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Bioprospecting of Endophytic Fungi from Certain Medicinal Plants

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Bioprospecting of Endophytic Fungi from Certain Medicinal Plants BIOPROSPECTING OF ENDOPHYTIC FUNGI FROM CERTAIN MEDICINAL PLANTS THESIS SUBMITTED TO BHARATI VIDYAPEETH DEEMED UNIVERSITY, PUNE FOR THE AWARD OF DOCTOR OF PHILOSOPHY (Ph. D.) IN MICROBIOLOGY UNDER FACULTY OF SCIENCE BY MONALI GULABRAO DESALE UNDER THE GUIDANCE OF DR. MUKUND G. BODHANKAR DEAN, FACULTY OF SCIENCE BHARATI VIDYAPEETH DEEMED UNIVERSITY YASHWANTRAO MOHITE COLLEGE PUNE May 2016 CERTIFICATE This is to certify that the work incorporated in the thesis entitled “Bioprospecting of Endophytic Fungi From Certain Medicinal Plants” submitted by Monali G. Desale for the award of the Degree of Doctor of Philosophy in Microbiology under the Faculty of Science of Bharati Vidyapeeth Deemed University, Pune was carried out in the Microbiology laboratory of Bharati Vidyapeeth Deemed University Yashwantrao Mohite College, Pune. Date:- ( Dr. K. D. Jadhav ) Principal, Bharati Vidyapeeth Deemed University Yashwantrao Mohite College, Pune CERTIFICATE This is to certify that the work incorporated in the thesis entitled “Bioprospecting of Endophytic Fungi From Certain Medicinal Plants” submitted by Monali G. Desale for the award of the degree of Doctor of Philosophy in Microbiology under the Faculty of Science of Bharati Vidyapeeth Deemed University, Pune was carried out under my supervision. Date: (Dr. Mukund G. Bodhankar) Dean, Faculty of Science Department of Microbiology Bharati Vidyapeeth Deemed University Yashwantrao Mohite College, Pune DECLARATION BY CANDIDATE I hereby declare that the thesis entitled “Bioprospecting of Endophytic Fungi From Certain Medicinal Plants” submitted by me to the Bharati Vidyapeeth Deemed University, Pune for the degree of Doctor of Philosophy (Ph.D.) in Microbiology under the Faculty of Science is original piece of work carried out by me under the supervision of Dr. M. G. Bodhankar. I further declare that it has not been submitted to this or any other university or institution for the award of any degree or diploma. I also confirm that all the material which I have borrowed from other sources and incorporated in this thesis is duly acknowledged. If any material is not duly acknowledged and found incorporated in thesis, it is entirely my responsibility. I am fully aware of the implications of any such act which might have been committed by me advertently or inadvertently. Date: (Monali G. Desale) Research Student Bharati Vidyapeeth Deemed University Yashwantrao Mohite College, Pune. ACKNOWLEDGEMENT I would like to place on record my deep sense of gratitude to my guide Dr. Mukund G. Bodhankar, Dean, Faculty of Science, Microbiology Department, Bharati Vidyapeeth Deemed University, Yashwantrao Mohite College, Pune for his support, guidance and encouragement without which this work would not have completed. I also take this opportunity to thank Prof. Dr. Shivajirao Kadam, Vice-Chancellor, Bharati Vidyapeeth Deemed University, Pune for creating an atmosphere conducive to research in all institutions of University which helped me a lot during the course of Ph. D. work. I also profusely thank Dr. K. D. Jadhav, Principal, Bharati Vidyapeeth Deemed University Yashwantrao Mohite College, Pune for his support and permission to perform experimental and thesis work in the laboratory of the college. I would like to express my sincere gratitude to Dr. Mrs. V. R. Sapre from Microbiology department of the college for her cooperation in completing the part of the work. I would also like to express my sincere gratitude to Dr. S. K. Singh, Scientist, Agharkar Research Institute (ARI), Pune for his support and guidance in morphological identification of endophytic fungi. I express my sincere thanks to Mr. Sachin Purohit, Managing Director, GeneOmbio Technologies, Pune and the team of Microbial Identification section specially Dr. Amol Raut, Mr. Yashawant Chavan, Mr. Asif Kawathekar for their prompt help in molecular identification. I place on record my sincere thanks to Dr. Niraj Vyawahare, Principal, Pad. D. Y. Patil College of Pharmacy, Pune and Dr. Santosh Gandhi, Professor, Department of Analytical Chemistry, AISSMS College of Pharmacy, Pune who lent a helping hand in chemical elucidation of the bioactive metabolite. I also thank Ms. Prajakta Pathade of Botanical survey of India, Pune for helping me in the part of the work. I would also like to express my sincere thanks to subject experts Prof. B. P. Kapadnis and Principal Dr. G. R. Pathade for their expert and critical suggestions and positive criticism. The work would have been incomplete without the help rendered in various ways by all staff of Department of microbiology including Mrs. Jape, Mr. Santosh Kharge, Mr. Patole and Mr. Golve. I would also like to express special thanks to all my colleagues, friends and teaching as well as non-teaching staff who kept my spirits high during the testing period of laboratory work. Though not