High Nitrogen Fertilization Modulates Morpho-Physiological Responses, Yield, and Water Productivity of Lowland Rice under Deﬁcit Irrigation

: Sustainability of rice production under ﬂooding conditions has been challenged by water shortage and food demand. Applying higher nitrogen fertilization could be a practical solution to alleviate the deleterious effects of water stress on lowland rice ( Oryza sativa L.) in semi-arid conditions. For this purpose, ﬁeld experiments were conducted during the summer of 2017 and 2018 seasons. These trials were conducted as split-split based on randomized complete blocks design with soil moisture regimes at three levels (120, 100 and 80% of crop evapotranspiration (ETc), nitrogen fertilizers at two levels (N 1 —165 and N 2 —200 kg N ha − 1 ) and three lowland Egyptian rice varieties [V 1 (Giza 178 ), V 2 (Giza 177 ) and V 3 (Sakha 104 )] using three replications. For all varieties, growth (plant height, tillers No, effective tillers no), water status ((relative water content RWC, and membrane stability index, MSI), physiological responses (chlorophyll ﬂuorescence, Relative chlorophyll content (SPAD), and yield were signiﬁcantly increased with higher addition of nitrogen fertilizer under all water regimes. Variety V 1 produced the highest grain yield compared to other varieties and the increases were 38% and 15% compared with V 2 and V 3 , respectively. Increasing nitrogen up to 200 kg N ha − 1 (N 2 ) resulted in an increase in grain and straw yields by 12.7 and 18.2%, respectively, compared with N 1 . The highest irrigation water productivity (IWP) was recorded under I 2 (0.89 kg m − 3 ) compared to (0.83 kg m − 3 ) and (0.82 kg m − 3 ) for I 1 and I 3 , respectively. Therefore, the new applied agro-management practice (deﬁcit irrigation and higher nitrogen fertilizer) effectively saved irrigation water input by 50–60% when compared with the traditional cultivation method (ﬂooding system). Hence, the new proposed innovative method for rice cultivation could be a promising strategy for enhancing the sustainability of rice production under water shortage conditions.


Introduction
Rice (Oryza sativa L.) is a staple food crop for about half of the world's population and ranks 2nd in production after wheat [1,2]. Globally, in 2018, the productivity of rice approximately amounted to 700 million tons, which was produced from 167 million hectares, by an average grain yield of 4.2 tons per hectare [2]. Rice (Oryza sativa L.) is being cultivated in various agro-ecosystems: irrigated rice, rainfed lowland rice, upland rice, and flood-prone rice. More than 75% of rice production is supplied by irrigated rates on the activity of photosynthesis and transpiration processes in rice. Application of N fertilizer resulted in a significant increase in grain yield of rainfed-lowland rice under water deficit, where observed the optimal timing of N application for continuously irrigated rice was when the rice was exposed to moderate water deficit before flowering [28]. The absorption and utilization of water and nitrogen nutrition are two coupled physiological processes [29,30]. Supplying plants with N can increase drought resistance by increasing root hydraulic conductivity (Lpr) through increased abscisic acid (ABA) and aquaporin expression [31][32][33][34].
Therefore, the current study hypothesized that exogenous application of N-fertilizer may positively affect the rice performance, irrigation water productivity, chlorophyll fluorescence, water status as well as yield of some drought-stressed lowland rice varieties. Accordingly, the recent investigation was conducted to evaluate the effect of new applied water and nutrition environment on water-saving capacity and productivity of some lowland rice varieties under water-scarce conditions in arid and semi-arid regions.

Experimental Design, Treatments, and Cultural Practices
The current investigation was conducted at the experimental station farm of Faculty of Agriculture, Fayoum University, Egypt, (30 • 56_N latitude, 75 • 52_ longitude). The site is described by arid climate type, hot in summer. The meteorological data in Table 1 show that the highest mean values of maximum temperature, minimum temperature and relative humidity % by 40.4 • C, 26 • C and 45%, respectively, were recorded during August. The maximum pan evaporation rate (mm day −1 ) 7.5 occurred in June month and decreased to its lowest value 5.3 in September during the two successive seasons of rice cultivation (2017-2018).  Table 2 shows that the soil is clay-textured. Soil moisture content % (at 0.33 bar and at 15 bar), available water %, bulk density (g cm −3 ) and saturated hydraulic conductivity (cm h −1 ) were determined at the surface soil layer (0-0.25 m) and amounted to 34.33, 14.60, 19.73, 1.40 and 1.2, respectively, and by 32.19, 13.06, 19.13, 1.36 and 0.9, respectively, a subsurface (0.25-0.5 m).

