Protective role of Spirulina platensis liquid extract against salinity stress e ﬀ ects on Triticum aestivum

: Salt stress is one of the most serious factors that in ﬂ uence the plant growth and yield. The extensive use of chemical fertilizers caused an increase in land contami nation and hence e ﬀ ects on human health. The use of biofertilizers can solve these problems. In the present work, di ﬀ erent concentrations of Spirulina platensis liquid extracts ( SLEs; 1%, 2%, and 4% ) were used to enhance seeds germination, seedling growth, length of radical, number of leaves, and plant height of T. aestivum L. S. platensis liquid extract ( SLE ) 2% was chosen to test against salt stress with 10% and 25% seawater in T. aestivum L. watered. The growth parameters such as shoot, root, length, fresh, dry weight, leaf width, number, and area were used to estimate the e ﬀ ects of 2% SLE against salt stress caused by seawater. Also the carbohydrate, protein, phenol, and total antioxidant contents were measured in T. aestivum L. treated with 2% SLE in combination with seawater and watered with di ﬀ erent concentrations with seawater alone. The results denote that the best concentration of SLEs was 2%, which promoted the germination growth of T. aestivum L. in comparison to control and other concentrations. SLE ( 2% ) enhanced the carbohydrates, protein, total phenol contents, and antioxidant contents of T. aestivum L., treated with seawaters ( SW10% and SW25% ) and SLEs in comparison to T. aestivum L. SW 10% and 25% alone. S. platensis liquid extract ( SLE ) can be used as bioferti lizer to enhance the growth and phytochemical contents of T. aestivum L. and to make tolerance of T. aestivum L. against salinity.


Introduction
Agriculture plays multiple tasks to yield more nutrition to face the development of the population, and chemical fertilizer is unwanted due to its being a costly process and it is made from nonrenewable fossil fuel, which causes substantial pollution [1,2]. The prolonged use of chemical fertilization has a negative influence on soil fertility and microbiota, leading to reduction in fertilizer effectiveness and amassing of its residues in soil [3]. Recent agricultural practices tend to look for unconventional biotechnology (biofertilizers) for control of the chemical fertilizers without any effect on crop yields and income with cost-effective feasibility [4,5]. Rengasamy et al. [6] reported the growing demand for organic food as well as increasing environmental consciousness, so the bio-stimulants marketplace is increasing all over the world. Algae are eco-friendly natural products commonly consumed as organic fertilizer; seaweed extracts represent the major group of biostimulants that advance seed germination, plant growth, and crop yields [7]. Spirulina is an edible blue-green microalga (cyanobacterium) that has major contents of beneficial quality protein as well as being valuable contents of vitamins, minerals, and other components that are helpful for health, such as principal fatty acids and antioxidant pigments such as carotenoids, chlorophyll, and phycocyanin [8]. Spirulina platensis is described as a simple cultivation and harvesting process and is also the most popular species in microalgae biotechnology research [9,10]. Spirulina contains a lot of photosynthetic pigments with high intensities that reach 20% of dry weight, which may be useful and have commercial values, and those pigments are chlorophylls, beta-carotene, phycocyanins, and xanthophylls [11,12]. Godlewska et al. [13] reported that algal extracts have a lot of potential in modern horticulture and agriculture. The usage of Spirulina-based products is in line with the concept of sustainable agriculture, which could assist to secure the production of enough human food to fulfill the rising population demands while also protecting the environment. Blue-green alga S. platensis significantly increase the protein contents in Trigonella foenum-graecum [14]. There were an increase in the seedling growth and an increase in phytochemicals content in leaves of Lettuce when S. platensis was applied as biofertilizers [15]. S. platensis is used to treat wastewater and to stimulate the growth of leafy vegetables such as Eruca sativa, Ameranthus gangeticus, and Brassica rapa sp. [16]. The yield of Florida prince peach trees was promoted quantitatively and qualitatively when S. platensis was used beside humic acid [17]. Today, about 20% of the world's cultivated land and approximately half of all irrigated lands are influenced by salinity [18,19]. Salinity is one of the highest environmental stresses that extremely decrease the crop yield and growth. Millions of hectares of working land will be unsuitable for plant cultivation due to the incessant highest salinity [20]. Salinity may influence the seedling growth by reducing the absorption of water and/or reducing the level of gibberellins in germinating seeds [21]. Applications of different types of marine algal had a significant enhancement in the growth parameters of wheat seedlings under salinity stress. The existence of some bioactive contents in marine algae, such as ascorbic acid, auxins, and gibberellins, alleviates the adverse effect of salinity on the wheat seedling growth [22][23][24]. The occurrence of inorganic phosphate algae extract makes a fundamental in the growth and protection of wheat seedlings from salt toxicity [18]. S. platensis displayed a worthy potential to advance the growth and yield of Vicia faba grown under salt stress [25]. Globally, wheat is the second most yielded food among the cereal crops after maize and rice, and wheat is a moderately salt-tolerant crop and its yield is noticeably reduced to the soil salinity level [26]. Wheat has a wide adaptation to diverse environmental circumstances, and it is cultured over a wide range of environments and adaptable to cultivation in moderate salty soils [27]. Wheat growth and yield parameters were negatively affected by salt, although cyanobacteria was applied as an extract and grains coating alleviated the salinity impacts. At all salinity levels studied, a combination of cyanobacteria extract and grain coating with 50% of chemical fertilizer (NP) generated the wheat growth and output comparable to a full dose of chemical fertilizer [28]. This study aimed to elucidate the enhancing potentiality of different concentrations of S. platensis liquid extracts on seed germination, plant growth parameters, pigments, and enzymes (α-amylase and protease) of Triticum aestivum L. and investigate the effects of S. platensis liquid extracts improving salt tolerance of T. aestivum L.

