Grain yield and N uptake of maize in response to increased plant density under reduced water and nitrogen supply conditions

Jingui Wei ,Qiang Chai # ,Wen Yin # ,Hong FanYao Guo,Falong Hu,Zhilong Fan,Qiming Wang

1 State Key Laboratory of Aridland Crop Science,Lanzhou 730070,China

2 College of Agronomy,Gansu Agricultural University,Lanzhou 730070,China

3 College of Life Sciences,Northwest Normal University,Lanzhou 730070,China

Abstract The development of modern agriculture requires the reduction of water and chemical N fertilizer inputs.Increasing the planting density can maintain higher yields,but also consumes more of these restrictive resources.However,whether an increased maize density can compensate for the negative effects of reduced water and N supply on grain yield and N uptake in the arid irrigated areas remains unknown.This study is part of a long-term positioning trial that started in 2016.A split-split plot field experiment of maize was implemented in the arid irrigated area of northwestern China in 2020 to 2021.The treatments included two irrigation levels: local conventional irrigation reduced by 20% (W1,3,240 m3 ha-1) and local conventional irrigation (W2,4,050 m3 ha-1);two N application rates: local conventional N reduced by 25% (N1,270 kg ha-1) and local conventional N (360 kg ha-1);and three planting densities: local conventional density (D1,75,000 plants ha-1),density increased by 30% (D2,97,500 plants ha-1),and density increased by 60% (D3,120,000 plants ha-1).Our results showed that the grain yield and aboveground N accumulation of maize were lower under the reduced water and N inputs,but increasing the maize density by 30% can compensate for the reductions of grain yield and aboveground N accumulation caused by the reduced water and N supply.When water was reduced while the N application rate remained unchanged,increasing the planting density by 30% enhanced grain yield by 13.9% and aboveground N accumulation by 15.3%.Under reduced water and N inputs,increasing the maize density by 30% enhanced N uptake efficiency and N partial factor productivity,and it also compensated for the N harvest index and N metabolic related enzyme activities.Compared with W2N2D1,the N uptake efficiency and N partial factor productivity increased by 28.6 and 17.6% under W1N1D2.W1N2D2 had 8.4% higher N uptake efficiency and 13.9%higher N partial factor productivity than W2N2D1.W1N2D2 improved urease activity and nitrate reductase activity by 5.4% at the R2 (blister) stage and 19.6% at the V6 (6th leaf) stage,and increased net income and the benefit:cost ratio by 22.1 and 16.7%,respectively.W1N1D2 and W1N2D2 reduced the nitrate nitrogen and ammoniacal nitrogen contents at the R6 stage in the 40-100 cm soil layer,compared with W2N2D1.In summary,increasing the planting density by 30% can compensate for the loss of grain yield and aboveground N accumulation under reduced water and N inputs.Meanwhile,increasing the maize density by 30% improved grain yield and aboveground N accumulation when water was reduced by 20% while the N application rate remained constant in arid irrigation areas.

Keywords: water and N reduction,plant density,maize,grain yield,N uptake,compensation effect

1.Introduction

N application is the most important factor driving crop production in agricultural practice (Chenetal.2018;Ahmadetal.2023).It has made a very large contribution toward ensuring food security and driving economic growth (Chenetal.2011;Fanetal.2012;Huangetal.2021).Because of the blind pursuit of high yields,the phenomenon of excessive N application is becoming more common (Gaoetal.2017;Daietal.2019).However,long-term application of massive N fertilizer rates leads to potential production and ecological risks,such as high production cost,more emissions,nitrate leaching,and production drawdown,which intensifies the contradiction between crop production and ecological protection(Erismanetal.2007;Maetal.2012;Sinclair and Rufty 2012).Meanwhile,water shortage has become a major obstacle to crop production in the arid and semi-arid regions,where poorly used irrigation water is a common source of soil environmental problems,which ultimately leads to lower N uptake efficiency (Yinetal.2015,2018;Wuetal.2021).Accordingly,it is urgent to study the technology that can achieve high N uptake efficiency with reduced water and N supply while maintaining grain yield.

In order to realize the greater grain yield and N uptake for crop production,many different field management practices have been proposed,such as the integrated management of water and N fertilizer,high planting density,improved tillage measures,and optimal cover methods (Hanetal.2015;Yinetal.2017;Yanetal.2021;MacLarenetal.2022).Among these agronomic measures,water and N management have the distinct advantage of the highly efficient utilization resources in farmland (Liuetal.2021).Previous results have shown that different irrigation quotas and N application rates had varying effects on grain yield and N uptake in the crops(Suietal.2018).The reasonable regulation of water and N can effectively improve the content of available N and enzyme activities in the soil,which ultimately plays a key role in enhancing grain yield and N uptake in the crops (Monetal.2016;Zhangetal.2017;Akhtaretal.2018).However,maize is a water-intensive crop,so insufficient irrigation leads to a reduction in dry matter accumulation,and thus diminishing N uptake (Chenetal.2014).On the one hand,under mild drought stress,increasing the N application rate enhances crop tolerance to drought,improving the N uptake capacity and yield formation (Brueck 2010;Sheshbahrehetal.2019).On the other hand,a moderate increase in N input promotes higher grain yield and N uptake in plants,and mitigates the negative impacts of water reduction supply in arid environments (Wangetal.2017;Daietal.2019).In conclusion,the reduction of water and N supply is detrimental for improving grain yield and N uptake (Gaoetal.2017;Chenetal.2018).Therefore,it is necessary to explore higher grain yield and N uptake under the reduction of water and N supply.

