An Assessment of Moringa (Moringa oleifera L.) Seed Extract on Crop Water Productivity and Physico-Biochemical Properties of Cancer Bush (Sutherlandia frutescens L.) under Deficit Irrigation

Abstract

Water deficit is a main abiotic stress limiting the cultivation of many plants including cancer bush (Sutherlandia frutescens L.), which is a traditional medicinal plant used to treat various diseases such as tuberculosis, cancer, diabetes and asthma. Natural plant growth hormones are a cost-effective and environmentally friendly alternative to synthetic growth regulators for plant production under favourable or adverse conditions. Thus, the current study investigated the biostimulant effect of moringa (Moringa oleifera L.) seed extract (MSE) on physiological and biochemical attributes, including crop water productivity (CWP) of cancer bush grown under deficit irrigation. The 2% MSE was foliar-sprayed to cancer bush plants subjected to full (100% of soil water holding capacity (SWHC)) and deficit irrigation (DI) (80, 60 and 40% of SWHC) in a pots experiment which was conducted and repeated twice consecutively in a tunnel. Plants that were not treated with MSE were considered as control. The results on water-deficit stress showed that the performance of cancer bush was significantly reduced in terms of growth and yield attributes, CWP, as well as physico-biochemical properties. Nevertheless, the foliar application of MSE on water-stressed plants effectively enhanced growth and yield characteristics, CWP, leaf photosynthetic pigments (chlorophyll “a”, chlorophyll “b”, total chlorophylls and total carotenoids), antioxidant activity (2′-Diphenyl-1-picrylhydrazyl and 2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonic acid), relative water content (RWC) and membrane stability index (MSI) of cancer bush plants compared to respective controls. Therefore, the 2% MSE application was effective in mitigating negative impact of drought stress in cancer bush plants by maintaining higher RWC, MSI, CWP and biochemical attributes.

1. Introduction

Cancer bush (Sutherlandia frutescens L.) is an important medicinal plant of the Fabaceae family [1]. It is commonly used in traditional medicine to treat various diseases such as immunodeficiency virus infection/acquired immune deficiency syndrome, tuberculosis, cancer, diabetes and asthma [2,3]. It has been documented that cancer bush contains anticancer, antimicrobial and antiviral pharmacological properties which could be attributed to the presence of phytochemicals such as antioxidants, flavonoids and amino acids. In addition, flavonoids such as kaempferol and quercetin have been reported as vital contributors to the various pharmacological activities of this species [1,3]. However, the cultivation of medicinal plants is severely limited by several environmental stresses including drought and heat stresses [1,4].

Water-deficit stress can directly affect photosynthesis, plant development, nutrient uptake and osmotic adjustment, thus consequently reducing plant yield [5,6]. Under water-deficit stress, plants overproduce the reactive oxygen species (ROS), superoxide ion (O₂^•−), hydrogen peroxide (H₂O₂), hydroxide ion (OH⁻) and singlet oxygen (¹O₂) in chloroplasts, peroxisomes and mitochondria [7]. The overproduced ROS negatively affects stomatal conductance and consequently transpiration and photosynthetic rates and plant growth, thus leading to the death of plant cells, crop failure and economic losses [8,9]. The elevated oxidative stress resulting from overproduced ROS requires the antioxidant mechanism of the plant species to be effective in order to strike a balance. Hence, interventions to mitigate the water-deficit stress may include among others the stimulation of the antioxidant response mechanism in plants [10].

Irrigation water especially for agricultural cultivation has been significantly reduced globally owing to climate change, rapid population growth and various human activities leading to water-deficit stress conditions for crops and plants [11,12]. Therefore, there is a need to explore environmentally friendly strategies such as plant-derived biostimulants to improve plant growth under stress conditions [8,13]. Furthermore, when adopting deficit irrigation (DI) strategy, the application of biostimulants helps plants to resist the negative impact of drought [13,14].

Biostimulants include plant extracts which comprise extensive variety of phytochemicals such as phytohormones, antioxidants, fatty acids, proteins, minerals and vitamins [15,16]. These compounds have the potential to improve plant growth and productivity and to alleviate the negative effects of abiotic stresses [4,14,17].

Moringa (Moringa oleifera L.) is one of the most nutritious, effective and current biostimulants used to improve plant growth and yield [15,17]. The moringa leaf extract (MLE) contains effective plant growth hormones such as auxins, gibberellins (GAs) and cytokinins (CKs) as well as a variety of phytochemicals including antioxidants and essential minerals [18,19]. These compounds are responsible for improving plant metabolism and thereby tolerance to environmental stress [17,19]. The MLE has been reported to improve growth and productivity of various crops when applied in both normal and abiotic stress conditions [14,20]. Abd El-Mageed et al. [11] reported that 3% MLE foliar spray effectively improved drought resistance in squash (Cucurbita pepo L.) compared to untreated plants grown under soil salt and drought stress conditions. Another study by Khan et al. [20] demonstrated that 3% MLE significantly enhanced growth and productivity including yield of wheat (Triticum aestivum L.) compared to untreated plants. Similarly, Abdalla [21] showed that MLE and twig extract (2% and 3%, respectively) effectively improved biomass production, photosynthetic pigments, total sugars, total proteins, growth promoting hormones and various essential mineral elements of rocket (Eruca vesicaria L.) compared to untreated plants. Although moringa aerial parts such as leaves, flowers and seeds are a rich source of plant growth hormones and phytochemicals and could be an effective source of biostimulant extracts, previous research has mainly focused on the potential of its leaves, whereas little is known about moringa seed extract (MSE) [22,23]. Therefore, the current study investigated the potential of MSE on enhancing water-deficit stress tolerance by evaluating growth and yield parameters and chemical composition in cancer bush plants grown under DI.

