MIAMI UNIVERSITY The Graduate School Certificate for Approving the Dissertation We hereby approve the Dissertation of Levi Yafetto Candidate for the Degree: Doctor of Philosophy ______________________________________ Director Dr. Nicholas P. Money ______________________________________ Reader Dr. Diana J. Davis ______________________________________ Reader Dr. John Z. Kiss ______________________________________ Reader Dr. Nancy Smith-Huerta ______________________________________ Graduate School Representative Dr. Richard T. Taylor ABSTRACT BIOMECHANICS OF RHIZOMORPH DEVELOPMENT IN ARMILLARIA MELLEA by Levi Yafetto Fungal rhizomorphs are complex, multicellular, root-like organs formed through the aggregation, interlacing, and adhesion of millions of tip-growing hyphae. There has been very little research on the invasive mechanism utilized by rhizomorphs to penetrate compacted soils and woody substrates. Initial studies with Meruliporia incrassata, a wood-decay fungus that decomposes wooden components of buildings with an annual value of destruction estimated in millions of dollars, was aimed at inducing rhizomorphs in vitro. This attempt was not very successful, as only mycelial cords were produced. The pathogen Armillaria mellea was therefore chosen because it readily forms rhizomorphs in culture and serves as an excellent model for developmental studies. This dissertation presents findings from experiments designed to study (i) comparative features of rhizomorph anatomy in M. incrassata and A. mellea that support its invasive behavior; (ii) the adaptive growth response of rhizomorphs subjected to mechanical stress; (iii) the biochemical basis of turgor generation within rhizomorphs, and (iv) novel measurements of the forces exerted by growing rhizomorphs. Anatomical studies of rhizomorphs of A. mellea cultured in potato dextrose agar (PDA) revealed zones of hyphal tissues namely, an outer layer of peripheral hyphae, radial hyphae, longitudinal hyphae, and a central cavity. A. mellea rhizomorphs were observed to have faster growth than mycelia in PDA. We determined that increasing concentration of agar stimulated the production of more rhizomorphs, with those in media having higher concentration of agar extending faster with tapered tips. Turgor generation within A. mellea rhizomorphs was shown to be partially due to the accumulation of osmolytes. Erythritol and mannitol were identified using Gas Chromatography/Mass Spectrometry (GC/MS) and quantitatively determined to be the most dominant osmolytes that contribute to turgor generation. Osmometric studies revealed that substantial portion of turgor generated was used to exert pressure at the tip of the rhizomorphs during invasive growth. The varying amounts of force that these A. mellea rhizomorph tips exert were measured, using a sensitive strain gauge. Our experiments provide the first clear picture of the mechanical processes that allow rhizomorphs to function as migratory, exploratory and invasive organs in low-moisture and nutrient-poor environments that present substantial obstacles to fungal colonization. BIOMECHANICS OF RHIZOMORPH DEVELOPMENT IN ARMILLARIA MELLEA A DISSERTATION Submitted to the faculty of Miami University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Botany by Levi Yafetto Miami University Oxford, Ohio 2008 Dissertation Director: Dr. Nicholas P. Money Table of Contents Page CERTIFICATE FOR APPROVING THE DISSERTATION ABSTRACT TITLE PAGE …………………………………………………………………………... i TABLE OF CONTENTS ……………………………………………………………… ii LIST OF TABLES ……………………………………………………………………... iv LIST OF FIGURES ……………………………………………………………………. vi DEDICATION ………………………………………………………………………….. x ACKNOWLEDGEMENTS ……………………………………………………………. xi Chapter 1: INTRODUCTION AND LITERATURE REVIEW ……………………. 1 Wood decay basidiomycetes and the ecology of mycelial cords and rhizomorphs ……... 1 Dry rot in buildings: Meruliporia incrassata and Serpula lacrymans …………………... 2 Armillaria mellea: Model for the study of rhizomorphs ………………………………… 3 Mycelial cord and rhizomorph structure ………………………………………………… 5 Translocation in mycelial cords and rhizomorphs ………………………………………. 7 Specific experimental aims …………………………………………………………….. 8 References ……………………………………………………………………………….. 9 Chapter 2: Ultrastructural studies of Meruliporia incrassata and Armillaria mellea using scanning electron microscopy ……………………………………………………. 18 Abstract ………………………………………………………………………………….. 18 Introduction …………………………………………………………………………….... 19 Materials and Methods ………………………………………………………………....... 21 Results ……………………………………………………………………………………. 25 Discussion ………………………………………………………………………………... 27 References ………………………………………………………………………………… 31 Chapter 3: In vitro studies of rhizomorph extension in Armillaria mellea…………….. 