Plant collection and isolation of endophytes
The plant was collected from Wadi Fatima, Makkah, Saudi Arabia, and identified by Dr. Hany Gouda, Department of Pharmacognosy, College of Pharmacy, Najran University, Saudi Arabia. A voucher specimen (UQU-2019-1) of the plant was kept at the herbarium of the Pharmacognosy Department, Faculty of Pharmacy, Umm Al-Qura University. Handling of the plant material was started by cutting it into small pieces, washing with demineralized, sterilized water and afterward treating its surface with 70% ethanol for 1–2 min followed by air-drying under a laminar flow hood. This aimed to eliminate contaminating microbes on the surface of the plant material. To confirm effective sterilization, 1 ml of the final rinse was placed onto nutrient agar media and incubated at 37 °C for 24 h. The outer tissues of the sterilized plant samples were removed with a sterile scalpel, and inner tissues were dissected under sterile conditions and planted onto malt agar (MA) plates containing 0.1 g streptomycin as an antibiotic. After an incubation time of about 3–4 weeks at room temperature, hyphal tips of the fungi were transferred to fresh malt agar medium. To eliminate the possibility of contamination, duplicate plates were prepared, and pure strains were isolated by repetition of inoculation. MA medium consisting of agar–agar (15.0 g), malt extract (15.0 g), distilled water (to 1000 mL), pH (7.4–7.8 adjusted with NaOH/HCl), was used for the storage of fungal cultures for a short term. Chloramphenicol or streptomycin (0.2 or 0.1 g, respectively) was added to the medium to inhibit bacterial growth during the isolation of endophytic fungi from plant tissues (Zhang et al. 2006).
Cultivation of endophytic strains
Each endophytic fungus was cultivated for 14 days on potato dextrose agar (PDA) at 23 °C. The mycelium was divided into 12 pieces, and each one was used to inoculate a 1-L Erlenmeyer flask that contained 250 mL of a culture medium composed of glucose 10 g/L, malt extract 20 g/L, Soybean flour 2 g/L, yeast extract 1 g/L, KH2PO4 1 g/L and MgSO4.7H2O 0.5 g/L. Incubation was carried out for 21 days (23 °C) under static conditions (Stevens 1981). For extraction, culture filtrate and mycelium were homogenized in each Erlenmeyer flask and the homogenized mixture was macerated in 200 mL EtOAc for 24 h, which was then collected by decantation. The obtained ethyl acetate extract was then evaporated to dryness and defatted with n-hexane to get the crude extract.
Identification of fungal strains
Identification of the fungal strains was carried out using the standard protocol based on their cultural and microscopic properties (Barnett and BB 1998) and was confirmed using molecular biological techniques through DNA extraction. Then, amplification was carried out by polymerase chain reaction (PCR), and finally, sequencing was performed using ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) primers. For DNA extraction from the fungal culture, a piece of 0.5 cm2 was cut in the MA plate and was freeze-dried in a 2-ml tube. One hundred microliters of rapid lysis solution from Sigma REDExtract-N-Amp™ Plant PCR Kit was added to it followed by 10-min incubation at 95 °C and vortex with glass beads was sometimes required. For neutralization after heating, 100 µL of dilution solution was added. DNA amplification was carried out by performing a polymerase chain reaction (PCR) in which Sigma REDExtract-N-Amp™ Plant PCR Kit’s polymerase and different primers were employed. To 46 µL of the polymerase master mixture, 4 µL of DNA template was added which consisted of 18 µL RNA (ribonucleic acid)-free water, 25 µL REDExtract-N-Amp™ Plant PCR solution and 3 µL of each primer (10 pmol/μL each). The 50 µL solution sample was then subjected to the PCR (Michaelsen 2006; Raja 2017).
PCR protocol, DNA extraction, amplification and sequencing
Denaturation was performed at 95 °C for 15 min followed by denaturation at 95 °C for 1 min, and finally, annealing was carried out at 56 °C for 0.5 min. The extension process was performed at 72 °C for 1 min followed by final extension at 72 °C for 10 min. All steps were carried out 35 times except initial denaturation and final extension. To confirm that PCR has been successfully carried out, the gel was transferred into the ultraviolet transilluminator after electrophoresis. By comparison with DNA ladder, PCR products were found to possess the right size of about 550 bp. The band was accurately excised from the gel and inserted into an Eppendorf tube. As indicated by the protocol of the manufacturer, the PCR product was isolated from the gel slice by the aid of Sigma GenElute™ Gel Extraction Kit. Three-gel volume of the solubilization solution was added to the gel and heated at 50–60 °C for 15 min. For precipitation of the DNA, one gel volume of isopropanol was added. Afterward, the solution was applied to the binding column followed by spinning at 13,000 rpm for 1 min. Then, the washing solution was added to remove any impurities which was followed by spinning at 13,000 rpm. The collection tube was finally changed, and the elution solution was added, followed by spinning for elution of DNA from the column. The concentration of DNA was determined using Nano drop 2000, and the PCR product was stored at − 20 °C until sequencing. DNA sequencing was carried out by the addition of 30 ng of PCR product to 3.2 pmol primer, 8 µL big dye and 20 µL RNA-free water. Afterward, this mixture was subjected to PCR (Bio-Rad, USA) starting with initial denaturation at 96 °C for 1 min followed by 35 cycles of denaturation at the same temperature for 10 s and annealing at 50 °C for 5 s followed by extension at 60 °C for 4 min. The obtained products were stored at 8 °C and then subjected to direct sequencing, and the sequences were aligned through Clustalw® and Boxshade® Web sites. Basic local alignment search tool (BLAST) search of the FASTA sequence was performed with the option “nr” on the BLAST homepage, National Center for Biotechnology Information (NCBI), Bethesda, USA (Elix et al. 1982; Fernandez 1998).