2. Materials and Methods

2.1. Experimental Site and Plant Materials

This experiment was conducted and repeated twice consecutively in the tunnel of the School of Science and Technology, Sefako Makgatho Health Sciences University (SMU), South Africa (latitude: 25°37′8″ S, longitude: 28°1′22″ E and altitude: 1276 m) during July ̶ September 2021 (first trial) and October–December 2021 (second trial). The tunnel was 4 m high, 8 m long and 4 m wide covered with a green photo-selective coloured net (40% shading) (ChromatiNet™, Carports and Pergola Builders (Pty) Ltd., Pretoria, South Africa). The seedlings of cancer bush were purchased from Plant & Palm Kwekery nursery in Akasia, Pretoria, South Africa (latitude: 25°39′50.6″ S, longitude: 28°08′01.1″ E and altitude: 1300 m). The seedlings were then placed in the tunnel at SMU where they were equally irrigated three times a day with 200 mL tap water per plant for a period of one week before they were transplanted into the pot plants. Thereafter, only healthy seedlings without any visible defects were selected and gently removed from seedlings trays and immediately transplanted into plastic terracotta pots (40 cm in diameter and 50 cm depth), filled with 5 kg of culterra potting soil per pot.

The culterra potting soil used in the experiment was purchased at Builders Express, north of Pretoria, South Africa (latitude: 25°40′28.49″ S, longitude: 28°6′31.22″ E and altitude: 1305 m). This product was formulated from raw organic material derived from one or more of the following products: COCO peat, forest products and water retentive agents. Specific ingredients of the culterra potting soil included general 2:3:2 (22), lawn 8:1:5 (25), LAN (28%), ammonium sulfate (21%), vita flora 5:1:5 (33) SRN and vital flora 3:1:5 (26) SRN per 30 kg of soil.

2.2. Microclimate and Photosynthetic Active Radiation (PAR) Quantifications

The photosynthetic active radiation (PAR) under the green shade net was recorded using PAR meter (LI-COR 250A Light Meter, LI-COR Biosciences, Cambridge, UK). The air temperature and relative humidity (RH) were measured using data loggers (Gemini data loggers Ltd., West Sussex, UK) ([24],

Table 1. Air temperature, relative humidity and photosynthetic active radiation measurements inside the tunnel during the growing season of the study (July–December 2021).

Air Temperature (°C)	Relative Humidity (%)	Photosynthetic Active Radiation (μmol m−2 s−1)
21.08 ± 0.60	43.01 ± 0.57	234.07 ± 0.07

).

2.3. Irrigation Water Applied

The utmost quantity of water that can be held by the soil is commonly referred to as soil water-holding capacity (SWHC), and was determined using Equation (1), according to Taha et al. [25] as follows:

$$SWHC \left(\%\right)=total porosity \left(\%\right)-air space \left(\%\right)$$

(1)

The four irrigation levels; 100, 80, 60 and 40% of SWHC were applied during the entire experiment after transplanting (all pots were well-watered three times per day with 200 mL of tap water before and after transplanting). The full irrigation level (100% of SWHC) was used as an ideal required irrigation water for optimum growth of cancer bush plants, whereas other treatments (80, 60 and 40% of SWHC) were considered as deficit irrigation levels. The DI levels were applied to cancer bush plants as stress (drought) levels one day after transplanting, three times per day, in the morning (8:00), noon (12:00) and afternoon (16:00). Soil moisture content of pots was monitored daily by HH2 moisture meter Version 4.0 (Delta-T Devices Ltd., Cambridge, UK) and maintained through water application where applicable.

2.4. Moringa Seed Extract (MSE) Preparation and Treatments

Moringa seeds were harvested from the commercial orchards of Afrinest Moringa Farm in Tzaneen, Limpopo, South Africa (latitude: 23°49′15.3″ S, longitude: 30°10′08.7″ E and altitude: 719 m). The seeds were ground into fine powder using a blender (RSH-080359 B, Game, Pretoria, South Africa) and were extracted using a method described by Taha et al. [25], with slight modifications. Each 100 g of moringa seed powder was accurately mixed with 1 L 80% aqueous ethanol and vortexed using vortex mixer (GS-GVM-AS, Air & Vacuum Technologies, Johannesburg, South Africa) for 30 s. Thereafter, the mixture was kept aside for about 6 h with constant starring and then left at ambient conditions overnight. Afterwards, the mixture was centrifuged (laboratory centrifuge-TD4C, Labtex Co., Ltd., Bangladesh, China) at 8000× g for 15 min and filtered through Whatman^® no. 1 filter paper. Subsequently, the supernatant was diluted with distilled water (v/v) to obtain the required concentration of 2% MSE to use as a foliar spray. The freshly prepared extract was then used within 24 h or kept in the refrigerator at −20 °C for further use. Cancer bush plants were sprayed with 2% MSE once per week after transplanting. To ensure optimal penetration of MSE into the leaf tissue, 0.1% (v/v) Tween-20 was added to the foliar spray as a surfactant while the control plants were sprayed with tap water comprising the same surfactant at the same time of MSE treatment. The analysed chemical composition of MSE is presented in

Table 2. Chemical constituents of M. oleifera seed extract (MSE; on a dry weight basis).