Increasing the density of maize is another way to increase plant competition for water and N in cropping systems,while also promoting N uptake (Duetal.2021).Many studies have demonstrated that reasonably increasing the density can mitigate the contradiction between groups and individuals of a crop,promoting the absorption of nutrient resources and N transportation(Huangetal.2013;Caoetal.2021).Lower N fertilizer input in the field management can reduce N uptake and crop growth,but higher planting density can raise the aboveground biomass yield,so that aboveground N accumulation is increased (Ciampittietal.2013;Duetal.2021).This is because densification may compensate for the negative effects of reduced N on crop productivity,thereby enhancing N uptake (Maetal.2012;Zhangetal.2019).In addition,due to interplant competition for mineral N,increasing the planting density can improve soil enzyme activity and the content of available N,decrease the loss of soil nitrate through leaching,and ultimately promote the target N uptake in agricultural systems (Hauggaardetal.2006;Fanetal.2019).Previous studies have shown the two-factor interactive effects of water,N,and planting density on grain yield and N uptake in maize,but there is no scientific basis for the compensatory effect of high density under reduced water and N inputs.Therefore,its feasibility needs to be studied further.

In this study,we carried out a field experiment with reduced water and N application rates based on the local conventional planting pattern and an increase in the planting density of maize.The objective of this study was to determine whether an increase in maize density could compensate for the negative effects of reduced irrigation water and N inputs on the reductions of grain yield and N uptake.Meanwhile,soil enzyme activities and N contents were also determined to confirm the mechanism of improved N uptake in maize in arid irrigation areas.The primary objective of this study was to explore different treatments of water,N,and density on (i) grain yield and aboveground N accumulation,(ii) N harvest index,N partial factor productivity,and N uptake efficiency and (iii) total N content,nitrate N content,ammonium N content,urease activity,nitrate reductase activity,and nitrite reductase activity in the soil.We hypothesized that increasing the planting density could compensate for the negative effects of reduced water and N supply on the lower grain yield and N uptake,while maintaining the conventional N application rate would further promote N uptake,thus improving maize productivity.

2.Materials and methods

2.1.Experimental site description

This research was conducted from April 2020 to October 2021 at the Oasis Comprehensive Agricultural Experimental Station (37°95´N,102°63´E) in Northwest China.This study is part of a long-term positioning trial that started in 2016.The field site annual average temperature is 7.2°C,the precipitation is 160 mm,and the annual potential evaporation is 2 400 mm.This area represents a typical maize ecological cultivation area,and it is also a typical oasis irrigation agricultural area.Here,severe water shortage is common and the local conventional N fertilization rate is relatively high,resulting in high production costs and potential ecological risks.The soil at this location is classified as an Aridisol (Chaietal.2014),where total N is 0.69 g kg-1,alkali-hydro N is 102.35 g kg-1,readily-available P is 26.93 mg kg-1,and readily-available K is 178.26 mg kg-1in the 0-20 cm soil layer.Rainfall and temperature during the maize growth period are shown in Fig.1.

Fig.1 Monthly precipitation and mean air temperature during the maize growth periods in 2020 and 2021 at the experiment station.

2.2.Experimental design and field management

The field experiment plots were set up in a split-split plot design with three replicates.Two irrigation levels,local conventional irrigation level (W2,4,050 m3ha-1)and a 20% reduction (W1,3,240 m3ha-1) as the main plots.Two N application rates of local conventional N(N2,360 kg ha-1) and a 25% reduction (N1,270 kg ha-1)formed the subplots.Three planting density levels,from 75,000 plants ha-1(D1) increased by 30% (D2,97,500 plants ha-1) and increased by 60% (D3,120,000 plants ha-1),represented the split-split plots.A total of 12 treatments were composed,and the plot area was 40 m2(5 m×8 m).

For the two years of the experiment in this study,the soil was plowed to a depth of about 30 cm with a moldboard plow in the previous fall for weed control.Before the subsequent sowing,the soil was fertilized,harrowed,smoothed,and compacted.Then plastic film (polyethylene film 0.01 mm thick and 120 cm wide) was placed on the soil surface in each plot.Herbicide was sprayed on the soil surface with a sprayer before covering it with a plastic film.A drone sprayed a mixture of insecticides and fungicides before the VT (tasseling) stage to control pests and diseases.The maize was planted on 20 April 2020 and 23 April 2021,and harvested on 27 September 2020 and 21 September 2021,respectively.The maize material was the variety ‘Xianyu 335’.All plots were plastic film mulched and had rows equally spaced at 40 cm.Planting density was adjusted by the plant spacings of 33,26,and 21 cm for D1,D2,and D3,respectively (Fig.2).In this experiment,only N fertilizer (urea,46.6% N) and P fertilizer (superphosphate,18% P2O5) were applied.No K fertilizer was needed,mainly because the K content was high in this area,which could meet the needs of crop growth.All the experimental plots received P fertilizers as a basal fertilizer of P2O5at 180 kg ha-1in each year.The maize received 30% of the N as base fertilizer before sowing,then 50 and 20% as topdressing at the V12 (12th leaf) and R2 (blister) stages.The irrigation regime was carried out using a drip irrigation system in which a flow meter was installed at the discharge end of the pipe to record the amount of irrigation received in each plot.The local conventional irrigation reduced by 20% (W1) was administered at rates of 720,600,720,600,and 600 m3ha-1at the V3 (3rd leaf),V6 (6th leaf),V12,VT,and R2 stages,respectively.The local conventional irrigation level (W2) was administered at rates of 900,750,900,750,and 750 m3ha-1at the V3,V6,V12,VT,and R2 stages,respectively.

Fig.2 Increased planting density tested at Wuwei Experiment Station in Northwest China.D1,local conventional density;D2,density increased by 30%;D3,density increased by 60%.

