The Efficiency of Nanoparticles on Improving Seed Germination and Mitigating Ammonium Stress of Water Spinach (Ipomoea aquatica Forssk.) and Hami Melon (Cucumis melo L.)

Abstract

Nitrogen, despite being essential for the growth of plants, can pose serious threats to the ecological environment when applied excessively as fertilizers. The application of nanomaterials has a catalytic effect on crop growth and a restorative effect on the environment. However, their effect on mitigating ammonium stress in crops is poorly understood. In the present study, the roles of nanoparticles of magnesium oxide (nMgO) and hydroxyapatite (nHA) with different application rates (0, 10, 100, 500, and 1000 mg L⁻¹) on seed germination and seedling growth in water spinach (Ipomoea aquatica Forssk.) and Hami melon (Cucumis melo L.) were investigated, and the ammonium stress mitigating capacity of nanoparticles with the optimal application rate on the two crops was analyzed. The results showed that the application of nMgO and nHA at an optimal rate of 100 mg L⁻¹ significantly promoted seed germination of water spinach, followed by the increase of germination potential, seed germination rate, and germination index, while alleviating the inhibitory effect of NH₄⁺ stress in water spinach. As for the Hami melon, nHA reduced the ammonium stress on seedlings by promoting antioxidant enzyme activity, while nMgO was found to be involved in reducing the root growth of Hami melon seedlings. This study provided a reference on how to select the appropriate type and optimize the application method of nanomaterials that will be used in agriculture in the future.

1. Introduction

Nitrogen (N) is one of the most essential elements, as it plays a vital role in the growth and development of plants. It also acts as a major limiting nutrient, which is being used excessively to improve crop yield [1]. Crops can only use 30–50% of nitrogen by absorbing fertilizer while the rest of the supplied nitrogen is leached out to the soil, thus causing acidification and erosion of soil [2]. Excessive application of nitrogen also leads to surface water eutrophication, groundwater pollution, ecological imbalance, and biodiversity loss [3,4].

Inorganic N from the soil is mainly utilized by plants as nitrate (NO₃⁻) and ammonium (NH₄⁺) because both forms serve as major sources of nitrogen for most plants. In C₃ plants, the net assimilation rate of NO₃⁻ is inhibited by increasing global CO₂ concentrations, while NH₄⁺ remains unaffected; that is why the supplication of NH₄⁺ for crop production is always preferred [5]. However, NH₄⁺ is a paradoxical nutrient ion as if provided moderately, it can promote growth, but in excess, it can have phytotoxic effects on plants [6]. The general symptoms of NH₄⁺ toxicity in plants are the cessation of root growth, yellowing, wilting of leaves, and reduction of biomass [7]. These symptoms are followed by changes in ion balance, inhibition of root respiration and photorespiration, interference with photosynthesis, increased oxidative stress, and inhibiting the uptake of cations in plants [8]. Water spinach at high levels of NH₄⁺ was stunted with few and short roots, old leaves were lost, and the growth of new ones was suppressed [9]. Therefore, it is particularly important to find ways to mitigate the effects of ammonium stress on the growth of crops. Recently, nanomaterials have been identified as the best option for replacing such nitrogenous substances used for the growth of crops.

Nanomaterials are substances ranging their size from 1 to 100 nm with one or more dimensions, characterized by a large active surface, and unique physical and chemical properties [10]. Due to having a smaller size and larger surface area, nanomaterials have cutting-edge applications in a variety of fields. In agriculture, a variety of nanomaterials used for crops have been developed successfully, including nano fertilizers, plant growth regulators, and pesticides [11,12]. These have the capacity to solve many key problems in agriculture, such as improving crop productivity, pests, and disease control [13]. Different morphological and physiological changes occur through the interaction of nanoparticles (NPs) and plants, based on the nature of NPs, including the chemical composition, size, surface covering, and reactivity of NPs [14]. It has been observed that low concentrations of ZnO NPs promoted germination and seedling growth in onion seeds, while its high concentrations showed inhibitory effects [15]. Similarly, low concentrations of multi-walled carbon nanotubes (MWCNTs) promoted seed germination in maize crops [16]. Currently, nanoparticles are being employed by many plant breeders for the sake of several beneficial effects on crops and can improve crop tolerance under various biotic and abiotic stresses [17]. Several authors have reported the applications of various nanoparticles in the field of crop cultivation. Li et al. [18] found that the application of hydroxyapatite NPs to cadmium-contaminated soil was effective in reducing the uptake of cadmium by enhancing the resistance of plants to cadmium stress. Similarly, Shah et al. [19] reported that TiO₂ NPs promoted maize seed germination and seedling growth by triggering antioxidant enzymes which effectively scavenged the reactive oxygen species (ROS) produced in excess under saline growth conditions.

