The certified Strawberry plantlets, cultivar Paros, were obtained from a commercial nursery in Sanandaj (Kurdistan province, western Iran). The plantlets were transferred into cylindrical plastic pots (20 cm × 18 cm) containing sterilized soil with a texture consisted of sand (37%), silt (39%), clay (20%), and organic matter (4.0%). The soil texture class was loam. No fertilization was applied to the pots during the experiment. A uonset greenhouse (height: 3 m, diameter: 6 m), with polyethylene cover and pad & fan cooling system located in Mollasani city [longitude (λ): 48.8648334, latitude (ϕ): 31.6242601], was used. The conditions of the greenhouse (temperature: 22 ± 3°C, relative humidity: 45 ± 5%, 12-h photoperiod) were regularly checked in terms of temperature and humidity. After 30 days, the first flowers were removed for better plant growth, and then the treatments were applied. The treatments were applied before the emergence of flowers, and after the fruits reached commercial maturity, they were harvested manually, wrapped in foil, instant frozen in liquid nitrogen, and stored at −70°C for further experiments.

The brown alga (S. angustifolium) was collected from the shores of Chabahar, southeastern Iran (25.300278°N 60.612778°E), and transferred to the laboratory. To remove impurities, the alga was first washed with deionized distilled water (ddH₂O) and impurities were removed. After air-drying for 10 days, the tissues were then ground using a grinder. To prepare the aqueous solution, 50 g of alga powder and 500 mL of ddH₂O were shacked at room temperature. Then it was boiled for 60 min and filtrated. A stock solution of the extract with a concentration of 5 mg/mL was prepared and used for the assay (Sivasankari et al., 2006; Ramarajan et al., 2012).

A virulent isolate (MP) of M. phaseolina was prepared from the Fungal Culture Collection of Shiraz University (Shiraz, Iran). Fungal microsclerotia were obtained without culture, by placing a block of agar culture containing active fungal mycelium in a flask containing potato extract and dextrose (Short and Wyllie, 1978). The flask was incubated for 3 months at room temperature until thick tangled strands of microsclerotia were formed. These thick tangled strands were removed and washed 3 times with ddH₂O and dried at 35°C and then it was ground gently with a mortar. Microsclerotia was mixed with 1,000 g of sterile sand and stored at 4°C. Before inoculation, this mixture was added to the soil required to be contaminated by the pathogen (Goudarzi et al., 2011).

The treatments were divided into four groups: control, pathogen (M. phaseolina), alga (S. angustifolium), and alga-pathogen. Control included non-treated pathogen-free plants irrigated with 180 mL of ddH₂O every 2 days. In the pathogen treatment, the soil next to the crown was replaced with 1,000 g of infested sand containing 100 viable of the fungus microsclerotia/g soil that was prepared in advance, and irrigation was applied. In case of non-inoculated plants, 1,000 g of pathogen-free sterilized sand was placed around their crown. In the alga-pathogen and alga treatment, 7.0 mL the algae extract (5 mg/mL concentration) was applied as a foliar spray every 3 days, and the application of this treatment continued until the appearance of flowers, and after the appearance of flowers and fruits, irrigation was conducted. The experiment was terminated 120 days post incubation in the greenhouse. A completely randomized design with total number of 4 replications per treatment was used and the experiments were performed two times.

The disease severity caused by M. phaseolina in strawberry plants was visually evaluated for the inoculated plants 92 days post inoculation. To this end, each plant was inspected and scored from 0 to 5 according the developed symptoms (Table 1). The percentage of disease severity index (DSI) was then measured using Equation (1) which has been previously described by Camara et al. (2013):

In which DSI = disease severity index; e = class; Re = number of plants in class €; N = total number of plants.

The vegetative and reproductive growth of strawberry plants were mainly determined according to Breen and Martin (1981). Vegetative traits included plant height, root length, aerial length, fresh weight of aerial organs, and dry weight of root. Also, reproductive indices included fruit number, fruit weight, fruit length and diameter, and fruit volume. Water displacement technique was used to measure the fruit size. The indices were measured at the end of the experiment.