2.3.Measurements and calculations

Plant sampling and data collectionAt the R6 stage(physiological maturity) of maize,the entire aboveground portions of 10 randomly selected maize plants were collected in each treatment.The oven-drying method was used at 105°C for the first 30 min and 80°C for the subsequent duration until the sampled material reached a constant weight.After oven drying,all the sample materials were pulverized using a micro plant sample shredder (Shanghai,China),and then put into Ziplock bags for the analysis of N content.Tissue N content was measured by dry combustion using an Elementar vario MACRO cube (Elementar,Hanau,Hessen,Germany).N accumulation (NA,kg ha-1) was determined by the product of N content (NC,%) in the plant organs and corresponding dry matter accumulation (DMA),while N harvest index was calculated by dividing the grain N accumulation (NG),and N partial factor productivity(NPFP) was defined as grain yield (GY) divided by N application rate (NR) using the following formulas (Huetal.2017;Guoetal.2018):

NA=DMA×NC

NHI (%)=NG/NA×100

NPFP=GY/NR

The N uptake efficiency (NUE,kg kg-1) was calculated by dividing the N accumulation (kg ha-1) by the NR and the sum the of nitrate N and ammonium N contents (NS,kg ha-1) in the 0-60 cm soil layer before sowing (Xueetal.2019):

NUE=NA/(NR+NS)

Soil sampling and data collectionAt the V6,VT,R2,and R6 stages,soil samples were collected using a soil drill made of an iron tube (150 cm length×6.5 cm internal diameter×7.0 cm external diameter) from depths of 0-20,20-40,40-75,and 75-100 cm in each plot.All the soil samples were air-dried,sieved to 0.425 mm and stored at Ziplock bags.Total N was measured using an Elementar vario MACRO cube (Elementar,Hanau,Hessen,Germany).Soil nitrate N and ammonium N contents were measured using a continuous flow analyzer (Autoanalyser 3,Bran-Luebbe,Norderstedt,Germany) after extraction with 100 mL of 0.01 mol L-1CaCl2by shaking for 60 min.

Determination of soil urease activityAt the V6,VT,and R2 stages,5 g of soil was suspended in 1 mL of C7H8in a conical flask for each sample,and then subjected to oscillatory mixing to homogeneity.After 15 min,10 mL of 10% urea solution and 20 mL of pH=6.7 citrate buffer solution were added to each block.The mixture was shaken and incubated at 37°C for 24 h in a constanttemperature incubator.Then the solution was filtered,1 mL of filtrate was added to a 50 mL volumetric flask,and 4 mL sodium phenol solution and 3 mL sodium hypochlorite solution were added.The resultant solution was allowed to stand for 20 min,then transferred to 50 mL volumetric flasks which were filled with distilled water to the scale line.Finally,the absorbance was measured at 578 nm using a multi-mode reader (SynergyHTX,Bio Tek,America),and urease activity was calculated based on the content of NH3-N (mg g-1) released from 1 g of soil.

Determination of soil nitrate reductase and nitrite reductase activitiesAt the V6,VT,and R2 stages,soil samples from 0-40 cm below the soil surface were analyzed for nitrate reductase and nitrite reductase activities using a detection kit and following the manufacturers of protocol(Beijing Solarbio Science &Technology Co.,Ltd.,China).Absorbance (OD) was determined at the corresponding wavelength with a multi-mode reader (SynergyHTX,Bio Tek,America),and the activity was calculated according to the standard curve equation.

Grain yieldAt the R6 stage,the maize was harvested in each plot individually.These grains were sun-dried,cleaned,and weighed after threshing by a small maize sheller.Grain yield data were calculated according to a 13% moisture content.

Economic benefitsThe net income for each treatment was determined by subtracting the inputs from the gross income.The gross income was calculated based on the economic value of both the grain and straw,and grain and straw prices were determined by local market prices in every year.The total inputs included plowing and harrowing,irrigation fees,labor,seeds,fertilizers,pesticides,plastic film,and harvesting fees.

Benefit:cost ratio=Gross income/Total input

2.4.Data statistics and analysis

The IBM SPSS Statistics 19.0 (SPSS Inc.,Chicago,IL,USA) software was used for the analysis the variance(ANOVA),using the Duncan’s multiple range test at a significant level ofP-value＜0.05.All the figures were drawn using Origin 2021 (OriginLab Corporation,Northampton,MA,USA).

3.Results

3.1.Compensating effects on grain yield and N accumulation by increased density under reduced water and N supply conditions

Grain yieldAs expected,the grain yield of maize was significantly lower with a reduced water and N supply,a higher grain yield was observed when the planting density increased,and interaction effects of irrigation×N,N×density,and irrigation×N×density had significant effects on grain yield (Fig.3).The grain yield of W1 was reduced by 3.0% compared with W2.The grain yield with N1 was 12.9% less than N2.Compared with D1,D2 and D3 increased the grain yield by 12.9 and 9.2%,respectively.However,it is notable that the grain yield of W1N1D1 was 12.3% less than W2N2D1,but there was no significant difference between W1N1D2 and W2N2D1.These results showed that lower water and N significantly reduced grain yield,and densification can compensate for the loss of grain yield.Compared with W2N2D1,W1N2D2 increased grain yield by 13.9%.As a result,increasing the density by 30% was a feasible measure to save water and enhance the grain yield of maize when the water was reduced by 20% while maintaining the N application rate.

Fig.3 The grain yield in maize as affected by different water,N,and density levels.W1,local conventional irrigation reduced by 20%;W2,local conventional irrigation.N1,local conventional N reduced by 25%;N2,local conventional N.D1,local conventional density;D2,density increased by 30%;D3,density increased by 60%.Bars mean SE (n=3).Different lowercase letters above the bars mean significant differences between treatments at P＜0.05.＊,＊＊,and ns indicate statistical significances of the variance at P＜0.05,P＜0.01,and no significance,respectively.

Grain N accumulationThe reduction of water and N significantly reduced grain N accumulation,but increased the density promoted grain N accumulation,and the interactions of irrigation×density,N×density,and irrigation×N×density had significant effects on grain N accumulation (Table 1).We found that the grain N accumulation of W1 was reduced by 10.7%,and N1 was 15.5% less than N2.Among the density management systems,D2 and D3 increased grain N accumulation by 20.3 and 15.7% compared to D1.Compared with W2N2D1,grain N accumulation was reduced by 26.7% with W1N1D1,but there was no significant difference between W1N1D2 and W2N2D1.These results showed that increasing the planting density by 30% can compensate for the negative effect on grain N accumulation under reduced water and N supply.Compared with W2N2D1,grain N accumulation was increased by 14.8% when W1N2D2 was used.These results showed that increasing the planting density by 30% further promoted grain N accumulation of maize when the water was reduced while maintaining the N application rate.

