Introduction

Salt stress affecting almost 20–33% of cultivated areas, 50% of irrigated areas and affects almost one billion hectares of global land^1,2. More than 397 million hectares of lands worldwide is affected by salinity and/or more than 434 million hectares affected by salinity³, which causing desertification around the world⁴. While the agricultural land, which is exposed to salinity, minimize, the food is demanded with the increase of the population⁵. By the year 2050, even more than 50% of the global agriculture land will be vulnerable to salt stress⁶. There is a general understanding, that salinity only occurs in arid and semi-arid regions, but there is no climatic area free from this problem⁷. Around 97.5% of the planet’s water is saline. Seawater is the most available source of water in the world. Thus, there are growing interested to use it in the agricultural sector to irrigate the plants^8,9. The major constituent of seawater is sodium chloride (NaCl)¹⁰.

Nanotechnology is a description of synthesis, fabrication, characterization and utilization of Nano-sized materials¹¹. The use of the applications of nanotechnology is increasing in different fields¹² such as industry, information technology, medicine, energy and agriculture which in turn impacts the environment, society and economy^13,14. There are different properties in the nanoparticles of metal oxides that are not found in their bulks counterparts like their shapes, size, surface reactivity, chemical stability and their large surface area to their volume ratio¹⁵.

Zinc (Zn) is a micronutrients and one of the essential nutrients for humans, animals and has an influential role in plant growth, development and protection. Generally, the plants uptake the Zn as a cation (Zn²⁺)¹⁶ .The appropriate concentrations of zinc oxide nanoparticles (ZnO NPs) improved the growth and protection of different plant species¹⁵. Using the nanoparticles, which are synthesized by green methods like (ZnO NPs) as a foliar application on the plants is one of the promising methods to reduce water and soil pollutions by putting less input and producing less waste than ordinary approaches¹⁷. Fertilizers at the nano size improve the plant’s growth because of their diminutive size, which in turn could enhance the uptake of micronutrients in a controlled and gradual manner in the plants compared to the regular fertilizers¹⁸.

Cowpea considers as one of the most important economically cultivated legumes worldwide which provides many economic, agronomic and environmental advantages to millions of people worldwide. It is a feed, food and forage crop¹⁹. This species is a herbaceous warm-season annual plant grown in tropical and subtropical regions and in the semiarid regions^20,21.

Okra is one of the most popular vegetables annually renewable crops cultivated during the hot summer seasons. It is a multipurpose crop which have been used in industrial and health applications, and it has nutritional quality²². It grows commercially in many countries²³.

Materials and Methods

Materials 

All chemicals employed in this study were of high purity, purchased from Sigma-Aldrich, USA. ZnO nanoparticles prepared by using [Phoenix dactylifera L. cv. Khalas] leaflets extract and characterized their formation and size by using the UV-visible spectroscopy [UV-1800] which demonstrated that the highest absorption peak was about [370 nm] using a transmission electron microscope (TEM) [Mic JEM 1011], and the size founded [from 16 to 35nm] (Fig.1). The concentration of seawater used to irrigate the plants prepared by diluted seawater to get (0, 10, 25, 50, 75 and 100 % seawater SW). The seeds of cowpea [Vigna unguiculata L. cv.California Blackeye NO.46] and okra [Abelmoschus esculentus L. Moench cv.Hasawi] were purchased from Modesto, California U.S.A and Altuajri, K.S.A. respectively. The powders of both ZnO types were mixed with deionized water.

Figure 1: TEM image of the biosynthesized (ZnO NPs) from [Phoenix dactylifera L. Click here to View Figure

Pot Experiment

Seeds of V.unguiculata and A. esculentus were surface sterilized by 4% for 1 min, then rinsed thoroughly with distilled water. The seeds then germinated in 15cm pots which contain 2.5kg of sand. The experimental pots were arranged in a simple randomized design and exposed to normal day length and natural temperature 25-28^⸰C.

All plants were irrigated 3 times per week with tap water for 15 days. After that, pots were treated with seawater concentrations (0, 10, 25, 50, 75 and 100%) with or without the foliar application of 10mg/L of bulk ZnO or ZnO NPs. The foliar treatments applied 2 times at 15 and 35 days after sowing using a hand-held sprayed separately after covering the surface of the pots with plastic film. Three vegetative stages were studied at 20, 40 and 60 days from planting dates for growth analysis.

Growth Parameters

After 20, 40 and 60days, the evaluation of shoot and root lengths had been determined by using a metric scale and expressed in centimeter (cm). After washed the plants with double distilled water to remove the sand particles, there had been separated into shoots and roots, then fresh weights and dry weights (DW) weighted by analytical balance [HR-200]. Dry weight was recorded by drying the plants at 65^⸰C until the weight became constant. The third leaf area measurement (cm²) was taken after 60 days since emergency by using [CI-202 AREA METER CID, INC]. The relative growth rate was measured  according to the formula of Hunt²⁴ and Hoffmann and Poorter²⁵:

R.G.R = ln W2 ˗ ln W1 / t₂ ˗ t₁

RGR: Relative Growth Rate (g g^-1 day ^-1)

In = natural logarithm

ln W₁ =The mean of the ln-transformed plant total dry weight

at time t₁.

ln W₂ = The mean of the ln-transformed plant total dry weight at time t₂

t₁ = number of days in the first time measurement (day)

t₂ = number of days in the last time measurement (day)

W₁ and W₂ are the dry weight of the plants at time t₁ and t₂ respectively.

