Using Nanomaterials and Arbuscular mycorrhizas to Alleviate Saline–Alkali Stress in Cyperus esculentus (L.)

Abstract

Saline–alkali (SA) stress is an abiotic stress that exists widely in the natural environment, seriously affecting the growth and development of crops. Tiger nut (Cyperus esculentus L.), a perennial herb of Cyperus in Cyperaceae, is considered a pioneer crop for growing and improving SA land due to its excellent adaptability and SA tolerance. This study is the first to evaluate the SA tolerance of tiger nut and the alleviative effects of nanomaterials (nano-selenium and multi-walled carbon nanotubes) and Arbuscular mycorrhizas (AMs) on SA stress. The results showed that the seedling fresh weight of tiger nut was the most suitable parameter to describe the dose–response effect of plant growth with increased SA concentration. Based on the log-logistic dose–response curve, the GR₅₀ values of NaCl and NaHCO₃ (the concentrations causing a 50% reduction in seedling fresh weight) were determined to be 163 mmol L⁻¹ and 63 mmol L⁻¹, respectively. Under these stresses, the exogenous application of MWCNTs at 100 mg L⁻¹ or Nano-Se at 10 mg L⁻¹ showed that the effect of SA on tiger nut was alleviated. Field evaluation further showed that the exogenous application of MWCNTs, Nano-Se, or AMs could effectively alleviate SA stress on tiger nut. Compared to the untreated control, the application of these substances significantly improved the plant photosynthesis-related parameter, antioxidant enzyme activity, plant height (height: 66.0–69.9 cm), tuber yield (yield: 23.4–27.4 g plant⁻¹), and oil quality of tiger nut under SA stress. The results of this study indicate that the application of MWCNTs, Nano-Se, or AMs, to tiger nut can alleviate SA stress and maintain seed yield, providing the possibility of using these nanoparticles to improve the SA tolerance of tiger nut in agricultural practice.

1. Introduction

Tiger nut (Cyperus esculentus L.), also known as yellow nutsedge, is a perennial Cyperus herb in Cyperaceae. Originating in northeast Africa, it developed rapidly in China, Xinjiang, and Inner Mongolia after 2006. Tiger nut not only has excellent botanical characteristics, such as strong adaptability, good stress resistance, and SA tolerance [1], but also has economic characteristics such as high oil content and rich nutritional value [2]. It is also a pioneer crop for planting and improving SA land [3]. Currently, the research on tiger nut mainly focuses on cultivation method [4], physical and chemical properties [5,6], etc., and few studies have been conducted on its response to SA stress. Tiger nut is more susceptible to SA stress at the seedling growth stage. Thus, improving the SA tolerance of tiger nut in this period is particularly important for plant growth, establishment, and development [7,8].

Due to many factors, such as ecological and climatic influences, the area of saline and alkaline land has been increasing, becoming a common concern worldwide [9]. The influence of soil salinization on plants includes salt and alkali stress. Salt stress (e.g., NaCl) mainly inhibits plant growth through osmotic stress and ionic toxicity [10], while alkali stress (e.g., NaHCO₃) could cause more serious damage to plants due to its high soil pH [11]. The cultivation of salinity-tolerant pioneer crops is significant in managing and improving SA land.

In recent years, numerous researchers have gradually applied nanomaterials to agricultural production [12], especially for the regulation of plant growth [13] and alleviating abiotic stresses (e.g., salinization, drought) [14,15], attracting great attention. Multi-walled carbon nanotubes (MWCNTs) are nanomaterials with unique physical and chemical properties that can be used as plant growth regulators to increase the absorption of nutrients and improve the stress resistance of plants [16]. Previous studies have shown that rice seedlings cultured on medium with MWCNTs grow to be healthier than those treated in control groups [17]. Martínez et al. [18] found that MWCNTs can improve the water absorption and utilization of plants under salt stress. Nano-selenium (Nano-Se) has also been reported in many studies to alleviate the stress from salt [19], drought [20], and heavy metals (e.g., Al³⁺) [21]. Additionally, the symbiotic relationship between plants and microorganisms can also be used to improve and restore SA land; for this, arbuscular mycorrhizals (AMs) are widely adopted. Some studies have found that AMs can form a symbiotic relationship with 80% of plants and promote the nutrient absorption and osmotic adjustment of plants under stress conditions [22]. Han et al. [23] found that AM inoculation in high-salt environments can increase soluble sugar and soluble protein content in cucumber leaves and roots. Ma et al. [24] found that AMs could reduce the level of membrane lipid peroxidation caused by low temperature and root rot in tomato neck. In recent years, MWCNTs, Nano-Se, and AMs have been used to improve abiotic stresses in various crops, but there is no report on tiger nut under SA stress. Therefore, the purposes of this study are (i) to evaluate the tolerance of tiger nut to SA stress and (ii) to test the potential of using MWCNTs, Nano-Se, and AMs to alleviate SA stress on tiger nut. The hypothesis is that treating tiger nut with MWCNTs, Nano-Se, or AMs could potentially alleviate adverse effects (e.g., plant growth inhibition, yield loss) through enhancing the biochemical processes of tiger nut plants, maintaining their tuber yield and oil content.

2. Materials and Methods

2.1. Materials and Reagents

The tiger nuts (not registered as a variety) used in this experiment were provided by the Institute of Grassland Research of Chinese Academy of Agricultural Sciences (CAAS). This plant species is native to Spain, with an initial germination rate ≥ 95%. Nano-Se (Se⁰ nanoparticles; mean size: 50–78 nm) was purchased from Guilin Jiqi Group Co., Ltd., Guilin, China. MWCNTs (outer diameter: <8 nm; inner diameter: 2–5 nm; length: 10–30 µm) were purchased from Shanghai Macklin Biochemical Co., Ltd., Shanghai, China. Arbuscular mycorrhizas (AMs) were purchased from Norman Lear Environmental Energy Technology Co., Ltd., Qingdao, China. The other chemicals and reagents used in this study were either purchased from Yangzhou Hongze Biological Co., Ltd. (Yangzhou, China) or other chemical companies.

