BIOPROSPECTS OF RHIZOSPHERE BACTERIA ASSOCIATED WITH COASTAL SAND DUNE VEGETATION, IPOMOEA PES-CAPRAE AND SPINIFEX LITTOREUS Thesis submitted to the GOA UNIVERSITY for the award of the degree of DOCTOR OF PHILOSOPHY In MICROBIOLOGY by Aureen Remedios Lemos Godinho, M. Sc., M. Phil. '.,---* V ,...-' '—"•-..,„, So 1\ .14 G.* 7 •.. , , `1, A. , , . r ......----- ..... Under the guidance of Li t. ri Dr.(Mrs.) Saroj Bhosle I *7sil Reader & Head - ------" ,4 Department of Microbiology ( CZ P‘,/;:41. Goa University, Goa AUGUST 2007 Certificate This is to certify that Ms. Aureen Remedios Lemos Godinho has worked on the thesis entitled "Bioprospects of rhizosphere bacteria associated with coastal sand dune vegetation, Ipomoea pes-caprae and Spinifex littoreus" under my supervision and guidance. This thesis, being submitted to the Goa University, Taleigao Plateau, Goa, for the award of the degree of Doctor of Philosophy in Microbiology, is an original record of the work carried out by the candidate herself and has not been submitted for the award of any other degree or diploma of this or any other University in India or abroad. Dr. (Mrs.) Saroj Bhosle RESEARCH GUIDE, Reader & Head, Department of Microbiology, \/ ,■ 1 Uct fre Goa University, Goa-403206 9 K Texaftc( e. eS Declaration I hereby state that this thesis for the PhD degree in Microbiology on "Bioprospects of rhizosphere bacteria associated with coastal sand dune vegetation, Ipomoea pes-caprae and Spinifex littoreus" is my original contribution and that the thesis and any part of it have not been previously submitted for the award of any degree/diploma of any University or Institute. To the best of my knowledge, the present study is the first comprehensive work of its kind from this area. Lk_ o „koi° Aureen Reme los Lemos Godinho Department of Microbiology Goa University Goa-403206 Acknowledgement This thesis was an experience that was challenging, illuminating and enjoyable. I would like to address my warmest acknowledgments to all the people who helped and supported me during the realization of this thesis work. I express my deep sense of gratitude and sincere thanks to my research guide and Head of the Department of Microbiology, Dr Saroj Bhosle, for her guidance, support, motivation, valuable discussions, patience and advice throughout the study. You are truly a mentor and a friend. Thanks for being such an enthusiastic and inspiring guide! I have learned a lot from you. I am also thankful to her for providing the necessary facilities as head of department and Research coordinator of Ocean and Atmospheric Science and Technology Cell (OAST). I also extend my sincere appreciation to Dr. B. Ingole, Scientist, NIO for suggestions and critical comments as member of my Faculty Research Committee and to Prof. G. N. Nayak, Dean, Faculty of Life Sciences, Goa University (GU) for the valuable comments and suggestions and for providing laboratory facilities for my work. My heartfelt gratitude to Prof U M X Sangodkar, Prof S Mavinkurve and Prof D J Bhat who have been instrumental in broadening my icnowledge of the subject and who have been sources of inspiration to carry out my research work. I am also grateful to the faculty of the Department of Microbiology, Goa University, Dr J D'souza, Dr S Nazareth, Dr I Furtado, Dr S K Dubey and Dr S Garg, for their advice and support throughout my work period. I am grateful to Dr. V. S. Korikanthimath , Director, Indian Council of Agricultural Research (ICAR), Goa for granting me permission to carry out part of my thesis work at ICAR and providing me with necessary facilities. I would like to express my deepest gratitude to Dr R Ramesh, Scientist, ICAR for guidance, constructive advice and providing me with the facilities to carry out my research work on plant growth promotion by sand dune bacteria at the Plant Pathology Laboratory at ICAR. I would like to acknowledge, Dr N B Bhosle, Scientist, National Institute of Oceanography (NIO) for his generous help and guidance in the isolation and characterization studies of the exopolymers and the Gas chromatographic analysis of the exopolymers. Special thanks to all my friends at the Marine Corrosion and Material Research Division(MCMRD), NIO, Dr. Rakhee Kakode, Anand Jain, Loreta Fernandes, Vishwas Khodse, Ranjita Harji, Mondher Noman and Sangeetha Jadhav for their help during the analysis. I am grateful to Dr Y Souche (NCCS, Pune) and Dr Upal Roy for the 16s rRNA sequencing of the sand dune bacterial cultures. I would also like to thank Dr P K Sharma, Department of Botany,G U for his help with the HPLC analysis of the pigment extracts and chlorophyll fluorimetry, Dr D J Bhat, Department of Botany, G U for granting permission to use the Lyophilizer, Dr P Sarode and Dr K R Priolkar, Department