1Title:

2 Screening of wheat varieties for yield performance and nitrogen assimilation under

3 different N levels in calcareous soil

4Abstract

5 The correlation between nitrogen (N) application and yield of different wheat varieties
6was determined at the farms of University of Agriculture Peshawar. Three different wheat
7varieties viz Pirsabaq 2103, Galaxy and Insaf were grown at five different N levels viz 0, 40, 80,
8120 and 160 kg N ha-1 under randomized complete block design with three replications in split
9plot arrangements. Mineral fertilizers such as Urea source was used for nitrogen (N), DAP
10source used for phosphorus (P) and SOP source used for potassium (K). Plant growth and yield
11as well as soil and plant N at different growth stages and its uptake by wheat significantly
12increased with N application. However, maximum 1000-grain weight, grains per spike, total
13biomass and straw yield was recorded at 80 kg N ha-1 whereas maximum grain yield was
14observed 120 kg N ha-1 when the data was averaged across the varieties. Insaf was the tallest
15variety with more straw yield whereas Galaxy was shortest with minimum straw yield. Pirsabaq
162013 superseded other varieties in term of grain weight, grains per spike and grain yield. Straw
17and grain [N] and as well as total uptake of N by wheat linearly increased with each increment of
18N level and showed correlation with soil N recorded at tillering, boot and grain filling stages.
19However, soil and plant N did not show significant correlation with wheat yield. This study
20indicated that owing to different N requirement and difference in yield potential a generalized
21correlation among the soil and plant N with yield potential could not be established.

22Keywords: Chlorophyll content, Grain Yield, Nitrogen concentration in soil and plant, Wheat
23varieties.

24Introduction

25 Nitrogen is one of the macro nutrients required by plants but mostly deficient in soil
26throughout the world and can be supplemented from fertilizers to the soil and also it can be also
27used as foliar to the leaves epidermis (Habtegebrial et al., 2007; Ran et al., 2016; Wang and

1
28Xing, 2017; Li et al., 2020a, Li et al., 2020b; Qi et al., 2020) which enhances vegetative growth
29and biomass (Xia et al., 2017; Luo et al., 2018; Zhang et al., 2019; Ogola et al., 2002). Being an
30integral constituent of chlorophyll, enzymes and proteins, its optimum supply favors the
31transformation of carbohydrates into proteins (Galloway et al., 2004). The succulence of
32protoplasm in cell is enhanced by nitrogen and hence provides the plant with succulence and
33turgidity as well dark green color (Xu et al., 2016). Maximum supplementation of N to plant
34causes the plant more susceptible to lodging, prolong vegetative growth and ultimately delay the
35maturity (Elayaraja and Singaravel, 2017). Labor cost increases when lodging in cereals occur
36with indiscriminate use of N fertilizers. It also contaminates ground water when used excessively
37(Hopkins et al., 2008). To fulfill the requirement of the crop and to avoid losses through leaching
38and volatilization proper management is necessary (Le Gouis et al., 2007). There must be a
39match in applying nitrogen fertilizer to the crop demand (Bijay et al., 2002).

40 It is unfortunate that the world soil is deficient in N, in the complete absence of which the
41production of crop is impossible (Zhang et al., 2019). In emerging agriculture production N
42fertilization is a common adoptable strategy for achieving the demandable production. In wheat
43the amount and time of N fertilization significantly affect grain yield (Sohail et al., 2013).
44Nitrogen in tillering stage enhances the green color and in late anthesis stage significantly
45improves the quality of wheat grains and protein content (Abedi et al., 2010). It is obvious from
46literature that about 70% of urea fertilization is not significantly utilizes by the cereals. The
47applied efficiency of cereals is 33%, the remaining N may be loss to the atmosphere through
48gaseous emission (volatilization), soil de-nitrification and leaching, (Matson et al., 1998; ICID,
49“International Commission on Irrigation and Drainage” 2018) and (Riley et al., 2001; Liu et al.,
502013; Abalos et al., 2014). The availability of the applied N and potential of the crop to take the
51fertilized N is the prime determinant of crop yield (Rothstein, 2007).

52 Green Revolution has two salient features, fertilization and dwarf genotype (Hirel et al.,
532007). For 2% increase in grain yield there is 7% increase is required in the fertilization (Shrawat
54et al., 2008). The nitrogen fertilizer consumption has a direct relation with wheat production
55(Dobermann, 2006). Today agriculture focuses on the NUE to maximize the demanding yield
56with optimum fertilizer application and reduce the environmental risks (Hirel et al., 2007),

2
57(Christensen et al., 2006). So nitrogen fertilization has key importance in wheat grains quality
58(Abedi et al., 2010).

59 The present research was established to study the demand of different varieties for N
60nutrition is also different. So the prime objective is to apply the correct dose of N without loss of
61production, reduce the environmental and economic risks. The excess application of N may loss
62through leaching, erosion, soil de-nitrification and volatilization.

63Materials and Methods

64Experimental Design

65 The experiment entitled “screening of wheat varieties for yield performance and
66nitrogen assimilation under different N levels in alkaline soil ” was plotted at New
67Developmental Research Farm at the University of Agriculture, Peshawar during fall session
682018-2019. The design used in this experiment was RCBD (2 Factorial) having three
69replications. The study was two factor including different N levels i-e (0, 40, 80, 120 and 160 kg
70N ha-1) and wheat genotypes i-e (Pir-sabak, Galaxy and Insaf). Wheat genotypes were assigned
71to the main plots and different N levels were allotted to sub plots. For N source urea was used at
72different levels. All the three wheat varieties (different in yield potential and leaf color) were
73grown at the mention N fertilizer levels starting from zero to optimum recommended level as per
74cultural practice of the area (Generally 120 to 140 kg N ha -1 is recommended for various wheat
75varieties, however, farming community especially in rain-fed areas apply much lower does). Half
76of each treatment plot was supplied with remaining N (the difference between recommended
77level and applied N to the treatment) and half were left untreated.

78Grain yield

79 To find grain yield in a hectare area four central row were selected for this purpose.
80These rows were harvested, dried in sun, threshed with the help of wheat thresher, cleaned from
81other impurities, weighted to find grain weight. Grain weight of each plot was recorded,
82converted to grain yield by using the following formula into kg ha -1. The same procedure was
83followed for all the plots receiving different treatments.

3
 Grain yield in four central rows
Grain yield (kg ha -1 )= x10000
84 Row − row distance x Row length x No . of rows

85Biomass

86 In order to find biological yield central four rows of each plot was taken. The plants in
87each plot was harvested and then dried. After drying the yield of the plot was found by weighting
88the dried mass. The weight obtained was taken in kg plot -1 and then converted to kg ha-1 by using
89the following formula.

Biological yield of randomly selected rows

Biological yield ( kg ha -1 )= x10000
90 Row − row distance x Row length x No. of rows

91Soil pH and EC

92 The soil pH was measured in 1:5 suspension, that was prepared by taking 10g soil and 50
93ml distilled water in shaking bottle. The suspension was mechanically shacked in mechanical
94shaker for 15 minutes and then analyze for pH by using pH meter (McLean, 1983). And EC was
95measured with the help of EC meter. The unit of EC was dS m-1 (Roades, 1982).

96Organic Matter Content in Soil

97 For analyzing the organic matter content in the soil, (Nelson and Sommers, 1996)
98procedure was followed.

99 % O.M = (Blank– Sample) x N x 0.69

100 Weight of soil

101Nitrogen Content in Soil

102 Soil mineral nitrogen content was found by Mulvany (1996). For this purpose, 10 g soil
103was taken in a conical flask and then 50ml of KCl added. The solution was shacked for 45
104minutes, after shacking the solution was filtered through filter paper (Whatman No. 42). Then 20
105ml from the extract was taken in a conical flask and for the purpose of indication 5 ml of
106indicator (boric acid mixed indicator) was added and 0.2 gm MgO and 0.1 g devarda alloy was
107added. The sample with these chemical then put to Kjeldhal apparatus for distillation purpose.

