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Isolation and Characterization of Bioactive Molecules from Endophytic Fungi

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Isolation and characterization of bioactive molecules from endophytic fungi A Thesis Submitted in fulfillment of the requirement for the award of the degree of DOCTOR OF PHILOSOPHY IN BIOTECHNOLOGY By M. Vasundhara (Reg. No. 950800006) Department of Biotechnology Thapar University Patiala 147004, India February 2017 -i- -ii- -iii- Acknowledgements Constant inspiration and encouragement given by all concerned was the driving force that enabled me to submit this thesis in the present form. Guidance, direction, cooperation, motivation and support came in my way from many people and it is a moment to acknowledge the same. First of all, I am thankful to the almighty God for blessing me and giving me the strength and perseverance to complete my work successfully. It is a moment of pride to put on record the professional guidance and valuable support that I have received from my guide, Dr. Anil Kumar, Associate Professor, Department of Biotechnology. My sincere thanks to Dr. Dinesh Goyal, Head, Department of Biotechnology for his cooperation and support throughout the research work. Also my special thanks to Dr. Sanjai Saxena, Head, CORE for extending the facilities to carry out my research work. I thank my doctoral committee members Dr. Rajesh Kumar, Dr. S.K. Pandey, Dr. Manoj Baranwal and Dr. Siddharth Sharma for their advice and suggestions. My great appreciation goes to Dr. M. S. Reddy for his valuable and constructive suggestions during the planning and development, execution of research work. I am particularly grateful for the assistance, valuable technical and moral support and encouragement provided by Dr. Manoj Baranwal during by research work. I am also thankful to Dr. N. Tejo Prakash for his continuous help while preparing thesis. I am also thankful to Dr. T.S. Suryanarayanan, Vivekananda Institute of Tropical Mycology, RKM Vidyapith, Chennai for providing the thermotolerant endophytic fungal isolates and Dr. Sunil Kumar Deshmukh, The Energy and Resources Institute, TERI Gram, Gual Pahari, Gurgaon for providing bacterial cultures. My special thanks of gratitude to Dr. N. Sivarasmaiah, Nano Temper Technologies, World Trade Centre, Bangalore, who provided insight and expertise that greatly assisted me in carrying out my research work and I came to know about many new things. -iv- -v- Publications The following publications are the outcome of the present research work 1. M. Vasundhara, Manoj Baranwal, N. Sivaramaiah, Anil Kumar. 2017. Isolation and characterization of trichalasins producing endophytic fungus from Taxus baccata. Annals of Microbiology. DoI: 10.1007/s13213-017-1256-4. 2. M. Vasundhara, Anil Kumar, M.S. Reddy. 2016. Molecular approaches to screen bioactive compounds from endophytic fungi. Front. Microbiol. 7:1774. DoI: 10.3389/fmicb.2016.01774. 3. M. Vasundhara, Manoj Baranwal, Anil Kumar. 2016. Fusarium tricinctum, an endophytic fungus exhibits cell growth inhibition and antioxidant activity. Indian Journal of Microbiology, 56:433-438. -vi- Table of Contents Chapters Page No. I. Introduction 1 II. Review of Literature 5 2.0 Natural products in drug discovery and development 5 2.1 Endophytic fungal diversity 6 2.1.1 Traditional techniques used in endophyte studies 6 2.1.2 Molecular identification of fungi 8 2.1.3 Internal transcribed spacers (ITS) 11 2.2 Antibacterials / Antimycobacterials from endophytic fungi 11 2.3 Antifungals from endophytic fungi 19 2.4 Anticancer, Immunosupressive, Antiinflammatory activities of endophytic 23 fungi 2.5 