To measure the amount of total phenol in the leaf, the Folin-Cicalito reagent was used (Singleton and Rossi, 1965). For this purpose, 1,500 μL of Folin-Cicalito reagent diluted with a ratio of one to ten (1:10) was added to 300 μL of the leaf homogenate (1 g) and placed at room temperature (25°C) for 5 min. 1,200 μL of 7.5% sodium carbonate were added to the resulting mixture and placed on a shaker in a dark place for 90 min. Six mL of ddH₂O were added to the solution and finally, the absorbance number at 765 nm wavelength was recorded with a spectrophotometer (made in the USA—model 2100-UV). The amount of total phenol was calculated using the standard curve of gallic acid and the results were expressed in terms of mg of gallic acid per 100 g of fresh fruit weight. To prepare gallic acid standard solutions, 0.1 g of gallic acid was dissolved in ddH₂O and the final volume was brought to 100 mL using distilled water. Then, to prepare 0, 10, 20, 30, 40, 80, 160, 320, and 480 mg/100 mL standards, 0, 0.1, 0.2, 0.3, 0.4, 8 0.0, 1.6, 3.2, and 4.8 mL of gallic acid solution were removed and made up to 10 mL using ddH₂O. Then, 300 μL were taken from the standards, and the rest of the steps were carried out like the samples.

For extraction, one g of strawberry leaf was kept with 8 mL of 80% methanol for 12 h at 4°C. Then it was centrifuged at 8,000 g for 25 min at 4°C. To measure the flavonoids, one mL of supernatant was mixed with 0.5 mL of 5% sodium nitrite. After 6 min, 0.5 mL of 10% aluminum chloride and after 6 min, 2 mL of one molar sodium hydroxide were added to the mixture and kept for 15 min. The absorption number at 510 nm wavelength was recorded with a spectrophotometer and the results were reported in terms of mg per 100 g of fresh fruit weight (Li et al., 2014).

The total antioxidant activity (TAA) was determined using the free radical reduction method (diphenylpicrylhydrazyl [DPPH]) with the method of Sanchez-Moreno (2002). First, DPPH solution with a concentration of 0.1 mM was obtained by dissolving 6 mg of DPPH in 100 mL of 80% methanol. This solution was prepared daily in a dark container to measure the inhibition percentage. To prepare the alcoholic extract, 0.3 g of leaf sample was pounded with 3 mL of solvent in the dark and shaken at 100°C for 30 min. After that, it was centrifuged for 10 min at 4°C at 5,000 rpm and the supernatant extract was used to measure TAA. For this purpose, 75 μL of the alcoholic extract was taken and 2,925 μL of DPPH solution was added to it. The samples were placed in the dark chamber for 30 min. The absorbance of the samples was measured at a wavelength of 517 nm (A₅₁₇) using a spectrophotometer. The control sample was read using DPPH solution without adding the extract. Finally, using Equation (2), the inhibition percentage of the samples was calculated:

In which CA is the absorbance of the control and SA is the absorbance of the sample.

To measure the activity of polyphenol oxidase (PPO) according to the proposed method (Worthington, 1988), one mL of 50 mM sodium phosphate buffer, one mL of 1 mM tyrosine, and 900 μL of ddH₂O were placed into the cuvette and 100 μL of enzyme extract was added to the cuvette. It was added and the increase in absorbance at 280 nm wavelength was recorded using a spectrophotometer at 2, 4, 6, 8, 10, and 12 min. By placing the absorbance changes (the difference between the highest and the lowest number read during 12 min, which is linear) in Equation (3), the amount of enzyme activity was calculated in mg/g/min.

Phenylalanine ammonia-lyase (PAL) activity was measured using the method of Zucker (1968). First, one g of leaf tissue was crushed with 5 mL of sodium borate buffer [pH 8.8]. Then the prepared solution was centrifuged at 12,000 rpm for 10 min. Five hundred microliter of the centrifuged extract, two mL of sodium borate buffer with 500 μL of 20 mM phenylalanine solution were poured into a tube and placed in a hot water bath for one h at 37°C was placed. Enzyme activity was determined by measuring the absorbance of the solution at a wavelength of 290 nm for one h with an interval of 15 min and was reported as units per mg of enzyme extract protein (U/mg protein).

All data collected during the experiment were transferred into Excel software and prepared for statistical analysis. Data analysis was performed using SAS software (v. 9.4) (Yuan, 2011). The statistical design used in this study was a completely randomized design with 4 replications and the means were compared based on Duncan's multiple range test with an alpha error level of 5%. In the case of biochemical experiments, technical (sample) and biological (plant) replications were considered per treatment. To analyze the data from disease severity index, Kruskal–Wallis H test was used and the significance at P= 0.01 level was determined.

