Estimation of Critical Nitrogen Concentration Based on Leaf Dry Matter in Drip Irrigation Spring Maize Production in Northern China

Background: The application of nitrogen (N) fertilizer not only increases crop yield but also improves the N utilization eciency. The critical N concentration (N c ) can be used to diagnose crops N nutritional status. The N c dilution curve model of maize was calibrated with leaf dry matter (LDM) as the indicator, and the performance of the model for diagnosing maize N nutritional status was further evaluated. Three eld experiments were carried out in two sites between 2018 and 2020 in Ningxia Hui Autonomous Region with a series of N levels (application of N from 0 to 450 kg N ha -1 ). Two spring maize cultivars, i.e., Tianci19 (TC19) and Ningdan19 (ND19), were utilized in the eld experiment. Results: The results showed that a negative power function relationship existed between LDM and leaf N concentration (LNC) for spring maize under drip irrigation. The N c dilution curve equation was divided into two parts: when the LDM < 1.11 t ha -1 , the constant leaf N c value was 3.25%; and when LDM > 1.11 t ha -1 , the N c curve was 3.33*LDM -0.24 . Conclusion: The LDM based N c curve can well distinguish data the N-limiting and non-N-limiting N status of maize, which was independent with maize varieties, growing seasons and stages. Additionally, the N nutrition index (NNI) had a signicant linear correlation with the relative leaf dry matter (RLDM). This study revealed that the LDM based N c dilution curve could accurately identify spring maize N status under drip irrigation. NNI can thus, be used as a robust and reliable tool to diagnose N nutritional status of maize.


Introduction
Nitrogen (N) is a key element for maintaining crop growth [1]. Under N de ciency, an increase of N application can dramatically stimulate the growth of crops and maintain a good grain yield [2]. However, the applied N in the soil cannot be fully absorbed by the crop, and consequently, about 60% of the N fertilizer is lost through ammoniation, nitri cation, denitri cation, leaching, and runoff [3]. The N utilization e ciency has become an important factor restricting sustainable development of agricultural production, since N utilization e ciency is generally low as a result of excessive N application for maintaining high grain yield [4,5]. The major challenge is how to achieve optimum water-fertilizer management, and obtain a continuous increase in maize production and e ciency in intensive planting [6,7]. At different growth stages of maize, optimizing its N application rate is especially helpful to further improve maize yield and quality [8,9].
Diagnosis of N nutrition in crops mainly includes the measurement of chlorophyll using chlorophyll meter [10], spectral diagnosis [11], remote sensors [12], as well as image processing [13]. One of the common disadvantages of these methods is that there are large variations in the results obtained especially under luxury absorption [14,15]. Greenwood et al. (1991) [15] summarized the characteristics of crop growth and N absorption and proposed the concept of critical N concentration (N c ), which is the minimum N required for the maximum crop growth. N c has received signi cant attention from numerous researchers related to reliable N diagnosis with su cient accuracy, and can thus, be used as an index to evaluate the crop N nutritional status of crops. Plant dry matter (PDM) based N c dilution curves have been successfully constructed and applied in rice [14,16,17], wheat [18,19], and other crops [20]. However, some previous reports show that N c dilution curves may vary with environmental conditions, crops, and varieties [21,22]. Therefore, a robust N c dilution curve is especially important for diagnosing N requirements during the growth of speci c crops.
Maize (Zea mays L.) is a globally main crop. Growth and yield of maize are signi cantly affected by the application of fertilizers, especially for N. Previous studies have reported calibrated N c curves model for maize in different regions using plant dry matter (PDM) [21,23]. Leaves are the main tissues and organs of maize, while the N c dilution curves model is also varied with tissues and organs [24]. Besides, leaves are extremely sensitive to N nutritional status. For instance, the leaf dry matter (LDM) and N of maize generally increase with increased N application. However, when N was applied above a certain dosage, LDM does not increase but the N concentration continues to increase [22]. With maize growth, the dry matter accumulation in leaves increases, but the N concentration decreases [22]. Therefore, the leaves N concentration (LNC) can be used as an important index for evaluating the status of crop growth. For instance, LDM based N c curve has been successfully established and applied in several main crops, such as rice [25], and winter wheat [26]. However, LDM based N c curve model in maize has not been calibrated under the integrated technology of water and fertilizer. Therefore, this study aimed (1) to calibrate and validate LDM based N c dilution curve in maize, (2) to compare the validated model with other crops N c curve models and to evaluate its reliability in spring maize, and (3) to offer a novel method for precisely managing N fertilizer application in spring maize cultivated under drip irrigation conditions.

