Isolation and Identification of Bacterial and Fungal Edophytes from Leaves and Stems of H. rosa-sinensis and Production of Secondary Metabolites from Isolated Endophytes

Abstract

Endophytes were first reported by the Freeman (1904) from dunel grass. 3,00,000 plant species known which exist on earth, all are host to one or more endophytes [1]. Endophytes are found in roots, leaves, stems, fruits, tubers, ovules etc. (Hallmann et al., 1997; Sturzet al., 1997: Hurek et al., 2002, Benhizia et al., 2004; Iniguez et al., 2004). Many different types of alkaloids, terpenoids, flavonoids and steroids are produced by endophytic fungi and these metabolites of endophytic fungi have antimicrobial, antiviral, anticancer, and antioxidant potential. The root of the Hibiscus rosa is used for cough treatment. The leaves of the H. rosa are used as a laxative and flowers has anti – diarrhea properties and are considered as demulcent, emollient, refrigerant, aphrodisiac and emmenagogue. The high level of antioxidants in H. rosa sinensis can slow down the aging process by eliminating the free radicals and neutralizing their harmful effects on the cells of human body. Hibiscus flowers are used as a remedy for relieving respiratory problem and to improve the health of respiratory system.
 This work reveals that the endophytic bacteria and fungi were isolated from healthy leaves and stem of medicinal plant, H. rosa-sinensis which were first sterilized in 70% ethanol. The bacterial endophytes were isolated on Nutrient Agar Medium and fungal endophytes were isolated on Potato Dextrose Agar Medium. Colonies showing similar morphology on one plate were considered as single colony and so the frequency of each colony is calculated and isolated in pure form. By pure culture isolation technique, 4 different isolates of bacterial endophytes and 2 different isolates of fungal endophytes from stem of H. rosa-sinensis and 3 different isolates of fungal endophytes from leaves of H. rosa-sinensis were obtained. Bacterial endophytes were identified on the basis of Gram Stain and biochemical characterization whereas the identification of fungal endophytes was carried out at Plant Pathology Department of Forest Research Institute (FRI), Dehradun. The secondary metabolites were isolated from isolated bacterial and fungal endophytes by using two different solvents (Ethyl acetate, chloroform) in the ratio 1:1 left for 15 minutes, the organic phase were collected and kept for drying at 37C. The antimicrobial activities were done against human pathogen (Stayphylococcus aureus and Klebsiella pneumonia) by using Disc diffusion method of isolated secondary metabolites.