E ﬀ ect of the Foliar Application of Microalgae Hydrolysate ( Arthrospira platensis ) and Silicon on the Growth of Pelargonium hortorum L.H. Bailey under Salinity Conditions

: Plant growth is limited by salinity stress. There are few strategies for alleviating it although Arthrospira platensis and silicon can stimulate plants to grow under stress conditions. The aim of this work was to study the e ﬀ ects of both a single and a joint application of Arthrospira platensis and silicon on the growth of Pelargonium hortorum L.H. Bailey under salt stress conditions. Plants were exposed to 2.0, 3.0, and 3.5 dS m − 1 EC (electrical conductivity), with and without the application of microalgae and silicon. At the end of the trial, the biometric parameters and the plant analysis were determined. The microalgae hydrolysate concentration was 5 g L − 1 and the silicon concentration was 150 mg L − 1 . Foliar spraying was applied weekly. Pelargonium can be grown in moderately saline irrigation water (3.0 dS m − 1 ). This bedding plant mitigates salt stress by avoiding the uptake of Cl − ions and by tolerating a high Na + concentration in the tissue. The joint foliar application of Arthrospira microalgae and silicon stimulates root, shoot, leaf, and ﬂower formation in the Pelargonium hortorum L.H. Bailey crop under salinity conditions (3.5 dS m − 1 ).


Introduction
There is a growing need to develop strategies aimed at reducing, or replacing, the use of chemicals with natural or biological substances, such as plant biostimulants [1]. In modern agriculture, plant biostimulants have attracted considerable interest as a way of enhancing crop performance, resilience to environmental stress, and efficient nutrient use [2]. The development of innovative technologies based on bioresources, which includes plant biostimulants, have proven to be effective methods at improving crop performance [3]. Plant biostimulants contain substances and/or microorganisms whose function, when applied to the plants or the rhizosphere, is to stimulate natural processes that enhance/benefit nutrient uptake, nutrient efficiency, tolerance to abiotic stress, and crop quality [4].
Microalgae, which comprise eukaryotic and prokaryotic cyanobacteria (blue-green algae), are of great interest to scientists, extension specialists, plant growers, and private industry because of their versatile nature; this includes their simple unicellular structure, high photosynthetic efficiency, potential for heterotrophic growth and valuable co-product yield, adaptability to domestic and industrial wastewater, and amenability to metabolic engineering [2]. Microalgae are classified mainly

Greenhouse Trial
The trials were carried out in a 170 m 2 greenhouse with zenith ventilation, and relative humidity and temperature control. The temperature and relative humidity were recorded every 15 min with a HOBO U12-013 data logger (Onset Computer Corporation, Bourne, MA, USA) placed at canopy height in the central part of the cultivation table where the plants are grown. External radiation was measured every 15 min with a Q20-B sensor (DGT-Volmatic, Barcelona, Spain). To estimate the internal radiation, the cover transmission coefficient was estimated as the ratio between the internal and external radiation using a manual quantum photoradiometer (Delta OHM, model RAD/PAR (Dicsa, Almeria, Spain)). The average temperature, humidity, and global radiation in the greenhouse were 17.5 • C, 68.6%, and 1.40 MJ m 2 day −1 , respectively; the maximum and minimum averages of the temperature and humidity were 24.6 and 12.4 • C, and 79.6% and 52.9%, respectively.
The species studied was Pelargonium hortorum L.H. Bailey. Pelargonium hortorum was chosen as a model plant because it has a rapid crop cycle and is easily grown in a greenhouse. Nonetheless, these results can be applied to other horticultural crops. Plant cuttings were transplanted into 2.5 L pots (one plant per pot). The substrate used was a mixture of peat and perlite 80:20 (v/v). The white peat substrate (Brill WPS medium) is a potting substrate with a high air capacity (41%), a pH of 5.7 and a 1.0 g L −1 salt content. Projar expanded perlite has a total porosity of 92-98% and a 1-5 mm fraction size.
Fertigation was applied manually until the leachate fraction reached 20%. The average dose for the Pelargonium hortorum was 85 mL per plant once every 2 days. For the C2, C3, and C3.5 treatments, leachates from three plants were collected in a tray with plastic saucers and covered with black film. The amounts of leached water, the EC, and the pH were determined every 3 days using a test tube, an EC-meter, and a pH-meter (Dicsa, Almería, Spain), respectively. Water uptake was estimated as the difference between the water applied and the water leached.

