AN ABSTRACT OF THE THESIS OF Thomas Joseph Michaels for the degree of Doctor of Philosophy in Botany and Plant Pathology presented on September 2, 1982 Title: IN VITRO CULTURE AND GROWTH MODELING OF TUBER SPP. AND INOCULATION OF HARDWOODS WITH T. MELANOSPORUM ASCOS PORES Abstract approved: In vitro growth of twelve isolates of Tither spp., the true truffles, was quantified by an agar-melt procedure. All isolates grew poorly on media commonly used for the culture of mycorrhizalfungi, but responded markedly to the addition of nitrate, as well as other inorganic ions, to malt and potato-dextrose basal media. The pH of all media containing nitrate rose during incubation, and maximum rates of growth occurred at these higher pH values.Growth response to eight polycarboxylic acid buffers was investigated; one in particular, /3-ntethylcarballylic acid is both nontoxic and has a buffering range from pH 2.5 - 8,2 and is suggested for use in subsequent in vitro Tuber studies. Phosphate buffer was also found to be non-inhibitory at moderate concentrations. Of the six isolates studied for temperature response, all had optima around 20 C and declinedaround 26.5 C. The possible response of other hard-to-culture ectomycorrhizal fungi to nitrate is discused. A few simple models of fungal growth in shallowPetri dishes were proposed, and the growth data obtained as mycelial dry weight for twelve Tuber isolates were tested for fit. Growth was hypothesied to be uniform and proportional to total biomass, or to occur only atthe colony periphery and proportional to the square root of the biomass. Each of these models was altered to include a time-dependent variation: growth was hypothesized to also depend upon the concentration of a metabolite produced by the fungus.Metabolite- dependent models gave better fit than metabolite-independent versions, but differences between peripheral and uniform growth models wereless clear. In inoculation studies of oaks and filberts with T. melanosporum ascospores, viability of spores was maintained either by freezing or 5 C storage of ascocarps. Liming improves mycorrhization, although not necessarily seedling vigor. The effect of soil organic matter is minimal on growth and mycorrhizal development of Corylus avellana. Soil inoculation with spores is an effective method of establishing the symbiosis, but a germling drench is less so. A method which nondestructively measures seedling root volume was proposed in order to study mycorrhizal colonization on the same seedling over time. 'Lopyright by Thomas Joseph Michaels September 2, 1982 All rights reserved IN VITRO CULTURE AND GROWTH MODELING OF TUBER SPP. AND INOCULATION OF HARDWOODS WITH T. MELANOSPORUM ASCOSPORES by Thomas Joseph Michaels A THESIS submitted to Oregon State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Completed September 2, 1982 Commencement June 1983 APPROVED: Professor of ByAfiIIA11t&' and P ant Pa''.gy in charge of major e. Head of Department of Botany and Plant Pathology Dean of Graduat School Date thesis is presented September 2, 1982 Typed by Phyllis Taylor-Hill for Thomas Joseph Michaels ACKNOWLEDGEMENTS and DEDICATION The active support and good will of many people made this thesis possible. My sincere appreciation and thanks... To Drs. Jim Trappe and Edward Trione, who developed the research proposal and obtained the funding to promulgate this work. They are most kindly acknowledged, since it was they who took me under their tutelage and had the confidence in my abilities and resolve to see this work through. And special thanks to Jim, who allowed me continued use of his lab facilities after grant funds ran out. To Messrs. Ralph Stone and Henry Trione of Tristo, Inc., who had the perspicacity to envision a truffle industry in the United States and provided Oregon State University with the seed money to begin the endeaver. Without them I would not have been able to do this work, nor would I have the knowledge I now possess. To Drs. Bill Denison, Dallice Mills, and Edward Starkey, committee members who offered encouragement and helped in making this a better manuscript. To Mr. Gerard Chevalier, whose hospitality toward my father and me on our visit to his laboratory will be long remembered and whose love of truffle cultivation is an inspiration. To Ms. Darr Duff, Ms. Phyllis Taylor-Hill, and all the other staff of RWU 2209 and 2210 of the Forestry Sciences Laboratory, who not only patiently endured my imposition on their time but willingly offered support, suggestions, and insight along the way. The slides prepared by Darr are a treasure. Phyllis' professionalism, efficiency and willingness to help in the typing and organization of this work are most deeply appreciated. To my friends, especially, John, Jerry, and Isabel, who never let me give up over the years. To Mom and Dad and Pat and Bob. Pat and Bob understand the pain and frustration, as well as the satisfaction, of putting a thesis together. Mom and Dad understand the sacrifice involved, because in raising and loving me they know far better than I what sacrifice is. This thesis is dedicated to all of them. TABLE OF CONTENTS PART I - IN VITRO CULTURE STUDIES CHAPTER 1. In Vitro Culture of Tuber, a Genus of Mycorrhizal Ascomycetes 2 Abstract 2 Introduction 3 Methods and Materials 5 Isolates 5 General Procedures 7 Experiment I. Growth on different media 8 Experiment II. Growth as a function of time . 