Antifungal Properties of Fucus vesiculosus L. Supercritical Fluid Extract Against Fusarium culmorum and Fusarium oxysporum

In this study, potential antifungal properties of a brown alga Fucus vesiculosus were evaluated. The algal extract was obtained with the use of supercritical fluid extraction (scCO2) at a temperature of 50 °C under a pressure of 300 bar. The aqueous solution of the extract at the concentration of 0.05%, 0.2%, 0.5% and 1.0% was studied against pathogenic fungi on a liquid RB medium. This study is the first report on antifungal properties of the brown algae F. vesiculosus scCO2 extract against Fusarium culmorum and Fusarium oxysporum phytopathogens. The concentrations of the studied extract (0.5% and 1.0%) were demonstrated to have an ability to inhibit 100% growth of macroconidia within 144 h, as well as an ability to cause their total degradation. As a result of the study, the antifungal effect of fucosterol against F. culmorum was also indicated. The total macroconidia growth was inhibited by 1.0% fucosterol. Moreover, at lower concentrations (0.05–0.2%) of fucosterol, macroconidia were characterized by shorter length and structural degradation was observed. The mycelial growth of Fusarium oxysporum (Fo38) by 1% scCO2 F. vesiculosus extract was analyzed at the level of 48% after 168 h of incubation, whereas 100% extract was found to be effective in F. culmorum (CBS122) and F. oxysporum (Fo38) growth inhibition by 72% and 75%, respectively after 168 h of incubation.


Introduction
Crops are exposed to diseases caused by pests and microorganisms during growth but also, after harvest. These factors can result in a large reduction in the annual level of food production worldwide, which depending on the source, is estimated at 25%-50% [1,2]. One-third of these losses are caused by fungal diseases. Currently, more than 10,000 species of fungi that can cause mycosis of plants have been classified, the most serious of which may be representatives of the genera Botrytis, Rhizopus, Alternaria, Penicillium, Aspergillus, Rhizoctonia and Fusarium [3,4].
Polyphagous fungi of the Fusarium genus, especially those belonging to the species of Fusarium culmorum and Fusarium oxysporum [5,6], are particularly dangerous for plants and human

Preparation of Fucus vesiculosus Extract
The extraction was performed with a supercritical fluid extraction (SFE) system on a laboratory-scale installation with a working temperature of up to 80 • C and pressure of up to 450 bar. The system was equipped with a 1 L extractor. Dried and milled brown algae (Fucus vesiculosus) (200 g) were extracted with carbon dioxide. The temperature and pressure were set at 50 • C and 300 bar, respectively. The method was used according to the one described in our previous study [40]. The extraction resulted in 2.83 wt% extraction efficiency.

Growth Rate of Fusarium Strains on the Medium with Extract
To determine the effect of the scCO 2 F. vesiculosus extract on the growth and development of phytopathogenic Fusarium strains, mycelia discs (0.8 cm) from initial cultures (grown on PDA medium for 7 days at 20 • C) were transferred to PDA medium with the addition of 100% extract (30 mg was spread on the agar surface using cell spreader) and 1% extract (1 mL of aqueous suspension of the extract was spread on the agar surface using cell spreader) in petri dishes (total diameter of 9.0 cm). At the same time, control versions (PDA medium without extract) were prepared for each studied strain. The petri dishes were incubated at 20 • C for 72 h, 96 h, 120 h and 168 h. After 72 h, 96 h, 120 h and 168 h of incubation, the diameters of the mycelium were measured and the R factor of the growth rate was calculated from the formula and was presented as mm 2 mycelium/day (1). The IC 50 (half-maximal inhibitory concentration) of Fucus vesiculosus scCO 2 extract and fucosterol was also provided. The percentage inhibition of mycelium growth of Fusarium strains in a comparison with the control was determined.

Preparation of RB Liquid Medium
A liquid RB medium was prepared by mixing the following components KH 2 PO 4 (1.0 g/L), MgSO 4 ·7H 2 O (0.5 g/L), KCl (0.5 g/L), (NH 4 ) 2 SO 4 (0.5 g/L), glucose (10.0 g/L) and then dissolving in 1 L of distilled water. The medium was then sterilized at a temperature of 121 • C and pressure of 0.75 atm for 30 min. After autoclaving, the medium was supplemented with a separately prepared sterile mixture of microelements, including Na 2 30.0 mg/L) was added to the medium upon cooling (less than 60 • C) in order to avoid its decomposition.

