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Entomopathocemc Nematodes NEMATICIDAL PROPERTIES OF XENOREMBDUS SPP. AND PHOTOiüX4BDUS SPP., BACTERIAL SYMBIONTS OF ENTOMOPATHOCEMC NEMATODES B.Sc., Northwestem College of Fonstry, Yangling, China, 1985 M.Sc., The Chinese Acaderny of Fonstry, Beijing, China, 1988 THESIS SUBMïlTED IN PARTIAL, FULFLMENT OF THE REQUlREMENTS FOR THE DEGREE OF DOCTOR OF PWSOPHY in the Department of Biological Sciences OKAIJI HU 1999 SIMON FRASER UNIVERSïïY Al1 rights reserved. This work may not be reproduced in whole or in part, by photocopy or other means, without permission of the author. National Library Bibliothèque nationale 1*1 of Canada du Canada Acquisitions and Acquisitions et Bibliographie Senrices seMces bibliographiques 395 Wdlington Sireet 395, rue Weltington OniwaôN KlAOW OttawaON KlAW Canada Canade The author has granted a non- L'auteur a accordé une licence non exclusive licence ailowing the exclusive permettant à la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or sell reproduire, prêter, distribuer ou copies of this thesis in microfom, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/nlm, 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 phted or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son pemission. autorisation. ABSTRACT Nematicidal properties of some secondary metabolites of Xenorhnbdus spp. and Photorhabdus spp. (Enterobacteriaceae), bacterial symbiunts of the entomopathogenic nematodes, Steinernema spp. and Heterorhabdiris spp., respectively, were identified and evaluated. Cell-free (CF) culture filtrates of X. bovienii, X. nematophilus and P. luminescens isolates were shown to be nematicidal against Meloidogyne incognita and Bursaphelenchus xylophilus. The degree of activity varied with the bacterial isolate/species and the culture conditions, such as media composition, ap, pH and aeration of the cultures. Three metabolites, namel y ammonia, 3,s-dihydroxy4isopmpylstilbene (ST) and indole, were identified from CF filtrates as having nematicidal properties. Ammonia was common to al1 bacterial cultures tested. but ST was produced (10 - 30 pg/ml) by only P. luminescens and indole was produced (10 - 50 pg/ml) by some straindspecies of both Xenorhabdus and Photorhabdus. ST and indole affected viability, mobility, egg hatch and dispersal khaviour of nematodes in vitro. ST was active against Aphelenchoides rhytium, Bursaphelenchus spp. and Caenorhabditis eleguns, but was not lethal to infective juveniles (Us) of H. megidis 90. or second stage juveniles (J2s) of M. incognita at 200 pg/ml. Indole was active in immersion tests against J2s (LOO - 400 pg/ml) of M. incognito, but failed to pievent infection of tomato seedhgs by M. inmgnita foîlowing a soi1 (a0 pdml) or foüar application (<1,000 pglml). Indole repelled Us of some species of both Steinememu and Heterorhabditis whereas ST repelled only some species of Steinemema. ST, but no< indole, was detected in variable quantities (-665.2 to 4,182 pg/g wet insect) in larval Galleria mellonella infected with Heterorhabditis spp. ST was produced after 24 h of infection (2S°C) of the larvae, increased rapidly in quantity by 48 h to 5 d, and nmained at a relatively hi@ and constant level even after the nematode symbiont had completed its reproduction. Bacterial symbionts built up high populations (-10' cellslg insect) within 24 h of entenng G. meilonella lame, and increased the cadaver pH to 7.4- 7.7. The early production and relatively large amount of ST in nematode-infected insect hosts, and the antibiotic, nematicidal and nematode-repelling properties of ST suggest that it play a significant role in the symbiotic nematode-bacterium association. The potential commercial application of these nematicidal metabolites may be limited by their relatively narrow spectnim and low activity. 1 would like to take this opportunity to express my heartfelt thanks for ail of the people who kindly offered their thoughts and help during my research. 