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The north American Pine mushroom tricholoma magnivelare (peck) redhead: in vitro mycelial culture, ectomycorrhizal… Fogarty, Fidel 1998

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THE NORTH AMERICAN PINE MUSHROOM Tricholoma Magnivelare (Peck) Redhead: IN VITRO MYCELIAL CULTURE, ECTOMYCORRHIZAL SYNTHESIS TRIALS AND PRELIMINARY SHIRO ANALYSIS by FIDEL FOGARTY B.Sc, University of Victoria, 1983 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Department of Soil Science We accept this thesis as conforming (tp the reViire&s^tandard THE UNIVERSITY OF BRITISH COLUMBIA May, 1998 Copyright, Fidel Fogarty, 1998. 0 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of ^ c t c &roax^i fate £ci free) The University of British Columbia Vancouver, Canada Date DE-6 (2/88) Abstract Tissue cultures of Tricholoma magnivelare (Peck) Redhead were isolated from basidiocarps and maintained on M M N agar media. The mycelial mass of resulting colonies was expanded by transfers to fresh media. Isolates were confirmed as T. magnivelare by employing polymerase chain reaction -restriction fragment length polymorphism (PCR-RFLP) analysis to compare DNA from the vegetative mycelium to DNA from sporocarp tissue. Isolates of T. magnivelare were tested in vitro for cellulose, l ignin and pectin decomposing abilities. As with many known ectomycorrhizal fungi, none of the isolates were able to degrade any of these substrates, indicating only limited, i f any, cellulase, lignase and pectinase production. In vitro ectomycorrhizal synthesis trials were performed by combining T. magnivelare isolates with lodgepole pine, Douglas fir and western hemlock seedlings. Mycelia from five isolates were inoculated onto root systems of 8-month-old seedlings grown aseptically in Erlenmeyer flasks. No ectomycorrhizae were formed. However, some of the lodgepole pine root systems developed slightly swollen, darkened root-tips, similar in appearance to the initial stages of rootlet colonization by a related ectomycorrhizal species, Tricholoma matsutake (Ito et Imai) Singer. The affected rootlets resembled what Japanese researchers described as a 'transient mycorrhization', and appeared similar in some respects to rootlets in the initial stages of a host-parasite relationship. Longer term studies are required to determine i f ectomycorrhizae can be synthesized from these isolates under laboratory conditions. Preliminary field investigations of basidiocarp production and the shiro (underground colony) of T. magnivelare were undertaken on three sampling plots located in a Pine Mushroom Study Area established in the Nass Valley, near Terrace, British Columbia. The numbers and grades of mushrooms in plots were determined and mapped during three sampling periods in autumn, 1994. Clusters of mushrooms were inferred from sporocarp maps however, several more years of intensive monitoring are required to assess spatial and temporal distributions. Soil samples were collected both directly underneath and away from T. magnivelare basidiocarps in order to examine the types and abundance of root-tip morphotypes. Initial macro and micromorphological descriptions were done for the seven root-tip morphotypes found. Preliminary results indicated that a charcoal-grey to black, slightly swollen, elongated and i i coarsely fissured morphotype was more common directly beneath T. magnivelare basidiocarps than further away from them. This morphotype probably represents one of the latter stages of T. magnivelare rootlet colonization. Another of the morphotypes (white mycelium covering light cinnamon-brown rootlet) may represent an earlier stage of T. magnivelare colonization. Molecular analysis of fungal DNA from excised root-tips is required to confirm the identity of these root-tip morphotypes. I f these were in fact T. magnivelare colonized rootlets then the presence of a mantle and Hartig net supports the theory that the T. magnivelare is ectomycorrhizal in nature. However, rootlet characteristics, similar in some respects to those of a host-parasite relationship (i.e. darkening, necrosis and eventual sloughing of cortical cells) were observed. As suspected, T. magnivelare has some trophic and morphological characteristics typical of an ectomycorrhizal fungus 111 Table of Contents Abstract i i Table of Contents iv List of Tables vi List of Figures vi i Acknowledgements i x Dedication i x Chapter One: General Introduction 1 Chapter Two: In vitro ability of Tricholoma magnivelare mycelium to degrade cellulose, l ignin and pectin. Literature Review - Enzyme Tests 8 Introduction 20 Methods and Materials 24 Cellulase Activity 24 Lignase Activity 25 Pectinase Activity 25 Experiment 1 25 Experiment 2 26 Experiment 3 27 Results 27 Discussion 31 Chapter Three: Ectomycorrhizal synthesis trials - in vitro inoculations of the roots of Pinus contorta, Tsuga heterophylla and Pseudotsuga menziesii seedlings with Tricholoma magnivelare mycelium. Introduction 38 Methods and Materials 43 Seedlings 43 Synthesis Vessels 44 Fungal Inoculum 47 Inoculations 47 Seedling Examination 49 Results 5 0 Discussion 61 Synthesis System and Controls 61 Seedlings and Root Systems 61 Mycelial Growth 62 Isolates and Inoculum 63 Anomalous Root-tips 66 Chapter Four: Nass Valley Pine Mushroom Study Area, establishment of sampling plots, preliminary Tricholoma magnivelare sporocarp assessments, shiro analysis and examination of the root-tip morphotypes beneath pine mushrooms. Literature Review - Mushroom Ecological Studies 72 Introduction 89 Objectives 91 iv Methods and Materials Study Site Location 92 Soils 96 Mushroom Counts Methodology 102 Root Core Sampling 103 Sample Preparation 104 Root-tip Morphological Descriptions 104 Root-tip Morphotype Counts 104 Examination of Primordia - Rootlet Connections and Mycelium 106 Data Analysis 106 Results Mushroom Counts 110 Root-tip Morphological Descriptions 115 Root-tip Morphotype Counts 119 Discussion Mushroom Counts 121 Root-tip Morphotype Counts 123 Chapter Five: General Conclusions 130 Literature Cited 132 Appendix 1. Clearing and staining procedures used for root systems from synthesis flasks 150 Appendix 2. Summary of site attributes for the three pine mushroom plots 151 Appendix 3. Summary of vegetation data for the three pine mushroom study plots 152 Appendix 4. Soil profile descriptions for the three pine mushroom study plots 153 Appendix 5. Pine mushroom grading system 156 Appendix 6. Root-tip morphotype descriptions 157 Appendix 7. Results of t-tests, sign, and sign-ranked tests of root-tip data 164 v List of Tables Table 1. Experiment 1 and 2 - In vitro cellulose, l ignin and pectin decomposition test results for Tricholoma magnivelare isolates (1-1-2, 2-1, M and N) and Pleurotus ostreatus 28 Table 2. Experiment 3 - In vitro cellulose, l ignin and pectin decomposition abilities for Tricholoma magnivelare (isolate P, isolate M (pectin only)) and Pleurotus ostreatus 28 Table 3. Seed lot numbers and source data for the tree species tested: Douglas f ir (Fd), western hemlock (Hw) and lodgepole pine (PI) 43 Table 4. Summary of Pinus contorta seedling inoculation data using five isolates of Tricholoma magnivelare (2-1-1, M, N, P, Q), one isolate of Laccaria laccata (L . l ) and uninoculated controls. (VM=visible mycelium; ECMhsigns of ectomycorrhiza formation) 51 Table 5. Summary of Tsuga heterophylla seedling inoculation data using five isolates of Tricholoma magnivelare (2-1-1, M, N, P, Q) and uninoculated controls. (VM=visible mycelium; ECM=signs of ectomycorrhiza formation) 52 Table 6. Summary of Pseudotsuga menziesii seedling inoculation data using five isolates of Tricholoma magnivelare (2-1-1, M, N, P, Q) and uninoculated controls. (VM=visible mycelium; ECM=signs of ectomycorrhiza formation) 53 Table 7. Numbers and grades of Tricholoma magnivelare sporocarps in Plots 1, 2 and 3 from three samplings in September and October, 1994 114 Table 8. The number per litre of each category of root-tip in soil core samples collected in the shiro (beneath T. magnivelare sporocarps) and outside the shiro (>3 m away from any visible T. magnivelare sporocarp) at each plot 120 v i List of Figures Fig. 1. Colony of T. magnivelare (90 days) (approx. 2 cm radius) 29 Fig. 2. T. magnivelare colony on cellulose medium (7 months) (approx. 2.25 cm radius) 29 Fig. 3. T. magnivelare colony on lignin medium (7 months) (approx. 2.0 cm radius) 30 Fig. 4. T. magnivelare colony on pectin medium (7 months) (approx. 2.25 cm radius) 30 Fig. 5. Sealed jar synthesis system tested 45 Fig. 6. Erlenmeyer flask synthesis system employed ; 46 Fig. 7. T. magnivelare mycelium employed as inoculum 48 Fig. 8. Reisolated T. magnivelare colony (approx. 