Isolation of fungal material
The plant samples were accumulated from conservation forest of Sundarban, Bangladesh in February, 2018. The plant material was recognized and authenticated by a taxonomist of Bangladesh National Herbarium (BNH), Dhaka, Bangladesh. A voucher specimen of this compilation was retained at BNH under the accession number DACB – 45,945. Fungal strains were isolated from the fresh plant parts following the procedure described by Khan et al. 2018 [12, 13].
Taxonomical identification of fungal cultures
Morphological identification
Slides were prepared from cultures by staining with lactophenol cotton blue examined with a bright-field and phase contrast microscope for the identification of endophytic fungal strains (Sadananda, Govindappa et al., 2014). Likewise, the standard taxonomic keys such as growth pattern, hyphae, the color of the colony and medium, margin character, aerial mycelium, surface, texture, sporulation and production of acervuli, the size and coloration of the conidia were used to identify morphological characteristics [14, 15].
Molecular identification
Fungal strains were identified by DNA amplification and sequencing of the internal transcribed spacer (ITS) region [16]. A section of fungal hyphae (0.5-1.0 cm2) was assembled from the Petridish and lyophilized in an Eppendorf tube (2 mL, Eppendorf, Germany). With the help of glass beads the lyophilized fungal mycelia were pulverized and agitated. In keeping with the manufacturer’s protocol, fungal DNA isolation was acquired by using DNeasy Plant Mini Kit (QIAgen, USA). Cell lysis, digestion of RNA by RNase A, removal of precipitates and cell debris, DNA shearing; precipitation and purification were assimilated in the method. The isolated DNA was then amplified by polymerase chain reaction (PCR). The PCR was conceded utilizing HotStarTaq Master Mix Kit (QIAgen, USA). ITS 4 (with base sequences TCCGTAGGTGAACCTGCGG) and ITS 5(with base sequences TCCTCCGCTTATTGATATGC) (Invitrogen, USA), as primers, were mixed with HotStarTaq Master Mix Kit and DNA template in a total volume of 50 µL. The mixture was then applied to the thermal cycler (BioRad, USA) using the programmed PCR cycle as outlined below: Initial activation step in 95 °C for 15 min to activate HotStarTaq DNA polymerase, cycling steps which were restated 35 times, denaturing: 1 min at 95 °C, annealing: 1 min at 56 °C, extension: 1 min at 72 °C, final extension for 10 min in 72 °C.The PCR product was purified using 2 % Agarose-Gel-Electrophoresis at 75 V for 60 min in 1 X TBE buffer. The agarose gel was then stained using 1 % ethidium bromide. An approximate size 550 bp stained DNA fragment was then wiped out from the agarose gel. By following manufacturer’s protocol, PCR product purification was carried out using Perfect Prep Gel Cleanup Kit (Eppendorf, USA). The amplified fungal DNA (PCR product) was then submitted for sequencing by a viable service and the base sequence was evaluated with publicly accessible databases such as GenBank with the help of Blast-Algorithms.
Bioactivity screenin
Brine shrimp lethality bioassay
According to Meyer et al. Brine shrimp lethality bioassay was implemented to inspect the cytotoxicity of the extract [17]. Serial dilution was then carried out in order to obtain the concentration of 1.25 µg/mL to 320 µg/mL. 5 mL of artificial sea water was added into all the test tubes. Artificial seawater (prepared by using sea salt 38 g/L and adjusted to pH 8.5 using 1 N NaOH) was used to hatch the eggs of the brine shrimps (Artemia salina) under constant aeration for 24 h under the light. To get shrimp larvae, the hatched shrimps were permitted to grow by 48 h. Suspension containing 10 nauplii was added into each test tube and was incubated at room temperature under the light. The tubes were then examined after 24 h and the number of surviving larvae in each test tube was counted with the aid of a magnifying glass. The percentage of mortality was plotted against the logarithm of concentration. The concentration that would kill 50 % of the nauplii (LC50) was determined from probit analysis as well as linear regression equation using the software “Microsoft Excel-2007”. Tamoxifen was used as standard in this bioassay [18].
Antimicrobial screening
The antimicrobial and antifungal activities were investigated by the procedure explained by Bauer et al. [19]. The test microorganisms used in the antimicrobial study incorporated four pathogenic bacterial strains Staphylococcus aureus (ATCC 25,923), Escherichia coli (ATCC 28,739), Bacillus megaterium (ATCC 259), Salmonella typhi (ATCC 28) and one fungal strains Aspergillus niger. After 18–24 h of incubation at 37 °C for bacteria and 48–96 h of incubation at 28 °C for fungi, the zones of growth inhibition around the discs were measured. By determining the diameter of inhibitory zones in millimeter compared to kanamycin (30 µg/disc) as a positive control and ketoconazole (30 µg/disc) as standard antibiotics for antibacterial and antifungal screening, correspondingly the sensitivities of the microorganism species to the fungal extract were calculated.
Initial screening by Thin Layer Chromatography (TLC)
This screening of the extract was performed by visual detection, UV light (in short and long wavelength) and vanillin- H2SO4 spray reagent.
Chemical screening
For identification of chemical constituents of endophytic fungal crude extracts the extracts were subjected to thin layer chromatographic technique. Commercially available precoated silica gel (Kiesele gel 60 PF 254) plates were used with solvent system of toluene: 10 % ethyl acetate for the initial chemical screening of the crude extracts.
Statistical analysis
IC50 values between groups were compared using independent student’s t-tests. For the analysis of the cytotoxicity results for each extract, continuous variables between groups were compared with one-way analysis of variance (ANOVA) with post hoc Tukey’s test. Mean values between groups were compared using independent student t-test for equality of variances. Statistical significance was accepted when P < 0.001.