Component	Unit	Value
Protein	g 100 g−1	30.44 ± 1.11
Total free amino acids		28.11 ± 0.56
Free proline		0.25 ± 0.03
Soluble sugars		10.11 ± 0.37
Vitamin B1	mg 100 g−1	0.12 ± 0.04
Vitamin B2		0.04 ± 0.01
Vitamin B3		0.12 ± 0.04
Vitamin C		2.92 ± 0.16
Vitamin E		490.35 ± 5.47
Calcium (Ca)		35.08 ± 0.13
Magnesium (Mg)		509.12 ± 0.64
Phosphorus (P)		59.96 ± 0.27
Copper (Cu)		4.19 ± 0.59
Sulphur (S)		0.02 ± 0.00
Cytokinins (CKs)	µg g−1	0.94 ± 0.04
Gibberellins (GAs)		0.85 ± 0.01

Values are the mean ± SE.

.

2.5. Experimental Design and Plant Management

Each experiment was repeated twice and was arranged in a randomised complete block design (RCBD). Intra-row pot spacing was 40 cm, whereas inter-row spacing was 50 cm [11]. Pots of each of the 4 irrigating treatments (fully irrigated plants; 100% of SWHC and DI-stressed plants; 80, 60 and 40% of SWHC) were arranged for MSE foliar spray as follows: (1) full irrigation with 100% of SWHC + foliar spray with tap water (control) and full irrigation with 100% SWHC + foliar spray with MSE, (2) DI with 80% SWHC + foliar spray with tap water and DI with 80% SWHC + foliar spray with MSE, (3) DI with 60% SWHC + foliar spray with tap water and DI with 60% SWHC + foliar spray with MSE and (4) DI with 40% SWHC + foliar spray with tap water and DI with 40% SWHC + foliar spray with MSE. The four treatments were replicated five times, making a total of 40 pots per experiment. Plants were trellised using trellis twine two weeks after transplanting to support and vertically uphold the main stem upright.

2.6. Measurements of Plant Growth and Yield Attributes and Crop Water Productivity (CWP)

Plant height was measured at harvest using a measuring tape placed at the base of the stem to the tip of the leaves, and stem diameter was measured at harvest using a caliper (150 mm LCD, Kawasaki, Japan) [26]. The leaf area index was determined using a portable leaf area meter (LAM-B, Biobase Biodustry (Shandong) Co., Ltd., Jinan, China) at harvest by taking four readings per plant (two reading above the canopy and the other two readings below canopy) [11]. The number of branches per plant were counted at harvest [25]. Cancer bush plants were harvested at 12 weeks for all the experimental treatments. Roots and shoots were separated and weighed for both fresh and dry mass using a digital scale (KB 10K0.05N, KERN, Midrand, South Africa). At harvest, the roots were thoroughly rinsed with distilled water. Thereafter, plant materials were oven dried (Oven AP 60L 230V-OGH60, Thermo Fisher Scientific, Randburg, South Africa) at 50 °C to constant weight to measure both shoot and root dry weight [11]. Yield and yield components such as number of fruit/plant and biomass yield/plant were measured after harvest [8]. In a gram of dry matter of cancer bush plant per litre of irrigation water applied, values of crop water productivity (CWP) were calculated at 84 days after transplanting following a method of Taha et al. [25] using Equation (2).

$$Crop water productivity \left(CWP\right)=\frac{cancer bush yield \left(g plan{t}^{-1}\right)}{water applied \left(L plan{t}^{-1}\right)}$$

(2)

2.7. Measurement of Relative Water Content (RWC)

Relative water content (RWC) was measured from three randomly selected plants per treatment (both none-stressed and DI-stressed plants) following a method described by Soltys-Kalina et al. [27] and using Equation (3).

$$RWC \left(\%\right)=\left[\frac{\left(FM-DM\right)}{\left(TM-DM\right)}\right]\times 100$$

(3)

where RWC is relative water content (%), FM is the fresh mass (g), TM is the turgid mass (g) and DM is the dry mass (g).

2.8. Measurement of Membrane Stability Index (MSI)

Measurement of membrane stability index (MSI) was determined using Equation (4), according to Abd El-Mageed et al. [11] as follows:

$$MSI \left(\%\right)=\left[1-\left(\frac{C_1}{C_2}\right)\right]\times 100$$

(4)

where MSI is the membrane stability index, C₁ is the electrical conductivity (EC) of the solution at 40 °C, and C₂ is the EC of the solution at 100 °C.

