The Antifeedant Action of Climacodon Septentrionalis and Two Species of Sphaerobolus to Hyphal Grazing by the Fungus-Feeding Nematode Aphelenchoides Sp
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Lakehead University Knowledge Commons,http://knowledgecommons.lakeheadu.ca Electronic Theses and Dissertations Electronic Theses and Dissertations from 2009 2012-11-10 The antifeedant action of climacodon septentrionalis and two species of sphaerobolus to hyphal grazing by the fungus-feeding nematode aphelenchoides sp. Tanney, Joey B. http://knowledgecommons.lakeheadu.ca/handle/2453/171 Downloaded from Lakehead University, KnowledgeCommons THE ANTIFEEDANT ACTION OF CLIMACODON SEPTENTRIONALIS AND TWO SPECIES OF SPHAEROBOLUS TO HYPHAL GRAZING BY THE FUNGUS-FEEDING NEMATODE APHELENCHOIDES SP. by Joey B. Tanney A Graduate Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Masters of Science in Forestry Faculty of Natural Resources Management Lakehead University January, 2011 ii LIBRARY RIGHTS STATEMENT In presenting this thesis in partial fulfillment of the requirements for the M. Sc. F. degree at Lakehead University in Thunder Bay, I agree that the University will make it freely available for inspection. This thesis is made available by my authority solely for the purpose of private study and research and may not be copied or reproduced in whole or in part (except as permitted by the Copyright Laws) without my written authority. Signature: _________________________________ Date: _____________________________________ iii A CAUTION TO THE READER This M. Sc. F. thesis has been through a semi-formal process of review and comment by at least two faculty members. It is made available for loan by the Faculty of Natural Resources Management for the purpose of advancing the practice of professional and scientific forestry. The reader should be aware that opinions and conclusions expressed in this document are those of the student and do not necessarily reflect the opinions of either the thesis supervisor, the faculty or Lakehead University. iv ABSTRACT Tanney, J.B. 2010. The antifeedant action of Climacodon septentrionalis and two species of Sphaerobolus to hyphal grazing by the fungus-feeding nematode Aphelenchoides sp. 90 pp. Keywords: Antifeedant, defence mechanism, fungivory, grazing responses, mycology, mycophagy, perennial mycelium. Fungi are ubiquitous in forest ecosystems and are, collectively, a major force in driving nutrient and organic matter availability and cycling. Some saprotrophic and mycorrhizal fungi are characterized by a perennial vegetative body, delayed sexual reproduction, and a relatively long lifespan. Such fungi are exposed to a barrage of antagonistic forces throughout their existence, one notable stress being invertebrate grazing. It is suggested that fungi with perennial mycelia must have developed means to deter grazing of their hyphal networks by pervasive mycophagist invertebrates. Controlled inoculation studies with a mycophagous nematode, Aphelenchoides sp., and isolates representing 78 fungal species were conducted to investigate the presence of possible antifeedants. These in vitro pairings resulted in the discovery of two novel antifeedant mechanisms in three species of fungi. In the presence of the saprotrophic fungi Sphaerobolus stellatus and S. iowensis, the anterior portion of nematodes became encapsulated in a material of unknown composition. This encapsulation phenomenon effectively prevented further hyphal grazing by obstructing stylet extension, which resulted in the eventual death of the nematode. Nematodes that died as a result of the encapsulation were never colonized or consumed by the fungus. It is hypothesized that the encapsulating material originates from modified hyphal cells, referred to as gloeocystidia, and is liberated when the cells are punctured by the nematode stylet. v The wood-decaying fungus Climacodon septentrionalis was found to produce tall, stalked secretory cells in abundance on the aerial mycelia of the colony. Nematodes were enveloped and immobilized by droplets produced at the apices of the secretory cells. Immobilized nematodes were rarely colonized by the fungus and dead individuals persisted for weeks. A media study was employed to investigate the effect of nutrient concentration on the in vitro production of secretory cells. The discovery of novel antifeedants which mitigate damage caused to the mycelial network by grazing invertebrates offers a stimulus for further investigation into the interactions between fungi and their co-inhabiting microfauna. vi CONTENTS GENERAL ABSTRACT iii TABLES vii FIGURES viii ACKNOWLEDGEMENTS ix GENERAL INTRODUCTION 1 CHAPTER ONE ABSTRACT 4 INTRODUCTION 5 FUNGI AS FOOD 6 MYCOPHAGOUS FAUNA BIODIVERSITY 7 Collembola 7 Acari 8 Nematodes 9 Feeding Behaviour and Style of Mycophagous Fauna 9 Selective Mycophagy 10 Gut Content Analysis 10 Food Preference Tests 11 The Dematiaceous Preference? 