4
108When the volume of the flask reach to 40 ml then it was removed from Kjeldhal and titrated with
1090.05 N HCl. Sample was titrated till the colour of the sample became pink. At that stage the
110reading of titration burette was noted. The same procedure was followed for a blank sample.

( sample−blank ) x N of HCL x meq N x 50

111 N min= x 1000000
Wt of sampe x volume distilled

112Nitrogen content in plant

113 Plant samples was taken at three stages (tillering, heading and grain filling) and samples
114were dried in the oven for 24 hours at 105 C 0, after drying the plant samples were grinded with
115electrical leaf grinder, digested in digestion tubes. For digestion 0.25 gm of sample was taken
116and then 1.10 g digestion mixture was added to the samples. After digestion the samples were
117transferred from the tubes to plastic bottle by making the volume with distil water up to 100 mL.
118The plant nitrogen content was found by kjeldhal apparatus of (Mulvaney and Page, 1982).

( sample−blank ) x N of HCL x meq N x 100

119 (% ) N = x 100
Wt of sampe x volume distilled

120Chlorophyll content determination

121 Chlorophyll was determined through (Lichtenthaler and Wellburn, 1983) method. The
122fresh and fully turgid leaves were collected and preserved in refrigerator for approximately 24
123hours. From preserved and cooled samples 100 mg (0.1 g leaf tissue) was crushed by mortar and
124pestle then added to test tube. 10 ml of 80% (v/v) aqueous Acetone (CH 3COCH3H2O) was added
125for the digestion of sample. The samples were covered with plastic material since the chlorophyll
126is degraded when exposed to light. The samples were kept in refrigerator at 4 0c for another 24
127hours for chlorophyll digestion. The extract was shaken for few minutes with acetone. That
128procedure was carried out for blank sample, analyzed by using spectrophotometer at 663 nm and
129646 nm wavelengths. The chlorophyll a and b was determined by using the following formula;

130Chlorophyll a= (12.21× A 663)-(2.81× A 646). ….mg g-1 FW

131Chlorophyll b= (20.13× A 646)-(5.03× A 663). ….mg g-1 FW

132Statistical analysis

5
133 In order to analyze the recorded data of the present study, statistix 8.1 software was used.
134All the data were analyzed by using analysis of variance techniques in randomize complete block
135design (RCBD). To find the best treatment and the best variety among all, least significant
136difference (LSD) at alpha 0.05 was done to specify the best level of nitrogen for variety (Steel
137and Torrie, 1986).

138Results

139 For the determination of nature, properties and soil nutrient status, a composite sample
140was collected. The laboratory analysis showed that the soil was silt loam in texture having
141calcareous pH, non-saline, organic content was <1%, bulk density of 1.28g cm -1 (Table 1). The
142Nitrogen and Phosphorus was less than the optimum range but potassium was moderate in soil.
143Over all the condition was optimum for the cultivation of wheat crop. Parameters recorded were
144analyzed statistically with the following results obtained.

145TABLE 1. Physicochemical properties of the field prior to sowing.

Properties Unit Concentration

Silt % 65.1

Sand % 28.9

Clay % 5.5

Texture Class - Silt loam

EC(e) dS m-1 0.27

pH - 7.8

Organic matter content % 0.73

Lime % 15.1

Total N content % 0.09

AB-DTPA extractable P mg/kg 3.21

146

147Straw yield

6
148 Data regarding straw yield was statistically significant at P<0.05 shown in (Table 2). It is
149clear from literature that nitrogen content increases the biomass and grain yield in response the
150straw yield also improves with the application of nitrogen. The highest straw yield was recorded
151at 160 kg N ha-1 that was 5411 kg ha-1 followed by 120 kg N ha-1 i-e 5389 kg ha-1. But the three
152levels (80, 120 and 160 kg N ha-1) were statistically similar while the lowest was recorded at 0 kg
153N ha-1 i-e 4724 kg ha-1. The percent increased among levels that of 160 and 140 kg N ha-1 were
15414.5% and 14% over control i-e 0 kg N ha -1 (Fig. 1a), the difference among different varieties
155was 20% and 12% of Insaf and Pirsabaq over Galaxy respectively.

156Grain yield

157 The statistical analysis about grain yield in response of nitrogen levels and varieties was
158observed significant at (P<0.05) shown in Table 2. The result showed that increasing the N levels
159grain yield increase in increasing trend. Among the treatment applied the highest grain yield was
160recorded at 160 kg N ha-1 i-e 3036.9 kg ha-1 followed by 120 kg N ha -1 that was 3021.9 kg ha-1.
161The result of 80 kg N ha-1 and 40 kg N ha-1 was statistically similar i-e 2883.9 and 2725.3 kg ha -1
162respectively, while the lowest grain yield was that of 0 kg N ha -1 that was 2553 kg ha -1. There
163was an increase of 18% in the grain yield with the application 160 kg N ha -1, same increase was
164also happening at 120 kg N ha -1 over control i-e 0 kg N ha -1. Treatment of 80 kg N ha -1 showed
165an increase of 12% in grain yield as compared to control treatment as shown in (Fig. 1b).

166Biomass

167 Statistical analysis of the data regarding biological yield showed that there was a
168significant effect of treatment observed at (P<0.05) on different wheat varieties as shown in
169Table 2. A linear trend was followed by biological yield in response to N levels and the highest
170biological yield was observed at 160 kg N ha -1 i-e 8448 kg ha-1 followed by 120 kg N ha-1 with a
171yield of 8411 kg ha-1then by 80 and 40 kg N ha-1. The lowest biological yield of 7277 kg ha-1 was
172recorded at control (0 kg N ha-1). According to (Fig. 1c), there was an increase of 16 % biomass
173of wheat at 160 kg N ha-1 application over control.

174Harvest Index

7
175 Statistical analysis of the data regarding harvest index was found non-significant at
176(P<0.05) when treated with various levels of nitrogen. However the difference among various
177varieties for harvest index was found significant (P<0.05) as shown in Table 2. The highest
178harvest index was reflected by Pirsabaq i-e 37% in precedence of Galaxy that was 36% and the
179lowest was shown by Insaf i-e 32%. The pirsabaq showed 15 percent increase over Galaxy,
180while Galaxy showed 12% increase over Insaf.

181TABLE 2. Shows the varietal differences in straw yield, grain yield, biological yield and
182 Harvest index affected by different N levels.

Straw yield Grain yield Biomass HI

Treatment
kg ha-1 %

Wheat Varieties

Pirsabaq 5354 a 3173 a 8527 a 37 a

Insaf 5545 a 2708 b 8254 b 32 b

Galaxy 4594 b 2650 b 7245 c 36 a

significance *** *** *** **

LSD 361 268 259 3.44

Nitrogen Levels (kg ha-1)

0 4724 b 2553 b 7277 c 35.1 a

40 4980 ab 2725 ab 7706 b 35.4 a

80 5317 a 2883 ab 8201 a 35.2 a

120 5389 a 3021 a 8411 a 35.9 a

160 5411 a 3036 a 8448 a 36.0 a

significance *** *** *** NS

LSD 466.6 346.5 334.4 4.4

H×N NS NS NS NS

183The means followed by different letters in each column are significantly different from each
184other at α = 0.05. * Significant at (P ≤0.05); ** Significant at (P ≤0.01): NS non-significant

8
185

186

187

188

189

190 (a) (b)

191

192

193

194

195

196 (c)

197Fig. 1 (a, b, c). a) Straw Yield, b) Grain Yield and c) biological yield comparison between
198 different varieties.