Among the nanoparticles being widely used for agricultural purposes, hydroxyapatite nanoparticles (nHA) are considered good one due to having good biocompatibility with large micro-interfaces, high microporosity, allowing photo-catalytic adsorption, specific adsorption, and surface adsorption during contact with pollutants, thus reducing the biotoxicity and migration capacity of pollutants. It is also considered an effective material in the remediation of soil contamination, especially heavy metal contamination [20,21]. Magnesium oxide NPs (nMgO) have also been a choice of interest due to their being non-toxic, safe, and easy to obtain. It functions as an outstanding bactericide against Ralstonia solanacearum and offers substantial promise to prevent root and stem diseases [22].

In southern China, water spinach (Ipomoea aquatica Forssk.) is one of the common leaf vegetables being cultivated on a large scale. Hami melon (Cucumis melo L.) is favored for its high nutritional value and pleasant flavor. Keeping given their nutritional values, as well as the lack of studies about the interaction of these crops with nMgO and nHA, this study was planned. It is hypothesized that the two nanoparticles could promote crop seed germination and alleviate ammonium stress. The main objectives of this study were to examine the effects of nMgO and nHA on seed germination in Hami melon and water spinach. NH₄⁺ is also one of the contributory factors for increased toxicity in case of its excessive doses, so the purpose of this study was also to know the ammonium stress mitigating capacities of NPs under their optimal applications in normal conditions of soils.

2. Materials and Methods

2.1. Nanomaterials and Crop Seeds

The nMgO with a purity of 99.0%, average particle size of 30 nm, and specific surface area of 42 m² g⁻¹ was synthesized by precipitation and supplied by Hangzhou Heng Na New Material Co., Ltd. (Hangzhou, China). The nHA was synthesized by the hydrothermal method and supplied by Beijing Deke Daojin Science and Technology Co., Ltd. (Beijing, China) with a purity of 99.9%, an average particle size of 20 nm, and a specific surface area of 50 m² g⁻¹. These nanoparticles were configured with deionized water and applied as a suspension after half an hour of sonication (JL-721DTH, Nanjing Kejie Analysis Instrument Lit., Nanjing, China). The zeta potential of the nanoparticles suspensions was tested by using Zetasizer Nano ZS90 (Malvern Instruments Ltd., Malvern, UK), which was −2.29 mV for nHA and 5.85 mV for nMgO, respectively.

The seeds of water spinach (Ipomoea aquatica Forssk.) and Hami melon (Cucumis melo L.) were collected by Shenzhen Fanji Seed Co., Ltd. (Shenzhen, China) and Xinjiang Annong Seed Co., Ltd. (Jichang, China), respectively. Before the experiment, seeds were soaked in 4% of sodium hypochlorite solution for 30 min in advance to maintain a sterile surface and washed thoroughly with deionized water 2–3 times for the removal of residual chemicals. Analytical graded ammonium sulfate ((NH₄)₂SO₄) and other chemicals used in the nutrient solution for ammonium stress were purchased from Xilong Scientific Co., Ltd. (Shantou, China).

2.2. Experimental Setup for Seed Germination

The seed germination experiment for water spinach or Hami melon was a completely randomized factorial design with two factors and three replicates, i.e., nanomaterial type (nMgO and nHA) and the application rates (0, 10, 100, 500, and 1000 mg L⁻¹). A qualitative filter paper with a diameter of 9 cm was placed bilayers in the bottom of plastic Petri dishes, and 20 sterilized seeds of water spinach and Hami melon were evenly placed on it. A total of 5 mL of nMgO and nHA suspensions with different applications of the two NPs were introduced into the Petri dishes containing the seeds of water spinach or Hami melon, respectively. The Petri dishes were placed in an artificial climate chamber with a day/night cycle of 12 h/12 h at 25 °C and 60% relative humidity.

