Potato dextrose agar (PDA) and broth (PDB), Mueller-Hinton agar (MHA) and broth (MHB), Sabouraud dextrose agar (SDA), gentamicin, nystatin, and sodium dodecyl sulfate (SDS) were purchased from HiMedia (Mumbai, India). Other materials were obtained from different manufacturers such as sodium hypochlorite solution (available chlorine 4%) from Fisher Scientific (Mumbai, India); Precoated aluminum-backed TLC silica gel plates (60 F254) from Merck (Darmstadt, Germany); HPLC grade solvents like ethyl acetate; hexane and methanol from SDFCL (Mumbai, India); and Silica gel 60–120 mesh from Qualigens (Mumbai, India). The ITS 1 and ITS 4 primers, 2,3,5-triphenyl tetrazolium chloride (TTC), and Thiazolyl Blue Tetrazolium Bromide (MTT) were procured from Sigma-Aldrich (Missouri, USA).
All the test microbial pathogens such as Staphylococcus aureus (MTCC 7443), Bacillus subtilis (MTCC 121), Bacillus cereus (MTCC 430), Staphylococcus epidermidis (MTCC 435), Escherichia coli (MTCC 7410), Klebsiella pneumonia (MTCC 7407), Salmonella typhi (MTCC 733), Shigella flexneri (MTCC 1457), Vibrio parahaemolyticus ((MTCC 451), Xanthomonas campestris (MTCC 7908), and Candida albicans (MTCC 183) were procured from Microbial Type Culture Collection and Gene Bank (MTCC; Chandigarh, India).
Plant collection and isolation of fungal endophytes
Healthy asymptomatic A. philippense was collected from the Western Ghats region near Virajpete (12.2° N, 75.8° E). The plant specimen was identified by Dr. S. Mahadevakumar (field taxonomist) and submitted to herbarium (Voucher specimen number: UOM-BOT20-AP02). The plant material was sealed in sterile polythene bags and transported to the lab and processed within 8 h of collection. The plant material was initially washed under tap water for removing unwanted debris and soil; followed by sterile double distilled water (Dw) washes for twice. The plant leaf material was separated and cut into segments of 0.5 cm2 size. The plant segments were subjected to surface sterilization with 75% (v/v) ethanol (1 min), followed by 4% (v/v) NaOCl (4 min) and 75% (v/v) ethanol (1 min). Finally, tissue bits were rinsed with sterile double distilled water to remove residual surface sterilizing agents and blot dried under aseptic conditions. The surface-sterilized and blot dried leaf segments were placed on potato dextrose agar amended with antibiotic chloramphenicol (100 mg/L) to eliminate bacterial growth followed by incubation in a light chamber for two weeks at a 12-h light/dark cycles at 25 ± 2 °C. The fungal colonies were picked and transferred onto fresh potato dextrose agar plates devoid of any antibiotics [15, 23].
Genomic DNA extraction
Bioactive mycoendophyte N. sphaerica isolated from A. philippense was cultured in potato dextrose broth for a week at 25 ± 2 °C for isolation of genomic DNA. The mycelial mat was harvested, grounded, and transferred to a microcentrifuge tube containing 1 ml of 2× CTAB extraction buffer and incubated at 65 °C water bath for 30 min, later centrifuged at 10,000 g (10 min; RT). The aqueous phase was mixed with equal volume phenol: chloroform: isoamyl alcohol (25:24:1) for total DNA extraction followed by the addition of propanol to precipitate DNA .
Polymerase chain reaction amplification
The PCR amplification was performed according to the protocol of White et al.  using ITS1 and ITS4 set of universal primers. The PCR reaction mixture (50 μL) was prepared with 5 μL PCR buffer (10X; 15 mM MgCl2), 5 μL of dNTPs (2 mM), 2 μL of forward and reverse primers (5 pmole/μL), 2 μL Taq polymerase (1 U/μL), and the final volume was made up for 50 μL using Nanopure water (30 μL). The PCR reaction was carried out for 30 cycles with the following conditions denaturation 94 °C (40 s), annealing at 54 °C (60 s), extension at 72 °C (60 s), and final extension for a 10-min interval at 72 °C. The sequence was analyzed using ABI 3730 sequencer.
Antimicrobial screening by agar plug method
The pure cultures of fungal endophytes isolated from A. philippense were preliminarily screened for antimicrobial activity using the agar plug diffusion method. Pure cultures of fungal endophytes were cultured in potato dextrose agar for 21 days at 25 ± 2 °C. The agar plugs of pure cultures were placed onto Mueller-Hinton agar preseeded with test organisms, followed by refrigeration at 4 °C for 1 h to facilitate diffusion of metabolites, and further plates were incubated at 37 ± 2 °C (24 h) for bacteria and 25 ± 2 °C (72 h) for fungi. After incubation, zones of inhibition around the agar plug were observed .