199Straw N concentration

200 Straw N concentration effected by different doses of N levels is shown in Table 3. The
201data was significantly affected by N levels at (P<0.05). Among the treatment the highest
202concentration of N was found in straw receiving 160 kg N ha -1 i-e 0.44% and at 120 kg N ha-1 that
203was 0.41%. The straw N content decreases with decreasing the N levels while the lowest N
204concentration was recorded at 0 kg N ha-1 i-e 0.19%. The percent increase of 131% and 115% at
205160 kg N ha-1 and 120 kg N ha-1 respectively was observed over control (0 kg N ha-1).

206Grain Nitrogen concentration

207 The most important parameter in cereal is to determine the N concentration in grains
208which was found significant at (P<0.05) when treated with different levels of N as shown in
209Table 3. The N content increased with ascending order of the N levels. Among treatment the

9
210highest result was obtained from the plot receiving 160 kg N ha -1 i-e 2.8% perused by 120 kg N
211ha-1 i-e 2.7%, while the lowest grain N was recorded by treatment receiving 0 kg N ha -1 i-e 1.9%.
212At 160 kg N ha-1 and 140 kg N ha-1 showed 47% and 42% an increase in grain N content over
213control.

214Soil mineral N tillering stage

215 Mineral N in soil was significantly (P<0.05) enhanced at tillering stage with the
216application of various doses of N feritilizers as shown in Table 3. An increasing trend was
217followed by mineral N with increasing levels of nitrogen. The highest N concentration was found
218in plot receiving 160 kg N ha-1 i-e 41.95 mg kg-1, pursued by 120 kg N ha-1 i-e 37.35 mg kg-1,
219while the least N concentration was shown by the plots receiving 0 kg N ha -1 has 14.84 mg kg-1.
220There was an increase of 182%, 151%, 114% and 80% by 160, 120, 80 and 40 kg N ha -1
221respectively over the plot receiving no treatment (control).

222Soil mineral N grain filling stage

223 When the amount of mineral N increases in the soil, the crop yield increases accordingly
224and simultaneously the N content in soil decreases. The data regarding mineral N content in soil
225at grain filling stage was highly significant at (P<0.05) shown in Table 3. Among the treatment
226the high concentration of mineral N was evident from 160 kg N ha -1 29.35 mg kg-1 that was
227followed by 120 kg N ha-1 i-e 25.41mg kg-1, 80 and 40 kg N ha-1 showed 21.74 mg kg-1 and 18.02
228mg kg-1 respectively, While the lowest mineral concentration was observed in control (0 kg N ha -
2291) i-e 10.0 mg kg-1. There was an increase of 193%, 178%, 154% and 110% by 160, 120, 80, 40
230kg N ha-1 over control respectively.

231TABLE 3. Shows the Straw and Grain N concentration at various N Levels.

Straw N Grain N Soil N till Soil N Grain fill

Treatment
----------------%-------------- ---------mg kg-1------------

Wheat varieties

Pirsabaq 0.39 2.3 ab 29.94 20.33

Insaf 0.33 2.5 a 31.26 21.13

10
 Galaxy 0.31 2.2 b 30.5 21.26

significance NS ** NS NS

LSD 0.06 0.16 1.67 1.29

Nitrogen Levels (kg ha-1)

0 0.19 c 1.9 c 14.84 e 10.0 e

40 0.31 b 2.1 c 26.83 d 18.02 d

80 0.37 ab 2.3 b 31.87 c 21.74 c

120 0.41 ab 2.7 a 37.35 b 25.41 b

160 0.44 a 2.8 a 41.95 a 29.35 a

Significance *** *** *** ***

LSD 0.08 0.2 2.16 1.67

V×N NS NS NS NS

232The means followed by different letters in each column are significantly different from each
233other at α = 0.05. * Significant at (P ≤0.05); ** Significant at (P ≤0.01): NS non-significant

234Plant N concentration tillering stage

235 A significant (P<0.05) effect was induced by different levels of N on plant N

236concentration (%) at tillering stage as shown in Table 4. Highest N content was recorded by the
237treatment receiving 160 kg N ha-1 i-e 3.1%, and only 3% by 120 kg N ha -1. While the least N
238content was found in control treatment 0 kg N ha-1 i-e 2.4%. Nitrogen contents was increased by
23929% by applying 160 kg N ha-1 while 120 kg N ha -1 shown an increase of 25%, 80 and 40 kg N
240ha-1 has the same increase of 12 % over control.

241Chlorophyll content in tillering stage

242 If we talk about nitrogen how we can ignore the chlorophyll, because both are mainly
243associated with each other, without N chlorophyll is incomplete. Chlorophyll content was also
244part of this experiment. Data regarding chlorophyll content in wheat crop at grain filling stage
245was highly significant at P<0.05. As N levels was increase the chlorophyll content was increased

11
246in linear sequence Table 4. The highest chlorophyll content was observed at 160 kg N ha-1 that
247was 31.5 SPAD Value closely followed by 120 kg N ha-1 i-e 30.4 SPAD value while the lowest
248was recorded at 0 kg N ha-1 i-e 28.11 SPAD value. Nitrogen has increase the chlorophyll content
249by 12% at 160 kg N ha-1 and 8% at 120 kg N ha-1 over control. While 80 and 40 kg N ha-1 shown
250an increase of 3% and <1% over control.

251 Data regarding chlorophyll content in wheat varieties was not that much effective
252because color may be a differential character among cultivar. The data was non-significant
253because of this trait but the highest chlorophyll content was recorded by Galaxy 29.8 SPAD
254value strive by Pirsabaq 29.4 SPAD value while the lowest was that of Insaf i-e 29.2 SPAD
255value. The percent difference was not that much high but can be considers. Noulas et al. (2018)
256result explain the possible pathway of enhancing the NUE but mainly dependent on season and
257genotype effects.

258 The interaction data revealed a non-significance result because as described above that
259the N requirement and chlorophyll content can be applicable within genotype however the high
260chlorophyll content was observed by Galaxy 32 SPAD value at 160 kg N ha -1 followed by
261Pirsabaq 31 SPAD value at 160 kg N ha-1 while the lowest SPAD value was shown by Insaf at 0
262kg N ha-1 28 SPAD value.

263Plant N content at booting stage

264 Green color is also an indication of N status; we can diagnose the N deficiency by color
265differences in cereal crops Table 4. The statistical analysis showed that the treatment effect was
266highly significant at P<0.05. Among the treatment applied the high concentration was found in
267the plants samples of the plots receiving high dose of the N levels. The results of 160 and 120 kg
268N ha-1 were statistically at par, revealed the concentration of 2.7% and 2.5% respectively. 80 and
26940 kg N ha-1 showed 2.3% and 2.2% while the lowest concentration was there in treatment of the
2700 kg N ha-1 i-e 1.9%.

271 While the data expressing the N concentration among different varieties was non-
272significant. But the high concentration of N in leaves was shown by Galaxy i-e 2.4% pursued by
273Pirsabaq i-e 2.38% while the lowest was resulted from Insaf that was 2.30%. The percent

12
274differences among these concentrations were very minute but also exist. The interaction among
275different treatment and varieties was also non-significant, but the highest concentration was
276observed at 160 kg N ha-1 i-e 2.8% followed by120 kg N ha-1 that was 2.6% of the same variety i-
277e Galaxy, while the lowest was observed at 0 kg N ha-1 in Insaf i-e 1.8 %.