2.3. Seed Germination and Seedling Growth Assay

Germination of seeds was marked as the radicle reached 1/2 of the seed length by following the method of Xiao et al. [23], and observation was ended on the 7th day of the germination experiment. The rest of the parameters, including seedling biomass, seed germination energy (GE), germination rate (GR), mean germination time (MGT), and germination index (GI), were measured with Equations (1)–(6) as follows [24]:

Germination energy (GE%) = Number of germinated seeds determined on the fourth day after setting up the germination test/total number of seeds × 100

(1)

Germination rate (GR%) = Number of seeds that germinated at the end of the experiment/total number of seeds for test × 100

(2)

Mean germination time (MGT) = ∑(Gt × Dt)/∑Gt

(3)

Gt = number of seeds germinated on different days

(4)

Dt = different days of germination

(5)

Germination index (GI) = ∑ (Gt/Dt)

(6)

For the observation of seedling growth, plants were harvested up to 14 days after the experiment, and roots were immersed in 1 mM EDTA-2Na, then were rinsed with deionized water to remove metal ions and nMgO/nHA from the nutrient solution. Fresh weights of root, stem, and plant length and antioxidant enzyme activity were also measured.

2.4. Experimental Setup for Ammonium Stress

The ammonium stress experiment for water spinach or watermelon was also a completely randomized factorial design with two factors and three replicates, i.e., NH₄⁺ concentration (0, 0.5, 2.5, and 5 mM) and nanomaterial treatment (no nanoparticles, nMgO and nHA addition). According to the results of the seed germination test, the optimal application rate of 100 mg L⁻¹ of nMgO or nHA was selected in the hydroponic experiments for ammonium stress. The seeds of water spinach and Hami melon were incubated for 3 days, and little seedlings wrapped in a sponge were placed in a custom basket containing a round-bottomed brown vial (5 cm × 4.5 cm) with 45 mL of the modified Hoagland nutrient solution. Then, seedlings were transferred to an artificial climate chamber (light time: 16 h d⁻¹, temperature: 25 ± 2 °C, relative humidity: 70%) for 14 days. The composition of the modified Hoagland nutrient solution was: 1 mmol KCl, 0.5 mmol CaCl₂, 0.625 mmol KH₂PO₄, 0.5 mmol MgSO₄•7H₂O, 0.025 mmol Fe-Na-EDTA, 12.5 mmol H₃BO₄, 1 mmol MnSO₄•H₂O, 1 mmol ZnSO₄•7H₂O, 0.25 mmol CuSO₄•5H₂O and 0.25 mmol H₂MoO₄. NH₄+ was added to the nutrient solution in the form of (NH4)₂ SO₄, and experiments with each NH₄⁺ level were conducted in ten replicates. The nutrient solution was changed approximately every 3 days to maintain a stable NH₄⁺ concentration.

2.5. Determination of N Content and Antioxidant Enzyme Activity

N contents of the plant were measured by grinding dried shoots and roots of seedlings into a fine powder, then poured into a tube having H₂SO₄–H₂O₂ for decoction until changed into transparent. The decocted material was transferred to a 50 mL volumetric flask, capacitated, filtered, and analyzed with a Kjeldahl nitrogen analyzer (K9840, Shandong Hanon Scientific Instruments Co., Ltd., Jinan, China).

For the antioxidant enzyme extraction, approximately 2 g of fresh seedling tissue was ground in liquid nitrogen and then extracted with 5 mL of 100 mmol L⁻¹ phosphate buffer (pH 7.8). After centrifuging at 10,000× g for 15 min, the supernatant was filtered and collected for the determination of catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) activity were measured by following the method as described by Nahakpam and Shah [25].

2.6. Statistical Analysis

The data were analyzed using two-way analysis of variance (ANOVA) and were further analyzed using one-way ANOVA with Duncan multiple-range test to show the significant differences between the treatments at p < 0.05. SPSS statistics 25.0 software was used to correlate seed germination with the growth of seedlings under ammonium stress, while Origin Pro 2021 (OriginLab Corporation, Northampton, MA, USA) was used for the evaluation of plots.