Treatments
Twelve treatments were performed, comprising single and joint foliar application with Arthrospira platensis and silicon, along with fertigation at EC 2.0, 3.0, and 3.5 dS m −1 (Figure 1).  nitric acid, calcium nitrate, potassium nitrate, ammonium nitrate, and potassium sulphate were used to prepare the nutrient solutions. The pH and EC of the standard nutrient solution was 6 and 2 dS m −1 , respectively. After the nutrient solution was prepared, NaCl (491 and 982 mg L −1 ) was added to obtain EC 3.0 and 3.5 dS m −1 , respectively. The potassium silicate was sprayed at a concentration of 150 mg L −1 . The microalgal hydrolysate concentration was 5 g L −1 . The density of algal cells used was 100 g L −1 . Foliar spraying was applied weekly. Arthrospira platensis was supplied by the Biorizon company (Almeria, Spain). The microalgae hydrolysate was produced using Arnon medium, with the addition of 16 g L −1 of bicarbonate, in a 100 m 2 raceway reactor. The cultures were performed controlling the pH (pH = 10) by CO 2 injection. The oxygen concentration was controlled and remained below 250% saturation. The biomass was harvested continuously at a dilution rate of 0.2 1/day.

Nutrient Solution Analysis
The parameters determined in the nutrient solutions tested were pH, EC, NO 3 − , SO 4

Biometric Parameters
Growth parameters and ornamental quality are particularly important for ornamental species since they can determine the plant price. Salinity can affect biomass production, leaf area, plant height, and water status. Therefore, the fresh weights (FW) and DW, the internode length, the leaf length and width, and the number of leaves and shoots were recorded at the end of the cultivation. The number of leaves and shoots per plant were counted. The internode length, and the leaf length and width were measured using a ruler. The number of flowers per plant was counted each week during the crop cycle. After removing the substrate, the plant material (comprising 12 plants per treatment) was separated into different vegetal fractions: absorption organs (roots), conductive organs (stems and petioles), photosynthetic organs (leaves), and flowers, which were weighed separately on a COBOS G M5-1000 scale (with a precision of 0.005 g) to determine the FW. All the samples were washed and dried in a Nüve EFN500 oven (with a 30 to 300 • C range) at 60 • C for 48 h to determine the DW. The total DW was calculated as the sum of the roots, stems and petioles, leaves, and flowers. The FW and DW totals were used to calculate the water content as (FW − DW) × 100/FW. The leaf area was estimated by a non-destructive method using the formula S = a + b LW, where S is the foliar surface area, L is the leaf length (cm), W is the leaf width, and the a and b coefficients are specific to each species. For Pelargonium, a is 0.07 cm 2 and b is 0.68 cm 2 . Leaf color was identified according to the Munsell chart for leaves. The Munsell color is based on a three-dimensional model in which each color is composed of three attributes: hue (the color itself), value (lightness/darkness), and chroma (color saturation or brilliance). The color of the leaves was visually matched [17].

Mineral Analysis
A subsample of the dry matter (0.2 g of DW) from the most significant treatments (C2, C3, C3.5, ASi2, ASi3, and ASi3.5) was ground up in a Wiley mill and digested (in 96% H 2 SO 4 ) in the presence of hydrogen peroxide (H 2 O 2 , 30%(w/v)) at 300 • C in a sand bath to analyze the organic N, P, K, Ca, and Mg. Each treatment was replicated four times. The total Na and K were directly measured by flame spectrophotometry using an Evans Electro Selenium LTB Flame Photometer (Halstead, Essex, England). The total Ca and Mg were analyzed by atomic-absorption spectrophotometry using a Perkin Elmer Atomic Absorption Spectrometer 3300 (Dicsa, Almeria, Spain). P was analyzed using the method proposed by [18], and nitrogen was analyzed using the method proposed by [19].