Alga used and growth condition
Cyanobacterium S. platensis was achieved from the Culture Collection of the Algal Laboratory, Faculty of Science, Alexandria University, Egypt. Spirulina was grown in Zarrouk's medium [29], with continuous illumination (35 µmol‧m −2 ‧s) at 30 ± 2.0°C in pH 9-10. After 24 days of growth (in the stationary phase), pellets were harvested by filtration, washed thoroughly with distilled water, and then dried at 60°C until constant weight. The characterization of S. platensis was previously published by El Din [30].

Spirulina liquid extracts
To obtain the 1%, 2%, and 4% of Spirulina liquid extract, approximately 1, 2, and 4 g of fine powder dry Spirulina was added to 100 mL distilled water, heated at 50°C with stirring for 60 min, and the extracts were cooling, filtered, and kept at 4°C until usage.

Bioassays for seeds germination using
Spirulina liquid extract (SLE) according to the relevant institutional, national, and international guidelines and legislation). Seeds were selected carefully and sterilized by using 0.01% HgCl 2 for 3 min, then washed under running tap water for 10 min. The number of germinated seeds, weight, and radical length were found. Ten seeds were spread on filter paper placed on glass Petridishes containing 5.0 mL of SLE (2%), and seeds without algal extract served as a control. The Petri-dishes were placed at natural illumination at 25°C [31,32]. The germination percentage (GP), germination index (GI), relative seed germination (RSG), seedling vigor index (SVI), and relative root elongation (RRE) were achieved according to the following equations [33]: Germination percentage GP Number of germinated seeds Total number of seeds 100 where Gt is the number of seeds germinated on day t and Tt is the number of days.

Crude extracts of enzymes
One gram of germinating seeds (hypocotyl) was blended and homogenized with potassium phosphate buffer (pH = 7) followed by centrifugation at 1,643g for 15 min, and the resultant crude preparation was used for the estimation of protease activity.

α-Amylase
The α-amylase activity was investigated based on the methodology described by Franco et al. [34]. Enzyme activity unit (U) was defined as the amount of enzyme releasing 1 µmol of glucose from the substrate in 1 min under the assay conditions.

Protease
The crude extracts of the enzyme (0.5 mL) were mixed with 1 mL of casein (1%) in 0.2 M phosphate buffer at pH 6 and incubated at 37°C for 30 min. The reaction was terminated by the addition of 0.5 mL of (10%) trichloroacetic acid (TCA). Enzyme activity was estimated at 660 nm after treating with Folin phenol reagent [35] and presented in terms of µmol amino acids released by unit time.

Pot experiments
Ten healthy seeds of T. aestivum (five seeds in each pot and three pots for each treatment) were cultivated in 1 L pots for 20 days. No fertilizer was applied, but the soil of the treated seedlings was sprayed with 200 mL of 1%, 2%, and 4% of Spirulina liquid extracts every 2 days. Weekly measurement of the plant growth was recorded in terms of leaf number per plant and plant height. At the end of the trials, the plants were harvested, and their leaf number, plant height, root length, fresh weight, and dry weight were determined, and pigment content was determined according to Inskeep and Bloom [36].

