Antimicrobial Activity of Xenorhabdus Sp. (Enterobacteriaceae), Symbiont of the Entomopathogenic Nematode, Steinernema Riobrave (Rhabditida: Steinernematidae)
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Antimicrobial activity of Xenorhabdus sp. (Enterobacteriaceae), symbiont of the entomopathogenic nematode, Steinernema riobrave (Rhabditida: Steinernematidae) BY Peter 3. lsaacson B. Sc., University of Victoria, 1994 THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF PEST MANAGEMENT ln the Department of BIOLOGICAL SCIENCES O Peter J. Isaacson 2000 SIMON FRASER UNIVERSITY April2000 All rights reserved. This work rnay not be reproduced in whole or in part, by photocopy or other means, without permission of the author National Library BibliotMque nationale 1+1 of Canada du Canada Acquisitions and Acquisitions et Bibliographie Services services bibliographiques 395 Wellington Street 395. nie Wellington OnawaON KlAûN4 OttawaON K1A ûN4 canada canada The author has granted a non- L'auteur a accordé une iicence non exclusive licence aiiowing the exclusive permettant à la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or seii reproduire, prêter, distribuer ou copies of this thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/£iim, de reproduction sur papier ou sur format électronique. The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis .nor substantial extracts fkom it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. ABSTRACT The entomopathogenic nematode, Steinernema riobrave, releases its bacterial symbiont, Xenorhabdus sp., into the haemocoel of an insect host resulting in bacterial septicemia and death of the insect. Gallena mellonella larvae infected with S. nobrave were killed within 72 h of infection by which time the growth of the bactenal symbiont had reached 1.1 x IO~CFUI~of wet insect tissue. These syrnbiotic bacteria praduced secondary metabolites that showed antimicrobial properties. These metabolites helped prevent contaminating microorganisms from becoming established in the insect cadaver and allowed the bacterial and nematode symbionts to grow optimally. However, Gram negative species other than Xenohabdus became established although no additional antibiotic activity was detected. This indicates that the later stages of nernatode development occur in a multixenic environment rather than a monoxenic environment populated only by the nematode's symbiont. The metabolites produced by the Xenorhabdus sp. showed significant broad spectrum antibacterial and antimycotic activities against fungal and bacterial species from different habitats. When tested against agnculturally important fungi in agar diffusion plate assays. the cell free culture broth completely inhibited the growth of many plant pathogenic fungi including, Botrytis cinerea, Diddymella bryoniae, Fusarium solani and Pjdhium ultimum. The majority of this activity was caused by water soluble metabolites rather than ethyl acetate soluble compounds. Further analysis showed that sorne of this water soluble activity was a result of extracellular proteins including ex* and significant endochitinase activity as deterrnined by the release of p-nitrophenol from the artificial substrates pnitrophenyCN-acetyl-PD-glucosaminide and p-nitrophenyl- P-D-N-N9-Nn-triacetyochitobiose,respectively. Gel filtration analysis of the water soluble proteinaceous fraction showed WO major activity peaks conesponding to (1) large molecular weight proteins and broth components and (2) small molecular weight, peptide or nonproteinaceous. heat tolerant biomolecule(s). Overall, the data showed that chitinases wntributed to but were not the dominant source of antimicrobial activity. These findings suggest that secondary metabolites from Xenothabdus sp. RIO may provide a source of novel compounds useful to the agncultural industry as pest management tools. FOR MY PARENTS ACKNOWLEDGMENTS I am grateful for the guidance, encouragement and support of my senior supervisor Dr. J. M. Webster who has persevered with me through the course of this project. Many thanks go to my committee member. Dr. J. E. Rahe for his valuable suggestions during my research and in my writing of this manuscript. The assistance of Drs. 2. Punja (Simon Fraser University), M. Moore (Simon Fraser University), R. Utkhede (PARC-Agassiz), and S. Berch (Glyn Road Research Station. Victoria) for supplying test cultures andfor laboratory facilities for my research was much appreciated. I would also Iike to thank Drs. J. Li and G.Chen (Welichem Technology Corp.) for sharing their knowledge and providing guidance; E. Urquhart (Simon Fraser University) for his invaluable technical assistance; B. Leighton (Simon Fraser University) for rearing and supplying nematode cultures; the students of both Drs. J. M. Webster and J. E. Rahe for their great friendship and support; and the many people in the Department of Biological Sciences who provided me with the opportunity to pursue this degree and who made my academic career much easier. Special gratitude goes out to my fnends and family who showed much patience and understanding through the course of my work, and rnost of all to my wife Sophie who has stood behind me al the way. I am forever indebted for their support. TABLE OF CONTENTS Approval .............. ............................................................................................... ii ... Abstract ................................................................................................................ III Acknowledgrnents ............................................................................................... vi .. Table of Contents ................................................................................................ vil List of Tables ...................................................................................................... ix List of Figures .............................. .................................................................... xi 1. 0 GENERAL INTRODUCTION ......................................................................... 1 2.0 GENERAL MATERIALS AND METHODS ..................................... ... ......... 18 2.1 Source and Maintenance of Xenortrabdus ......................................... 18 2.2 Source and Maintenance of Test Organisms ...................... ... ....... 23 2.2.1 Test Bacteria ....................................................................... 23 2.2.2 Test Fungi ............................................................................ 23 2.3 Bacterial Fermentation ....................................................................... 24 2.4 Measurement of Antimicrobial Activity ................................................ 25 2.4.1 Bactenal Bioassays .............................................................. 25 2.4.2 Fungal Bioassays ................................................................. 26 2.5 Satistical Analysis .............................................................................. 27 3.0 CHARACTERISTICS OF XENORHABDUS SP. RIO .................................. 28 3.1 Introduction ....................................................................................... 28 3.2 Materials and Methods ....................................................................... 28 3.2.1 Morphological Characteristics.............................................. 29 vii 3.2.2 Carbohydrate Metabolism .................................................... 30 3.2.3 Enzyme Production ............................................................ 30 3.3 Results .............................................................................................. 31 4.0 IN VIVO ANTlMlCROBlAL ACTlVlTY ........................................................... 37 4.1 Introduction .......................................................................................37 4.2 Materials and Methods ....................................................................... 38 4.2.1 In vivo Development of Microflora ....................................... 38 4.2.2 In vivo Development of Antimicrobial Activity ....................... 39 4.3 Results .............................................................................................. 39 5.0 CHARACTERIZATION OF ANTlMlCROBlAL ACTIVITY ..................... ....... 43 5-1 Introduction .......................................................................................43 5.2 Materials and Methods .......................................................................44 5.2.1 h vitro Development of Xenorhabdus sp . RIO ................... ..44 5.2.2 Spectnim of Antimicrobial Activity ........................................ 45 5.2.3 Fractionation of Antimicrobial Activity ................................... 46 5.2.4 Analysis of the Exo-Enzymatic Activity .................................47 5.2.5 Partial Purification of Chitinase and Bioactive Proteins ........ 50 5.2.6 Characteristics of Partially Purified Bioactive Proteins ....... -51 5.3 Results .............................................................................................. 52 6.0 DISCUSSION ........................................................................................ 72 7.0 REFERENCES ............................................................................................86 LIST OF TABLES Table 1. Estimated 1993 worid