2.2. Experimental Design

2.2.1. Study I: Determination of GR₅₀ for NaCl and NaHCO₃

This experiment was conducted in the greenhouse of the Grassland Science Experimental Station of Yangzhou University (119°23′ E, 32°20′ N) in March 2021. NaCl and NaHCO₃ were used to simulate salt and alkali stress, respectively. A range of concentrations, including 20, 40, 80, 160, and 320 mmol L⁻¹ for NaCl (pH = 7.02–7.32), and 40, 60, 80, 100, and 120 mmol L⁻¹ for NaHCO₃ were used in this study. Tiger nut tubers were firstly placed in a tray covered with a layer of wet filter paper. During this period, the tuber materials were kept in a light incubator (25 °C, 10/14 h photoperiod, 70% relative humidity, Changzhou Haibo Equipment Co., Ltd., Changzhou, China) to induce germination. The trays were sprayed with water twice a day to replenish water. After germination, three healthy tiger nut seedlings were each transplanted into a plastic pot (diameter: 25.5 cm; height: 29 cm) filled with organic horticultural potting soil (Yiyuan Agriculture and Forestry Ltd., Suzhou, China) and raised in a greenhouse (growth condition: 25 °C, 12/12 h photoperiod supplemented by an overhead sodium lamp) up to 10 cm in height before NaCl or NaHCO₃ treatment. The treatment is described in

Table 1. Summary of various solutions treatments in this study.

Group	Pot-Planting Test	Field Test
	Various Solution Treatments
1	Control (only clean water)	Control (only clean water)
2	163 mM NaCl	163 mM NaCl
3	63 mM NaHCO3	63 mM NaHCO3
4	50 mg L−1 MWCNTs	100 mg L−1 MWCNTs
5	100 mg L−1 MWCNTs	10 mg L−1 Nano-Se
6	5 mg L−1 Nano-Se	AM
7	10 mg L−1 Nano-Se	100 mg L−1 MWCNTs + 163 mM NaCl
8	50 mg L−1 MWCNTs + 163 mM NaCl	10 mg L−1 Nano-Se + 163 mM NaCl
9	100 mg L−1 MWCNTs + 163 mM NaCl	AM + 163 mM NaCl + 163 mM NaCl
10	5 mg L−1 Nano-Se + 163 mM NaCl	100 mg L−1 MWCNTs + 63 mM NaHCO3
11	10 mg L−1 Nano-Se + 163 mM NaCl	10 mg L−1 Nano-Se + 63 mM NaHCO3
12	50 mg L−1 MWCNTs + 63 mM NaHCO3	AM + 63 mM NaHCO3
13	100 mg L−1 MWCNTs + 63 mM NaHCO3
14	5 mg L−1 Nano-Se + 63 mM NaHCO3
15	10 mg L−1 Nano-Se + 63 mM NaHCO3

. Either NaCl or NaHCO₃ solution (50 mL) was root-applied to the plants. The controls were treated with the same amount of distilled water. This experiment was set up as a randomized complete block design (RCBD) with 3 replications of each treatment. After 24 h of treatment, the chlorophyll content and chlorophyll fluorescence of the tiger nuts were determined. Chlorophyll content was determined by DMSO rapid extraction [25]. Chlorophyll fluorescence was measured by a chlorophyll fluorescence meter (FP110-LM/D, Czech Republic) [26]. Three months after transplanting, the plant height, aboveground fresh weight, tiller number, and tuber number of all treated plants were assessed. Then, nonlinear regression analysis (log-logistic dose–response curve) was carried out on the measured data, and the GR₅₀ values of NaCl and NaHCO₃ were calculated for all parameters. Among these parameters, the most suitable parameter for describing the dose–response of tiger nut to NaCl and NaHCO₃ was determined.

2.2.2. Study II: Screening the Effect of Alleviation Concentration of MWCNTs or Nano-Se on GR₅₀ of Tiger Nut

Based on the determined GR₅₀ for tiger nut fresh weight, we further screened the effect of the concentration of the exogenous application of MWCNTs or Nano-Se on the alleviation of NaCl or NaHCO₃ stress. In this experiment, the concentrations of MWCNTs were 50 and 100 mg L⁻¹, and the concentrations of Nano-Se were 5 and 10 mg L⁻¹, respectively. A total of 15 treatments were included (n = 3). The preparation of the germinated tiger nut seedlings and plant growing conditions was carried out as described before. When the tiger nut seedlings grew up to 10 cm, the plants were first treated with 50 mL of NaCl (163 mmol L⁻¹) or NaHCO₃ (63 mmol L⁻¹). After 4 h of treatment, 100 mL of MWCNTs (50 and 100 mg L⁻¹) or Nano-Se (5 and 10 mg L⁻¹) was further applied to the treated plants. The leaf chlorophyll content and chlorophyll fluorescence were determined after 24 h of MWCNT or Nano-Se treatment. At maturity, all plants were measured for plant height, aboveground fresh weight, and dry weight.