278Plant N concentration (grain filling stage)

279 Statistical data regarding plant N concentration in response to the applied treatment was
280significant at (P<0.05). Among the treatment the maximum concentration of N was recorded at
281160 kg N ha-1that was 1.69% followed by 120 kg N ha-1 i-e 1.65%, and 80 and 40 kg N ha-1 have
2821.6% and 1.5% N concentration at grain filling stage respectively, while the least concentration
283of recorded by 0 kg N ha-1 1.4% Table 4.

284TABLE 4. Shows the concentration of N in different wheat varieties at different levels of N.

Till-stage Chlorophyll Boot-stage Grain fill-stage

Treatment
-------------------------------%-------------------------------------

Wheat Varieties

Pirsabaq 2.7 a 29.4 ab 2.3 a 1.5 a

Insaf 2.8 a 29.2 b 2.3 a 1.5 a

Galaxy 2.6 a 29.85 a 2.4 a 1.5 a

significance NS NS NS NS

LSD 0.12 0.6 0.15 0.1

Nitrogen Levels (kg ha-1)

0 2.45 d 28.1 d 1.98 c 1.4 b

40 2.73 c 28.3 cd 2.2 b 1.5 ab

80 2.73 c 29.1 c 2.3 b 1.6 a

120 3.0 b 30.4 b 2.5 a 1.6 a

160 3.1 a 31.5 a 2.7 a 1.6 a

13
 Significance *** *** ***

LSD 0.16 0.7 0.2 0.12

v×N NS NS NS NS

285The means followed by different letters in each column are significantly different from each
286other at α = 0.05. * Significant at (P ≤0.05); ** Significant at (P ≤0.01): NS non-significant

287Correlation (r value) of soil mineral N with grain yield of all varieties

288 Statistical analysis regarding correlation (r value) is shown in Table 5. The correlation of
289soil mineral N with wheat growth and grain yield in different varieties was influential but not
290good. The r value of soil mineral N with grain yield and biomass yield was positively correlated
291in all the stages of wheat growth. Which indicates that with increase in soil N in different stage
292increase in yielding parameters observed. However, in plant N showed positive significant
293correlation with grain yield only in tillering stage (Fig. 2). Thousand grain weight and grains per
294spike was significantly influenced by soil mineral N i-e r value >0.5, but showing a weak relation
295with grain yield. The data of soil mineral N in relation with plant N content showed a positive
296correlation with 1000 grain weight and N uptake. The correlation of SPAD value with soil
297mineral N has shown a positive significant correlation with 1000 grain weight, grain spike -1,
298grain yield, biomass yield and N uptake.

299TABLE 5. Correlation (r value) of soil mineral N with plant height, 1000 grain weight,
300 grains spike-1, grain yield, biological yield and N uptake.

Plant Grain Grain Grain Biomas N

height wt spike-1 yield s uptake

Soil N Tillering 0.12 0.77** 0.60** 0.42** 0.55** 0.79**

Boot 0.10 0.73** 0.55** 0.46** 0.59** 0.83**

Grain 0.08 0.71** 0.60** 0.40** 0.51** 0.79**

filling

14
 Plant N Tillering 0.15 0.62** 0.42** 0.31** 0.44** 0.71**

Boot 0.03 0.53** 0.43** 0.20 0.34** 0.69**

Grain 0.05 0.42** 0.31* 0.15 0.37** 0.55**

filling

SPAD -0.11 0.56** 0.58** 0.37** 0.34** 0.62**

301

15
302

303

304

305

306

307

308

309Fig. 2. Correlation of grain yield of all tested varieties with soil N at tillering stage
310 induced by different fertilizer N levels.

311Correlation (r value) of soil mineral N with grain yield of Pirsabaq-2013 cultivar.

312 The correlation data of soil mineral N in all stages was positive significantly correlated
313with all the mentioned traits which suggest that with increase in soil N increase in all these stages
314were observed in the verity pirsabaq 2013. Non-significant correlation of thousand grain weight
315with plant N at tillering stage and with grain yield at tillering and grain filling stage was
316observed while in rest of the case plant N was significantly positive correlated with all the
317parameters.

318TABLE 6. Shows the r value of soil mineral N with plant height, 1000 grain weight, grains
319 pike-1, grain yield, biological yield and N uptake in Pirsabq-2013.

Plant Grain Grain Grain Biomas N

height wt spike-1 yield s uptake

Soil N Tillering 0.86** 0.81** 0.55** 0.83** 0.82** 0.86**

Boot 0.85** 0.73** 0.42** 0.73** 0.86** 0.89**

Grain 0.80** 0.74** 0.51** 0.81** 0.83** 0.91**

filling

Plant N Tillering 0.49** 0.19 0.28 0.62** 0.58** 0.74**

Boot 0.69** 0.64** 0.38* 0.80** 0.77** 0.84**

16
 Grain 0.49** 0.33** 0.28 0.68** 0.47** 0.76**
filling

SPAD 0.53** 0.72** 0.60** 0.67** 0.74** 0.80**

320

321

322

323

324

325

326

327

328

329Fig. 3. Shows the correlation of grain yield of Pirsabaq-2013 variety with soil N at
330 tillering induced by different N levels.

331Discussion

332 Straw yield affected by the genetic makeup of the specified varieties. Varieties also
333showed significant response to N levels. The data regarding Varieties was observed significant at
334p<0.05. The highest straw yield was recorded by Insaf followed by Pirsabaq, while the lowest
335straw yield was observed by Galaxy Table 2. Our result was in conformity to (Maqsood et al.,
3362014) who stated that the interaction was non-significant among treatments and local cultivar.
337(Gao et al., 2015), also support our findings that the nitrogen significantly increase the length of
338crop roots which uptake the nutrients in optimum amount and that increase was observed in the
339straw, grain and biological yield of cereal crops. The same trend among the N levels and increase
340was also observed by (Yi et al., 2015) that N is attributed with the increase of straw yield.
341Among the cultivars the highest grain yield was given by Pirsabaq i-e 3173.6 kg ha -1 followed by
342Insaf that give 2708.3 kg ha-1 of the grain yield while the lowest yield was that of Galaxy i-e

17
3432650.8 kg ha-1. Cultivar Insaf and Galaxy produced statistically similar grain yields. Pirsabaq
344was 19% and 2.1% enhanced as compared to Insaf and Galaxy Table 2. The variation among
345grain yield of different wheat cultivar was also reported by (Maqsood et al., 2014; Yi et al.,
3462015; Saleem et al., 2010) concluded that with N application the increase of biological yield the
347increase in grain yield was observed clearly. The interaction result among N levels and varieties
348was non-significant. Akmal et al. (2018) and Gao et al. (2015) also find the same trend of
349increasing grain yield with increment in applied N. In terms of various varieties tested in this
350experiment there was also a significant difference observed in biological yield. Among varieties
351the highest biological yield was recorded by Pirsabaq (8527kg ha -1) followed by Insaf (8254 kg
352ha-1) and Galaxy (7245 kg ha-1). There was an increase of 17% and 13.9% of Pirsabaq and Insaf
353over Galaxy. The difference among biomass between different varieties may be the genetic
354potential. The decrease among varieties may be associated with lodging and high disease
355susceptibility in galaxy variety. The interaction between N levels and varieties of wheat was
356found non-significant at (P<0.05). Similar finding was noted by (Maqsood et al., 2014). The
357trend of increasing N levels to crop can increase the biomass of the crop is mentioned by
358(Sadeghi et al., 2014; Akmal et al., 2018; Gao et al., 2015; Khan et al., 2020a). The application
359of N fertilizer can also enhance the biological yield in Maize crop (Ikram ullah et al., 2015; Khan
360et al., 2020b). The harvest index of wheat was not significantly affected by the application of
361nitrogen fertilizers while the effect was significant among varieties Table 2. The findings of
362Ahmad et al. (2018) was in support of our results who confirmed the harvest index was linearly
363enhanced among different cultivars of maize while there was no effect shown by nitrogen
364fertilizers. Abera et al. (2017) confirmed the same effect on harvest index was visualized in
365maize crop at former field at Ethiopia. The nitrogen response in maize crop to harvest index was
366also highlight by (Ahmad et al., 2018). The interaction among treatments and varieties was found
367statistically non-significant.

