Chlamydomonas sp. extract meliorates the growth and physiological responses of ‘Camarosa’ strawberry (Fragaria × ananassa Duch) under salinity stress

Abstract

Microalgae like Chlamydomonas are beneficial organisms employed as biological stimulants to improve plants’ growth, fruit quality, and stress tolerance. In the current study, the effects of Chlamydomonas sp. foliar spraying (0, 20, and 40 ml L⁻¹) were assayed on Camarosa strawberry plants under salinity stress (0, 40, and 80 mM NaCl). The results showed that the foliar application of Chlamydomonas extract influenced strawberry’s morphological, physiological, and biochemical characteristics under salinity stress. Foliar treatment of Chlamydomonas extract with and without salinity stress increased the leaf number and leaf area, the leaf relative water content, and photosynthetic pigments content. Moreover, the foliar application of Chlamydomonas extract decreased lipid peroxidation and hydrogen peroxide content and, on the other hand, enhanced the antioxidant enzymes activity (superoxide dismutase, guaiacol peroxidase, and peroxidase), phenolics, flavonoids, and anthocyanins content under salinity stress. For instance, the highest total antioxidant capacity was found in the plants foliar treated with 40 ml L⁻¹ of Chlamydomonas algae extract under 80 mM salinity stress, which increased by 102.4% compared to the controls, as well as the highest total phenolic compounds and anthocyanin’s content were 30.22, and 7.2% more than the control plants, respectively. Overall, the foliar application of Chlamydomonas algae extracts, especially at a concentration of 20 ml L⁻¹ enhanced the strawberry’s growth, yield, and physiological traits under saline conditions. The results with more detailed evaluations will be advisable for the pioneer farmers and extension section.

 Introduction

Strawberry (Fragaria×ananassa Duch.) is one of the world’s most prominent small fruits with high nutritional and medicinal values¹. The fruits have nutritious compounds such as minerals, vitamins, organic acids, fatty acids, and fiber. Also, they have a wide range of biologically active compounds, including polyphenols, tannins, and flavonoids². Iran is one of the world’s most important producers of strawberries with a production of approximately 59,136 tons per year³.

The salinity of soil and water resources is increasing worldwide. Salinity is a critical environmental limitation that reduces the yield and productivity of various crops, including strawberries, especially in arid and semi-arid regions⁴. On the other hand, the strawberry is one of the most sensitive plant species to salinity among horticultural crops⁵. Notable decreases in morphological attributes as well as yield of strawberry plants grown under saline conditions were recorded in different studies⁶. The high salt concentration in soils affects plants’ diverse responses and causes water stress, ion toxicity, nutritional disorders, oxidative stress, membrane disruption, and finally declines the cell division⁷. One of the most acute destructive effects of salt stress on plants is damaging the leaf tissue, ultimately leading to leaf senescence and it is believed that leaf senescence is mainly due to the reduction of chlorophyll content under saline conditions⁸. Salinity reduces the water potential of soil and leaves, disturbs the plant’s water relations, and hence the cell turgor decreases, ultimately leading to osmotic stress⁹. Salinity reduces the growth and development of plants through osmotic stress, enormously declines gas exchange, and leads to the over-generation of reactive oxygen species¹⁰. Plants employ several mechanisms including ions and sugar accumulation, and the activity of enzymatic and non-enzymatic antioxidants to deal with salinity, which restricts the proliferation of reactive oxygen species¹¹. The toxic effects of ROS include lipid peroxidation, membrane destruction, and DNA and protein damage¹². The activity of antioxidant enzymes [superoxide dismutase (SOD), ascorbate peroxidase (APX), and peroxidase (POX)] is stimulated to resist damages caused by the accumulation of ROS.

Algae extract is a natural biological stimulant. These extracts act as growth stimulants by providing compounds such as signaling molecules, amino acids, vitamins, cytokinins, auxins, and carbohydrates¹³. The presence of these bioactive compounds in plants treated with algae extracts ameliorates tolerance to environmental stresses¹⁴. Seaweed extract is widely used to improve plant growth and fruit quality, especially under stress. In addition, microalgae showed good potential as biofertilizers and biostimulants and they contain large amounts of micro- and macro elements for crops’ optimal growth and development¹⁵. The genus Chlamydomonas is a unicellular green microalga from the Chlamydomonadaceae family. It is a flagellated motile that can live in stagnant water, wet soils, freshwater, seawater, and snow as “snow algae”¹⁶. Some studies show the positive effect of microalgae extract on the growth of different plants such as eggplant, garlic, pepper, tomato, and parsley^17–19. Chlamydomonas spp. is a genus that produces extracellular mucilage that exhibits pseudo-auxin and pseudo-cytokinin activity and acts as a plant growth stimulator²⁰. The application of algae extracts increased the yield and the content of Fe, Zn, and Mn elements in cucumber²¹. Moreover, the extracts improved fruit size, and total anthocyanin content in strawberries²². Extracts of Dunaliella spp. and Phaeadactylum spp. reduced the salinity stress damage during seed germination of Capsicum annuum L. due to a significant decrease in the production of superoxide radicals and lipid peroxidation²³. Foliar application of Green algae significantly improved photosynthetic pigments content, osmolytes, and antioxidant enzyme activity under normal and salt stress conditions. Findings by Al-Taisan, et al.²⁴ Showed showed a notable increase in SOD, CAT, and APX, particularly in GPX enzyme activity in two Mentha species (Mentha piperita; Mentha longifolia) seedlings. Another study cleared that the foliar treatment with Ulva intestinalis extract positively affected antioxidant metabolites including polyphenols and carotenoids in parsley²⁵.

Biostimulants have received much attention in modern agriculture due to their high efficiency. Following their use, plants have shown different reactions, such as enhanced growth and yield, improved nutrient absorption, and increased tolerance to biotic and abiotic stresses. Owing to the high incidence of salinity, and on the other hand, due to the importance of strawberries, this study for the first time aimed to utilize a novel environment-friendly methodology to combat the salinity damage on Camarosa strawberries. In detail, this research aimed to; (1) investigate the negative effects of salinity stress at different concentrations (0, 40, and 80 mM NaCl) on the development and yield potential of strawberries, and (2) test the efficacy of Cllamydomanas sp. extract, as a biostimulant in modulating salt stress on morphological and physiological attributes of Camarosa strawberry.