3. Results

3.1. Seed Germination Parameters

The application of nMgO or nHA significantly affected GE, GR, and GI for water spinach seeds, while no effect was observed on MGT (

Table 1. Table 1 Effects of nano magnesium oxide (nMgO) and nano-hydroxyapatite (nHA) on seed germination energy (GE), germination rate (GR), mean germination time (MGT), and germination index (GI) of water spinach and Hami melon.

Nanomaterial Types	Nanomaterial Concentration	GE	GR	MGT	GI
	mg L−1	%	%	Day	—
			Water spinach
Control	0	33.33 ± 4.41 c	70.00 ± 5.77 b	3.53 ± 0.07 a	4.60 ± 0.44 b
nMgO	10	35.00 ± 2.89 bc	71.67 ± 1.67 ab	3.47 ± 0.07 a	4.57 ± 0.12 b
	100	45.00 ± 2.89 ab	73.33 ± 1.67 ab	3.40 ± 0.35 a	5.07 ± 0.28 ab
	500	41.67 ± 4.41 abc	70.00 ± 2.89 b	3.60 ± 0.06 a	4.37 ± 0.17 b
	1000	41.67 ± 4.41 abc	70.00 ± 2.89 b	3.40 ± 0.17 a	4.57 ± 0.22 b
nHA	10	43.33 ± 3.33 abc	75.00 ± 2.89 ab	3.40 ± 0.06 a	4.97 ± 0.23 b
	100	48.33 ± 1.67 a	81.67 ± 4.41 a	3.40 ± 0.10 a	5.83 ± 0.29 a
	500	46.67 ± 3.33 a	73.33 ± 1.67 ab	3.47 ± 0.09 a	4.80 ± 0.23 b
	1000	45.00 ± 2.89 ab	71.67 ± 1.67 ab	3.57 ± 0.18 a	4.57 ± 0.29 b
			Hami melon
Control	0	73.33 ± 3.84 abc	88.90 ± 2.20 ab	2.70 ± 0.08 bc	5.34 ± 0.04 abcd
nMgO	10	78.90 ± 4.85 ab	91.10 ± 2.20 ab	2.66 ± 0.13 bc	5.53 ± 0.35 abcd
	100	78.90 ± 1.10 ab	93.33 ± 3.84 ab	2.59 ± 0.14 c	5.87 ± 0.31 ab
	500	55.57 ± 5.88 d	88.90 ± 2.20 ab	3.12 ± 0.06 a	5.05 ± 0.26 cde
	1000	53.33 ± 3.84 d	86.67 ± 3.84 b	3.26 ± 0.17 a	4.39 ± 0.30 e
nHA	10	75.53 ± 2.23 abc	91.13 ± 4.43 ab	2.63 ± 0.09 bc	5.62 ± 0.30 abc
	100	80.00 ± 3.87 a	97.77 ± 2.23 a	2.58 ± 0.04 c	6.10 ± 0.05 a
	500	67.90 ± 1.20 bc	93.30 ± 0.00 ab	3.00 ± 0.04 ab	5.23 ± 0.05 bcd
	1000	64.47 ± 2.23 cd	88.87 ± 4.43 ab	3.15 ± 0.21 a	4.82 ± 0.19 de

Note: the same letter (column) does not statistically differ (Duncan multiple-range test; p < 0.05). Each data is expressed as mean ± standard error (n = 3).

). As compared to the control, the nMgO application at the rate of 100 mg L⁻¹ significantly increased GE by 35% (p < 0.05), while nHA application at the rate of 100 mg L⁻¹ significantly increased GE, GR, and GI by 45%, 16.7% and 26.7% (p < 0.05), respectively.

As for Hami melon seeds, the application of nMgO or nHA at the rate of 100 mg L⁻¹ significantly affected the GE, GI, and MGT of the Hami melon while producing no effect on GR (Table 1). The GE and GI of Hami melon seeds gradually increased with the increase of concentration of NPs and reached a maximum at the concentration of 100 mg L⁻¹. However, when the application rates of nMgO or nHA crossed 100 mg L⁻¹, the GE and GI of Hami melon seeds showed an obvious decreasing trend with prolonged MGT.