Preparation of Fusarium Strains Macroconidia
Macroconidia of studied F. culmorum (FcCBS122 and DEM Fc37) and F. oxysporum (FoCBS129 and DEM Fo38) strains used for the preparation of inocula were obtained from a culture grown on a liquid RB medium with 1.0% glucose as a carbon source. The isolates were cultivated in darkness at 20 • C and 60% relative humidity in an Innova 4900 growth chamber (New Brunswick Scientific, Edison, NJ, USA) at 120 rpm for 7 days. Then, the fungal cultures were filtered through 5 layers of a sterile cotton gauze. The macroconidia were obtained by centrifuging the supernatants (10,000 g for 15 min). The supernatant was then collected and the resulting conidia pellet was resuspended in sterile distilled water. The density of the macroconidia suspension was determined in a hemocytometer using a light microscope (Olympus BX53 Upright Microscope) and then diluted with sterile distilled water to a desired concentration [38].

Preparation of Final Samples
The extract of F. vesiculosus was prepared with a concentration of 2.0% (w/v) by suspending 400 mg of extract in distilled water (20 mL). Correspondingly, F. vesiculosus extract at concentrations of 0.05%, 0.2%, 0.5% and 1.0% was also prepared. A macroconidia suspension (500 µL) of the F. culmorum DEM Fc37 strain (at a concentration of 2.0 × 10 6 per mL) was transferred to four separate sterile Eppendorf tubes, followed by centrifugation (10,000 g for 15 min) on a centrifuge. The supernatant was then collected and the macroconidia pellet was supplemented with 500 µL of the extract at concentrations of 0.05%, 0.2%, 0.5% and 1.0%. Similarly, the samples of F. culmorum CBS122, F. oxysporum DEM Fo38, F. oxysporum CBS129 were prepared. In the experimental version with the RB medium, a macroconidia suspension (500 µL) of the F. culmorum DEM Fc37 strain (at a concentration of 2.0 × 10 6 per mL) was also transferred to four separate sterile Eppendorf tubes, followed by centrifugation (10,000 g for 15 min) on a centrifuge. The supernatant was then collected and the macroconidia pellet was supplemented with 250 µL of 2.0% extract and 250 µL of the RB medium to obtain a concentration of 1.0%. Similarly, the samples of 0.5%, 0.2% and 0.05% extract was obtained by mixing 125 µL of 2.0% extract + 375 µL of the RB medium, 50 µL of 2.0% extract + 450 µL of the RB medium and 12.5 µL of 2.0% extract + 487.5 µL of the RB medium, respectively.

Preparation of Fucosterol Standard
The fucosterol standard was prepared with a concentration of 2.0% (w/v) by suspending 2.0 mg extract in distilled water (98 µL). A macroconidia suspension (100 µL) of the F. culmorum DEM Fc37 strain (at a concentration of 2.0 × 10 6 per mL) was transferred to four separate sterile Eppendorf tubes, followed by centrifugation (10,000 g for 15 min) on a centrifuge. The supernatant was then collected and the macroconidia pellet was supplemented with 50 µL of the RB medium and 50 µL of 2.0% fucosterol to obtain a concentration of 1.0%. Similarly, samples of 0.5%, 0.2% and 0.05% fucosterol were obtained by mixing 75 µL RB medium + 25 µL 2.0% fucosterol, 90 µL RB medium + 10 µL 2.0% fucosterol and 97.5 µL RB medium + 2.5 µL 2.0% fucosterol, respectively. Figure 1 presents the schema of studied samples. The samples names are used throughout the text.

