Isolation and purification of Fusarium equiseti from a marine sediment sample

A sediment sample was collected from the Mediterranean coast of northern Egypt for fungal isolation using the serial dilution technique on potato dextrose agar (PDA) plates. To enhance fungal recovery, we suspended 1 g of wet sediment in 9 mL of sterilized distilled water and incubated it at 60 °C for 40 min to minimize bacterial contamination. A 100 µL aliquot was spread onto PDA plates containing potato extract (200 g/L), glucose (10 g/L), and agar (16 g/L), supplemented with streptomycin (50 µg/mL) and penicillin (100 µg/mL) to suppress bacterial growth. Serial dilutions was prepared (10⁻1, 10⁻2, 10⁻3) with sterilized seawater to ensure fungal isolation, incubated the plates at 25 °C in the dark, and monitored fungal growth for seven days. Individual colonies were subcultured onto fresh PDA plates for purification, transferring actively growing hyphal tips to isolate a single strain. The purified isolate was stored at 4 °C on PDA slants for subsequent studies (Farouk et al. 2024).

Morphological and molecular characterization of Fusarium equiseti

The identification of the isolated fungal strain was performed using a combination of morphological and molecular approaches. Initial morphological characterization was carried out using the mycological keys of Arifah et al. (2023), along with cultural and conidial characteristics. The micromorphological features of the fungal isolate were examined using slide culture techniques and observed under a light microscope (Optika Microscope, Italy). The isolate was maintained on Sabouraud dextrose agar (SDA) at 28 °C and stored at 4 °C for further studies.

For molecular identification, Internal Transcribed Spacer (ITS) region sequencing was employed to confirm the morphological classification of Fusarium equiseti. Genomic DNA was extracted using the Patho-gene-spin DNA/RNA extraction kit (Intron Biotechnology, Korea) following the manufacturer’s protocol. The fungal strain was cultivated on Czapek's yeast extract agar (CYA) for Penicillium species and V8 juice agar for Alternaria species, with incubation at 28 °C for seven days (Al Mousa et al. 2021). The extracted DNA was submitted to SolGent Company (Daejeon, South Korea) for PCR amplification and sequencing of the ITS region.The amplification reaction was performed using universal fungal primers ITS1 (5'-TCC GTA GGT GAA CCT GCG G-3') and ITS4 (5'-TCC TCC GCT TAT TGA TAT GC-3'). Following amplification, Sanger sequencing was performed using the same primers. The obtained sequences were analyzed using the Basic Local Alignment Search Tool (BLAST) from the National Center for Biotechnology Information (NCBI) database to determine sequence similarity. Phylogenetic analysis was conducted using MegAlign (DNA Star) software version 5.05 to assess the evolutionary relationships of Fusarium equiseti with closely related species (Cheruiyot et al. 2024).

Extracellular biosynthesis of silver nanoparticles using Fusarium equiseti

The extracellular biosynthesis of silver nanoparticles (AgNPs) was conducted utilizing the culture filtrate of F. equiseti. The term "extracellular" denotes that nanoparticle formation occurred in the fungal culture supernatant, external to the fungal biomass, facilitated by the secreted bioactive metabolites and enzymes. Specifically, F. equiseti is known to release extracellular reductase enzymes (e.g., NADH-dependent nitrate reductase), proteins, phenolic compounds, and polysaccharides into the surrounding medium, which collectively serve as reducing and stabilizing agents. For biosynthesis, F. equiseti was cultured in Sabouraud dextrose broth (SDB) at 28 ± 2 °C under shaking conditions (150 rpm) for 5 days to promote maximum secretion of extracellular metabolites. The culture broth was subsequently filtered using Whatman No. 1 filter paper to obtain a sterile cell-free supernatant. To this filtrate, an aqueous solution of silver nitrate (AgNO₃) was added to a final concentration of 1 mM and incubated in the dark at 28 ± 2 °C under static conditions. The biosynthetic reaction was monitored periodically, with the visual transition from pale yellow to dark brown confirming the extracellular reduction of Ag⁺ ions to elemental silver nanoparticles (Ag⁰) (Hulikere and Joshi 2019).

To ensure the validity of the biosynthesis process and exclude abiotic factors, a negative control was included, consisting of 1 mM AgNO₃ added to uninoculated, autoclaved SDB medium under identical incubation conditions. No color change or nanoparticle formation was observed in this control. All synthesis experiments were performed under aseptic conditions, with autoclaved media and sterile equipment, to prevent external contamination. The extracellular approach was selected for its advantages over intracellular synthesis, including ease of nanoparticle recovery, reduced processing steps, and the inherent functionalization of AgNPs by fungal biomolecules, enhancing their colloidal stability and biological activities (Rai et al. 2021).

