Plant growth conditions.

This experiment was conducted under conditions similar to our previous studies (Buthelezi et al. 2022, 2023, 2024). It was carried out twice sequentially in the tunnel at Sefako Makgatho Health Sciences University, South Africa (25°37′8″S, 28°1′22″E), during the winter to summer seasons of 2021. Plants were grown in the tunnel that was 4 m high, 8 m long, and 4 m wide covered with a green colored net with 40% shading [ChromatiNet™; Carports and Pergola Builders (Pty) Ltd., Pretoria, South Africa]. During the experiment, the average temperature and relative humidity in the tunnel were 21.08 °C and 43.01%, correspondingly.

About 150 seedlings of cancer bush were purchased from the Plantland Akasia Garden Centre Nursery in Pretoria, South Africa (25°39′41.2″S, 28°04′34.5″E). The seedlings were then transported and kept in the tunnel at Sefako Makgatho Health Sciences University, where they were watered three times a day with an average of 200 mL of tap water per plant for a week. Afterward, healthy seedlings of about 30 cm long were transplanted into plastic terracotta pots (40 cm in diameter and 50 cm depth), containing 5 kg of Culterra potting soil per pot.

The Culterra potting soil used in the current study was obtained from Builders Express, Pretoria, South Africa. This product is composed of raw organic material such as general 2:3:2 (22), lawn 8:1:5 (25), limestone ammonium nitrate (28%), vita flora 5:1:5 (33) slow release nitrogen, and vital flora 3:1:5 (26) slow release nitrogen per 30 kg of soil (pH 7.7 and EC 2.01 dS·m⁻¹) (Buthelezi et al. 2023). The soil also had total porosity of 48.90% and infiltration rate of 30.54 mm·h⁻¹ and consisted of ammonium sulfate (21%), available potassium (120.32 mg·kg⁻¹), available phosphorus (15.32 mg·kg⁻¹), and total nitrogen (1.98 mg·kg⁻¹).

Irrigation water applied.

Soil water-holding capacity (SWHC) was evaluated according to Taha et al. (2020) using Eq. [1] as follows:$$\mathit{SWHC} \left(\%\right)\hspace{0.17em}=\hspace{0.17em}\mathit{total} \mathit{porosity} \left(\%\right)-\hspace{0.17em}\mathit{air} \mathit{space} \left(\%\right)$$ [1] Two irrigation treatments were used during the experiment after transplanting: 80% (160 mL of irrigation water), which was calculated as the ideal irrigation water for optimum growth of cancer bush plants, and 60% (120 mL of irrigation water) of SWHC. All pots were well irrigated with 200 mL of tap water before transplanting. The ideal irrigation treatment (80% of SWHC) was used as a control, whereas 60% of SWHC was regarded as a drought stress treatment. In our preliminary study (data not shown), water deficiency in 40% of SWHC significantly reduced growth and yield of cancer bush even when treated with MSE. The drought stress levels were applied to cancer bush plants three times a day. Culterra potting soil moisture content per terracotta pots was observed daily (HH2 moisture meter, version 4.0; Delta-T Devices Ltd., Cambridge, UK) and maintained through water application where applicable.

Moringa seed extract preparation and treatments.

Moringa seeds were harvested in bulk from commercial orchards of Afrinest Moringa Farm in Tzaneen, Limpopo, South Africa (23°49′15.3″S, 30°10′08.7″E). The harvested seeds were then ground into fine powder using a blender (Mellerware Fusion 1L 300 W; Makro, Pretoria, South Africa) and extracted according to El-Sappah et al. (2023). Moringa seed powder (100 g) was mixed with 1 L of 80% aqueous ethanol and vortexed (Oxford LP Benchmate digital vortex mixer, VM-D; Selectech Laboratory Equipment Supplier, Pretoria, South Africa) for 1 min. Afterward, the mixture was kept at ambient conditions overnight. Then, the solution was centrifuged (laboratory centrifuge, TD4C; Labtex Co., Ltd., Bangladesh, South Asia) at 8000 g_n for 20 min and filtered twice using Whatman^® no. 1 filter paper and then with nonabsorbent cotton. Consequently, the supernatant was mixed with distilled water (v/v) to attain the desired extract at 6% MSE to use as a foliar spray. In our preliminary study (data not shown), 6% and 8% MSE successfully enhanced plant growth and development of cancer bush with no and little significant differences compared with 2 and 4% MSE. Freshly prepared extract was then used within 24 h or kept in the refrigerator at −20 °C for further use. About 200 to 500 mL of 6% MSE foliar spray, depending on the plant growth stage, was applied to a single cancer bush plant/pot/irrigation level per week. Untreated plants, which were considered to be control, were sprayed with tap water at the same time of MSE treatment.

