11% greater than zero-N, namely, N fertilizer is necessary to increase yield when indigenous N sources are insufficient to meet the growing demand(La Menza et al.2017).Intercropping of cereals and legumes are efficient cropping systems, e.g., promoting nitrogen fixation, enhancing biodiversity,increasing and stabilizing yields, improving resource use and reducing the damage caused by diseases and pests (Awal et al.2006; Hauggaard-Nielsen et al.2008; Gao et al.2010; Ghanbari et al.2010; Wang et al.2017; Yang et al.2017).Previous studies pointed out that compared with sole cereal,intercropped cereal with legume reduces N fertilizer input through biologicalnitrogen fixation, while planting patterns and fertilization treatments significantly affect legume nitrogen fixation (Li et al.2001; Ghosh et al.2006; Salvagiotti et al.2008; Hauggaard-Nielsen et al.2009; Wu et al.2014).

Table 5 Leghemoglobin content of soybean under different nitrogen application treatments in 2013 (mg plant-1)1)

In the maize-soybean relay intercropping system, soybean was sown at the wide rows when maize was at the 12th leaf stage.The topdressing for maize was integrated with soybean base fertilizer, and strips-placed at interspecific rows.The optimized fertilizer application methods increased NUE by sharing N fertilizer.Traditionally, fertilizers are hole-placed or strip-placed near plants stems.However, fertilizer placement near to plants may not be advantageous to nutrients efficient utilization in intercropping systems.In this study, LN treatments were advantageous to the soybean root system, nodule growth, and biological nitrogen fixation.Substantially, compared with intraspecific interactions, the interspecific interactions and facilitations are more important, namely, balancing interspecific and intraspecific interactions is the key fator to maximize the nutrients use efficiency of intercropping systems (Zhang and Long 2003).Compared with fertilizers placement at the plant stems, fertilizer placed at interspecific rows can promote the interspecific interactions and facilitation (Wu et al.2014).However, if the fertilizers are placed far away from plants, the availability of nutrients is low before the roots reaching the fertilizers (Nakamura et al.2008).

After maize harvest, soybean performed better in LND1 and LND2 compared with LND3.It demonstrated the previous finding that starter N fertilizer input can influence soybean growth (Osborne and Riedell 2006).Additionally, the previous study also indicated that high N input has a negative effect on nodule nitrogen fixation (Gan et al.2002).High soil mineral N concentration is adverse to legume biological N fixation and thereby decreasing N transfer in legume-nonlegume mixtures (Thilakarathna et al.2016).Lower N input significantly increased soybean nitrogen fixation and promoted fixed N transfer (Stern 1993; Yong et al.2015).

Fertilizer-placed strips were far from the soybean rows in LN treatments, which decreased soil N concentration and mitigated the N suppression of soybean nitrogen fixation compared with CN.The variation of biological nitrogen fixation could be explained by reduced N competition in the intercropping system (Li et al.2011).Moreover, compared with NN, the increased soil N concentration in LND1 and LND2 promoted soybean root growth compared with NN, thereby enhancing the potential for N acquisition.Maize nitrogen uptake led to decreased available N in the soybean rhizosphere, which forced soybeans to fix more nitrogen from the air (Li et al.2006).It also meets the soybean starter nitrogen needs during early growth stages and effectively promoted soybean root growth (Osborne and Riedell 2006).Therefore, crops performed better when fertilizers were placed at interspecific rows in the maize-soybean relay intercropping system.Namely, lower N input with 15-30 cm between the fertilization locations and the maize rows promoted competitive nitrogen uptake and met crop N demand in the growing stage.

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4.2.Saving fertilizer and improving yield by optimizing N fertilization rates and locations

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Abuse and low efficiency of chemical N fertilizer pollute the environment.Recently,new theories and technologies for N reduction that maintain crop yield and improve NUE have been reported (Caliskan et al.2008; Constantin et al.2010; Ruan et al.2013; Gaudin et al.2015; Li et al.2017).There are two ways to reduce chemical N input: breeding N-efficient cultivars and optimizing N management strategies.N management strategies are based in part on the use of efficient cultivars.Therefore,both approaches are essential to developing sustainable agriculture.However, breeding efficient cultivars may take many years,while optimizing nutrient management can be implemented quickly.