2.9. Determination of Leaf Photosynthetic Pigments

Chlorophylls and carotenoids contents were evaluated based on Lima et al. [28], with minor alterations. A sample of 2 g of fresh leaves was homogenized in 50 mL 80% (v/v) acetone. Afterwards, the mixture was centrifuged at 10,000× g for 15 min and the supernatant was filtered through Whatman^® no. 1 filter paper. The measurement of pigments was performed using a UV–visible spectrophotometer (UV-1700, Shimadzu, Johannesburg, South Africa) at the following wavelengths: 663 nm (chlorophyl-a), 647 nm (chlorophyl-b) and 470 nm (carotenoids). Total chlorophyll and carotenoids contents were calculated using Equation (5). Pigment concentration was expressed in micrograms of pigment per gram of tissue fresh weight (μg g⁻¹ FW).

$$Ch{l}_{a }=12.25\times A_{663.20-2.79}\times A_{646.80}$$

(5a)

$$Ch{l}_{b }=21.50\times A_{646.80-5.10}\times A_{663.20 }$$

(5b)

$$Ch{l}_{a+b }=7.15\times A_{663.20+18.71}\times A_{646.80}$$

(5c)

$$C_X=\frac{1000\times A_{470.00-1.82}\times Ch{l}_{a-85.02}\times Ch{l}_b}{198}$$

(5d)

where Chl_a is Chlorophyll a, Chl_b is Chlorophyll b, Chl_a+b is total chlorophylls, and C_x is total carotenoids.

2.10. Sample Extraction

Freeze-dried leaves were ground into fine powder using pestle and mortar. Sample extraction was then carried out according to Khatri et al. [29], with some alterations. A 1 g of leaf powder was mixed with 10 mL 80 % (v/v) methanol. Afterwards, the mixture was vortexed and sonicated (ultrasonic cleaning baths-702, SCIENTECH, Boulder, CO, USA) for 15 min and centrifuged at 20,000× g for 30 min. The supernatant was then filtered through Whatman^® no. 1 filter paper and used for antioxidants assays.

2.10.1. 2,2′-Diphenyl-1-picrylhydrazyl (DPPH) Assay

The 2,2′-diphenyl-1-picrylhydrazyl (DPPH) assay was measured according to Ehteshami et al. [30], with slight modifications. Briefly, 50 µL of the methanol extract was accurately mixed with 950 µL DPPH solution by dissolving 0.025 g DPPH in 100 mL 85% (v/v) methanol. The solution was then incubated in the dark at room temperature for 30 min, and the absorbance was read at 517 nm using the UV–VIS spectrophotometer. The antioxidant activity was calculated using Equation (5).

$$Antioxidant activity \left(\%\right)=\left[1-\frac{Abs sample}{Abs control}\times 100\right]$$

(6)

where Abs is the absorbance.

2.10.2. 2,2′-Azinobis-3-ethylbenzothiazoline-6-sulfonic Acid (ABTS) Assay

The 2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS⁺) assay was assessed according to Tesfay et al. [31], with minor modifications. The ABTS was dissolved in purified water to reach 7 mM concentration. The ABTS radical cation was produced by reacting the 7 mM ABTS solution with 2.45 mM ammonium persulfate for about 6 h. Afterwards, the solution was diluted with ethanol until it reached the absorbance of 0.70 ± 0.02 at 734 nm. Subsequently, 1.0 mL activated ABTS solution was added to 10 μL sample solution from extracts of freeze-dried material in acetate buffer (pH 4.0). The mixture was then incubated in the dark at ambient conditions for 15 min before the absorption was measured at 734 nm, and the results were expressed as the clearance rate as ABTS radical scavenging (%).

2.11. Statistical Analysis

GenStat statistical software (GenStat^®, 18.1 edition, VSN International, Hertford, UK) was used to perform the analysis of variance (ANOVA) of the collected data. The noted differences at p < 0.001 were considered statistically significant according to Duncan’s multiple range test. Mean (±S.E) values of all the studied variables were presented.

3. Results

3.1. Microclimate and Photosynthetic Active Radiation (PAR)

The recorded microclimate data (air temperature and relative humidity) and photosynthetic active radiation (PAR) data are presented in Table 1. The temperature, relative humidity (RH) and PAR under the green shade net were 21.08 °C, 43.01% and 234.07 μmol m⁻² s⁻¹, respectively.

3.2. Growth and Yield Parameters

The growth and yield parameters in fully irrigated and water deficit plants are shown in

Table 3. Response of growth and yield attributes of S. frutescens to foliar spray application of M. oleifera seed extract under deficit irrigation conditions.