Ameliorating effect of SLEs on T. aestivum seedling grown under salinity stress
Seeds of T. aestivum that were treated with 2% SLE were watered with tap water and 10% and 25% seawater (collected from the Mediterranean Sea, at Alexandria), and other seeds that remained untreated were also watered with tap water and 10% and 25% seawater. After 20 days of sowing, the growth, carbohydrates, proteins, phenol, and antioxidant contents were investigated [25].

Bio-stimulant assays
After 20 days of sowing, ten replicates of the growing plant were used to estimate morphological criteria of the growing plant (shoot height, root length, leaves the area, and width of leaves).
The equation was computed as suggested by Quarrie and Jones [37]. The dry biomass of the grown plants was estimated after drying at 80°C for 3 days.
Total protein content was investigated related to the method by Lowry et al. [35]. Carbohydrate fractions were determined following the colorimetric phenol method described by Dubois et al. [38], using glucose as a standard. Total phenolic contents were estimated using Folin-Ciocalteu and gallic acid reagents as standards [39]. The total antioxidant capacity was determined by the reaction of antioxidants in the sample with a definite amount of exogenously provided hydrogen peroxide as performed by Koracevic et al. [40].

Statistical analysis
All results were exposed to the analysis of variance (ANOVA) [41]. Significance of the variable was investigated using Duncan's multiple range tests (P ≤ 0.05). All analyses were performed using SPSS 16 software.

Seeds germination under the influence of Spirulina liquid extract (SLE)
During the experiment, the germination percentage of T. aestivum was significantly increased when treated with SLE, compared with the corresponding control. Results presented in Table 1 indicate that germination indices (GP, GI, RSG, SVI, and RRE) of T. aestivum were increased when soaking in 2% SLE compared to control (without algal extract). The germination percentage was 97.77% relative to control 88.88%, GI (2.09), RSG (110.07), RRE (113.85), and SVI (378.48). The increased seed germination percentage may be due to the existence of growth-promoting substances like gibberellins in cyanobacteria [42]. The growth stimulations of pepper using Spirulina extract may be related to nutrient contents of Spirulina, which are simply absorbed by plants [43]. The highest germination of chickpea and soybean was obtained when treated with Spirulina [44]. The highest growth of Indian mustard was obtained when fertilized with 3 g‧L -1 Spirulina suspension [45]. The growth of cowpea was enhanced using Spirulina suspension as a biofertilizer [46]. Crouch and Van Staden [46] detected that Spirulina liquid extracts enhanced the nutrient uptake by roots with amended water and nutrient efficiency. Kumari et al. [47] reported that S. platensis can promote root growth and plant development. S. platensis extracts are an important source of plant nutrition in sustainable agricultural production as they contain vitamins, various trace elements, and amino acids that are beneficial to the plant growth and development [48]. The promotion of maize plants was obtained with applied red algae as biofertilizers [49]. Seaweed algae Ulva lactuca and Sargassum vulgare are used to biocontrol nematode and promote the growth banana plants [50]. Selvam and Sivakumar [51] reported that seaweed extract of Ulva reticulata increased the crop productivity of Arachis hypogea by spraying 2% concentration. In particular, the growth of V. mungo plant when treated with seaweed extracts like Caulerpa scalpelliformis [52] and U. reticulate [53] increased.

Enzyme activities
Concerning the start of germination, the cotyledon reserves (polysaccharides and proteins) are degraded under the action of amylases and proteases that yield respiratory substrates. Generally, amylase and protease activities demonstrate significant positive responses under the SLE treatments relative to control (Figures 1 and 2  increased the amylase and protease activities of leaves of T. aestivum. Sivasankari et al. [53] reported that Sargassum wightii and Caulerpa chemnitzia promoted the α-amylase and β-amylase activities of Vigna sinensis. The increase in amylase and protease activities at the concentration (2% SLE) may be due to the existence of growth-promoting substances like gibberellins. During seed germination, gibberellic acid encourages the synthesis and secretion of amylase, which decomposes starch into monosaccharides in the endosperm, and this enzyme is mainly responsible for the hydrolysis of starch in the endosperm [54].