 Materials and methods

Plant material, experimental design, and treatments

The Camarosa strawberries (Fragaria × ananassa Duch., cv. ‘Camarosa’) were obtained from a commercial greenhouse in Maragheh, Iran (north latitude 37°1’ to 37°45’ and east longitude 46°9’ to 46°44’) and the research was conducted from early November 2018 to the end of April 2019. The plants were grown in 5-liter plastic pots with cocopeat and perlite in the ratio of 1:1 (V/V) as the culture medium. The Hoagland’s nutrient solution (pH = 6) was used to feed the plants. The night/day temperature of the greenhouse was 24/18°C, and the relative humidity was about 65–75%. The experiment was conducted as a factorial based on a completely randomized design with three replications (three pots in each replication, one plant per pot as an experimental unit). Salinity stress (0, 40, and 80 mM NaCl) was applied one month after cultivating and establishing daughter plants (December 8th) via the nutrient solution. The plants were nourished with Hoagland’s nutrient solution every other day until the fruit harvest (April 30th). Ten days after salinity initiation, the Chlamydomonas algae extract (supplied by Agricultural Biotechnology Center, Tabriz, Iran) was treated as foliar spraying at three levels (0, 20, and 40 ml L^-1). The control plants were nourished with Hoagland’s solution without salinity stress and foliar sprayed with distilled water. Foliar applications were performed 3 times every 3 days. Spraying the leaves was done in such a way that the surface of the leaves was completely wet.

The compositional analysis of used alga (Chlamydomonas sp.) extract.

Total protein (%)	Chlorophyll a (mg/L)	Chlorophyll b (mg/L)	Total carotenoids (mg/L)
45.6	25.5	4.06	9.75

 Growth and yield parameters

The fruits were harvested in March 2018 at the optimal physiological maturity stage to measure the biochemical parameters. The developed leaves were collected at the end of April 2019. Immediately after harvesting, the leaf samples were frozen in liquid nitrogen and kept at a suitable temperature (-80 °C) for further experiments. The number of leaves in each plant was counted at the end of the growth period, the petiole length was calculated using a caliper, and the Image J software was used to measure the leaf area index (LAI).

Electrolyte leakage (EL)

EL was recorded according to Zhao, et al.²⁶ Equal pieces of leaves were transferred into falcons containing 20 ml of distilled water, and each sample’s EC was measured (EC0). To measure the initial EC (EC1), the samples were kept at 4 °C for 24 h. In the next step, the test tubes containing the samples were placed in an autoclave at 121 °C for 15 min, and (EC2) was determined. Finally, the EL percentage was obtained using the following formula:

EL = [(EC1- EC0)/(EC2- EC0)] ×100.

Relative water content (RWC)

The fresh weight (FW) of 3 leaves was recorded, then turgid weight (TW) was obtained by maintaining leaf samples in distilled water. Afterward, they were kept in an oven for 48 h at 70 °C to obtain dry weight (DW). RWC of leaves was computed according to the below Eq. 2⁷:

RWC = [(FW-DW)/(TW-DW)] × 100.

Photosynthetic pigments and SPAD index

The total chlorophylls (TChl) and carotenoids (Car_s) content were calculated using the Arnon method²⁸. Fresh leaves (0.5 g) were digested by adding 5 mL of 80% acetone. After centrifugation at 6000 rpm for 10 min, the absorbance of the separated supernatant was read by a spectrophotometer (UV-1800 Shimadzu, Japan) at wavelengths of 663, 645, and 470 nm. The photosynthesis pigments were presented as mg g^-1 FW.

The SPAD index of leaves was measured by the method of Ling, et al.²⁹ using a SPAD device (502 Plus Chlorophyll Meter, Minolta Camera Co., Osaka, Japan).

Total soluble carbohydrates content (TSC)

The TSC of the strawberry plant was recorded by the method of Schlegel³⁰. The leaf sample (0.2 g) was heated in 10 ml of 96% ethanol for one hour at 80 °C. 1 ml of 0.5% phenol and 5 ml of 98% sulfuric acid were added to the ethanolic extract. Finally, the absorbance was read at 483 nm with a spectrophotometer. The TSC was reported as mg g⁻¹ FW.

Total soluble proteins (TSP) contents

Fresh leaves (0.5 g) were homogenized with potassium phosphate buffer (pH = 6.8, 100 mM) including 1% polyvinyl polypyrrolidone (PVP) and ethylenediaminetetraacetic acid (EDTA) (4 mM) using a magnetic stirrer for 10 min and, then the homogenate was centrifuged at 6000 rpm for 20 min. The supernatant was separated and used for TSP content and antioxidant enzyme activity. The TSP content of strawberry leaves was measured by the method of Bradford³¹, To measure TSP, 50 µl of enzyme extract was mixed with 1000 µl of Bradford solution, and the absorption was spectrophotometrically read at 595 nm. The TSP was stated as mg g⁻¹ FW.

The activity of antioxidant enzymes

The superoxide dismutase (SOD) activity was measured by recording the enzyme’s inhibition of nitroblue tetrazolium (NBT) photoreduction³². The activity of guaiacol peroxidase (GPX) was measured by the method of Mencarelli, et al.³³, as absorbance increasing pattern in min at 490 nm. The peroxidase (POX) activity was calculated based on the method of Polle, et al.³⁴.

Total antioxidant capacity (TAC)

The TAC was determined through the free radical inhibitory potential of DPPH (2.2-diphenyl-1-picrylhydrazyl) using the spectrophotometric method³⁵. Doing this, 1000 µl of methanolic leaf extract was mixed with 1900 µl of 1% DPPH and placed in the dark for 30 min. The absorbance of the samples was read using a spectrophotometer at 517 nm and the data were calculated based on the following formula:

Inhibition of DPPH = [(Abs control − Abs sample)/Abs control] × 100

where Abs control is the absorbance of DPPH solution without extract.