Preparation of Fucosterol Standard
The fucosterol standard was prepared with a concentration of 2.0% (w/v) by suspending 2.0 mg extract in distilled water (98 μL). A macroconidia suspension (100 μL) of the F. culmorum DEM Fc37 strain (at a concentration of 2.0 × 10 6 per mL) was transferred to four separate sterile Eppendorf tubes, followed by centrifugation (10,000 g for 15 min) on a centrifuge. The supernatant was then collected and the macroconidia pellet was supplemented with 50 μL of the RB medium and 50 μL of 2.0% fucosterol to obtain a concentration of 1.0%. Similarly, samples of 0.5%, 0.2% and 0.05% fucosterol were obtained by mixing 75 μL RB medium + 25 μL 2.0% fucosterol, 90 μL RB medium + 10 μL 2.0% fucosterol and 97.5 μL RB medium + 2.5 μL 2.0% fucosterol, respectively. Figure 1 presents the schema of studied samples. The samples names are used throughout the text. control; E-extract; F-fucosterol; CON-conidia; CON.W-conidia suspended in water).

Evaluation of Macroconidia Germination Capacity of Fusarium spp.
After 24 h, 48 h, 72 h, 96 h, 120 h and 144 h of incubation in darkness at 20 °C and 60% relative humidity in an Innova 4900 growth chamber (New Brunswick Scientific, Edison, NJ, USA) at 120 rpm, 30 μL of each sample was applied on a sterile slide, observed and photographed using an Olympus BX53 Upright Microscope equipped with an Olympus XC30 camera. The presented results are the percentage of germinated macroconidia and the average length of hyphae of 300 conidia from 3 independent experiments/replicates (100 spores per one repetition) observed in 10 different microscopic fields. The obtained results were calculated in relation to an appropriate control (water, RB).

Statistical Analysis
The statistical analyses were conducted using Statistica 12.5 (StatSoft Inc., Kraków, Poland). All assays were performed in three independent experiments and the data were expressed as means ± SD calculated from these experiments. The data were subjected to one-way analysis of variance (ANOVA) followed by a Tukey's post hoc test, with the significance evaluated at p < 0.05.

Evaluation of Macroconidia Germination Capacity of Fusarium spp.
After 24 h, 48 h, 72 h, 96 h, 120 h and 144 h of incubation in darkness at 20 • C and 60% relative humidity in an Innova 4900 growth chamber (New Brunswick Scientific, Edison, NJ, USA) at 120 rpm, 30 µL of each sample was applied on a sterile slide, observed and photographed using an Olympus BX53 Upright Microscope equipped with an Olympus XC30 camera. The presented results are the percentage of germinated macroconidia and the average length of hyphae of 300 conidia from 3 independent experiments/replicates (100 spores per one repetition) observed in 10 different microscopic fields. The obtained results were calculated in relation to an appropriate control (water, RB).

Statistical Analysis
The statistical analyses were conducted using Statistica 12.5 (StatSoft Inc., Kraków, Poland). All assays were performed in three independent experiments and the data were expressed as means ± SD calculated from these experiments. The data were subjected to one-way analysis of variance (ANOVA) followed by a Tukey's post hoc test, with the significance evaluated at p < 0.05.

