Impact of ZnO Nanoparticles on Growth of Cowpea and Okra Plants under Salt Stress Conditions

Salt stress causes a serious threat to agricultural productivity and global food security. It is one of the most pervasive crops limiting factor. This study examined the effect of six salinity concentrations (0, 10, 25, 50, 75 and 100% of seawater); on the growth of two crop species, cowpea (Vigna unguiculata L. var.california blackeye NO.46) and okra (Abelmoschus esculentus L. Moench var.Hasawi) in the presence or absence of (10 mg/L) of the green synthesized zinc oxide nanoparticles (ZnO NPs) or zinc oxide (bulk ZnO), as a foliar spray after (20, 40 and 60 days) from sowing. The results showed a gradual decrease in shoot and root lengths, fresh and dry weights of shoot, leaf area and relative growth rate (RGR) with the increase of seawater concentrations in both plants. However, application of ZnO enhanced the growth parameters compared to the control plants, but better results were observed in the plants treated with (ZnO NPs). Thus, nanoparticles of (ZnO) environmentally friendly, cheap cost, and can be considered as a promising application to alleviate the effects of salt stress on plants.

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 salinity 3 , 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 population 5 . By the year 2050, even more than 50% of the global agriculture land will be vulnerable to salt stress 6 . There is a general understanding, that salinity only occurs in arid and semi-arid regions, but there is no climatic area free from this problem 7 . 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 materials 11 . The use of the applications of nanotechnology is increasing in different fields 12 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 ratio 15 .
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 species 15 . 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 approaches 17 . 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 fertilizers 18 .
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 crop 19 . 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 quality 22 . It grows commercially in many countries 23 .  Significance of values at p<0.05, a= (highly significant), b= (significant), c= (not significant).

MaTerials and MeThods
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 [

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 Effect of different concentrations of seawater (SW) in the presence or absence of (bulk ZnO) or (ZnO NPs) on relative growth rate (RGR; g g -1 day -1 ) of (a)Vigna unguiculata and (b)Abelmoschus esculentus plants RGR: Relative Growth Rate (g g-1 day -1) In = natural logarithm ln W 1 =The mean of the ln-transformed plant total dry weight at time t1. ln W 2 = The mean of the ln-transformed plant total dry weight at time t 2 t 1 = number of days in the first time measurement (day) t 2 = number of days in the last time measurement (day) W 1 and W 2 are the dry weight of the plants at time t 1 and t 2 respectively.

statistical analysis
All experiments were carried out using the statistical package SPSS software, version 20 Significance of values at p<0.05, a= (highly significant), b= (significant), c= (not significant).
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.

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 nonsignificant 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).
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) Significance of values at p<0.05, a= (highly significant), b= (significant), c= (not significant).
increased these measures high significantly as compared to their corresponding controls (Fig. 3a).

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

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 tissue 27 . 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 stress 33 . Also, Kaya et al 34 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 division 27 , 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 rate 35 . The reduction in fresh and dry weight is due to the formation of smaller and fewer leaves and a decrease in plant height 33 .
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 health 36 41 . 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 production 43 . The uptake of Zn through the leaves is influenced by environmental factors, type of the leaf, stress level and plants health 44 . 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 growth 45 . 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 growth 46 .
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 membrane 47 . Furthermore, the high specific surface area and a higher rate of uptake explain the better efficiency of the application of nanoparticles compared to bulk forms 48 . This helps to raise the rate of dissolution of zinc oxide (ZnO) which has low solubility in water 49 . 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 concentrations 50 . 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 iron 52 .

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.