1 am deeply grateful to Dr. J. M. Webster, rny senior supervisor, for his encouragement, guidance and support throughout the course of this snidy. I would also like to thank Drs. J. R. Sutherland and A. Plant for their helpful suggestions and comrnents during my research and during the revision of the thesis. My thanks are also given to the following people who offered their thoughts, encouragement and help during my research: Dr. J. Li for his work on chemicai characterization and for his help and invaluable suggestions: Dr. G. Chen and Mr. K. Ng for their discussion and help; Dm. V. L. Bourne and G. Gries, Mrs. R. Gries and Mrs. M. SieWUnen as well as Mr. B. Leighton and Mr. M. Yang for their technical support; Mr. Ian Bercovitz for statistical consulting; those mentioned in the text for their kindness and generosity in providing some of the test materials (nematodes, bacteria and plant seeds); finaily, my colleagues and friends for their discussion and help. I acknowledge the financial support of five Graduate Fellowships, a President's Ph.D. Research Stipend and a Prototype Developrnent Fund for Student Entrepreneurs from Simon Fraser University and of support ihrough research gants to my senior supervisor, Dr. J. M. Webster, from the National Science and Engineering Resemh Council of Canada. Finally, I would like to express my heartfelt gratitude to my wife and son for their love, patience and support throughout the course of this study. TABLE OF CONTENTS 2.4. Biossssys for nematiciàai actidty..... .............................................................. ...33 2.4.1. Activity of celi-free culture filtrates .............................. ............................33 2.4.2. Activity of metabolic compounds ........................ .......................................33 2.4.3. Mortality of the test nematodes .............. .......*.....**...*.......*..*.***.........*.....34 2.4.4. Antibacterial activity .....*........... .. ............. 2.5. Staüsücal andysis.. ............. ............................................................................... ..35 CHAITER 3. NEMATICIDAL PROPERTIES OF IN VZTRO CULTURES OF THE BA~RIAeoooiooem~omooaoooooooomoomooooo~emmoomm~oooooooooeoooo oeooo 0036 3.1. htrod~~tl~~ooooooeooo~oooo~oooeooee~mooooeommmoommoeooomomoe~om~omooommooeoaooooooooooe0 sommomoooeommooom 0 l0 0 oo.36 3.2. Materials and ~~thodsbmbmmbbb~~0bb~mb~bb0bb~b~bbbbbbb~bb~b~~~~~bm~b~~b~0~~b0bb~b~b~0~b~~b~bbbmm~0~00l *, b*bm m37 3.2.1. Bacteria and nematodes ..... .............................. .. ................ ..... ....... 37 3.2.2. Preparation of cell-free filtrates.. .. .37 3.2.3. Nematicidal activity of bacterial strains and species .............................. .. .. .38 3.2.4. Nematicidal activity of the bacterial cultures against different nematode species............... .. + .. .. .. ... 39 3.2.5. Influence of bacterial culture conditions on nematicidal activity of the culture filtrates...... .. .. .. .,. .. .. .. .. .. .. .. .,.. ..39 3.2.6. Nematicidal activity of organic and aqueous fractions of the culture filtrates ... ................. ....... ....... ........... ..................... ... ...... ..... .. .4 1 3.2.7. Nematicidal activity of some known antibiotics produced by Xenorhabdus spp ..... ..... .. .. .... .. .. .. ...... .. ... ...... ... .. ....... .. ..... ...... .42 3.30 ~e~~~~~o~~~~o~.o~~e~oo~~obnmbom~bb~oo~~mb~.bb~oo~bom~bmm~mmbmbmmbmmmmmmbmbmbmmmmmommmbmmmmmmbmommll memoa*mm ll l l l l ll e43 3.3.1. Nematicidai activity of bacteriai strains and species. ... .. .. .. ........................ .43 3.3.2. Nematicidal activity of different cultures against different nematodes species............... ..... .................................... ............ ...... ..... ... ...... ..... .. ........ .44 3.3.3. Influence of bacterial culture conditions on filtrate nematicidal activity..... .. .44 3.3.4. Nematicidal activity of the organic and aqueous fractions of the bacterial cultures .................... ..... + .............. ............ ...+........ ...... .... .... ......... .. ...$ 1 3.3 S. Nematicidal activity of some known antibiotics produced by Xenorhabdw spp........................ .................................................. .. S4 3m4m Dk~~~mmommmmom~~~~~ommoe~omommmmm~mmoommmo~w~momm~~mmmmmomommoomwmmmmmmmll l l l l moS4 CHAPTER 4. ISOLATION, IDENTIFICATION AND IN WTRO vii PRODUCTION OF NEMATICIDAL METABOLITES FRûM BACTERIAL CULTURES ............................................................................SS 4.2. Materiah md metbods.... ................................................................................... .Sa 4.2.1. Bacteria and their broth cultures ...............,.......... ......................................... 58 4.2.2. Generai procedures for isolation and identification of nematicidai metabolites.......... ., ....................................................................................... S9 4.2.3. Isolation and identification of nematicidal metabolites €rom cultures of Photorhabdus luminescens and Xenorhabdus spp ........................................ 62 4.2.4. In vitro production of the nematicidal substances identified from the ....................... bacterial cultures ...........,.. ......................
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