The Effect of F. vesiculosus Extract on the Mycelial Growth of Fusarium Strains
The scCO 2 F. vesiculosus extract significantly limited the mycelial growth of phytopathogenic Fusarium strains. The value of R factor of the CBS122, Fc37, CBS129 and Fo38 strains was lower by about 1.2, 1.5, 1.4 and almost 2 times lower on the medium with the addition of 1% extract, as well as 5-, 2-, 2.3-and 4-times lower on the medium with the addition of 100% extract in a comparison with the control, respectively (Table 1). Figure 2 presents the effect of 1% and 100% F. vesiculosus extract on the inhibition of mycelial growth of F. culmorum and F. oxysporum. In the case of 1% F. vesiculosus, the inhibition of mycelial growth of F. culmorum was at the level of 17% (72 h) to 11% (168 h) and 29% (72 h) to 39% (168 h), respectively, for CBS122 and Fc37. The inhibition of CBS129 and Fo38 was at the level of 28%-16% and 40%-48% from 72 h to 168 h of incubation, respectively. The mycelial growth of F. culmorum was strongly and significantly inhibited by 100% F. vesiculosus extract as compared to 1% extract. For instance, the inhibition of F. culmorum and F. oxysporum after 72 h was 70%, 53%, 57%, and 60%, respectively, for CBS122, Fc37, CBS129 and Fo38. The highest inhibition of CBS122 (80%) was observed after 96 h of incubation treated with 100% extract, whereas Fc37 was inhibited at the highest rate (53%) after 72 h of incubation. As for F. oxysporum, the treatment with 100% extract resulted in the highest inhibition of CBS129 (57%) and Fo38 (75%) after 72 h and 168 h of incubation, respectively. Nowadays, the most common method for plant protection against phytopathogenic fungi is the use of chemical fungicides. These products are characterized by a high effectiveness, however, concerns regarding the safety and health of food mean that alternative methods of combating phytopathogens are being sought [41]. In order to limit the negative effects of the use of non-biological plant control agents, studies are being conducted to develop environmentally friendly crop technologies based on the use of soil microorganisms or their metabolites and natural products (plants extracts) in accordance with the assumption of integrated pest management (IPM) and organic farming [42,43]. Manni et al. [44] studied the influence of five chemical fungicides, such as Prodazin (Carbendazim), Dithane (mancozeb), Alliette Express (Fosetyl-Al), Tachigazol (Hymexazol) and Beltanol (Chinosol) in the concentration range of 10, 25, 50 and 100 ppm against Fusarium oxysporum. The best results were observed for Tachigazol and Beltanol, with the highest F. oxysporum inhibition ability among all the tested fungicides being that of Beltanol. After 144 h of incubation, F. oxysporum was inhibited by over 90% and 70%, respectively, by Beltanol (100 ppm) and Tachigazol (100 ppm). The least effective was Alliette Express (Fosetyl-Al) with F. culmorum inhibition by 7.03% (10 ppm) and 0.61% (100 ppm). In another study, the increase of Fusarium growth inhibition was noticed with the increase of the concentrations of fungicides, such as propiconazole (250 g/L), metconazole (60 g/L) and tebuconazole (250 g/L) [45]. In the triazole group, metconazole showed the strongest inhibitory effect on the growth of all tested fungi. The growth of Fusarium culmorum and Fusarium oxysporum was inhibited by 100% and 70%-100%, respectively. The results provided in this study, indicate that F. vesiculosus scCO 2 extract may be used as a potential additive to biological plant protection products as the extract was characterized by a high inhibitory effect on Fusarium culmorum and Fusarium oxysporum, similar to the effect caused by the chemical fungicides. Moreover, the extracts obtained with the use of supercritical carbon dioxide are free of solvents, as carbon dioxide is removed by depressurization [30]. Figure 3 presents the effect of 100% F. vesiculosus extract on F. culmorum (CBS122, Fc37) and F. oxysporum (CBS129, Fo38) on PDA medium after 168 h of incubation. Nowadays, the most common method for plant protection against phytopathogenic fungi is the use of chemical fungicides. These products are characterized by a high effectiveness, however, concerns regarding the safety and health of food mean that alternative methods of combating phytopathogens are being sought [41]. In order to limit the negative effects of the use of non-biological plant control agents, studies are being conducted to develop environmentally friendly crop technologies based on the use of soil microorganisms or their metabolites and natural products (plants extracts) in accordance with the assumption of integrated pest management (IPM) and organic farming [42,43]. Manni et al. [44] studied the influence of five chemical fungicides, such as Prodazin (Carbendazim), Dithane (mancozeb), Alliette Express (Fosetyl-Al), Tachigazol (Hymexazol) and Beltanol (Chinosol) in the concentration range of 10, 25, 50 and 100 ppm against Fusarium oxysporum. The best results were observed for Tachigazol and Beltanol, with the highest F. oxysporum inhibition ability among all the tested fungicides being that of Beltanol. After 144 h of incubation, F. oxysporum was inhibited by over 90% and 70%, respectively, by Beltanol (100 ppm) and Tachigazol (100 ppm). The least effective was Alliette Express (Fosetyl-Al) with F. culmorum inhibition by 7.03% (10 ppm) and 0.61% (100 ppm). In another study, the increase of Fusarium growth inhibition was noticed with the increase of the concentrations of fungicides, such as propiconazole (250 g/L), metconazole (60 g/L) and tebuconazole (250 g/L) [45]. In the triazole group, metconazole showed the strongest inhibitory effect on the growth of all tested fungi. The growth of Fusarium culmorum and Fusarium oxysporum was inhibited by 100% and 70%-100%, respectively. The results provided in this study, indicate that F. vesiculosus scCO2 extract may be used as a potential additive to biological plant protection products as the extract was characterized by a high inhibitory effect on Fusarium culmorum and Fusarium oxysporum, similar to the effect caused by the chemical fungicides. Moreover, the extracts obtained with the use of supercritical carbon dioxide are free of solvents, as carbon dioxide is removed by depressurization [30]. Figure 3 presents the effect of 100% F. vesiculosus extract on F. culmorum (CBS122, Fc37) and F. oxysporum (CBS129, Fo38) on PDA medium after 168 h of incubation.