30 days) 48 Fig. 9. Douglas f ir seedling showing coiled root system 54 Fig. 10. Uninoculated lodgepole pine rootlet (#25 - control) (100X magnification) 54 Fig. 11. Lodgepole pine root system showing lateral roots 55 Fig. 12. Laccaria laccata colonized lodgepole pine rootlet (#18) (100X magnification) 55 Fig. 13. Mantle L. laccata inoculated rootlet (#18) (400X magnification) 56 Fig. 14. Mantle and Hartig net L. laccata inoculated rootlet (#18) (1000X magnification) 56 Fig. 15. T. magnivelare inoculated (M-isolate) lodgepole pine #8 rootlet with swollen apex (100X magnification) 58 Fig. 16. Lodgepole pine seedling #8 root system 58 Fig. 17. Hemlock rootlet with repetitive ring pattern (100X magnification from seedling #3 inoculated with isolate 2-1-1 60 Fig. 18. Douglas f ir root-tip from seedling #11 inoculated with isolate N (100X magnification). Note substrate attached and root hairs in background 60 Fig. 19. General location of Nass Valley Pine Mushroom Study Area (reprinted with permission from dc Geus, 1995b) 93 Fig. 20 Nass Valley Pine Mushroom Study Area location 94 Fig. 21. Looking south over study area - Shumal River in center, study area to north and east of Shumal drainage 95 Fig. 22. Study area - typical pine mushroom habitat 95 Fig. 23. Nass Valley Pine Mushroom Study Area sampling plots (approximate locations) 97 v i i Fig. 24. Plot 1 soil pit site prior to excavation 98 Fig. 25. Plot 1 soil pit during excavation. Note uncovered primordia adjacent to pine mushroom...98 Fig. 26. Plot 2 soil pit during excavation showing mycelial mat in upper horizons 99 Fig. 27. Plot 2 soil pit during excavation showing exposed colonized rootlets and mycelium 99 Fig. 28. Canopy showing lodgepole pine and western hemlock 101 Fig. 29. Portion of plot 1 site showing T. magnivelare sporocarps 101 Fig. 30. Root mat during preliminary sorting showing rootlets embedded in dense, white mycelium (8X magnification) 105 Fig. 31. Root-tips extracted from mycelial mat for examination (12X magnification) 105 Fig. 32. Tricholoma magnivelare primordium with rootlets embedded in its base (12X magnification) 107 Fig. 33. Hyphae with slightly swollen 'ball-joint-like' septae in mycelium taken from base of T. magnivelare primordium 107 Fig. 34. Hyphal branching in the form of 'H-shapes' (anastamoses) in mycelium taken from the base of T. magnivelare primordia 108 Fig. 35. Nass Valley Plot #1: Location of T. magnivelare sporocarps on three sampling dates in fall, 1994 I l l Fig. 36. Nass Valley Plot #2: Location of T. magnivelare sporocarps on three sampling dates in fall, 1994 112 Fig. 37. Nass Valley Plot #3: Location of T. magnivelare sporocarps on three sampling dates in fall, 1994 113 Fig. 38. Root-tip morphotype #4 ('matchstick') showing proximal constriction and darkened surface with whitish-gray remnant mycelium (32X magnification) 116 Fig. 39. Longitudinal section through morphotype #4 rootlet showing loose mycelial sheath (remnant 'mantle'?) and palmate invaginations (poorly developed 'Hartig net') on epidermal and outer cortical cells (1000X magnification) 116 Fig. 40. Root-tip morphotype #5 (white/tan) (8X magnification) 117 Fig. 41. Morphotype #4 root-tip. Whole mount unsquashed (120X magnification) 117 Fig. 42. Morphotype #4 root-tip. Whole mount squashed (120X magnification) 118 V l l l Acknowlegements I would like to thank my wife, Doris, for all her assistance. Dedication To my three daughters, Emily, Oonagh and the New Baby - Finola Frances Fogarty - who, in her first weeks of life, slept through the final drafting of this manuscript. ix Chapter 1 General Introduction British Columbia has long been recognized for its rich macrofungal diversity. Fungi perform important ecological roles in forests. Wood and litter decomposers, mycorrhizal (tree-root symbionts), pathogenic and parasitic fungi interact with other organisms and play integral parts in ecosystem stability and biodiversity maintenance. The extensive commercial harvest of wild edible mushrooms and recent resource conflicts have led to concern regarding the sustainability of macrofungal populations and have heightened the awareness amongst resources managers and the public for forest fungi. Foresters and ecologists now recognize that the health of forests also depend on organisms and processes below ground. The North American pine mushroom {Tricholoma magnivelare (Peck) Redhead) is found in many parts of British Columbia, generally in association with stands of lodgepole pine {Pinus contorta Dougl.), western hemlock (Tsuga heterophylla, (Raf.) Sarg.) and Douglas fir (Pseudotsuga menziesii (Mirb.) Franco) (Redhead, 1989). The pine mushroom has been described as a robust mushroom with a 5-20 cm (rarely up to 35 cm), white dry to subvisid cap that with age, develops yellowish to pale, brown streaks or stains. Initially, the fruiting bodies are phallic in shape and subterranean. The cap is rounded when young, becoming flatter, often covered with delicate brownish scales at maturity. The flesh is f i rm and white with a distinctive, somewhat sweet, pungent aroma similar to cinnamon. The stem is up to 15 cm long, 5 cm thick, solid, tough and fibrous. It is smooth above and scaly below the ample annulus that flares out in young specimens leaving a thick, soft veil which usually forms a conspicuous ring. Detailed descriptions of T. magnivelare (as Armillaria ponderosa (Peck) Sacc.) were given by Zeller and Togashi (1934) and Hotson (1940). More recent descriptions of the mushroom are provided by Arora (1986) and Phillips (1991). The pine mushroom has become a biological forest product of importance in British Columbia's economy (Meyer, 1995) and as harvesting increases so also does concern over its long-term sustainability. Although preliminary pine mushroom harvesting guidelines have been developed, 1 pine mushroom management strategies have not yet been implemented in British Columbia (de Geus, 1995). Also referred to as the American matsutake, T. magnivelare is commercially harvested in British Columbia, Washington, Oregon, California and Mexico. Some professional pine mushroom harvesters follow the production which usually begins in early autumn in northern British Columbia and proceeds south through the western United States to Mexico. In British Columbia the majority of the large scale commercial harvesting of pine mushrooms occurs near Terrace, Bella Coola, Powell River, Pemberton, Duncan and in the Nahatlatch area near Boston Bar. Commercially the pine mushroom is the most sought after mushroom in British Columbia with virtually the entire crop being shipped to Japan. In British Columbia there were an estimated 5,000 pine mushroom pickers in 1994 (de Geus, 1994, pers. comm.). Each year more people are becoming involved in the harvest of pine mushrooms. According to the Kootenay Business Journal (Chesik, 1994) the value of pine mushrooms has reached as much as $350 per pound. In 1994 Seiyo Trading Company, a major buyer of British Columbia pine mushrooms, paid a minimum of $20 and a maximum of $150 per pound for 'number one' grade mushrooms (Appendix 5). In some areas pine mushroom harvesters have made as much as $2000 per day during the height of fruiting season (Chesik, 1994). Meyer (1995) reported anecdotal estimates on average of 30-40 tons of pine mushrooms, worth about 1 mill ion dollars, being harvested from the Nass Valley, near Terrace, each year. According to that author, 1994 was a good year for pine mushroom production and an estimated 350,000 pounds worth just under 4 mil l ion dollars were collected in the Nass Valley alone. Fresh mushrooms are exported to Japan where they are a highly prized delicacy. In 1997, prices paid in Japan for Canadian pine mushrooms averaged just under $100 per kilogram. Japanese pine mushrooms were worth as much as ten times that value (Wills, 1998, pers. comm.). The Japanese pine mushroom Tricholoma matsutake (Ito et Imai) Singer, which grows in stands of Pinus densiflora Sieb. et Zucc. in Japan, has been intensively harvested since the early 19th century (Ogawa, 1977a). The biology, ecology and growth of T. matsutake have been studied extensively 2 (Tominaga, 1978). Much of our current understanding of T. magnivelare is inferred from studies on T. matsutake. In Japan and Korea many researchers have suggested that over-harvesting of T. matsutake has resulted in diminished populations (Tominaga, 1978; Lee, 1991; and others). Large scale commercial harvesting of pine mushrooms is relatively recent in British Columbia (15-20 years) and now occurs throughout the range of pine mushroom producing areas in the province. Remote areas are being accessed by helicoptor and boat. Researchers from the University of British Columbia, the Ministry of Forests, the Ministry of Environments, Lands and Parks, the Vancouver Mycological Society, First Nations, environmental organizations, and private industry have all expressed concern about the potential effects of commercial pine mushroom harvesting on long-term sustainability of pine mushroom producing sites. Mycologists, foresters, and commercial pickers and buyers have presented anecdotal observations that pine mushroom populations have been depleted in areas that are intensively harvested (Kovacs, 1995, pers. comm.; Lahai, 1997, pers. comm.). Soil disturbance and compaction due to repeated visits to the same site are factors that may contribute to diminishing populations. Excessive picking and site degradation by some pine mushroom harvesters (i.e. forest floor raking) may have significantly impacted pine mushroom habitats. Habitat disturbance goes well beyond the direct influence on sustainability of the mushroom. Wildlife disturbance, increased risk of forest fire and garbage left in the woods are some other problems associated with mushroom harvesting. The need to develop protocols for macrofungal studies is great. Survey methodology and monitoring techniques for forest fungi are in the early stages of being field tested. Macrofungal data bases in British Columbia are extremely limited and information regarding the biology and ecology of most forest fungi is scant. Basic questions remain unanswered regarding reproduction methods, rates of growth and resilience of pine mushroom populations after disturbance. The management of the pine mushroom industry in British Columbia has been discussed by Eligh (1989); de Geus et al. (1992); de Geus (1993, 1994, 1995, 1995b); and de Geus and Berch (1995). These authors describe a 'gold rush mentality' amongst pine mushroom pickers due to the high 3 market value of these mushrooms. The general consensus amongst mycologists appears to be that a concerted, integrated effort must be initiated to inventory this species and study the impacts of mushroom harvesting, timber harvesting and other anthropogenic influences on its long-term sustainability. Recently there has also been concern over the impacts of timber harvesting on pine mushroom producing areas. Anecdotal information indicates that in British Columbia pine mushrooms have not been found in stands less than 40 years old. Pine mushrooms have however been found in second growth stands aged greater than 40 years indicating it is probably a mid-late stage colonizer. It may take a number of decades before the underground mycelium develops enough biomass or reaches a stage of maturity capable of producing fruit