Effects of Microalga (Chlorella vulgaris Beijerinck) on Seconder Metabolites and Antioxidative Defense System Improve Plant Growth and Salt Tolerance in Guar [Cyamopsis tetragonoloba (L.) Taub.]

In nature, abiotic stressors, more commonly than biotic stressors, commonly result in a reduction of crop yield and limit potential production by up to 70% (Yamauchi, 2018). Salinity in growth media, which could presumably be caused by the existence of salt in the soil, causes an unfavorable impact on plant growth and development and thus reduces the plant’s water uptake (low osmotic potential).
       
This, in turn, results in high levels of salt being absorbed into the plant via transpiration, which causes damage to transpiring leaf cells (specific ion toxicity) (Parihar et al., 2015). The result of this is excessive reactive oxygen species (ROS) accumulation, which may result in lipid peroxidation, protein oxidation, enzyme inactivation, DNA damage and/or interaction with other essential plant cell components. In order to minimize the toxic effects caused by ROS, plants possess various kinds of enzymatic and non enzymatic antioxidative systems (Kusvuran et al., 2013).
       
Microalga are the world’s largest oxygen-producing organisms and are crucial for planetary functions and sustainability of the ecosystem. Following the application of microalga products, plants have been reported to have exhibited different responses, such as robust growth, increased yield, improved nutrient uptake, increased biotic and abiotic stress resistance (fungal infections, pest attacks and frost), increased quality and fruit with a longer shelf life (Plaza et al., 2018; Singh et al., 2018a; Righini et al., 2018). Algae are comprised of active compounds, like organic and free amino acids, enzymes and phytohormones, as well as bioactive secondary metabolites, vitamins, vitamin precursors (Plaza et al., 2018), essential nutrients and plant growth regulators like auxins and cytokinins (Righini et al., 2018).
       
Guar has gained significant commercial status as a result of its gum and is now the most highly exported product in the farming sector. It a highly-valued cash crop in both arid and semiarid regions because of its drought tolerance and vast array of uses (Pathak, 2015).
       
The objective of this study was to investigate the effect of Chlorella vulgaris on the growth and biochemical and physiological responses of guar seedlings under salt stress. In addition, it was aimed to determine the relationship between the application of microalga and improved salt tolerance.

The guar seedlings were grown in 12-L plastic pots containing a 2:1 ratio of peat to perlite medium and housed in a greenhouse that was maintained at day/night temperatures of 26 ± 2°C and 18 ± 2°C and a relative humidity of 65% ± 5. Growth chamber and nutrient solution irrigation (following that of Kusvuran and Dasgan, 2017) was used to grow the plants. The experiment was performed with three replications. Each pot contained five plants and each of the three replications also included five pots. At 39 days after sowing, 100 mM sodium chloride (NaCl) was used to induce saline stress. The amount of water applied during the experiment was calculated according to the ratio of drained water to applied water (Schröder and Lieth, 2002). For the untreated control plants, this was around 30%. In this study, the treated (experimental) and untreated (control) plants were both grown for 17 days. For the experiment, three groups were formed: C group (control): nutrient solution irrigation void of  NaCl, S group (salt application): nutrient solution irrigation containing 100 mM NaCl and S+MA group (salt application + microalga) (5%): nutrient solution irrigation containing 100 mM NaCl ⁺ foliar microalga application.
       
For the microalga, Chlorella vulgaris, in the form of a liquid organic commercial product, was used in this experiment (number of viable algae 2 × 10⁷ alg mL^–1; pH: 7; density: 1; vitamins: A, B1, B2, C, E and biotin and amino acids: arginine, cystin, histidine, leucine, lysine, methionine, phenylanine, tryptophan and valine content). The microalga solution was sprayed on the plant foliage and allowed to run off every there days (a total of six times).
       
The total phenolic content was determined using a Folin-Ciocalteu reagent. The phenolic content of the leaves was expressed in milligrams. Gallic acid was used as a standard (Singleton et al., 1999). The colorimetric assay was used to establish the flavonoid content (Medina-Juárez et al., 2012). The entire enzyme analyzes were carried out according to the method of Karanlik (2001). Determination of the CAT, APX and GR activities were performed according to the method of Cakmak and Marschner (1992). The amount of malondialdehyde (MDA) ascertained via the thiobarbituric acid reaction was used to measure the lipid peroxidation (Heath and Packer, 1968).
       