The symptoms of the disease including blackening of root and crown, reduction of leaf area, leaf necrosis, reduction of fruit size, and decreased number of fruits were observed on M. phaseolina-inoculated plants 92 days post inoculation (dpi) (Figure 1a). Strawberry plants inoculated with the fungus were treated with the alga extract. In these plants, there are fewer symptoms compared to the non-treated plants (Figure 1b). Healthy strawberry plants treated with the alga extract showed improved vegetative growth (Figure 1c). Figure 1D shows the comparison of strawberry fruits among different treatments at 92 dpi. Accordingly, in plants treated with the alga extract, the size of the fruits was larger than those of non-treated plants (Figure 1d). In contrast, the infected plants which had not been treated with the alga extract produced the smallest fruits. Also, infected plants treated with the alga extract produced larger fruits compared to infected non-treated plants. Evaluation of disease severity showed that alga-treated infected plants had a significantly lower disease severity index (20%) than non-treated infected plants (80%). These results showed that foliar application of S. angustifolium extract to strawberry plants can reduce the disease severity caused by M. phaseolina.

Statistical analysis of the data from the effect of different treatments on strawberry plant length showed that the highest plant length (29.75 cm) was related to the plants under alga extract treatment, while the lowest plant length was found in non-treated infected plants (17 cm) (Table 2). Also, the length of the plant treated with alga extract was significantly higher than the other treatments, and the length of the infected plants was significantly lower than other treatments. The difference between the length values of non-infected alga-treated plants and infected alga-treated plants was not significant.

Similarly, the highest root length (18 cm) was found in non-infected alga-treated plants, while non-treated infected plants showed the lowest root length (8 cm) (Table 2). The root length of infected alga-treated plants was significantly higher than infected non-treated plants.

The highest value of aerial length (14 cm) was recorded for infected alga-treated plants (Table 2). The second highest value of aerial length (12 cm) belonged to non-inoculated alga treatment plants. The remaining plants did not show any significant difference in aerial length.

The non-inoculated alga-treated plants showed the highest fresh weight of the aerial organs (12.025 g), while non-treated infected plants exhibited the lowest fresh weight of aerial organs (5.84 g) (Table 2). Furthermore, this index in infected alga-treated plants was significantly higher than in non-treated infected plants.

The dry weight of roots in control plants was determined as the highest value (10.97 g), while non-treated infected plants showed the lowest value (5.58 g) (Table 2). Also, this index in alga-treated infected plants was significantly higher than in non-treated infected plants. The difference between the dry weight of roots of non-infected alga-treated plants and infected alga-treated plants was not significant.

Table 3 shows the effect of alga treatment on infected and non-infected strawberry plants. The mean number of fruits (5.50) in the alga-treated plants was significantly larger than that of other plants. Also, the significantly smallest mean number of fruits (3.00) was found in the non-treated infected plants compared to other treatments. Similarly, the highest value of fruit weight (8.86 g) and fruit volume (10.37 g/mL) were observed in alga-treated plants. These indices in non-treated infected plants were significantly lower than in plants under other treatments. Also, the highest fruit diameter was found in alga-treated plants and alga-treated infected plants (16.87 mm and 16.56 mm, respectively). The non-treated infected plants showed the lowest fruit diameter (12.5 mm) compared to other plants. The highest fruit length (27.287 mm) belonged to the alga-treated plants. The non-treated infected plants showed the lowest fruit diameter (12.50 mm) compared to plants under other treatments. There was no significant difference between the fruit diameter of alga-treated infected plants and control plants.

The results showed that alga-treated plants had a significant effect on the total phenol, flavonoids, and TAA of strawberries (Figure 2). Alga-treated infected plants had significantly more phenol content (1.60 mg/g) and flavonoids (1.44 mg/g) than plants under other treatments (Figures 2A,B). The lowest phenol content and flavonoids were found in control plants (0.93 mg/g and 0.51 mg/g, respectively). Phenol content and flavonoids were not significantly different between alga-treated and non-treated infected plants (Figures 2A,B). The highest level of TAA (0.2) was recorded for alga-treated infected plants (Figure 2C). In contrast, non-treated infected plants showed the lowest level of TAA (0.06).

The results from the effect of different treatments on the PAL content in strawberry plants showed that the alga-treated infected plants had the highest enzyme content (0.89 U/mg) found while the lowest amount of PAL enzyme was recorded for alga-treated non-infected plants (0.54 U/mg) (Figure 3A). The content of the PAL enzyme was not significantly different between non-treated infected and control plants (Figure 3A).

In the alga-treated infected plants, the highest amount (0.65 mg/g/min) of PPO enzyme was found while the lowest value was determined in the alga-treated non-infected plants (0.34 mg/g/min) (Figure 3B). These results showed that foliar application of alga extract can significantly increase the content of PAL and PPO in infected plants.