Experimental Layout and Treatments
Seeds of the three studied lowland rice varieties [V 1 (Giza-178), V 2 (Giza-177) and V 3 (Sakha-104)] were sown manually on 16 May 2017 and 10 May 2018 in rows with seed rate 130 kg h −1 with rows spaced distance 0.25 m. The characteristics of the tested rice varieties were reported by [37] as follows: V 1 (Giza-178): Pedigree (Giza175/Milyang 49), Properties (Indica/Japonica type-tolerant to drought-medium maturing-high yield), grain yield (10 t ha −1 ) growth period (135 days) V 2 (Giza-177): Pedigree (Giza 171/Yomjo No. 1//PiNo.4,) Japonica type-sensitive to drought-short stature-early duration-,grain yield (9 t ha −1 ) and the growth period (125 days) V 3 (Sakha-104): Pedigree (GZ4096-8-1/GZ4100-9-1), Properties (Japonica type-sensitive to drought-), grain yield (10 t ha −1 ) growth period (135 days) Each sub-subplot (experimental unit) involved 5 rows. Agronomic practices for crop management viz fertilizers, pesticides and herbicides applications were implemented according to the recommended practices described by [38]. Fertilizers were manually broadcast then incorporated within the basal application (35 kg P and 50 kg K per hectare). The experiments were conducted in a randomized complete block design (Split-split Plot) in three replicates including three factors. Using a surface irrigation system, three different irrigation regimes as follows: I 1 (120% of ETc), I 2 (100% of ETc) and I 3 (80% of ETc) were applied and allocated to the main plots and two N fertilization levels N 1 (100% of recommended dose (RD) by 165 kg N ha −1 ) and N 2 (125% of RD by 200 kg N ha −1 ), were broadcasted in three equal splits and added at basal, mid-tillering and panicle initiation of rice developing stages and allocated to the sub main plots, where the three lowland rice varieties seeded and allocated to sub-sub main plots. The total experimental area specified for each year was 1134 m 2 divided into 54 experimental plots of 21 m 2 for each. To protect against irrigation and nitrogen fertilizer treatment effects, three meters were utilized to isolate the experimental units.
To convey water for each plot plastic pipe (spiles) of 2 inch diameter was used, and the amount of water delivered through a plastic pipe was calculated according to [40].
where: Q is the discharge of irrigation water (m 3 ), C is the coefficient of discharge, A is a cross-sectional area of irrigation pipe (cm 2 ), g is gravity acceleration (cm s −2 ) and h is the average of the effective head of water (cm) above the pipe.
where E pan : is the evaporation from a class A (mm day −1 ) and K p : is the pan coefficient.

Chlorophyll a Fluorescence (Fv/Fm) and Performance Index (PI) Values
Chlorophyll a fluorescence was determined by plant efficiency analyzer, Handy PEA (Hansatech Instruments Ltd., Norfolk, UK). From each plot, ten fully expanded flag leaves were randomly selected, the leaf samples were dark-adapted for 15 min before being illuminated with irradiance intensity of 3000 µmol/(m 2 ·s) [41,42]. While PI was measured as reported by [43].

Relative Water Content (RWC)
Leaf samples for RWC measurement were randomly collected in the morning (8:00 to 9:00 a.m.). RWC was estimated according to the method described by [44].
where: FW: Fresh weight was measured within two hours after excision of leaves. Turgid weight (TW) was computed by soaking leaves in distilled water and left at room temperature for (16-18 h) then rapidly and carefully blotted dry by tissue paper to determine turgid weight. The small leaf pieces were later oven-dried for 48 h at 70 • C to estimate the dry weight (DW).

Relative Chlorophyll Content (SPAD)
The SPAD meter (SPAD502, KONICAMINOLTA. Inc., Tokyo, Japan) was used to estimate the relative chlorophyll content of the rice.