Treatments	Plant Height (cm)	Stem Diameter (cm)	Leaf Area Index	Number of Branches	Shoot Dry Weight (g)	Root Dry Weight (g)
100% of SWHC (C)	35.05 ± 0.61 bc	3.61 ± 0.36 bc	52.12 ± 1.57 d	24.00 ± 2.65 c	12.33 ± 0.88 b	2.02 ± 0.06 b
100% of SWHC (MSE)	50.12 ± 0.99 e	4.80 ± 0.33 d	60.38 ± 1.43 e	35.33 ±1.45 d	18.33 ± 1.45 c	3.01 ± 0.26 c
80% of SWHC (C)	33.19 ± 1.44 b	3.08 ± 0.25 abc	40.11 ± 1.07 c	26.00 ± 1.73 c	13.04 ± 0.20 b	1.81 ± 0.11 b
80% of SWHC (MSE)	49.04 ± 2.53 e	4.68 ± 0.32 d	54.16 ± 1.07 d	34.00 ±0.58 d	17.16 ± 0.54 c	2.89 ± 0.19 c
60% of SWHC (C)	25.03 ± 1.15 a	2.92 ± 0.43 ab	34.07 ± 0.49 b	18.00 ± 2.08 b	8.05 ± 0.30 a	1.61 ± 0.31 ab
60% of SWHC (MSE)	40.35 ± 0.89 d	4.02 ± 0.29 cd	40.34 ± 2.67 c	23.00 ± 1.15 c	11.14 ± 0.19 b	1.80 ± 0.06 b
40% of SWHC (C)	20.38 ± 2.63 a	2.52 ± 0.45 a	29.37 ± 0.89 a	13.00 ± 1.15 a	6.36 ± 0.64 a	1.03 ± 0.24 a
40% of SWHC (MSE)	39.24 ± 0.68 cd	3.52 ± 0.08 abc	34.08 ± 1.75 b	18.00 ± 0.58 b	8.26 ± 0.39 a	1.62 ± 0.16 ab

SWHC, soil water holding capacity; C, control; MSE, moringa seed extract. Values are presented as mean ± SE. Different letters among treatments for each attribute are significantly different (p < 0.001) according to Duncan’s multiple range test.

and Figure 1. The treatment of cancer bush plants with MSE under full irrigation (100% of SWHC) effectively (p < 0.001) increased all evaluated growth and yield attributes in comparison to the respective control (Table 3; Figure 1). This treatment effectively enhanced plant height (50 cm), stem diameter (4.08 cm), leaf area index (60), number of branches (35), shoot dry weight (18.01 g), root dry weight (3 g), number of fruit/plant (30) and biomass yield/plant (680 g) compared to corresponding control. Deficit irrigation gradually (p < 0.001) decreased all assessed growth and yield attributes in contrast to full irrigation (100% of SWHC). Nevertheless, applying MSE effectively (p < 0.001) increased all measured growth and yield parameters of water-stressed plants contrary to respective controls. The application of MSE on D1-stressed plants (80, 60 and 40% of SWHC) significantly (p < 0.001) increased plant height (49, 40 and 38 cm), stem diameter (4.6, 4.0 and 3.5 cm), leaf area index (54, 40 and 34), number of branches (34, 23 and 18), shoot dry weight (17.1, 11.2 and 8.4 g), root dry weight (2.8, 1.8 and 1.6 g), number of fruit/plant (28, 22 and 15), biomass yield/plant (651, 500 and 460 g) compared to corresponding controls. The positive influence of MSE application was more evident under DI compared to its application under full irrigation. In addition, there were no or minor significant differences between all evaluated growth and yield attributes of fully irrigated plants and those of water-stressed plants sprayed with MSE.

3.3. Membrane Stability Index (MSI), Relative Water Content (RWC) and Crop Water Productivity (CWP)

Foliar application of MSE on the fully irrigated plants (100% of SWHC) significantly (p < 0.001) improved MSI (67%) and RWC (80%) compared to respective controls. However, this treatment resulted in lower CWP (1.38 g DW/L of irrigated water) compared to corresponding control (

Table 4. Crop water productivity (CWP) and plant water status of S. frutescens plants treated with M. oleifera seed extract (MSE) under deficit irrigation conditions.

Treatment	CWP (g DW/L of Irrigated Water)	RWC (%)	MSI (%)
100% of SWHC (C)	1.17 ± 0.44 ab	76.11 ± 2.21 d	58.33 ± 3.84 bcd
100% of SWHC (MSE)	1.39 ± 0.15 b	80.03 ± 0.93 d	67.33 ± 4.91 d
80% of SWHC (C)	1.087 ± 0.06 ab	64.06 ± 3.55 bc	55.33 ± 3.28 bc
80% of SWHC (MSE)	1.483 ± 0.02 b	78.39 ± 0.89 d	63.33 ± 1.45 cd
60% of SWHC (C)	0.957 ± 0.03 ab	60.40 ± 0.98 b	50.33 ± 1.76 ab
60% of SWHC (MSE)	1.603 ± 0.21 b	69.35 ± 0.52 c	56.33 ± 3.76 bc
40% of SWHC (C)	0.617 ± 0.13 a	44.94 ± 3.02 a	44.33 ± 0.88 a
40% of SWHC (MSE)	1.623 ± 0.38 b	58.05 ± 1.57 b	49.33 ± 0.88 ab

C, control; SWHC, soil water holding capacity; MSE, moringa seed extract; CWP, crop water productivity; RWC, relative water content; MSI, membrane stability index. Data presented as mean ± SE. Different letters among treatments for each attribute are significantly different (p < 0.001) according to Duncan’s multiple range test.