2.2.3. Study III: Effects of MWCNTs, Nano-Se, and AMs on Tiger Nut Under SA Stress

After determination of the concentrations of GR₅₀ for MWCNTs and Nano-Se under SA stress, a field experiment was conducted at the experimental site of Yangzhou University, Yangzijin Campus, Jiangsu Province (119°26′ E, 32°23′ N), in April 2023 to evaluate the effects of MWCNTs and Nano-Se on tiger nut under SA stress. In this experiment, the alleviative effect of inoculating tiger nut with AMs on SA stress was also assessed. Meteorological data, including monthly air temperature, soil temperature, and accumulated precipitation during the experimental period of 2022–2023 are shown in Figure 1. Prior to the field study, all tiger nut tubers were germinated as described in the previous section. After that, the germinated tiger nut tubers were transplanted into the experimental field plots. For AM treatment, prior to transplanting, the tiger nut tubers were inoculated with AMs. Following the product preparation instructions, 1.6 g of AM was suspended in 50 mL distilled water. Each plot (0.5 m × 0.5 m) consisted of 3 rows, with 3 seedlings in each row. The depth was about 2–3 cm. The transplanted seedlings were irrigated manually. When the plants grew up to 10 cm, they were treated with 50 mL of NaCl or NaHCO₃, while the control groups were treated with distilled water. Four hours after treatment, the plants, except for AM-inoculated ones, were applied with of 2 L MWCNTs (100 mg L⁻¹) or Nano-Se (10 mg L⁻¹) (Table 1). The leaf chlorophyll content and chlorophyll fluorescence were determined at 24 h after NaCl or NaHCO₃ treatment. Meanwhile, five leaves of the treated plants from each plot were sampled for the determination of the enzyme activity of superoxide dismutase (SOD) and catalase (CAT), and the content of malondialdehyde (MDA), soluble protein, and proline. The enzyme activity of SOD and CAT were determined by nitroblue tetrazole photooxidation [27] and ultraviolet absorption [28], respectively. MDA was determined by the thiobarbituric acid method [27]. Soluble protein content was determined by the Coomassie brilliant blue chromogenic method [27], and the content of free proline was determined by the acid ninhydrin method. During the experiment, each treatment and index determination was repeated three times with an interval of one week. At 45 days after treatment, all plants were harvested to assess plant height, relative conductivity, and forage nutritional value. The tuber yield and oil content were also determined. The relative conductivity was measured by the conductivity meter method [29]. Briefly, we weighed 0.1 g of leaves, removing the main veins and leaf edges. After that, we added 10 mL of deionized water; this was fully oscillated and left to stand for 12 h. Conductivity R1 was measured first, and then the solution was treated with a boiling water bath for 30 min to determine conductivity R2.

$$\mathrm{R}\mathrm{e}\mathrm{l}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{e}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{d}\mathrm{u}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{i}\mathrm{t}\mathrm{y}(\%)=(\mathrm{R}1/\mathrm{R}2)\times 100\%$$

(1)

The forage nutritional values, including the contents of neutral detergent fiber (NDF) and acidic detergent fiber (ADF), were determined according to the procedure described regarding the analysis and evaluation of forage feed [30]. The crude protein content was determined by total nitrogen × 6.25 (kjeldahl method). Additionally, the oil content was determined by the Soxhlet extraction method [31]. Firstly, tiger nuts were dried in an oven at 130 °C for 1.5 h, and then the tubers were cooled and ground into a fine powder. Secondly, 0.5 g tuber powder was added into a Soxhlet extraction device containing 200 mL anhydrous ether. After refluxing for 12 h, the sample was taken out, dried at 45 °C for 12 h, and weighed. The oil content was determined by comparing the difference in sample weight before extraction (m₀) and after extraction (m₁).

$$\mathrm{O}\mathrm{i}\mathrm{l}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{e}\mathrm{n}\mathrm{t}\left(\%\right)={\displaystyle \frac{{\mathrm{m}}_0-{\mathrm{m}}_1}{{\mathrm{m}}_0}}\times 100\%$$

(2)

2.3. Statistical Analysis

Before variance analysis, the Shapiro–Wilk test was used to test the normal distribution of the measured data. The residual normal distribution was determined, and then the Levene test was used to test the homogeneity of variance. In Study I, the statistical software R (version r x64.1.2) was used to analyze the variance (ANOVA) of the data, which showed that there was a significant (p < 0.05) dose–response effect between the treatments and the fresh weight of the tiger nuts. To estimate the GR₅₀ of the fresh weight of tiger nuts, the measured fresh weight value was fitted to the log-logistic dose–response curve [32,33]:

$$\mathrm{y}={\displaystyle \frac{\mathrm{c}}{1+{\left(\frac{\mathrm{x}}{{\mathrm{GR}}_{50}}\right)}^{\mathrm{b}}}}$$

(3)

y is the predicted aboveground fresh weight of tiger nut, c is the upper limit of the model, and b is the slope when passing GR₅₀. In Study II and III, all the parameters measured were the average of three replicates, and the data were analyzed and processed by Microsoft Excel 2019 and SPSS 26.0. Drawings were completed with R and Excel 2019.

3. Results

3.1. Dose–Response Effect and Tolerance of Tiger Nut to SA Stress

In general, with increasing SA concentration, all the growth parameters (e.g., plant height, fresh weight, chlorophyll) of tiger nut showed a downward trend, and the three-parameter log-logistic dose–response curve described the relationship between dose and growth parameters well (R²: 0.76–0.99; p < 0.05) (Figure 2;

Table 2. Summary of parameters estimated from the three-parameter log-logistic model ^a for shoot length, SPAD, Fv/Fm, fresh weight, and tiller number of tiger nut under saline and alkaline stress in the pot-planting test.

