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Materials and methods
Downloaded by Gothenburg University Library at 04:36 15 November 2017
Accepted Manuscript
Plant material and experimental site
Seeds of maize (Three Ways Cross (TWC) 329) were obtained from the National Research
Centre at Dokki, Giza, Egypt. Two field experiments were undertaken at the Experimental
Farm, Faculty of Agriculture, Tanta University, Tanta, Egypt (30° 47? N: 31° 0? E) during
the 2015 and 2016 successive summer seasons. The hydro-physical and chemical properties
of the experimental soil are detailed in Table 1. Data on the weather during the whole
course of study are given in Table 2.
Preparation of MLE and SA solutions
Moringa fresh leaves were collected from trees grown in the Experimental Farm, Faculty of
Agriculture, Tanta University, Egypt. Immediately after collection, aqueous extract of
moringa leaves was prepared by mixing 100 g of fresh leaves with a litre of distilled water
(1:10 w/v) in a household blender for 15 min and then filtered through muslin cloth. The
obtained solution was diluted at a ratio of 1:30 (w/v) immediately before application
(Yasmeen et al. 2013 b).
Salicylic acid (SA) solution at 0.5 mmol (Elgamaal & Maswada 2013) was prepared
by dissolving about 69 mg of SA in a little of warm water and supplementing the volume
with a litre of distilled water or MLE at the ratio of 1:30 (w/v). Tween-20 at 0.1% (v/v) was
added, as a surfactant, to all foliar spray treatments immediately before application.
Crop husbandry, treatments, and experimental design
Maize seeds were sown on 25th of May in 2015 and 2016 seasons. The experimental plot
was 20 m2 including 5 rows of 5 m length each and 85 cm width with one plant per hill 25
cm plant spacing (100 plants per plot). Maize plants were subjected to two furrow irrigation
treatments (full irrigation and deficit irrigation). Deficit irrigation was applied by cutting
one irrigation at the vegetative stage, i.e. maize plants under deficit irrigation were
subjected to drought stress by withholding water at the vegetative stage of development (12
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Accepted Manuscript
to 48 days after sowing “DAS”). The applied irrigation water (m3 ha-1) in each irrigation
treatment every season was quantified by using water counter apparatus that attached with
the irrigation machine. The management of each irrigation treatment at each development
stage of maize plants over both seasons (2015 and 2016) is shown in Table3. Nitrogen and
phosphorus fertilizers were applied to maize as recommended by the Egyptian Ministry of
Agriculture (285 kg N ha-1; urea 46.5% N and 357 kg superphosphate ha-1 (15.5% P2O5).
Foliar spray treatments comprising SA at 0.5 mmol, MLE at 1:30 (w/v) ratio and their
combinations, as well as tap water as a control were applied twice, at 30 and 45 DAS, (at
2.5-3.0 L plot-1; i.e. 25 and 30 ml, respectively for each plant) in both seasons. The
experiment was laid out in a randomized complete block design (RCBD) using split-plot
arrangements. Two furrow irrigation treatments (full and deficit irrigation) and four spray
treatments (tap water, MLE, SA, and MLE+SA) were assigned as main plots and sub-plots,
Determination of growth attributes
Five plant samples were randomly collected from each plot at 30 and 48 DAS (before and
after spray treatments, respectively), then divided into roots, leaf blades and aerial non-leaf
blades to estimate growth parameters including root, shoot and plant biomass (determined at
70 °C for 72 h), total leaf area (determined by leaf discs), as well as relative growth rate
(RGR) and net assimilation rate (NAR) that were calculated according to Salisbury (1996)
as follows:
RGR (mg g-1 day-1) = (In m2 – In m1) / (t2-t1)
NAR (mg cm-2 day-1) = (m2 – m1)/ (t2-t1) x (In LA2 – In LA1)/ (LA2 – LA1)
Where m1 and LA1 refer to total plant biomass and leaf area at 30 DAS (t1), respectively;
and m2 and LA2 refer to total plant biomass and leaf area at 48 DAS (t2), respectively. Other
growth parameters including plant height and number of leaves were estimated at 48 DAS.
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Physiological and biochemical attributes
At 48 DAS, the 3rdfully expanded leaf from the top (Anjum et al. 2011a) from each
randomly five plant samples in each plot was collected and separated to estimate
physiological and biochemical parameters including photosynthetic pigments, relative water
content (RWC), proline, membrane permeability (MP) and lipid peroxidation
(malondialdehyde; MDA).
Determination of photosynthetic pigments concentration
Leaf photosynthetic pigments (chlorophyll a, b and carotenoids) were extracted with cold
acetone (80%) according to Maswada & Abd El-Kader (2016). The absorbance of pigments
extract was recorded at 470, 646 and 663 nm wavelengths for carotenoids, chlorophyll a
and b, respectively using UV-VIS Spectrophotometer (Model SM1200; Randolph, NJ,
USA). The concentration of photosynthetic pigments (mg g-1 FW) was calculated using
equations described by Wellburn (1994).
Relative water content and proline estimation
Relative water content (RWC %) of leaf samples was determined according to Weatherley
(1950). Fresh leaves were weighed (FW), then floated on water for 24 h. After removing
superficially adhering water droplets using tissue paper, the turgid weight (TW) was
measured. Afterwards, the leaf discs were dried at 70 °C for 72 h until have a constant
weight (DW). Relative water content was calculated as follows:
RWC (%) = (FW-DW)/ (TW-DW) × 100
Leaf proline concentration was determined spectrophotometrically using 3%
aqueous sulphosalicylic acid and ninhydrin reagent and expressed as ?mol g-1 FW (Bates et
al. 1973).
Determination of membrane permeability and lipid peroxidation
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Membrane permeability of maize leaves was estimated by electrical conductivity method
according to Yan et al. (1996). Briefly, a portion of fresh leaf samples was cut into 1cm
pieces, and then put in a beaker containing distilled water at 30 ºC for 3 h, then the
conductivity of the solution was measured (EC1) using CMD 830 WPA conductivity. After
boiling the samples for 2 min, their conductivity (EC2) was measured again when the
solution was cooled to room temperature. The percentage of membrane permeability (MP
%) was calculated as follows:
MP (%) = (EC1/EC2) × 100.
Malondialdehyde (MDA) concentration was measured by using the thiobarbituric acid
(TBA) protocol (Peever & Higgins 1989) with a slight modification. Briefly, 500 mg of
fresh leaves was homogenized in 10 mL of 0.1 % (w/v) trichloroacetic acid (TCA) in an ice
bath, and centrifuged at 12,000 × g for 15 min. Two mL supernatant was mixed with 2 mL
of 0.5% (w/v) TBA and incubated at 95 ºC for 30 min, and cooled. The absorbance of the
mixture was recorded at 450, 532, and 600 nm wavelengths. The MDA concentration was
calculated and expressed as nmol g-1 FW (Bao et al. 2009).
Yield, yield-related components and water use efficiency
At harvest, ten maize plants from each plot were collected randomly to quantify grain yield
and aboveground biomass (Mg ha-1), plant height (cm), the number of rows per ear, the
number of grains per row, 100-grain weight (g) and harvest index. Irrigation water use
efficiency (WUE) was calculated according to Jensen (1983) as follows:
WUE (Kg m-3) = Grain yield (Kg ha-1) / water applied (m3 ha-1)
Statistical analysis

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