The experimental plot design was randomized, comprising three replications. A comparison of the parameter mean values was performed via the least significant difference test. Statistical significance was determined as P < 0.05 using JMP statistical software, v.5.1 (SAS Institute Inc., USA) (Anonymous, 2002). The data are presented as the mean ± standard deviation and error bars represent the standard errors of the means in all of the figures.

Growth characteristics
 
In plants treated with NaCl, the shoot fresh and dry weights, length and diameter; steam and leaf number and leaf area were reduced by 62%, 53%, 38%, 11%, 26%, 27% and 81% when compared to control group, respectively (Table 1). However, in the S+MA group, significantly enhanced growth components, such as the shoot fresh and dry weights, diameter and length; number of leaves per plant and leaf area per plant were observed when compared to plants under salt stress alone. These increases ranged between 11% and 81%. When the S+MA group was compared to the S group, S+MA enhanced growth amelioration by 10%-66%. In this study, the application of microalga successfully limited the effects of 100 mM NaCl on guar seedling growth and development. The favorable effect of microalgae might be the result of its success in providing plants with necessary nutrients and phytohormones. The use of Chlorella vulgaris with NaCl could have enhanced the nutrient uptake of the plants, which means it may have aided amelioration of the growth parameters. Foliar sprays provide more rapid nutrient utilization and enable faster correction of nutrient deficiencies when compared to soil fertilizer. Generally, the results have indicated a positive correlation between foliar extract applications and greater plant growth (Garcia-Gonzalez and Sommerfeld, 2016). Thus, when compared to the C group, the above mentioned growth parameters decreased by 11%-81%; however, in the S+MA group, these parameters were decreased by 10%-66%. In fact, this hypothesis was previously proven in wheat, maize, bean and lettuce (Hajnal-Jafari et al., 2016). In addition to this, the growth medium and cellular extracts of some species of microalgae have been reported as containing phytohormones, such as abscisic acid, cytokinins, auxins, salicylic acid and gibberellins, all of which play a significant role in the development of plants. With regards to other growth parameters, such as the stem and leaf number and leaf area, they were observed to have decreased in the S group by 26.5%, 27.1% and 81.2%, respectively. In the S+MA group, the stem and leaf number and leaf area increasing by 6.1%, 5.4% and 68%, respectively. However, when compared to the S group, an improvement of 27%-66% was observed. This may have been the result of increased access to the nutrients responsible for augmenting protein synthesis, leading to an increased accumulation of carbohydrates, as a result of the application of Chlorella vulgaris (Dineshkumar et al., 2018).
 

 
Photosynthetic pigments
 
Growth inhibition in plants is the result of decreased chlorophyll content, possibly due to ROS-induced chlorosis, photo-reduction and triplet chl formation, which causes serious damage to photosystems I and II and the formation of chlorophyll in plants (Singh et al., 2018b). In this study, the favorable effects of the microalgae were identified on the chlorophyll components and were increased by 16%-32% when compared to the S group (Table 2). The other component, the carotenoid content, was increased the S+MA group by 68.42%. Carotenoids play a specifically critical part in light harvesting and oxidative damage protection through the deactivation of singlet oxygen, satisfactorily meeting the chlorophyll excited triplet state, as well as the enhancement of carotenoid synthesis as a way of protecting itself from photo damage caused by cell division arresting when under saline stress (Singh et al., 2018a). Dineshkumar et al., (2018) reported that increased chlorophyll accumulation in organic fertilizers, such as microalga, even at a decreased rate, could be the result of the cooperative effects of consortium, which better facilitates plant N, P and K uptake, resulting in increased chlorophyll accumulation.
 

 
Malondialdehyde (MDA) content
 
Salt-induced oxidative stress was confirmed via an intercellular evaluation of the MDA levels (Table 2). A significant increase was observed in the MDA content in the S group, by 6.85 μmol g^-1 fresh weight, when compared to the C group, where it was lowest. However, the effects of salt stress were mitigated by the microalga in the S+MA group, which further decreased the MDA content by 49.63%. Salt stress results in the formation of free radicals, which cause irreversible lipid and protein damage. MDA, which plays the role of a cellular toxicity bioindicator, is a well-known oxidation that is caused by lipid peroxidation during oxidative stress (Singh et al., 2018b). Cell membrane integrity is destroyed by lipid peroxidation, eventually resulting in cellular death (Dolatabadian et al., 2008). In this study, the lipid peroxidation of the guar plants increased with salt stress. The results showed that the application of microalga reduced the MDA levels by 49%, presenting a favorable effect in reducing the oxidative stress resulting from salt stress.
 