Indicator	Parameter				Statistical Data
	b	d	GR50	DF	MS	R2	p
ShootLength-Na+	1.37 (0.52) b	83.68 (1.47)	1097.06 (537.55)	5	81.51	0.94	**
SPAD-Na+	0.58 (0.07)	48.00 (0.80)	3234.41 (803.42)	5	31.99	0.99	***
Fv/Fm-Na+	4.69 (2.06)	7.86 (1.83)	864.25 (3743.28)	5	119.60	0.86	***
FreshWeight-Na+	0.47 (0.12)	44.70 (2.57)	163.10 (61.69)	5	293.08	0.96	***
TillerNumber-Na+	1.77 (0.70)	95.01 (7.90)	259.06 (69.77)	5	2518.50	0.95	***
ShootLength-HCO3−	2.56 (0.54)	84.90 (1.86)	176.94 (18.41)	5	258.44	0.95	***
SPAD-HCO3−	1.97 (0.50)	48.12 (0.76)	253.10 (49.36)	5	36.32	0.96	***
Fv/Fm-HCO3−	5.77 (2.12)	7.83 (1.50)	233.34 (57.25)	5	168.60	0.76	***
FreshWeight-HCO3−	1.52 (0.33)	44.96 (2.66)	63.78 (7.32)	5	442.40	0.99	***
TillerNumber-HCO3−	3.86 (1.00)	74.04 (3.42)	116.92 (6.72)	5	787.60	0.98	***

^a Parameters estimated using Equation (2); b, the slope of the dose–response curve through GR₅₀; d, the upper limit of the dose–response curve; GR₅₀, the saline and alkaline concentrations causing a 50% reduction in shoot length, SPAD, fresh weight, F_v/F_m, and tiller number of tiger nut; ^b, values in parentheses in each column are the standard errors of the parameters estimated; ** and *** significant at 0.01 and 0.001 probability levels, respectively.

). Additionally, a lack of fitting evidence in the fitting model was not detected (p < 0.05), which indicated that the model was applicable and could be used to estimate the reliable GR₅₀ value for tiger nut.

Among them, the estimated GR₅₀ values of plant height (1097 mmol L⁻¹), chlorophyll content (3234 mmol L⁻¹), and chlorophyll fluorescence (864 mmol L⁻¹) for the NaCl treatment were greater than the range of NaCl concentration (0–320 mmol L⁻¹). The GR₅₀ values estimated by plant height (177 mmol L⁻¹), chlorophyll content (253 mmol L⁻¹), and chlorophyll fluorescence (233 mmol L⁻¹) for the NaHCO₃ treatment also exceeded the concentration range of NaHCO₃ (0–120 mmol L⁻¹). Compared to tiller number, the model generated by fresh weight showed that this was the most suitable parameter, with the smallest mean square value (MS). These results showed that fresh weight can be used as a reliable parameter to describe the relationship between SA dose and plant growth response. The GR₅₀ values of NaCl and NaHCO₃ estimated using fresh weight were 163 mmol L⁻¹ and 63 mmol L⁻¹ for tiger nut, respectively (Table 2).

3.2. Alleviation of SA Stress in Tiger Nut Using MWCNTs, Nano-Se, or AMs

3.2.1. Pot-Planting Study

The alleviation effects of different concentrations of MWCNTs and Nano-Se on tiger nut under SA stress were tested at the whole plant level using NaCl and NaHCO₃ (GR₅₀ determined in Study I). As far as F_v/F_m is concerned, under NaCl stress (Figure 3A), F_v/F_m in the leaves above the ground increased significantly (p < 0.05) by nearly 4% with the exogenous application of 50 or 100 mg L⁻¹ of MWCNTs. The F_v/F_m in the leaves of tiger nut with 10 or 5 mg L⁻¹ of Nano-Se increased by 3.9% and 5.06%, respectively. Under NaHCO₃ stress (Figure 3B), the F_v/F_m ratio in the aboveground leaves of tiger nut treated with MWCNTs or Nano-Se was significantly increased (p < 0.05). After 50 or 100 mg L⁻¹ MWCNT treatments, the F_v/F_m increased by 4.74% and 5.56%, respectively. After Nano-Se treatment, the F_v/F_m ratio of tiger nut increased by 5.79% with the 10 mg L⁻¹ treatment and 4.87% with the 5 mg L⁻¹ treatment. These results demonstrate that the application of MWCNTs (100 mg L⁻¹) or Nano-Se (10 mg L⁻¹) could potentially alleviate SA effects on the F_v/F_m ratio of tiger nut.

In terms of chlorophyll content, under SA stress, the values decreased significantly (p < 0.05) to 20% and 23.84% lower than that of the untreated control (1.3 mg g⁻¹), respectively. Under NaCl stress (Figure 3C), the exogenous application of 50 or 100 mg L⁻¹ of MWCNTs resulted in a significant (p < 0.05) increase in chlorophyll content by 14.42% and 23.08%, respectively. The exogenous application of 10 or 5 mg L⁻¹ of Nano-Se increased values by 22.12% and 18.27%, respectively, in tiger nut compared to the NaCl-treated plants. Under NaHCO₃ (Figure 3D), the chlorophyll content increased by 18.18% and 25.5%, respectively, after treatment with 50 or 100 mg L⁻¹ of MWCNTs. Nano-Se treatment increased the chlorophyll content of tiger nut by 28.28% with the 10 mg L⁻¹ treatment and 23.23% with the 5 mg L⁻¹ treatment. Overall, the exogenous application of MWCNTs (100 mg L⁻¹) and Nano-Se (10 mg L⁻¹) had an effective impact on the chlorophyll content of tiger nuts.

