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Article

Green Synthesis of Zinc and Iron Nanoparticles Using Psidium guajava Leaf Extract Stimulates Cowpea Growth, Yield, and Tolerance to Saline Water Irrigation

by
Mohamed H. Sheta
1,
Ahmed H. M. Abd El-Wahed
2,
Mohammed A. Elshaer
3,
Hala M. Bayomy
4,*,
Nawal A. Ozaybi
4,
Mohamed A. M. Abd-Elraheem
3,
Abdel-Nasser A. El-Sheshtawy
5,*,
Rasha S. El-Serafy
6 and
Mahmoud M. I. Moustafa
2
1
Soils and Water Department, Faculty of Agriculture, Al-Azhar University, Cairo 11884, Egypt
2
Horticulture Department, Faculty of Agriculture, Al-Azhar University, Cairo 11884, Egypt
3
Agricultural Biochemistry Department, Faculty of Agriculture, Al-Azhar University, Cairo 11823, Egypt
4
Food Science and Nutrition Department, Faculty of Science, University of Tabuk, Tabuk 71491, Saudi Arabia
5
Environment and Bio-Agriculture Department, Faculty of Agriculture, Al-Azhar University, Cairo 11884, Egypt
6
Horticulture Department, Faculty of Agriculture, Tanta University, Tanta 31527, Egypt
*
Authors to whom correspondence should be addressed.
Horticulturae 2024, 10(9), 915; https://doi.org/10.3390/horticulturae10090915
Submission received: 3 July 2024 / Revised: 21 August 2024 / Accepted: 23 August 2024 / Published: 28 August 2024
(This article belongs to the Special Issue New Insights into Stress Physiology and Resistance Regulation in Horticultural Plants)
Browse Figures
Versions Notes

Abstract

:
Plants use a variety of physiological, biochemical, and molecular mechanisms to mitigate salt stress impacts. Many techniques, including the application of nanoparticles (NPs), are being used to increase plant stress tolerance. To assess the growth and productivity of Vigna unguiculata L. (cowpea) plants exposed to salt stress, cowpea has been cultivated using different saline water levels and subjected to green synthesized zinc NPs (ZnNPs) and iron NPs (FeNPs) applied via foliar spraying. The cowpea plants that grew under the lowest saline water level showed the best leaf traits, leaf water content per area (LWCA), pods, and seed yields, but when salinity levels increased, the plants’ growth and productivity slightly declined. ZnNP and FeNP treatments slow down the degradation of photosynthetic pigments and greatly mitigate the negative effects of salt stress. In both stressed and unstressed plants, ZnNP treatments produced the highest osmoprotectant concentrations (proline, protein, and total carbohydrates). As a result of salt stress, cowpea seeds showed a marked decrease in dry matter and protein content, but ZnNP and FeNP treatments increased it. Conclusively, the results obtained indicated that ZnNPs and FeNPs foliar application to cowpea plants stimulated leaf pigment and polyphenol production, which in turn increased seed dry matter, seed yield, protein content, and the plants’ ability to withstand saline stress.

1. Introduction

Cowpea (Vigna unguiculata L.) is one of the Leguminosae family and is a main legume plant in sub-Saharan Africa, the Americas, and many regions of Asia [1]. The tropical lowlands and warm temperate regions are favorable regions for cowpea cultivation. Cowpea is a nutritious food containing 24% protein, 1112 mg/100 g potassium, and 11% dietary fiber, in addition to a variety of essential amino acids [2]. It is used for human food, soil coverings, and green manure. Cowpea is classified as having a moderate sensitivity to salinity [3]. It has not exhibited any yield reduction despite salt in the root zone reaching an electrical conductivity of 4.9 dS m−1, but every additional dS m−1 caused a reduction in the seed yield by 21% [4]. On the other hand, Al-Hayany [5] stated that cowpea plants subjected to saline water treatments experienced a reduction in pod weight, the number of seeds per pod, and dry matter accumulation.
Water shortage is a worldwide crisis that continues to grow increasingly [6,7,8,9]. To compensate for the lack of water, it is necessary to use lower-quality water, such as seawater. After the Second World War, many efforts were made to replace potable water in agriculture with seawater. The high salinity is the main obstacle to using seawater for agricultural crop irrigation. The most common signs of foliage damage in plants irrigated with saline water are leaf necrosis and burns [10]. Salinity disrupts a number of physiological, biochemical, and molecular processes, inhibiting and impeding crop growth and development; it also reduces economic output and degrades product quality [11]. Salinity hinders plant growth through water stress, cytotoxicity, and nutritional imbalance, affecting photosynthesis, antioxidant capacity, and ion homeostasis [12]. A lot of experiments were performed for salt tolerance inducement, and nanotechnology is among the bright methods for this achievement.
Nanotechnology is a promising and effective technology for reducing salt stress in plants, with excellent results [13]. Nanoparticles (NPs) are compounds with at least one dimension and a size less than 100 nm [14]. Despite the harmful impacts of nanomaterials on the soil and groundwater, nanoscale fertilizer has been greatly employed in the creation of fertilizers due to the effective delivery of nutrients and the possibility that their small size may allow them access to a range of plant surfaces and transport channels [15]. Zinc (Zn) is an essential micronutrient that is required for the optimal growth of cowpeas, including seed yield and biomass production. It activates over 300 enzymes and improves photosynthesis by participating in processes related to glucose metabolism [16]. Zinc deficiency causes physiological stress, decreased growth and yield, photosynthetic activity, and elevated ROS levels in plant cells [17]. Zinc NPs (ZnNPs) are ideally soluble, accessible, and susceptible relative to ZnNP bulk-size fertilizer due to their high specific surface area and nanoscale size [18]. ZnNPs significantly increased the growth, leaf photosynthetic pigment content, and grain yield of wheat under salt stress [19]. Iron (Fe) is the third limiting element for plant growth and plays crucial roles in the electron-transport pathways of photosynthesis and respiration [20]. So, it plays a vital role in chlorophyll production, photosynthesis, antioxidant enzymes, and the respiration process in plants [21]. Iron oxide nanoparticles (FeNPs) can enhance the salt tolerance of trees by increasing the activity of antioxidant enzymes [22]. Iron application boosted the salt tolerance of pea plants through sustaining antioxidant activities, reducing Na toxicity, and conserving nutrient homeostasis in soil solutions [23].
Plant extracts have been employed to generate Zn and Fe oxide nanoparticles as a part of the nanocomposite’s synthesis. These extracts have active compounds that serve as stabilizers and reducing agents, enabling the synthesis of eco-friendly and long-lasting nanoparticles [24]. Numerous studies have been conducted on the synthesis of zinc and iron oxide nanoparticles using plant extracts from plants like Psidium guajava [25], Artemisia absinthium [26], and Salvia officinalis [27]. Green zinc oxide and iron oxide nanoparticles can enhance plant tolerance to salinity, promote plant development [20], and improve resilience against environmental stresses like heat, salt, drought, and pollution [28]. ZnNP treatments at lower concentrations boosted the growth and development of faba beans under saline conditions [29]. Fe treatments improved wheat growth grown under salt-affected soils, and the highest yield was obtained by FeNPs as compared with the other sources of Fe [30].
The objective of the current study was to evaluate the influence of foliar application with green synthesized ZnNPs and FeNPs on the growth, yield, photosynthesis, osmolytes, and antioxidant enzymes in cowpea grown under saline water levels. Therefore, the hypotheses of this study are as follows:
  • Psidium guajava leaf extract (GLE) can be used as a stabilizer and reducing agent for zinc and iron oxide nanoparticle green synthesis.
  • Foliar application of ZnNPs and FeNPs may enhance cowpea growth, yield, and yield parameters under saline conditions.
  • Promotion of polyphenol and osmoprotectant levels in stressed plants as affected by ZnNP and FeNP foliar application.