Hydrogen peroxide (H2O2) and malondialdehyde (MDA) content

The method of Velikova, et al.³⁶ was used for H₂O₂ content determination. 0.5 g of fresh leaf sample was digested with 1% trichloroacetic acid (TCA) solution. After centrifugation, 0.5 ml of the supernatant was separated, and 0.5 ml of phosphate buffer and 1 ml of potassium iodide (KI) were added. The absorbance of the samples was read at 390 nm using a spectrophotometer. The content of H₂O₂ was calculated using a hydrogen peroxide standard curve and stated as µmol g^-1 FW.

A fresh leaf sample (0.5 g) was digested with 1.5 ml of TCA to measure MDA content. The resulting extracts were centrifuged at 10000 rpm for 10 min. After centrifugation, 1 ml of thiobarbituric acid (TBA) solution was added to the supernatant. The mix was heated in a hot water bath (95 °C) for 30 min and cooled down immediately. Finally, the absorbance of the admixture was measured by a spectrophotometer at two wavelengths of 532 and 600 nm. MDA was calculated using the following formula³⁷.

MDA= ([(532–600 nm) × 20]/155) × 100.

Titratable acidity (TA) of fruits

To measure the TA of the strawberry fruits, 3 ml of fruit juice was diluted to 20 ml with distilled water, then titrated with 0.1 normal sodium hydroxide until the pH reached 8.1. The TA was calculated using the following formula, the TA of fruits was stated in mg 100 ml^-1 of citric acid³⁸.

TA= (ml NaOH×N NaOH× acid Meq. Factor /ml juice titrated) ×100.

Total phenolics content (TPC) of fruits

The total phenolics content was determined according to the Folin-Ciocalteu procedure. First, the fruit tissue was digested with 0.01% acidic methanol and centrifuged at 12,000 rpm for 10 min. The supernatant was mixed with 600 µl of Folin Ciocalteu reagent for 5 to 10 min at room temperature and sodium carbonate (7.5%) was added to each of the extracts. The reaction mixture was kept in a dark place for 2 h and then its absorbance was recorded at a wavelength of 760 nm³⁹. The TPC was stated as mg gallic acid 100 g^-1 FW.

Total flavonoids content (TFC) of fruits

The TFC of the plant fruits was recorded using the method described by Kaijv, et al.⁴⁰, The fruit tissue was homogenized with 80% methanol and centrifuged at 14000 rpm for 15 min. The reaction mixture consisted of 200 µl of fruit extract, 75 µl of 5% potassium acetate, 150 µl of 1% aluminum chloride, and 500 µl of 1 M NaOH at room temperature for 40 min. The absorbance of the solution was read at 570 nm. The fruit TFC was presented as mg quercetin equivalent to 100 g^-1 FW.

The anthocyanins content of fruits

The fresh fruit tissue (1 g) was digested with 10 ml of acidic methanol and centrifuged at 10000 rpm for 10 min. The reaction mixture consisted of 100 µl of extract and 1900 µl of acidic methanol. The absorbance of the solution was read at 550 nm by a spectrophotometer. The anthocyanin content was expressed in mg g^-1 FW⁴¹.

Statistical analysis

The data were subjected to a standard analysis of variance using the SAS software (ver. 9.1), and the means were compared with the least-significant-difference (LSD) test at p ≤ 0.05. The figures were drawn with Microsoft Excel software. Principal component analysis (PCA), and Pearson correlation analysis were carried out using R v3.4.3.

Results

Morphological traits, EL and RWC

The results showed that salinity stress reduced the evaluated morphological traits while the algae extract mitigated these features (Fig. 1). The highest number of leaves was recorded with algae extract foliar spray (40 and 20 ml L^-1) under normal conditions, while the lowest number of leaves was obtained in 80 and 40 mM NaCl treatment without any foliar application as well as in 20 and 40 ml^-1 of algae foliar spraying under salinity stress. Foliar treatment improved the trait up to 35% at 40 ml L^-1 in normal conditions compared with the control. On the other hand, the number of leaves was reduced in salinity of 40 and 80 mM by up to 46% and 65% with no foliar treatment, compared to the control plants, respectively.

Fig. 1.

Effect of Chlamydomonas foliar treatment on morphological parameters of strawberry plant (Fragaria× ananassa Duch., cv. ‘Camarosa’) under salinity stress. Ch (Chlamydomonas); S (Salinity).

The highest leaf area index (LAI) and the petiole length (PL) were recorded in 20 ml L^-1 of algae foliar application and increased by 73% and 16.2%, over to the control, respectively. The lowest was obtained at 80 mM NaCl treatment with no and also with 20 ml L^-1 algae application. The LAI was lessened by 23% under 80 mM NaCl without foliar spraying.

The results revealed that EL increased under salinity stress, but the algae foliar spraying reduced EL. Hence, the highest EL was obtained in 80 mM NaCl treatment with no foliar treatment. Whereas, the lowermost was achieved in the control plants. The highest salinity level caused an increment in EL up to 283.17% compared to the plants without salinity exposure. Algae extract (20 ml L^-1) caused 78.7 and 59.3% in EL data under 40 and 80 mM NaCl treatment compared to plants sprayed with distilled water, respectively (Table 1).

Table 1.

Effect of Chlamydomonas sp. spraying on the morphological characteristics, electrolyte leakage, and relative water content in leaves of the strawberry plants under salinity stress.