The same trend was shown in the effects of different treatments on the plant height, fresh weight, and dry weight of tiger nut under SA stress. As for plant height (Figure 4A,B), there was no statistical difference in the treatment effects of different concentrations of MWCNTs or Nano-Se on tiger nut under SA stress. However, compared with the control group (Con. = 82.33 cm), the effects of the exogenous application of 100 mg L⁻¹ of MWCNTs and 10 mg L⁻¹ of Nano-Se on plant height were better than those of the applications of 50 mg L⁻¹ of MWCNTs and 5 mg L⁻¹ Nano-Se. For the dry weight (Figure 4E,F), MWCNTs (100 mg L⁻¹) and Nano-Se (10 mg L⁻¹) showed a better effect on tiger nut under SA stress, and there was no statistical difference between the treatment effect and the growth of tiger nuts. However, in terms of fresh weight (Figure 4C,D), there was no significant difference (p < 0.05) between the concentrations of MWCNTs and Nano-Se on alleviating the effect of SA stress on tiger nut. The results showed that both MWCNTs and Nano-Se could promote the growth of tiger nut, but 100 mg L⁻¹ of MWCNTs and 10 mg L⁻¹ of Nano-Se had better alleviation effects.

3.2.2. Field Study

In this study, effective alleviation concentrations of MWCNTs and Nano-Se were used to verify the alleviation effects of nanomaterials on tiger nut under SA stress in field tests at the whole plant level, and Arbuscular mycorrhizas (AMs) were added to explore their alleviation effects.

Effects of Using MWCNTs, Nano-Se, or AMs on Photosynthesis

The effects of different treatments on the F_v/F_m of tiger nut are shown in Figure 5A,B. Under SA stress, the F_v/F_m in the leaves of tiger nut decreased significantly (p < 0.05), with decreasing rates of 7.79% and 9.09%, respectively. Compared to the untreated control (0.77), the effects of the exogenous application of MWCNTs, Nano-Se, or AMs on F_v/F_m in the leaves of tiger nut had no significant difference, all being about 0.785. Under NaCl stress (Figure 5A), the F_v/F_m of tiger nut treated with MWCNTs, Nano-Se, or AMs increased by 3.23%, 1.83%, and 4.49%, respectively, compared with the NaCl group (0.712), among which the effect of AM treatment was the most significant (p < 0.05). Under the stress of NaHCO₃ (Figure 5B), the F_v/F_m of tiger nut sprayed with MWCNTs, Nano-Se, or AM was significantly increased (p < 0.05), reaching 1.04, 1.08, and 1.06 times higher than the values of the NaHCO₃ (0.703) treatment, respectively. Generally, the exogenous application of Nano-Se or AMs had relatively good effects on alleviating F_v/F_m in tiger nut under SA stress.

As seen in Figure 5C,D, the chlorophyll content of tiger nut under SA stress decreased significantly (p < 0.05) compared to the untreated group (1.25 mg g⁻¹), with values of 29.37% and 30.95%, respectively. In addition, under normal conditions, the chlorophyll content in the shoot of tiger nut with MWCNTs, Nano-Se, or AMs was relatively stable, and there was no significant difference. Under SA stress, the exogenous application of MWCNTs, Nano-Se, or AMs increased the chlorophyll content of tiger nut. Under NaCl stress (Figure 5C), when MWCNTs, Nano-Se, or AMs were applied, the chlorophyll content in the shoot increased significantly (p < 0.05), increasing by 41.57%, 20.22%, and 33.71%, respectively. Under NaHCO₃ (Figure 5D), the chlorophyll content in the tiger nut leaves was increased by spraying MWCNTs, Nano-Se, or AMs. Among them, the chlorophyll content of tiger nut treated with MWCNTs and AMs increased significantly (p < 0.05), reaching 1.24 and 1.38 times higher than the values of the NaHCO₃-treated plants (0.87 mg g⁻¹).

Effects of Using MWCNTs, Nano-Se, or AMs on Antioxidant Activity

As shown in Figure 6A,B, the SOD activity in tiger nut increased significantly (p < 0.05) under SA stress, showing values of 25.15% and 22.43%, respectively. Compared to NaCl (Figure 6A), the exogenous application of MWCNTs, Nano-Se, or AMs increased the SOD activity in tiger nut by 8.01%, 10.23%, and 7.73%, respectively. Under NaHCO₃ (Figure 6B), the spraying of MWCNTs, Nano-Se, or AMs increased SOD activity by 17.99%, 7.44%, and 8.71%, respectively. However, regardless of NaCl or NaHCO₃, the exogenous application of MWCNTs, Nano-Se, or AMs had no significant effect on the SOD activity of tiger nut (p < 0.05), and there was no significant difference in the SOD activity of tiger nut treated with exogenous mitigating substances under SA stress at about 135 U g⁻¹.

The CAT activity in the tiger nut leaves showed the same trend as SOD. As shown in Figure 6C,D, the CAT activity in the tiger nut leaves increased significantly under SA stress, reaching 31.74% and 38.45% higher than the values of the control group (101.29 U g⁻¹), respectively, particularly in the leaves of tiger nut treated with NaHCO₃ treatment (p < 0.05). Under NaCl stress (Figure 6C), the activity of CAT in tiger nut leaves treated with MWCNTs, Nano-Se, or AMs increased by 21.3%, 13.44%, and 3.67%, respectively. Compared to the NaHCO₃ group (Figure 6D), the CAT activity of tiger nut treated with MWCNTs, Nano-Se, or AMs increased by 16.7%, 11.1%, and 0.92%, respectively. Similarly, under NaCl or NaHCO₃ stress, the exogenous application of MWCNT, Nano-Se, or AMs had no significant effect on the CAT activity of tiger nut (p < 0.05).

