Isolation, maintenance and Fermentation of the Fungal Endophyte: A young and healthy plant material of Catharanthus roseus (L.) G. Don (EC 120837) was obtained from herbal garden of Jamia Hamdard University, New Delhi, India. The leaves, stem and root of the plant were cut in to small pieces (approximately 2mm size) with the help of a surgical blade, and washed in reverse osmosis water using cetrimide. Afterwards, the plant segments were surface sterilized (using optimised method) by adding plant material in 70% ethanol for 1minute, rinsed twice with autoclaved water, and then the plant material was added to 0.1% mercuric chloride for 90 seconds, finally washed thrice with sterile water. The effectiveness of surface sterilization was determined by observing no bacterial growth in the rinsed water on potato dextrose agar (PDA) media after 7days of incubation at 28˚C.The segments were transferred onto petri plates containing PDA media (Himedia, Mumbai, India) supplemented with antibiotic mixture of streptomycin sulphate 250 mg/L and penicillin G 250 mg/L in order to grow endophytic fungus without any contamination. Plates were sealed with parafilm and incubated at 28oC until the fungal colonies started oozing out of the explants.All of the isolated endophytic fungal cultures were maintained in PDA slant and sub-cultured in every 30 day interval. Isolated endophytes were cultured in liquid medium (20g dextrose, 1g yeast extract, 3g potassium dihydrogen sulphate, 1.5g magnesium sulphate.7H2O, pH 6.0) in 250mL Erlenmeyer flasks at 28oC, 110 rpm in a shaker incubator. After 6 days, the culture (fungal biomass) were separated and analysed for monomeric/dimeric Vinca alkaloids by qualitative analysis using high performance liquid chromatography (HPLC) and mass spectrometry separately.
Extraction & analysis of vinca alkaloids: Various endophytes isolated from the explants were analysed for the presence of the secondary metabolites through qualitative analysis mode using high performance liquid chromatography (HPLC) technique. The fungal biomass was obtained by filtration process using Whatman No 1 filter paper and dried at 50oC for 4 hr in a hot air oven. To the dried fungal biomass, methanol was added (25 ml per 5 g of dried biomass) and sonicated (Pulse-30Khz, Gap-10 seconds, amplitude-100%) for 10 min using probe sonicator (VCX 130, Vibra Cell, Sonics, USA). After the sonication process, supernatant was collected by centrifugation (1537g for 5min), evaporated to dryness in a vacuum evaporator at 60°C. For HPLC-UV analysis, the dried extract was dissolved in equal volume of methanol and filtered through 0.45 µm membrane. Elution was carried out by the mobile phase consisting of methanol and water with 0.1% triethylamine at a flow rate of 1ml/min under gradient mode (55% v/v methanol at 0 min, 65% v/v methanol at 5 min, 70% v/v methanol at 15 min, 80% v/v methanol at 18 min, 90% v/v methanol at 35 min, 55%v/v methanol at 40 min).[10, 27] Detection was carried at 260nm. The mass analysis was carried out on UPLC MS/MS analyser of “WATERS” and data were acquired using MassLynx4.1 software. Source temperature was set at 80°C and the gas flow at 500l/hr.
Identification of the endophytic fungal strain: The vindoline producing endophytic fungus was identified and characterized by using molecular biology techniques as per the manufacturer protocol (Bhat Biotech, Bangalore, India). Fungal genomic DNA was extracted from fungus using genomic DNA extraction kit from fresh culture of MPBL 105 (desired endophytic fungus). Amplification of the nuclear internal transcribed spacers (ITS) region was performed using the universal primers ITS1 (5’-TCCGTAGGTGAACCTGCGG-3’) and ITS4 (5’-TCCTCCGCTTATTGATATGC-3’). The amplification was carried out in a Master cycler® Thermocycler (Eppendorf, Germany) using the following program. Polymerase chain reaction (PCR) was performed with initial denaturation of 94oC for 2 minutes followed by 40 cycles of denaturation at 94oC for 1 min, annealing at 55.5oC for 1 min and extension at 72oC for 30 sec. Final extension was carried out at 72oC for 10 min. Amplified PCR products were separated in 1% agarose gel containing ethidium bromide in 0.5 µg/ml concentration (for detection of bands under UV) in Tris-Borate –EDTA (TBE) buffer. The purified PCR product was used for sequencing. Sequencing was carried by automated DNA sequence machine − 3037xl DNA analyzer from Applied Biosystems using BigDye® Terminator v3.1 cycle sequencing Kit (Applied Biosystems). Sequence data were aligned and dendrograms were generated using sequence analysis software version 5.2 from Applied Biosystems. Sequences were compared to the non-redundant NCBI database by using BLASTN, with the default settings used to find the most similar sequence and were sorted by the E score. A representative sequence of 10 most similar neighbours was aligned using CLUSTAL W2 for multiple alignments with the default settings. The multiple-alignment file was then used to create phylogram using Molecular Evolutionary Genetic Analysis (MEGA 5) software. On the basis of the results of the BLAST search, the isolated endophytic fungal strain MPBL 105 was identified as Alternaria sesami (Pleosporaceae).
Establishment of C. roseas callus culture: Healthy and immature leaves of Catharanthus roseus (L.) G.Don.were collected from the herbal garden, Jamia Hamdard University, New Delhi and used as explants for callus culture. The explants were washed in running reverse osmosis water for 10 minutes using cetrimide and were subjected to surface sterilization using 70% ethanol and 0.1% mercuric chloride.For C. roseas callus culture MS (Murashige and Skoog) media was used as basal media with 3% sugar, 1.1% agar, 0.01% myoinositol and combination of different growth hormones [naphthalene acetic acid (NAA), indole acetic acid (IAA), kinetin, 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzyladenine (6-BA)]. The incubation temperature was 25°C and light intensity of 1600 lux with 16 hr photoperiods per day. The callus was sub-cultured after every 21days and grown and maintained for 90 days. After 90 days,C. roseas callus was treated with methanolic or aqueous extract of fungal endophyte (A. sesami) at a concentration of 25µl/g of media, 50µl/g of media, and 100µl/g of media and grown for another 15days under same culture condition. After a total 105 days of growth, each callus was collected and fresh weight was noted. Further the callus was dried at 60oC. The drying was carried out up to the constant weight of the callus. To the dried callus, methanol was added (5% v/w) and sonicated (Pulse-30Khz, Gap-10 seconds, amplitude-100%) for 15 min using probe sonicator (VCX 130,Vibra Cell, Sonics, USA). After the sonication, biomasswas centrifuged for 5min at 1537g and the supernatant was collected and evaporated to dryness in a vacuum evaporator at 60°C. The dried extract was dissolved in equal volume of methanol and filtered through 0.45 µm membrane and analysed by HPLC –UV using same procedures as described in above section.