2. Materials and Methods

2.1. Experimental Site, Plant Material, and Experimental Design

This pot investigation was performed at Al-Sadat Experimental Farm, Agriculture Faculty, Al-Azhar University, Menoufia Governorate, Egypt, in the two seasons of 2022 and 2023. The climate data for the experimental site are exhibited in Table S1. The seeds of cowpea (Vigna unguiculata L.) variety Kafr El Sheikh-1 were provided from the Agricultural Research Center, Giza, Egypt. On February 25th, the seeds were sown in plastic pots (35 cm in depth and 28 cm in diameter), which were filled with 10 kg of dried loamy sand collected from the surface layer (0.0–0.3 m) of the experimental farm. Before sowing, soil samples were collected, air-dried, crushed, and sieved through a 2.0 mm sieve for physical and chemical analysis, according to Page et al. [31] and Klute [32], and their results are presented in Table 1.
This investigation was arranged in a factorial, randomized complete block design. Saline water was the first factor, while green synthesis ZnNP and FeNP foliar application was the second. The experiment consisted of 12 treatments, which were replicated three times; each replicate consisted of twelve pots.

2.2. Saline Water Treatments

Tap water was used for pot irrigation until the appearance of the two true leaves. After that, seedlings were thinned to one plant per pot, and saline water treatments were applied. The pots were divided into four groups: the first group was irrigated with (1) tap water with an EC of 0.75 dS m−1 as a control (S1); (2) the second group was subjected to saline water with an EC of 2.00 dS m−1 (S2); (3) the third group received 4.00 dS m−1 saline water (S3); and (4) the fourth group received 6.00 dS m−1 saline water (S4). The saline water used in this experiment was prepared for each treatment by mixing seawater with tap water at different ratios (v/v) to obtain the required salinity levels using a digital conductivity meter (AD3000-EC/TDS; Adwa). The soil moisture was maintained at around 100% field capacity for all treatments by weighing the pots. The chemical characteristics of the used irrigation water are shown in Table 2.

2.3. Collection and Preparation of Plant Extract

Guava (Psidium guajava) leaves were collected from trees cultivated in a private nursery at El-Mamoura, Alexandria, Egypt. The leaves were entirely washed using tap water and then with distilled water to remove any dust. After that, the leaves were oven-dried at 60 °C until a constant weight was obtained, and then a stainless-steel grinder (Moulinex 700 W, France) was used to grind the dried leaves to a fine powder. The method of Ramya et al. [33] was used to prepare a guava leaf extract (GLE) with some modifications as follows: 150 g of leaf powder were stirred on a magnate stirrer and heated in 1500 mL of distilled water for 1 h at 70 °C. Then the mixture was allowed to cool to room temperature. Using Whatman filter paper No.102, the obtained extract was filtered and stored at 4 °C.

2.4. Phytochemical Screening and Estimation

Active ingredients of aqueous GLE were identified according to Harborne [34] and Pasdaran and Hamedi [35] descriptions. Total phenol and flavonoid levels in GLE were estimated spectrophotometrically using the Folin–Ciocalteu method according to Kupina et al. [36] for total phenols and the methods described by Pękal and Pyrzynska [37] for flavonoids determination.