43 ii Abstract …………………………………………………………………………………… 43 Introduction ……………………………………………………………………………..... 44 Materials and Methods ………………………………………………………………........ 45 Results …………………………………………………………………………………….. 48 Discussion ……………………………………………………………………………….... 50 References ………………………………………………………………………………… 54 Chapter 4: Osmolyte accumulation in Armillaria mellea rhizomorphs: Its role in turgor pressure generation ……………….……………………………………………………… 63 Abstract …………………………………………………………………………………… 63 Introduction ……………………………………………………………………………..... 64 Materials and Methods ………………………………………………………………........ 68 Results …………………………………………………………………………………….. 75 Discussion ……………………………………………………………………………….... 76 References ………………………………………………………………………………… 82 Chapter 5: The biomechanics of invasive growth in Armillaria mellea rhizomorphs … 108 Abstract …………………………………………………………………………………… 108 Introduction ……………………………………………………………………………..... 109 Materials and Methods ………………………………………………………………........ 111 Results …………………………………………………………………………………….. 115 Discussion ……………………………………………………………………………….... 117 References ………………………………………………………………………………… 122 Chapter 6: General discussion, Conclusions and Future Studies ……………………… 131 Research background ……………………………………………………………………... 131 General discussion ……………………………………………………………………….. 134 Conclusions……………………………………………………………………………....... 140 Future studies …………………………………………………………………………....... 142 References …………………………………………………………………………………. 144 iii List of Tables Page Table 3.1 Summary of rhizomorph length measurements in Armillaria mellea 62 in PD medium solidified with different concentrations of agar Table 4.1 Wet and dry weight of Armillaria mellea rhizomorphs before and after 97 cellular extraction Table 4.2 Mean concentrations of glycerol, erythritol and mannitol determined in 98 Armillaria mellea rhizomorphs Table 4.3 Data of ion intensity from characteristic 103 ion from different 99 concentrations of glycerol standard solutions Table 4.4 Data of ion intensity from characteristic 217 ion from different 100 concentrations of erythritol standard solutions Table 4.5 Data of ion intensity from characteristic 139 ion from different 101 concentrations of mannitol standard solutions Table 4.6 Data of ion intensity from characteristic 103 ion of glycerol in 102 rhizomorph samples cultured in PD broth with corresponding x values obtained from glycerol standard curve Table 4.7 Data of ion intensity from characteristic 103 ion of glycerol in 103 rhizomorph samples cultured on cellophane with corresponding x values obtained from glycerol standard curve Table 4.8 Data of ion intensity from characteristic 217 ion of erythritol in 104 rhizomorph samples cultured in PD broth with corresponding x values obtained from erythritol standard curve iv Table 4.9 Data of ion intensity from characteristic 217 ion of erythritol in 105 rhizomorph samples cultured on cellophane with corresponding x values obtained from erythritol standard curve Table 4.10 Data of ion intensity from characteristic 139 ion of mannitol in 106 rhizomorph samples cultured in PD broth with corresponding x values obtained from mannitol standard curve Table 4.11 Data of ion intensity from characteristic 139 ion of mannitol in 107 rhizomorph samples cultured on cellophane with corresponding x values obtained from mannitol standard curve Table 5.1 Individual measurements of applied forces and pressures from 129 Armillaria mellea rhizomorphs Table 5.2 Mean values of applied force and pressure by rhizomorphs pressing 130 against strain gauge v List of Figures Page Fig 1.1 The development of fungal mycelium from a spore 15 Fig 1.2 Formation of mycelial strands in Phymatotrichum omnivorum 16 Fig 1.3 (Top) Apical region of rhizomorph of Armillaria mellea 17 (Below) Apical region of rhizomorphs of Armillaria mellea growing in agar medium Fig 2.1 Apical region of rhizomorphs of Armillaria mellea growing 33 in agar medium Fig 2.2 Field-collected rhizomorphs of Meruliporia incrassata from an 34 infested home in California Fig 2.3 Scanning electron micrograph of field-collected Meruliporia 35 incrassata rhizomorph showing constituent hyphae lying parallel to one another Fig 2.4 (A) Scanning electron micrograph showing longitudinal hyphae 36 of Meruliporia incrassata rhizomorph (arrow). (B) Longitudinal hyphae are tightly packed, protecting the inner layers of hyphae in the middle portion of the organ. (C) Middle portion of loosely packed hyphae, m Fig 2.5 (A) A vessel hypha of Meruliporia incrassata with thin wall and 37 wide diameter lumen (arrows) in close association with tightly packed regular hyphae. (B) Vessel hyphae embedded in and surrounded by hyphae and soil particles. (C) Vessel hyphae with remnants of degenerated internal wall components vi Fig