Effects of Using MWCNTs, Nano-Se, or AMs on Membrane Permeability

The effects of different treatments on the relative conductivity of tiger nut are shown in Figure 7A,B. Under normal conditions, the relative conductivity of tiger nut is 22.32%. Under SA stress, the relative conductivities of tiger nut are 36.43% and 37.24%. Compared to the control group, SA stress significantly (p < 0.05) increased the relative conductivity of tiger nut by 63.22% with NaCl and 66.85% with NaHCO₃. However, the relative conductivity of tiger nut sprayed with MWCNTs, Nano-Se, or AMs was 24.66%, 25.65%, and 24.34%, respectively, and there was no significant difference compared with the untreated control group. Under SA stress, the relative conductivity of tiger nut was significantly decreased (p < 0.05) by applying MWCNTs, Nano-Se, or AMs. Under NaCl stress (Figure 7A), the relative conductivity of tiger nut was significantly decreased (p < 0.05) by applying MWCNTs, Nano-Se, or AMs, with values 28.22%, 30.66%, and 25.14% lower than that of the NaCl group, respectively. Under the stress of NaHCO₃ (Figure 7B), the relative conductivities in tiger nut leaves were significantly (p < 0.05) lower than in the NaHCO₃ group, with decreases of 35.2%, 22.42%, and 25.97%, respectively.

Under SA stress, the MDA content in the leaves of tiger nut increased to 16.34 μmol L⁻¹ and 17.13 μmol L⁻¹, respectively, as shown in Figure 7C,D. Compared to the control, SA stress significantly (p < 0.05) increased the MDA content in the leaves of tiger nut by 92.46% and 101.77%, respectively. However, the MDA contents of tiger nut sprayed with MWCNTs, Nano-Se, or AM, were 8.97 μmol L⁻¹, 7.16 μmol L⁻¹, and 10.01 μmol L⁻¹, respectively, and there was no significant difference compared to the control group. Under SA stress, the exogenous application of MWCNTs, Nano-Se, or AMs significantly reduced the MDA content of tiger nut (p < 0.05). Under NaCl stress (Figure 7C), the MDA content of tiger nut sprayed with MWCNTs, Nano-Se, or AMs decreased significantly (p < 0.05), with values 30.48%, 47.55%, and 42.22% lower than the NaCl stress group, respectively. Under NaHCO₃ (Figure 7D), the exogenous application of MWCNTs, Nano-Se, or AMs had the same trend, and significantly (p < 0.05) reduced the MDA content of tiger nut, with values of 36.14%, 48.57% and 23.12%, respectively.

Effects of Using MWCNTs, Nano-Se, or AMs on Osmoregulator

In terms of soluble protein, compared to the control group (25.16 mg g⁻¹), SA stress significantly increased the soluble protein content of tiger nut, increasing by 15.31% with NaCl and 9.78% with NaHCO₃ (Figure 8A,B). The soluble protein content of tiger nut can be improved by applying MWCNTs, Nano-Se, or AMs alone. Among them, the soluble protein content of tiger nut treated by AMs alone was 26.93 mg g⁻¹; the effect was the most significant (p < 0.05), increasing by 7.08%. The soluble protein contents in the leaves of tiger nut treated with MWCNTs and Nano-Se were 30.67 mg g⁻¹ and 29.27 mg g⁻¹, respectively; these values were significantly higher than the NaCl group (p < 0.05) (Figure 8A). Under NaHCO₃ (Figure 8B), the soluble protein contents after the exogenous application of MWCNTs, Nano-Se, or AMs all exceeded 30 mg g⁻¹.

Proline concentration in tiger nut increased by 60.1% (NaCl) and 68.12% (NaHCO₃), respectively, in response to SA stress (Figure 8C,D). Under the control condition, the proline contents of MWCNTs, Nano-Se, or AMs sprayed alone were 6.23 μg g⁻¹, 6.56 μg g⁻¹, and 5.88 μg g⁻¹, among which Nano-Se treatment had the most significant improvement effect (p < 0.05). Under NaCl (Figure 8C), the proline contents of plants sprayed with MWCNTs, Nano-Se, or AMs were all over 9 μg g⁻¹, with values 14.56%, 11.34%, and 11.83% higher than under NaCl stress, and the differences were significant (p < 0.05). Under NaHCO₃ stress (Figure 8D), the proline content significantly increased (p < 0.05) by 9.1% and 7.04% in plants with MWCNTs and Nano-Se exogenously applied. However, the AM treatment had no significant effect on increasing the proline content in tiger nut.

Effects of Using MWCNTs, Nano-Se, or AMs on Forage Value

At maturity, the forage value of the aboveground parts of the tiger nut under each treatment was measured (Figure 9). Generally, under SA stress, the content of neutral detergent fiber (NDF) and acidic detergent fiber (ADF) in tiger nut increased significantly (p < 0.05), while the content of crude protein decreased significantly. The application of MWCNTs, Nano-Se, or AMs alone has little effect on the fiber content of tiger nut, but MWCNTs or Nano-Se can significantly increase the crude protein content of tiger nut. Among them, the changing trends of NDF and ADF were similar. For example, the content of ADF in tiger nut was significantly (p < 0.05) reduced by applying MWCNTs, Nano-Se, or AMs under SA stress. Under NaCl (Figure 9C), the ADF content of tiger nut sprayed with MWCNTs, Nano-Se, or AMs decreased to 61.35%, 66.72%, and 62.4%, respectively. Similarly, under NaHCO₃ (Figure 9D), the exogenous application of MWCNTs, Nano-Se, or AMs significantly (p < 0.05) reduced the ADF content of tiger nut, with values 32%, 19.57%, and 17.4% lower than the NaHCO₃ group, respectively.