Field experiment design:
Three experiments were carried out in two experimental sites, Pingjipu (38°25′N, 106°1′E) and Yongning  Table 1, including sowing and harvesting date information. The integrated technology of water and fertilizer was adopted in eld experiments. The total irrigation amount applied during the maize growing season in each plot was 400 mm, including 20, 100, 140, 120, and 20 mm at the seedling, jointing-bell, tasseling-silk, lling, and maturity stage, respectively. Urea dissolved in drip irrigation was applied as N fertilizer, with 10%, 45%, 20%, and 25% of the N applied at seedling, jointing-bell, tasseling-silk, and lling stage, respectively. And 138 kg potassium dihydrogen (P 2 O 5 ha −1 ) and 120 kg potassium sulfate (K 2 O ha −1 ) were applied as the phosphorus and potassium fertilizer for each plot. Other management followed the practices of local farmers.

Sampling and measurement
Three spring maize plants with uniform growth were randomly selected from each plot at the fourth leaf collar (V4), the sixth leaf collar (V6), the tenth leaf collar (V10), the twelfth leaf collar (V12), tasseling stage (VT) and silking stage (R1). The plants were separated into stems and leaves. Plant tissues were oven-dried at 105℃ for 30 min and then at 80℃ until reaching a constant weight. All sampled leaves were weighted to calculate the LDM and stored in hermetic bags for the downstream chemical analysis. The leaves N content was measured by using the micro-Kjeldahl method [27].

Critical Nitrogen Dilution Curve
The critical nitrogen dilution (N c ) was calculated following the method reported by Justes et al. [28]. A typical power-law function was adopted to regress the correlation between N c and LDM. The two-year data (i.e., experiment 1 and 2) were used to calibrate the N c model for the two spring maize cultivars. The calibrated curve model was further validated by an independent dataset from experiment 3. N c was derived from the equation described by Plénet and Lemaire [29]: Where N c means the N c concentration concentration (%), LDM represents the leaf dry matter (t ha −1 ), a indicates the N c concentration concentration when LDM equalts to 1 t ha −1 , while b is the statistical parameter controlling the slope of the curve, representing the ratio of the relative dry matter accumulation rate to the relative N content accumulation rate.

Nitrogen Nutrition Index
The N nutrition index (NNI) was derived from the equation reported by Plénet and Lemaire [29]: NNI = LNC/N c 2 NNI = 1, > 1, and < 1 mean plant N nutritional status is optimal, excessive, and de cient, respectively.

Relative Ldm
Relative LDM (RLDM) was calculated by dividing the LDM to the maximum LDM at each growth stage.
The following equation was used: where RLDM i is the relative leaf dry matter within growth stage i, LDM i is the measured leaf dry matter within growth stage i (t ha −1 ), and LDM max represents the maximum leaf dry matter at each growth stage (t ha −1 ).

Statistical Analysis
ANOVA was used to analyze the signi cant difference between LDM and LNC in different nitrogen fertilizer application rates. Means were determined using the least signi cant difference at the P<0.05 level and analytical data were classi ed to the N-limiting and non-N-limiting group. The non-linear relationship between NNI and RLDM were tted using Origin 2018 (Origin Lab Corporation, USA).

Results
The dynamic change in the status of both spring maize cultivar's LDM and LNC The leaf dry matter (LDM) of both spring maize cultivars increased as the growth stage increased in all the six N fertilizer application rates. The increase followed an S-shaped pattern (Fig. 1). The LDM of TC19 and ND19 ranged from 0.32 to 4.06 t ha −1 (Fig. 1a) and 0.23 to 3.73 t ha −1 (Fig. 1b) in 2018, and from 0.39 to 4.30 t ha −1 (Fig. 1c) and 0.34 to 3.97 t ha −1 (Fig. 1d) in 2019, respectively. Signi cant differences in LDM were observed from N0 to N3 rates in both 2018 and 2019. However, no signi cant differences in LDM of both maize cultivars were observed between N4 and N5 rates.
The leaves nitrogen content (LNC) of both spring maize cultivars also increased as the growth stage increased in all the six N fertilizer application rates. In general, for all the crop growth stages, decrease of LNC accompanied by an increase in LDM (Fig. 2). The LNC of TC19 and ND19 ranged between 1.38-3.74% and 1.85-3.53% in 2018 (Fig. 2a, b), and between 1.42-3.61% and 1.43-3.48% in 2019 (Fig. 2c,  d), respectively. The LNC of the same cultivar had a similar variation tendency.