Effect of different concentrations of
Spirulina liquid extract (SLE) on growth parameters, and pigments contents of T. aestivum

Growth parameters
Compared to the control, the shoot height and root length of T. aestivum were significantly increased at all concentrations 1%, 2%, and 4% (ST1, ST2, and ST3, respectively) of the SLE treatment ( Figure 3). ST2 induced the maximum increases in shoot height, root length, and leaf area of 25.72 cm, 4.81 cm, and 9.29 cm 2 of T. aestivum, respectively. In the case of seed treatment with SLE, all morphological parameters were amplified significantly in the following descending order ST2 > ST3 > ST1 > control. The lowest promoting effect was recorded in control giving the following increments 21.23 cm, 3.23 cm, and 5.32 cm 2 for shoot height, root length, and leaf area, respectively, of T. aestivum. The best concentration of SLE was 2%, which enhanced all growth characters in comparison with other treatments and control. The application of Spirulina liquid extract on T. aestivum increased the seed germination rate at a lower concentration, while the superior concentrations of these extracts decreased the rate of germination. The lower concentrations of Sargassum polycystum extract 1% promoted seed germination and the growth rate of Cajanus cajan [55]. S. platensis extract promoted seed germination and the plant growth of wheat and barley plants [56]. The application of S. platensis and antitranspirant weekly on chili pepper improved the marketable product by 2.1% [57]. Seaweed concentrate prepared from Ecklonia maxima was found to increase the root growth of tomato at 0.4% concentration [46]. Table 2 presents the results of pigment contents of T. aestivum L. that were treated with different concentrations (1%, 2%, and 4%) of SLE. The same trend appears in the case of growth. All measured pigments were increased with concentrations of 1% followed by 2% SLE. Meanwhile, 4% SLE reduced the pigment contents of T. aestivum L. compared with 2% SLE, but the levels of pigment contents of T. aestivum L. treated with 4% SLE were still in the upper  levels than 1% SLE and control. The lower concentration of the Spirulina liquid extracts also promoted the total chlorophyll and carotenoid contents of T. aestivum seeds up to 2% concentration of SLEs when compared to the control. Enhanced chlorophyll, carotenoid, and fucoxanthin contents at lower concentrations of SLEs have been reported for Vigna catajung [58], Dolichos biflorus [59], Vigna sinensis [53], Cajanus cajan [60], Brassica nigra, Abelmoscus esculentus, and Cyamopsis tetragonoloba [61].

Effect of Spirulina liquid extract (SLE) on
the T. aestivum L. was grown under salinity 3.4

.1 Growth parameters
The results presented in Table 3 demonstrate that the maximum dry weight value of T. aestivum was recorded under the SLE treatment and tap water and was recorded at 1.45 g/seedling after 20 days of treatments. The highest promotion effect on growth parameters of T. aestivum was evident in treatment with 2% SLE consortium with 10% SW supplementation, given the following enhancement of 28.31 cm, 5.52 cm, and 8.20 cm 2 for shoot height, root length, and leaf area, respectively. The results demonstrated that when T. aestivum was treated with 2% SLE with 10% or 25% seawater, all growth parameters were increased when compared with seawater without SLE. The significant results were obtained with different treatments on shoot length, root length, total fresh weight, total dry weight, and leaf area and leaf numbers, but in the case of leaf width and leaf length, there are no significant effects on seeds treated with 10% SLE and SLE besides SLE. All morphological criteria increased in the following descending order: SLEs-T.W > Control > SLE-10% S.W > 10% S.W > SLE-25% S.W > 25% S.W. S. platensis (100 mg‧L −1 ) enhanced the growth of V. faba grown under salt-stressed environment, which proved that S. platensis was more beneficial against salinity stress [25]. Spirulina is considered to be good dietary addition and one of the best solutions for treating malnourishment problems in developing countries [62].

Total carbohydrate contents
The total carbohydrate content of T. aestivum seedlings decreased significantly under the influence of increased salinity, but SLE triggered an interaction with SW (seawater) supplementation that resulted in the accumulation of carbohydrates, compared to water-treated seeds and seedlings under salt stress. The highest total carbohydrate accumulation was recorded for SLE conjugated with tap water to trigger a consortium T. aestivum 77.04 mg‧g FW −1 (Figure 4). After treatment with S. platensis extract, the soluble carbohydrate content increased. Sridhar and Rengasamy [63] reported that the induction of S. platensis was associated with an increase in total soluble carbohydrates in T. aestivum and L. termis. Therefore, the increase in carbohydrates in the current results may be due to the effect of the extract of S. platensis on photosynthetic efficiency, which may be related to the cytokinins, auxins, macro, and micronutrients in the extract of cyanobacteria [64]. The carbohydrate and protein content of the seeds increases when plants are treated with a higher seaweed dosage and more than 2 g of chemical fertilizer. At higher doses, the carbohydrate and the protein content of algae plus chemical fertilizers will decrease [65]. The carbohydrate content of Vigna radiata L. was promoted at 2.0% concentration Sargassum wightii grev [66]. The treatments of different crops with Spirulina extract were of great importance due to higher levels of organic matter, microelements, vitamins, and fatty acids and also amusing in growth regulators such as auxin, cytokinins, and gibberellins [46].