2.5. Green Synthesis of Nanoparticles

Zinc acetate (Zn (CH3COO)2.2H2O), iron chloride hexahydrate (FeCl3.6H2O), and sodium hydroxide (NaOH) were brought from El-Gamhouria Trading Chemicals and Drugs Company, Egypt.
Zinc oxide nanoparticles (ZnNPs) were synthesized according to the Faisal et al. [38] protocol, with some modifications. A total of 6.0 g of zinc acetate (ZnC4H6O4) was added to 100 mL of aqueous GLE and put on a magnetic stirrer at 60–70 °C for 2 h. Then 0.2 M NaOH was added dropwise to reach pH 12. The mixture was stirred for an additional hour until a light-yellow solid product was obtained, let cool down to 20 °C, and centrifuged at 4000 rpm for 10 min. The float was neglected, and the residual bead was washed with distilled water three times, teeming into a spotless Petri plate, and oven-dried at 90 °C. The dried matter was then melded into a fine powder with a hammer and mortar. The solid powder was stored in an airtight glass vial and was additionally used for physical characterization and applications.
Iron oxide nanoparticles (FeNPs) were synthesized by a modified protocol of Syarifah et al. [39]. Briefly, 2.704 g of FeCl3.6H2O (0.01 M) was mixed with 1000 mL of distilled water. The solution is then stirred by a magnetic stirrer at 35 °C for 15 min. Slowly, 1000 mL of ferric chloride solution was mixed with 1000 mL of GLE. FeNPs were instantly acquired with the reduction operation. The mixture was continued to be stirred at 70–80 °C for 2 h. The formation of FeNPs is shown by a modification in the color of the mix solution from light brown to dark brown and finally dark black. The feasible technique for the formation of FeNPs may be due to the iron ion reduction that occurred with phenolic compounds and flavonoids in the guava leaf extract.

2.6. Characterization of ZnO and Fe Nanoparticles

The green synthesized ZnNPs and FeNPs from the aqueous extract of guava leaves were confirmed and characterized as follows:

2.6.1. Transmission Electron Microscope Analysis (TEM)

Samples were prepared by placing a drop of the green ZnNP and FeNP solution on basic carbon-coated copper grids [40]. Images were analyzed by the ImageJ software (GEOL, GEM-1010 Transmission Electron Microscope at 70 kV).

2.6.2. Zeta Potential

A zeta voltage test was carried out on a Zetasizer (Malvern Instruments, Malvern, UK) to estimate the electrical charge and verify the stability of the green ZnNP and FeNP solution [41].

2.6.3. Energy Dispersive Analysis of X-rays (EDX)

Confirmed the presence of the elements through EDX. EDX microscopic analysis was performed by an X-ray precision analyzer (Oxford 6587 INCA) connected to a JEOL JSM-5500 LV electron microscope at 20 kV [42].

2.7. ZnNP and FeNP Treatments

Each group of S1, S2, S3, and S4 was divided into three groups for foliar spray application. The first group was subjected to foliar spray with distilled water as a control (without), the second group was foliar sprayed with 3 mM green ZnNPs, and the third group was foliar sprayed with 3 mM FeNPs. Foliar spray treatments were applied three times; the first application was performed after seedling thinning, while the second and third times were performed 15 days later, and the total volume of the solution used was 48 mL per pot.

2.8. Leaf Traits and Yield Performance

After 70 days from sowing, six pot samples were randomly chosen for leaf fresh weight (g), leaf dry matter (g), specific leaf area (SLA), and leaf water content per leaf area unit (LWCA) and dry matter (%) measurements.
The SLA was determined using the following formula:
SLA (cm2 g−1) = leaf area ÷ leaf dry weight
The LWCA was determined and calculated using the formula given by Sepehri and Golparvar [43].
LWCA (g cm−2) = leaf fresh weight − leaf dry weight/leaf area
At the harvesting stage, the pods were harvested, and pod number plant−1, pod length (cm), 100 seed weight (g), and seed yield pod−1 (g) were determined.

2.9. Physiological and Biochemical Analysis

2.9.1. Photosynthetic Pigments

Leaf pigment content was estimated spectrophotometrically (6800 UV/Vis spectrophotometer, Jenway, Bibby Scientific Ltd., Staffordshire, UK) after 70 days from sowing using the methods of Lichtenther [44]. Briefly, 0.2 g of fresh leaf samples were blended with 15 mL of acetone (80%) and filtered. Chlorophyll a, b, and carotenoids contents were estimated at wavelengths of 663.2, 646.8, and 470 nm and calculated in mg g−1 FW using the following formula:
Chl. a = 12.25A663.2 − 2.79A646.8
Chl. b = 21.50A646.8 − 5.1A663.2
Total Chl = 7.15A663.2 + 18.71A646.8
Cart = ( 1000 A 470   −   1.8 Chl . a   −   85.02 Chl . b ) 198

2.9.2. Proline Content

The proline content (µg g−1 FW) in cowpea leaves was estimated as Bates et al. [45] described. In brief, a dried leaf sample of 1 g was homogenized in 5 mL with aqueous sulfosalicylic acid (3%), then centrifuged at 14,000× g for 150 s using a Neya 10R, REMI, Mumbai, India. A mixture of 2 mL of acetic acid and 2 mL of ninhydrin was heated for 1 h at 100 °C with 2 mL of supernatant added. Using an ice bath, the mixture was quickly cooled, and 4 mL of toluene was then added. A spectrophotometer was used to measure the absorbance at the 525 nm wavelength.