Experimental design and plant management.

The two experiments were each conducted on 50 individual plants using a randomized complete block design. Plant pots were set up with an intrarow pot of 40 cm and interrow spacing of 50 cm. The treatments of this experiment were irrigation with 80% of SWHC + foliar spray with distilled water (control), irrigation with 80% of SWHC + foliar spray with MSE application, irrigation with 60% of SWHC + foliar spray with distilled water (control), and irrigation with 60% of SWHC + foliar spray with MSE. Overall, each treatment had 25 pot plants, making a total of 100 pots plants of the entire experiment. The MSE foliar pray was applied to cancer bush plants at 7 d intervals during the 10 weeks of the experiment.

Measurements of plant growth, ornamental, and root morphology characteristics.

At harvest (10 weeks after transplanting), the growing medium (Culterra potting soil) was carefully washed from the roots, and the plants were divided into stem, leaves, flowers, and roots. Five plants per treatment were used to determine shoots and roots fresh weight using a precision balance [ME3002T; Labotec (Pty) Ltd., Johannesburg, South Africa]. Afterward, these were oven dried (DON-H; Bioevopeak, Co., Ltd., Shandong, China) at 70 °C until a constant weight was attained (dry weight). The number of inflorescences was counted. Fruit yield attributes such as fruit number, individual fruit mass, and overall fruit yield per plant were also recorded. Root scanning was performed following a method of Ertani et al. (2018), with minor adjustments. Fresh root systems were gently washed with tap water after harvest, placed on a transparent tray, and scanned before the sampling process with a root scanner (Epson Expression 10000XL 1.0 system; Regent Instruments Inc., Québec, Canada) to measure total root length, average diameter, volume, tips, forks, crossings, projected area, and surface area. The roots of three plants were scanned per replicate and treatment.

Photosynthetic parameters quantification.

Leaf gas exchanges based on the CO₂ net assimilation rate (A), stomatal conductance (Gs), transpiratory rate (E), and intercellular CO₂ concentration (Ci) were performed on fully expanded leaves (2 to 6 weeks after transplanting) using an infrared gas analyzer (portable model LI-6400xt; Thermo Fisher Scientific Inc., Johannesburg, South Africa). Measurements were taken between 9:00 and 11:30 am with constant photosynthetically active radiation (1000 μmol photons m⁻²·s⁻¹), atmospheric CO₂ concentration, room temperature, and humidity (Agliassa et al. 2021; Kałużewicz et al. 2017). These data were used to calculate water use efficiency (WUE = A/E) and carboxylation efficiency (CE = A/Ci) (Agliassaet al. 2021).

Determination of soluble protein.

The total soluble protein concentration was determined according to Contartese et al. (2015). Briefly, 1 g of fresh leaf sample was extracted in 5 mL of 50 mM Tris-HCl buffer [pH 7.4; containing 0.2 M NaCl, 20 mM MgSO₄, 1 mM ethylenediaminetetraacetic acid (EDTA), 5 mM β-mercaptoethanol, 0.5 mM phenylmethylsulfonic fluoride, 10 mM leupeptin, and 10% (v/v) glycerol]. The mixture was allowed to stand on ice for 10 min and then centrifuged at 20,000 g_n for 15 min. The supernatant was filtered through Miracloth^® and then used for a Bradford (1976) assay, with bovine serum albumin as a standard. Bradford dye reagent was prepared by mixing the dye concentrate with distilled water 1:4. Afterward, 1 mL of sample extract was added to 0.1 mL of water and 2 mL of Bradford reagent in a test tube, mixed, and incubated at ambient temperature for 3 min. The absorbance was read at 595 nm using a spectrophotometer (UV-2700; Shimadzu, Kyoto, Japan).