Salinity (mM)	Chlamydomonas (ml L−1)	No. of leaves	LA (cm2 )	PL (cm)	El (%)	RWC (%)
0	0	25.33 ± 3.293b	46.53 ± 2.962d	5.41 ± 1.372b	5.41 ± 0.692e	73.21 ± 1.572b
	20	33.00 ± 1.626a	80.52 ± 6.588a	6.29 ± 1.266a	7.58 ± 0.959de	77.79 ± 3.068a
	40	34.33 ± 1.691a	60.52 ± 1.420b	5.69 ± 2.534b	7.58 ± 1.372de	66.45 ± 0.611c
40	0	17.33 ± 2.624d	46.13 ± 3.731d	4.37 ± 2.407c	16.53 ± 0.968b	68.29 ± 2.033c
	20	22.00 ± 2.163bc	50.74 ± 3.629dc	4.49 ± 2.593c	9.25 ± 0.760 cd	72.28 ± 1.055b
	40	25.66 ± 0.470b	55.84 ± 2.233bc	3.73 ± 1.123d	10.80 ± 1.247c	68.20 ± 2.604c
80	0	15.33 ± 1.238d	37.58 ± 1.960e	3.60 ± 2.390d	20.73 ± 1.302a	57.47 ± 0.692e
	20	16.00 ± 1.412d	44.18 ± 0.931de	3.40 ± 0.453d	11.30 ± 0.576c	70.25 ± 0.655bc
	40	18.33 ± 0.940dc	44.45 ± 2.123d	3.38 ± 2.567d	18.00 ± 1.413b	61.85 ± 2.160d
LSD		4	6.852	4.225	2.258	3.830
S.O.V.		significance
S		**	**	**	**	**
Ch		ns	**	**	**	**
S × Ch		**	**	**	**	**

The data displayed are the means (±SE) of the three replications (n = 3), and dissimilar letters are significantly different at the p ≤ 0.05 level of the LSD test. ** and ns show significance at p ≤ 0.01 level and no significant difference, respectively. The number of leaves (No.leaves); Leaf area index (LAI); Petiole length (PL); Electrolyte leakage (EL); Relative water content (RWC). S.O.V., S, and Ch refer to the source of variance, salinity, and Chlamydomonas treatments.

The findings revealed that with increasing salinity stress, RWC declined and algae extract of 20 ml L^-1 ameliorated the trait under saline conditions. The RWC decreased by 27.38% under 80 mM NaCl treatment without algae extract foliar use. The highest RWC was noted in algae extract of 20 ml L^-1 and it improved more in comparison with the strawberry plants both with and without salinity stress in comparison with 40 ml L^-1 concentration (Table 1).

Photosynthetic pigments content and chlorophyll index (SPAD)

The findings revealed that algae extract foliar treatment improved total chlorophyll (TChl) and carotenoids content, especially at 20 mg L^-1 under both saline and no-saline conditions (Table 2). The moderate and severe salinity reduced the content of TChl by 84.7 and 104.8% compared to non-stressed plants, respectively. While algae extract treatment at the highest concentration improved TChl in moderate and severe salinity up to 10.8 and 20.5% over to the strawberry plants that were sprayed with distilled water under the salinity stress conditions. The severe salinity reduced carotenoid content by 144.8%. In the moderate and intense salinity stress, the higher algae extract treatment alleviated carotenoids content by 133.3 and 82.7% compared to without algae extract (Fig. 2A, B). The highest SPAD value was recorded in 40 ml L^-1 algae extract (Fig. 2C).

Table 2.

The analysis of variance of salinity and Chlamydomonas foliar use on photosynthesis pigments and SPAD index.

S.O.V.	Total Chlorophyll	Carotenoids	SPAD
S	**	ns	ns
Ch	**	ns	**
S × Ch	**	*	**

**, * and ns show significance at p ≤ 0.01 and p ≤ 0.05 level and no significant difference, respectively. Also, S.O.V., S, and Ch refer to the source of variance, salinity, and Chlamydomonas treatments.

Osmolytes, oxidative markers, and antioxidant enzymes of leaves

Total soluble carbohydrates content (TSC)

Based on these results, the TSC of strawberry plants was lessened using salinity stress, while it was ameliorated with the algae extract treatments. Hence, the highest TSC was observed at 40 ml L^-1 algae foliar utilization, and the lowest was achieved at 40 and 80 mM NaCl treatment with no foliar treatment and along with 20 ml L^-1 algae application. The foliar spraying of algae extracts at 20 and 40-ml L^-1 concentrations increased TSC by 56.18 and 129.76% compared to the control plants, respectively. The intense salinity stress caused a reduction of 70.8% in TSC content in comparison with untreated strawberry plants. Also, algae extract at 40 ml L^-1 improved TSC in moderate and severe stress by 91.4 and 73.7%, respectively, compared to the plants with no foliar spraying (Table 3).

Fig. 2.

Effect of Chlamydomonas spraying (0, 20, and 40 ml L^-1) on the content of total chlorophyll (A), carotenoids, and SPAD index in leaves of strawberry plants grown under salt stress. Dissimilar letters on the columns are significantly different based on the LSD test.

Table 3.

Effect of Chlamydomonas foliar treatment on osmolytes and oxidative markers in leaves of the strawberry plant under salt stress.

Salinity (mM)	Chlamydomonas (ml L−1)	TSC (mg g−1 Fw)	Pro (mg g−1 Fw)	TAC (%)	H2O2 (µmol g−1 Fw)	MDA (µmol g−1 Fw)
0	0	2.99 ± 0.093c	1.67 ± 0.018b	43.17 ± 2.023f	22.91 ± 0.863e	0.54 ± 0.211d
	20	4.67 ± 0.944b	1.70 ± 0.013b	57.36 ± 0.721e	22.73 ± 1.850e	0.62 ± 0.146 cd
	40	6.87 ± 0.835a	2.32 ± 0.022a	59.75 ± 3.587de	26.11 ± 1.886e	0.51 ± 0.129d
40	0	1.74 ± 0.492d	1.52 ± 0.024bc	66.81 ± 2.320c	54.96 ± 0.931b	1.42 ± 0.202a
	20	2.50 ± 0.065 cd	1.67 ± 0.013b	65.20 ± 3.352 cd	35.68 ± 1.090d	0.66 ± 0.124bcd
	40	3.35 ± 0.284c	1.47 ± 0.340bc	70.60 ± 2.218b	47.02 ± 1.164c	0.72 ± 0.041bcd
80	0	1.75 ± 0.243d	1.27 ± 0.014c	45.37 ± 1.823f	64.63 ± 2.911a	1.46 ± 0.140a
	20	2.59 ± 0.880 cd	1.63 ± 0.027b	59.75 ± 4.855de	55.44 ± 3.360b	0.91 ± 0.046bc
	40	3.04 ± 0.161c	1.45 ± 0.271bc	87.40 ± 0.975a	57.69 ± 0.313b	0.96 ± 0.117b
LSD		0.29	3.9	5.73	0.31	1.17
Significance
S		**	**	**	**	**
Ch		ns	*	**	**	**
S × Ch		**	**	**	**	**

The data displayed are the means (± SE) of the three replications, and columns with dissimilar letters differ significantly at the p ≤ 0.05 level of the LSD test. FW, TSC, Pro, H₂O₂, and MDA refer to Fresh weight, Total Soluble Carbohydrates, Protein, Hydrogen peroxide, and Malondialdehyde, respectively.