of Physics, G U for X-ray diffraction studies of the exopolymers. Thanks to Mr Aareef Sardar and Mr Khedekar, NIO for capturing the images of my culture slides on the Scanning Electron microscope. I would also like to thank Leena Verenkar, SESA Goa Mines, for providing me with mine reject soil samples to carry out my experiments. A heartfelt thanks to the non teaching staff members of the Department of Microbiology, Mr Shashikant Parab in particular who helped me find chemicals and other materials that I needed for my research from time to time. I would also like to thank Sir Anant, Ladoo, Budhaji, Dominic and Sumati for their kind help in my laboratory work. I would like to thank Anna Fernandes, Saraswati Naik and Deepa Chari for their friendship and for the fond memories of the good times we have had together. I would like to thank Rajeshwari and Kumud Phadte, who helped me with my experimental work at ICAR. Special thanks to Gauri Phatke, who helped me whenever I needed her assistance in nursery studies. A heartfelt thank you to the staff at ICAR who helped me a lot with my field work, especially Prafula and Ankush, from assisting me to filling the pots with soil to watering the pots daily. I am thankful to the Department of Ocean Development (DOD),Govt. of India for providing financial support for this investigation. I am also thankful to the Librarian of the Indian Institute of Science(IISc), Bangalore and the Librarian of University of Agricultural Sciences, Gandhi Krishi Vignyan Kendra(GKVK), Bangalore for helping me in procuring required reference papers which have helped me in the compilation of my thesis. In a special way, I would like to thank my fellow PhD colleagues and good friends, Rasika Gaokar and Naveen Krishnamurthy, for all their help and support throughout these years. Your friendship is truly priceless. I would like to thank my fellow research colleagues, Tabitha Marbaniang, Christina De Souza, Lakshangy Chari, Nimali Prabhu, Celisa Santimano, Lorna Silveira, Vidya Ramachandran, Anju Pandey, Neelam Singh, R Krishnamurthy and Prakash Munnoli for their practical support, friendship and all the fun times we have had. I would also like to acknowledge the suggestions and advice given to me by my senior colleagues and friends, Dr Chanda Parulekar, Dr Trupti Rawte, Dr Judith Braganca, Dr Upal Roy, Dr Madhan Raghavan, Dr Yogita Sardessai, Dr Sunita Borkar and Dr V Bramhachari which at some point or the other have helped me and led me to the successful completion of my thesis. I would also like to thank my friends at the Department of Botany, Janet Vaz, Dr Rupali, Dr Pratibha and Pooja for their help whenever I needed Thank you to my dear friend, Girija Parvatkar, for efficient organisation of numerous field trips and for always being there for me anytime. My wonderful and dear friends Deepa Nair, Bina Bhobe, Rohan Fernandes, Sheetal Naik and Ambuja Bhingi, I will always have a special place for you, filled with great memories and laughter. My sincere thanks to all my past and present MSc friends whom I have not included here for want of space. A special note of thanks to Mr. Sachin Patil for designing the cover page of my thesis. I want to thank my parents, Mama and Papa, I want you to know how grateful I am for all the love, patience and support you have given me over the years. Thanks for standing by me and supporting me in all the major decisions in my life. A special note of appreciation to my dearest husband, Lawrence, I want to thank you for being a friend and a loving companion. Thank you for your understanding, encouragement, patience and consistent support which has led me to the successful completion of my thesis. Your motivation and unwavering support towards the completion of my work has made this endeavor possible. I would also like to thank my brothers and my in-laws for their constant encouragement and support without whom this would not have been possible. Most importantly, Almighty God who brought me to this path and led me through it, nurturing me and pruning me to grow into what pleases Him. This work is to His honour. Aureen Godinho August 2007 D•eolizateoi To vu. pa newts with Love PIAA erPtItt/Ote CONTENTS Chapter No. Chapter Page No. List of abbreviations List of figures and tables Introduction 1 - 12 Chapter 1 Literature survey 13 - 62 Chapter 2 Biodiversity of bacteria associated with sand dune 63 - 134 vegetation, Ipomoea pes-caprae and Spinifex littoreus Chapter 3 Isolation, extraction and characterization of 135 - 180 exopolysaccharide production by Microbacterium arborescens Chapter 4 Plant growth promoting characteristics of four 181 - 238 potential sand dune rhizobacterial isolates Chapter 5 Growth promoting ability of sand dune bacteria in 239 - 266 eggplant