Table 1 Grain N accumulation,aboveground N accumulation,N harvest index,N partial factor productivity,and N uptake efficiency as affected by the levels of water,N,and density

Aboveground N accumulationWe found that reduced water and N had negative effects on aboveground N accumulation,while improving the density had a positive effect,and the interactions of irrigation×density,N×density,and irrigation×N×density had significant effects on aboveground N accumulation (Table 1).Aboveground N accumulation was 5.5% lower in W1 than W2,and it was reduced by 8.7% in N1 compared to N2.Aboveground N accumulation was increased by 11.9 and 10.8% with D2 and D3 compared to D1.Aboveground N accumulation was reduced by 11.2%with W1N1D1,but W1N1D2 was not significantly different from W2N2D1.These results indicated that increasing the density by 30% had a positive compensating effect on aboveground N accumulation under reduced water and N inputs.Aboveground N accumulation was improved by 15.3% with W1N2D2 compared to W2N2D1.Therefore,from the perspective of reduced water and the standard N application rate,increasing the density by 30% further improved the aboveground N accumulation.

3.2.N harvest index,N partial factor productivity,and N uptake efficiency of maize in response to high planting density under reduced water and N conditions

N harvest indexAs shown in Table 1,irrigation,N,density,and the N×density interaction had significant effects on the N harvest index of maize.The N harvest index with W1 was lower by 5.6% than with W2,and N1 decreased it by 7.4% compared to N2.In terms of the density,D2 increased the N harvest index by 8.0%compared to D1.When compared with W2N2D1,the N harvest index was reduced by 17.4% for W1N1D1,but there was no significant difference between W1N1D2 and W2N2D1.Accordingly,based on the above analysis,the N harvest index was significantly lower under the reduced water and N inputs,but increasing the density by 30%effectively compensated for that negative effect.

N partial factor productivityThe ANOVA results showed that the N partial factor productivity of maize had significant effects from N,density,and N×density,but irrigation had no significant effect (Table 1).N partial factor productivity for N1 was increased by 15.9%compared to N2.Compared with D1,the N partial factor productivity was increased by 12.4 and 8.9% with D2 and D3,respectively.The N partial factor productivity values of W1N1D2 and W1N2D2 were 28.6 and 13.9% more than W2N2D1,respectively.These results suggested that increasing the planting density by 30% can improve the N partial factor productivity with the simultaneous reduction of water and N inputs.

N uptake efficiencyIrrigation,N,density,irrigation×N,and N×density had significant effects on the N uptake efficiency of maize (Table 1).W1 reduced the N uptake efficiency by 5.7% in comparison to W2,but N1 increased it by 8.9% over N2.For the density management systems,D2 and D3 increased N uptake efficiency by 9.3 and 8.3% when compared with D1,respectively.The N uptake efficiency of W1N1D2 was 17.6% higher than that of W2N2D1.Compared with W2N2D1,the N uptake efficiency was increased by 8.3% when W1N2D2 was used.These results indicated that under the simultaneous reduction of water and N,increasing the density of 30% was beneficial for improving the N uptake efficiency,and the conventional N application rate can lead to even higher N uptake efficiency.

3.3.N metabolism-related enzyme activities of soil under various water,N,and density levels

Urease activityThe urease activities in different growth stages of maize were significantly affected by irrigation,N,density levels,and the interactions of irrigation×N,irrigation×density,N×density,and irrigation×N×density(Fig.4).W1 reduced the urease activity by 6.5% in stage V6 and 5.2% in stage VT when compared with W2.Similarly,N1 reduced the urease activity by 11.7%at stage V6,9.3% at stage VT,and 5.6% at stage R2 relative to the N2.Using D2 enhanced the urease activity by 7.0% at stage V6,9.8% at stage VT,and 5.3% at stage R2 when compared with D1.Compared with W2N2D1,W1N1D1 resulted in lower urease activity by 17.2% in stage V6,14.4% in stage VT,and 5.8% in stage R2;but for W1N1D2 and W2N2D1 at the maize VT and R2 stages,no significant differences were observed.The above results showed that the reduction of water and N reduced the urease activity,but increasing the density by 30% can compensate for the negative effects on urease activity at the VT and R2 stages.Under the N2 level,W1 combined with D2 resulted in urease activity that was 5.4%higher at stage R2 compared to W2N2D1.This result further suggested that increasing the planting density by 30% effectively increased the urease activity under water reduction and a conventional N application rate.

Fig.4 The activity of soil urease as affected by the levels of water,N,and density.W1,local conventional irrigation reduced by 20%;W2,local conventional irrigation.N1,local conventional N reduced by 25%;N2,local conventional N.D1,local conventional density;D2,density increased by 30%;D3,density increased by 60%.V6,VT,and R2 stages,6th leaf,tasseling,and blister stages,respectively.Bars mean SE (n=3).Different lowercase letters indicate significant differences within one year (P＜0.05).＊,＊＊,and ns indicate statistical significances of the variance at P＜0.05,P＜0.01,and no significance,respectively.