368 Concentration of N in wheat straw was significantly affected by the application of

369mineral nitrogen. Our finding was supported by Zhang et al. (2017) where they claimed that the
370uptake of nitrogen enhanced with the application of nitrogen, which leads to increases the straw
371N concentration in wheat. The result of our research also matches with Rutkowska (2009) where
372he applied N15 to the winter wheat at anthesis stage on top of the wheat to calculate the

18
373accumulation and allocation of N. Among the cultivars used, there was no-significant difference
374observed in straw N Table 3. The interactive influence of applied N levels and varieties on straw
375N was also found non-significant. This linear increase of grain N content was also in the
376conformity of Noulas et al. (2018). Saleem et al. (2010) claimed that the protein yield of wheat
377increases up-to the highest level of N applied i-e 150 kg N ha -1 in his experiment. The statistical
378analysis of data regarding varieties were observed significant at P<0.05. The highest N content in
379grain was shown by Insaf i-e 2.5% followed by Pirsabaq i-e 2.3% while the lowest grain N
380content was recorded by Galaxy i-e 2.2%. Insaf showed 13% and pirsabaq has 4% more grains N
381over Galaxy. Scharf, (2001) also relates the soil N with the plant N and concluded that the N
382requirement is different for different field and varieties. The main objective was to evaluate the
383soil and plant test for potential production and to predict optimum N recommendation in maize.
384The same trend among N levels and varieties was also observed by Khan et al. (2012). Statistical
385data about the combine effect of varieties and N levels on grain N was found non-significant.
386This increased in biological yield is in conformity with that of (Okubay, 2019) where he
387concluded that application of various levels of N increase biological yield accordingly. The
388deficient soil N is responsible for exploitable production in wheat keeping the importance of N
389(Smith et al., 2019) performed an experiment to match the N fertilizer with the potential yield of
390wheat. We have same result like Staugaitis et al. (2007) they also highlight the importance of
391mineral N for the optimum production of wheat crop. The tillering stage N concentrations of
392varieties were not significantly enhanced with nitrogen application Table 3. Like that trend Gao
393et al. (2017) test eight-four varieties under different N condition (sufficient and deficient) and
394correlate the N status with N content, grain yield and biological yield and classify the varieties
395into different groups i-e high-responsive, responsive and least responsive to soil N fertilization.
396In line with our finding (Noulas et al., 2018) also test four wheat cultivar with N fertilizer in two
397years have the same aim to assess variation and select a superior genotype in NUE and correlate
398that N to the yielding component of the wheat crop and concluded that N after anthesis can
399preferably accumulate in the grain and increase the grain yield. The interactive effect of varieties
400and nitrogen was found non-significant (P<0.05) in terms of plant tillering N concentration.

401 The efficient utilization of N was measured by different wheat varieties. The statistical
402data regarding the soil N concentration in different cultivar was observed significant at P<0.05.

19
403Among varieties the highest uptake was shown by Pirsabaq and Galaxy i-e 29.94 and 30.5 N mg
404kg-1 that was clear from the soil analysis while Insaf shows less uptake i-e 31.26 mg kg -1 N was
405remain in the plots. The remaining high concentration of soil mineral N shows the less uptake of
406N from the soil. The statistical data regarding the interaction between treatment and varieties was
407non-significant. There was a non-significant influence laid by different varieties on mineral N
408content in soil at grain filling stage. The interaction among varieties and N levels was also found
409non-significant at P<0.05. The application of nitrogen fertilizer can increase the N concentration
410of plant. The treatment showed 42%, 31%, 21% and 15% increase over control Table 4. Germa
411et al. (2006) used mid-season prediction of soil and plant N concentration for winter wheat. They
412used different levels of N with the recommended dose of P&K. The potential levels among N
413levels was 135 kg N ha-1 that were observed for different parameters i-e chlorophyll content, soil
414NH4-N, soil NO3-N and N mineralization data were collected. The correlation among these
415factors was studied for the final prediction of winter wheat biomass and grain yield. They
416suggested that the correlation and regression of chlorophyll content and plant N uptake be the
417best predictors of the final production of wheat crops. Our result also in line with Durani et al.
418(2016) where they claimed that N fertilization can enhance the soil mineral N. there was a
419positive significant correlation among N uptake and crop yield. (Mendizábal et al., 2012)
420revealed that the late fertilization of N effect the use of pre-anthesis N during grain filling stage.
421The same split doses of N cannot improve the N but enhancing the dose can improve the N
422concentration. The application in the late grain filling stage all the N goes to the grain without
423improving the vegetative growth of the plant. In grain filling stage the physiological process and
424photosynthesis process slow down and the N concentration in leaves become less moving to the
425grain, in response the grain color of the crop become light and at last the physiological maturity
426start and all the N translocate to grains. Varieties showed no significant (P<0.05) enhancement in
427N concentration at grain filling stage at various N doses application. The interactive influence of
428different varieties and various levels of N didn’t induce significant change in N contents of
429wheat at grain filling stages as shown in Table 5.

430 Noulas et al. (2018) have the same finding but they focus on the interrelation of yielding
431components to N levels with SPAD indices. Dunna et al. (2018) conducted the finding of their
432research on poinsettia leaves where they concluded that the highest value was achieved at the

20
433application of 250 mg N fertilizer was applied. Our result was in line of Hebbara et al. (2016)
434where they conducted results on chlorophyll meter reading and nitrogen fertilization in coconut
435crop they also claimed that estimating the chlorophyll content within the genotype is acceptable
436while across the genotype the chlorophyll meter reading was not challengeable. Feng et al.
437(2014) conducted a research in china on wheat genotype for the rapid diagnosing of the rapid N
438requirement in the field by leaf chlorophyll fluorescence parameter where best result regarding
439chlorophyll content was shown by 270 and 360 kg N ha-1 within the same genotype.

440Conclusions

441 It was concluded from this research that the application of N significantly influenced wheat
442yield, plant N concentration, N uptake and use efficiency. Different varieties, however, due to
443different genetic character and yield potential responded differently to applied N levels. Pirsabaq
4442013 and Insaf yielded higher at 120 kg N ha -1 whereas Galaxy yielded higher at 80 kg N ha -1.
445Soil application of N also increased soil and plant N at tillering, boot and grain filling stage but
446difference among varieties were non-significant. Regarding yielding potential, Pirsabaq was
447found the most productive whereas Galaxy yielded the lowest. Similarly, Insaf was observed the
448tallest variety whereas the Galaxy was found to be the shortest one. When data was calculated
449based on all varietal values, poor correlation was observed among the soil and plant N with plant
450growth and yield but taking into account any single variety, a significant correlation was
451observed among the soil and pant N and crop yield that could be associated to different yielding
452potential of various varieties.

453Conflict of Interest

454 The authors declare that they have no competing interests.

455Authors Contributions

456  Muhammad Ibrar conceived and designed the experiments, prepared figures and/or
457 tables, and approved the final draft.
458  Dost Muhammad conceived and designed the experiments, analyzed the data, prepared
459 figures and/or tables, and approved the final draft.