). Furthermore, DI (80, 60 and 40% of SWHC) gradually (p < 0.001) decreased all the above-mentioned attributes. However, the application of MSE on DI-stressed plants (80, 60 and 40% of SWHC) effectively (p < 0.001) enhanced MSI (63%, 56% and 49%), RWC (78%, 69% and 58%) and CWP (4.48 g DW/L of irrigated water, 0.96 g DW/L of irrigated water and 1.62 g DW/L of irrigated water) in comparison to respective controls. The effectiveness of MSE application on enhancing MSI, RWC and CWP was more distinctive on DI-stressed plants compared to none-stressed plants. There were minor significance variations amongst the aforementioned attributes of none-stressed plants and those of water-stressed plants sprayed with MSE.

3.4. Photosynthetic Pigments

Table 5. Leaf photosynthetic pigments of S. frutescens plants treated with M. oleifera seed extract (MSE) under deficit irrigation conditions.

Treatment	Chlorophyll “a” (mg g−1 FW)	Chlorophyll “b” (mg g−1 FW)	Total Chlorophylls (mg g−1 FW)	Total Carotenoids (mg g−1 FW)
100% of SWHC (C)	1.13 ± 0.12 ab	0.50 ± 0.12 abc	1.46 ± 0.03 abcd	0.42 ± 0.05 bc
100% of SWHC (MSE)	1.45 ± 0.26 b	0.80 ± 0.12 d	1.82 ± 0.05 df	0.69 ± 0.05 d
80% of SWHC (C)	1.04 ± 0.12 ab	0.47 ± 0.03 abc	1.43 ± 0.12 abc	0.39 ± 0.01 abc
80% of SWHC (MSE)	1.32 ± 0.16 b	0.73 ± 0.13 cd	1.72 ± 0.07 cdef	0.64 ± 0.11 d
60% of SWHC (C)	0.90 ± 0.23 ab	0.43 ± 0.04 ab	1.28 ± 0.08 ab	0.33 ± 0.02 ab
60% of SWHC (MSE)	1.20 ± 0.12 ab	0.61 ± 0.06 bcd	1.59 ± 0.24 bcdef	0.54 ± 0.04 cd
40% of SWHC (C)	0.70 ± 0.15 a	0.32 ± 0.07 a	1.11 ± 0.12 a	0.23 ± 0.05 a
40% of SWHC (MSE)	1.11 ± 0.06 ab	0.51 ± 0.01 abc	1.46 ± 0.05 abcde	0.41 ± 0.01 bc

C, control; SWHC, soil water holding capacity; MSE, moringa seed extract; FW, fresh weight. Data presented as mean ± SE. Different letters among treatments for each attribute are significantly different (p < 0.001) according to Duncan’s multiple range test.

shows that the MSE foliar spray effectively (p < 0.001) enhanced leaf photosynthetic pigments of none-stressed cancer bush plants (100 of SWHC) in comparison to corresponding control. This treatment resulted in higher chlorophyll “a” (1.45 mg g⁻¹ FW), chlorophyll “b” (0.80 mg g⁻¹ FW), total chlorophylls (1.82 mg g⁻¹ FW) and total carotenoids (0.69 mg g⁻¹ FW) compared to controls. Furthermore, DI significantly (p < 0.001) reduced leaf photosynthetic pigments compared to full-irrigated plants. Nonetheless, the application of MSE on DI-stressed plants (80, 60 and 40% of SWHC) effectively increased leaf photosynthetic pigments: chlorophyll “a” (1.31, 1.2 and 1.10 mg g⁻¹ FW), chlorophyll “b” (0.72, 0.6 and 0.51 mg g⁻¹ FW), total chlorophylls (1.71, 1.59 and 1.46 mg g⁻¹ FW) and total carotenoids (0.64, 0.51 and 0.41 mg g⁻¹ FW), compared to corresponding controls. The ability of MSE foliar application to increase leaf photosynthetic pigments was distinctively evident under DI compared to full irrigation. Furthermore, there were slight significant differences between all evaluated leaf photosynthetic pigments of none-stressed and DI-stressed plants treated with MSE.

3.5. Antioxidants Activity

The data shown in Figure 2 demonstrate that the application of MSE improved (p < 0.001) antioxidant concentration of none-stressed cancer bush plants (100 of SWHC) compared to the corresponding control. None-stressed plants treated with MSE had higher (p < 0.001) DPPH (78.15%) and ABTS (80.01%) compared to the respective control. The DI significantly (p < 0.001) reduced antioxidants activity. However, MSE foliar application on DI-stressed plants (80, 60 and 40%) significantly (p < 0.001) improved DPPH (79.92, 70.91 and 45.21%) and ABTS (82.95, 75.09 and 49.97%), as opposed to respective controls. The positive influence of MSE on increasing antioxidants activity was more evident on DI-stressed plants compared to none-stressed plants. There were no or slight significance differences between assessed antioxidants activity (DPPH and ABTS) of none-stressed plants and those of water-stressed plants particularly 80 and 60% of SWHC, respectively.