2.9.3. Peroxidase Activity Assay (POD)

The protocol of El-Argawy and Adss [46] was used to estimate the peroxidase activity (POD) as follows: 1 g of leaf sample and 1 mL of 0.01 M sodium phosphate solution (pH 6.5) were homogenized in a pre-cooled pestle and mortar. An enzyme source was the floating material from the centrifuge at 10,000 rpm after a 15 min homogenization at 4 °C. A total of 1.5 mL of pyrogallol (0.05 M), 0.5 mL of H2O2 (1%), and 0.5 mL of extract were used to grate the reaction mixture. The reaction mixture was incubated at 28 °C. The mixture’s absorption was estimated at 420 nm, and the enzyme activity level was calculated as U min−1 g−1 FW.

2.9.4. Total Phenolic Content

The Folin−Ciocalteu technique was utilized to determine the total phenolic content in the cowpea leaves according to the protocols of Singleton and Rossi [47]. The absorbance was measured in a spectrophotometer at a wavelength of 725 nm in comparison to a blank. Total phenol content was expressed as mg gallic acid equivalents (GAE) g−1 dry weight.

2.9.5. Seed Analysis

Total carbohydrate (%) and protein (%) content in cowpea seeds were determined using A.O.A.C. [48]. Also, seed dry matter (%) was calculated.

2.10. Statistical Analysis

COSTAT version 6.4 software (CoHort Software, Monterey, CA, USA) was used to perform the statistical analysis of the data. The Tukey’s test was used to calculate the significant difference between mean values at 5% probability. Results are presented as the average means ± standard error (SE). n = 6.

3. Results

3.1. Qualitative Detection of GLE

The qualitative detection of GLE was performed, and the results presented in Table 3 reveal the presence of phenols, saponins, flavonoids, coumarins, carbohydrates, alkaloids, terpenes, and tannin active compounds in crude GLE, and the results are all positive. Total phenols and flavonoids estimation indicates that the GLE is abundant in phenolic and flavonoid compounds (15.80 mg mL−1 and 10.30 mg mL−1 for total phenols and flavonoids, respectively), which are distinguished for their capacity to reduce Zn and Fe particles to a nanoscale dimension (Table 4).

3.2. Characterization of ZnNPs and FeNPs

TEM image analysis showed the different shapes and confirmed that an organic layer derived from the guava extract covers the nanoparticles. In addition, TEM analysis also revealed the different shapes for ZnNPs with a size from 25.42 to 61.37 nm (Figure 1) and FeNPs with a size from 9.03 to 14.71 nm (Figure 2). EDX analysis was performed, and its results are presented in Figure 3 and Figure 4. The chemical composition of samples indicates the presence of Zn, Fe, O, and others. The spectra emphasize the impurities in the samples, but the C peak coming from the tape was used to fix the sample. Also, the elements other than Zn and Fe are due to the extracts that were used in the synthesis of ZnNPs and FeNPs. The zeta potential of green ZnNPs and FeNPs exhibited negative values of −7.67 and −8.71, respectively, as indicated in Figure 5 and Figure 6.

3.3. Leaf Traits

Salinity treatments negatively affected cowpea leaf traits, as the leaf fresh weight, SLA, LWCA, and leaf dry matter were significantly decreased by saline water treatments, and their values gradually decreased with increasing saline water concentration (S4), while S1 plants significantly exhibited the highest values in this respect for both seasons (Table S3). The leaf traits of cowpea plants were significantly enhanced following foliar application with ZnNPs or FeNPs relative to untreated plants. Regarding the interaction, for both seasons, the best leaf traits of cowpea plants were treated by ZnNP plants under S1 salinity. However, the lowest values were given by untreated plants exposed to S4 salinity (Figure 7).

3.4. Yield Traits

The pod traits (pod number plant−1 and pod length) of cowpea plants were significantly lowered by salinity treatments, and increasing salinity levels from S1 to S4 significantly showed a gradual decrease in the pod values (Table S4). Concerning NP treatments, ZnNP-treated plants exhibited superior pod traits to FeNP-treated plants without foliar application. The mean values of pod number and length were increased following ZnNPs × S1 treatment by 100 and 175% for pod number and 35.6 and 30.3% for bod length for the first and second seasons, respectively, as compared with without × S4 treatment (Figure 8).
Increasing the salinity level from S1 to S4 led to a significant reduction in the 100 seed weight and seed yield plant−1 values in both seasons. Foliar application with ZnNPs showed the best seed traits relative to FeNPs and untreated plants. When it comes to the interaction, the ZnNPs × S1 treated plants significantly exhibited the heaviest seed weight (17.2 and 15.8 g for the first and second seasons, respectively), with an increase in the seed yield plant−1 by about 31.4 and 25.9% as compared with without × S1 plants (Figure 8).

3.5. Physiological and Biochemical Analysis

3.5.1. Photosynthetic Pigments

Photosynthetic pigments (chlorophyll a, b, total chlorophyll, and carotenoids) presented a negative correlation with saline water concentration, as their mean values significantly depressed at the higher salinity level (S4) to reach the least mean values (0.93, 0.36, 1.29, and 0.18 mg g−1 FW and 0.88, 0.34, 1.22, and 0.14 mg g−1 FW for chlorophyll a, b, total chlorophyll, and carotenoids in the first and second seasons, respectively), as indicted in Table S5. The subjected plants to ZnNPs exhibited the highest mean values of total chlorophyll by about 9.8 and 4.9% higher than FeNP-treated plants, while the untreated plants showed the least mean values in this respect. The subjected plants to ZnNPs × S1 significantly exhibited the highest photosynthetic pigment level in both seasons (Figure 9).