The Effect of F. vesiculosus Extract on the Fusarium Macroconidia
The present investigations demonstrated that the F. vesiculosus scCO2 extract tested had a high in vitro antifungal activity against F. culmorum and F. oxysporum macroconidia. The susceptibility of studied pathogens towards the brown algae extract was reflected in a trend received from two quantitative bioassays (fungal macroconidia germination and hyphae length). The best efficacy was when pathogens were treated with the F. vesiculosus extract at concentrations of 0.5% and 1.0%. The same effect was observed for all tested pathogens (F. culmorum Fc37, F. culmorum CBS122, F. oxysporum Fo38, F. oxysporum CBS129). Figure 4 presents the microscopic view of F. culmorum

The Effect of F. vesiculosus Extract on the Fusarium Macroconidia
The present investigations demonstrated that the F. vesiculosus scCO 2 extract tested had a high in vitro antifungal activity against F. culmorum and F. oxysporum macroconidia. The susceptibility of studied pathogens towards the brown algae extract was reflected in a trend received from two quantitative bioassays (fungal macroconidia germination and hyphae length). The best efficacy was when pathogens were treated with the F. vesiculosus extract at concentrations of 0.5% and 1.0%. The same effect was observed for all tested pathogens (F. culmorum Fc37, F. culmorum CBS122, F. oxysporum Fo38, F. oxysporum CBS129). Figure 4 presents the microscopic view of F. culmorum DEMFc37 macroconidia treated with the F. vesiculosus extract at the concentration of 0.05%-1.0% after 120 h of incubation.  Fc37 (B)) and F. oxysporum (CBS129 (C), Fo38 (D)) on PDA medium after 168 h of incubation.