Antioxidants from endophytic fungi 33 2.6 Analysis of bioactive metabolites 38 2.6.1 Mass Spectrometry 39 2.7 Industrial enzymes from endophytic fungi 40 III. Materials and Methods 43 3.1 Collection of bark samples 43 3.1.1 Isolation of endophytic fungi from Taxus baccata bark 44 samples 3.2 Preparation of fungal extracts for biological assays 44 3.3 Assay for antimicrobial activity 45 3.3.1 Test microorganisms and growth conditions 45 3.3.2 Antimicrobial agent (as control) 46 3.3.3 Turbidity standard for inoculum preparation 46 3.3.4. Prescreen assay: Agar-well diffusion assay 47 3.3.5 Screening: Microplate broth dilution assay 48 3.4 Cell growth inhibition assay 49 3.4.1 Isolation of peripheral blood mononuclear cells 50 3.4.2 Counting of cells 50 -vii- 3.4.3 Lymphocyte proliferation assay 50 3.4.4 Measurement of TNF-α 51 3.4.5 Antioxidant assay 52 3.5 Identification of endophytic fungi 52 3.5.1 Morphological characters 52 3.5.2 Molecular characterization 53 3.5.2.1 Isolation of fungal genomic DNA 53 3.5.2.2 CTAB method for DNA extraction 53 3.5.2.3 Checking of DNA (Agarose Gel Electrophoresis) 54 3.5.2.4 Quantification of DNA using Nano drop 54 3.5.2.5 Amplification of Internal Transcribed Spacer (ITS) 55 region 3.5.2.6 Purification of PCR products and sequencing 55 3.5.2.7 Sequence analysis 56 3.6 Extracellular enzymes production by thermotolerant endophytic fungi 56 3.6.1 Agar Plate Assays 56 3.6.1.1 Amylase activity 56 3.6.1.2 Cellulase activity 56 3.6.1.3 Laccase activity 57 3.6.1.4 Lipase activity 57 3.6.1.5 Pectinase activity 57 3.6.1.6 Proteinase activity 57 3.6.1.7 Tyrosinase activity 57 3.6.1.8 L-asparaginase activity 58 3.7 Quantitative Assay 58 3.7.1 Cellulase Activity 58 3.7.2 Culture conditions for cellulase production 58 3.7.3 Cellulase assay 58 3.7.4 Characterization of enzyme 59 3.7.4.1 Optimization of time for cellulase production 59 3.7.4.2 pH 60 3.7.4.3 Temperature and thermostability 60 3.7.4.4 Lipase Activity 60 -viii- 3.7.4.5 Culture condition for lipase production 61 3.7.4.6 Lipase assay 61 3.7.4.7 Protein assay 62 3.7.4.8 Characterization of enzyme 63 3.7.4.8.1 Optimimization of time for lipase 63 production 3.7.4.8.2 pH 63 3.7.4.8.3 Temperature and thermostability 64 3.8 Analysis of bioactive molecules 64 3.8.1 Sample preparation 64 3.8.2 Mass spectrometry 64 3.8.3 UHPLC-QTOF-MS/MS analysis 65 3.9 Statistical analysis 66 IV. Results and Discussion 67 4.0 Isolation, identification and biological activities of endophytic fungi 67 4.1 Isolation of endophytic fungi from Taxus baccata bark samples 67 4.2 Preliminary screening for bioactivity 68 4.3 Identification of endophytic fungal isolates 68 4.3.1 T1 isolate 69 4.3.2 T2 isolate 74 4.3.3 T5 isolate 78 4.3.4 T6 isolate 82 4.4 Antimicrobial activity 87 4.5 Cytotoxic activity 93 4.6 Antioxidant assay 95 4.7 Cell growth inhibition and antioxidant activity of Diaporthe sp. T1 96 4.7.1 Cytotoxicity 96 4.7.2 Antioxidant activity 68 4.8 Cell growth inhibition and antioxidant activity of Fusarium tricinctum T6 100 4.8.1 Cytotoxicity 100 4.8.2 Anti-proliferative activity of peripheral blood 102 mononuclear cells 4.8.3 TNF- production 103 -ix- 4.8.4 Antioxidant activity 105 5.0 Identification and characterization of putative bioactive molecules 108 5.1 UHPLC-MS analysis of extract of T1 109 5.1.1 Cytosporones C & E 109 5.1.2 Cytochalasins 112 5.2 UHPLC-MS analysis of extract T6 115 5.2.1 Gniditrin 115 5.2.2 7-hydroxyheptaphylline 116 5.2.3 Tirandamycin 118 5.2.4 Fumitremorgin C 120 5.2.5 Paclitaxel (Taxol) 121 5.2.6 10-deacetyl baccatin III (10-DAB) 123 6.0 Extracellular enzymes production by thermotolerant endophytic fungi 126 6.1 Screening for extracellular enzymes 126 6.2 Quantitative