connected directly with the work, my acknowledgment would be incomplete without mentioning all my family members. I express my deep gratitude for my parents in law, husband, parents and specially kids who allowed me to complete the work uninterruptedly. Date: (Monali G. Desale) TABLE OF CONTENTS Page Abstract I Chapter 1 : Introduction 1 Chapter 2 : Objectives 6 Chapter 3 : Review of Literature 2.1 Definition 7 2.2 Distribution and biodiversity of endophytic fungi 9 2.3 Bioprospecting of microbial endophytes and their natural products for 11 various pharmaceutical activities 2.4 Antibacterial activities of endophytic fungi 14 2.5 Antifungal activities of endophytic fungi 15 2.6 Anticancer activities of endophytic fungi 15 2.7 Antiviral activities of endophytic fungi 16 2.8 Diversity and studies of endophytic fungi in India 17 2.9 Fungal endophytes from medicinal plants with reference to India 19 2.10 Therapeutic properties and endophytic fungi of Ocimum sanctum 19 2.11 Therapeutic properties and endophytic fungi of Vitex negundo 20 2.12 Therapeutic properties and endophytic fungi of Barleria prionitis 22 Chapter 4 : Materials and Methods 3.1 Selection of plant species 51 3.2 Localities/Sites for collection of plant samples 54 3.3 Collection of plant samples 54 3.4 Isolation of endophytic fungi 54 3.5 Media used for isolation of endophytic fungi 57 3.6 Calculation of colonization rate, isolation rate and density of colonization 57 3.7 Preservation of culture 58 3.8 Identification of endophytic fungi 58 3.8.1 Morphological identification 58 3.8.2 Molecular identification 58 3.9 Cultivation of fungi in different media for the production of metabolites 60 3.10 Fermentation, extraction and isolation of secondary metabolites 61 3.11 Screening of endophytic fungi for antibacterial activity 61 3.11.1 Microorganisms used for antibacterial activity 61 3.11.2 Preparation of test sample 61 3.11.3 Agar well diffusion method 61 3.12 Process optimization of fermentation conditions for production of active 62 metabolites from selected endophytic fungi 3.13 Extraction and purification of secondary metabolites of selected endophytic 62 fungi for evaluation of antibacterial activity 3.14 Determination of minimum inhibitory concentration (MIC) 63 3.15 Chemical screening of selected endophytic fungal extract 63 3.15.1 Chromatographic analysis by High performance thin layer 63 chromatography (HPTLC) 3.15.1.a Instrumentation 63 3.15.1.b Chromatographic conditions 63 3.15.1.c Mobile phase 63 3.15.1.d Calculation of Rf values 64 3.15.2 Structural elucidation 64 Chapter 5 : Results 4.1 Biodiversity and taxonomical study of the endophytic fungi isolated from 65 Ocimum sanctum, Vitex negundo and Barleria prionitis 4.1.1 Plant-wise and plant part-wise distribution of endophytic fungi 65 4.1.2 Species-wise distribution of fungi among the three medicinal plants 68 4.1.3 The species, genera and other taxonomical details of the endophytic 71 fungi isolated from the three medicinal plants 4.1.4 Colonization rate and isolation rate of plant part samples 71 4.1.4.a Colonization rate and isolation rate of endophytic fungi in case of 74 Ocimum sanctum 4.1.4.b Colonization rate and isolation rate of endophytic fungi in case of 74 Vitex negundo 4.1.4.c Colonization rate and isolation rate of endophytic fungi in case of 74 Barleria prionitis 4.1.4.d Colonization rate and isolation rate of endophytic fungi for all three 76 medicinal plants taken together 4.1.5 The density of colonization (rD%) or colonization frequency (CF%) of an 77 individual endophytic species in three medicinal plants 4.1.5.a The density of colonization (rD%) or colonization frequency (CF%) 77 of different species of endophytic fungi isolated from Ocimum sanctum 4.1.5.b The density of colonization (rD%) or colonization frequency (CF%) 78 of different species of endophytic fungi isolated from Vitex negundo 4.1.5.c The density of colonization (rD%) or colonization frequency (CF%) 79 of different species of endophytic fungi isolated from Barleriaprionitis 4.1.5.d The density of colonization (rD%) or colonization frequency (CF%) of different species of endophytic fungi isolated from all three medicinal 79 plants together 4.2. The identification of endophytic fungi 81 4.2.1 Molecular identification of Phomopsis sp. aff. P. archeri B. Sutton isolated 83 from culture no. 505 of Vitex negundo 4.2.2 Molecular identification of Phomopsis sp. aff. P. archeri B. Sutton isolated 86 from culture no. 582 of Barleria prionitis 4.2.3 Molecular identification of Alternaria raphani J.W. Groves and Skolko 89 isolated from culture no. 536 of Vitex negundo 4.3 Brief taxonomy of the other major endophytic fungi isolated from the different 92 parts of the three medicinal plants 4.4 Antibacterial screening of Fungal Endophytes 94 4.4.1 Analysis of antibacterial screening of fungal endophytes 108 4.4.2 Salient features of analysis of antibacterial screening of fungal endophytes 113 4.5 Process optimization for production of active metabolites from Phomopsis archeri B.
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