Antimicrobial Potential of Endophytic Fungi Isolated from Wild Plants in the Qassim Region, Saudi Arabia

Plant material selection

Plant samples were not chosen randomly; rather, the selection was based on a targeted search for the most ecologically and traditionally significant wild plants in the Qassim region of Saudi Arabia. Wild thyme (Thymus serpyllum) was collected from the Al-Arifiyah area, while ramth (Haloxylon salicornicum), athl (Tamarix nilotica), and besbas (Anisosciadium isosciadium) were gathered from the Nafud desert region. All plant materials were identified and collected in their natural habitats to ensure authenticity and relevance to the local flora.^[8]

Isolation of endophytic fungi

Plant samples were first thoroughly washed under running tap water to remove soil and debris, then separated into leaves, stems, and roots. Each plant part was cut into small segments (approximately 0.5–1 cm in size) and subjected to surface sterilization to eliminate epiphytic microorganisms. Surface sterilization was carried out by immersing the segments in a 0.01% (w/v) mercuric chloride (HgCl₂) solution, prepared by dissolving 0.01 g of mercuric chloride in 100 mL of sterile distilled water, for 30 s. The segments were then rinsed 3-4 times with sterile distilled water to remove any residual sterilant. To ensure the effectiveness of sterilization, an aliquot (100 µL) of the final rinse water was plated onto sterile Potato Dextrose Agar (PDA) and incubated at 27°C. Plates with no microbial growth confirmed successful surface sterilization.

Sterilized plant segments were then aseptically transferred onto PDA plates supplemented with chloramphenicol (100 mg L^-1) to suppress bacterial contamination. The plates were incubated at 27 ± 1°C for 3 to 5 days, and monitored daily for the appearance of fungal colonies emerging from the inner tissues. Emergent fungal colonies were carefully subcultured onto fresh PDA to obtain pure isolates, which were maintained for further studies.^[9]

Identification of endophytic fungi

Fungal isolates were initially identified based on morphological characteristics. A small portion of each pure fungal colony was transferred onto a clean microscope slide using a sterile inoculation needle. A drop of sterile distilled water was added to the sample, and the fungal material was gently spread. A coverslip was placed over the preparation, and the slide was observed under a compound microscope at 40× and 100× magnifications. Morphological features such as hyphal structure, spore shape, septation, and arrangement were noted, and identification was performed using standard mycological references and identification keys.^[10]

Molecular identification of 24 representative fungal isolates was performed by amplifying the internal transcribed spacer (ITS) region of fungal rDNA.^[11] Fresh fungal mycelia were ground in a mortar and pestle using liquid nitrogen and mixed with CTAB extraction buffer (2% CTAB, 100 mM Tris-HCl pH 8.0, 20 mM EDTA, 1.4 M NaCl, and 1% polyvinylpyrrolidone). The homogenate was transferred to sterile 1.5 mL microcentrifuge tubes and incubated in a water bath at 65°C for 30 min, with gentle mixing every 10 min. Following incubation, 500 µL of chloroform:isoamyl alcohol (24:1) was added to each tube. The mixture was centrifuged at 12,000 rpm for 8 min. The upper aqueous phase containing the DNA was carefully transferred to a new tube, followed by the addition of 500 µL of 3M sodium acetate and 500 µL of ice-cold absolute ethanol to precipitate DNA. Samples were centrifuged at 12,000 rpm for 5 min, and DNA pellets were incubated at –20°C for 30 min. After cold incubation, the samples were centrifuged again (12,000 rpm for 5 min), the supernatant was discarded, and the DNA pellets were air-dried. The dried pellets were then resuspended in 50–110 µL of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). DNA concentration and purity were determined using a NanoDrop spectrophotometer (Thermo Scientific).^[12]