Determination of free proline.

Free proline was assessed according to Agliassa et al. (2021) and Hamedeh et al. (2022). Ground leaf tissue (0.05 g) was homogenized in 0.5 mL of ethanol (v/v) and incubated overnight at 4 °C in the dark. The homogenates were centrifuged [FC5706 230 V, United Scientific (Pty) Ltd., Johannesburg, South Africa] at 20,000 g_n for 15 min at 4 °C. Samples of 100 μL of the extract were added to 200 μL of acidic ninhydrin solution, and the mixture was incubated at 100 °C for 1 h. After cooling at ambient temperature, the absorbance was measured at 520 nm using a spectrophotometer (UV-2700). After preparing a calibration curve standard curve with l-proline (Sigma-Aldrich, Johannesburg, South Africa), free proline content is expressed as mg·g⁻¹ dry weight (Contartese et al. 2015).

Determination of lipid peroxidation.

Lipid peroxidation was measured by the quantification of malondialdehyde (MDA) in leaf tissues. The MDA concentration was analyzed by the reaction with thiobarbituric acid (Jacomassi et al. 2022). Briefly, the MDA was extracted with 10% trichloroacetic acid, and then the extract was mixed with 0.6% thiobarbituric acid and centrifuged at 15,000 g_n at 4 °C for 25 min. Afterward, 0.5 mL aliquots of the supernatants were added to 1.5 mL of 0.5% thiobarbituric acid solution in 20% trichloroacetic acid and incubated at 90 °C in a shacking water bath (Monochrome LCD; Thermo Fisher Scientific, Johannesburg, South Africa) for 30 min, and the reaction was stopped in an ice bath (17763; Sigma-Aldrich) for 15 min. Thereafter, the samples were centrifuged at 10,000 g_n for 5 min, and the absorbance was read at 532 and 600 nm. The MDA concentration was measured by subtracting the nonspecific absorption 600 nm from the absorption at 532 nm. The value for nonspecific absorption at 600 nm was subtracted using an absorbance and absorptivity coefficient of 155 mM⁻¹·cm⁻¹. The results are expressed in MDA nmol·g⁻¹ fresh weight.

Determination of antioxidant enzyme activities.

To measure the activities of antioxidant enzymes, the total proteins were extracted using a method of Rahimi et al. (2022), with some alterations. Briefly, 0.3 g samples of leaf tissue were macerated in liquid nitrogen, homogenized with 0.1 M potassium phosphate buffer (pH 7.8), 0.1 mM EDTA, 1 mM phenylmethylsulfonic fluoride, and 1% (w/v) polyvinylpyrrolidone for extraction of the enzymes SOD (EC 1.15.1.1), CAT (EC 1.11.1.6), POX (EC 1.11.1), and APX (EC 1.11.1.11). The mixture was centrifuged for 20 min at 12,000 g_n at 4 °C, and the supernatant was used as crude enzyme extract.

Superoxide dismutase activity.

SOD (EC 1.15.1.1) activity was evaluated using the method outlined by Repke et al. (2022) and Rahimi et al. (2022) with slight modifications. Briefly, SOD activity was determined in a reaction medium consisting of 50 mM sodium phosphate buffer (pH 7.8) containing 13 mM methionine, 75 μM p-nitro blue tetrazolium, 0.1 mM EDTA, and 2 μM riboflavin together with the enzyme extract. The absorbance was detected at 560 nm (UV-2700) after 15 min of light exposure (4000 lx). One unit of total SOD activity was calculated as the amount of protein per milligram causing 50% inhibition of p-nitro blue tetrazolium reduction. Enzymatic activity results are expressed as U·mg⁻¹ proteins.

Catalase activity.