studies on extracellular enzymes 134 6.2.1 Cellulase 134 6.2.2 Lipase 139 6.3 Cellulase 146 6.4 Laccase 149 6.5 Amylase 150 6.6 Lipase 151 6.7 Protease 152 6.8 L-asparaginase 153 Summary 155 References 160 Appendices ( I to III) 183 -x- List of Tables Sl. No. Table Description 1 3.1 McFarland Standard 2 4.1 Antimicrobial activity of fungal extracts of T1, T2, T5 and T6 tested with different microorganisms 3 4.2 Effect of the fungal extracts (500 µg/ml) of T1, T2, T5 and T6 isolates on growth inhibition (%) of different microorganisms by MTT assay 4 4.4a Effect of fungal extract of different endophytic fungi on the growth inhibition of MCF cell lines 5 4.4b Effect of different concentration of fungal extract of T1 and T6 isolates on the growth inhibition of MCF cell lines 6 4.5 Effect of different concentrations of fungal extracts of T1, T2, T5 and T6 on the antioxidant activity 7 4.6 Cytotoxic effect of Diaporthe sp. extract against human breast cancer (MCF-7) and human cervical (HeLa) cancer cell lines 8 4.7 Antioxidant effect of Diaporthe sp., extracts based on free radical scavenging activity. Ascorbic acid (AA) (100 µg/ml) was used as positive control 9 4.8 Cytotoxic effect of F. tricinctum T6. extract against human breast cancer (MCF-7) and human cervical (HeLa) cancer cell lines 10 4.9 Immunosuppressive effect of Fusarium tricinctum extract on concanavalin stimulated peripheral blood mononuclear cells 11 4.10 Effect of Fusarium tricinctum extract on TNF-α production in MCF-7 concanavalin A stimulated peripheral blood mononuclear cells (PBMCs) 12 4.11 Antioxidant effect of Fusarium tricinctum extracts based on free radical scavenging activity 13 5.1 LC/ESI-MS analysis data of cytosporone C & cytosporone E from Diaporthe sp. (T1) and their literature reported data 14 5.2 LC/ESI-MS analysis data of trichalasin E, trichalasin F and trichalasin H from Diaporthe sp.
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  • Fungal Pathogens of Proteaceae

    Fungal Pathogens of Proteaceae

    Persoonia 27, 2011: 20–45 www.ingentaconnect.com/content/nhn/pimj RESEARCH ARTICLE http://dx.doi.org/10.3767/003158511X606239 Fungal pathogens of Proteaceae P.W. Crous 1,3,8, B.A. Summerell 2, L. Swart 3, S. Denman 4, J.E. Taylor 5, C.M. Bezuidenhout 6, M.E. Palm7, S. Marincowitz 8, J.Z. Groenewald1 Key words Abstract Species of Leucadendron, Leucospermum and Protea (Proteaceae) are in high demand for the interna- tional floriculture market due to their brightly coloured and textured flowers or bracts. Fungal pathogens, however, biodiversity create a serious problem in cultivating flawless blooms. The aim of the present study was to characterise several cut-flower industry of these pathogens using morphology, culture characteristics, and DNA sequence data of the rRNA-ITS and LSU fungal pathogens genes. In some cases additional genes such as TEF 1- and CHS were also sequenced. Based on the results of ITS α this study, several novel species and genera are described. Brunneosphaerella leaf blight is shown to be caused by LSU three species, namely B. jonkershoekensis on Protea repens, B. nitidae sp. nov. on Protea nitida and B. protearum phylogeny on a wide host range of Protea spp. (South Africa). Coniothyrium-like species associated with Coniothyrium leaf systematics spot are allocated to other genera, namely Curreya grandicipis on Protea grandiceps, and Microsphaeropsis proteae on P. nitida (South Africa). Diaporthe leucospermi is described on Leucospermum sp. (Australia), and Diplodina microsperma newly reported on Protea sp. (New Zealand). Pyrenophora blight is caused by a novel species, Pyrenophora leucospermi, and not Drechslera biseptata or D.