3.5.2. Proline Content

The data presented in Table S6 indicate a significant and positive correlation between salinity level and proline content in the cowpea leaves, as proline content significantly increased with increasing salinity level. Foliar spraying with green NPs significantly enhanced proline content, and ZnNPs showed the highest proline content, followed by FeNP application. When it comes to the interaction influence, the greatest proline values (16.29 and 17.74 µg g−1 FW for the first and second seasons, respectively) were exhibited by ZnNPs × S4 plants, while the lowest proline mean values (11.56 and 11.76 µg g−1 FW for the first and second seasons, respectively) were produced by the un-foliar sprayed plants cultivated under the S1 conditions (Figure 10).

3.5.3. Peroxidase Activity

The activity of the peroxidase enzyme was significantly influenced by salinity and NP applications (Table S6). In this regard, peroxidase activity significantly recorded the lowest values under the S4 treatment (4.48 and 4.72 U min−1 g−1 FW for the first and second seasons, respectively). Untreated plants with NPs produced the highest mean values of POD activity by about 36 and 14.8% higher for ZnNPs and 41 and 42.9% higher for FeNP plants for the first and second seasons, respectively. The cowpea plants exposed to ZnNPs × S1 treatment revealed the highest mean values in this respect (5.48 and 5.57 U min−1 g−1 FW for the first and second seasons, respectively), as given in Figure 10.

3.5.4. Total Phenols

Growing salinity levels caused a growth in total phenol levels in cowpea leaves to reach the highest values when the plants were exposed to the S4 level in both seasons (Table S6). Total phenols were significantly induced, as influenced by NP applications, and ZnNP treatment exhibited the highest content of total phenols, giving 15.4 and 13.5% increases for the first and second seasons, respectively, as compared with untreated plants. The maximum phenol concentration (288.14 and 277 mg GAE g−1 DW for the first and second seasons, respectively) was obtained in cowpea plants that were subjected to S4 × ZnNP treatment (Figure 10).

3.5.5. Seed Analysis

Results in Table S7 show the seed dry matter, total carbohydrate, and total protein content in cowpea seed as influenced by salinity and NP applications. There was a significant and negative relation between seed dry matter and total protein with saline water concentrations, as seed dry matter and total protein content significantly decreased with salinity level increase, while the correlation between total carbohydrates and salinity level was positive. ZnNP foliar application revealed significant elevations in seed dry matter, total carbohydrate, and total protein content in cowpea seeds, and FeNPs ranked second in this respect. Concerning the interaction effect, the maximum increase in seed dry matter and protein content was observed by ZnNPs × S1 treatment (3.8 and 4.3% for seed dry matter and 39.9 and 35.6% for protein content in the first and second seasons, respectively) relative to without × S4 treatment, while the highest carbohydrates values were obtained by ZnNPs × S4 treatment (54.64 and 54.53% for the first and second seasons, respectively) (Figure 11).

