Molecular characterization and evaluation of phytochemical constituents of endophytic fungi derived from Mitracarpus scaber Zucc. (Rubiaceae)

In quest to ameliorate the issues of recurring incidences of multidrug-resistant organisms and over-exploration of plants, researchers have delved into researches involving endosymbiotic microorganisms, known as endophytes. These are microorganisms inhabiting the internal part of plants, which have been verified to possess great potentials of bioengineering novel products for therapeutic purposes. The aim of this study was to isolate and identify endophytic fungal species present in Mitracarpus scaber, molecularly characterize the pure isolates, and test for phytochemical compounds present. Freshly collected non-diseased leaves of M. scaber were subjected to a four-step surface sterilization. Thereafter, they were sliced into fragments of 1 cm-length, exposing the leaf blades and the midribs, and were aseptically inoculated on sterilized malt extract agar, containing chloramphenicol (500 mg/l), in Petri dishes. Hyphal tips of actively growing fungi from the plant material were harvested and further sub-cultured for purification and isolation. Segments were aseptically cut from the actively growing pure isolates, on malt extract agar and inoculated onto the fermentation medium (sterilized local rice), in 500 ml Erlenmeyer flasks, which were properly sealed with sterile cotton and kept at static condition at 25 oC  for 21 days. Ethyl acetate was used to extract the metabolites from the end products of the fermentation processes. Four isolates obtained from the endophytic fungi (EDF), present in the leaf of M. scaber, were subjected to molecular identification. The DNA extraction was done using Zr fungal/bacterial DNA miniprep. The extracted DNA was amplified through PCR, and sequenced. The resultant sequences were compared with GenBank database, using Basic Local Alignment Search Tool. The result of phytochemical screening of the extracts revealed the presence of flavonoids, tannins and phenols, in large amounts; terpenoids, alkaloids and cardiac glycosides, in moderate amounts; steroids, hydrogen cyanide and saponins in very low quantities, while that of molecular characterization identified four organisms: Penicillium sclerotiorum, Clasporium cladosporiodies, Cryptococcus nemorosus, and Phyllosticta capitalensis.