Statistical Analysis

All experiments were carried out using the statistical package SPSS software, version 20 with three replicates (n=3) ±SE by a completely randomized design (CRD). Statistical analysis was carried out according to Snedecor and Cochran²⁶, using T test. Significant differences were obtained by calculating (LSD) at p<0.05.

Results

Growth of cowpea (Vigna Unguiculata)  

The results revealed that in V.unguiculata plants the shoot and root lengths, shoot and root fresh and dry matter decreased with the increase seawater concentrations at the three vegetative stages, (bulk ZnO) improved the growth parameters non-significantly and significantly. While these parameters increased significantly and high significantly with (ZnO NPs) relative to control plants except at (20 days) the increase was non-significant in root length with (75 and 100% SW) treatments, and the fresh weight of shoot with (10, 25 and 50 % SW) treatments (Tables 1,2,3). After 60 days, V.unguiculata leaf area was measured; seawater treatment showed a non-significant decrease in leaf area with increasing salinity. When applying (bulk ZnO) non-significantly increased the leaf area in all seawater concentrations, while with (ZnO NPs) showed a better significant increase as compared to (bulk ZnO) and control treatments (Fig. 2a).

Figure 2: Effect of different concentrations of seawater (SW) in the Click here to View Figure

Table 1: Effect of different concentrations of seawater (SW) in the presence or absence of (bulk ZnO) or (ZnO NPs) on shoot length and root length (cm), fresh and dry weights of shoot and root (g) of Vigna unguiculata plants after 20 days of age.

Significance of values at p<0.05, a= (highly significant), b= (significant), c= (not significant).

Table 2: Effect of different concentrations of seawater (SW) in the presence or absence of (bulk ZnO) or (ZnO NPs) on shoot length and root length (cm), fresh and dry weights of shoot and root (g) of Vigna unguiculata plants after 40 days of age.

Significance of values at p<0.05, a= (highly significant), b= (significant), c= (not significant).

Table 3: Effect of different concentrations of seawater (SW) in the presence or absence of (bulk ZnO) or (ZnO NPs) on shoot length and root length (cm), fresh and dry weights of shoot and root (g) of Vigna unguiculata plants after 60 days of age.

Significance of values at p<0.05, a= (highly significant), b= (significant), c= (not significant).

The relative growth rate (RGR) decreased gradually with the increasing seawater concentrations. The non-fertilized V.unguiculata plants (control) showed non-significant decrease in (RGR) in the lower seawater concentrations (10 and 25% SW), while the decrease was significant in (50, 75 and 100% SW). The addition of (bulk ZnO) increased the (RGR) non-significantly in all seawater concentrations. However, (ZnO NPs) increased these measures high significantly as compared to their corresponding controls (Fig. 3a).

Figure 3: Effect of different concentrations of seawater (SW) in the presence Click here to View Figure

Growth of Okra (Abelmoschus Esculentus)

In A.esculentus plants, all the growth parameters decreased gradually with the increase of seawater levels. After 60 days there was high significant inhibition reached (51.70, 55.90, 67.00, 71.43, 74.7 and 75.56%) in shoot and root lengths, shoot and root fresh weights, shoot and root dry weights respectively, compared to control treatments. It is worth mentioning that the plants treated with the green synthesized (ZnO NPs) give the best results to enhance the growth measurements compared to the plants treated with (bulk ZnO), (Tables 4,5,6). Present results show that the leaf area of A.esculentus plants treated with different concentrations tend to decrease non-significantly in (10% SW), while the decrease was high significant at all the other concentrations. The leaf area increased non-significantly above the different controls when (bulk ZnO) was used, while (ZnO NPs) increased high significantly the leaf area in all concentrations except the higher concentration (100% SW), (Fig. 2b).

Table 4: Effect of different concentrations of seawater (SW) in the presence or absence of (bulk ZnO) or (ZnO NPs) on shoot length and root length (cm), fresh and dry weights of shoot and root (g) of Abelmoschus esculentus plants after 20 days of age.

Table 5: Effect of different concentrations of seawater (SW) in the presence or absence of (bulk ZnO) or (ZnO NPs) on shoot length and root length (cm), fresh and dry weights of shoot and root (g) of Abelmoschus esculentus plants after 40 days of age.

Table 6: Effect of different concentrations of seawater (SW) in the presence or absence of (bulk ZnO) or (ZnO NPs) on shoot length and root length (cm), fresh and dry weights of shoot and root (g) of Abelmoschus esculentus plants after 60 days of age.