Total soluble protein (pro) content

The findings showed that algae extract utilization alleviated the protein content under saline conditions and also improved it under normal conditions. So, algae extract treatment at a higher concentration (40 ml L^-1) enhanced the content of Pro by 38.92% compared to the control, but the highest salt stress decreased Pro content by 31.49% compared to the plants grown in normal conditions (Table 3).

Total antioxidants capacity (TAC)

The results showed that salinity stress and algae extract increased the TAC of strawberry plants, especially without and with 40 mM NaCl treatment. TAC was increased by 54.76 in plants under moderate salinity in comparison with the strawberries with no salinity treatment. The highest TAC was found in the plants foliar treated with 40 ml L^-1 of Chlamydomonas algae extract under 80 mM salinity stress, which increased by 102.4% compared to the controls (Table 3).

Hydrogen peroxide (H2O2) and malondialdehyde (MDA) content

H₂O₂ and MDA contents were enhanced with salinity stress, while the traits were reduced using the algae extract foliar application in saline conditions. The Highest salinity levels without any foliar treatments caused an enhancement of up to 182.1 and 270.3% in the content of H₂O₂ and MDA compared to the control, respectively. Algae extract of 20 ml L^-1 decreased H₂O₂ and MDA content by 115.15 and 60.43% in the plants under moderate and severe salt stress conditions compared to the plants foliar sprayed with distilled water, respectively (Table 3).

Antioxidant enzymes

Salinity and the algal extract foliar application had significant influences on GPX, SOD, and POX activity (Table 4) and those enzyme activities was increased by the mentioned treatments. The highest GPX and SOD activity was observed in 80 mM NaCl along with 40 ml L^-1 of algae extract, while their activity increased by 216.6 and 22.8% compared to the control plants, respectively (Fig. 3A, B). The highest POX enzyme activity was observed in the plants treated with 40 ml L^-1 algae extract application under no-saline and with moderate and severe salinity stress and, in the strawberries sprayed with 20 ml L^-1 under moderate stress. While the lowest was recorded in the control plants (Fig. 3C).

Table 4.

The analysis of variance for the effects of salinity and Chlamydomonas foliar use on guaiacol peroxidase (GPX), superoxide dismutase (SOD), and peroxidase (POX) activity of strawberries.

S.O.V.	GPX	SOD	POX
S	**	ns	*
Ch	**	ns	*
S × Ch	**	**	*

**, * and ns show significance at p ≤ 0.01 and p ≤ 0.05 level and no significant difference, respectively. Also, S.O.V., S, and Ch refer to the source of variance, salinity, and Chlamydomonas treatments.

Fig. 3.

Effect of Chlamydomonas algae foliar spray (0-, 20-, and 40-ml L^-1) on the activity of superoxide dismutase (SOD; A), guaiacol peroxidase (GPX; B) and peroxidase (POX; C) in strawberry leaves under salinity stress. Dissimilar letters on the columns are significantly different based on the LSD test.

Fruit biochemical traits (TA, TPC, TFC, and anthocyanins)

Moderate salinity (40 mM) increased TA by 69.09% compared to the control plants, while severe salinity (80 mM) led to a reduction of 84.7% in TA without any foliar spraying. Algae extract treatment (20 ml L^-1) without salinity stress caused the highest TA and increased the percentage of TA by 112.7% compared to the control plants (Table 5). The highest TPC was observed in the plants grown under severe salinity stress along with 0 and 20 ml L^-1 algae extract, as well as in the plants under moderate salinity with 40 ml L^-1 algae extract spraying. Also, the highest salinity along with 40 ml L^-1 algae extract produced the highest TFC and anthocyanins content: 30.22, and 7.2% more than the control plants, respectively (Table 5).

Table 5.

Effect of Chlamydomonas foliar treatment on the biochemical characteristics of strawberry fruits under salinity stress.

Salinity (mM)	Chlamydomonas (ml.L−1)	TA )mg/100 g acid citric(	TPC (Mg 100 g−1 FW)	TFC (Mg 100 g−1 FW)		Anthocyanin (Mg 100 g−1 FW)
0	0	0.55 ± 0.044de	55.12 ± 3.922c	38.84 ± 2.180e		0.12 ± 0.001d
	20	1.17 ± 0.126a	57.50 ± 3.056c	58.03 ± 4.653bc		0.12 ± 0.002d
	40	0.65 ± 0.031de	60.43 ± 5.054c	47.48 ± 5.518d		0.13 ± 0.001b
40	0	0.93 ± 0.052b	66.04 ± 3.444b	46.04 ± 1.548de		0.12 ± 0.002 cd
	20	0.70 ± 0.127 cd	66.08 ± 3.143b	61.32 ± 3.350b		0.13 ± 0.002b
	40	0.60 ± 0.055de	72.58 ± 0.808a	51.62 ± 2.5664 cd		0.13 ± 0.002bc
80	0	0.51 ± 0.023e	73.16 ± 0.723a	50.58 ± 1.204 cd		0.13 ± 0.003b
	20	0.62 ± 0.040de	74.87 ± 3.018a	61.32 ± 2.277b		0.13 ± 0.003b
	40	0.82 ± 0.067bc	55.87 ± 1.084c	72.93 ± 7.843a		0.19 ± 0.002a
LSD		1.16	6.40	8.44		0.005
Significance
S		**	**	**	**
Ch		ns	*	**	**
S × Ch		**	*	**	**

The data displayed are means (± SE) of three replicates. The dissimilar letters in the columns are significantly different at the p ≤ 0.05 level of the LSD test. **, *, and ns were different at the level of 1 and 5%, and no significant difference, respectively. Fresh weight, Titratable acidity, Total phenolic content, and Total flavonoids content refer to FW, TA, TPC, and TFC, respectively.