The Effect of F. vesiculosus Extract on the Fusarium Macroconidia
The present investigations demonstrated that the F. vesiculosus scCO2 extract tested had a high in vitro antifungal activity against F. culmorum and F. oxysporum macroconidia. The susceptibility of studied pathogens towards the brown algae extract was reflected in a trend received from two quantitative bioassays (fungal macroconidia germination and hyphae length). The best efficacy was when pathogens were treated with the F. vesiculosus extract at concentrations of 0.5% and 1.0%. The same effect was observed for all tested pathogens (F. culmorum Fc37, F. culmorum CBS122, F. oxysporum Fo38, F. oxysporum CBS129). Figure 4 presents the microscopic view of F. culmorum DEMFc37 macroconidia treated with the F. vesiculosus extract at the concentration of 0.05%-1.0% after 120 h of incubation.  The antifungal properties exhibited by marine algae may be attributed to the presence of biologically bioactive compounds, including polysaccharides and derived oligosaccharides, lipids, fatty acids, sterols, phenolic compounds, pigments, lectins, alkaloids, and terpenes, as well as halogenated compounds (furanones, bromoditerpenes, bromophenols) [46]. Antifungal activities of particular bioactive compounds have been reported previously [47][48][49]. In the case of brown algae, in particular, fucoidans are the main group belonging to polysaccharides [50]. In a review on Fucus spp. by Catarino et al. [25], the content of fucoidans, alginic acid and laminaran in F. vesiculosus may be in the range of 3.4%-25.7%, 8.4%-58.5% and 0.6-7.0 dry weight, respectively. According to our previous study [40], Fucus vesiculosus scCO 2 extract contains fucosterol in the amount of 8.05 wt%. The microwave-processed extract of F. vesiculosus was studied in terms of the production of fucoidanase enzymes by a solid-state fermentation with two fungal strains, such as Aspergillus niger and Mucor sp. [51]. However, according to Alexeeva et al. [52], fucoidans are described as low-activity compounds.
Two experiments were carried out in order to examine the behavior of fungi in two environments. The water control (W.C) for the first experiment contained distilled water with particular Fusarium macroconidia, whereas for the second one, the control (RB.E) constituted macroconidia on a liquid RB medium. The aim of the second bioassay was to evaluate the influence of the F. vesiculosus extract on Fusarium conidia when the conditions were favourable for their growth as they could have taken up nutrients from the RB medium. The visual effect is shown in Figure 5. The obtained results of the percentage of germinated macroconidia, as well as the percentage of the average length of hyphae, showed a consistent fungistatic and antifungal effect. The extract concentrations of 0.5% and 1.0% were more effective than that of 0.05% and 0.2%. After 10 days of the experiment, F. vesiculosus scCO 2 extract at a concentration of 1.0% still showed a strong inhibitory effect on all studied Fusarium conidia.
described as low-activity compounds.
Two experiments were carried out in order to examine the behavior of fungi in two environments. The water control (W.C) for the first experiment contained distilled water with particular Fusarium macroconidia, whereas for the second one, the control (RB.E) constituted macroconidia on a liquid RB medium. The aim of the second bioassay was to evaluate the influence of the F. vesiculosus extract on Fusarium conidia when the conditions were favourable for their growth as they could have taken up nutrients from the RB medium. The visual effect is shown in Figure 5. The obtained results of the percentage of germinated macroconidia, as well as the percentage of the average length of hyphae, showed a consistent fungistatic and antifungal effect. The extract concentrations of 0.5% and 1.0% were more effective than that of 0.05% and 0.2%. After 10 days of the experiment, F. vesiculosus scCO2 extract at a concentration of 1.0% still showed a strong inhibitory effect on all studied Fusarium conidia. In the case of F. culmorum Fc37, the germination of macroconidia was strongly inhibited when 0.5% and 1.0% extracts were applied, which also corresponded to no increase of average length of hyphae in an experiment with the extract and conidia (Fc37 W.E). It was observed that 0.5% extract in Fc37 RB.E samples was effective after 48 h of incubation. It may be assumed that the spores In the case of