3.2. Seedling Growth

As shown in (Figure 1A), nMgO or nHA significantly affected the shoot and root growth of water spinach seedlings. It was observed that nMgO application at the rate of 100 mg L⁻¹ promoted the growth of water spinach while it produced an inhibitory effect when its concentration exceeded over 100 mg L⁻¹. As compared to the control, the shoot length of water spinach increased by 30.7% and 44.4% at the nMgO application rate of 10 mg L⁻¹ and 100 mg L⁻¹, respectively. However, root length decreased by 38.1% and 42.5% when the nMgO application rate was up to 500 mg L⁻¹ and 1000 mg L⁻¹. Similarly, the shoot length of water spinach increased by 29.4% at the nHA application rate of 100 mg L^−1, while it decreased by 34% at the application of 1000 mg L⁻¹.

The effects of nMgO and nHA on Hami melon have been shown in (Figure 1B). The two nanoparticles did not produce any significant effect on the shoot length of the Hami melon. However, the root length of Hami melon increased by 42.4% at the nHA application rate of 100 mg L⁻¹ as compared to that of the control (p < 0.05).

3.3. Ammonium Stress Alleviation

There was a significant effect of NH₄⁺ concentrations on the growth of root and shoot of water spinach (Figure 2A). At 5 mmol of NH₄⁺, root growth was significantly decreased, while shoot growth did not show any clear changes by increasing NH₄⁺ concentrations. Compared with NH₄⁺ alone, the addition of nMgO or nHA at 100 mg L⁻¹ boosted the shoot length by 87% and 84%, respectively, while root length increased by 39.5% and 37.9%, respectively (p < 0.05). There were also significant effects of NH₄⁺ on the fresh weight of the shoot and root of water spinach (Figure 2C). NH₄⁺ supplied at the rate of 2.5 mmol and 5 mmol NH₄⁺ decreased the fresh weight of the shoot by 28.3% and 35%, respectively (p < 0.05), but the addition of nHA significantly increased the fresh weight by 60.5% as compared to the control. Similarly, there was also a significant increase in root fresh weight after treating with 100 mg L⁻¹ of nMgO or nHA less than 5 mmol of NH₄⁺ stress as compared to the control.

As mentioned in (Figure 2B), NH₄⁺ stress beyond the concentration of 2.5 mmol also produced adverse effects on the growth of root and shoot of Hami melon seedlings, but root length increased after the addition of nHA. The addition of nHA under 2.5 mmol and 5 mmol of NH₄⁺ alleviated ammonium stress by increasing root length by 1.2-fold and 1.3-fold, respectively. The addition of 100 mg L⁻¹ of nHA increased shoot length by 11% more than the control. However, the addition of 100 mg L⁻¹ of nMgO caused an inhibitory effect on the root growth of Hami melon seedlings (p < 0.05). Compared with the control, shoot fresh weight treated with 2.5 mmol and 5 mmol of NH₄⁺ decreased by 14.1% and 42.3%, while fresh root weight decreased by 32.4% and 40.5%, respectively (Figure 2D).

3.4. Plant N Content

The total nitrogen content (TN) of water spinach seedlings increased significantly with increasing NH₄⁺ concentration (Figure 3A). Compared with the control, the TN content of water spinach seedlings under the NH₄⁺ concentration of 2.5 mmol and 5 mmol increased by 24.4% and 26.4%, respectively. The addition of 100 mg L⁻¹ of nMgO or nHA did not significantly affect the TN content of water spinach seedlings (p < 0.05).

As shown in Figure 3B, the TN content of the Hami melon seedlings also increased significantly with the increasing NH₄⁺ concentration and was up to the maximum under the 5 mmol NH₄⁺, which increased by 74.3% than the control. Seedlings treated with the addition of nHA showed no significant increase in TN content as compared to NH₄⁺ treated alone, while the TN content of Hami melon seedlings treated with nMgO was found to be significantly lower than that of NH₄⁺ supplied (p < 0.05).

3.5. Antioxidant Enzyme Activities

The effects of NH₄⁺ and nanoparticles on the enzymatic activity of water spinach seedlings showed two different results, as shown in (Figure 4A). At the NH₄⁺ concentration of 5 mmol, the CAT activity increased by 40.5% as compared to that of the control (p < 0.05). While seedlings treated with the composite solution of nHA had no significant effect, CAT activity after the addition of 100 mg L⁻¹ of nMgO was found to be significantly lower (p < 0.05) as compared to that of NH₄⁺ alone. As for Hami melon seedlings, CAT activity had a rapid increase of 76.5–114% to the control (Figure 4B). CAT activity of seedlings treated with 100 mg L⁻¹ of nHA and NH₄⁺ was found to be higher, while nHA along with 2.5 mmol and 5 mmol of NH₄⁺ increased the seedling by 75% and 112% as compared to that of sole treatment of NH₄⁺, respectively.