21
460  Ishaq Ahmad Mian performed the experiments, prepared figures and/or tables, and
461 approved the final draft.
462  Shadman Khan performed the experiments, analyzed the data, prepared figures and/or
463 tables, approved the final draft, and response the comments.
464  Muhammad Ismail performed the experiments, analyzed the data, and approved the final
465 draft.
466  Maria Mussarat performed the experiments, analyzed the data, prepared figures and/or
467 tables.
468  Farmanullah Khan performed the experiments, prepared figures and/or tables.
469  Aizaz Ali performed the experiments, and approved the final draft.
470  Beenish Ali performed the experiments, authored or reviewed drafts of the paper, and
471 approved the final draft.
472  Taufiq Nawaz performed the experiments, reviewed drafts of the paper, and approved the
473 final draft.

474References

475Abalos, D., Jeffery, S., Sanz-Cobena, A., Guardia, G. and Vallejo, A. (2014) Meta-analysis of
476 the effect of urease and nitrification inhibitors on crop productivity and nitrogen use
477 efficiency. Agriculture, Ecosystems & Environment, 189, 136-144.

478Abedi, T., Alemzadeh, A. and Kazemeini, S.A. (2010) Effect of organic and inorganic fertilizers
479 on grain yield and protein banding pattern of wheat. Aust. J. crop sci. 4 (6) pp.384-389.

480Ahmad, S., Khan, A.A., Kamran, M., Ahmad, I., Ali, S. and Fahad, S. (2018) Response of maize
481 cultivars to various nitrogen levels. Eur. Exp. Biol. 8 (1) pp.1-4.

482Akmal, M., Shah, A. and Ali, J. (2018) Growth, radiation use efficiency and grain yield of wheat
483 as influenced by nitrogen, tillage, and crop residue management. Journal of Plant
484 Nutrition. 41 (16) 2032-2047.

22
485Argenta, G., Silva, P.R.F.D. and Sangoi, L. (2004) Leaf relative chlorophyll content as an
486 indicator parameter to predict nitrogen fertilization in maize. Ciência Rural. 34 (5) 1379-
487 1387.

488Bojović, B. and Marković, A. (2009) Correlation between nitrogen and chlorophyll content in
489 wheat (Triticum aestivum L.). Kragujevac J.Sci. 31 (5827) 69-74.

490Christensen, L., Riley, W.J. and Ortiz-Monasterio, I. (2006) Nitrogen cycling in an irrigated
491 wheat system in Sonora, Mexico: measurements and modeling. Nutrient Cycling in
492 Agroecosys. 75 (1) 175-186.

493Dobermann, A. (2006) September. Nitrogen use efficiency in cereal systems. In Ground

494 breaking stuff. Proceedings of the 13th ASA Conference. 10-14.

495Dong, S., Zhang, J., Zha, T., Dai, X. and He, M. (2020) Increased plant density with reduced
496 nitrogen input can improve nitrogen use efficiency in winter wheat while maintaining
497 grain yield. Arch. Agro. and Soil Sci. 66 (12) 1707-1720.

498Dunn, B.L., Singh, H. and Goad, C. (2018) Relationship between chlorophyll meter readings and
499 nitrogen in poinsettia leaves. J. Plant Nutr. 41 (12) 1566-1575.

500Durani, A., Brar, B.S. and Dheri, G.S. (2016) Soil nitrogen fractions in relation to rice-wheat
501 productivity: Effects of long-term application of mineral fertilizers and organic
502 manures. J. Crop Impro, 30 (4) 399-420.

503Elayaraja, D. and Singaravel, R. (2017) Effect of vermicompost and micronutrients fertilization

504 on the growth, yield and nutrients uptake by sesame (Sesamum indicum L.) in coastal
505 saline soil. Inter. J. Agri. Sci, 13 (2) 177-183.

506Engström, L. and Bergkvist, G. (2009) Effects of three N strategies on tillering and yield of low
507 shoot density winter wheat. Acta Agriculturae Scandinavica Section B–Soil and Plant
508 Science, 59 (6) 536-543.

23
509Feng, W., He, L., Zhang, H.Y., Guo, B.B., Zhu, Y.J., Wang, C.Y. and Guo, T.C. (2015)
510 Assessment of plant nitrogen status using chlorophyll fluorescence parameters of the
511 upper leaves in winter wheat. European J. Agro. 64, 78-87.

512Fuertes‐Mendizábal, T., González‐Murua, C., González‐Moro, M.B. and Estavillo, J.M. (2012)
513 Late nitrogen fertilization affects nitrogen remobilization in wheat. J.Plant Nutr. and
514 Soil Sci. 175 (1) 115-124.

515Galloway, J.N., Dentener, F.J., Capone, D.G., Boyer, E.W., Howarth, R.W., Seitzinger, S.P.,
516 Asner, G.P., Cleveland, C.C., Green, P.A., Holland, E. and Karl, D.M. (2004) Nitrogen
517 cycles: past, present, and future. Biogeochemistry. 70, 153e226.

518Gao, S., Zhang, F., Zhi, Y., Chen, F. and Xiao, K. (2017) The yields, agronomic, and nitrogen
519 use efficiency traits of wheat cultivars in north China under N-sufficient and-deficient
520 conditions. J. Plant Nutr. 40 (7) 1053-1065.

521Gao, S., Zhang, F., Zhi, Y., Chen, F. and Xiao, K. (2017) The yields, agronomic, and nitrogen
522 use efficiency traits of wheat cultivars in north China under N-sufficient and-deficient
523 conditions. J. Plant Nutr. 40 (7), 1053-1065.

524Ghasemi, M., Arzani, K., Yadollahi, A., Ghasemi, S. and Khorrami, S.S. (2011) Estimate of leaf
525 chlorophyll and nitrogen content in Asian pear (Pyrus serotina Rehd.) by CCM-
526 200. Notulae Scientia Biologicae, 3 (1) 91-94.

527Giday, O. (2019) Effect of type and rate of urea fertilizers on nitrogen use efficiencies and yield
528 of wheat (Triticum aestivum L.) in Northern Ethiopia. Cogent Environmental Science, 5
529 (1) 1655980.

530Girma, K., Martin, K.L., Anderson, R.H., Arnall, D.B., Brixey, K.D., Casillas, M.A., Chung, B.,
531 Dobey, B.C., Kamenidou, S.K., Kariuki, S.K. and Katsalirou, E.E. (2006) Mid-season
532 prediction of wheat-grain yield potential using plant, soil, and sensor measurements. J.
533 Plant Nutr. 29 (5) 873-897.

24
534Hebbar, K.B., Subramanian, P., Sheena, T.L., Shwetha, K., Sugatha, P., Arivalagan, M. and
535 Varaprasad, P.V. (2016) Chlorophyll and nitrogen determination in coconut using a non-
536 destructive method. J. Plant Nutr. 39 (11) 1610-1619.

537Herrera, J.M., Rubio, G., Häner, L.L., Delgado, J.A., Lucho-Constantino, C.A., Islas-Valdez, S.
538 and Pellet, D. (2016) Emerging and established technologies to increase nitrogen use
539 efficiency of cereals. Agronomy, 6 (2) 25.

540Hirel, B., Le Gouis, J., Ney, B. and Gallais, A. (2007) The challenge of improving nitrogen use
541 efficiency in crop plants: towards a more central role for genetic variability and
542 quantitative genetics within integrated approaches. J experi. Botany, 58 (9) 2369-2387.

543Hlisnikovský, L., Kunzová, E., Hejcman, M. and Dvořáček, V. (2015) Effect of fertilizer
544 application, soil type, and year on yield and technological parameters of winter wheat
545 (Triticum aestivum L.) in the Czech Republic. Archives of Agronomy and Soil
546 Science, 61 (1) pp.33-53.