In terms of crude protein, the crude protein content of tiger nut is 5.61% under normal conditions. During SA stress, the crude protein content of tiger nut decreased to 4.53% and 4.31%, respectively. The crude protein content of tiger nut increased by 6.64%, 6.26%, and 5.61%, respectively, upon the application of MWCNTs, Nano-Se, or AMs alone. The addition of MWCNTs, Nano-Se, or AMs raised the crude protein content of tiger nut under SA stress (p < 0.05). Tiger nut sprayed with MWCNTs, Nano-Se, or AMs showed an increase in crude protein content under NaCl stress (Figure 9E) to 5.09%, 5.19%, and 5.39%, respectively, with all values significantly higher than the NaCl group (p < 0.05), which increased by 12.36%, 14.57%, and 18.98%, respectively. Under NaHCO₃ (Figure 9F), the exogenous application of MWCNTs, Nano-Se, or AMs can significantly (p < 0.05) increase the crude protein content of tiger nut, reaching values 20.65%, 21.81%, and 13.69% higher than that of NaHCO₃.

Effects of Using MWCNTs, Nano-Se, or AMs on Yield and Quality

In terms of plant height, compared to the control group (82.67 cm), the plant height of tiger nut under SA stress decreased by 17.34% (NaCl) and 19.56% (NaHCO₃), respectively. Tiger nut treated with MWCNTs, Nano-Se, or AMs alone showed improvements in plant height, whereas those treated with Nano-Se or AM showed significant (p < 0.05) increases in plant height of 9.68% and 10.8%, respectively. Under NaCl (Figure 10A), the exogenous application of MWCNTs, Nano-Se, or AMs significantly (p < 0.05) increased the plant height of tiger nut under stress by 25.37%, 25.86%, and 10.74%, respectively. Similarly, under NaHCO₃ (Figure 10B), the exogenous application of MWCNT, Nano-Se, or AMs significantly (p < 0.05) alleviated the stress-induced damage of tiger nut, increasing plant height by 22.3%, 23.3%, and 11.77%, respectively.

The total grain weight of tiger nut was calculated in terms of yield (Figure 10C,D). Under SA stress, the total grain weight of tiger nut decreased significantly (p < 0.05), especially under the treatment of NaHCO₃, which decreased the total grain weight by nearly half compared to the untreated control (41.32 g). However, the application of MWCNTs, Nano-Se, or AMs alone had little effect on the total grain weight of tiger nut, which was about 40 g. Under NaCl (Figure 10C), the total grain weight of tiger nut treated with exogenous MWCNTs increased significantly (p < 0.05) by 26.34%, but the effect of Nano-Se or AMs on the total grain weight was not significant. Under NaHCO₃ (Figure 10D), the exogenous application of MWCNTs, Nano-Se, or AMs increased (p < 0.05) the total grain weight of tiger nut by 42.17%, 37.46%, and 21.4%, respectively.

In addition, the oil content (Figure 10E,F) of tiger nut was also determined. It can be seen that the oil content of tiger nut decreased significantly (p < 0.05) to 10.04% and 10.27% when it was subjected to SA stress. Under NaCl stress (Figure 10E), the oil content of tiger nut significantly increased (p < 0.05) by 18.92%, 31.27%, and 20.21% more than the NaCl group after spraying with MWCNTs, Nano-Se, or AMs, respectively. Under NaHCO₃ (Figure 10F), the oil content of MWCNTs, Nano-Se, or AMs increased to different degrees, with values of 11.48%, 14.01%, and 13.28%, respectively. Among these treatments, the exogenous application of Nano-Se or AMs significantly (p < 0.05) increased the oil content of tiger nut under SA stress, reaching values 36.41% and 29.31% higher than under NaHCO₃.

4. Discussion

For most plants, SA stress has a certain inhibitory effect on plant growth [34]. SA stress makes it difficult for plants to absorb water through ion toxicity and osmotic stress, interferes with the dynamic balance of ions in plants, and then causes plant metabolism disorder, resulting in the slow growth and development of plant seedlings [35]. As a plant growth regulator, multi-walled carbon nanotubes (MWNTs) have been used in recent years to regulate plant growth and development [36], resist stress environments [18], and improve soil fertility [37]. Nano-selenium (Nano-Se) is often used to improve plant quality and stress resistance because of its environmental safety [38,39]. Arbuscular mycorrhizas (AMs) is a kind of symbiont that can establish symbiotic relationships with 80% of plants [22], promote the absorption of nutrients by plant roots [40], and enhance the stress resistance of plants. These results may provide new possibilities for improving the stress resistance of tiger nut in agricultural practice. The purpose of this study was to evaluate the salt and alkali tolerance of tiger nut and further test the application potential of MWCNTs, Nano-Se, and AMs in alleviating SA stress. The results showed that the growth of tiger nut was inhibited under SA stress, but that the application of MWCNTs, Nano-Se, or AMs could effectively alleviate the growth inhibition induced by SA stress and maintain the yield and oil quality of tiger nut.

The chlorophyll content and chlorophyll fluorescence coefficient of plants are important indicators reflecting the process of photosynthesis and the transformation and accumulation of organic matter [41]. When plants are subjected to SA stress, the structure and function of chloroplasts are damaged, the activities of enzymes are decreased, and the total amount of chlorophyll and carotenoids in leaves is altered [42,43]. This study showed that the chlorophyll content and F_v/F_m of tiger nut decreased significantly under SA stress (p < 0.05; Figure 3 and Figure 5), which is consistent with the research of Wu [44] and Liu [45]. However, the exogenous application of MWCNTs, Nano-Se, or AMs significantly increased the chlorophyll content and F_v/F_m of tiger nut under SA stress, which shows that the addition of exogenous substances can alleviate the degradation of chlorophyll, effectively alleviate declines in photosynthesis [46], and also maintain good PSII photochemical activity [47].