12 Food Preference Conclusions 14 EFFECTS OF GRAZING ON FUNGI 14 Physiological Effects 15 Mycelial Growth Responses to Grazing 16 The Fungal Community 18 FUNGAL DEFENCE 19 Mechanical Antifeedant Structures 20 Adhesive Structures 20 Constricting Rings 21 Crystalline Structures 21 Chemical Antifeedants 23 Exotoxins 23 Secretory Cells 24 Endotoxins 26 CONCLUSION 26 vii LITERATURE CITED 28 CHAPTER TWO ABSTRACT 41 INTRODUCTION 42 MATERIALS AND METHODS 45 Fungi 45 Nematodes 45 Fungus-Nematode Pairing 46 Scanning Electron Microscopy 47 Light Microscopy 48 Statistics 48 RESULTS 49 Nematodes-Sphaerobolus Observations 49 Rate of Encapsulation 53 Effect of Encapsulation on Nematode Movement 61 DISCUSSION 62 LITERATURE CITED 67 CHAPTER THREE ABSTRACT 73 INTRODUCTION 74 MATERIALS AND METHODS 76 Source of Fungal Cultures 76 Nematode Isolation and Collection 76 Media Study 77 Microscopy 77 RESULTS 78 Secretory Cell Observations 78 Nematode-Fungus Interactions 78 Media Study 82 DISCUSSION 83 LITERATURE CITED 86 APPENDIX I LIST OF FUNGI USED IN NEMATODE PAIRINGS 88 viii TABLES CHAPTER TWO Table 2.1. Nematode encapsulation rates observed with time treatments. 53 Table 2.2. Normality test statistics for the proportion of encapsulated 55 nematodes data. Table 2.3. Normality test statistics for the transformed proportion of 57 encapsulated nematodes data. Table 2.4. ANOVA results for proportion of encapsulated nematodes data. 58 Table 2.5. Mann-Whitney U-Test results for effects of encapsulation on 61 number of lateral head turns. CHAPTER THREE Table 3.1. Kruskal-Wallis H-test results for the effect of medium on 82 secretory cell production. Table 3.2. Media subgroups categorized via Mann-Whitney U tests. 83 ix FIGURES CHAPTER TWO Figure 2.1. SEM micrograph of control Aphelenchoides sp. reared on C. 52 macrocephala (bar = 5 µm). Figures 2.2-2.3. Gloeocystidia on aerial hyphae of S. stellatus 52 (bar = 20 µm). Figure 2.4. Encapsulated nematode with adhering material along the body 52 (bar = 120 µm). Figure 2.5. SEM micrograph of encapsulated nematode head 52 (bar = 10 µm). Figure 2.6. Mean frequency histogram for proportion of encapsulated 54 nematodes. Figure 2.7. Normal Q-Q plot for proportion of encapsulated nematodes 55 data. Figure 2.8. Mean frequency histogram for transformed proportion of 56 encapsulated nematodes data. Figure 2.9. Normal Q-Q plot for transformed proportion of encapsulated 57 nematodes data. Figure 2.10. Proportion of encapsulated nematodes over 3 sample periods. 59 Figure 2.11. Proportion of encapsulated nematodes over time for 60 each Petri dish count. Figure 2.12. Effect of encapsulation on nematode movement 62 (error bars = 2 standard errors). CHAPTER THREE Figures 3.1, 3.3. Nematodes immobilized within droplets exuded from 81 secretory cells (bar = 100 µm). Figure 3.2. Secretory cell produced on aerial mycelia of C. septentrionalis 81 (bar = 10 µm). Figure 3.4. Hyphal growth enveloping dead nematode (bar = 100 µm). 81 x ACKNOWLEDGMENTS I wish to thank Dr. Hutchison for the guidance, freedom, and financial support which made this thesis an enjoyable and thought-provoking experience. My aspirations to become a mycologist are wholly the result of his enthusiasm and consideration as a professor. I am grateful to Lynne Sevean for his enthusiasm, optimism, and assistance in the laboratory. This project would not have been possible without the working space that Lynne provided me with. I would also like to thank my committee members, Dr. Chander Shahi and Dr. Lada Malek, for their valuable input and efforts. Thanks are extended to my external reviewer, Dr. Richard Summerbell, for his insightful comments and kind words. The following people are gratefully acknowledged for their contributions to this project: Al MacKenzie, of the Lakehead University Instrumentation Laboratory, provided technical expertise in the preparation of specimens and operation of the scanning electron microscope. Dr. R.G. Thorn generously provided a Climacodon septentrionalis isolate. Alexis McEwan collected and isolated the Aphelenchoides sp., an essential component of this thesis. Dr. Qing Yu, curator of The Canadian National Collection of Insects, Arachnids and Nematodes, verified the identification of Aphelenchoides sp. Thanks to Carolyn Babcock for the accession of the Sphaerobolus and Climacodon cultures into the Canadian Collection of Fungal Cultures. I wish to thank my parents for their support and patience through the years. I am grateful to Helena Spizarsky for always being there for me and also having a good eye for mushrooms and grouse during our forays. 1 GENERAL INTRODUCTION Fungi with perennial mycelia may be characterized by a relatively long existence with delayed sexual reproduction. As a consequence of the ubiquity of fungus-feeding