There was no significant difference between the POD activity of water spinach under the addition of nMgO or nHA (Figure 5A). However, the POD activity of Hami melon seedlings under the ammonium stress at the rates of 0.5 mmol, 2.5 mmol, and 5 mmol significantly increased by 48.7%, 123.7%, and 208.4% as compared to the control, respectively. The addition of nHA significantly increased the POD activity of Hami melon seedlings except under 2.5 mmol NH₄⁺, while nMgO addition reduced the POD activity compared to that of NH₄⁺ alone (p < 0.05).

The SOD activity of water spinach seedlings at the rates of 0.5, 2.5, and 5 mmol NH₄⁺ significantly increased by 55.4%, 119%, and 98% as compared to the control, respectively (Figure 6A). The addition of nHA under 2.5 mmol NH₄⁺ significantly improved the SOD activity of water spinach seedlings by 39.8% more than that of the NH₄⁺ supplied alone. As compared with the control, both the increasing NH₄⁺ and the addition of NPs significantly increased the SOD activity of Hami melon seedlings (p < 0.05). The SOD activity for the treatment of nHA addition under 5 mmol NH₄⁺ was found to be the maximum, which was significantly higher than that of only NH₄⁺ treatment (p < 0.05).

4. Discussion

4.1. Effects of NPs on Seed Germination and Growth

The seed germination test is a basic method to determine the effect of nanoparticles on plant growth. The effect of nanoparticles on the growth of plants depends upon their concentrations, as well as changes from plant to plant. In earlier studies, it was found that MgO NPs at the concentration of 100 mg L⁻¹ significantly promoted seed germination and seedling vigor in green gram (Vigna radiata), and biogenic (betel leaf extract) synthesized MgO NPs at 50 mg L⁻¹ and 100 mg L⁻¹ concentrations improved biomass, shoot root growth [26,27]. However, Sharma et al. [28] reported that MgO NPs reduced stem length by 33–55% and relative root length by 27–59% as the concentration increased from 10 to 150 mg L⁻¹ in black gram. The present study also supports such findings at the optimum application rate of nMgO (100 mg L⁻¹) for the germination and growth of water spinach and Hami melon.

Analysis of root elongation is the simplest method for determining whether NPs have beneficial, harmful, or no effects on plant growth [29,30]. According to Wang et al. [31], hydroxyapatite nanoparticles suspension promoted root growth of lettuce with the concentration range of 100–2000 mg L⁻¹ while retarded root growth of tomato with the concentration range of 500–2000 mg L⁻¹. In the present study, the best optimal dose of nMgO or nHA in the present study is 100 mg L⁻¹ for seed germination and seedling growth of water spinach and Hami melon, and adding high concentrations of nMgO or nHA exerted negative effects, which was probably caused by producing or releasing oxhydryl, changing the solution pH and allowing nanoparticles to enter the plant cells [21]. Moreover, nHA was also found to have a more positive effect on the growth of two crops, which was probably related to the nanoparticle charge and size [32].

4.2. Effects of NPs on Ammonium Stress Alleviation

When NH₄⁺ is treated as the sole N, high concentrations of NH₄⁺ are toxic to plants, and its accumulation in cells leads to tissue damage, with retarded root growth and leaf discoloration being the main manifestations of ammonium toxicity in plants [7]. The results of the present study also relate to such comprehensive reasons by showing that root length and biomass of water spinach seedlings were severely inhibited at 5 mmol of NH₄⁺ (Figure 2A,C), while in Hami melon seedlings, no such changes were observed at 0.5 of mmol NH₄⁺ but as crossed the threshold limit, seedling growth was seen to reduce due to NH₄⁺ stress (Figure 2B,D). A significant toxic effect on both plants was observed under 2.5 mmol and 5 mmol NH₄⁺ stress corresponding to Li et al. [33] that Myosoton aquaticum showed high uptake and accumulation of TN with the increase of exogenous NH₄⁺ concentration. In the current study, the TN content of both crops increased with the increase of NH₄⁺ concentration, indicating that these crops had a high capacity for NH₄⁺ uptake in a high NH₄⁺ concentration environment.