F. culmorum Fc37, the germination of macroconidia was strongly inhibited when 0.5% and 1.0% extracts were applied, which also corresponded to no increase of average length of hyphae in an experiment with the extract and conidia (Fc37 W.E). It was observed that 0.5% extract in Fc37 RB.E samples was effective after 48 h of incubation. It may be assumed that the spores of F. culmorum Fc37 were able to feed themselves with RB-medium nutrients within 48 h. Similarly to Fc37, the spores of F. culmorum CBS122 were proven to be inhibited by the addition of 0.5% and 1.0% extract in FcCBS122 W.E samples, whereas FcCBS122 RB.E samples were characterized by the percentage of germinated spores in the range of 7%-21% (24-144 h of incubation), as well as the average percentage of hyphae length at the level of 6%-8% (24-144 h of incubation) (Figure 6(A1, B1, A2, B2)).
The growth of F. oxysporum Fo38 was fully inhibited by the F. vesiculosus scCO 2 extract at the concentration of 0.5% and 1.0%. The samples of Fo38 W.E were characterized by no increase of both germinated macroconidia and mean length of hyphae upon treatment with the extract, according to an appropriate control (W.C, RB.C). Similarly to Fo38 W.E, the same effect of the 0.5% and 1.0% extracts was observed for the FoCBS129 W.E and FoCBS129 RB.E samples. In the case of Fo38 RB.E, the most effective concentration of F. vesiculosus extract in the inhibition of macroconidia growth was 1.0%. The treatment of Fo38 RB.E with 0.5% extract resulted in an increase by 0%-37% of germinated spores and by 0%-21% of mean length of hyphae within 0-144 h of incubation ( Figure 6(A3, B3, A4, B4)).  Like other Fusarium species, F. culmorum and F. oxysporum are known for their ability to survive in the soil, even for several years, especially on post-harvest residues, due to inoculum production [53,54]. Moreover, both fungi species (F. culmorum and F. oxysporum) are characterized by a low degradation risk as they have an ability to survive in adverse conditions by using defense mechanisms. Furthermore, Fusarium species are responsible for causing fusariosis, which is one of the most serious infections of plants, especially cereal plants [55]. According to Lionakis and Kontoyiannis [56], biological plant protection products are effective to some extent due to an innate resistance of pathogens to such preparations. On the other hand, more drastic methods include the use of chemical protection products that have a negative impact on the environment [57]. The promising agents for combating these pathogens may be algal-based preparations. Apart from its properties to inhibit F. culmorum and F. oxysporum macroconidia growth, the F. vesiculosus scCO 2 extract was also found to be effective in terms of total degradation and lysis (Figure 7) of Fc37, FcCBS122, Fo38 and FoCBS129 macroconidia. Our study is the first to prove the degradation and lysis of macroconidia caused by the F. vesiculosus scCO 2 extract. The lysis was performed using extract even at the concentration of 0.2%, as presented in Figure 7. Like other Fusarium species, F. culmorum and F. oxysporum are known for their ability to survive in the soil, even for several years, especially on post-harvest residues, due to inoculum production [53,54]. Moreover, both fungi species (F. culmorum and F. oxysporum) are characterized by a low degradation risk as they have an ability to survive in adverse conditions by using defense mechanisms. Furthermore, Fusarium species are responsible for causing fusariosis, which is one of the most serious infections of plants, especially cereal plants [55]. According to Lionakis and Kontoyiannis [56], biological plant protection products are effective to some extent due to an innate resistance of pathogens to such preparations. On the other hand, more drastic methods include the use of chemical protection products that have a negative impact on the environment [57]. The promising agents for combating these pathogens may be algal-based preparations. Apart from its properties to inhibit F. culmorum and F. oxysporum macroconidia growth, the F. vesiculosus scCO2 extract was also found to be effective in terms of total degradation and lysis (Figure 7) of Fc37, FcCBS122, Fo38 and FoCBS129 macroconidia. Our study is the first to prove the degradation and lysis of macroconidia caused by the F. vesiculosus scCO2 extract. The lysis was performed using extract even at the concentration of 0.2%, as presented in Figure 7.