547ICID (International Commission on Irrigation and Drainage). (2018) Sprinkler and Micro-
548 Irrigated Areas in Some Participating Members of ICID. From ICID Database. http
549 ://www.icid.org/database.html.

550Kassie, M. and Tesfaye, K. (2019) Influence of variety and nitrogen fertilizer on productivity and
551 trait association of malting barley. J. Plant Nutr. 42 (10) 1254-1267.

552Khan, S., Ismail, M., Ibrar, M. and Ali, Z. (2020b) The effect of biochar on soil organic matter,
553 total N in soil and plant, nodules, grain yield and biomass of mung bean. Soil &
554 Environment, 39 (1).

555Khan, S., Shah, Z., Mian, I.A., Dawar, K., Tariq, M., Khan, B., Mussarat, M., Amin, H., Ismail,
556 M., Ali, S. and Shah, T. (2020a) Soil Fertility, N2 Fixation and Yield of Chickpea as
557 Influenced by Long-Term Biochar Application under Mung–Chickpea Cropping
558 System. Sustainability, 12 (21), p.9008.

25
559Kumar, D., Patel, R.A. and Ramani, V.P. (2019) Assessment of precision nitrogen management
560 strategies in terms of growth, yield and monetary efficiency of maize grown in Western
561 India. J. Plant Nutr. 42 (20) 2844-2860.

562Li, G., Zhao, B., Dong, S., Zhang, J., Liu, P. and Lu, W. (2020a) Controlled-release urea
563 combining with optimal irrigation improved grain yield, nitrogen uptake, and growth of
564 maize. Agricultural Water Management, 227, 105834.

565Li, Y., Ma, L., Wu, P., Zhao, X., Chen, X. and Gao, X. (2020b) Yield, yield attributes and
566 photosynthetic physiological characteristics of dryland wheat (Triticum aestivum L.) /
567 maize (Zea mays L.) strip intercropping. Field Crops Research, 248, 107656.

568Lichtenthaler, H.K. and Wellburn, A.R. (1983) Determinations of total carotenoids and
569 chlorophylls a and b of leaf extracts in different solvents. 591-592

570Liu, C., Wang, K. and Zheng, X. (2013) Effects of nitrification inhibitors (DCD and DMPP) on
571 nitrous oxide emission, crop yield and nitrogen uptake in a wheat–maize cropping
572 system. Biogeosciences, 10 (4) 2427-2437.

573Liu, X., Ju, X., Zhang, F. and Chen, X. (2003) Nitrogen recommendation for winter wheat using
574 Nmin test and rapid plant tests in North China Plain. Communications in soil science
575 and plant analysis, 34 (17-18), 2539-2551.

576Loh, F.C., Grabosky, J.C. and Bassuk, N.L. (2002) Using the SPAD 502 meter to assess
577 chlorophyll and nitrogen content of benjamin fig and cottonwood
578 leaves. HortTechnology, 12 (4) 682-686.

579Luo, G., Li, L., Friman, V.P., Guo, J., Guo, S., Shen, Q. and Ling, N. (2018) Organic
580 amendments increase crop yields by improving microbe-mediated soil functioning of
581 agroecosystems: A meta-analysis. Soil Biology and Biochemistry, 124, 105-115.

582Makary, T., Schulz, R., Müller, T. and Pekrun, C. (2020) Simplified N fertilization strategies for
583 winter wheat. Part 1: plants: compensation capacity of modern wheat varieties. Archives
584 of Agron. and Soil Sci. 66 (6), 847-857.

26
585Maqsood, M., Shehzad, M.A., Ramzan, Y. and Sattar, A. (2014) Effect of nitrogen nutrition on
586 growth, yield and radiation use efficiency of different wheat (Triticum aestivum L.)
587 cultivars. Pakistan J. Agri. Sci. 51 (2).

588Matson, P.A., Naylor, R. and Ortiz-Monasterio, I. (1998) Integration of environmental,

589 agronomic, and economic aspects of fertilizer management. Science, 280 (5360), 112-
590 115.

591McLean, E.O. (1983) Soil pH and lime requirement. Methods of soil analysis: Part 2 Chem.
592 microbial. prop. 9, 199-224.

593Metho, L.A., Hammes, P.S., De Beer, J.M. and Groeneveld, H.T. (1997) Interaction between
594 cultivar and soil fertility on grain yield, yield components and grain nitrogen content of
595 wheat. South African Journal of Plant and Soil, 14 (4) 158-164.

596Mulvaney, B.J. and Page, A.L. (1982) Nitrogen-total. Methods of soil analysis: Part, 2, 595-624.

597Mulvaney, R.L. (1996) Nitrogen—inorganic forms. Methods of soil analysis: part 3 chemical

598 methods, 5, 1123-1184.

599Nakano, H., Morita, S. and Kusuda, O. (2008) Effect of nitrogen application rate and timing on
600 grain yield and protein content of the bread wheat cultivar ‘Minaminokaori’in
601 southwestern Japan. Plant production science, 11 (1), 151-157.

602Nelson, D.W. and Sommers, L.E. (1996) Total carbon, organic carbon, and organic
603 matter. Methods of soil analysis: Part 3 Chemical methods, 5, pp.961-1010.

604Noulas, C., Herrera, J.M., Tziouvalekas, M. and Qin, R. (2018) Agronomic assessment of
605 nitrogen use efficiency in spring wheat and interrelations with leaf greenness under field
606 conditions. Commun. soil sci. plant analysis. 49 (7) 763-781.

607Onasanya, R.O., Aiyelari, O.P., Onasanya, A., Nwilene, F.E. and Oyelakin, O.O. (2009) Effect
608 of different levels of nitrogen and phosphorus fertilizers on the growth and yield of
609 maize (Zea mays L.) in Southwest Nigeria. Inter. J. of Agri. Res. 4 (6), 193-203.

27
610Peltonen, J., Virtanen, A. and Haggren, E. (1995) Using a chlorophyll meter to optimize nitrogen
611 fertilizer application for intensively‐managed small‐grain cereals. Journal of Agronomy
612 and Crop Science, 174 (5) 309-318.

613Phullan, N.K., Memon, M., Shah, J.A., Memon, M.Y., Sial, T.A., Talpur, N.A. and Khushk,
614 G.M. (2017) Effect of organic manure and mineral fertilizers on wheat growth and soil
615 properties. Journal of Basic and Applied Sciences, 13.

616Qi, D., Hu, T., Song, X. and Zhang, M. (2019) Effect of nitrogen supply method on root growth
617 and grain yield of maize under alternate partial root-zone irrigation. Scientific reports, 9
618 (1) 1-10.

619Rahman, M.A., Sarker, M.A.Z., Amin, M.F., Jahan, A.H.S. and Akhter, M.M. (2011) Yield
620 response and nitrogen use efficiency of wheat under different doses and split application
621 of nitrogen fertilizer. Bangladesh J. Agri. Res. 36 (2) 231-240.

622Ran, H., Kang, S., Li, F., Du, T., Ding, R., Li, S. and Tong, L. (2017) Responses of water
623 productivity to irrigation and N supply for hybrid maize seed production in an arid
624 region of Northwest China. Journal of Arid Land, 9 (4) 504-514.

625Ran, H., Kang, S., Li, F., Tong, L. and Du, T. (2016) Effects of irrigation and nitrogen
626 management on hybrid maize seed production in north-west China. Frontiers of
627 Agricultural Science and Engineering, 3 (1) 55-64.

628Rasmussen, I.S., Dresbøll, D.B. and Thorup-Kristensen, K. (2015) Winter wheat cultivars and
629 nitrogen (N) fertilization—effects on root growth, N uptake efficiency and N use
630 efficiency. European Journal of Agronomy, 68, 38-49.