4. Discussion

Green synthesis is currently attracting a lot of interest in the creation of metal nanoparticles (NPs) due to its simplicity of synthesis, environmental friendliness, and ability to use plant waste products [49]. Several studies have reported that guava leaf extract can be employed in several NP green syntheses, including Zn, Cu, Fe, and Ni [25,50,51]. Phytochemical screening of GLE referred to the presence of several phytochemicals, including phenols, saponins, flavonoids, coumarins, carbohydrates, alkaloids, terpenes, and tannins, which support the effective photosynthesis of NPs by serving as a reducing and stabilizing agent [52]. Also, the data reported in Table 3 show that GLE has significant quantities of phenolic and flavonoid compounds, which are known for their capacity to reduce Zn and Fe particles to nanoscale dimensions.
Nanotechnology is emerging to elevate nutrient efficiency in stressed environments [53]. Nanoparticles enable nutrients to penetrate the cell wall pores and stomata, can easily cross plant tissues when foliar sprayed, and arrive at all plant organs faster as compared with bulk materials due to their smaller diameter [54].
Additionally, nanoparticles have a large surface area and surface area-to-weight ratio, which enables them to be a sustainable option for salt stress mitigation [6]. The benefits of ZnNP and FeNP supplementation on strawberries and wheat under stressful conditions were previously reported by Mozafari et al. [55] and Merinero et al. [56].
Salinity stress influence is a complicated phenomenon that impacts plant growth via its impacts on physiological and biochemical processes, including osmotic stress, which causes ion toxicity and nutritional imbalance [57]. Under the current study, cowpea plants subjected to the lowest salinity level (2 dS m−1) exhibited better growth with more leaf and pod yield, while increasing salinity levels decreased the vegetative and reproductive growth of cowpea plants, exhibiting a depression in the leaf’s fresh and dry weights, SLA, and LWCA traits. Salinity impacts plant growth and productivity negatively via its impacts on osmotic stress, as the increase in the salinity of the soil solution is followed by a depression in the soil water potential, restricting water absorption by the roots and causing a water deficit in plant cells [58]. Salinity decreases physiological and metabolic activity speed and reduces cell growth, expansion, and division, as well as cell turgor [59]. Moreover, it negatively impacts stomatal conductance, causing a depression in carbon fixation and assimilation and, ultimately, lowering the biomass yield [60].
In the current study, higher saline treatments (S3 and S4) exhibited a significant reduction in the photosynthetic pigment level in the cowpea leaves (Figure 3). High salt concentrations limit the growth and development of plants [61] and exacerbate the harmful cytoplasmic sodium and ion imbalances caused by salt stress. Under saline conditions, absorbed nitrogen is lowered, and enzymes of chlorophyllase degradation are stimulated, causing a reduction in chlorophyll content, photosynthetic rate, and plant health, growth, and productivity [62]. A significant reduction in the growth and productivity of tomato [63] and bean [64] plants affected by salt stress was reported. It is worth noting that increasing the saline water level in this experiment led to an increase in the carbohydrate content in cowpea seeds, and this may be explained by the nutrients Mg, K, and Ca found in sea water, which are important for different physiological activities, including the production of carbohydrates [6].
On the contrary, ZnNP and FeNP foliar sprays maintained the growth and yield of stressed cowpea plants relative to untreated plants. Zinc (Zn) is a vital item for the synthesis of thousands of proteins; it also stimulates the synthetase of tryptophan and its enzymes, which are responsible for indoleacetic acid (IAA) biosynthesis. Plants treated with ZnNPs exhibited more leaf growth with the heaviest leaf fresh weight and higher SLA under saline and non-saline treatments. Zn deficiency resulted in small leaves with very slow growth, which is strongly linked to troubles in auxin metabolism [65]. Zinc promotes the elongation and enlargement of plant cells, photosynthetic pigments, nitrogen metabolism, the accumulation of phospholipids, and the maintenance of the structural stability of plant cell membranes [66]. In the current study, treated plants with ZnNP foliar application significantly showed higher LWCA values. Increasing LWCA values are an indicator of good leaf water status and membrane integrity. Maintaining good water status in plant cells promotes resistance to salinity stress, osmotic adjustments, and metabolic process activity [67]. Zinc contributes to the maintenance of membrane integrity where it links with the sulfhydryl groups and membrane phospholipid-forming complex groups of tetrahedral with cysteine residues of polypeptide chains, which finally contributes to enhancing the water status of plant cells under saline conditions. The LWCA of cowpea leaves under saline water treatments steadily decreased as salinity levels rose. Salts have an adverse influence on plant roots’ ability to absorb water because they lower the osmotic pressure within plant cells [68]. Our findings are supported by the results obtained by Alabdallah and Alzahrani [69] on cowpea.
Iron (Fe) is a vital microelement, serving as the structural component of chlorophyll and numerous antioxidant enzymes [70]. Under stressful conditions, Fe application promoted the activity of the photosynthetic process, leading to an increase in chlorophyll content [70]. Cowpea plants subjected to foliar sprays with FeNPs exhibited higher leaf fresh and dry weights and higher SLA under saline treatments. The increment in the fresh and dry weights of treated cowpea plants was likely linked to the improvements in photosynthetic pigments, proteins, and total carbohydrates in the leaves. In response to salt stress, Fe stimulates the synthesis of several proteins, enzymes, and antioxidants that counteract the damaging effects of ROS. This improves plant health and facilitates root absorption, enabling plants to take up essential nutrients [30]. Fe application improves the stability of the cell membrane of stressed pea plants, which controls water transport in the plant cells [23].
These findings supported our results, which revealed that under saline water treatments, LWCA values were enhanced following FeNP treatment. Iron has an irreplaceable role in producing antioxidant enzymes that alleviate the harmful effects of environmental stress. Increasing osmoprotectant compounds and antioxidant activity in plant cells are evidence of salt stress tolerance [71]. The main roles of osmoprotectants and antioxidants are to combat free radicals and protect cell membrane stability and turgor, which improve water flux into plant cells and organs [72]. Proline, total phenols, total protein, and total carbohydrates are osmolytes that accumulate in plants when exposed to stress conditions. In this regard, cowpea plants foliarly sprayed with ZnNPs or FeNPs presented an increase in proline, total carbohydrate, total protein, and total phenol levels as compared with S1 plants. Under stress conditions, accumulated osmoprotectant compounds in plant cells are vital mechanisms, as they have osmoprotective roles and increase the amount of water and nutrients absorbed, which are needed for stimulating photosynthesis, metabolic activities, and their metabolites for osmotic adaptation under stressful conditions [6,73].
Proline has a vital role in chlorophyll and protein maintenance by preventing degradation and promoting the activities of many enzymes [74]. The enhancement detected in the proline, total protein, total phenol, and carbohydrate content of cowpea leaves following ZnNP and FeNP applications is evident in good growth, leaves, pods, and seed yield, accompanied by increasing dry matter accumulation. Increasing biomass yield points to a plant-stress tolerance against stressful conditions [75]. It is worth noting that increasing total phenols and carbohydrates in cowpea plants following green ZnNPs or FeNPs may be due to the GLE utilized, as it is a good source of carbohydrates, protein, total phenols, and other active compounds and nutrients that are necessary for cowpea growth and productivity. Our results were in harmony with those reported by Sturikova et al. [76] and Rehman et al. [30], who illustrated that ZnNP and FeNP treatments increased proline, total phenol, and carbohydrate levels in wheat plants grown under saline conditions.
Better antioxidant defenses in salt-stress-tolerant plants restricted ROS generation and increased the plant’s ability to resist stressful conditions [77]. More antioxidants generated contributed to higher ROS combating. Cowpea plants subjected to ZnNP and FeNP foliar applications under saline water treatments revealed an enhancement in non-enzymatic antioxidants (total phenols) and a decrease in enzymatic antioxidants (peroxidase). The increments in total phenols of cowpea leaves may be due to the GLE utilized for green nano-synthesis, which is rich in phenolic compounds, as well as the role of Zn and Fe in stimulating phenolic biosynthesis pathways. FeNP application is a sustainable approach for the mitigation of salt stress due to FeNPs’ higher surface area and surface area-to-weight ratio [78]. FeNPs’ large surface area and reactivity allow them to act as adsorption sites and immobilize sodium and chloride ions [79].
Increasing enzymatic antioxidants is a defense mechanism against ROS production in stressed plants [80]. These previous findings agreed with our results that revealed an elevation in the POD level with increasing the level of saline water treatments, as the S4 plants exhibited the highest POD values. Whereas these POD values decreased following ZnNP and FeNP foliar applications in stressed cowpea plants, which means that these plants were less exposed to stress than untreated plants. The reduction in POD values following ZnNP and FeNP foliar applications may be explained by the role of Zn and Fe in ROS scavenging, lessening the damage that occurs in cell membranes [81].