Experimental Design and Statistical Analysis
The experimental design comprised a completely randomized block with 12 treatments. There were four replicates per treatment and three plants (pots) per replicate (12 plants per treatment). The effect significance of the treatments (salinity, and the microalgae and silicon application) was examined using the standard analysis of variance (one-way ANOVA) and Fisher's least significant difference (LSD) test, performed using Statgraphics Centurion XVI.II software (Statpoint Technologies, Inc., Warrenton, VA, USA). Differences were considered significant at p < 0.05.

Salinity Effect
No differences in stems (SDW), leaves (LDW), flowers (FDW), or total dry weight (TDW) were found between the salinity treatments (Table 1). However, Pelargonium underwent a negative root growth response when the salinity level rose from 2 to 3.5 dS m −1 . The treatments with an EC of 3.0 and 3.5 dS m −1 resulted in a reduction of more than 20% compared to an EC of 2.0 dS m −1 . The number of shoots and flowers, the width and length of the leaves, and the internode length showed no significant differences (Table 2, Figure 2). However, the leaf area and leaf number improved in the treatment with an EC of 3.0 dS m −1 . The Na concentration in the leaves was high in all the treatments [10], even in the C2 treatment where sodium was not added. This is due to the relatively high sodium concentration present in irrigation water. The Na levels were significantly higher in the leaves of plants that were given the salinity treatments (Table 3) although no differences were found between the C2.0 and C3.0 treatments; the similar concentration in the leaves was probably due to the effect of reduced root DW. The concentrations of N, P, S Cl, Ca, and Mg were not affected by the salinity treatments. The K concentration in the leaves decreased as the salinity increased, reducing by 20% in treatment C3.5 compared to C2. The evaluation of leached solution over the cultivation period is shown in Figure 3. The leachate pH did not present significant differences between the treatments (6.6-7.4). Nonetheless, the salinity treatment did affect the leachate EC, indicating that salt accumulation varied with the treatment [20] 59 days after transplanting, the leachate EC was 3.47, 5.10, and 6.06 dS m −1 for C2, C3, and C3.5, respectively. In all the treatments, the leachate EC increased during the assay, but this increase was greater in treatments C3 (50%) and C3.5 (60%) than in the C2 treatment (30%). However, no difference in the plant water content was found in treatments C3 and C3.5 compared to C2; the water content was 90.9%, 91.1%, and 91.0%, respectively. Differences in the water uptake were not found until 55 days after transplanting. At this point, plant water uptake reduced in the NaCl treatments. The water uptake was 17% and 12% lower in C3 and C3.5, respectively, than in C2 (58 days after transplanting) ( Figure 4).

Silicon and Microalgae Application
The combined application of microalgae and Si improved the LDW, RDW, IDW, and TDW in the treatment with an EC of 3.5 dS m −1 whereas it improved the IDW in the treatment with an EC of 3.0 dS m −1 (Figures 5-7). However, no effect was observed on the RDW, IDW and between the treatments with an EC of 2.0 dS m −1 . The Si treatments improved LDW compared with the control in the treatments with an EC of 2.0 and 3.5 dS m −1 . A single application of microalgae improved the IDW in treatments with an EC of 3 dS m −1 although a reduction in TDW was observed in treatments A2 and A3.5. In treatments A2 and Si2, the number of shoots increased with an EC of 2 compared to the control, as it also did in treatments Si3.5 and ASi3.5 with an EC of 3.5 dS m −1 (Figure 8a). The leaf number increased compared to the control (by 22% and 28%) as did the flower number (by 24% and 51%) in treatments Si3.5 and ASi3.5, respectively (Figures 8b and 9a). Furthermore, applying Arthrospira platensis and silicon increased the flower number in the treatments with an EC of 3.0 and 3.5 dS m −1 . No differences were found in the internode length or the leaf area (Figures 9b and 10). The Munsell chart was used to define color for all the treatments at the end of the assay. No differences were found between treatments; the leaves were identified as 5GY 4/4 for color, value, and chroma, respectively. The Cl and Na concentrations increased by 15% in treatment ASi3.5 compared to C3.5, as did the P and Ca concentrations in treatment ASi3 compared to C3 ( Figure 11). The Si levels were significantly higher in plant leaves that had been treated with the Arthrospira and silicon foliar application. In contrast, the nitrogen, magnesium, and sulfur contents were not affected by the microalgae and silicon application. Table 1. DW (g) of S, R, L, I and T at the end of the trial for the C2 (fertigation EC 2.0 dS m −1 ), C3.0 (fertigation EC 3.0 dS m −1 ), and C3.5 (fertigation EC 3.5 dS m −1 ) treatments. Different letters indicate significant differences between treatments at the p < 0.05 level using the LSD test. Values are the means of 12 plants.  Table 3