CAT (EC 1.11.1.6) activity was determined according to the decomposition of H₂O₂ using the method of Rahimi et al. (2022), with some alterations. Briefly, an aliquot of 50 µL of crude extract was added to 950 µL of reaction medium containing 50 mM sodium phosphate buffer (pH 7.0) and 12.5 mM H₂O₂. The absorbance was obtained at 240 nm using a spectrophotometer (UV-2700) after 1 min. CAT activity was calculated as μmol of decomposed H₂O₂ per minute. Enzymatic activity results are expressed as nmol H₂O₂ min⁻¹·mg⁻¹ proteins.

Peroxidase activity.

POX (EC 1.11.1) activity was measured in aliquots of crude extract added to the reaction medium consisting of 25 mM potassium phosphate buffer (pH 6.8), 20 mM pyrogallol, and 20 mM H₂O₂. The production of purpurogallin was measured at 420 nm using a spectrophotometer (UV-2700). Enzyme activity was calculated using the absorbance and molar extinction coefficient of 2.47 mM⁻¹·cm⁻¹ (Akensous et al. 2022; Panda et al. 2003) and is expressed as μmol of purpurogaline min⁻¹·mg⁻¹ protein.

Ascorbate peroxidase activity.

APX (EC 1.11.1.11) activity was determined according to Akensous et al. (2022), with slight modifications. An aliquot of 100 µL of crude extract was added to 900 µL of reaction medium, consisting of 0.05 M sodium phosphate buffer (pH 7.0), 0.8 mM ascorbic acid, and 1.0 mM H₂O₂. Enzymatic activity was measured at 290 nm using a spectrophotometer (UV-2700), considering the molar extinction coefficient of 2.8 Mm⁻¹·cm⁻¹. The results are expressed as µmol of ascorbic acid min⁻¹·mg⁻¹ protein.

Statistical analysis.

The collected data were subjected to analysis of variance using GenStat^® statistical software (18.1 edition; VSN International, Hemel Hempstead, UK). Fischer’s least significant differences were calculated and used to separate means at 5% significance level.

Plant growth and yield characteristics.

The foliar application of MSE under ideal irrigation (80% of SWHC) significantly (P < 0.05) improved all measured growth and yield parameters of cancer bush plants compared with the corresponding control (Fig. 1). Drought stress (60% of SWHC) significantly (P < 0.05) decreased shoot fresh weight, shoot dry weight, number of inflorescences per plant, and number of fruit per plant compared with normal irrigation (80% of SWHC). Individual fruit mass per plant and overall yield per plant of plants under both 80 and 60% of SWHC were not statistically significant. The application of MSE effectively (P < 0.05) increased shoot fresh weight, shoot dry weight, and number of inflorescences per plant of drought-stressed plants compared with the corresponding controls. Although not statistically significant, the number of fruit per plant, individual fruit mass per plant, and overall yield per plant of drought-stressed plants treated with MSE were improved compared with respective controls. As shown in Fig. 1, MSE significantly (P < 0.05) increased shoot dry and fresh weight, root fresh and dry weight, number of inflorescences per plant, number of fruit per plant, individual fruit mass per plant, and overall yield per plant of drought-stressed plants by 50.04%, 60.75%, 30.29%, 74.22%, 36.22%, 23.44%, 36.98%, and 19.70%, respectively, in comparison with corresponding controls. In addition, Fig. 2 shows that roots parameters such as total length, volume, tips, crossings, forks, and fresh and dry weight of drought-stressed plants treated with MSE were increased by 49.45%, 35.81%, 43.19%, 41.47%, 72.76%, 30.29%, and 82.03%, respectively compared with corresponding controls. Although not statistically significant, the root diameters of drought-stressed plants treated with MSE were increased by 37.27% compared with control. The positive effect of MSE treatment was more evident under drought stress than its application under normal irrigation and yielded minor to no significant differences between most evaluated traits of ideal-irrigated plants and those of drought-stressed plants treated with MSE.

View Figure

• Download as Powerpoint
• Download Figure

View Figure

• Download as Powerpoint
• Download Figure

Photosynthetic parameters.