The decrease in RGR was significant in plants treated with (10%) of seawater and highly significant in plants treated with (25, 50, 75 and 100 % SW). Addition of (bulk ZnO) increased the relative growth rate significantly at (0 and 10%) of seawater, while it increased non-significantly at (25, 50, 75 and 100% SW). The addition of (ZnO NPs) gave positive increases than (bulk ZnO). The increase was highly significant in plants treated with all seawater concentrations (Fig. 3b).

Discussion

Salinity affects plant growth by ionic stress, oxidative stress, reducing cell enlargement and cell division and osmotic stress, which depends on the concentration of salts and the type of plant tissue²⁷. Salt stress can strongly affect the plants morphology ^28,29, it has a great inhibition influence which can lead to apparent stunting of plant growth^29,30.

The growth of roots decreases when soil salinity exceeds (40mM)^31,32, thus inhibition of root growth leads to reduction in water use efficiency, water uptake capacity, leaf water potential and transpiration rate under salt stress³³.  Also, Kaya et al³⁴ pointed out that stressed plants resorted to close the stomata to retain the amount of water in the leaves and thus less entry of CO₂ and rate of photosynthesis, which leads directly or indirectly to a decrease the amount of photosynthetic products.

Salt stress causes a reduction in turgor pressure, which leads to a major reduction in cell growth, cell elongation, cell division²⁷, and consequently the whole plant growth. The decrease in leaf area is a result of cell water relations, changes in cell wall features and reduction in photosynthetic rate³⁵. The reduction in fresh and dry weight is due to the formation of smaller and fewer leaves and a decrease in plant height³³.

The morphological parameters in the plants such as shoot and root lengths, shoot and root weights, leaf area as well as, relative growth rate (RGR) are indicate the plant health³⁶. The measured growth parameters in cowpea (V.unguiculata) and okra (A.esculentus) plants increased with the foliar application of (ZnO bulk) and (ZnO NPs) under salinity stress. (ZnO NPs) showed better results than other treatments. These data are in agreement with other studies such as Sah et al³⁷ on  Borago officinalis L.; Sabaghnia and Janmohammadi³⁸ on Lens culinaris Medik.; Luksiene et al³⁹ on Strawberries and Shinde et al⁴⁰ on maize. Zinc applications have a positive impact on the plants salt tolerance. (ZnO NPs) can potentially alleviate the negative effects of abiotic stress on plants⁴¹. This enhancement influence of foliar application might be attributed to the crucial role of zinc on the biological and metabolism activity of plants such as stimulating enzymes activities, cell elongation and enlargement, nitrogen metabolism, photosynthetic pigments, maintaining the membranes structural stability of the plant cells and accumulation of the phospholipids^16,42.

The efficiency of (ZnO NPs) also, relates to their ability to penetrate in the plant cell through the natural Nano pore (stomata) in the leaves which may improve metabolic activities and consequently higher plant production⁴³. The uptake of Zn through the leaves is influenced by environmental factors, type of the leaf, stress level and plants health⁴⁴. In addition, Zn applications positively improve biosynthesis of the growth regulator IAA which promotes cell division, cell elongation and absorption of minerals, thus increased plant growth⁴⁵. The addition of micronutrient is more economical and beneficial than soil fertilization, because of the nutrients are more actively to reach the cells and be obtainable for plant growth⁴⁶.

Nanoparticles forms with their smaller size, have more ability and dynamic to be absorbed, translocate, assimilate and accumulate in the plant than their bulk forms. Nanoparticles can pass through the cell wall and plasma membrane⁴⁷. Furthermore, the high specific surface area and a higher rate of uptake explain the better efficiency of the application of nanoparticles compared to bulk forms⁴⁸. This helps to raise the rate of dissolution of zinc oxide (ZnO) which has low solubility in water⁴⁹. The various physiological effects of the foliar supply of (ZnO NPs) may be due to the slow release of Zn ion from the nanoparticles, which supplies a long-term provenance of Zn, and help to avoid toxicity by sudden uptake of Zn by plants at high concentrations⁵⁰. The increase in plants growth with nanoparticles application might be due to rising of the efficiency of nutrient usage diminish soil toxicity which produces by over dosage of the addition of fertilizers and enhance the activities of antioxidant enzymes which help to protect the plants from injury caused by (ROS)⁵¹. Rising in the plant height may because of the improvements of auxin biosynthesis and synergistic relation between both nutrients nitrogen and iron⁵².

Conclusion

The results of this study showed that both treatments of (bulk ZnO) and (ZnO NPs) enhanced the growth parameters in the salt-stressful plants cowpea (V.unguiculata) and okra (A.esculentus). Notably, both of these plants showed good tolerance to salt stress. The nanoparticles of (ZnO) gave better results by improving plant salinity tolerance than their bulk size. The foliar application of the green synthesize (ZnO NPs) can be a good alternative to their bulks because they are ecologically friendly approaches with low-priced.

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