bodies. When carefully planned, forest management can be made to be more conducive to the management of other resources including macrofungi. Scientific information regarding the effects of various forest management practices on T. magnivelare is scant. Recently a number of models have been developed to examine long-term pine mushroom productivity in managed stands (Weigand, 1998; Olivotto, 1998, unpublished). Many parameters and assumptions are involved and it wi l l be several decades before the longer term hypotheses presented in these models can be tested. In their closing remarks Hosford et al. (1997) said that the pine mushroom cannot be managed in isolation from the plant communities and ecosystem to which it is ecologically adapted. Time and energy must be invested to conserve this poorly understood, but valuable resource. The present author is currently undertaking research in a coastal Douglas fir-western hemlock ecosystem (CWHdm) examining the impacts of alternative silviculture systems on 120 species of macrofungi including the pine mushroom (Fogarty, 1997, unpublished). At the 1994 Ecosystem Management for Forest Fungi conference in Corvallis, Oregon, the monitoring and assessment of forest fungi was discussed. One of the objectives of the workshop was to discuss potential methods to inventory and monitor forest fungi in order to examine the effects of mushroom harvesting and forestry management practices on macrofungal populations. Another objective of the conference was to prioritize the range of concerns, initiate long-term ecological 4 studies and to develop a regional data base on fungal productivity and diversity. In recent years, numerous macrofungal studies have been initiated by the U.S. Forest Service to monitor and assess mushrooms, especially economically valuable species (Hosford et al., 1997). The Forest Renewal British Columbia Provincial Forest Soils Research Strategy (1995-draft) points out some major areas of related research which need to be initiated in British Columbia including: • Detailed studies and descriptions of the biology and ecology of commercially valuable mushrooms and other soil microorganisms. • How different levels of forest canopy retention influence soil microclimates, carbon, organic matter, nutrient dynamics, and soil organisms, including macrofungi. • The development of in vitro and in vivo techniques to enhance production of botanical forest products, including macrofungi. According to the 1995 British Columbia Forest Practices Code Biodiversity Guidebook, management for biodiversity must be flexible and adaptive. The long-term goals of mycologists and forest managers in British Columbia should be to develop mushroom management strategies that help sustain key macrofungal values in the province. Zeller and Togashi (1934) published the first ecological studies of T. magnivelare, then called Armillaria ponderosa, in western North America. They noted its association in Washington and Oregon with lodgepole pine and western hemlock and traced "rhizomorphic connections between the basidiocarps and ectotrophic mycorrhizas on rootlets of lodgepole pine". They referred to associated shrub and herb species such as salal (Gaultheria shallori) and princes pine (Chimaphila umbellata) as well as fungi including members of the genera Amanita, Boletus, Rhizopogon, Russula and Tricholoma. Kinugawa and Goto (1978) published preliminary studies of T. magnivelare which described its occurrences in western hemlock stands in Washington State. According to Molina et al. (1993) T. magnivelare forms ectomycorrhizae with a broad range of hosts. Although for many years the North 5 American pine mushroom has been reported as an ectomycorrhizal species, this has not been proven. Recently in the U.S., Hosford et al. (1997) summarized work undertaken over the past decade in Washington State describing pine mushroom producing sites, with a focus on the shiro (underground mycelium or colony) of T. magnivelare and sporocarp production (Hosford and Ohara, 1986, 1990, 1995). This research is ongoing and other facets of pine mushroom ecology are also being examined. Hosford et al. (1997) indicated that considerable disagreement exists about the trophic character of the pine mushroom. Characteristics of saprobic, parasitic and ectomycorrhizal fungi have been reported. Although it is possible that differing strains of T. magnivelare vary in terms of potential parasitic abilities, they also indicated that differing reports by various researchers may be attributable to the examination of ectomycorrhizae at different stages of colonization. Most reports agreed however that the latter stages of T. magnivelare infection, and indeed T. matsutake and related species, appeared on rootlets as slightly swollen, charcoal grey to black, coarsely fissured root-tips, similar in some respects to parasitized rootlets. Despite the increasing economic importance of T. magnivelare, widespread, integrated research has only recently been proposed to elucidate its ecological role in British Columbia forests. Concern over sustainability of pine mushroom populations and appreciation of the potential role of the pine mushroom in the British Columbia economy has stimulated interest in increasing our understanding of its field and laboratory behavior and growth. Tricholoma magnivelare has been presumed to be ectomycorrhizal, but its trophic character remains uncertain and very little is known about its in vivo requirements. In 1994, a joint pilot project involving the Nisga'a Tribal Council and the British Columbia Ministry of Forests was initiated. A Pine Mushroom Study Area was established north of the Village of New Aiyansh in the Nass Valley, 80 km north of Terrace. The Pine Mushroom Project had both scientific and socio-economic components. The study area was selected because it had been a renowned pine mushroom producing area over the past decade and is geographically isolated (boat 6 or helicopter across the Nass River). Pine mushroom harvesting in the area was 'restricted' to persons registered in the pine mushroom study. Pine mushroom sampling plots described later in this thesis were established in this study area. Objectives The overall objective of this research was to enhance our knowledge of the biology and ecology of the North American pine mushroom and to provide further information which may help elucidate its putatively ectomycorrhizal nature. The primary objectives of these studies were: 1. To isolate mycelium of T. magnivelare from tissues excised from fresh, young pine mushrooms collected in the Nass Valley Pine Mushroom Study Area. These isolates and others from Mesachie Lake and Boston Bar were maintained for use in laboratory studies. 2. To examine in vitro the ability of pine mushroom mycelium to degrade cellulose, l ignin and pectin. 3. To attempt in vitro the synthesis of ectomycorrhizae between isolates of pine mushroom mycelium and root systems of lodgepole pine, Douglas f i r and western hemlock seedlings. 4. To establish permanent T. magnivelare sporocarp sampling plots in the Nass Valley Pine Mushroom Study Area and collect preliminary ecosystem information and mushroom production data. 5. To undertake preliminary investigations, descriptions and comparisons of the types and abundances of root-tip morphotypes extracted from soil cores collected both directly beneath and away from pine mushroom sporocarps. 7 Chapter 2 In Vitro Ability of Tricholoma Magnivelare Mycelium to Degrade Cellulose, Lignin and Pectin. Literature Review - Enzyme Tests The in vitro detection of extracellular enzymes produced by fungi in pure culture on solid media is a useful technique for determining their enzymatic capabilities (Taylor, 1974; Hankin and Anagnostakis, 1975; Egger, 1986; Hutchison, 1990a). Care is needed in transposing laboratory results to the field, and it cannot be assumed that just because an organism is capable of util izing a specific substance in a test tube or petri dish it wi l l utilize that compound as a substrate in the soil (Richards, 1987). In vitro tests frequently underestimate the capacity of a fungus to hydrolyse substrates because laboratory conditions may not be appropriate for optimal enzyme production or substrate hydrolysis (Jakobsen, 1991). As well, depending on the sensitivity of the tests, trace amounts of enzyme activity may not be detected (Hutchison, 1990a). Conversely, strong in vitro hydrolysis of substrates would suggest the fungus is capable of producing the enzyme under field conditions (Egger, 1986). For saprophytic fungi, enzymatic organic matter decomposition provides nutrients and an energy source. For pathogenic fungi, degradative enzymes are important in host infection (Hankin and Anagnostakis, 1975). Plant cell walls are constructed primarily from microfibrils of cellulose embedded in a matrix of lignin, pectin and hemicelluoses (Curtis, 1983). Soil organic matter consists of a myriad of different substances originating from the remains of dead plants and animals, and their excretory products in various stages of decomposition (Richards, 1987). Thus, polysaccharides, polypeptides and lignin are major components of soil organic matter. The production of extracellular enzymes by saprophytic and pathogenic fungi is essential for the decomposition of organic matter and overcoming host resistance (Chang et al., 1992). In order to utilize polysaccharides from soil organic matter, fungi must contend with physical and chemical barriers, including the molecular stability and rigidity of carbon polymers such as cellulose, lignins and pectin. 