4. Discussion

The current study showed that MSE is rich in osmoprotectants such as total free amino acids, free proline and soluble sugars, mineral nutrients such as vitamins including vitamin C also known as ascorbic acid as well as vitamin B₁, B₂, B₃ and E, copper (Cu), calcium (Ca), magnesium (Mg), phosphorus (P) as well as sulphur (S) and phytohormones such as CKs and GAs. The MSE could be considered as having antioxidants activity since vitamin E, which includes both tocopherols and tocotrienols, comprises lipid-soluble antioxidants that modulate lipid peroxidation [32]. In addition, ascorbic acid acts a main redox and cofactor for enzymes involved in regulating photosynthesis, hormone biosynthesis and regenerating other antioxidants [33]. Ascorbic acid also neutralizes reactive oxygen species (ROS) directly using secondary antioxidants during the reduction of the oxidized form of ɑ-tocopherol and is a significant plant metabolite and acts as a cell signalling modulator in many physiological processes such as mitosis [11,33]. Therefore, the nutrient composition of MSE makes it a vigorous plant growth biostimulant and has certain vital mechanisms for water-stressed plants to enhance their tolerance to drought. The current study used MSE as a foliar application for DI-stressed cancer bush plants (80, 60 and 40% of SWHC). Foliar application of MSE significantly enhanced plant growth and yield attributes, CWP, RWC and MSI, leaf photosynthetic pigments and antioxidants activity of DI-stressed cancer bush plants. Therefore, it is a good environmentally friendly and cost-effective alternative for eliminating and reducing the use of commercially synthesised mineral nutrients, vitamins, antioxidants, or plant hormones, which are associated with adverse impacts on the environment and human health when consumed; it is also expensive [34].

The current study demonstrated that the application of MSE as a foliar spray succeeded as a growth biostimulant against water-deficit stress for cancer bush plants. This could be attributed to the biostimulant or effective ingredients of MSE which could have easily penetrated or translocated through leaf stomata to active parts such as meristematic cells, supplying plants with stimuli against the negative impact of water-deficit stress [35]. Our findings suggest that the major stimuli of MSE, particularly phytohormones, such as CKs and Gas and antioxidants or vitamins including vitamin C and E and other biostimulants, played an important role in DI-stressed cancer bush plants by enhancing plant metabolism. These MSE stimuli are responsible for promoting cell division, elongation, plant nutritional composition and accumulation of dry matter [35,36], thus allowing plants to overcome the negative impact of water-deficit stress as observed in the findings of the current study. The enhanced growth and yield characteristics could also be attributed to the mineral nutrients of MSE which may have promoted the ability of cancer bush plants to adapt to water-deficit stress levels through the signalling pathways that effect the adaptive responses of plants to harsh environmental conditions and/or expression and regulation of stress-induced genes that contribute to drought tolerance [37]. Moreover, plant hormones are vital endogenous modulators that regulate multiple physiological processes required for plant growth and development and play a major role in inducing tolerance to plants against various biotic and abiotic stresses [38]. Cytokinins are key hormones regulating cell division and differentiation, root and shoot architecture, senescence and responses to environmental stresses [39]. The GAs promote plant part elongation, including the hypocotyl and stem [40]. Our results are similar to Abd El-Mageed et al. [11] who reported that the application of moringa leaf extract (MLE) (3%) effectively enhanced growth attributes of squash plants subjected to DI (80 and 60% of evapotranspiration (Etc) compared to control (100 % of Etc or none-stressed plants), due to the high content of essential mineral nutrients, antioxidants and phytohormones in MLE [8]. Moreover, MSE significantly enhanced CWP in DI-stressed cancer bush plants compared to the control, which could be attributed to its high content of essential mineral nutrients such as vitamin E, which is a family of antioxidants in plants and consists of tocochromanols which are involved in the scavenging of lipid peroxyl radicals responsible for accelerating lipid peroxidation [41]. The current study also demonstrated that foliar application of MSE effectively enhanced yield characteristics such as the number of fruit/plant and biomass yield/plant of DI-stressed and none-stressed cancer bush plants compared to respective controls. Our results are in agreement with Elzaawely et al. [14], who reported that foliar application of MLE (5.00%, 3.30% and 2.50%) effectively enhanced yield attributes by up to 35% of snap bean plants (Phaseolus vulgaris L.) compared to corresponding controls. Another study by Abd El-Mageed et al. [11] demonstrated that the use of MLE (3%) successfully improved yield characteristics of DI-stressed squash plants (80 and 60% of ETc) compared to control. The increased yield by MSE could be due to increased growth attributes and the biostimulant characteristics such as CKs which have important roles in enhancing plant growth as well as chlorophyll content leading to improved photosynthesis, consequently enhancing plant yield [14].

In the present study, the application of MSE improved leaf photosynthetic pigments in cancer bush plants grown under DI conditions compared to corresponding controls. This could be attributed to MSE components such as vitamins or antioxidants and phytohormones which promote tolerance to environmental stress conditions and improve plant productivity [8]. In addition, MSE components such as Mg, P and Cu may effectively penetrate the stomata and improve plant metabolism such as photosynthesis, respiration and organic compounds [11]. Magnesium is required by plants to help capture solar energy for growth and production through photosynthesis [42], whereas Cu acts as a catalyst in photosynthetic and respiratory electron transport chains, ethylene sensing, cell wall metabolism, oxidative stress protection and biogenesis of molybdenum cofactor [43]. In addition, improved photosynthesis and metabolic processes contributed to maintaining cell health and turgidity in terms of increased RWC and MSI, providing tissue health and vigorous plant growth under DI stress conditions.