Structural and morphological characterization of silver nanoparticles

The formation of silver nanoparticles (AgNPs) was initially confirmed by a visible color change in the reaction mixture, where the fungal filtrate and AgNO₃ solution transitioned from pale yellow to brown, indicating the bioreduction of Ag⁺ ions into elemental silver (Ag⁰) nanoparticles. The synthesis of AgNPs was further verified using ultraviolet–visible (UV–Vis) spectroscopy with a Shimadzu UV-2600 spectrophotometer (Shimadzu Corporation, Japan), which detected a distinct surface plasmon resonance (SPR) peak within the 300–500 nm wavelength range, characteristic of AgNPs. The synthesized AgNPs were purified by centrifugation at 15,000 × g for 15 min using an Eppendorf 5804R centrifuge (Eppendorf, Germany) and repeatedly washed with deionized water to eliminate residual ions and impurities. The purified nanoparticles were then subjected to comprehensive physicochemical characterization. The crystallinity and phase composition of the AgNPs were analyzed using X-ray diffraction (XRD) with a Rigaku RINT2000 vertical goniometer (Rigaku Corporation, Japan), operated at 70 kV and 200 mA with Cu Kα radiation (λ = 1.5405 Å), scanning over a 2θ range of 10°–70°. Morphological and surface characteristics were evaluated by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). For SEM, the AgNP suspension was drop-cast onto carbon-coated copper grids, air-dried, and examined using a Quanta FEG 250 SEM (Thermo Fisher Scientific, USA) to assess particle size distribution and surface morphology. High-resolution imaging was performed via transmission electron microscopy (TEM) using a HITACHI H-800 TEM (Hitachi, Japan) at 200 kV to confirm the shape and structural integrity of the synthesized nanoparticles. Additionally, Fourier-transform infrared (FTIR) spectroscopy (PerkinElmer Spectrum Two, USA) was employed to identify functional groups involved in nanoparticle stabilization and surface functionalization. Zeta potential analysis was carried out using a Malvern Zetasizer Nano ZS (Malvern Instruments, UK) to determine the colloidal stability of the AgNPs in aqueous suspension (Fath-Alla et al. 2024).

Optimization of silver nanoparticle biosynthesis parameters

One-variable-at-a-time (OVAT) approach was used to assess the influence of temperature, pH, and silver nitrate (AgNO₃) concentration on nanoparticle formation. The effect of temperature was evaluated by incubating the reaction mixture at 15, 20, 25, 30, 35, 40, and 45°C while maintaining constant pH and AgNO₃ concentration. Similarly, the impact of pH on AgNP biosynthesis was assessed by adjusting the reaction medium to pH 3, 4, 5, 6, 7, 8, and 9 using 0.1 M HCl or 0.1 M NaOH. The influence of silver ion concentration was examined by testing AgNO₃ at different concentrations (1, 1.5, 2, 2.5, 3, and 3.5 mM). The formation and stability of AgNPs under different conditions were monitored by measuring surface plasmon resonance (SPR) at 420 nm using a Shimadzu UV-2600 spectrophotometer (Shimadzu Corporation, Japan) (Salem et al. 2024).

Antimicrobial activity of silver nanoparticles

The antimicrobial activity of the biosynthesized silver nanoparticles (AgNPs) from Fusarium equiseti was evaluated using a broth microdilution assay to determine the minimum inhibitory concentration (MIC) against bacterial and fungal pathogens, including Fusarium solani, Bacillus subtilis, Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, and Candida albicans. Bacterial strains (P. aeruginosa ATCC 25619, B. subtilis ATCC 6633, S. aureus LC189114, and E. coli OK087362) were cultured overnight in nutrient broth at 37 °C. The bacterial suspensions were adjusted to an optical density (OD) of 0.2 (~ 2 × 104 CFU/mL). Spore suspensions of F. solani and C. albicans were also adjusted to 2 × 104 CFU/mL. AgNPs were serially diluted to final concentrations ranging from 0.125 to 64 μg/mL in Czapek's Yeast Broth (CYB) for fungi and in nutrient broth for bacteria. In sterile 96-well microtiter plates, 100 µL of each AgNP dilution was mixed with 100 µL of microbial suspension. Plates were incubated at 37 °C for bacteria and 28 °C for fungi. Streptomycin (for bacteria) and fluconazole (for fungi) were used as positive controls, while silver nitrate (AgNO₃) was included as a comparative control. After 24 h (bacteria) or 72 h (fungi), microbial growth was assessed, and MIC values were recorded as the lowest AgNP concentration that visibly inhibited microbial growth (Mwangi et al. 2024).

Evaluation of the antioxidant activity of silver nanoparticles

The antioxidant activity of biosynthesized silver nanoparticles (AgNPs) was assessed using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay. A 0.5 mL solution of 0.15 mM DPPH was prepared and mixed with AgNPs dissolved in methanol. Ascorbic acid was used as a positive control. The reaction mixture was incubated at room temperature for 30 min in the dark to allow the reduction of the DPPH radical by AgNPs. After incubation, absorbance was measured at 517 nm using a UV–Vis spectrophotometer (Shimadzu UV-2600, Shimadzu Corporation, Japan). The percentage of DPPH radical scavenging activity was calculated using the following equation: Reduction of the DPPH radical (%) = [(Control absorbance—Control sample absorbance)/ (Control absorbance)] × 100. Higher scavenging activity indicated a stronger antioxidant potential of the AgNPs (Bhavi et al. 2024).