Membrane Stability Index (MSI %)
The (MSI %) was measured by the method described by [45]. The small leaf strips (0.2 g) of equal size were prepared and taken in two sets of test tubes containing 10 mL of distilled water. The test tubes arranged in one set were maintained at 40 • C in a water bath for 30 min then the ECe of the water covering the leaf samples was estimated (C 1 ). While the test tubes of the other set were incubated in a bath of boiling water at 100 • C for 15 min then measured ECe (C 2 ). The MSI was computed: MSI = [1 − (C 1 /C 2 )] × 100.

Growth, Yield and Yield Components Measurements
All the studied three rice varieties were harvested after 135 days from the sowing date. From each plot, ten plants were selected randomly to determine the plant growth and yield components parameters, i.e., tillers and productive tillers number, plant height (from ground level to the tip of panicle, was measured by meter-scale), length and weight of panicle, number and weight of grains per panicle and 100-grain weight. The grain and straw yield were measured by using digital balance from all plants collected from 1m 2 sampling area.

Grain N Content
Digestion process was performed for the dried grain samples with a mixture consisting of perchloric and nitric acids (at 1:3, v/v, respectively). Using the previous digestion solution, an assessment of N content was performed. Determination of N was performed using the micro-Kjeldahl apparatus (Ningbo Medical Instruments Co., Ningbo, China) following [46] methods.

Statistical Analysis
The obtained data for each variable were subjected to two-way analysis of variance (ANOVA) using GenStat statistical package (12th Ed., VSN International Ltd., Oxford, UK). In case of significant effects, the treatment means were separated using LSD test at p ≤ 0.05 probability level.

Rice Water Status
Results of plant water status (RWC and MSI) in response to irrigation, nitrogen and variety are displayed in Table 3. In both seasons, the highest MSI, RWC values were recorded when rice was subjected to irrigation at 120% of ETc and received 200 kg N ha −1 ; on the other hand, the integrative application of irrigation at 80% of ETc and plants received 165 kg N ha −1 recorded the lowest values over all varieties.
Regarding the effect of varieties, data enumerated in Table 3 revealed that Giza-178 (V 1 ) recorded the highest MSI, RWC values while Giza-177 (V 2 ) gave the lowest values in both seasons. Application of 200 kg N ha −1 to drought-stressed plants up to 20% compensated for this shortage of irrigation and recorded similar values to well-irrigated plants (I 120% ) and received 165 kg N ha −1 . RWC and MSI declined remarkably in both I 100% and I 80% treatment compared with well-watered treatment (I 120% ) [49,50]. We found that RWC and MSI had positive relationships with IWA irrespective of nitrogen applications. The RWC and MSI values were decreased as drought increased. Interestingly, leaf RWC and MSI in higher N treatment were 3.2 and 5.2% higher than that low N treatment, irrespective of the variety effect. In this investigation, N-supply decreased the detrimental effects of water stress on rice plants and kept their RWC and MSI values at close levels as in well-watered plants (Table 3). In the present study, the adverse changes that occurred in the health of cell membranes under drought stress were assessed. Our results revealed that higher N-application plays an important role in stabilizing membrane integrity and maintaining cell turgor of rice leaves under drought stress. In this respect, increases of tissue RWC and MSI as metabolically available water seems to maintain tissue health and may reflect on the metabolic processes in rice under water stress, which agrees with the results of [34,51], who stated that, N supply under drought stress improved RWC and MSI as well as enhanced the photosynthetic efficiency with increased grain yield of wheat plants. Our results agree with those of [48,52]. They reported that drought stress severely affected and reduced rice growth, photosynthesis, stomatal conductance. Ref. [53] reported drought adversely affects physiological responses of plants through a reduction in gas exchange especially stomatal conductance, photosynthetic pigments and overall crop water status.

Leaf Physiological Traits
For all varieties, physiological traits like relative chlorophyll content (SPAD value), chlorophyll fluorescence (Fv/Fm) and performance index (PI) were significantly improved in drought stress-treated rice plants under high N application (Table 3). Drought stress (80% of ETc) reduced the values of SPAD (18.7%), Fv/Fm (5.5%), and PI (46.1%) as compared to plants irrigated at 120% of ETc (Table 3). All physiological traits were significantly decreased under drought [17,54]. However, rice plants that received high N concentrations (200 kg ha −1 ) significantly increased SPAD (7.3%), Fv/Fm (3.8%), and PI (45.3%), as compared to low N concentration, over all varieties as average for both seasons. Moreover, results in Table 3 showed that the highest SPAD, Fv/Fm, and PI values were recorded in Giza 178 (V 1 ), while the lowest values were recorded under Giza 177 (V 2 ) for both seasons. The best results of these traits were recorded in Giza 178 with application of 200 kg N ha −1 + I 120 of ETc. Our results suggest that the application of high N concentration can mitigate the negative effects of water stress on SPAD, Fv/Fm, and PI and as a result of increasing the photosynthetic efficiency of rice plants. Similar trends were reported by [16,[55][56][57][58].