Essential mineral nutrients such as vitamins C and E which comprise lipid-soluble antioxidants that modulate lipid peroxidation are important components in MSE. Vitamin C acts as ROS-scavenging activity through enzymatic and non-enzymatic detoxification, thus minimizing the oxidative damage to cells [44]. Vitamin E can eliminate ROS and prevent the progression of lipid peroxidation, hence playing an important role in the adaptation to several stresses and enhancing plant tolerance to drought and heat stresses [45]. Therefore, MSE as a rich source of antioxidants enabled cancer bush plants to perform well by enhancing growth and yield characteristics, photosynthetic pigments, RWC and MSI under DI conditions. These antioxidants play an important role in increasing plant resistance to environmental stresses by enhancing the scavenging activities to eradicate ROS [46]. Moreover, antioxidants function as a mechanism to alleviate and repair damages caused by ROS, and they allow plants to gradually develop complex defence systems to improve the cellular defence strategies to overcome water-deficit oxidative stress [8].

The improved tissue RWC and MSI by MSE application in DI-stressed plants could be attributed to the ability of MSE to repair damages that may have developed in cell membranes of plants. Furthermore, this could be attributed to MSE as a productive biostimulant to preserve membrane integrity [14]. In addition, Darvishan et al. [37] reported that RWC is an important measurement of plant water status as a physiological consequence of tissue water deficit, whereas water potential is an evaluation of plant water transport in the soil–plant atmosphere continuum. In addition, osmotic adjustment is the main physiological adaptation mechanism that plants use to cope with osmotic stress such as salinity and drought [9]. The findings of the present study showed that the enhanced osmotic adjustment contributed to maintaining higher RWC and MSI in DI-stressed cancer bush plants treated with MSE, thus alleviating the negative impact of water stress. The enhanced plant RWC status by the application of MSE is correlated with the increase in CWP in DI-stressed cancer bush plants. This aided in preserving healthy metabolic processes in cancer bush foliage as a productive mechanism produced in water-stressed plants.

Our study showed that the application of MSE effectively increased antioxidant activity (DPPH and ABTS) of DI-stressed cancer bush plants which could be attributed to the crucial role of particularly phytohormones as well as vitamins with antioxidants properties present in MSE, helping plants to maintain high antioxidant activities to survive the negative impact of DI. Antioxidant properties such as vitamins C and E present in MSE may have enhanced the effectiveness of antioxidant defence system constituents in water-stressed cancer bush plants. Abd EL-Mageed et al. [11] and Spicher et al. [45] reported that antioxidants mitigate and repair the deterioration initiated by ROS under stress, helping plants to promote complex antioxidant defence systems to support the cellular defence strategies versus oxidative stress. Thus, antioxidants and phytohormones are mechanisms whereby MSE application helped cancer bush plants to alleviate the negative influence of water stress.

The elevation of antioxidants activity in DI-stressed cancer bush plants may be due to phytohormones present in MSE such as CKs and GAs which could be responsible for enhancing stress tolerance genes in plants [37]. Moreover, CKs act as direct free radical scavengers that might be involved with antioxidant mechanisms associated with the preservation of purine breakdown [47]. The GAs are quickly increased when plants are exposed to harsh environmental conditions to induce stomatal resistance and improve plant water use. In addition, free radical-induced lipid peroxidations are repressed by Gas. They catabolize other phytohormones such as abscisic acid, which is a pivotal regulator in plants, and they coordinate a complex regulatory network enabling plants to cope with abiotic stresses, such as drought, salinity, and temperature fluctuation [48]. Consequently, phytohormones could be a possible mechanism of plant resistance to harsh environmental conditions [37]. Since MSE is rich in plant growth hormones, it can promote hormonal balance in water-stressed plants to effectively resist the negative influence of water stress [49].

5. Conclusions

The findings of the current study demonstrated that MSE is a rich source of essential mineral nutrients including those that act as antioxidants such as vitamin C and E and phytohormones including CKs and GAs. Therefore, MSE can be used as a productive bioactive stimulant to enhance production and productivity of cancer bush plants. The MSE foliar spray was effective in mitigating physiological response of water stress damages, and its positive influence was more noticeable under DI at 80, 60 and 40% of SWHC. The water-stressed cancer bush plants treated with MSE had improved RWC, CWP, leaf photosynthetic pigments and antioxidant activity, suggesting that MSE played an important role in plant growth, metabolism and responses to water stress conditions due to its antioxidant and phytohormone components that helped plants to perform well under water stress. Water-stressed cancer bush plants sprayed with MSE had enhanced growth and yield characteristics as those of fully irrigated plants. It could be recommended that MSE may be used as a cost effective and environmentally friendly natural biostimulant for growing plants under favourable or water stress conditions. Therefore, MSE can be used as a natural source of plant growth stimuli rather than the synthetic growth regulators. However, the current study investigated the impact of MSE on only water-stressed plants; therefore, future studies should involve other abiotic stress conditions such as salt and high temperature or heat stresses. Moreover, previous studies have demonstrated that other plant parts of moringa such as leaves, twigs and roots are also a rich source of essential minerals, antioxidants and phytohormones; therefore, future research should explore the biostimulant potential of these plant parts, particularly roots, since very little is known about its biostimulant potential on plant growth.