Macroalgae collection
Green seaweeds were collected from Port Said and Alexandria coasts, Egypt. The algae collected immediately were washed carefully under running fresh water to remove the sand and the other extraneous matter. After that, algae were drained and wiped by blotting sheet, then air-dried at 45 °C for 5 days. Algae were dried entirely and ground in a mechanical grinder, according to Soliman et al. (2018).
Pathogenic fungi
The two plant pathogenic fungi caused significant economic losses on the Cucumber crop, namely, F. solani (Mart.) Sacc., and M. phaseolina (Tassi) Goid. were obtained from the culture collection of the Mycology Research and Diseases Survey Department.
Macroalgae identification
The collected green macroalgae, U. fasciata Delile, and E. flexuosa (Wulfen) were identified according to (Papenfuss 1968; Gribb 1983; Womersley 1984; Aleem 1993; Madkour and El-Shoubaky 2007).
Preparation of algal extracts
Five solvents were experimented by ethyl acetate, methanol, benzene, acetone, and chloroform in addition to water by adding 200 ml of each solvent to 50 g of algal powder (V/W). After that, mixtures were shaken for 10 days on an arbitral shaker at room temperature (25 °C), then extracts were filtered using cheesecloth, followed by Whatman paper No. 2 (Kumar et al. 2008).
In vitro assay of fungal growth reduction
Dual cultural plates with potato dextrose agar (PDA) medium were used to study the reduction effect of the 2 algal extracts against M. phaseolina and F. solani, as described by (Kumar et al. 2008). In each plate, 2 wells (5 mm in diameter) were made 4 cm apart. One well was inoculated with (100 μl) each of the tested algal extract. The opposite well was inoculated by a disk (5 mm) of each pathogen (4 days old culture). For each treatment, 3 plates were used. The control plates were inoculated only with each of the pathogenic fungi. All plates were incubated at 25 ± 2 °C for 6–10 days. When the control mycelial fungal growth covered the entire medium surface in plates, all plates were then examined, and the linear growth of the pathogens was measured. The growing cultures were observed visually and microscopically for evidence of a reduction. The percentage of reduction in mycelial growth (X) of the fungal pathogens was calculated using the following the formula of (Nikam et al. 2007):
$$ X=\left[\mathrm{G}1\hbox{-} \mathrm{G}2/\mathrm{G}1\right]\times 100 $$
Where X, % of reduction in growth G1, linear growth of pathogenic fungus in control plates
G2, linear growth of pathogenic fungus in dual plates with algae extract.
Analysis of crude extracts (chloroform and ethyl acetate) by GC-mass and infra-red (IR)
The gas chromatography-mass spectrometry analysis (GC) for derivatives of chloroform and ethyl acetate extracted filtrates of U. fasciata and E. flexuosa was chosen in function of the percentage of reduction in mycelial growth from in vivo experiment by using an Agilent 6890 series II gas chromatograph. An Agilent 5973 mass spectrometer with electron ionization, mode (EI) generated at 70 eV (ion source at 230 °C and transfer line at 280 °C). The GC was performed using a HP5-MS capillary column (30 m × 0.25 mm, the film thickness of 0.25 μm). Operating conditions were as follows: carrier gas, helium with a flow rate of (1 ml min−1). The initial temperature was programmed from 80 to 280 °C (at 8 °C min−1) and maintained at 280 °C for 5 min. all compounds were identified by comparison of both the mass spectra (Wiley and Nist library) (Liu et al. 2008; Soliman et al. 2018). The infrared (IR) absorption spectrum of the purified fraction was evaluated to determine the possible functional groups in the algal extracts responsible for bio-reduction for fungal pathogens. This work was estimated at the National Center for Research (He et al. 2016).
In vivo assay of greenhouse experiment
Two algal powders were used in the present study and tested for their potentiality as biocontrol agents against the 2 tested pathogenic cucumber seeds fungi, M. phaseolina, and F. solani, in greenhouse experiments. Preparations of the algal powders were used in this investigation to evaluate their effect on disease incidence of the 2 fungal pathogens as follows: autoclaved sterilized sandy-loam soil and was infested with sorghum-grain inoculums of each pathogen M. phaseolina and F. solani at the rate 3% (w/w)/kg soil. Infested pots were irrigated and kept for 7 days to ensure fungi dispersal in the soil before seed sowing. The infested soil was amended with dry algae powders in the ratio (1 g powder of each algal powder: kg soil) w/w at seed sowing. Three seeds were sown in each pot, 4 replicates were used for each treatment. The control treatment was carried out by the chemical fungicide vitavax as seed coating 3 g vitavax/kg seeds before seed sowing. Pots were kept in the greenhouse until the end of the experiment (60 days), and the cucumber’s yield was weighted for all treatments. Seeds of cucumber were obtained from the commercial sector in Egypt. Seeds were surface-sterilized in a 2% sodium hypochlorite solution for 2 min before sowing (Sultana et al. 2011).
Statistical analysis
The results of all experiments were statistically analyzed using one-way analysis of variance (ANOVA) to test for significance, and the Fisher test was used for mean separations by the GENSTAT computer package system. Means were made following Fishers LSD (LSD at 0.01 for in vitro experiment, while it was at 0.05 for in vivo experiment) (Gajardo et al. 2017).