8 Cellulose is a long chain polymer of glucose (Morrison and Boyd, 1979). It is the principal structural component of plant cell walls and is the most abundant substrate in soils (Richards, 1987). The linear nature of the cellulose molecule combined with strong beta-(l-4) linkages between the individual D-glucopyranosyl units make cellulose chemically stable, and its decomposition relatively slow, in nature (Richards, 1987). Cellulose biodegradation is brought about by the combined activities of three different enzymes: endoglucanases, exoglucanase and cellobiase (beta-glucosidase) (Burns, 1983). Lignin, another major component of woody substrates (23-33% in conifers), (Richards, 1987), is composed mainly of coniferyl alcohol and other polymerized phenyl propane units (Morrison and Boyd, 1979). Lignin acts as a cementing agent binding cellulose and other polysaccharides together. Lignin is relatively inaccessible to microorganisms because of its large molecular size, low solubility and complex cross linked structure (Richards, 1987). Microbial l ignin degradation is brought about primarily through the action of polyphenol oxidases (laccase and tyrosinase) (Hutchison, 1990b). Tyrosinase has been characterized as an endoenzyme (Stalpers, 1978) and its role in l ignin degradation other than detoxification of breakdown products is unlikely. Laccase, on the other hand, is an exoenzyme and has been correlated with white-rot (preferential l ignin decomposition). Piatt et al.(1984) concluded that the preliminary actions of laccase is a prerequisite for l ignin degradation. Pectin occurs mainly in the middle lamella and helps bind adjacent plant cell walls together (Lindeberg and Lindeberg, 1977). Pectin compounds are made up of residues of alpha-galacturonic acid. Commonly pectin exists as calcium and magnesium salts of pectic (polygalacturonic) acid residues (Raven et al., 1978). Endopolygalacturonase is the enzyme which assists in pectin decomposition of the middle lamella (Lindeberg and Lindeberg, 1977). The ability of fungi to degrade cellulose, l ignin and pectin has been studied extensively under field and laboratory conditions. Many wood and litter decay fungi in both the Aphyllophorales (Nobles, 1958; Kaarik, 1965) and Agaricales (Zadrazil, 1978; Trojanowski, 1984; Bassous et al., 1989) have been shown to enzymatically decompose one or a combination of cellulose, l ignin or pectin. 9 The oyster mushroom, Pleurotus ostreatus Fr. was used as a positive control fungus in the present enzyme assays to ensure the treatment media were reacting to extracellular carbohydrases. Carbohydrase production by P. ostreatus and other saprophytic fungi wi l l be discussed prior to reviewing enzyme production by ectomycorrhizal fungi. Pleurotus species have been studied extensively because of their commercial value and ability to colonize a vast array of substrates. Pleurotus ostreatus and related species (eg. P. sajor-caju (Fr.) Singer) have been shown to degrade cellulose, l ignin and pectin in vitro. P. ostreatus is considered a white-rot fungus because of its preferential hydrolysis of l ignin (Kaneshiro, 1977). Brown-rot is the result of cellulose decomposition by a small group of basidiomycetes, while white-rot results from the utilization of both cellulose and lignin by a wide range of basidiomycetes and a few ascomycetes (Richards, 1987). Kaarik (1965) was able to show that white-rot fungi produced laccase while brown-rot fungi could not. Numerous authors have reported in vitro polyphenol oxidase production by members of the genus Pleurotus (eg. Molitoris, 1979; Wojtas-Wasilewska and Trojanowski, 1975) as well as on natural media such as straw (Zadrazil, 1978) and wood-based media (Kirk and Moore, 1972). Toyama and Ogawa (1974) demonstrated the in vitro ability of P. ostreatus, and other Pleurotus species to produce cellulase and polyphenol oxidases by growing mycelium in liquid culture in flasks on a number of lignin and cellulose-containing substrates including carboxymethylcellulose. Kaneshiro (1977) showed that P. ostreatus mycelia degraded lignocellulosic solid wastes. He found a 10-40% decrease of l ignin and a 15-40% decrease of cellulose during fermentaion of feed lot wastes. Zadrazil (1977) also reported the decomposition of cellulose and lignin by Pleurotus species while studying the potential to upgrade waste straw to animal feed. Bassous et al. (1989) grew P. sajor-caju mycelium in aseptic submerged culture on a medium containing 30% lignin and 70% polysaccharides. They found that P. sajor-caju mycelium was capable of hydrolysing celluloses and hemicelluloses and some lignin from the lignocellulosic substrates. 10 Bushnell and Chang (1994) showed that P. sajor-caju produced cellulases in vitro. In their study, they measured sugar released from carboxymethylcellulose (filter paper) by mycelium of P. sajor-caju growing in submerged culture. Enzyme activity was defined by these authors as the amount of enzyme produced necessary to release 1 umole of reducing sugar (glucose or xylose) per minute at 25°C. A diverse array of saprophytic fungi have been shown to have in vitro cellulolytic capabilities including Lentinus edodes (Berk.) Pegler (Nakazawa et al., 1974), some members of the genus Pholiota (Klan et al., 1989) and the edible fungus Volvariella volvacea (Bull:Fr.) Sing. (Hobbs, 1995). The ability of P. ostreatus mycelium to digest pectin in vitro has been shown (Chang and Quimo, 1982). Many other saprophytic fungi have also been demonstrated to have pectinolytic abilities (eg. Mirasmius species) (Lindeberg and Lindeberg, 1977). The ability of ectomycorrhizal fungi to decompose cellulose, l ignin and pectin remains a controversial question. Most authors agree that wood and litter decomposers have a greater ability to produce cell wall decomposing enzymes than ectomycorrhizal fungi. In 1975, Singer proposed that the ectomycorrhizal fungus Laccaria laccata (Scopoli:Fries) Cooke has facultative saprophytic abilities. Indeed, mycorrhizal species have been shown to produce cellulases (Dighton et al., 1987; Entry et al., 1991), lignases (Giltrap, 1982; Trojanowski et al., 1984; Haselwandter et al., 1990; Entry et al., 1991) and pectinases (Lamb, 1974; Lindeberg and Lindeberg, 1977; Giltrap and Lewis, 1982). Hutchison and Malloch (1988) suggest a number of criteria and methods which help to determine whether a putatively ectomycorrhizal isolate is in fact truly ectomycorrhizal. Combined with macro- and micro- morphological characteristics, in vitro enzymatic activity is proposed as another aid in determining identity of isolates. In vitro carbohydrate utilization by ectomycorrhizal fungi has been studied extensively. The ability of an ectomycorrhizal fungus to utilize different carbon sources can be tested by growing the fungus on agar or in a liquid medium containing different carbon polymers (Jakobsen, 1991). 11 According to Hacskaylo (1973), Melin (1925) found that ectomycorrhizal fungi utilize only simple sugars, primarily glucose, as a carbon source. At that time, evidence of the absence of carbohydrases in ectomycorrhizal fungi was based on the inability of isolates to grow in culture with carbon polymers as the sole carbon source. More than twenty years later Mikola (1948) showed that Cenococcum graniforme (Ferd. and Winge) was able to use glucose, sucrose, maltose and mannitol in vitro. Norkrans (1950) showed that some putatively ectomycorrhizal Tricholoma species could also use fructose, mannose, maltose, sucrose and starch. Norkrans (1950) also reported that one of her ectomycorrhizal isolates of Tricholoma vaccinum (Pers. ex Fr.) Kummer was able to decompose cellulose in vitro and that cellulase production was enhanced in the presence of a small amount of "starter" glucose added to the growing medium. Norkrans (1950) summarizes that some ectomyccorhizal fungi may be capable of producing cellulases. She proposes the difference between litter decomposing and ectomycorrizal fungi is probably quantitative rather than qualitative with respect to extracellular enzyme production. In reviewing the literature on ectomycorrhizal carbohydrase production, Hacskaylo (1973) contends that, although Norkrans' (1950) purported cellulase-producing T. vaccinum isolate may have been an exception, as a rule ectomycorrhizal fungi were unable to decompose litter and other naturally occurring complex carbohydrates. Palmer and Hacskaylo (1970) had previously found that, although monomers such as D-glucose provided good in vitro growth for a wide range of common ectomycorrhizal fungi, their strains were unable to decompose cellulose. However, significant in vitro pectinase activity was shown in their ectomycorrhizal isolates. These authors suspected that as the hyphae of ectomycorrhizal fungi grow between the cortical cells, pectinase is secreted, hydrolysing the pectin. They contended that free pectin would rarely be available to ectomycorrhizal fungi in soil due to its rapid utilization by saprophytes. Palmer and Hacskaylo (1973) also reported that, in the presence of free glucose, pectinase activity was suppressed, the opposite of a starter effect. They suspected that supression of 12 pectinase in the presence of soluble carbohydrates (eg. glucose) could have resulted in this enzyme remaining undetected in previous assays. Lamb (1974) showed that, even in the absence of added starter glucose, 21 species of ectomycorrhizal fungi were able to use a variety of sugars as sole carbon sources including D-glucose, D-mannose, fructose, cellobiose, trehalose, sucrose, dextrin, glycogen, and to a lesser extent starch and pectin. Some of Lamb's isolates showed abilities to degrade cellulose after a 4-6 week lag phase even without added starter glucose. He proposed that by adding starter glucose at low concentrations (0.1 g/1) some ectomycorrhizal fungi were able to get over the lag phase and 'switch on' cellulolytic and pectinolytic enzyme systems. Lindeberg and Lindeberg (1977) tested Lamb's (1974) hypothesis by comparing dry weights of Boletus edulis Bull, ex Fr. Steinpilz, Suillus luteus (Fr.) S. F. Gray and S. grevillei (Kl.) Singer grown on glucose versus glucose plus pectin. Contrary to Lamb (1974) they found that those species were unable to use pectin with or without starter glucose. However, Lindeberg (1986) later conceded that strain differences could have explained significant within-species differences in extra-cellular carbohydrase production. Giltrap and Lewis (1982) examined