The internal transcribed spacer (ITS) region was amplified using the universal fungal primers: ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′), ITS4 (5′-TCCTCCGCTTATTGATATGC-3′). The PCR reaction mixture (25 µL total volume) contained: 12.5 µL of 2× PCR Master Mix (containing Taq polymerase, dNTPs, MgCl₂, and buffer), 1 µL of ITS1 primer (10 µM), 1 µL of ITS4 primer (10 µM), 2 µL of genomic DNA, 8.5 µL of nuclease-free water. The PCR program was set as follows: Initial denaturation at 95°C for 5 min, 35 cycles of: Denaturation at 94°C for 30 s, Annealing at 55°C for 30 s, Extension at 72°C for 1 min, final extension at 72°C for 10 min.^[13]

PCR products were analyzed using 1.5% agarose gel electrophoresis prepared in Tris-EDTA (TE) buffer. Ethidium bromide (0.5 µg mL^-1) was added to the gel for DNA staining. After solidification, 5 µL of each PCR product was loaded into the wells along with a 100 bp DNA ladder. The gel was run at 100 V for 30-40 min, and DNA bands were visualized under UV transillumination using a gel documentation system.^[14]

Preparation of fungal extracts

Dried fungal biomass (1g) was accurately weighed and placed into a sterile mortar. A solvent mixture consisting of 15 mL methanol and 15 mL ethyl acetate was added to the biomass. The mixture was thoroughly ground using a pestle to ensure complete homogenization of the fungal tissue with the solvents. The homogenate was transferred to a sterile container and incubated on a vibratory shaker at room temperature for 24 h to enhance extraction. Following the incubation period, the mixture was left under ambient conditions until the solvents had completely evaporated, leaving behind a dry fungal extract. To recover the extract, another 15 mL of methanol and 15 mL of ethyl acetate were added directly to the dry residue. The contents were gently stirred using a sterile glass rod to ensure solubilization. The mixture was then filtered using Whatman No. 1 filter paper and a standard filtration funnel to obtain the final crude fungal extract. The extract was collected in sterile tubes and stored at low temperature until further analysis.^[15]

Determination of total phenolic content using Folin-Ciocalteu method

The total phenolic content of the fungal extract was estimated using the Folin-Ciocalteu reagent. Preparation of Reagents and Samples: Sodium carbonate (Na₂CO₃) solution: Prepared by dissolving 20 g of Na₂CO₃ in 100 mL of distilled water. Fungal fluid extract: Prepared by dissolving the crude fungal extract in methanol. Folin-Ciocalteu’s reagent: Commercially available reagent used directly in appropriate dilution. A total of 84 test tubes were prepared. Each tube received the following: 0.5 mL of fungal fluid extract, 2.5 mL of distilled water, 0.5 mL of Folin-Ciocalteu reagent. Tubes were gently shaken several times to mix the components, then 2.5 mL of Na₂CO₃ solution was added to each. After a final gentle shaking, the tubes were placed in a boiling water bath for 2 min to develop color. After cooling, the total volume in each tube was 5.5 mL. The absorbance was measured at an appropriate wavelength (typically 765 nm) using a UV-Vis spectrophotometer. Phenolic content was calculated using a gallic acid standard curve, and results were expressed as mg gallic acid equivalent (GAE) per mL of extract.^[16]

Dual culture resistance assay

The resistance of bacterial and fungal pathogens against endophytic fungal isolates was evaluated using the dual culture method. Fungal plugs (6 mm in diameter) were aseptically excised from the edge of actively growing fungal colonies using a sterile cork borer. A single disc was inoculated near the edge of a Petri dish containing: Nutrient Agar (NA) for bacterial pathogens, Sabouraud Dextrose Agar (SDA) for Candida albicans. Plates were incubated for 3 days to allow pre-growth of the fungal colony. After fungal growth was established, a straight streak of bacterial or Candida culture was inoculated parallel to and near the fungal colony. The plates were incubated under the following conditions: Bacterial plates: 27°C for 24 h and Candida plates: 37°C for 3 days.^[17]

The antagonistic property of each endophyte was expressed as percentage inhibition of the Candida and bacterial streak using the formula:

$\text{Percentage of growth inhibition }\left(\%\right)=\left(\left(\text{R1}-\text{R2}\right)/\text{R1}\right)\times \text{1}00$

where: R1 = The width of pathogen streak in control plates

R2 = The width of pathogen streak at 10 days of antagonism trials

Selection and collection of plant samples

Total of four wild plant species were successfully collected from distinct regions within the Qassim area of Saudi Arabia, selected based on their ecological prevalence and traditional relevance. Thymus serpyllum was harvested from the Al-Arifiyah area, whereas Haloxylon salicornicum, Tamarix nilotica, and Anisosciadium isosciadium Plants were sourced from the arid Nafud desert region. The collection process was conducted in situ on [03/02/2024], ensuring that samples were obtained directly from their natural habitats. Each species was taxonomically verified to ensure accurate identification before further processing.

Preparation of plant material

All collected plant samples were successfully processed into dry powders, with plant parts (leaves, stems, and roots) separated prior to grinding. The air-drying process yielded approximately 25–30 g of dry material per plant, with an average distribution ratio of 50% leaves, 30% stems, and 20% roots. The powders were uniform in texture and free from visible contaminants. All samples were stored in airtight glass containers under dry, dark conditions and remained stable throughout the study.

Isolation of endophytic fungi

Endophytic fungi were successfully isolated from all tested plant species and tissues (leaves, stems, and roots). A total of 24 fungal isolates were obtained across the four plant species. Colonies began to emerge within 3 to 5 days of incubation on PDA media supplemented with chloramphenicol. The isolates displayed variable colony morphologies, growth rates, and pigmentation. Control plates inoculated with the final rinse water showed no microbial growth, confirming the efficiency of surface sterilization and the true endophytic origin of the isolates.

Morphological and molecular identification of fungal isolates

A total of 24 fungal isolates were obtained from various tissues of the selected plant species. Morphological examination revealed distinct colony characteristics in terms of pigmentation, texture, and radial growth on PDA medium after 5–7 days of incubation at 28 ± 2°C. Microscopic observation using lactophenol cotton blue staining highlighted diagnostic features such as conidiophore structure, spore morphology, and arrangement. Based on these features, the isolates were preliminarily assigned to four genera: Penicillium, Aspergillus, Alternaria, and Curvularia.

For molecular confirmation, genomic DNA was extracted and the ITS region was successfully amplified in all isolates using ITS1 and ITS4 primers. PCR products were sequenced, and BLAST analysis of the sequences revealed ≥98% similarity with reference sequences in the NCBI GenBank database. Molecular data fully corroborated the morphological classification, allowing identification of the isolates at both genus and species levels within the same four genera. The identified isolates, along with their corresponding accession numbers submitted to the GenBank database have been listed in Table 1.

Quantification of total phenolic content in endophytic fungal extracts

The total phenolic content of 24 endophytic fungal extracts was quantitatively assessed using the Folin-Ciocalteu assay, with absorbance measured at 765 nm as an indicator of phenolic compound concentration. Results in Table 2 and Figure 1 revealed considerable variability among the isolate. Notably, isolates 1, 3, 8, and 16 exhibited the highest phenolic contents recording from 1.206 to 1.263 mg GAE/100mL, indicating substantial production of phenolic metabolites. Conversely, isolates numbered 9 through 15 and 17 through 24 generally showed lower phenolic levels, with values typically below 0.8 mg GAE/100mL, suggesting comparatively reduced phenolic synthesis.

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Figure 1:

Total phenolic content of endophytic fungal extracts (ethanol extracts).

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This variation in phenolic content may partially explain the differences observed in the antimicrobial activities of the fungal extracts. Phenolic compounds are well-recognized for their antioxidant and antimicrobial properties and isolates with higher phenolic content often correlate with enhanced antimicrobial efficacy. For instance, isolate 16, which demonstrated elevated phenolic content, correspondingly exhibited notable antimicrobial activity in previous assays.