Plant Growth Characteristics
Results presented in Table 4 showed that the applied irrigation regimes, nitrogen fertilization levels and their interaction significantly affected the plant height and the number of effective tillers. Applying water stress level (I 80%) significantly reduced plant height, tillers No, effective tillers no and panicle length by 9.45, 21.54, 21.12 and 10.58% compared by control (I 120%). Increasing the amount of the applied irrigation water and nitrogen dose at (I 120 × N 2 ) treatment gave the greatest values of plant height and effective tillers. Tillers number in particular fertile tillers is considered one of the most important components of yield. The observed increase in number of fertile tillers in the current investigation might be related to the higher availability of the nitrogen element that played a vital role in cell division. On the contrary, the lowest estimation for these parameters was observed at (I 80 × N 1 ) treatment. Drought stress may cause various structural and functional disruptions in reproductive organs [20]. Among the grown rice varieties (Giza 178 ) recorded the maximum plant height (76.14 cm) and effective tillers number (2.75) as compared with other varieties. The increases in both plant height and produced effective tillers under (I 1 × N 2 ) treatment could be due to the availability of water and nitrogen resulting in better translocation of photosynthesis, higher cell deviation and there by favored highest yield attributes under these treatments. The obtained results were in line with [59] who observed that increasing nitrogen fertilizer resulted in drier matter accumulation. Ref. [60,61] found that the irrigation regime and N application significantly affected rice yield and yield traits. In addition, increasing the N uptake may have beneficial effects on plants grown under drought conditions, where the plant drought resistance increased with increasing N supply resulted in increasing root hydraulic conductivity (Lpr) through increased abscisic acid and aquaporin expression [30,31].

Yield Components
Yield components of, i.e., panicle length (cm), grains number per panicle, panicle weight, weight of grain per panicle (g) and weight of 100 grain (g), were significantly affected by irrigation and nitrogen nutrition treatments. Results in Table 5 showed that rice variety Giza 178 yielded the highest values of these traits under irrigation and nutrition level (I 120 × N 2 ), while the lowest values were yielded by Giza 177 under (I 80 × N 1 ). Among irrigation regimes (I 120 ) produced the highest averages of these traits, that gradually reduced by increasing drought level. The reduction percent amounted to 5.48, 7.78, 13.63, 18.41 and 11.07% respectively, as compared with those obtained at moderate stress level (I 100 ) and by 10.57, 17.83, 29.66 32.61and 18.38% respectively compared to (I 80 ). The obtained results were in line with those reported by [52] who found that yield components of rice crop (panicle number, panicle length, 100-grain weight and grain yield) decreased with increasing water stress especially if it occurred at the stage of panicle initiation. Ref. [17,62] noted that depending upon the stress severity the plant physiological responses, the apical development rate, biomass production, panicle development, spikelet number, and grain yield were decreased.
However, applying nitrogen fertilization at N 2 resulted in an increase in panicle length (cm), grains number per panicle, panicle weight, weight of grain per panicle (g) and weight of 100 grain (g) by 5.72, 10.81, 12.85, 11.91 and 5.21% as compared to N 1. Inter varietal comparison showed that, yield component traits were significantly differed in both seasons. Results are in agreement with those revealed by [63,64] who noted that the application of N fertilizers significantly increased the yield and yield components of rice.
However, nitrogen application gave an appositive effect and improved the productivity of all grown rice varieties. Data in Table 6 showed that increasing the nitrogen fertilization dose from N 1 to N 2 resulted in an increase in grain, straw and biological yield by 12.66, 18.20 and 15.99%, respectively . Nitrogen application could produce promoted root growth, enhanced water and nitrogen extraction from soil, resulting in better crop growth and higher yield productivity. Similar trend was noted by [68] who observed an increase in grain yield with the increasing of nitrogen application levels. The combined effect of (I × N) on grain and straw was found to be significant during both seasons except their effect on straw yield in the second year was non-significant. The effect of (V × N) during two seasons was non-significant. Meanwhile, the effect of (I × V × N) was significant.