Discussion
Salinity can reduce growth in plants; this growth reduction may result from the effects of salt on physiological processes, biochemical relationships, or a combination of such factors [21]. However, salinity affects the biomass and biochemical parameters differently, depending on the species [22].
The results from this assay indicate that salinity caused a reduction in root growth. The root/shoot ratio for treatments C2, C3, and C3.5 were 0.065, 0.058, and 0.058 g g −1 , respectively. Root growth appears to be limited by increasing Na concentrations in the growth medium [10]. However, the C3 treatment, which had an EC of 3 dS m −1 , improved the leaf area and leaf number compared to C2 and C3.5. A number of herbaceous perennials, groundcover plants, floriculture crops, and landscape woody shrubs became more compact yet showed little visual damage when irrigated at low to moderate salinity compared to the non-saline control [20]. Pelargonium may be considered a plant that is moderately tolerant to salinity [21].
Water uptake was affected 56 days after transplantation in treatments that had an EC of 3.0 and 3.5 dS m −1 . Furthermore, no differences in water content were found. In spite of the high salt concentration in the soil causing a decrease in the water uptake, salt stress did not cause changes in the water status of the pelargonium leaves, which shows that the plants did not suffer from water deficit stress [23]. There are three salt tolerance mechanisms in plants: osmotic stress tolerance, tissue tolerance to accumulated Na and/or Cl ions, and minimizing the entry of Na and/or Cl ions [24]. Both the proline and anthocyanin contents increased significantly in the pelargonium leaves growing in peat substrate with the two highest NaCl doses [23]. These compounds may function as osmotic adjusters, and as scavengers of oxygen radicals protecting cytoplasmic structures and chloroplasts from the adverse effects of toxic ions [23]. According to our results, the ability to maintain turgor in the plant probably also resulted from Na + accumulation in the vacuoles after Na + transport from the root to the aerial parts; this is an important mechanism in salinity tolerance [23]. Soil salinity can affect nutrient acquisition by severely reducing root growth [25] and causing deficiencies or imbalances in the plants, which depend on the ionic composition of the external solution [25]. In accordance with our results, the concentration of potassium ions in the leaves was found to change significantly with increased NaCl concentration in the growing medium [23]. Moreover, the leaf concentrations of P, N, Ca, Mg, and S did not present any differences. Some species tolerate salt stress by avoiding the uptake of certain ions or by tolerating a high ion concentration in the tissue [20]. A plant's ability to tolerate excessive amounts of Na + varies widely between species [25]. Pelargonium seems to tolerate high Na concentrations in the tissue. Despite observing an increased Cl concentration in the external solution, no statistical difference in the Cl concentration of the leaves was found. One of the most important traits associated with tolerance is the ability to restrict the entry of saline ions through the roots [26]; however, a good Cl − ion excluder is not necessarily a good Na + excluder, and vice versa [22]. Pelargonium hortorum L.H. Bailey seems to be a good Cl − ion excluder.
The results obtained by [11] found that silicon could be used as a fertilizer additive to improve the growth and quality of silicon-responsive ornamental plants. In our assay, LDW and leaf area were positively affected by the single application of silicon in treatments with an EC of 2.0 dS m −1 . A single application of Arthrospira increased the number of shoots in treatments with an EC of 2.0 dS m −1 , and the IDW and number of flowers in treatments with an EC of 3.0 dS m −1 . Regarding ornamental plants, the authors of [9] showed that a foliar application of Arthrospira platensis improved the RDW and the number of flowers per plant. In petunias, applying microalgae stimulates bud formation in the mother plant and induces flowering in commercial petunia plants [9,27]. In our assay, the combined application of Arthrospira and silicon gave