Principal components analysis and correlation between parameters

The heatmap shows a correlation between parameters. There was a positive correlation between morphological parameters (PL, LA, no. of leaves) with photosynthetic pigments (TChl, SPAD) and biochemical parameters of fruit (TFC, Anthocyanin, TA). Antioxidant parameters, TFC, and antioxidant enzymes (SOD, GPX) positively correlated with anthocyanin content. A positive correlation was observed between leaf biochemical traits (MDA, H₂O₂) and electrolyte leakage (EL), but on the other hand, there was a negative correlation between EL and RWC. A negative correlation was observed between morphological traits, total chlorophyll, and protein content with MDA, H₂O₂, and EL (Fig. 4A).

Fig. 4.

(A) Correlation analysis and cluster dendrogram of the Chlamydomonas extract treatment on strawberry plants grown under saline conditions. (A) Heatmap indicates the positive (blue) and negative (red) correlations. (B) Principal component analysis (PCA) of Chlamydomonas extract treatments on strawberry plants under saline conditions. PCA individual plots of 20 ml.L^-1 and 40 ml.L^-1 Chlamydomonas extract treatments on strawberry plants subjected to non-saline (control, 0 mM NaCl), moderate (40 mM), and severe (80 mM) salinity. PCA biplots of the treatment variable association where the lines originating from the center indicate positive or negative correlations of different variables.  The tested variables included the Number of leaves, No.leaves; Leaf area, LA; Petiole length, PL; Electrolyte leakage, EL; Relative water content, RWC; Total chlorophyll, Tchl; Carotenoids, CARs; Total Soluble Carbohydrates, TSC; Protein, Pro; Hydrogen peroxide, H₂O₂; Malondialdehyde, MDA; Titratable acidity, TA; Total phenolic content, TPC; Total flavonoids content, TFC; superoxide dismutase (SOD); guaiacol peroxidase (GPX) and peroxidase (POX).

All 20 morphological, physiological, and biochemical parameters are divided into two principal components (PC1, PC2). The parameters (RWC, TChl, PL) were positively related to the plants sprayed with 20 ml.L^-1 algae extract under the conditions of no salinity. In contrast, the morphological parameters (No. of Leaves, LA) were related to the plants sprayed with 40 ml.L^-1 algae extract under no salinity. Biochemical parameters (SOD, TFC, antioxidants) were positively correlated to strawberry plants treated with 40 ml.l^-1 algae extract under severe salt stress. MDA, TPC, CARs, and POX parameters positively correlated with plants grown under moderate salt stress without foliar applications (Fig. 4B).

Discussion

The findings revealed that salinity increased EL, while algal foliar application reduced the trait. On the other hand, algae extract spraying mitigated RWC. Salinity stress through the production of reactive oxygen species damages cell membranes’ stability, As a result, it leads to an increase in EL. Also, salinity strongly affects the water potential of the soil. Therefore, plants face a problem absorbing water, which causes a decrease in the RWC of plants^42,43. Maintaining the integrity of the cell membrane under salinity stress is considered an integral part of the salt tolerance mechanism, so plants with a low EL index are tolerant to stress^44,45. The decrease in RWC is caused by the reduction of water absorption to regulate osmosis under the influence of salinity⁴⁶. Hegazi, et al.⁴⁷, showed that the use of seaweed extract had positive effects on membrane permeability maintained the cell membrane from the harmful effects of salinity stress, and increased the RWC of plants under salinity stress. Moreover, the foliar application of Chlamydomonas extract decreased the adverse effects of salinity on chlorophyll content and subsequently improved the RWC. The algal extract improves the growth responses and even the metabolism of plants via the biochemical and physiological attributes which in turn enhance the RWC and the water relations in favor of growth and productivity enhancement. Salinity enhanced the osmolyte levels in the leaves of common bean plants⁴⁸. In agreement with our results, treating pepper with 100 mM NaCl increased the accumulation of proline and glycine betaine that triggered the accumulation of osmolytes for better protection of cellular functions⁴⁹. Plants facing NaCl toxicity often exhibit symptoms associated with oxidative stress and membrane lipid peroxidation, which can result in the accumulation of ROS and MDA⁵⁰. Salinity stress causes a burst of oxidative stress markers such as H₂O₂, MDA, and EL⁴⁹. In this study, high salinity triggered overproduction of H₂O₂, increased electrolyte leakage, and MDA in common bean leaves, whereas caused a reduction in RWC content. Plants produce antioxidant enzymes to keep ROS levels constant, and when ROS levels rise dramatically under stress, the antioxidant enzyme system is triggered. Similar studies found that H₂S declined salt damage by lowering H₂O₂, and MDA levels and generating reactive oxygen species through an increased antioxidant system in rice⁵¹ and cucumber seedlings⁵².

Based on these results, algae extract foliar spraying recovered the adverse effect of salinity stress on growth parameters in strawberry plants. Intense salt concentration in soil or irrigation water caused adverse effects on plant metabolism, disturbing cellular homeostasis⁵³. The researchers related the decrease in the number of leaves under salinity stress to the reduced photosynthesis and dry matter production, as well as the decrease in water absorption by the roots, which ultimately leads to a reduction of vegetative growth⁵⁴. Also, salinity reduced leaf area, increased leaf thickness, leaf abscission, necrosis of roots and shoots, and caused a reduction in internode length⁵⁵. On the other hand, it has been shown that algae extract foliar treatment improved vegetative growth, plant height, the number of leaves, and the leaf area in plants under salinity and normal conditions⁵⁶ which is in line with our results. Algae extracts contain several valuable compounds, including nutrients, amino acids, vitamins, polysaccharides, and hormones that improve vegetative growth and plant performance⁵⁷. Under salinity, any enhancement in growth attributes may be traceable in the related physiological responses. The promotions induced in the growth and physiological potential by the foliar application of algae extract are directly and indirectly related to the beneficial effects of algae extract which influence diverse metabolic pathways and the photosynthetic efficiency of plants.