Irrigation Water Applied and Water Productivity
Results in Table 7 show that total water applied was varied between the applied irrigation treatments. The lowest amount of irrigation water (646 mm ha −1 ) required was at (I 80 ), while the highest (970 mm ha −1 ) was needed for (I 120 ). According to the grain yield obtained under each watering treatment, the applied irrigation water and crop water consumption, irrigation water productivity (IWP) and crop water productivity (CWP) were significantly differed (p ≤ 0.05) in both growing seasons. Among irrigation treatments watering at (I 100 ) gave the highest values of IWP and CWP by 0.89 and 1.28 (kg m 3 ), respectively, when comparing with other irrigation treatments. In addition, increasing nitrogen application dose up to N 2 resulted in higher IWP and CWP by 12.50 and 12.17%, respectively, than N 1 (as average for both seasons). Between rice varieties, Giza 179 corresponding to its high grain productivity which resulted in the highest value as averages of IWP (0.99 Kg m −3 ) and CWP (1.41 kg m −3 ) meanwhile, the lowest IWP and CWP amounted 0.71 and 1.02 kg m −3 , respectively, noted for Giza 177 . Table 6. Effect of deficit irrigation, nitrogen fertilizers varieties, and their interaction on grain yield, straw yield, biological yield, and grains N content of rice plants in 2017 (S I ) and 2018 (S II ) seasons.  Table 7. Effect of deficit irrigation, nitrogen fertilizer varieties, and their interaction on grain yield, straw yield, biological yield, irrigation water productivity and crop water productivity of rice plants in 2017 (S I ) and 2018 (S II ) seasons. Many studies have been summarized that rice cultivation under flooded condition consumes approximately 20,000 m 3 ha −1 [7]. In North Delta, Egypt [69] estimated water requirements for flooded-rice by 19,000 m 3 ha −1 and water utilization efficiency by 48%. Similar results reported by [70] who found that rice normally needs under traditional methods in Egypt a water application of about 20,000 m 3 ha −1 . Studies conducted by [11] concluded that the water requirement of paddy fields in Egypt is about 1800-2200 mm ha −1 . Therefore, compared with previous studies, the current investigation aimed to create new environmental cultivation conditions to grow lowland rice varieties (aerobically) in nonpuddled fields under non-flooded conditions and maintain a profitable grain yield could save irrigation water by 60%. Moreover, results concluded that lowland rice variety Giza 178 was observed to be more tolerant than other varieties, Giza 177 and Sakha 104 , while v. Giza 177 was much affected by deficiency of irrigation water. A similar trend was reported by [3]. Tables 8 and 9 illustrated the correlation coefficients between rice grain yield and the other yield components. This type of examination could be used as an appropriate instrument to indicate which one of them is positive and greatly associated with the obtained yield of grains.  It was observed that grain yield was positive and strongly correlated with plant height, spike length, grain no/spike then followed by spike weight, which validates their economic importance. Data in Table 9 show that plant height and spike weight in both seasons were significantly (p ≤ 0.01) contributed to variations in rice grain yield.

Conclusions
Cultivating rice aerobically under deficit irrigation has been observed to be used as an effective and efficient approach in saving irrigation water and improving IWP compared with the flooding system. Additionally, improving nitrogen supply enhanced rice crop productivity under deficit irrigation conditions. The combined effect of (I 120 × N 2 ) treatment achieved the greatest values of all agronomic and physiological traits, grain and straw yield of rice. Applying of irrigation water at (I 2 ) produced the maximum (IWP) and (CWP) by 0.89 and 1.28 kg m −3 , respectively, compared with other irrigation schedules. Rice variety (Giza 178 , V1), gave the highest CWP and grain yield which amounted to 1.41 and 7.97, respectively. Consequently, it could be a suitable genotype for improving rice productivity under drought conditions as compared with other tested rice varieties. Thus, based on the results of the current study the incorporating deficit irrigation technique at level (100 of ETc) with nitrogen application at 120% of recommended dose could be a valuable agro-management strategy for maintaining relatively high yields of some sensitivedrought Egyptian rice varieties and saving irrigation water supply by 50-60% compared to the conventional rice cultivation method.

Data Availability Statement:
The data presented in this study are available upon request from the corresponding author.