Evaluation of the cytotoxic effects of silver nanoparticles on MCF-7 Cells

The cytotoxic activity of biosynthesized silver nanoparticles (AgNPs) against MCF-7 human breast cancer cells was assessed using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. MCF-7 cells were seeded at a density of 1 × 104 cells per well in a 96-well microtiter plate and allowed to adhere overnight. The cells were then exposed to varying concentrations of AgNPs (0, 10, 20, 30, 40, 50, and 60 μg/mL) and incubated for 24 h at 37°C in a humidified atmosphere containing 5% CO₂. Following incubation, 100 µL of MTT solution (0.5 mg/mL in phosphate-buffered saline, PBS, pH 7.2) was added to each well, and the plate was further incubated for 3 h at 37 °C to allow the formation of insoluble purple formazan crystals. The MTT solution was carefully removed, and 50 µL of dimethyl sulfoxide (DMSO) was added to dissolve the formazan. Absorbance was measured at 570 nm using a microplate reader (Bio-Rad iMark™, USA). Cell viability was expressed as a percentage relative to the untreated control group, and cytotoxicity was determined based on the decrease in viability in response to increasing AgNP concentrations (Al-Ziyadi et al. 2024).

Molecular docking study of silver nanoparticles against microbial and human target proteins

A spherical silver nanoparticle (AgNP) model, with a diameter of 50 nm, was constructed using Materials Studio 2020 based on the face-centered cubic (FCC) crystal structure of bulk silver. To ensure the structural and energetic stability of the model, energy minimization was performed using the density functional theory (DFT) approach. The nanoparticle surface was subsequently functionalized with citrate ions to improve solubility, biocompatibility, and colloidal stability by preventing nanoparticle aggregation in aqueous environments. The stability of the citrate-functionalized AgNPs was further verified through molecular dynamics simulations under simulated physiological conditions. Molecular docking simulations were performed using Molegro Virtual Docker (MVD, version 6.0) to evaluate the interactions between the citrate-functionalized AgNP model and selected microbial and human proteins. Protein structures were retrieved from UniProt and the Protein Data Bank (PDB) and were prepared by removing water molecules, adding polar hydrogens, and assigning partial atomic charges using standard MVD protocols. The active sites of each protein were automatically predicted and grid boxes were generated to focus docking runs on biologically relevant regions (Nayel et al. 2024).

The protein targets were chosen based on their essential roles in microbial survival, cell wall biosynthesis, and human cellular processes. For bacterial strains, targets involved in peptidoglycan biosynthesis and remodeling were selected, including the cell wall-associated protease (P54423) in B. subtilis, peptidoglycan D,D-transpeptidase PbpC (Q9I1K1) in P. aeruginosa, glycyl-glycine endopeptidase LytM (O33599) in S. aureus, and peptidoglycan D,D-transpeptidase MrdA (P0AD65) in E. coli. These enzymes are critical to bacterial cell wall integrity, making them attractive targets for antimicrobial action (Hugonneau-Beaufet et al. 2023; Piatek et al. 2023; Darrouzet et al. 2024; Zhao et al. 2025). For fungal pathogens, 2-methylcitrate synthase, mitochondrial (C7C435) in F. solani, and the cell wall integrity transcriptional regulator CAS5 (Q5AMH6) in C. albicans were selected due to their roles in fungal metabolism and structural maintenance (Xiong et al. 2021).

In addition to microbial targets, human proteins were included to evaluate the potential antioxidant and apoptotic activities of AgNPs. For the antioxidant mechanism, pyridoxine 5'-phosphate synthase (PdxS, P0A794), a key enzyme in oxidative stress resistance pathways, was selected to assess how AgNPs might disrupt cellular redox homeostasis (Rivero et al. 2024). For the cytotoxicity mechanism, the anti-apoptotic protein Bcl-2 (P10415) was selected due to its involvement in regulating mitochondrial-mediated apoptosis in cancer cells (Harikrishnan et al. 2021). The inclusion of these targets was intended to provide mechanistic insights into the dual antimicrobial and anticancer effects of the biosynthesized AgNPs.

Statistical analysis

All experiments were performed in triplicate, and the data are presented as mean ± standard deviation (SD). Statistical significance was assessed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test for multiple comparisons. A p-value of < 0.05 was considered statistically significant. The IC₅₀ values for DPPH radical scavenging activity and MCF-7 cytotoxicity assays were calculated using nonlinear regression analysis fitted to a sigmoidal dose–response model. All statistical analyses were performed using GraphPad Prism (version 9.0) and SPSS software (version 22.0).