It is well documented that excessive N input results in a reduction of maize yield(Abbasi et al.2012), yield loss may result from soil acidification.The previous study confirmed that the soil pH of maize rows significantly decreased in the no partition condition compared with a partition in a maize-soybean relay intercropping system(Wang et al.2017).In addition, soil pH significantly decreased with the increase in N applied rate (Zhao et al.2014).In this study,lower N application rate increased both crop yield and NUE, which supported previous findings by Ju et al.(2015) and Caliskan et al.(2008).Under CN treatment, maize yield loss may result from soil acidification,and soybean vigorous growth results in decreased grain yield.Under LN treatment,N topdressing for maize, and soybean N,P and K fertilizers were mixed and shared with each other.Although the N input of LN treatments decreased compared with CN,the base fertilizers of soybean that shared by maize could be another reason for the maize yield increase in LN treatments.This confirmed the previous finding that intercropped crops perform better when fertilizer is supplied at interspecific rather than intraspecific rows (Wu et al.2014).

Zhang et al.(2003) pointed out that adjusting the relative roles of interspecific and intraspecific interaction is the key factor to improved NUE and yield in intercropping systems.A recent study shows that fertilizers placed at interspecific rows can promote interspecific interaction and facilitation,and can increase yield compared with intraspecific placement in an intercropping system (Wu et al.2014).In the present study, under LN treatments, crops performed better in LND1 and LND2 than in LND3.Optimized fertilization locations further promoted root growth, improving the potential to utilize soil resources and thereby increasing resource use efficiency and crop yield.The increased distance between the fertilization locations and maize rows resulted in decreased maize yield, crop NUE, and NAE.However, soybean yield presented a unimodal curve and peaked at LND2.This suggested that the optimized fertilization location could be 15 or 30 cm, or between 15 and 30 cm, because optimized fertilization locations can alleviate interspecific nutrient competition, and can promote interspecific interaction and facilitation.Therefore, the optimized methods can increase fertilizer use efficiency and grain yield.It is well documented that the N competition ability of maize is stronger than that of soybean in maizesoybean relay intercropping system (Wang et al.2017).Interspecific competitive (Li et al.2001; Zhang et al.2011)and complementary N use (Chalk 1997; Xiao et al.2004)in cereal-legume intercropping system, decrease soybean“nitrogen suppression” effects and promote N fixation.In addition, maize root exudates increased faba bean biological nitrogen fixation by promoting nodule formation and N2fixation (Li et al.2016).Barley promoted nodule formation of pea and decreased interspecific N competition(Hauggaard-Nielsen et al.2009).In the present study, N accumulation of soybean organs in LND1 and LND2 was higher than that in other treatments, respectively, suggesting a more favorable balance between fixed atmospheric N and absorbed fertilizer N.

5.Conclusion

The results showed that the NAE, NUE, and yield of maize peaked at LND1, which were 242.60, 76.97, and 23.60%respectively higher than LND3.Similarly, the NUE of soybean peaked at LND1, was 85.23% higher than that of LND3.The NAE and yield of soybean peaked at LND2,were increased by 59.85 and 16.20% compared with LND3.Moreover, the nitrogen fixation ability of soybean was greater under LND1 and LND2.On the whole, crop performed better under LND1 and LND2 treatments.The NUE and total grain yield of the maize-soybean relay intercropping system were significantly higher in LND1 and LND2 than that in LND3 and CN, respectively.Those findings suggested that lower N application at 15-30 cm from the fertilizer application location to the maize row was optimal.This study contributes to improved nutrient management for legume-cereal relay intercropping system and helps to develop an efficient, sound agricultural system which is environmentally friendly.Nevertheless, this N application method requires assay using more legume-cereal relay intercropping systems and fertilizer types and rates.

Acknowledgements

The research was supported by the National Key Research and Development Program of China (2016YFD0300202),and the National Natural Science Foundation of China(31671625, 31271669).Thanks also go to two anonymous reviewers for their constructive comments.

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