Total phenolic and total flavonoid contents
 
Under salt stress, the total phenolic and flavonoid contents decreased in the S (8.15%) and S+MA (64.32%) groups when compared to the C group (Table 2). On the other hand, the application of microalga in the S+MA group resulted in significant increases in the mean total phenolic (60.70%) and flavonoid (174.80%) contents when compared with the S group. During extreme environmental stress conditions, microalgae multiply and a variety of secondary metabolites are synthesized and produced, which is assumed to be an endeavor by microorganisms at retaining their rate of growth or increasing their likelihood of survival (Markou and Nerantzis, 2013). As was clearly seen, the results showed that microalga caused a stimulatory effect on the phenolic accumulation in guar.
 
Ion contents (Na⁺, K⁺, Ca²⁺ and Cl^–)
 
When compared to the C group, the Cl^– and Na⁺ and contents were seen to have increased as a result of saline stress in the S group by 892% Cl^– and 296.9% in Na⁺, respectively (Table 3). However, with the application of microalga in the S+MA group, decreased levels of Na⁺ and Cl^– were observed, by 37% and 41%, respectively, when compared to the S group. On the contrary, Na⁺, Cl^–, K⁺ and Ca²⁺ ion accumulation was observed to have decreased in the S group. Foliar microalga application reduced the effects of stress on these parameters, while significantly increasing the Ca²⁺ and K⁺ contents when compared to the S group. Microalga application ensured an increase in the K⁺ content by 30.6% and the Ca²⁺ content by 36.1%. Ion toxicity in plant cells is the result of salt stress caused by a significant Na⁺ and Cl^– cell influx, as well as the reality that the majority of plants amass a high concentration of Cl^- and Na⁺ ion in their shoots when cultivated under salt stress, which is a significant cause of decreased growth (Parihar et al., 2015). The uptake of K⁺ and Ca²⁺ was reduced by Na⁺ in guar genotypes under salinity conditions. A decreased K⁺ content is a response commonly observed under salt stress because it directly competes for binding sites that are charge-dependent with Na⁺ (Chen et al., 2007). The results herein showed that the application of microalga limited toxic ion accumulation, thus enabling increased K⁺ and Ca²⁺ accumulation, by 30% and 36%, respectively. The microalga may have potentially prevented the loss of nutrients as the result of N, P and K being released slowly, as an organic fertilizer, based on the plant’s needs. Plaza et al., (2018) found that a foliar application of scenedesmus hydrolysates was also seen to have increased the leaf and shoot number and improve foliar Ca, Mg, K and P levels.
 

 
Antioxidative enzyme activities
 
Levels of antioxidative enzyme activity, including APX, CAT, GR and SOD, were evaluated in the C, S and S+MA groups (Fig 1). It is evident from Fig 1 that the application of microalga had a serious effect on the SOD, CAT, GR and APX activities of the treated guar plants. In the S+MA group, these enzyme activities increased by 113.58%, 256.45%, 63.57% and 55.98%, respectively, when compared with the C group. When examined in the S group, the increases were determined as 29.59%, 113.18%, 30.35% and 57.55%, respectively. A direct consequence of salinity in plants is the induction of stress antioxidant enzymes to minimize the damage caused by reactive oxygen species (Amar and Nourredine, 2016). Singh et al., (2018a) reported that plants possess defenses against oxidative damage that include physiological and biochemical status changes, using plant growth-promoting rhizobacteria to facilitate protection against losses due to pathogens or abiotic stress and improved plant tolerance against abiotic stress, as a result of physical and chemical changes. This is an approach that is rather new and overlaps a great deal with the process of systemically-induced resistance in plants.
 

The application of microalga under salt stress appeared to be favorable to growth and development, in addition to biochemical and physiological processes in guar. These effects were observed clearly in the salt-sensitive genotypes and the integrative microalgae application supported salt.