The Effect of Fucosterol on the Fusarium Macroconidia
The experiment to determine the influence of the fucosterol standard on F. culmorum Fc37 on an RB medium was carried out in order to evaluate the antifungal properties of one of F. vesiculosus scCO2 extract components. According to our previous study, the content of fucosterol in the extract was 8.06 wt% [40]. On the basis of the results from this study, it may be concluded that fucosterol significantly affected the growth of F. culmorum strains. The germination of F. culmorum Fc37 macroconidia was fully inhibited by fucosterol at the concentration of 1.0% in the Fc37 RB.F sample. In the case of the Fc37 RB.F sample with 0.5% fucosterol, the percentage of germinated macroconidia was in the range of 5.95%-21.95%, whereas the percentage of hyphae length was in the range of 6.25%-28.12%. After 24 h of incubation of Fc37 RB.F 0.5% sample, the highest germination of macroconidia (21.95%) was observed, which was further decreased over two times (9.47%) after 144 h of incubation ( Figure 8. (1A, 1B)).

The Effect of Fucosterol on the Fusarium Macroconidia
The experiment to determine the influence of the fucosterol standard on F. culmorum Fc37 on an RB medium was carried out in order to evaluate the antifungal properties of one of F. vesiculosus scCO 2 extract components. According to our previous study, the content of fucosterol in the extract was 8.06 wt% [40]. On the basis of the results from this study, it may be concluded that fucosterol significantly affected the growth of F. culmorum strains. The germination of F. culmorum Fc37 macroconidia was fully inhibited by fucosterol at the concentration of 1.0% in the Fc37 RB.F sample. In the case of the Fc37 RB.F sample with 0.5% fucosterol, the percentage of germinated macroconidia was in the range of 5.95%-21.95%, whereas the percentage of hyphae length was in the range of 6.25%-28.12%. After 24 h of incubation of Fc37 RB.F 0.5% sample, the highest germination of macroconidia (21.95%) was observed, which was further decreased over two times (9.47%) after 144 h of incubation ( Figure 8A,B).
Similarly to the F. vesiculosus scCO 2 extract, fucosterol at a concentration of 1.0% caused total degradation, as well as lysis of F. culmorum Fc37 conidia, in the Fc37 RB.F sample. In the case of lower concentrations of fucosterol (0.05%-0.5%), the deformation of Fc37 macroconidia was observed in all samples (Fc37 RB.F 0.05%, Fc37 RB.F0.2%, Fc37 RB.F0.5%). The percentage of germinated macroconidia treated with fucosterol in Fc37 RB.F 0.2% and Fc37 RB.F 0.05% samples was 44.21% and 67.37%, respectively. Even though the growth of macroconidia was partially inhibited by fucosterol, the conidia were observed to grow. However, they were much shorter and deformed in a comparison with the control (Figure 9). According to Newman et al. [58], sterols (cholesterol, ergosterol) may take part in lipid bilayer modifications in terms of both structural and thermodynamic properties, causing a liquid-ordered (lo) phase separation. Moreover, sterols are said to react with membrane components and enzymes, enabling their activation or causing their inactivation [59]. The literature reports the antifungal properties of β-sitosterol, campesterol and stigmasterol form the extract of Dispacus asper against phytopathogenic fungi, such as Botrytis cinerea, Puccinia recondita and Rhizoctonia solani [60]. The fungal pathogens are suggested to survive the effect of active compounds (for instance, azole) due to the ability to decrease the accumulation of these compounds by different defense mechanisms [61].
Both Papadopoulou et al. [62] and Upadhyay et al. [63], indicated the potential antifungal effect of saponins. Similarly to the F. vesiculosus scCO2 extract, fucosterol at a concentration of 1.0% caused total degradation, as well as lysis of F. culmorum Fc37 conidia, in the Fc37 RB.F sample. In the case of lower concentrations of fucosterol (0.05%-0.5%), the deformation of Fc37 macroconidia was observed in all samples (Fc37 RB.F 0.05%, Fc37 RB.F0.2%, Fc37 RB.F0.5%). The percentage of germinated macroconidia treated with fucosterol in Fc37 RB.F 0.2% and Fc37 RB.F 0.05% samples was 44.21% and 67.37%, respectively. Even though the growth of macroconidia was partially inhibited by fucosterol, the conidia were observed to grow. However, they were much shorter and deformed in a comparison with the control (Figure 9). According to Newman et al. [58], sterols (cholesterol, ergosterol) may take part in lipid bilayer modifications in terms of both structural and thermodynamic properties, causing a liquid-ordered (lo) phase separation. Moreover, sterols are said to react with membrane components and enzymes, enabling their activation or causing their inactivation [59]. The literature reports the antifungal properties of β-sitosterol, campesterol and stigmasterol form the extract of Dispacus asper against phytopathogenic fungi, such as Botrytis cinerea, Puccinia recondita and Rhizoctonia solani [60]. The fungal pathogens are suggested to survive the effect of active compounds (for instance, azole) due to the ability to decrease the accumulation of these compounds by different defense mechanisms [61]. Both Papadopoulou et al. [62] and Upadhyay et al. [63], indicated the potential antifungal effect of saponins.  Table 2 presents the half-maximal inhibitory concentration for Fucus vesiculosus scCO2 extract and fucosterol. The IC50 values for the extract in the RB.E experiment were in the range of 0.02%-0.25%, with the highest concentration of the extract against CBS129 and DEMFo38. In the case of W.E experiment, the highest IC50 value (1.82%) was obtained against CBS129. The IC50 for fucosterol against DEMFc37 was calculated at the level of 0.06%.   Table 2 presents the half-maximal inhibitory concentration for Fucus vesiculosus scCO 2 extract and fucosterol. The IC 50 values for the extract in the RB.E experiment were in the range of 0.02%-0.25%, with the highest concentration of the extract against CBS129 and DEMFo38. In the case of W.E experiment, the highest IC50 value (1.82%) was obtained against CBS129. The IC 50 for fucosterol against DEMFc37 was calculated at the level of 0.06%.

Conclusions
Due to its strong inhibition properties against pathogenic fungi, supercritical carbon dioxide extract of F. vesiculosus may be used as a potential component of a biological plant protection product, especially against phytopathogenic fungi of Fusarium spp. These fungi are particularly dangerous for various crops in all geographical regions.