13 Experiment III. Growth at different temperatures 13 Experiment IVa and b. Effects of minerals on growth 15 Experiment V. Effects of inorganic phosphate buffer on growth 16 Experiment VIa and b. Effects of polycarboxylate buffers on growth 17 Results and Discussion 17 Experiment I. Growth on different media 17 Experiment II. Growth as a function of time. 20 Experiment III. Growth at different temperatures 25 Experiment IVa and b. Effects of minerals on growth 28 Experiment V. Effects of inorganic phosphate buffer on growth 34 Experiment VIa and b. Effects of polycarboxylate buffers on growth 36 Summary and Conclusions 40 Literature Cited 44 CHAPTER 2. Models of Mycelial Growth, Measured as Dry Weight, of Tuber spp. on Agar 52 Abstract 52 Introduction 52 Fungal growth models 54 Materials and Methods 65 The fungi 66 The culture medium 67 Incubation and weight determination 68 Statistical analysis 69 Results and Discussion 69 Summary 76 Literature Cited 78 PART II -MYCORRHIZAL COLONIZATION STUDIES CHAPTER 3. A Simple, Nondestructive Method for Measuring Seedling Root Volume and Estimating Dry Weight. 83 Introduction 83 Materials and Methods 87 Results and Discussion 88 Summary 90 Literature Cited 91 CHAPTER 4. Inoculation of Hardwoods with Ascospores of Tuber melanosporum: Some Simple Experiments . 92 Materials and Methods 93 Experiment 1. Two methods of spore storage treatment by two fertility levels 93 Experiment II. Effect of liming 95 Experiment III. Effect of organic matter 96 Experiment IV. Effect of soil inoculation vs. germling drench 97 Results and Discussion 98 Experiment I. Two methods of spore storage treatment by two fertility levels 98 Experiment II. Effect of liming 99 Experiment III. Effect of organic matter 99 Experiment IV. Effect of soil inoculation vs. germling drench 100 Summary 104 Literature Cited 105 LITERATURE CITED (Cumulative) 108 LIST OF TABLES Table Page CHAPTER 1 I Sources of Tuber isolates used in this study. 6 II Concentrations of principal moieties in test media of 14 experiment I. III Effect of selected undefined or partially defined 18 media of mycorrhizal and/or general mycological utility on growth of Tuber isolates in experiment I. IV Final pH of media after growth of 40 d by Tuber 21 isolates in experiment I. V Final pH of working medium (PDMA) on growth of twelve 26 Tuber isolates in experiment III. VI Effect of deletion of mineral ions from Hewitt's 29 mineralsupplement to PDMA on growth of twelve Tuber isolates in experiment IVa. VII Effect of deletion of mineral ions from Hewitt's 30 mineral supplement to PDMA on pH change from 5.7 of nutrient medium by twelve Tuber isolates in experiment IVa. VIII Effect of nitrate addition as NaNO3 to PDMA on 32 growth of Tuber isolates and final medium pH in experiment IVb. IX Effect of concentration of added phosphate on 35 growth of Tuber isolates and final medium pH in experiment V. X Polycarboxylic acid buffers tested in experiment VIa 37 XI Effect of selected buffers on growth of Tuber isolates 39 and final pH of media. XII Effect of concentration of 2-methylpropane-1,2,3-tri- 41 carboxylic acid ( -MTCarbA) on growth of Tuber isolates and final medium pH in experiment VIb. CHAPTER 2 XIII Desirable properties of a fitting equation. 55 LIST OF TABLES (continued) Table Page CHAPTER 2 XIV Summary of assumptions and equations of proposed models. 66 XV Estimated parameters and statistics of regressions 70-71 from transformations y-.Y. XVI Definition of symbols. 77 CHAPTER 4 XVII Effect of spore storage method and fertility on 98 mycorrhizal colonization of j. garryana. XVIIIEctomycorrhiza development and growth of two oak 100 species inoculated with T. melanosporum spores with or without lime. XIX Ectomycorrhiza. development and growth of C. avellana 101 inoculated with T. melanosporuin spores at three levels of organic matter. XX Ectomycorrhiza development and growth of C. avellana 103 inoculated with T. melanosporum spores in soil vs. germling drench. LIST OF FIGURES Figure Page CHAPTER 1 Scatter diagram of growth vs. final medium pH 22 of 12 isolates of Tuber growing on PDMxnA. The three-digit numbers plotted are the culture collection S-number identifiers. 2 Growth of 12 Tuber isolates on PDMmA at 20 C. 23-24 3 Effect of temperature on growth of six Tuber 27 isolates on PDMmA: 20 d, 40 d, and 60 d.