Our findings showed that salinity stress declined photosynthesis pigments content, while algae extract foliar spraying improved them. Chlorophyll content and leaf photosynthesis are considered the prominent physiological indices of salinity tolerance in plants. Salinity stress reduces photosynthetic pigments content. Moreover, salinity reduces transpiration and stomatal conductance, leading to a declined gas exchange and, consequently, a decreased photosynthesis rate⁵⁸. Severe salinity caused a reduction in NADPH consumption through the C3 cycle and decreased the efficiency of leaf photosynthesis⁵⁹. It can be said that salinity reduces the photosynthesis pigments content by closing the stomata and lowering carbon metabolism⁶⁰. Microalgae extracts increased the nutrient capacity of the soil for crop growth, improved water-holding capacity, increased the antioxidant capacity and cell metabolism, and even chlorophyll content⁶¹. It has been shown that seaweed extract from Ascophyllum nodosum increased the content of chlorophyll in the leaves, which is attributed to the presence of aminobutyrate, glycine betaine, and betaine in the extract. Green microalgae Chlorella ellipsoida also improved wheat plants’ carotenoids content and antioxidant activity under salt stress⁶³ which is in agreement with our study. Foliar application with organic compounds like algae extracts seemingly triggers or enhance several biochemical pathways involved in the metabolism of several bioactive constituents and may even accelerate the activity of divergent enzymes responsible for tolerance under stressful environments. The microalga extract improved the growth attributed of the non-stressed plants. These responses could be associated with the algae extracts as plant growth-promoting compounds, antioxidants, osmoprotectants, and secondary metabolites that benefit the plant’s growth⁴⁸. Many studies have recommended that different algal extracts are rich in phytohormone-like plant-growth molecules, which boost the growth and yield of plants^64,65. Of these molecules, Martini, et al.⁶⁶ reported that the physical treatment of algal cells as a bio-stimulator agent ascribed to the liberation of specific peptides or proteins which has bio-stimulant properties. The extracts were rich in sugars, proteins, and amino acids, which may stimulate tolerance against various stresses.

The present results showed that the amount of TSC increased under moderate salinity stress. Still, under high stress, its content decreased, as the result observed in Queen Eliza strawberries under salinity stress, that during a long-term salinity stress period, a decrease in leaf area, photosynthetic capacity, and plant growth are observed, leading to a decrease in carbohydrate production in the plant⁶⁷. Salinity stress affects the biosynthesis of carbohydrates, their transport, and the use of these compounds in plant tissues⁶⁸. In another research, foliar spraying of seaweed extract increased the content of carbohydrates in strawberry plants. This increase in total carbohydrates content may be due to improved plant growth indicators such as plant height, number of leaves, leaf area, wet and dry weight of roots, and aerial organs⁶⁹.

Proteins are another biochemical indicator of salinity stress. Salinity decreases nitrate reductase activity and since protein synthesis depends on nitrate absorption, it is obvious that the reduction of nitrogen absorption can be the reason for the decline of proteins content⁷⁰. The decrease in the protein content under salt stress can be due to a reduction in protein synthesis or an increase in the pretentious compounds hydrolysis⁷¹. Microalgae increase the remaining nitrogen and carbon in the soil and improve soil pH and electrical conductivity⁷². As already mentioned, foliar treatment with algae extract influences the biochemical and physiological efficacy of plants in such a way as to enhance the secondary metabolites pool in favor of quality attributes and stress tolerance.

In our study, salinity stress and algae extracts increased the total antioxidant activity. Salinity stress significantly increased the activity of antioxidants, which is a protective mechanism in stressed plants⁷³. Antioxidants are compounds that effectively and in different ways prevent the action of free radicals and protect macromolecules like proteins, amino acids, lipids, and DNA infrastructure⁷⁴. ROS induced by salinity greatly declines by enzymatic and non-enzymatic antioxidant activities⁷⁵. Studies over the past few years showed that sulfated polysaccharides in seaweed have considerable antioxidant capacity. These compounds (galactans and sulfated fucans) induce defense responses against stressors⁷⁶.

The primary effect of ROS accumulation in the cell system is lipid peroxidation (high MDA content), which is a sign of oxidative stress⁷⁷. In saline conditions, the increase in free radicals leads to the breakdown of membrane lipids and the production of H₂O₂ and MDA⁷⁸. Hydrogen peroxide inside the cell can act as a secondary messenger and increase the activity and the expression of some antioxidant compounds⁷⁹. MDA is used as a marker to evaluate the damage to cell membranes under environmental stresses. The use of algae extract reduces the activity of antioxidant enzymes and the content of malondialdehyde, as well as significantly decreases ROS accumulation⁸⁰. Algae extracts can strengthen the plant’s defense systems and facilitate the synthesis of compounds that protect cells and organs. Recent studies show that algae extract can provide different biochemical, physiological, and molecular mechanisms to increase plant tolerance to salinity^81,82. Moreover, foliar treatment with stimulative compounds enhances the antioxidant potential of plants aligns with growth promotion, and hence accelerates the scavenging of ROS radicles for better tolerance under stress. However, the variation pattern of antioxidants shows diverse responses with different foliar treatments. The severity of salt stress is commonly associated with photosynthetic pigment catabolism. This could be associated with the negative salinity stress on chlorophyll and has been linked to the activation of Chl-degradative enzymes and the suppression of enzymes responsible for Chl biosynthesis^64,83. These deteriorations were followed by the reduction of proteins and amino acids biosynthesis. Furthermore, algal extract treatment efficiency increased free amino acids and soluble proteins parallel to the increment of nitrate reductase activity.