the in vitro ability of S. luteus to utilize pectin and found that its pectinolytic ability was reduced in the presence of glucose confirming Palmer and Hacskaylo's (1970) earlier supposition. Giltrap and Lewis (1982) postulate that the inhibitory effect of available glucose on pectinolytic ability prevents extensive intracellular fungal infection when ectomycorrhizal fungi are colonizing intercellularly. Lewis (1986) states that although growth on structural polymers is variable, that on pectin is commonly good but this polymer has not always been free of low molecular weight sugars. Pectin-containing substrates have been found to release soluble sugars during autoclaving. Released sugars may result in erroneous positive pectinase activity test results on pectin-containing media (Egger, 1986). Todd (1979) examined the abilities of Cenococcocum geophilum, Laccaria laccata (Fr.) Berk, and Br., Rhizopogon vinicolor Smith, Suillus lakei (Murr.) Smith and Thiers and Amanita 13 pantherina (D. C. ex Fr.) Schumm. to decompose organic matter in microcosms. Decomposition was measured by determining the 14CC>2 respired by the fungi grown in axenic culture with Douglas f ir in peat-vermiculite to which ^C-labelled cellulose, hemicellulose, litter and humus were added. The five ectomycorrhizal fungi tested were all able to some degree to degrade all substances. However, humus was degraded appreciably less than litter and cellulose less than hemicellulose. Taber and Taber (1984) showed by measuring respiration and ^^COj production of mycelia in submerged culture that Pisolithus tinctorius Pers. was unable to utilize cellulose. Other similar studies (Cromack, 1984) have shown that some ectomycorrhizal fungi may hve the ability to decompose complex carbon substances. Trojanowski et al. (1984) examined the abilities of five known ectomycorrhizal fungi: Cenococcum geophilum, Amanita muscaria (Fr.) S. F. Gray, Tricholoma aurantium (Schaeff. ex Fr.) Rick, Rhizopogon luteolus Fr. and Nordh. and Rhizopogon roseolus (Corda) Hollos to metabolize 1 4 C -labelled lignin, lignocellulose and holocellulose in pure culture. Al l 5 species exhibited significant rates of degradation on the three substrates however, the saprophytic fungi Heterobasidion (Fomes) annosus (Fr.) Cooke and Sporotrichum pulveralentum (Ander and Eriksson) degraded the substrates more rapidly. These authors suggest that under the conditions in the top layer of forest soils, where a mixed population of various litter decomposing microorganisms is to be expected, mycorrhizal fungi may be able to contribute to the decomposition of litter material. Thus, evidence has accumulated to suggest that a degree of saprophytic ability may be present in the mycorrhizal community. Egger (1986) examined substrate hydrolysis by post-fire ascomycetes including two mycorrhizal fungi (Sphaerosporella brunnea (A. and S.) Svreek and Kubicka and E-stmin-WiIcoxina species). Both species produced polyphenol oxidases and S. brunnea was found to also produce cellulase.. Egger (1986) concludes that the ability to utilize complex polysaccharides under post-fire conditions would give early successional mycorrhizal species a competitive advantage over other mycorrhizal or saprophytic soil fungi prior to the re-establishment of potential hosts and successful colonization of their root systems. 14 Dighton et al. (1987) have shown, by measuring the loss in tensile strength of cotton cellulose in a microcosm experiment, that Suillus luteus (Fries) S.F.Gray and Hebeloma crustuliniforme (Bull, ex Saint-Amans) Quel, have cellulolytic abilities. Dighton (1987) states that the saprotrophic ability of S. luteus was greater than that of H. crustuliniforme and attributes this to the fact that S. luteus is a late stage colonizer and exists in closed canopy or forest ecosystem where a greater amount of recalcitrant, tree-derived litter accumulates. As with cellulose it is also of interest to know whether in fact the dominance of certain ectomycorrhizal types is also associated with an ability to mobilize lignin. In order to further examine this question Haselwandter et al. (1990) selected representatives of ectomycorrhizal and ericoid mycorrhizal fungi and carried out comparative analysis of their in vitro abilities to degrade and utilize 1 4C-labelled lignin. The results of this study showed that the ericoid fungi {Oidiodendron griseum Robak and Hymenoscyphus ericae (Read) Korf et Kernan degraded lignin at a significantly higher rate than the ectomycorrhizal fungi. However, Paxillus involutus (Batsch ex Fr.) Fries degraded lignin more readily than those fungi which were normally considered to be obligately ectomycorrhizal (Suillus bovinus L. ex Fr. and Rhizopogon roseohts (Corda) Hollos). These authors concluded that because P. involutus is most often found in mor-humus types, where recalcitrant carbon compounds of the lignin type accumulate, an ability to degrade lignin would increase its saprophytic potential. In ecological terms these environments are comparable to, and indeed overlap with, those occupied by ericoid fungi which have been shown to degrade lignin (Jakobsen, 1991). In summing up carbon nutrition in ectomycorrhizae, Jakobsen (1991) suggests that heterotrophic fungal carbon assimilation may account for 10% and 50% of the carbon gained by ectomycorrhizal and ericoid mycorrhizal fungi respectively. Haselwandter et al. (1990) suggested that such lignolytic ability may be of direct benefit to the fungus in providing it with an additional carbon source. While provision of such access would be of potential benefit to both fungus and host, the expression of ligninolytic potential within the mycorrhizal root could pose a threat to the plant. Regulation of ligninolytic activity is thus a matter of considerable biological importance (Haselwandter et al., 1990). 15 Hutchison and Malloch (1988) used an array of biochemical and physiological tests to differentiate between cultures of over 100 ectomycorrhizal fungi including cellulase, pectinase and polyphenol oxidase tests. None of the ectomycorrhizal fungi tested were able to break down pectin. These authors attributed contamination by saprophytic fungi to any purportedly ectomycorrhizal isolates which showed positive cellulase activity. They also found that, though members of the Amanita, Laccaria and to a certain extent Paxillus and Rhizopogon were tyrosinase dominant, all Tricholoma and Hebeloma species tested appeared to be laccase dominant. The function of laccase and tyrosinase in ectomycorrhizal fungi is unclear. Egger (1986) proposes that the production of polyphenol oxidases may assist ectomycorhizal fungi in detoxifying antifungal phenolic compounds produced as defense barriers by host roots. Hutchison (1990b) suggests polyphenol oxidases may aid ectomycorrhizal fungi in detoxifying phenolic compounds in forest humus released through organic matter decomposition. It is difficult to ascertain exactly what the implications of the production of small amounts of in vitro carbohydrases are to the fungus under field conditions (Richards, 1987). Hutchison (1990a) repeated the series of biomechanical and physiological tests on 96 species of ectomycorrhizal fungi representing 30 genera. He found that none of his 169 isolates tested were able to break down cellulose or lignin and only one species (Cortinarius brunneus) broke down pectin. Hutchison (1990a) concluded that putatively ectomycorrhizal fungal isolates, which in the past have shown positive cellulase or pectinase activity, were often saprophytes misidentified as ectomycorrhizal cultures. He also said that current taxonomic changes and ecological findings have placed putatively ectomycorrhizal fungi into saprophytic groups. This, he claims, explains some of the false positives reported for purportedly ectomycorrhizal isolates. Hutchison (1990b) again examined the potential to use polyphenol oxidase (laccase and tyrosinase) activity tests on the 96 species of ectomycorrhizae to determine i f laccase and tyrosinase dominance patterns were taxonomically or ecologically significant. He used a spot test in which fungal colonies grown for 5 weeks on modified Melin-Norkrans agar (Marx, 1969) were tested by 16 placing a drop of 1-napthol on one side and a drop of p-cresol on the other side of the colony edge. 1-napthol turns blue when laccase is present and p-cresol turns reddish-brown when tyrosinase is present. By grouping different species into 'no activity', 'laccase-dominant' or'tyrosinase-dominant' groups, based on a subjective no activity (-) to intense reaction (+++++) colour change at the colony edge, Hutchison (1990b) was able to show that patterns of polyphenol oxidase activity observed among ectomycorrhizal isolates were generally consistent with genus. He concludes that the polyphenol oxidase test is a valuable tool for differentiating ectomycorrhizal isolates into initial groupings. In the case of Thcholoma species for example subgeneric differentiation could be made among the species examined which correlated with the taxonomic scheme of Singer (1986) (ie. subgenus Tricholoma sect. Genuina were laccase and tyrosinase negative while Tricholoma sect. Tricholoma were (laccase-dominant) (Hutchison, 1990b). The North American pine mushroom {Tricholoma magnivelare (Peck) Redhead) is purported to form ectomycorrhizae on a variety of conifer hosts in British Columbia, but so far, no direct evidence of this has been reported. No reports have been published on the in vitro ability of T. magnivelare to degrade cellulose, l ignin or pectin. However, the physiology and nutritional requirements of the mycelium of the Japanese pine mushroom {Tricholoma matsutake (Ito and Imai) Sing.) has been investigated by many researchers (Kawai and Ogawa, 1981). Oyama et al. (1974) examined the dry weight yield of mycelium on various carbon sources for 5 species of ectomycorrhizal fungi including T. matsutake. Suprisingly, T. matsutake mycelium grew significantly better on the polysaccharides pectin and starch than on the