Overall, these findings emphasize the significant role of endophytic fungi as producers of bioactive phenolic compounds, which could contribute to their observed biological activities and potential applications in antimicrobial agent development.

Antifungal and antibacterial potential of endophytic fungi

To evaluate the anticandidal and antibacterial potential of the isolated endophytic fungi, dual culture bioassays were conducted against three different pathogens indicators: Candida albicans, Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. The following Figure 2 summarizes the growth performance of fungal isolates and their inhibition capacity against each pathogen.

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Figure 2:

Dual culture assay showing antagonistic activity of endophytic fungal isolates against Candida albicans.

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A marked variability was recorded among the examined endophytic fungi in terms of their growth and anti-Candida activity. Isolates No. 11 and 19 recorded the highest growth inhibition percentage (61.23 and 57.61, respectively), revealing a strong antagonistic behavior, whereas isolates such as 1, 6, 8, 13, 17, and 20 showed no inhibition. Several isolates no. (3, 4, 7, 9, 14, 15, 16, 18, 21, 22, and 23) displayed low to moderate inhibition. These findings suggest that only a few endophytic strains possess promising anti-Candida potential, which may be linked to differential secretion of antifungal metabolites.

The dual culture assay demonstrated variable antifungal activity of endophytic fungal isolates against E. coli [Figure 3]. Isolate 19 exhibited the highest inhibition percentage (67.42%), indicating strong antibacterial potential. Isolate 11 also showed notable activity with 48.63% inhibition percentage. Most other isolates displayed minimal to moderate inhibitory effects, while isolate 9 showed no measurable growth or inhibition. These results suggest considerable diversity in antibacterial capabilities among the tested fungal endophytes, possibly due to differential metabolite production.

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Figure 3:

Dual culture assay showing antagonistic activity of endophytic fungal isolates against E. coli.

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As shown in Figure 4, the results from the dual culture assay against S. aureus showed generally low to moderate inhibition among the fungal isolate. The highest inhibition percentage was recorded by isolates 15 and 16 with 49.18% and 48.25%, respectively. Isolate 23 demonstrated the minimal inhibitory activity reaching 4.9% whereas, isolate 20 showed no measurable inhibition. This pattern suggests a relatively weak antibacterial effect of the tested endophytic fungi against S. aureus compared to other pathogens.

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Figure 4:

Dual culture assay showing antagonistic activity of endophytic fungal isolates against Staphylococcus aureus.

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In dual culture with Pseudomonas aeruginosa, most fungal isolates exhibited weak inhibitory activity [Figure 5]. Isolate 2 was the most active, displaying a 55.12% inhibition percentage, while isolates 4 and 14 showed moderate inhibition effect recording 41.73% and 42.38%, respectively. Most isolates produced only slight inhibition, indicating limited antibacterial effectiveness against Pseudomonas aeruginosa. This suggests that only certain endophytic fungi possess metabolites capable of suppressing this Gram-negative pathogen.

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Figure 5:

Dual culture assay showing antagonistic activity of endophytic fungal isolates against Pseudomonas aeruginosa.

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Overall, comparison of the dual culture assays against the four tested pathogens revealed clear differences in the inhibitory capabilities of the endophytic fungal isolates. The most pronounced antibacterial activity was observed against E. coli, where several isolates exhibited sizeable inhibition zones, followed by Staphylococcus aureus, which showed generally weak suppression. Pseudomonas aeruginosa appeared to be the most resistant, displaying only minimal inhibition in most cases. These results indicate that the antibacterial potential of endophytic fungi is highly strain-dependent and varies according to the target microorganism, with a greater antagonistic effect exerted against Gram-negative E. coli compared to S. aureus and Pseudomonas aeruginosa.