As concerned the role of nanoparticles in ammonium stress alleviation, the addition of nHA at 100 mg L⁻¹ alleviated the inhibitory effect of NH₄⁺ toxicity on both plants, but the addition of nMgO produced an inhibitory effect, which accorded with the views of Elizabath et al. [34] that the effects of NPs can also vary from plant to plant. The high application rate of MgO NPs has adverse effects on crop nutritional growth, such as reducing stem and root length, weakening antioxidant capacity, and decreasing carbohydrate and protein accumulation [28]. In the present study, it was also examined that the addition of nMgO caused damage to the growth of Hami melon seedlings and reduced plant N content.

4.3. Effects of NPs on Antioxidant Enzyme Activity

The increase in the plant’s antioxidant enzyme activity may contribute to the enhancement of antioxidant toxicity. Plants have evolved defense strategies to reduce oxidative damage, including enhanced enzymatic antioxidants (SOD, POD, and CAT) that can scavenge reactive oxygen species (ROS) and mitigate the resulting negative effects [35,36]. According to studies by Xie et al. [37], the higher tolerance capacity of rice to NH₄⁺ was achieved by activating antioxidant defense signals and regulating ROS homeostasis to neutralize the excess oxygen produced under NH₄⁺ stress. Similarly, the antioxidant enzymes glutathione reductase, guaiacol peroxidase, and superoxide dismutase activities were found to be higher in ammonium-treated roots of Arabidopsis thaliana, indicating that ammonium-induced oxidative stress [38]. In the present study, it was noted that ammonium stress enhanced the antioxidant enzyme activities of water spinach and Hami melon. However, this may not be sufficient to scavenge excess ROS, as evidenced by the reduction in biomass of the two crops at high NH₄⁺ concentrations.

Fe₃O₄ NPs were reported to significantly activate antioxidant enzyme activity in wheat seedlings to attenuate heavy metal-induced oxidative stress [39]. Nanocoatings containing SiO₂ NPs improved drought tolerance in wheat by regulating ROS production and maintaining higher antioxidant activity [40]. It was also confirmed in the present study that the addition of nHA increased the level of induced antioxidant enzyme activity and alleviated the effects of ammonium stress on Hami melon seedlings compared to NH₄⁺ alone. It was also reported that exposure of mustard to AgNPs inhibited antioxidant enzyme activity and caused oxidative damage to mustard seedlings [41]. However, the treatment of Brassica napus with 2000 and 4000 mg L⁻¹ of Co₃O₄ NPs reduced its antioxidant potential by inhibiting the accumulation of non-enzymatic polyphenols and antioxidant enzyme activities [42]. The findings of the present study also showed a similar pattern of changes in which the addition of nMgO attenuated the CAT activity of water spinach and the POD activity of Hami melon seedlings. The current study provides data elucidating the comparative effectiveness of nMgO and nHA in mitigating ammonium stress in water spinach and Hami melon through hydroponic experiments, while its application in real soils environment to reduce ammonium concentrations remains controversial. This is mainly because the resistance strategies of plants to abiotic stresses and the potential of nanomaterials in different plants are not fully explored. The present study provided a reference on how to select the appropriate type of nanomaterials and optimize the application method that will be used in agriculture in the future.

5. Conclusions

Both nMgO and nHA significantly affected seed germination and seedling growth of both plants. The optimal application rate of nMgO or nHA for promoting the seed germination of water spinach and Hami melon was observed as 100 mg L⁻¹ in. NH₄⁺ stress with concentrations of 2.5 mmol and 5 mmol caused severe inhibitory effects on the seedling growth of Hami melon and water spinach, respectively. The addition of nMgO or nHA at 100 mg L⁻¹ alleviated the inhibitory effect of NH₄⁺ stress on water spinach by enhancing the antioxidant enzyme activity. However, the nMgO addition produced an inhibitory effect on the root growth of Hami melon seedlings. Due to the potential toxicity, the ecological risk of nanomaterials must be assessed before the large-scale application.