monosaccharides glucose or fructose. However, it is possible that soluble sugars released from the polysaccharides during autoclaving (Egger, 1986; Lewis, 1986) supported mycelial growth. Contrary to Oyama et al. (1974), Kawai and Abe (1976) showed that only glucose and fructose supported significant growth of T. matsutake while Tominaga (1978) found that as well as glucose and fructose, two other sugar monomers, mannose and maltose, supported good growth of T. matsutake mycelium. Both Kawai and Abe (1976) and Tominaga (1978) showed that dissacharides, 17 including sucrose, and polysaccharides such as pectin and cellulose are not utilized by T. matsutake in vitro. Yang et al. (1983) reported that a crude acetone extract of polyphenol oxidase was isolated from T. matsutake. However, as mentioned earlier, the production of small amounts of polyphenol oxidases does not necessarily incur saprophytic abilities. Instead it may function to assist ectomycorrhizal fungi in overcoming antifungal phenolic compounds produced as defense barriers by tree roots (Egger, 1986) or to detoxify phenolics released during litter decomposition (Hutchison, 1990b). However, according to Iwase (1992), Ogawa (1978) reported that mycelium of T. bakamatsutake (a relative of T. matsutake) may have some capacity to decompose litter. Ogawa (1981) showed that T. bakamatsutake inhabits the upper mineral layer of soil beneath the litter and frequently colonizes the litter layer. However, no f i rm evidence of litter decomposition was put forth. Lee (1991) and others have suggested that T. matsutake is 'slightly' parasitic, surrounding the roots of Pinus densiflora and turning them dark brown (necrotic). He cites poor performance of mature host trees as further evidence of this. The potential parasitic nature of T. matsutake is supported by reports of necrosis of the roots of young pine seedlings exposed to T. matsutake (Ogawa, 1975a; Ogawa, 1975b; Kawai and Ogawa, 1981). Lee (1991) also found using biochemical spot tests (alpha-napthol drops on the colony edge) similar to those used by Hutchison (1990b) that a number of isolates of T. matsutake were positive for laccase. He speculated that T. matsutake may also have limited saprophytic abilities, but no direct experimental evidence of this was reported. One characteristic of ectomycorrhizal fungi is slow mycelial growth on agar media (Hutchison and Malloch, 1988). Kawai and Ogawa (1981) state that mycelial growth of T. matsutake was extremely slow both in solid and in liquid media. Indeed Ohta (1990) reported a growth rate of only 1 cm/month for T. matsutake on generally employed agar media. Isolates of the North American pine mushroom (T. magnivelare) also grow slowly in culture (Fogarty, unpublished). Although no reports have been published regarding the ability of the North American pine mushroom (T. magnivelare) to produce carbohydrases, Ho and Trappe (1992) have shown that 18 T. magnivelare mycelium has the ability to produce other enzymes such as phosphatases, nitrate reductase, and extracellular growth regulators such as giberellins, cytokinins and indole acetic acid. Production of these compounds is common to many ectomycorrhizal fungi (Ho and Trappe, 1987). This is not f i rm evidence of its ectomycorrhizal nature, however, because the production of phosphatases and nitrate reductases is common to all fungi and the production of plant growth regulators has been reported for numerous non-mycorrhizal fungi including many parasitic species such as Armillaria ostoyae (Kendrick, 1985). More information is required to determine the nutritional mode and ecological role of the North American pine mushroom. One of the objectives of the present work is to determine the in vitro ability of T. magnivelare to decompose cellulose, l ignin and pectin, which wi l l assist in elucidating its purported ectomycorrhizal habit. 19 Introduction The North American pine mushroom (Tricholoma magnivelare (Peck) Redhead) is purported to be an ectomycorrhizal fungus and is found in many parts of British Columbia in association with stands of lodgepole pine (Pinus contorta Dougl.), western hemlock (Tsuga heterophylla, (Raf.) Sarg.) and Douglas f ir (Pseudotsuga menziesii (Mirb.) Granco (Redhead, 1989). It is generally accepted that ectomycorrhizal fungi obtain their carbon and energy sources from living roots whereas saprophytic fungi utilize the organic substance in the litter and humus (Lindeberg, 1986). Harley (1986) agreed with this view because i f ectomycorrhizal fungi obtain their carbohydrate in the form of simple sugars from host roots, they should have little requirement for enzyme systems to degrade complex polymers. At that time Harley (1986) indicated that there was no good evidence that any ectomycorrhizal fungi produce enzymes, either wall attached or secreted externally, that are capable of hydrolyzing the carbon polymers that make up plant cell walls. On the other hand, many litter and wood decomposing fungi have been shown to produce one or a combination of cellulase, lignase and pectinase. For example, the saproproph Trichaptum abietinus (Fr.) Ryv. is one of the most important delignifiers of coniferous slash in North America (Aurora, 1986). Another common wood rotter Pleurotus ostreatus Fr. has been found to degrade cellulose (Toyama and Ogawa, 1974; Bassous et al., 1989), l ignin (Zadrazil, 1978; Molitoris, 1979; Bassous et al., 1989) and pectin (Chang and Quimio, 1982). The in vitro production of carbohydrases by Pleurotus species and other saprophytic fungi is discussed by Chang et al. (1992) and in the Literature Review, at the beginning of this thesis. Many common ectomycorrhizal fungi have been tested for cellulase, lignase and pectinase ability, including some members of the genera Tricholoma (Norkrans, 1950); Suillus and Boletus (Lamb, 1974; Lindeberg and Lindeberg, 1977; Giltrap and Lewis, 1982); Hebeloma (Dighton et al., 1987) and Paxillus (Haselwandter, 1990). Although results varied, most ectomycorrhizal species showed only l imit or no ability to utilize complex carbon sources. 20 Richards (1987) says that there is no evidence of enzymatic degradation of cortical cell walls by ectomycorrhizal fungi in nature, and any lytic activity of the mycobiont would have to be suppressed by the host. Thus to maintain a compatible relationship it seems unlikely that ectomycorrhizal fungi can obtain the sugars essential for growth from any source other than the living roots of their host. The utilization of host-supplied carbon by the mycobiont has been shown for many species of ectomycorrhizae by exposing the shoots of the host plant to an atmosphere containing labeled 14CC»2. Radioactivity is subsequently detected in the fungal (mycobiont) tissue (Jakobsen, 1991). This was first done for ectomycorrhizal fungi by Melin and Nilsson (1957) and numerous researchers have developed and used 14CC>2 techniques since (e.g. Ho and Trappe, 1973). Other methods have been developed to show that ectomycorrhizal fungi utilize host supplied carbon. For example, Reiger et al. (1991) examined the longitudinal distribution of sugars in Picea abies (L.) Karsten fine roots colonized by the ectomycorrhizal fungus Amanita muscaria (Fries) S.F. Gray. They found that in areas of the root most heavily colonized by A. muscaria (i.e. the Hartig-net region) the level of glucose was lowest. They attribute this to glucose being delivered to and utilized by the fungus. Uncolonized P. abies fine roots did not show longitudinal variations in glucose concentration. These types of studies have confirmed the passage of simple sugars from host to fungus in an ectomycorrhizal relationship. However, ectomycorrhizae are a diverse group of fungi. Trappe (1977) points out that forests host a wide array of species of mycorrhizal fungi and likely a wide diversity of enzyme potentials. A better understanding of mycorrhizal enzyme systems may help elucidate some of the patterns of distribution and abundance of ectomycorrhizal fungal species in forest ecosystems. Indeed according to Jakobsen (1991) some ectomycorrhizal fungi and especially ericoid mycorrhizal fungi, have a well developed ability to utilize complex organic compounds. Heterotrophic fungal carbon assimilation may account for 10 and 50% of the carbon gained by ectomycorrhizal and ericoid mycorrhizal fungi respectively. 2 1 Read (1991) says that though recent studies have largely confirmed that most ectomycorrhizal fungi, unlike their ericoid counterparts, fail to degrade the most complex polymers such as lignin, it has become increasingly clear that there is a wider range of potential enzyme activity than previously realized. Thus, there is mounting evidence that at least some ectomycorrhizal fungi are capable of cellulase and pectinase production. It is generally accepted, however, that although some ectomycorrhizal fungi may have limited capacity to produce extracellular carbohydrases, in nature, they obtain the majority of their carbon from the living roots of hosts (Lindeberg, 1986). Physiological and biochemical tests have been performed on 96 species of North American ectomycorrhizal fungi, eleven of which were in the genus Tricholoma but not including T. magnivelare (Hutchison, 1990a). These tests include the enzymatic degradation of cellulose, l ignin and pectin. Hutchison found that none of the 96 ectomycorrhizal fungi he tested were able to appreciably degrade cellulose or l ignin and only one species (Cortinarius brunnens) degraded pectin. He suggests that it is unlikely that significant quantities of these enzymes are produced in vivo as the balanced symbiotic relationship with plants would be negatively affected. He concludes that tests for enzymes can assist in determining whether or not a basidiomycetous fungus is ectomycorrhizal. There has been extensive research done on the carbohydrate biochemistry and physiology of the Japanese pine mushroom {Tricholoma matsutake) (e.g. Oyama et al., 1974; Kawai and Ogawa, 1981; Yang et al., 1983; Ohta, 1990; Lee, 1991; Iwase, 1992). Though Lee (1991) claims T. matsutake may have some saprophytic or even "slightly parasitic" abilities, no f i rm evidence of its ability to degrade cellulose, l ignin or pectin was reported. However, T. matsutake is presumed ectomycorrhizal (Ogawa, 1981) and according to some authors may have limited cellulase, lignase and pectinase production abilities (Lee, 1991; Tominaga, 1978; Oyama et al., 1974). Prior to this study, the in vitro ability of the North American pine mushroom (T. magnivelare) to degrade cellulose, l ignin or pectin had not been tested. Thus, despite its putative ectomycorrhizal nature, there is limited scientific evidence to support this. 22 The objective of this study is to examine the in vitro abilities of 5 isolates of T. magnivelare to degrade cellulose, lignin and pectin. These results wi l l be compared to those of a known saprophytic wood decomposing fungus {Pleurotus ostreatus). Results wi l l also be compared with other ectomycorrhizal fungi reported in the literature with special reference to the Japanese pine mushroom {T. matsutake). This wi l l assist in determining the nutritional mode and ecological role of the North American pine mushroom. 