Nitrate reductase activityAs shown in Fig.5,irrigation,N,density,and irrigation×density had significant effects on nitrate reductase activity according to three-way ANOVAs.Compared with W2,the nitrate reductase activity was 5.2% lower in stage V6 and 5.5% lower in stage VT under W1.Just like the irrigation level,when compared with N2,nitrate reductase activity was reduced by 7.2,6.5,and 7.5% at the V6,VT,and R2 stages with N1,respectively.Compared with D1,nitrate reductase activity was promoted by 7.6% at stage V6,5.5% at stage VT,and 6.4% at stage R2 with D2.In terms of the interaction effect,at the N2 level,the nitrate reductase activity of W1D1 was reduced respectively by 6.6,6.9,and 11.2% at the V6,VT,and R2 stages compared with W2D1.There was no significant difference between W1N1D2 and W2N2D1.The nitrate reductase activity of W1N2D2 was 19.6 and 5.1% higher at the V6 and VT stages than those of W2N2D1.These results suggested that increasing the density by 30% can compensate for the negative effects caused by reduced water and N inputs on nitrate reductase activity,and increasing the maize density by 30% further increased the nitrate reductase activity under the conditions of reduced water while maintaining the N application rate.

Fig.5 The activity of nitrate reductase in soil with different levels of water,N,and density.W1,local conventional irrigation reduced by 20%;W2,local conventional irrigation.N1,local conventional N reduced by 25%;N2,local conventional N.D1,local conventional density;D2,density increased by 30%;D3,density increased by 60%.V6,VT,and R2 stages,6th leaf,tasseling,and blister stages,respectively.Bars mean SE (n=3).Different lowercase letters indicate significant differences within one year(P＜0.05).＊,＊＊,and ns indicate statistical significances of the variance at P＜0.05,P＜0.01,and no significance,respectively.

Nitrite reductase activityThe nitrite reductase activity of maize had significant influences from irrigation,N,density,and irrigation×N (Fig.6).The nitrite reductase activity of W1 was depressed by 7.0% at stage V6,5.8%at stage VT,and 5.7% at stage R2 as compared with W2.The nitrite reductase activity with N1 was 6.6,6.0,and 8.6% lower than N2 at the V6,VT,and R2 stages,respectively.The nitrite reductase activity of D2 was improved by 8.4% at stage V6,7.8% at stage VT,and 6.3% at stage R2,compared with D1.As compared with W2N2D1,the nitrite reductase activity was reduced by 12.4,11.6,and 12.9% in W1N1D1 at the V6,VT,and R2 stages,respectively.Most importantly,nitrite reductase activity was not significantly different between W1N1D2 and W2N2D1.These results suggested that nitrite reductase activity was reduced by water and N reduction,but increasing the density of 30% can compensate for this negative effect.

Fig.6 The activity of soil nitrite reductase as affected by different levels of water,N,and density.W1,local conventional irrigation reduced by 20%;W2,local conventional irrigation.N1,local conventional N reduced by 25%;N2,local conventional N.D1,local conventional density;D2,density increased by 30%;D3,density increased by 60%.V6,VT,and R2 stages,6th leaf,tasseling,and blister stages,respectively.Bars mean SE (n=3).Different lowercase letters indicate significant differences within one year(P＜0.05).＊,＊＊,and ns indicate statistical significances of the variance at P＜0.05,P＜0.01,and no significance,respectively.

3.4.Soil total N,nitrate N,and ammonium N contents in the different treatments

Soil total N contentIn this study,the soil total N content was significant influenced by irrigation,N,density,irrigation×N,N×density,and irrigation×N×density(Fig.7).W1 had reductions of 5.6% at stage V6 and 5.3% at stage R2 in the total N content compared to W2 in 0-40 cm soil.At the V6,VT,R2,and R6 stages in 0-40 cm soil,N1 resulted in lower total N contents by 10.8,5.5,9.4,and 5.6%,relative to N1,respectively;and total N contents were reduced by 8.0,7.8,and 7.3% at the V6,VT,and R2 stages in 40-100 cm soil,respectively,compared with N2.The total N content of D2 was reduced by 5.9 and 5.7% at the VT stage,and by 8.0 and 7.4% at the R2 stage compared to D1 in 0-40 and 40-100 cm soil,respectively.Similarly,W1N1D2 had lower total N contents respectively by 21.7 and 12.4% at stage V6,19.9 and 17.0% at stage R2,and by 6.6 and 6.8% at stage R6 compared to W2N2D1 in the 0-40 and 40-100 cm soils.W1N2D2 reduced the total N content respectively by 14.0 and 8.6%,by 15.1 and 9.8%,and by 12.7 and 11.6% at the V6,VT,and R2 stages compared to W2N2D1 in the 0-40 and 40-100 cm soils.At stage R6,increasing the density by 30% significantly reduced the total N content under the concurrent reduction of water and N,but increasing the density by 30% showed no significant difference under reduced water and the conventional N application rate.

Fig.7 Total N content distribution in soil as affected by different levels of water,N,and density.W1,local conventional irrigation reduced by 20%;W2,local conventional irrigation.N1,local conventional N reduced by 25%;N2,local conventional N.D1,local conventional density;D2,density increased by 30%;D3,density increased by 60%.V6,VT,R2,and R6 stages,6th leaf,tasseling,blister,and physiological maturity stages,respectively.＊,＊＊,and ns indicate statistical significances of the variance at P＜0.05,P＜0.01,and no significance,respectively.

Soil nitrate N contentIrrigation,N,density,irrigation×N,and irrigation×density had significant effects on the nitrate N content (NO3--N) of maize (Fig.8).W1 reduced the NO3--N respectively by 5.0,14.3,and 11.1% at the VT,R2,and R6 stages in 40-100 cm soil compared to W2.The NO3--N with N1 was respectively 12.4,19.7,14.9,and 12.8% lower than that with N2 at the V6,VT,R2,and R6 stages in 40-100 cm soil.Compared with D1,D2 reduced it by 6.0% at stage V6,7.4% at stage VT,10.2% at stage R2,and 11.2%at stage R6 in 40-100 cm soil.The NO3--N was 9.0 and 14.2%,4.8 and 21.5%,7.4 and 25.6%,and 4.9 and 21.7%lower with W1N1D1 than W2N2D1 at the V6,VT,R2,and R6 stages in 0-40 and 40-100 cm soil,respectively.The NO3--N of W1N1D2 was 11.9 and 11.5% less than W2N2D1 at the V6 and R2 stages,respectively.Similarly,NO3--N was reduced by 18.1,22.2,34.2,and 32.9% at the V6,VT,R2,and R6 stages under W1N1D2 compared to W2N2D1 in 60-100 cm soil.For W1N2D2,NO3--N was reduced by 7.5% at stage V6,9.9% at stage VT,24.0% at stage R2,and 25.3% at stage R6 in 40-100 cm soil,compared with W2N2D1.Therefore,these results suggested that either the simultaneous reduction of water and N or the single reduction of water significantly reduced the NO3--N,and increasing the maize density by 30% further reduced the NO3--N in the 40-100 cm soil layers.