Fermentation and extraction of secondary metabolites
Bioactive N. sphaerica was cultured in 2 L Erlenmeyer flasks (10) containing 450 ml potato dextrose broth at 25 ± 2 °C for 4 weeks under static conditions. The culture broth was filtered, and the liquid supernatant was extracted thrice with an equal volume of ethyl acetate and concentrated using a rotary flash evaporator (Rotavapor R® R-3, Buchi) (50 °C) and it was stored at 4 °C. The ethyl acetate extract concentrate was fractionated by column chromatography over a silica gel column (50 g, 60–120 mesh) using stepwise gradient elution from hexane: ethyl acetate (100:0 to 0:100) mixtures with increasing polarity, to afford different fractions (eluates). The fractions obtained were developed in TLC for pooling the similar fractions using optimized solvent system ethyl acetate: hexane (1:1; v/v) .
Detection of antimicrobial metabolite by TLC-bioautographic assay
Analytical TLC was employed for detecting antimicrobial metabolites. Ten microliters of flash evaporated ethyl acetate fraction of bioactive mycoendophyte N. sphaerica was spotted on TLC silica gel plates and developed in an optimized solvent system of ethyl acetate/hexane (1:1; v/v). TLC sheets were observed under UV light (254 nm) and dried in aseptic conditions. The developed TLC sheets were placed onto sterile Petri plates and overlaid with Mueller-Hinton agar amended with TTC (1 mg/ml) for bacteria and Sabouraud Dextrose agar for fungi previously seeded with 1% standardized (McFarland standard) microbial inoculum and incubated. After incubation, for fungi, the Petri plate was flooded with 10 ml of soft agar (agar 1% w/v) amended with MTT (0.05% w/v). Inhibition zones were observed as clear spots, and the active bands and corresponding retention factor (Rf) value was measured [19, 23].
Antimicrobial activity by disc diffusion method
The disc diffusion method was used to determine the antimicrobial activity of the endophytic fungal extract. Sterile media plates (MHA for bacteria and SDA for fungi) were seeded with predetermined test microbial inocula as described by Rakshith et al. . Sterile discs were impregnated with purified bioactive metabolites from the stock solution (10 mg/ml). The concentration of 30, 60, 150, 300, 450, and 600 μgdisc-1 were placed on respective agar media with gentamicin (10 μg/disc; for bacteria) and nystatin (100 U/disc; for fungi) as a positive control, whereas ethyl acetate served as a negative control. Inoculated plates were incubated at 37 ± 2 °C (24 h) for bacteria and 25 °C ± 2 (72 h) fungi. After the incubation, the zone of inhibition was measured around discs in millimeter and results were expressed as mean ± SD.
The purity of the bioactive metabolite was analyzed with reference to the chromatogram of the crude ethyl acetate extract of N. sphaerica with Shimadzu UFLC – LC – 20 AD series. An amenable 5 μm C18 120 Å, 250 × 4.6 mm LC (A8-ST5C18G120-98) column was used for the detection of metabolites. The injection volume of 20 μL (1 mg/ml) was developed with the mobile phase using methanol with a flow rate of 1 ml/min. The chromatogram was recorded at 260 nm using Lab solutions (Shimadzu corp., Japan) software.
The mass of the purified bioactive metabolite was measured using liquid chromatography coupled with Q-TOF in positive electrospray ionization-mass spectrometry (Waters Acquity UPLC Synapt G2 HDMS) with BDS HYPERSIL C18 column (250 × 4.6 mm × 5 μm). The LC separation was recorded using Quattro Premier XE with the Mass Lynx 4.1 software with a gradient solvent system A: 0.1 vol. % formic acid–water and B: 0.1 vol. % formic acid–acetonitrile; 2–98 % B in 50.0 min of run time with a controlled column temperature at 30 °C. The Q-TOF–ESI–MS data were recorded in the range of 200 and 400 nm and positive ESI full MS (60.00–1000.00).
The 1H and 13C NMR spectra of the purified bioactive metabolite (35 mg/ml) were recorded using deuterated chloroform (d-CDCl3) on an Agilent 400-MHz WB (Widerbore) NMR magnet (Santa Clara CA, USA) spectrometer using the VJ3.1 software at 400 MHz and 100 MHz, respectively.
Fourier transform infrared spectra of the bioactive purified fraction (1 μLof 1 mg/ml) was recorded using PerkinElmer spectrum TwoTM ATR MIRacle Diamond S2PE (Norwalk, CT, USA) spectrophotometer with the scanning range of 4000–600 cm-1.
Determination of minimal inhibitory concentration
The MIC was determined by the microdilution method according to Clinical and Laboratory Standards Institute (CLSI) by modifications by . Briefly, the stock solutions of purified phomalactone (10 mg/ml), Gentamicin, and Nystatin (2 mg/ml) were prepared. Two-fold dilutions were carried out in the concentration range of 100–0.19 μg/ml, in 100 μL of sterile Mueller Hinton broth for bacteria and Sabouraud’s dextrose broth for fungi. Twenty microliters of standard suspension of bacterial and fungal inocula were added, except for sterility control well. Gentamicin for bacteria and Nystatin for fungi used as a standard positive control along with growth and sterility controls. Minimal inhibitory concentrations were determined by absorbance at 600 nm after incubation with compounds at 37 ± 2 °C (24 h) for bacteria and 25 °C ± 2 (72 h) fungi .
All the experiments were conducted in triplicates and statistical significance such as one-way ANOVA was evaluated using IBM SPSS (Version 25) software.