23 Methods and Materials In October, 1994, 20 isolatesof the pine mushroom {Tricholoma magnivelare (Peck) Redhead) were obtained using tissue excised from the interior of fresh, young sporocarps collected in the Nass Valley, near Terrace, British Columbia. Pieces of excised pine mushroom tissue were aseptically placed on a basal medium of modified Melin Norkrans agar (MMN) (Hutchison, 1990a) in standard (90 mm) plastic petri plates. M M N (Marx, 1969) was made up of 1.0 g glucose, 2.0 g malt extract, 1 g yeast extract, 0.5 g K H 2 P 0 4 , 0.25 g ( N H 4 ) 2 H P 0 4 , 0.15 g M g S 0 4 . 7 H 2 0 ) , 0.05 g CaCl 2 , 0.012 g FeCl3.5H 2 0, 15.0 g agar and 1 L distilled water. This is a modification of the medium used by Marx (1969) in that glucose was included as a carbon source as well as malt extract (Hutchison, 1990a). Nass isolates were labeled A-T. Cultures of T. magnivelare isolated from sporocarps collected in October, 1992, near Mesachie Lake (isolate 1-1-2) and Boston Bar (isolate 2-1-1) were also obtained (Berch, pers. comm., 1993). Five of the resulting T. magnivelare isolates were selected for testing: 1-1-2, 2-1-1, M, N and P. Al l isolates except 2-1-1 were confirmed by DNA RAPD analysis as being T. magnivelare (Egger, pers. comm., 1996). Isolates were aseptically transferred to new petri-plates containing modified M M N and incubated at 20°C in darkness for 90 days prior to inoculations onto petri-plates containing cellulose, l ignin and pectin media. An isolate of Pleurotus ostreatus Fr. (a known, wood-decomposing saprophyte) was grown on M M N and maintained at 20°C in darkness. Resulting P. ostreatus colonies were grown to approximately 5 cm diameter and stored at 4°C in darkness prior to testing. The five T. magnivelare isolates and the control, P. ostreatus, were tested by aseptically transferring small (approximately 3 mm x 3 mm) pieces of colonized agar cut from the actively growing colony edges onto petri-plates containing each of the 3 treatment media described below. Cellulase activity: A solution of modified M M N (MMN without the glucose or malt extract) was prepared, autoclaved and poured into petri-plates. The agar medium was allowed to cool and solidify. A 2 g/1 cellulose-azure preparation (Sigma Chemical Co. cat# C-1052) in modified M M N was autoclaved 24 separately and a 1-3 mm thick layer was poured over the solidified modified MMN medium in each petri-plate. This second layer was allowed to cool and solidify. The degradation of cellulose (cellulase activity) is detected when azure dye is released from the cellulose substrate and diffuses into the lower clear layer of MMN agar. This test is a modification of the techniques used by Hutchison (1990a) and by Egger (1986). Lignase activity: Lignin utilization was tested using the modified MMN medium to which was added 0.2 g/1 of poly R-478 (Sigma Chemical Co. cat# P-1900). Poly R-478 is a polymeric dye which has been chemically bound to lignin. Lignin utilization (laccase production) causes the dye to turn from pinkish to colourless. If lignin decomposition takes place a clear zone occurs around and beneath the colony in an otherwise pink background. This test is similar to the technique used by Hutchison (1990a) and Glenn and Gold (1983). Pectinase activity: Modified MMN containing 5.0 g/1 of citrus pectin (Sigma Chemical Co. cat.# P9135) was prepared, heated to dissolve the pectin, then autoclaved and poured into standard (90 mm) plastic petri-plates and allowed to cool and solidify. Pectin utilization is determined by flooding the colonized petri-plates with a 1% aqueous solution of hexadecyltrimethylammonium bromide (Sigma Chemical Co. cat.# H5882). Fungal colonies were covered with this solution for several hours after which it was poured off. If pectinase was produced, a clear zone occurred around the colony in an otherwise opaque background. This test is similar to that used by Egger (1986) and Hutchison (1990a). The five T. magnivelare isolates (1-1-2, 2-1-1, M, N and P) and the control fungus, P. ostreatus were tested as follows: Experiment 1 Eleven petri-plates of each medium were inoculated as follows: one plate with 1-1-2, one with 2-1-1, two with M, two with N, three with P. ostreatus and two uninoculated controls. P. ostreatus inoculated plates were incubated for 10 days at 20°C in darkness prior to examining for enzyme 25 activities. T. magnivelare isolates were allowed to grow under similar conditions for 60 days prior to testing. Uninoculated control plates were examined after 60 days. Experiment 2 Experiment 2 was set up in a similar manner to Experiment 1 using the same T. magnivelare isolates. However, prior to re-testing the T, magnivelare isolates were transferred to new MMN plates to maintain colony vigour. The P. ostreatus isolate was refrigerated (4°C) on MMN. Experiment 3 Eight petri-plates of each medium were inoculated as follows: three with P (a previously untested T. magnivelare isolate), three with P. ostreatus and two uninoculated controls. An additional three pectin plates were inoculated with T. magnivelare isolate M to re-test its pectinase-producing ability. All plates were incubated at 20°C in darkness. For T. magnivelare isolates, one plate of each medium was examined at 20, 43 and 53 days after inoculation. Pleurotus ostreatus plates were examined after 10 days incubation. Uninoculated control plates were examined after 10 and 53 days incubation. The results from the three experiments were tabulated and isolates compared for carbohydrase production. Plates of each medium inoculated with the 'M' isolate were incubated at 20°C for 7 months to examine colony growth. 26 Results Tables 1 and 2 show the results for experiments 1, 2 and 3. Throughout the study none of the uninoculated control plates showed any diffusion or clearing. The control fungus Pleurotus ostreatus showed positive enzymatic activity for cellulase, lignase and pectinase on all media in all experiments. Definitive diffusion of colour from the upper azure layer to the lower clear layer was seen on P. ostreatus inoculated cellulose plates. The blue colour diffused completely through the lower layer beneath the colonies. The portions of the plate which had not been colonized by P. ostreatus mycelium (i.e. the edges) at the time of examination showed no diffusion of azure dye. Clearing was evident adjacent to colonies of P. ostreatus growing on lignin plates. Zones of clearing beneath the colonies also extended a short distance (1-2 mm) beneath and beyond colony edges. A l l of the P. ostreatus inoculated pectin plates showed clear zones adjacent to mycelial colonization on an otherwise opaque medium. None of the T. magnivelare isolates showed any detectable cellulase, lignase or pectinase activity with the exception of one petri-plate of isolate M in the first Experiment (Table 1). The T. magnivelare plate which showed pectinase activity was examined earlier (after 40 days incubation) than other plates (which were examined at 60 days). The distinct zone of clearing extended 3-5 mm around the colony edges. No signs of contamination were seen on this or any of the other plates. Isolates of T. magnivelare used as inoculum grew relatively dense colonies at a rate of approximately 0.6 cm/month on M M N agar (Figure 1). Colonies grew approximately 0.3 cm/month on each of the test media. On the test media colony growth appeared relatively dense adjacent to the inoculum plugs, however, became relatively sparse, appearing to diminish in density further away. Figures 2, 3 and 4 show 7 month old colonies of isolate 'M' on cellulose, l ignin and pectin media respectively. 27 Table 1. In vitro cellulase, lignase and pectinase test results for Tricholoma magnivelare isolates ( 1 -1-2, 2-1-1, M and N), Pleurotus ostreatus and uninoculated controls. Fungal Isolate Tested Experiment 1 Experiment 2 Cellulase Lignase Pectinase Cellulase Lignase Pectinase Control - - _ _ Control - - - - _ P. ostreatus + + + + + + P.ostreatus + + + + + + P. ostreatus + + + + + + 1-1-2 - - _ 2-1-1 - - - - _ _ M - - *+ _ _ M - _ _ _ _ _ N - _ _ _ _ N - - - - - -* anomalous false positive (see text) Table 2. In vitro cellulase, lignase and pectinase test results for Tricholoma magnivelare isolate P, isolate M (pectin only), Pleurotus ostreatus and uninoculated controls. Fungal Isolate Tested Cellulase Experiment 3 Lignase Pectinase # Days Incubated Control - _ 7 Control - - - 53 P. ostreatus + + + 7 P. ostreatus + + + 7 P. ostreatus + + + 7 P - - - 20 P - - - 43 P - - - 53 M - 20 M - 43 M - 53 28 Fig. 1. Colony of T. magnivelare (90 days) (approx. 2 cm radius). 2. T. magnivelare colony on cellulose medium (7 months) (approx. 2.25 cm radius). 29 Fig. 4. T. magnivelare colony on pectin medium (7 months) (approx. 2.25 cm radius). 