Fig.8 Soil nitrate N content distribution of maize in different water,N,and density levels.W1,local conventional irrigation reduced by 20%;W2,local conventional irrigation.N1,local conventional N reduced by 25%;N2,local conventional N.D1,local conventional density;D2,density increased by 30%;D3,density increased by 60%.V6,VT,R2,and R6 stages,6th leaf,tasseling,blister,and physiological maturity stages,respectively.＊,＊＊,and ns indicate statistical significances of the variance at P＜0.05,P＜0.01,and no significance,respectively.

Soil ammonium N contentThree-way ANOVAs showed that irrigation,N,density,irrigation×density,and irrigation×N×density had significant influences on the ammonium N content (NH4+-N) of soil (Fig.9).W1 reduced NH4+-N respectively by 8.7 and 5.0% at the V6 and R2 stages compared to W2.The NH4+-N was reduced by 9.7,12.6,11.0,and 8.1% at the V6,VT,R2,and R6 stages,respectively,in N1 compared with N2 in 40-100 cm soil.The NH4+-N was reduced respectively by 5.4 and 6.3% for D2 and D3 at the V6 stage.Meanwhile,the NH4+-N of D2 had reductions of 5.6% at stage V6,6.2%at stage VT,6.7% at stage R2,and 5.5% at stage R6 than those of D1 in 40-100 cm soil.Integrating the three test factors,compared with W2N2D1,W1N1D1 reduced NH4+-N respectively by 10.3 and 13.1% at stage V6,8.6 and 14.5% at stage VT,12.7 and 11.3% at stage R2,and 5.8 and 11.0% at stage R6 in the 0-40 and 40-100 cm soils.W1N1D2 had a lower NH4+-N by 5.1% at the VT stage than that of W2N2D1 in 0-40 cm soil;while NH4+-N was reduced by 24.1,19.4,21.7,and 14.1% at the V6,VT,R2,and R6 stages under W1N1D2 compared to W2N2D1 in 40-100 cm soil,respectively.For W1N2D2,NH4+-N was reduced respectively by 12.9,8.7,and 8.7% at the V6,R2,and R6 stages compared to W2N2D1.Overall,these results indicated that increasing the planting density by 30% under the simultaneous reduction of water and N or the reduction of only water reduced the NH4+-N in the 40-100 cm soil layers.

Fig.9 Soil ammonium N content distribution of soil under different water,N,and density levels.W1,local conventional irrigation reduced by 20%;W2,local conventional irrigation.N1,local conventional N reduced by 25%;N2,local conventional N.D1,local conventional density;D2,density increased by 30%;D3,density increased by 60%.V6,VT,R2,and R6 stages,6th leaf,tasseling,blister,and physiological maturity stages,respectively.＊,＊＊,and ns indicate statistical significances of the variance at P＜0.05,P＜0.01,and no significance,respectively.

3.5.Principal component analysis of grain yield,N accumulation,N harvest index,N uptake efficiency,N partial factor productivity,soil N,and soil N metabolic enzymes

Principal component analysis (PCA) revealed that the grain yield,N harvest index,N accumulation,urease activity,nitrate reductase activity,and nitrite reductase activity had similar loading contributions to the first axis of PC1 (Fig.10),implying that soil urease activity,nitrate reductase activity,and nitrite reductase activity had the main contributions for N accumulation and grain yield.Additionally,N uptake efficiency and N partial factor productivity had high relative contributions to the second axis of PC2,and they had strong negative correlations with total nitrogen,nitrate nitrogen,and ammonium nitrogen.These results indicated that soil N contents effectively regulated the N uptake efficiency and N partial factor productivity of maize plants.In other words,these results indicated that soil N increased the N uptake and N metabolism enzyme activities,which was beneficial for improving the N uptake efficiency and N partial factor productivity,thus further promoting N accumulation and grain yield.

Fig.10 Principal component analysis (PCA) of grain yield(GY),grain N accumulation (GNA),aboveground nitrogen accumulation (ANA),N harvest index (NHI),N uptake efficiency(NUE),N partial factor productivity (NPFP),soil N (total nitrogen,TN;nitrate nitrogen,NO3--N;ammoniacal nitrogen,NH4+-N)contents,and soil N metabolism enzymes (urease activity,UR;nitrate reductase activity,NR;nitrite reductase activity,NiR)under the different treatments.

3.6.Gross income,net income,and the benefit:cost ratio of different treatments in maize

The ANOVA results showed that the gross income and net income of maize were significantly lower with a reduced water supply (Table 2).In addition,gross income,net income,and the benefit:cost ratio of maize had significant effects from N,density,and their interaction effect.Gross income and net income with W1 resulted in reductions of 5.5 and 6.4% compared to W2.N1 reduced gross income,net income,and the benefit:cost ratio by 10.6,13.6,and 7.0% compared to N2,respectively.Gross income was increased by 13.1 and 10.3%,net income was improved by 18.7 and 13.2%,and the benefit:cost ratio was enhanced by 10.9 and 5.3% with D2 and D3 compared to D1,respectively.Compared with W2N2D1,gross income,net income,and the benefit:cost ratio were reduced by 10.9,12.4,and 4.9% when W1N1D1 was used.W1N1D2 increased the benefit:cost ratio by 4.6%,but gross income and net income showed no significant differences,compared with W2N2D1.Gross income,net income,and the benefit:cost ratio of W1N2D2 were 14.0,22.1,and16.7% higher than those of W2N2D1,respectively.These results further suggested that under reduced water and N inputs,increasing the planting density by 30% can compensate for the negative effects on gross income and net income,and the benefit:cost ratio was promoted.Meanwhile,increasing the planting density by 30% further improved the gross income,net income,and benefit:cost ratio when the water was reduced while maintaining the N application rate.