631Riley, W.J., Ortiz-Monasterio, I. and Matson, P.A. (2001) Nitrogen leaching and soil nitrate,
632 nitrite, and ammonium levels under irrigated wheat in Northern Mexico. Nutrient
633 Cycling in Agroecosystems, 61 (3) 223-236.

634Roades, J.D., 1982. Soluble salts. P 167-178. Methods of soil analysis. Part, 2.

28
635Rothstein, S.J. (2007). Returning to our roots: making plant biology research relevant to future
636 challenges in agriculture. The Plant Cell, 19 (9) 2695-2699.

15
637Rutkowska, A. (2009) Nitrogen study on accumulation and allocation of nitrogen applied at
638 anthesis on top of early nitrogen applications in winter wheat. Journal of plant
639 nutrition. 32 (8), 1306-1320.

640Sadeghi, S.M., Noorhosseini, S.A. and Damalas, C.A. (2018) Environmental sustainability of
641 corn (Zea mays L.) production on the basis of nitrogen fertilizer application: the case of
642 Lahijan, Iran. Renewable and Sustainable Energy Reviews, 95, 48-55.

643Saleem, M.F., Ma, B.L., Voldeng, H. and Wang, T.C. (2010) Nitrogen nutrition on leaf
644 chlorophyll, canopy reflectance, grain protein and grain yield of wheat varieties with
645 contrasting grain protein concentration. J. plant nutr. 33 (11), 1681-1695.

646Scharf, P.C. (2001) Soil and plant tests to predict optimum nitrogen rates for corn. Journal of
647 Plant Nutrition, 24 (6) 805-826.

648Shrawat, A.K., Carroll, R.T., DePauw, M., Taylor, G.J. and Good, A.G. (2008) Genetic
649 engineering of improved nitrogen use efficiency in rice by the tissue‐specific expression
650 of alanine aminotransferase. Plant biotechnology J. 6 (7) 722-732.

651Smith, C.J., Hunt, J.R., Wang, E., Macdonald, B.C., Xing, H., Denmead, O.T., Zeglin, S. and
652 Zhao, Z. (2019) Using fertiliser to maintain soil inorganic nitrogen can increase dryland
653 wheat yield with little environmental cost. Agriculture, Ecosystems & Enviro. 286,
654 106644.

655Sohail, M., Hussain, I., Abbas, S.H., Qamar, M. and Noman, M. (2013) Effect of split N
656 fertilizer application on physio-agronomic traits of wheat (Triticum aestivum L.) under
657 rainfed conditions. Pakistan J. Agri. Res. 26 (2).

658Spaner, D., Todd, A.G., Navabi, A., McKenzie, D.B. and Goonewardene, L.A. (2005) Can leaf
659 chlorophyll measures at differing growth stages be used as an indicator of winter wheat

29
660 and spring barley nitrogen requirements in eastern Canada?. Journal of Agronomy and
661 Crop Science, 191 (5) 393-399.

662Staugaitis, G., Vaisvila, Z., Mazvila, J., Arbaciauskas, J., Adomaitis, T. and Augustine Fullen,
663 M.A. (2007) Role of soil mineral nitrogen for agricultural crops: Nitrogen nutrition
664 diagnostics in Lithuania. Archives of Agron. and Soil Sci. 53 (3) 263-271.

665Steel, R.G. and Torrie, J.H. 1986. Principles and procedures of statistics: a biometrical
666 approach. McGraw-Hill.

667Tian, Z., Ge, Y., Zhu, Q., Yu, J., Zhou, Q., Cai, J., Jiang, D., Cao, W. and Dai, T. (2019) Soil
668 nitrogen balance and nitrogen utilization of winter wheat affected by straw management
669 and nitrogen application in the Yangtze river basin of China. Archives of Agro. and Soil
670 Sci. 65 (1) 1-15.

671Tremblay, N., Wang, Z. and Belec, C. (2009) Performance of Dualex in spring wheat for crop
672 nitrogen status assessment, yield prediction and estimation of soil nitrate content. J.
673 Plant Nutr. 33 (1) 57-70.

674Ullah, M.I., Khakwani, A.A., Sadiq, M., Awan, I. and Munir, M. (2015) Effects of Nitrogen
675 Fertilization Rates on Growth, Quality and Economic Return of Fodder Maize (Zea
676 mays L.). Sarhad J. Agri. 31 (1).

677Wang, X. and Xing, Y. (2017) Effects of irrigation and nitrogen on maize growth and yield
678 components. In Global Changes and Natural Disaster Management: Geo-information
679 Technologies 63-74. Springer, Cham.

680Woldesenbet, M. and Haileyesus, A. (2016) Effect of nitrogen fertilizer on growth, yield and
681 yield components of maize (Zea mays L.) in Decha district, Southwestern
682 Ethiopia. International Journal of Research-Granthaalayah, 4 (2) 95-100.

683Wood, C.W., Reeves, D.W. and Himelrick, D.G. (1993) Relationships between chlorophyll
684 meter readings and leaf chlorophyll concentration, N status, and crop yield: a review.
685 In Proceedings of the Agronomy Society of New Zealand (Vol. 23, pp. 1-9).

30
686Wu, W., Ma, B.L., Fan, J.J., Sun, M., Yi, Y., Guo, W.S. and Voldeng, H.D. (2019) Management
687 of nitrogen fertilization to balance reducing lodging risk and increasing yield and protein
688 content in spring wheat. Field Crops Res. 241, 107584.

689Xia, L., Lam, S.K., Yan, X. and Chen, D. (2017) How does recycling of livestock manure in
690 agroecosystems affect crop productivity, reactive nitrogen losses, and soil carbon
691 balance?. Environmental science & technology, 51 (13), 7450-7457.

692Xu, L., Yan, D., Ren, X., Wei, Y., Zhou, J., Zhao, H. and Liang, M. (2016) Vermicompost
693 improves the physiological and biochemical responses of blessed thistle (Silybum
694 marianum Gaertn.) and peppermint (Mentha haplocalyx Briq) to salinity
695 stress. Industrial Crops and Products, 94, 574-585.

696Yi, Q., He, P., Zhang, X.Z., Yang, L. and Xiong, G.Y. (2015) Optimizing fertilizer nitrogen for
697 winter wheat production in Yangtze River region in China. Journal of Plant
698 Nutrition, 38 (11), 1639-1655.

699Yue, X., Hu, Y., Zhang, H. and Schmidhalter, U. (2019) Optimizing the nitrogen management
700 strategy for winter wheat in the North China Plain using rapid soil and plant nitrogen
701 measurements. Communications in Soil Science and Plant Analysis, 50 (11) 1310-1320.

702Zhang, M., Wang, H., Yi, Y., Ding, J., Zhu, M., Li, C., Guo, W., Feng, C. and Zhu, X. (2017)
703 Effect of nitrogen levels and nitrogen ratios on lodging resistance and yield potential of
704 winter wheat (Triticum aestivum L.). PLoS One, 12 (11) e0187543.

705ZHANG, S.Q., Liang, Y.U.A.N., Wei, L., LIN, Z.A., LI, Y.T., HU, S.W. and ZHAO, B.Q.
706 (2019) Effects of urea enhanced with different weathered coal-derived humic acid
707 components on maize yield and fate of fertilizer nitrogen. Journal of Integrative
708 Agriculture, 18 (3) 656-666.

709Zhang, X., Fang, Q., Zhang, T., Ma, W., Velthof, G.L., Hou, Y., Oenema, O. and Zhang, F.
710 (2020) Benefits and trade‐offs of replacing synthetic fertilizers by animal manures in
711 crop production in China: A meta‐analysis. Global change biology, 26 (2) 888-900.

31