better results than using each compound alone. The increase in root growth, and the stimulation of flower and shoot formation, may be due to the synergistic effect of microalgae and silicon, and their effect on certain hormones such as abscisic acid and cytokines [9,28]. Cytokinin promotes bud development and abscisic acid promotes root growth. In addition, the authors of [16] suggested that the advancement effect of silicon and seaweed on the flowering time might be attributed to their essential role in balancing the ratio between carbohydrates and nitrogen in favor of flowering. In accordance with our results, in [13,27] the authors showed that the positive effect of microalgae and silicon was particularly beneficial under stress conditions. A wide array of chemicals has been identified in seaweed extracts; these include polysaccharides, phenolics, fatty acids, vitamins, osmolytes, phytohormones, and hormone-like compounds involved in the plant-signaling response to abiotic stress [2]. In Spirulina spp., carbohydrates may account for up to 46% of the DW extract [2], and these molecules can play an important role in osmotic adjustment [26]. In this sense, the major role played by sugars in stress mitigation involves osmoprotection, carbon storage, and ROS scavenging [29]. Amino acids can also contribute to mitigating the damage caused by abiotic stress [5]. Furthermore, the application of Si has been shown to alleviate the negative effects of numerous abiotic stresses, including salt. Several potential mechanisms associated with stress alleviation in higher plants have been identified: increased structural reinforcement, an altered photosynthetic rate, changes in stomatal conductance, and enhanced water use efficiency [28]. Other possible mechanisms may also include osmotic adjustments through increased water potential and water content, reductions in oxidative stress, alterations in mineral uptake, and accumulation and alterations in phytohormone concentrations [28].
Notably, all the essential minerals (about 7%) are available in Spirulina, including potassium, calcium, chromium copper, iron, magnesium, manganese, phosphorus, selenium, sodium, and zinc [6]. The nutrient analysis of Arthrospira spp. dry biomass revealed that it contained 6.70%, 2.47%, and 1.14% (on a dry basis) of N, P, and K, respectively, whereas the microalgae's calcium (Ca) content was lower, relative to the other minerals [5]. In contrast to the results of other authors, no clear effect on nutrient concentration in the leaves was found after applying only the microalgae. The joint application of the microalgae and Si increased the P and Ca concentrations in the leaves in the treatment with an EC of 3.0 dS m −1 , whereas it increased the Cl and Na concentrations in the EC 3.5 dS m −1 treatment. Conversely, the authors of [30] observed that applying silicon significantly decreased the Na + concentration in the leaves while increasing the K + concentration, compared with no saline treatment. In accordance with our results, it was found that the nitrogen, potassium, magnesium and sulfur contents were not affected by applying Arthrospira to Petunia x hybrida [16].

Conclusions
The Pelargonium hortorum L.H. Bailey plant can be considered moderately tolerant to salinity. The mechanisms that confer salinity tolerance to Pelargonium are its ability to avoid Cl − uptake and its moderate tolerance to high internal Na + . Our results show that the joint application of Arthrospira platensis and silicon improves the FDW and the number of flowers in treatments with an EC of 3.0 dS m −1 , while improving the LDW, RDW, IDW and TDW, and the number of flowers, shoots, and leaves in treatments with an EC of 3.5 dS m −1 . The foliar application of Arthrospira platensis and Si mitigates the negative effect of NaCl on Pelargonium hortorum L.H. Bailey crops at an EC of 3.5 dS m −1 . Combined applications of microalgae and silicon can improve plant growth and yield by stimulating vegetative growth and promoting plant flowering. This strategy can be an alternative to using chemicals in agriculture.

Conflicts of Interest:
The authors declare that they have no competing financial or personal interests that might have influenced the work reported in this manuscript.