Like many abiotic stresses; salinity causes oxidative damage to plant cells by over-generation of reactive oxygen species (ROS)⁸⁴. The enzymatic antioxidant system can be divided into two categories; One reacts with ROS and keeps them at a low level (peroxidase, superoxide dismutase, and catalase), and the other regenerates oxidized antioxidants (ascorbate peroxidase and glutathione reductase)⁸⁵. Therefore, high antioxidant enzymes inhibit free radicals’ levels and reduce oxidative damage. SOD is the first line of defense against reactive oxygen species; by increasing the activity of SOD, ROS, especially the inhibition of superoxide radicals and their conversion to hydrogen peroxide and water which ultimately reduces the oxidative stress and damage to membranes⁸⁶. GPX enzyme is active in the cytosol, and its cofactor is glutathione. This enzyme breaks down hydrogen peroxide and turns it into water. Increasing its activity along with other cytosol defense mechanisms significantly helps maintain the membranes’ stability⁸⁷. Peroxidase is mainly located in the apoplastic space and vacuole and plays a vital role in the catalysis of H₂O₂ into H₂O and O₂⁸⁸. Peroxidases are distributed outside the cell and accelerate the conversion of H₂O₂ into water. The balance between the activity of antioxidant enzymes is an essential factor in determining the level of stability of superoxide and hydrogen peroxide radicals. This balance is necessary to prevent the excessive formation of hydroxyl radicals⁵³. Using seaweed extract as a foliar spray increases the antioxidant potential in plants under salt stress, which can explain the reduction of the cell damage caused by ROS⁸⁹. The use of seaweed extract increases tolerance to oxidative stress by enhancing the activity of superoxide dismutase, which scavenges superoxide radicals⁹⁰. Enzymes such as SOD, POX, CAT, and APX help protect tomato plants against oxidative damage and increase salt stress tolerance⁹¹.

The decrease in titratable acid compounds under severe salinity stress triggers high cellular respiration and the oxidation of more organic acids as substrate. This causes the consumption of more fruit acids and a decrease in the percentage of titratable acidity in the fruit⁹³. The increase in total phenolics content and antioxidant activity is a protective mechanism against oxidative damage to protect cellular structures caused by salinity. Under stress conditions, the activity of PAL (Phenylalanine Ammonio-Lyaze) as a critical enzyme in the biosynthesis of phenylpropanoid compounds increases. The enzyme stimulates the generation of phenolic compounds, including anthocyanins⁹⁴. The concentration of phenylpropanoids, especially flavonoids increases significantly in plants exposed to a wide range of environmental stresses⁹⁵. Anthocyanins are the most important antioxidant compounds that destroy free radicals and prevent their further production in the plant. Anthocyanins availability directly reduces stress and protects the plant⁹⁶. In general, algae extracts balance the plant’s endogenous hormone content and ratios, change the expression of genes for transporting nutrients through the cell membrane, stimulate photosynthesis, and reduce stress-related responses⁹⁷. The increase in the content of total phenolics using algae extract can be explained by the enzyme activity. So, the use of seaweed extract significantly improved the activity of the PAL, the most crucial enzyme responsible for the biosynthesis of polyphenols⁹⁸. Recently, researchers have reported a high concentration of phenolic substances in seaweed extracts. These compounds usually accumulate under stress and can inhibit reactive oxygen species, chelate metal ions, and stabilize proteins and membranes⁹⁹. Investigating Ulva rigida green algae extract on wheat plants under salinity stress exhibited an increase in total phenolics content⁹⁹. Similar results were obtained using Ascophyllum nodosum algae extract on the strawberry plants¹⁰⁰. The strawberry plant is a herbaceous species with a wide range of growth potentials and so capable of growth in many environments. However, the plant is quite sensitive to salinity as outlined in our study. The foliar algae extract treatment was potentiated to mitigate the adverse effects of salinity. Moreover, biostimulats like algae extracts contain several substrate can be utilized as the skeleton or as a mediator for the biosynthesis of several group of primary and secondary metabolites. These recently generated biomolecules enhance the tolerance to stressors and simultaneously maintain the growth potential. Besides, biostimulants may trigger or stimulate several stress responsive metabolic pathways which directly or indirectly are involved in the stress tolerance and may fortify the growth related traits as well. Salinity damaged several aspects of growth and physiological responses of strawberry plants, however, foliar treatments were able to partially or totally neutralize the sharp effect of salinity. Our findings are in agreement with many other research themes that salinity defects can be smoothened by a reliable methodology based on biostimulants. As already known, alge extracts are also containing several hormonal substrates or hormone analogoues which may onset diverse sponteounous metabolic cascades to overcome the salinity damages. Several other biomoleculaes involed in algae extrats such as plyamines may even intensify the stress responsive feedback.

Conclusion

Salt stress adversely affected the growth and the physiological responses of plants. The results showed that salinity reduced photosynthetic pigments and osmolytes content, antioxidant enzymes activity, and phenolic compounds content. On the other hand, the foliar application of Chlamydomonas algae extracts mitigated the adverse effects of stress. In general, algae extract increased the photosynthetic pigments content and the activity of antioxidant enzymes by reducing the content of hydrogen peroxide and malondialdehyde. Overall, the application of Chlamydomonas extract (especially 20 ml.L^-1) improved the growth potential of plants under salinity. In general, algae extract promisingly enhanced the stress tolerance in plants and would be a reliable candidate for future studies in stress-prone environments. The foliar algal treatment is a bright idea and has the potential to enhance the productivity of strawberry plants under marginal saline lands or in greenhouse conditions with low-quality water resources. The limitations behind the commercial application of algae extracts are the compositional content of the extract and, the exact formulation of the final product. Pilot experiments with some other related algae genus or species are essential to decide on the commercialization of the desired extract. Moreover, detailed studies with several other strawberry varieties are necessary to reach a solid opinion for advising the results to the extension section and pioneer producers.

Author contributions

Conceptualization, A.E., S.J.; data curation, S.J., F.H. and F.R.; formal analysis and methodology, F.R., S.J., and M.B.H.; project administration, F.R.; visualization, F.R., S.J., M.B.H., S.J.B., and A.E.; writing – original draft, S.H., F.R., and A.E.,; writing – review and editing, M.B.H., S.J.B., and A.E. All authors have read and agreed to the published version of the manuscript.

Funding

The authors wish to thank the University of Maragheh, Iran, for the financial support of the study.

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Ethics approval and consent to participate

All procedures were conducted according to the relevant institutional, national, and international guidelines and legislation.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.