Calibration Of N Curves
The N c values were calculated from V6 to R1 for the two spring cultivars (TC19 and ND19) using experimental data 1and 2 between 2018 and 2019. The N c dilution curves for the two spring cultivars (TC19 and ND19) were calibrated using twenty data points (LDM range from 1.11 to 4.25 t ha −1 ), during the vegetative growth stages of spring maize (Fig. 3a). The N c values decreased with an increase in LDM.
TC19: N c =3.41LDM −0.22 (4) ND19: N c =3.28 LDM −0.27 (5) The coe cients of the two N c curves for TC19 and ND19 (Fig. 3a) were analyzed based on the method described by Mead and Curnow [30]. We did not observe a signi cant difference (t slope = 0.548< t (0.05, 20) = 2.086, and t intercept = 0.435 < t (0.05, 20) = 2.086) at the 95% con dence level. Therefore, the N c curves of the two spring cultivars were combined and tted to obtain the uni ed N c curve of maize under drip-irrigated (Fig. 3b). However, we could not obtain a robust N c curve the early growth stages (V4) of spring maize, since LNC does not change signi cantly with the increase of the low LDM. There is no obvious competition between plants' utilization of water, fertilizer, light, and other resources. When LDM increases, LNC does not be signi cantly reduced. The low LDM data were acquired in the early growth stage of maize when the plants were very small. In this study, the N c curves were not applied to the early growth of maize. Instead, a minimum LNC value (3.31%) was de ned for the non-N-limiting group, while a maximum LNC value (3.19%) for the N-limiting groups. The LNC value of 3.25% was de ned as the constant leaf N c value for the early growth stage of maize (Fig. 3b), when LDM < 1.11 t ha −1 (Fig. 3b).
Therefore, the N c curve can be described as follows:

Validation Of The N Curve
The LDM based N c model was validated using data from a one-year independent experiment 3 in two spring maize cultivars (TC19 and ND19), in 2020 (n=72) (Fig. 4). The results showed that the non-Nlimiting and N-limiting group data were located above and below the N c curve, respectively. The N c curve effectively distinguished the N-limiting from the non-N-limiting groups in the independent experimental data and was not affected by the growing seasons, growth stages, and cultivars. Therefore, the calibrated LDM based N c curve in this study can be applied for evaluating and diagnosing the N nutritional status of spring maize.

Changes Of Nni Values Under Various N Levels
There was substantial variation in NNI between different N levels, cultivars growing seasons and stages (Fig. 5). NNI increased with an increase of N application. NNI values of TC19 and ND19 ranged between 0.61 to 1.45 and 0.60 to 1.32, respectively (Fig. 5a, b) in 2018, and between 0.55 to 1.29 and 0.62 to 1.37 (Fig. 5c, d) in 2019, respectively. Furthermore, the NNI values were N0, N1, and N2 rates less than 1.0 in 2018 and 2019, which indicated that LNC was low and that the N fertilizer application rates were insu cient. However, the NNI values in N4 and N5 rates in 2018 and 2019 were greater than 1.0, which indicated that LNC was high and that the N fertilizer rates were excessively applied. NNI values in the N3 rate were around 1.0, which indicated the N application dose of 270 kg ha −1 not only ensured the accumulation of LDM but also prevented N extravagant absorption.

Relationships Between Nni And Rldm
The correlation between NNI and RLDM was studied with data from experiments 1 and 2, obtained in 2018 and 2019. NNI and RLDM showed a signi cant linear correlation at different growth stages, and RLDM increased with an increase in NNI (Fig. 6). The coe cients of determination from V4 to R1 were 0.84, 0.89, 0.83, 0.87, 0.65 and 0.67, respectively.

Critical Nitrogen Dilution Curves Compared with Other Crops
Precise estimation of crops N nutritional status is essential for improving their N utilization e ciency. The N c has been broadly utilized for diagnosing crops N nutritional status [31]. In the present study, the N c value in the vegetative growth stage of spring maize gradually decreased with increased LDM (Fig. 3a), and this is consistent with that reported in rice (either the whole plant or the speci c organ) [14,25] and wheat [18,26]. The LNC based N c dilution curve of spring maize under drip irrigation consisted of two parts: when LDM < 1.11 t ha −1 , the N c value was de ned as 3.25%, and when LDM ≥ 1.11 t ha −1 , the N c curve was de ned as 3.33*LDM −0.24 (Fig. 3b). Due to the little individual plants and minimal dry matter accumulation in leaves before the early growth stages (V4) of spring maize, there was no obvious competition for water, fertilizer, light, and other resources. The increase in LDM did not signi cantly reduce LNC. Therefore, the leaf N c value was relatively stable in the early growth stage of spring maize.
After the jointing stage (V6), leaf area index and leaf number increased, stalk growth occurred due to the phenomenon of shading, and this led to N dilution [32].
From a mathematical view, parameter a means the LNC when LDM equals to 1.0 t ha −1 and represents the inherent N demand characteristics of the spring maize at the early growth stage. Parameter b indicates the change in LNC with LDM, and its value is determined by the proportional ratio between leaf N absorption and dry matter. In the present study, the value of parameter b (0.24) was less than that of the N c dilution curve based on PDM for maize [33] (Fig. 3b). Low parameter b means that the leaf N dilution process is slow. The differences in PDM and LDM curves were mainly re ected in the calibrated model. Based on the N c curve determined by LDM, the leaves were regarded as the central locations of crop growth. The N absorbed by the crop met the leaf growth, photosynthesis, and respiration needs, and led to a slow decline in LNC [34,35]. For PDM based N c curve, the stem N concentration was lower than that of leaf during the vegetative growth stages, while the dilution rate was lower than that of the stem [21], which was related to the leaf being the main organ of photosynthesis, and requires a certain N concentration to ensure e cient photosynthesis and yield formation [36]. Additionally, the N c dilution curve b value of spring maize leaves under drip irrigation was higher than the reported N c dilution curve b value of winter wheat and rice leaves (0.15, 0.22) [25,26] (Fig. 7). The b value of N c curve varies with crop types.

