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Investigating the Novel Use of Seaweed Extracts As Biopesticides

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Investigating the Novel Use of Seaweed Extracts As Biopesticides Investigating the Novel Use of Seaweed Extracts as Biopesticides Submitted to the Waterford Institute of Technology for the Degree of Doctor of Philosophy By Emma O Keeffe Eco-Innovation Research Centre (EIRC) Waterford Institute of Technology Waterford Ireland Prepared under the supervision of Dr Nick McCarthy, Dr Helen Hughes, Dr Peter McLoughlin and Dr Shiau Pin Tan DECLARATION I hereby certify that this material, which I now submit for assessment is entirely my own work and has not been taken from the work of others, save to the extent that such work has been cited and acknowledged within the text of my work. Signed: _________________________ ID No.: _________________________ Date: _________________________ ii ACKNOWLEDGEMENTS It’s hard to believe four years has passed since I began my studies in WIT which has had both its highs and lows but have always being grateful of the opportunity that was given to me. I could not of completed this journey without the help of others who offered me guidance, support and friendship throughout the years of study and also the WIT scholarship programme for funding this research. Foremost, I would like to express my sincere gratitude to Nick, Helen, Peter and Shiau Pin Tan (Graece) for their continuous support, patience and freedom to pursue my research, while silently and non-obtrusively ensuring that I stayed on course and do not deviate from the core of my research. Without this guidance, this thesis would not have been possible and I shall eternally be grateful to my supervisors for their assistance. I would like to give a special thanks to Graece who was an incredible mentor particularly in the area of microbiology which I had very little experience in. This was an essential part of my research and I thank you Graece for getting me through it and apologies for all the head-wrecking questions I asked along the way. My thesis wouldn’t be near as good without your support and guidance. A big thanks to the technicians, postgraduate and postdoctoral friends for all their help and encouragement. The lunch-time bants were a great way to distract from the pressures of research as we were all on this journey together. To the “Glenmore girls” ye have being an incredible group of friends through our viber conversations and nights out which are an essential part of the research life in order to maintain that all in important work-life balance!!! Annabel where can I start who would of thought I would of made such an amazing friend throughout my studies with your incredible smarts and your never ending encouragement. You have made me a much more open-minded person. And not forgetting the countless tea breaks and chats about the weekend gossip which have made my time here at WIT so much more enjoyable. Thank you ;). Michael you have being my rock throughout this journey. Your emotional support and understanding especially through the turmoil times have made you one of my greatest supporters in this thesis. I can’t wait to see what the next chapter in life has for us. Last but not the least, I would like to thank my family; my parents Declan and Eileen and my sisters Orla and Elaine for their continuous support over the years. I know it took awhile and I love that the bank of Declan never charged interest lol! So therefore it only seems right that I dedicate this dissertation to my parents for their endless support and encouragement, as without them this journey would not of been possible. iii TABLE OF CONTENTS Page number Title Page…...…………………………………………………………………………….i Declaration ........................................................................................................................ ii Acknowledgements .......................................................................................................... iii Table of Contents ............................................................................................................. iv List of Figures ................................................................................................................... x List of Tables................................................................................................................ xviii List of Equations ........................................................................................................... xxii List of Abbreviations.................................................................................................... xxiii Abstract ........................................................................................................................ xxvi Chapter 1: Introduction ................................................................................................. 1 1.1 Introduction ........................................................................................................... 2 1.2 Seaweeds ............................................................................................................... 3 1.2.1 Morphology and ecology of seaweeds ............................................................ 3 1.2.2 Classification of seaweeds ............................................................................... 5 1.2.2.1 Chlorophyta (green seaweed) .................................................................... 5 1.2.2.2 Phaeophyta (brown seaweed) .................................................................... 6 1.2.2.3 Rhodophyta (red seaweed) ........................................................................ 6 1.2.3 Applications of seaweed .................................................................................. 8 1.2.3.1 Food source................................................................................................ 8 1.2.3.2 Agriculture fertiliser .................................................................................. 9 1.2.3.3 Industrial applications.............................................................................. 10 1.2.3.4 Cosmetic industry .................................................................................... 10 1.2.3.5 Medicinal uses ......................................................................................... 12 1.2.3.6 Biopesticides ............................................................................................ 12 1.3 Investigation into the Antimicrobial Compounds in Seaweeds .......................... 13 1.3.1 Collection and processing of seaweeds ......................................................... 13 1.3.2 Extraction of the bioactive compounds ......................................................... 14 1.3.2.1 Selection of extracting solvents ............................................................... 14 1.3.2.2 Extraction techniques .............................................................................. 18 1.3.3 In vitro assessment of the antimicrobial properties of the crude extracts ..... 20 1.3.3.1 Disk diffusion assay................................................................................. 20 iv 1.3.3.2 Well diffusion assay ................................................................................ 21 1.3.3.3 Broth dilution assay ................................................................................. 22 1.3.3.4 Bioautography assay ................................................................................ 22 1.3.3.5 Poisoned food technique .......................................................................... 24 1.3.3.6 Comparison of the various antimicrobial test methods ........................... 24 1.3.4 Investigation into the efficacy of the most potent extracts ............................ 26 1.3.4.1 Minimum inhibitory concentration and minimum bactericidal concentration/minimum fungicidal concentration .................................................. 26 1.3.4.2 Causes of biofilm formation and problems associated ............................ 27 1.3.4.3 Toxicity testing ........................................................................................ 32 1.3.5 Separation and purification of crude extracts ................................................ 34 1.3.5.1 Solvent partitioning ................................................................................. 34 1.3.5.2 Chromatographic techniques ................................................................... 36 1.3.6 Identification and structural elucidation of purified antimicrobial compounds.……………............................................................................................39 1.4 Biopesticide Properties of Seaweed Extracts ...................................................... 45 1.4.1 Antimicrobial activity of seaweed extracts against plant pathogens ............. 45 1.4.2 Insecticidal and larvicidal activity of seaweed extracts ................................ 48 1.4.3 Herbicidal activity of seaweed extracts ......................................................... 50 1.5 Applications of Biopesticides .............................................................................. 51 1.5.1 Factors contributing to the emergence of plant pathogens ............................ 51 1.5.2 Economically problematic pathogens ........................................................... 53 1.5.2.1 Armillaria spp. ......................................................................................... 53 1.5.2.2 Heterobasidion annosum ......................................................................... 56 1.5.2.3 Quarantine bacterial plant pathogens .....................................................
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