Protein contents
Compared with seedlings without hydroprime stress, a significant decrease in the protein contents of the studied The different letters within a column are significant (P ≤ 0.05) according to Duncan's multiple range tests ± standard error of means.
plant was observed under salinity treatments (10% and 25% SW). The conjugation effect of seawater and SLE preparations leads to a different increase in protein storage with seawater supplementation. The results obtained in Figure 5 demonstrated that the 2% SLE promoted the protein contents of T. aestivum more than the protein contents in control. The increase in protein content of T. aestivum exits after treatment with S. platensis extract, which indicates that S. platensis extract may contain substances that cause this increase. According to reports, S. platensis extract contains amino acids and zinc [25]. The highest protein, total carbohydrate contents, phenols, and antioxidant of T. aestivum were observed at a concentration of SLE of S. platensis (2%). There was such an increase in protein, amino acid, and total carbohydrate contents. Foliar applied S. platensis reduced adverse effects of salinity by improving the total protein level, N, P, K, and photosynthetic activity of V. faba L. [25].

Phenol contents
There was a remarkable induction of significant increments in total phenol contents of T. aestivum treated with SLE, the phenol content of T. aestivum irrigated with 10% and 25% seawater was reduced, but when treated with SLE, the phenol contents were increased but were still less than the control value ( Figure 6).

Antioxidant activities
There was a remarkable initiation of significant increases in total antioxidant and phenol contents relative to the control of T. aestivum treated with SLE. Results in Figure 7 denote the maximum value of total antioxidant contents in T. aestivum that was treated with SLE and irrigated with tap water (0.75 mg‧g FW −1 ). Results demonstrate the treatments T. aestivum by seawater concentrations reduced the antioxidant contents, but the T. aestivum that seeds were treated in 2% SLE, showed more growth enhancement than those treated with seawater alone. The total content of phenol and antioxidants can be attributed to the greater availability and absorption of essential elements (Ca, Na, K, Mg, N, and Zn) in seaweed extracts [60]. Salinity is one of the main environmental factors that limit the plant growth and productivity [47]. Salinity is a physiological parameter to investigate the capability of organisms to survive in their environment. The cyanobacterial retort to salinity includes many physiological and biochemical processes such as nucleic acid synthesis, carbohydrates and protein metabolism, photosynthesis, and respiration [50]. The increase in salinity initiated a decrease in the growth [49]. Many previous studies have reported that high salt agronomy reduces chlorophyll and protein content [12]. S. platensis may be binding to many elements, such as potassium and calcium, and keeping in natural levels ratio and hence the cells remain alive [67]. Spirulina maxima and Chlorella ellipsoida water extracts improved wheat tolerance to salinity and enhanced the antioxidant capacity of the whole grains [68]. Scenedesmus obliquus and S. platensis increased the activities of SOD, APX, CAT, POD, chlorophyll, and carotenoid content of T. aestivum grown under salt stress [69]. Ulva lactuca t induced plant growth and yield and antioxidant enzyme activity (SOD, CAT, APX, and GR) of T. aestivum grown under salt stress [70]. Ulva rigida extract promoted leaf pigments, total phenolics, and antioxidant enzymatic activities of Triticum durum grown with salinity [71].

Conclusion
Spirulina liquid extracts successfully alleviated the harmful effects of salt stress on tested plants by significantly reducing the inhibition of the affected parameters, total protein, total carbohydrate accumulation, total phenol contents, and antioxidant activity. In this study, T. aestivum treated with S. platensis liquid extract (SLE) (biological fertilizer) significantly increased branch length, root length, number of leaves, fresh weight, and weight dried. Seedlings that received the application of biofertilizer for 20 days resulted in the maximum plant growth, which could theoretically mean higher growth parameters.