Application And Feasibility Analysis Of Lnc Based N Curve
The main purpose of calibrating the N c dilution curve was to assess the N nutritional status of maize through agronomic research methods in this study. This scienti c N application technology can reduce the cost of production and anthropogenic N pollution [37]. The N c curve provides an alternative to assess the N nutritional status in the growth stage of maize under drip irrigation. Previous studies indicate that only the LDM and LNC of the maize can re ect its N nutritional status using the N c curve. For example, when LNC values were on the N c curve, which indicated that the N application rate was optimum; LNC values ranged between N min and N c , indicating a de ciency in the N application rate. LNC values were between N c and N max , which indicated that excessive N was applied. The calibrated N c curve can be used to distinguish the N de ciency from the surplus states of crops, and the Crop-Syst model [38] is used to calculate the N demand with the assistance of the N c curve [39].
NNI can serve as an ideal indicator to diagnose N nutritional status of crops [22]. In this study, the NNI values of leaves in the two spring maize cultivars ranged between 0.55 and 1.45 under different N rates (Fig. 5). The NNI value per N rate was lowest in V12, which increased with an increase in N fertilizer application rate from V12 to R1 stage. This was associated with the vigorous spring maize growth and development in V12 to R1 stage, the high absolute and relative quantities of nutrients required by maize, and the high absorption rate during this stage [40]. Secondly, the NNI was recovered in R1 (Fig. 5), which was linked to the N status of spring maize converting from vegetative growth to reproductive growth stage, and the demand for N fertilizer was not as urgent as that in V12 (Fig. 5). This indicates that the N status of spring maize can be affected by the amount of N fertilizer application at different fertilization periods, which NNI can con rm, and this was in line with the previous reports [41]. This conclusion could be adopted to obtain a quantitative NNI and to diagnose N de ciency in spring maize with su cient e ciency and effectiveness.
The N c dilution curve of crops are calibrated under different N rates in most studies, and other conditions are found to be relatively suitable. However, crops are subjected to stress caused by different factors in real agricultural production. For example, severe drought stress severely affected the nal yield of spring wheat during the growth stage in Northern China [42]. Previous studies show that N c dilution curves and NNI values of crops under drought stress are lower than normal [43]. This can be associated with the lack of water, which limits plant growth and causes a reduction in dry matter, thus caused a decrease of N accumulation. Besides, because water content has a close relationship with the N availability, water de ciency reduces the bio-available N in soils, thus caused a decrease in the soil N supply capacity [44]. Therefore, the crop N de ciency was seen when NNI was lower than 1.0, and topdressing with N was needed. Besides water, the interaction between other elements such as phosphorus and potassium with N Therefore, the N3 rate (270 kg ha −1 ) may be used as the reference of N application for spring maize under drip irrigation, and water and fertilizer integration in Northwest China.

Declarations
The authors are grateful to the members of Ma Kun's laboratory for constructive suggestions. We also appreciate the editor and reviewers for their valuable suggestions and comments to improve this scienti c study.
Authors' contributions BJ conceived the project and co-wrote the manuscript. JPF, HFL and ZHL collected training data, models and co-wrote the manuscript. YL, XW,YQZ, JZM and HZ collected training data and models. All authors read and approved the nal manuscript.

Funding
The nancial support of the Natural Science Foundation of Ningxia (2020AAC02012, 2021AAC03025) is acknowledged.
Ethics approval and consent to participate  Validation of the established leaf Nc dilution curve. The data were collected from an independent experiment using two maize cultivars (TC19 and ND19), which was conducted in 2020.

Figure 6
The correlation between NNI with relative leaf dry matter (RLDM) for two maize cultivars (TC19 and ND19) at various growth stages (V4 to R1) during the 2018 and 2019 growing seasons. (a: V4, V6, and V10 stages of maize; b: V12, VT and R1 stages of maize).