5. Conclusions

This investigation was carried out to evaluate the possibility of positive effects of foliar application of green synthetized zinc NPs (ZnNPs) and iron NPs (FeNPs) for enhancing the plant growth, seed yield and quality, and antioxidant content of cowpea plants grown under saline conditions. Saline conditions caused a depression in the growth and quality of cowpea seeds, but foliar applications with ZnNPs or FeNPs significantly maintained the damaging influences of salinity. In this regard, ZnNP application decreased photosynthetic pigment degradation and exhibited higher osmoprotectants, total phenols, and decreased POD activity than FeNP treatment. The ZnNPs × S1 treatment showed the highest seed weight, yield, and quality.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/horticulturae10090915/s1.

Author Contributions

Conceptualization, M.H.S., A.H.M.A.E.-W. and M.M.I.M.; methodology, M.H.S., A.H.M.A.E.-W., M.A.E., M.A.M.A.-E. and M.M.I.M.; software, M.H.S., A.-N.A.E.-S. and M.M.I.M.; validation, M.H.S., A.H.M.A.E.-W., H.M.B., N.A.O., A.-N.A.E.-S. and R.S.E.-S.; formal analysis, M.H.S., A.-N.A.E.-S. and M.M.I.M.; investigation, M.H.S. and A.H.M.A.E.-W., M.A.E., M.A.M.A.-E. and M.M.I.M.; resources, M.H.S., A.-N.A.E.-S., M.A.E., M.A.M.A.-E. and M.M.I.M.; data curation, M.H.S., H.M.B., N.A.O. and A.-N.A.E.-S.; writing—original draft preparation, M.H.S., A.H.M.A.E.-W., M.A.E., M.A.M.A.-E. and M.M.I.M.; writing—review and editing, M.H.S., H.M.B., N.A.O., A.-N.A.E.-S., M.A.M.A.-E. and R.S.E.-S.; visualization, M.H.S. and M.A.E.; supervision, M.H.S. and M.M.I.M. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