Well-watered (80% of SWHC) cancer bush plants treated with MSE had effectively (P < 0.05) improved Gs, E, and CE in comparison with control (Fig. 3). Well-irrigated (80% of SWHC) cancer bush plants treated with MSE had lower Ci than control. Although not statistically significant, Fig. 2A and 2F shows that plants treated with 80% SWHC + MSE treatment had improved A (16.91 µmol CO₂ m⁻²·s⁻¹) and WUE (5.12 mmol CO₂ H₂O⁻¹) compared with corresponding controls (80% SWHC) (15.81 µmol CO₂ m⁻²·s⁻¹ and 4.25 mmol CO₂ H₂O⁻¹, respectively). Nevertheless, drought stress (60% of SWHC) treatment significantly decreased A, Gs, E, CE, and WUE and increased Ci compared with well-irrigated plants (80% of SWHC). However, MSE treatment significantly (P < 0.05) enhanced A, Gs, E, CE, and WUE and decreased Ci of drought-stressed plants compared with the corresponding controls. The A, Gs, E, CE, and WUE were increased by 56.81%, 92.31%, 31.96%, 99.59%, and 84.62%, respectively, and Ci was decreased by 30.02% compared with corresponding controls. Overall, the application of MSE was effective in improving photosynthetic parameters including A, Gs, E, CE, and WUE and decreasing Ci of cancer bush plants, which was demonstrated more under drought stress than under normal irrigation. In addition, there were no significant differences between all assessed parameters of well-irrigated plants and those of drought-stressed plants treated with MSE.

View Figure

• Download as Powerpoint
• Download Figure

Soluble protein and free proline concentration.

Although not statistically significant, MSE induced the accumulation of soluble protein content, which was increased by 7.45% and significantly (P < 0.05) increased free proline content by 49.38% in well-watered (80% SWHC) cancer bush plants compared with corresponding controls (Fig. 4). Subjecting plants to drought stress (60% of SWHC) significantly reduced the above-mentioned parameters in comparison with well-irrigated (80% of SWHC) plants. However, MSE effectively (P < 0.05) increased the soluble protein (31.88%) and free proline (66.67%) of drought-stressed plants in comparison with corresponding controls. The effectiveness of MSE was more apparent under drought stress than under ideal irrigation. This is further supported by no significant differences between the contents of soluble protein and free proline of well-irrigated plants and that of drought-stressed plants treated with MSE.

View Figure

• Download as Powerpoint
• Download Figure

Lipid peroxidation.

As shown in Fig. 5, the application of MSE under ideal irrigation (80% of SWHC) significantly (P < 0.05) reduced MDA concentration of cancer bush plants compared with control. The MDA content was reduced by 48.75% compared with control. In addition, MSE treatment under drought stress (60% SWHC) further reduced the MDA content of cancer bush plants by 62.28% compared with control. Overall, plants that were well-irrigated (80% of SWHC), as well as those who were drought stressed (60% of SWHC), had high MDA content, whereas MSE significantly (P < 0.05) reduced MDA content of plants that were well irrigated and those that were drought stressed. The positive effect of MSE foliar application was more noticeable under drought stress than under ideal irrigation, with no or slight significant differences between well-irrigated plants and drought-stressed plants treated with MSE.

View Figure

• Download as Powerpoint
• Download Figure

Antioxidants enzyme activities.

Although not statistically significant, an enhancement of SOD and significantly (P < 0.05) higher activities of CAT, POX, and APX were observed in well-irrigated (80% of SWHC) MSE-treated plants, which were increased by 14.53%, 36.79%, 95.68%, and 29.58% in comparison with respective controls (Fig. 6). However, drought stress (60% of SWHC) significantly (P < 0.05) reduced all the above-mentioned antioxidant enzyme activities compared with well-irrigated (80% of SWHC) plants. The foliar application of MSE had a positive effect on SOD, CAT, POX, and APX activities of drought-stressed plants, which were significantly (P < 0.05) increased by 16.54%, 21.33%, 73.13%, and 65.85%, respectively, compared with respective controls. The positive effect of MSE treatment was evident under drought stress than under ideal irrigation, with no or minor statistical differences between well-irrigated plants and drought-stressed plants treated with MSE.

View Figure

• Download as Powerpoint
• Download Figure