Table 2 Gross income,net income,and benefit:cost ratio in the different treatments

4.Discussion

4.1.Grain yield and aboveground N accumulation in maize were compensated by increased density under reduced water and N inputs

Water shortages and lower N use efficiency have become the primary stress factors for crop production,and they are not conducive to the improvement of grain yield and N uptake (Lietal.2022;Ibrahimetal.2013).The water deficit can depress the accumulation of nutrients in crops,and the lack of nutrients may also affect water availability,both of which lead to lower grain yield (Brueck 2010;Wangetal.2017;Daietal.2019).The results of this study are consistent with those findings,in that grain yield was significantly lower under reduced water and N inputs.Planting density is another dominant factor in the grain yield of maize (Duetal.2021).Moderate densification leads to competition among plants for limited resources such as water and nutrients,and reinforces the balance of the synergistic benefits between population structure and individual functions (Li and Wang 2010;Huangetal.2017).Additionally,interaction effects between ecological factors,such as water and N,as well as N and density,are effective measures for increasing crop yield (Suietal.2018;Ahmadetal.2023).Also,the densification effect is mainly driven through the interception of more solar radiation,which makes the population productivity higher and increases production (Ciampitti and Vyn 2011;Huangetal.2017).However,excess planting density causes mutual shading among adjacent plants,and limits the interception and utilization efficiency of light energy for a single plant,which decreases dry matter accumulation,and the plants can even show signs of premature aging,thereby reducing grain yield (Sheshbahrehetal.2019;Guoetal.2021).In this study,increasing the density by 30% showed better performance over increasing the density by 60% or the local conventional density.Meanwhile,increasing the maize density by 30%compensated for the negative effects of water and N input reductions on grain yield.The appropriate N application rate could increase the amount of active oxygen scavenging enzymes synthesized by the plants,maintain the photosynthetic performance of leaves at a higher level,provide sufficient organic matter for grain formation,and ultimately increase the yield of maize (Sheshbahrehetal.2019;Liuetal.2021),thereby strengthening the synergistic effect between density and N application to achieve the purpose of water saving.

The results demonstrated that aboveground N accumulation in maize was significantly lower with the reduced water and N supply,and increased planting density significantly improved the aboveground N accumulation.Most importantly,increasing the planting density by 30% can relieve the negative effects of reduced water and N supply on aboveground N accumulation.Increasing the density by 30% was more conducive to N accumulation under reduced water and the local conventional N application rate.The main reasons include several aspects.(i) The reduction in water or N application continuously depressed the aboveground N accumulation,and increasing the planting density played a positive role in the reduction of N accumulation,which ensured the utilization of available N and promoted N accumulation in maize (Zhuetal.2016,2021).(ii)Increasing the planting density contributed to enhanced root viability,thus maintaining better N metabolism and promoting soil N absorption by the crops (Brayetal.2020).(iii) A high planting density could absorb enough water and nutrients from the soil in early period,which would lay the foundation for aboveground N accumulation in plants in the reproductive growth period (Duetal.2021).(iv) A lower water and N supply reduced the accumulation of N for the aboveground organs of plants,and the moderate densification improved N accumulation for the population of plants,which ultimately led to the compensation of the N accumulation (Yanetal.2021).(v)Under the mild water deficit,a coordinated relationship between N fertilizer and density can be used to meet the growth and development requirements of maize,thereby collaborating to achieve improved N accumulation in plants (Wangetal.2017;Daietal.2019).

4.2.Compensation effect of densification on N uptake efficiency in different water and N reduction management regimes

The N accumulation potential of plants is related to N transport and uptake efficiency,which lays the foundation for high efficiency N uptake by the crops (Zhaoetal.2021;Qi and Pan 2022).In addition,the N source in the kernel organ is N transportation and remobilization from the vegetative organs (Jingetal.2013;Kongetal.2016).At a lower water and N supply,N accumulation is limited for plants,resulting in insufficient transportation from vegetative organs to reproductive organs and lower N uptake efficiency (Wangetal.2012;Wangetal.2021).However,reasonable densification effectively promotes the use of more nutrients,thereby improving N accumulation in the early growth period and increasing N transportation for grain at the later growth period(Chengetal.2015;Wangetal.2020).On the one hand,the spike number per unit area increases with an increase in planting density to a certain extent,so that indirectly improves the N uptake (Duetal.2021;Ahmadetal.2023).On the other hand,the increased density enhances root activities and root exudates,improving microbial richness and diversity index values,and promoting nutrient cycling and crop uptake,thus indirectly boosting N uptake (Coskunetal.2017;Chenetal.2021).In addition,under high density,the two uppermost leaves could enhance the light intensity,but this reduces the net photosynthetic rate of the ear leaves and the amount of the photosynthetically active radiation reaching the lower leaves,thus indirectly decreasing N uptake (Caoetal.2021;Guoetal.2021).Consistent with these findings,the present study revealed that increasing the density by 30 and 60% increased uptake efficiency by 9.3 and 8.3%,respectively,compared with the local conventional density.Under the simultaneous reduction of water and N inputs,increasing the density by 30% improved N uptake efficiency by 17.6% and enhanced N partial factor productivity by 28.6%,compared with local conventional water,N,and density levels.N stress depresses N uptake efficiency,and many studies have shown that maintaining a higher N fertilization rate improves N uptake efficiency (Teixeiraetal.2014;Chengetal.2015;Huetal.2017).In accordance with this observation,increasing the planting density by 30% is a feasible measure for achieving higher N uptake efficiency,when water is reduced while maintaining the N application rate in arid irrigation regions.