Wild medicinal plants and their associated endophytic fungi represent a largely untapped reservoir of bioactive compounds, particularly in arid regions where environmental stress drives unique metabolite production. Investigating these systems can provide insights into novel antimicrobial and anticancer agents. The selection of wild plants from ecologically diverse regions of the Qassim area provides a strong basis for investigating their bioactive potential. Species such as Thymus spp., Haloxylon salicornicum, and Tamarix spp. are well recognized in ethnomedicine and are adapted to arid environments, which often drives the accumulation of secondary metabolites as part of their stress tolerance mechanisms. This ecological adaptation may contribute to their antimicrobial and antioxidant activities observed in later stages of the study. The systematic processing of plant materials, including separation of leaves, stems, and roots followed by standardized drying and grinding, ensured reproducibility and preserved the phytochemical integrity of the samples. Previous reports have emphasized that careful preparation of plant materials is critical for maintaining the stability of bioactive compounds and for achieving consistency in biological assays.^[18]

Isolation trials confirmed that all investigated plant tissues harbored fungal endophytes, reflecting their wide ecological distribution in desert species. From the four plant hosts, a total of 24 isolates were recovered, demonstrating a considerable diversity of fungal associates. The ability of these endophytes to colonize plants thriving under arid conditions suggests that they may play an adaptive role in stress tolerance. Moreover, the absence of microbial growth on control plates validated the efficiency of the surface sterilization process, confirming the true endophytic origin of the isolates. Comparable observations have been reported in recent surveys of endophytes from desert plants, which highlight their ecological significance and potential as reservoirs of bioactive metabolites.^[19]

The identification process combining classical and molecular tools revealed that the isolates belonged to Penicillium, Aspergillus, Alternaria, and Curvularia. While colony morphology and microscopic features provided initial insights, ITS rDNA sequencing was essential for accurate resolution, showing ≥98% identity with GenBank references. The agreement between morphological and molecular data reinforced the reliability of the classification. These four genera are frequently encountered as dominant endophytes in arid-zone plants and are recognized producers of bioactive secondary metabolites. Such findings not only authenticate the taxonomic placement of the isolates but also provide a solid framework for correlating their identity with antimicrobial and antioxidant properties in subsequent experiments.^[20]

Endophytic isolates from Alternaria and Penicillium showed the highest phenolic content, which correlated with their enhanced antimicrobial activity. Lower phenolic levels in other isolates were associated with reduced bioactivity, highlighting the key role of phenolic metabolites in mediating antimicrobial effects. These findings suggest that targeting specific fungal genera can optimize the discovery of bioactive compounds for potential antimicrobial applications.^[21]

The pronounced antimicrobial activity observed in the Penicillium underscores the capacity of certain endophytic fungi to produce potent bioactive compounds capable of inhibiting bacterial growth. Endophytes such as Penicillium are well-documented for their synthesis of diverse secondary metabolites, including phenolics, alkaloids, and polyketides, which contribute to their antimicrobial properties. The significant antifungal potential of Thymus serpyllum extracts further highlights the complementary role of plant-derived compounds, suggesting that combining plant and endophytic fungal metabolites could enhance the overall antimicrobial spectrum.^[22]

The observed differences in susceptibility between Gram-negative and Gram-positive bacteria suggest that microbial cell wall architecture significantly influences the efficacy of fungal metabolites. Gram-negative bacteria, such as E. coli, were generally more susceptible to selecting fungal compounds, potentially due to the specific targeting of outer membrane structures. In contrast, Gram-positive bacteria demonstrated relative resistance, likely reflecting their thicker peptidoglycan layers and innate defensive mechanisms. These patterns indicate that the interaction between fungal metabolites and microbial physiology is a key determinant of antimicrobial effectiveness.^[23]

Generally, the combined exploration of wild medicinal plants and their associated endophytic fungi represent a highly promising avenue for discovering novel antimicrobial agents. These species serve as reservoirs for endophytes capable of producing chemically diverse and biologically potent metabolites with significant therapeutic potential. Conservation of wild plant biodiversity is therefore crucial, not only for ecosystem stability but also as a strategic means to harness these bioactive compounds for therapeutic applications.