Under normal conditions, the cell membrane is the key barrier to maintaining the normal life activities of cells [48], and the cell membrane can be judged by measuring conductivity [49] and malondialdehyde (MDA) [50]. In this study, the relative conductivity in the leaves of tiger nut were significantly (p < 0.05; Figure 7) higher than those in the control group, which is consistent with the research results of Li et al. [51] and Zhang [52]. This shows that SA stress breaks the metabolic balance of reactive oxygen species in cells and the cell membrane is seriously damaged. The exogenous application of MWCNTs, Nano-Se, or AMs can significantly (p < 0.05) reduce the MDA and relative conductivity content of tiger nut under SA stress, which may be attributed to the fact that these exogenous substances could inhibit the oxidation of the cellular plasma membrane in the tiger nut under stress and attenuate the extravasation of the cellular electrolytes of the leaves [53]. Chu [54] found that selenium application can improve the antioxidant capacity of seedlings, and an appropriate concentration of selenium can reduce the production of free radicals and membrane lipid peroxidation, which is consistent with the results of this study.

Soluble protein and proline are key components of the osmoregulatory system and can reduce the osmotic potential of cells and maintain the normal function of cell membranes, thus improving the SA tolerance of plants [55]. It has been reported that the contents of soluble protein and proline in leaves of oats [56], mung bean [57], and other crops are significantly increased (p < 0.05) under SA stress. This study showed that SA stress significantly increased the soluble protein and proline content of tiger nut (Figure 8). The increase in both soluble protein and proline content could result from the responses of plants to stress, i.e., attempting to resist adverse stress [58]. It has also been pointed out that the content of free proline and soluble sugar in tomatoes increased with the increase in stress degree under SA stress [59]. Exogenous MWCNTs, Nano-Se, or AMs can increase the soluble protein and proline content of tiger nut to different degrees, which may further strengthen the self-protection mechanism of tiger nut with exogenous mitigating substances and reduce oxidative damage by increasing the soluble protein and proline content [60]. This is consistent with the conclusion that AMF can relieve cucumber and tomato from low-temperature and weak-light stress [24].

When plants are stressed, a large amount of reactive oxygen species (ROS) will be produced in the rapid senescence of plant cells [61]. To reduce the toxic effect caused by ROS accumulation, plants themselves will eliminate active oxygen by increasing the activities of SOD and CAT. In this study, through the study of SOD and CAT, it was found that the contents of SOD and CAT in the leaves of tiger nut increased significantly (p < 0.05; Figure 6) under SA stress, which is consistent with previous research results on oats [56]. It is possible that plants activate their own protection mechanism in response to stressful environments to reduce the damage of ROS to plants [62]. Under SA conditions, the exogenous application of MWCNTs, Nano-Se, or AMs also improves SOD and CAT activities to some extent, but no significant difference was found compared with the stress group. This may be due to the slow response of tiger nut to foreign substances, or it may be related to the fact that tiger nut has a certain degree of SA tolerance. Therefore, it is necessary to conduct a more comprehensive study on the alleviation effect of the exogenous application of MWCNTs, Nano-Se, or AMs on tiger nut under SA conditions.

Tiger nut is an oil-bearing herb energy plant with tuber, and its oil yield exceeds 170 kg, which is about 4 times that of soybean, 2 times that of rape, and 1.5 times that of peanut [63]. In addition, tiger nut has a high yield, and its stems and leaves are rich in crude fat and crude protein, so it is an excellent animal feed. The quality of forage depends on the nutrient content of the forage, and crude protein, ADF, and NDF are important indexes for evaluating forage quality [64]. This study showed that the oil content of tiger nut decreased significantly (p < 0.05; Figure 10E,F) and the contents of ADF and NDF increased significantly (p < 0.05; Figure 9A–D) under SA stress, which suggests that SA stress will lead to slow plant development, inhibit the growth and differentiation of tissues and organs, shorten the vegetative period and flowering period, advance the development process [65], and result in a lack of sufficient nutrition to maintain growth. The exogenous application of MWCNTs, Nano-Se, or AMs can significantly reduce the fiber content and increase the crude protein and oil content of tiger nut. This may be related to the fact that these substances can improve the absorption of nutrients by plants and promote the accumulation of dry matter [16,40].

In addition, the effects of MWCNTs or Nano-Se on plants are obviously concentration-dependent. Lower concentrations promoted the growth of the plant, while high concentrations inhibited the growth and development of plants. For example, the germination rate of mustard seeds treated with MWCNTs in the range of 2.3–23 mg L⁻¹ is higher than those treated with distilled water; however, the higher concentration of 46 mg L⁻¹ obviously inhibited the seed germination [66]. Additionally, MWCNTs at 10–40 mg L⁻¹ can increase and prolong the germination time of tomatoes and onions, but MWCNTs at a higher concentration (>40 mg L⁻¹) show toxicity to the two plant species [67]. For Nano-Se, Qin [68] reported that when the concentration of selenium is 0.05–0.5 mg L⁻¹, it can promote the growth of rice; however, when a high concentration of Nano-Se is involved, the growth of rice is inhibited. Chen [69] also reached the same conclusion regarding the treatment of oats.

5. Conclusions

The exogenous application of MWCNTs, Nano-Se, or AMs can alleviate the adverse effect of SA-induced stress in tiger nut while maintaining tuber yield and oil quality. A higher concentration of the two nanomaterials should be avoided, as a higher concentration could adversely affect plant metabolism and reduce plant growth. Further study should be conducted on the mechanisms involved and the beneficial effect of each of them on tiger nut under SA stress. Furthermore, the residue of nanoparticles in soil may pose potential ecological harm; thus, the evaluation of the ecological risks of nanomaterials and their impact on agricultural ecosystems is required.