4.3.Effect of densification on soil N and soil N metabolism-related enzyme activities with reductions in the water and N supply

Different agronomic cultivation practices have diverse effects on N formation,content,and distribution in the soil (Maetal.1999).Previous studies have shown that water and N play important roles in soil N cycling,promoting N mineralization and nitrification processes to produce mineral N,and regulating the contents of nitrate N and ammonium N in soil,and thus promoting N uptake by the crops (Murphyetal.2003;Kuypersetal.2018).Reasonable water and N inputs can improve soil physical and chemical properties and increase the N fertilizer holding capacity of soil,which lays the foundation for absorption and utilization by the crop to maintain its viability (Shisanyaetal.2009;Zhangetal.2017;Liuetal.2018).However,mild water deficit enhances the extension of plant roots to deep soil,which improves the potential absorption of available N (Coskunetal.2017;Kenobietal.2017).Also,the lower soil water content could lead to more nitrate being retained in the shallow soil,which further reduces N loss (Erismanetal.2007;Colmer and Pedersen 2008).Nonetheless,under reduced water or N supply,markedly depressed crop utilization of soil available N and increased planting density could improve the utilization of mineral N by the plant population,which would further increase N uptake in the aboveground organs (Colmer and Pedersen 2008;Lietal.2017;Yanetal.2017).Additionally,higher density can be used to enhance interspecific competition stimulated gross mineralization of labile organic N in sparse crops,which could potentially reduce the N losses from the system at the cost of minimal recovery (Corre-Hellouetal.2006;Ciampitti and Vyn 2011;MacLarenetal.2022).The results of our study showed that the nitrate N and ammonium N contents were significantly lower when water and N were reduced.Increasing the maize density by 30% further reduced the nitrate N and ammonium N contents under reduced water and N supply in the 40-100 cm soil at the R6 stage.Meanwhile,increasing the planting density by 30% significantly diminished the nitrate N content of the R6 stage when the water was reduced by 20% while maintaining the N application rate in the 40-100 cm soil.

Soil N metabolic enzymes play an important role in soil biochemical processes and nitrogen cycles,and they are also the main indicator for reflecting the utilization efficiency of soil nitrogen (Chenetal.2018).Urease,nitrate reductase,and nitrite reductase are indispensable enzymes in soil N transfer,and their activities are closely related to indicators of soil quality and fertility,thus indirectly impacting N uptake by crops (Chenetal.2018;Koyamaetal.2020).The results of this experiment showed that the urease,nitrate reductase,and nitrite reductase activities were significantly lower under a reduced water and N supply,but increasing the maize density can compensate for this negative effect.The main reasons for these results were that: (i) The reduced supply of water and N could significantly decrease root exudates and suppress soil microbial activity (Lietal.2010);(ii) increased maize density can promote soil mineral N uptake into plant roots,improve soil N conversion,and enriche the structure and abundance of the soil microbial community,thereby enhancing soil N metabolism enzyme activities (Daietal.2013),and (iii)the mild water stress reduces the soil enzyme activities,but increasing the planting density promotes nutrient absorption by the crop roots and improves soil microbial activity and reproduction,which further enhances the soil enzyme activities (Muhammadetal.2022;Yangetal.2022).Furthermore,the adequate N supply promotes the growth and development of maize roots,increases the release of root exudates,and improves the reproductive ability of microorganisms,thus enhancing soil enzyme activity (Barabásetal.2000;Rockeletal.2002).Meanwhile,previous studies have shown that N and density have synergistic effects,improving the activity of the maize root system and its exudates,and sufficient substrate stimulates soil microbial activities,which could promote the enzymatic activities in the soil (Barabásetal.2000;Sheshbahrehetal.2019;Zhaoetal.2020).Therefore,under the simultaneous reduction of water and N,increasing the density by 30% was beneficial for alleviating the ecological risks.When water was reduced while maintaining the conventional N application rate,increasing the density by 30% could not only promote grain yield and N uptake but also save water resources in arid irrigated areas.

5.Conclusion

In the arid irrigated area,the reduction of water and N inputs lowered the grain yield and aboveground N accumulation of maize,but increasing the maize density by 30% can compensate for this negative effect.W1N1D2 improved N partial factor productivity by 28.6%and N uptake efficiency by 17.6%.Meanwhile,W1N1D2 compensated the N harvest index,soil N metabolic enzyme activities,and economic benefits.Increasing the planting density by 30% enhanced the grain yield and aboveground N accumulation by 13.9 and 15.3%,respectively,when water was reduced while maintaining the N application rate.Additionally,W1N2D2 enhanced N partial factor productivity and N uptake efficiency by 13.9 and 8.4%,improved urease and nitrate reductase activities by 5.4% at the R2 stage and 19.6% at the V6 stage,and promoted net income and the benefit:cost ratio by 22.1 and 16.7%,respectively,as compared with W2N2D1.Both W1N1D2 and W1N2D2 reduced the nitrate N and ammonium N contents compared to W2N2D1 in the 40-100 cm soil at the R6 stage.Therefore,increasing the maize density by 30% can be regarded as a viable option for increasing grain yield and aboveground N accumulation,which can not only increase the efficient utilization of resources,but also improve economic benefits in the arid irrigated regions.

Acknowledgements

We are very grateful for financial support of the National Natural Science Foundation of China (U21A20218 and 32101857),the ‘Double First-Class’ Key Scientific Research Project of Education Department in Gansu Province,China (GSSYLXM-02),the Fuxi Young Talents Fund of Gansu Agricultural University,China (Gaufx-03Y10),and the “Innovation Star” Program of Graduate Students in 2023 of Gansu Province,China (2023CXZX-681).

Declaration of competing interest

The authors declare that they have no conflict of interest.