The original contributions presented in the study are included in the article/Supplementary Materials, further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. TEM analysis of green zinc oxide nanoparticles.
Figure 1. TEM analysis of green zinc oxide nanoparticles.
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Figure 2. TEM analysis of green iron oxide nanoparticles.
Figure 2. TEM analysis of green iron oxide nanoparticles.
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Figure 3. EDX analysis of green zinc oxide nanoparticles.
Figure 3. EDX analysis of green zinc oxide nanoparticles.
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Figure 4. EDX analysis of iron oxide nanoparticles.
Figure 4. EDX analysis of iron oxide nanoparticles.
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Figure 5. Zeta potential analysis of green zinc oxide nanoparticles.
Figure 5. Zeta potential analysis of green zinc oxide nanoparticles.
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Figure 6. Zeta potential analysis of green iron oxide nanoparticles.
Figure 6. Zeta potential analysis of green iron oxide nanoparticles.
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Figure 7. Effects of irrigation water salinity and foliar applications of ZnNPs and FeNPs on (a) leaf fresh weight; (b) SLA, specific leaf area; (c) LWCA, leaf water content area; and (d) leaf dry matter of cowpea during seasons 2022 and 2023. Different bars indicate the values of means ± SE, n = 6; different letters indicate significant differences (p ≤ 0.05) between the treatments after performing Tukey’s test.
Figure 7. Effects of irrigation water salinity and foliar applications of ZnNPs and FeNPs on (a) leaf fresh weight; (b) SLA, specific leaf area; (c) LWCA, leaf water content area; and (d) leaf dry matter of cowpea during seasons 2022 and 2023. Different bars indicate the values of means ± SE, n = 6; different letters indicate significant differences (p ≤ 0.05) between the treatments after performing Tukey’s test.
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Figure 8. Effects of irrigation water salinity and foliar applications of ZnNPs and FeNPs on (a) pod number; (b) pod length; (c) 100 seed weight; and (d) seed yield plant−1 of cowpea during seasons 2022 and 2023. Different bars indicate the values of means ± SE, n = 6; different letters indicate significant differences (p ≤ 0.05) between the treatments after performing Tukey’s test.
Figure 8. Effects of irrigation water salinity and foliar applications of ZnNPs and FeNPs on (a) pod number; (b) pod length; (c) 100 seed weight; and (d) seed yield plant−1 of cowpea during seasons 2022 and 2023. Different bars indicate the values of means ± SE, n = 6; different letters indicate significant differences (p ≤ 0.05) between the treatments after performing Tukey’s test.
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Figure 9. Effects of irrigation water salinity and foliar applications of ZnNPs and FeNPs on (a) chlorophyll a; (b) chlorophyll b; (c) total chlorophyll; and (d) carotenoids of cowpea during seasons 2022 and 2023. Different bars indicate the values of means ± SE, n = 6; different letters indicate significant differences (p ≤ 0.05) between the treatments after performing Tukey’s test.
Figure 9. Effects of irrigation water salinity and foliar applications of ZnNPs and FeNPs on (a) chlorophyll a; (b) chlorophyll b; (c) total chlorophyll; and (d) carotenoids of cowpea during seasons 2022 and 2023. Different bars indicate the values of means ± SE, n = 6; different letters indicate significant differences (p ≤ 0.05) between the treatments after performing Tukey’s test.
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Figure 10. Effects of irrigation water salinity and foliar applications of ZnNPs and FeNPs on (a) proline; (b) peroxidase; and (c) total phenols of cowpea during seasons 2022 and 2023. Different bars indicate the values of means ± SE, n = 6; different letters indicate significant differences (p ≤ 0.05) between the treatments after performing Tukey’s test.
Figure 10. Effects of irrigation water salinity and foliar applications of ZnNPs and FeNPs on (a) proline; (b) peroxidase; and (c) total phenols of cowpea during seasons 2022 and 2023. Different bars indicate the values of means ± SE, n = 6; different letters indicate significant differences (p ≤ 0.05) between the treatments after performing Tukey’s test.
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Figure 11. Effects of irrigation water salinity and foliar applications of ZnNPs and FeNPs on (a) seed dry matter; (b) total carbohydrates; and (c) total protein of cowpea during seasons 2022 and 2023. Different bars indicate the values of means ± SE, n = 6; different letters indicate significant differences (p ≤ 0.05) between the treatments after performing Tukey’s test.
Figure 11. Effects of irrigation water salinity and foliar applications of ZnNPs and FeNPs on (a) seed dry matter; (b) total carbohydrates; and (c) total protein of cowpea during seasons 2022 and 2023. Different bars indicate the values of means ± SE, n = 6; different letters indicate significant differences (p ≤ 0.05) between the treatments after performing Tukey’s test.
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Table 1. Some physical and chemical properties of soil at the experimental site before sowing during growing seasons 2022 and 2023.
Table 1. Some physical and chemical properties of soil at the experimental site before sowing during growing seasons 2022 and 2023.
Soil PropertyValue
Particle size distribution:
Coarse sand (%)6.99
Fine sand (%)78.71
Silt (%)4.43
Clay (%)9.87
Texture classLoamy sand
Field capacity (%)13.02
Permanent wilting point (%)5.36
Available water (%)7.66
Bulk density (Mg m−3)1.70
Total porosity (%)35.85
pH (1:2.5 soil water suspension)8.49
ECe (soil paste extract, dS m−1)0.69
CaCO3 content (g kg−1)43.37
Available nutrients (mg kg−1)
N18.95
P4.58
K67.33
Fe2.60
Zn0.62
Mn1.18
Cu0.47
Table 2. Chemical features of the saline water used.
Table 2. Chemical features of the saline water used.
TreatmentspHECW
(dS m−1)		Cations (mmolc L−1)Anions (mmolc L−1)SAR
Ca2+Mg2+Na+K+CO32−HCO3−Cl−SO42−
S17.120.751.501.743.840.420.002.094.950.463.02
S27.442.003.455.0210.980.550.003.6312.943.435.34
S37.514.006.868.4923.551.100.006.6320.8812.498.50
S47.556.0010.2612.4434.752.550.009.4728.9821.5510.31
ECW: electrical conductivity of saline water; SAR: sodium adsorption ratio.
Table 3. Qualitative detection of guava leaf extract.
Table 3. Qualitative detection of guava leaf extract.
Active CompoundsPhenolsSaponinsFlavonoidsCoumarinsCarbohydratesAlkaloidsTerpenesTannins
Inference++++++++
Table 4. Total phenols and flavonoids in guava leaf extract.
Table 4. Total phenols and flavonoids in guava leaf extract.
CompoundsConcentration
Phenols (mg gallic acid equivalent mL−1 of extract)15.80 ± 0.48
Flavonoids (mg of quercetin equivalent mL−1 of extract)10.30 ± 0.39
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Sheta, M.H.; Abd El-Wahed, A.H.M.; Elshaer, M.A.; Bayomy, H.M.; Ozaybi, N.A.; Abd-Elraheem, M.A.M.; El-Sheshtawy, A.-N.A.; El-Serafy, R.S.; Moustafa, M.M.I. Green Synthesis of Zinc and Iron Nanoparticles Using Psidium guajava Leaf Extract Stimulates Cowpea Growth, Yield, and Tolerance to Saline Water Irrigation. Horticulturae 2024, 10, 915. https://doi.org/10.3390/horticulturae10090915

AMA Style

Sheta MH, Abd El-Wahed AHM, Elshaer MA, Bayomy HM, Ozaybi NA, Abd-Elraheem MAM, El-Sheshtawy A-NA, El-Serafy RS, Moustafa MMI. Green Synthesis of Zinc and Iron Nanoparticles Using Psidium guajava Leaf Extract Stimulates Cowpea Growth, Yield, and Tolerance to Saline Water Irrigation. Horticulturae. 2024; 10(9):915. https://doi.org/10.3390/horticulturae10090915

Chicago/Turabian Style

Sheta, Mohamed H., Ahmed H. M. Abd El-Wahed, Mohammed A. Elshaer, Hala M. Bayomy, Nawal A. Ozaybi, Mohamed A. M. Abd-Elraheem, Abdel-Nasser A. El-Sheshtawy, Rasha S. El-Serafy, and Mahmoud M. I. Moustafa. 2024. "Green Synthesis of Zinc and Iron Nanoparticles Using Psidium guajava Leaf Extract Stimulates Cowpea Growth, Yield, and Tolerance to Saline Water Irrigation" Horticulturae 10, no. 9: 915. https://doi.org/10.3390/horticulturae10090915

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