30 Discussion The in vitro degradation of cellulose, l ignin and pectin by fungi has been used to determine qualitatively their enzymatic capabilities (Hankin and Anagnostakis, 1975; Egger, 1986; Hutchison and Malloch, 1988). Ectomycorrhizal fungi are notoriously difficult to identify in pure culture (Hutchison, 1990a). The in vitro cellulase, lignase and pectinase activity tests used in the present study were chosen because they had previously been shown to aid in distinguishing a wide range of ectomycorrhizal isolates from saprophytic ones (Hutchison, 1990a) and can assist in determining the ecological niches and nutritional modes of fungal species (Egger, 1986). The saprophytic fungus, Pleurotus ostreatus, was chosen as a positive control because of its known ability to degrade cellulose, l ignin and pectin in vitro. Indeed, the major carbon compound is cellulose in most substrates used to cultivate P. ostreatus commercially. Numerous authors have demonstrated in vitro l ignin utilization by P. ostreatus and related species (e.g. Wojtas-Wasilewska and Trojanovvski, 1975; Molitoris, 1979). The ability of P. ostreatus mycelia to digest pectin has been shown (Chang and Quimio, 1982). As expected, the results of this study indicate that P. ostreatus was able to produce enzymes to degrade cellulose, lignin and pectin in vitro. The results indicate that none of the five isolates of T. magnivelare tested were able to degrade cellulose, lignin or pectin in amounts detectable by these tests. The absence of cellulase and pectinase activity has been proposed by Hutchison and Malloch (1988) as a diagnostic criterion in determining i f a putatively ectomycorrhizal isolate is in fact ectomycorrhizal. Many studies have been done with other ectomycorrhizal fungi (Hutchison, 1990a; Egger, 1986) including the Japanese matsutake (Ogawa, 1977b; Yang et al., 1983; Ohta, 1990). Most of these studies have shown that ectomycorrhizal fungi have very limited or no ability to produce cellulase, lignase or pectinase. The results of this study therefore provide increased evidence to support the purportedly ectomycorrhizal nature of T. magnivelare because T. magnivelare was also shown not to utilize cellulose, lignin or pectin appreciably. However, results are preliminary and there are drawbacks to the enzyme tests employed here. 31 Although the two-layered cellulase test employed in the present study is more sensitive than the agar plate technique used by Hankin and Anagnostakis (1975), this test still only gives a qualitative approximation of the enzymatic capabilities of an isolate (Hutchison, 1990a). Results are likely to underestimate the hydrolytic capacity of the fungal isolate. Negative test results can either mean the fungus is not able to decompose the substrate appreciably or the in vitro conditions were not appropriate for substrate degradation (Egger, 1986). Hutchison (1990a) says that minute quantities of enzymes may not be detected by these tests and the tests should not be viewed as being final indications of what each fungus can potentially produce in vivo. No attempts were made to quantify the tests used here, however, according to Hutchison (pers. comm., 1995) the cellulose-azure test could potentially be modified using colorimetric studies to quantify cellulase production. By producing standard spectrophotometric curves from colour changes induced by cellulose degradation using known amounts of purified cellulase, a measurement of intensity of cellulose depolymerization could be determined. However, Hutchison (pers. comm., 1995) believes that the cellulose test used in this experiment is adequate to qualitatively determine potential in vitro cellulase production. Indeed, Hutchison (1990a) states that his saprophytic fungal isolates (species unnamed) produced positive results on all test media indicating that the tests were functional. As found with the five T. magnivelare isolates in the present study, Egger (1986) and Hutchison (1990a) found little or no lignase activity for any of their known ectomycorrhizal isolates. Hutchison (pers. comm., 1995) also indicated that the release of soluble sugars from the pectin medium or changes in pH due to autoclaving could potentially cause anomalous results with the pectinolytic activity test. This was also suggested by Lewis (1986) as an explanation for erroneous positive pectinase tests for ectomycorrhizal fungi. The positive pectin plate inoculated with isolate M observed in Experiment 1 of this study (Table 1) may indeed have been an erroneous result or false positive due to the precipitation of the nutrient agar by hexadecyltrimethylammonium bromide (Egger, 1986; K. Egger, pers. comm., 1995). Another possible explanation for a positive pectinolytic test is that upon autoclaving, the pectin medium released sugar monomers (Egger, 1986) which acted as starter sugar (Norkrans, 1950) and 32 allowed the initiation of some some pectinolytic activity by the 'faster' growing, glucose enhanced isolate M colony. Palmer and Hacskaylo (1970) and subsequently Lamb (1974) reported that this might be the case with isolates of ectomycorrhizal genera including Amanita, Russula, Suillus, Boletus, Coenococcum and Rhizopogon. The positive pectin plate could also have been the result of pectinase production by a contaminant. However, the macroscopic appearance of the colony on the positive pectin plate resembled T. magnivelare the colonies on other pectin plates in the same replicate. As well, the colony on the positive M plate smelled like pine mushroom. The odor of mushrooms has been shown to be an important diagnostic characteristic of species (Arora, 1986). Thus it was unlikely to have been a contaminant, unless it was an unseen pectinolytic bacterial contaminant growing in conjunction with or obscured by the T. magnivelare isolate. Another potential problem with the lignin and pectin tests is that faster growing isolates can overgrow the zone of clearing around the colony obscuring its presence. This can be controlled by employing a polycarbonate membrane beneath the colony which can be peeled off to expose overgrown zones of clearing (Chang et al., 1992). Without such controls erroneous negative results can occur (Egger, 1986). This was not a problem in the present study as P. ostreatus did not overgrow the obvious zones of clearing and T. magnivelare isolates grew slowly (approximately 0.3 cm/month on the test media). Enzyme activity is affected by factors such as pH. Ogawa and Akama (1983) found that the optimum pH for T. matsutake was between 4 and 5.5 for some isolates and between 4 and 8 for others. The in vitro growth rate of T. magnivelare has been found not to vary significantly between pH 4 and 8 (Ho and Trappe, 1992). The pH (after autoclaving) of the modified M M N medium used in the present study was 5.5-5.8 (Hutchison, 1990a). Thus, in the present study, it is unlikely that pH was a contributing factor in the inability of T. magnivelare mycelium to produce measurable quantities of carbohydrases. The addition of a small amount of starter glucose (1 g/1) to the medium when testing ectomycorrhizal isolates for cellulose degradation has been found to initiate cellulase activity 33 (Norkrans, 1950; Lamb, 1974). Norkrans (1950) used starter glucose to initiate cellulose decomposition by an isolate of Tricholoma vaccinum (Pers. ex Fr.) Lamb (1974) also indicated adding glucose at low concentrations to the test medium was sufficient to initiate carbohydrase production by his Suillus isolates. The basal (pre-treatment) medium used to grow T. magnivelare inoculum contained glucose (1 g/1) and malt extract (2 g/1). As noted in the methods section of this chapter, small (approximately 3 x 3 mm) pieces of the basal medium were transferred along with mycelium from the colony edges onto the treatment media (cellulose, l ignin and pectin). The small amount of residual sugar remaining in the transferred pieces supported growth of the transferred mycelium over the lag phase and allowed some colonization of the test media. Colony density however appeared to diminish substantially as the mycelium grew from the original inoculum source medium (MMN) onto the test media. The T. magnivelare isolates in the present study had access to less starter glucose than Norkrans' (1950) or Lamb's (1974) isolates, however, sufficient to allow some colonization of the test media. Experiments using varying amounts of starter glucose should be undertaken to test whether higher levels of glucose enhancement would initiate carbohydrase activity in T. magnivelare. Harley (1985) reports that ectomycorrhizal fungi have very limited or negligible abilities to hydrolyze carbohydrate polymers enzymatically. It is therefore possible that the penetration of the host root tissues by mycorrhizal fungi is not necessarily associated with the possession of carbohydrase enzymes. Carbohydrase enzymes produced by some mycorrhizal fungi in the saprophytic or parasitic modes must be inhibited in the symbiotic mode in order to minimize negative impacts on host root systems. Evidence of the absence of carbohydrase enzymes in ectomycorrhizal fungi is often based on their inability to grow in culture with carbon polymers as the sole source of carbon (Richards, 1987; Ohta, 1990). This was the case for T. magnivelare in the present study. Lindeberg and Lindeberg (1977) postulate that it is still possible that the apical region of the hyphae of ectomycorrhizal species may contain minute amounts of wall-attached enzymes that assist in host root penetration. According to Hutchison (pers. comm., 1995) the 34 cellulase, lignase and pectinase tests used in the present study are probably not sensitive enough to detect such minute amounts of carbohydrases. Minute amounts of cellulase and pectinase have been detected in ectomycorrhizal fungi using more sensitive 1 4C-labeled enzyme assays (Dahm et al., 1987). The production of small amounts of hydrolytic enzymes could assist ectomycorrhizal fungi to colonize host roots (Harley and Smith, 1983). The ability to degrade complex carbon compounds may also give some ectomycorrhizal fungi 'facultative saprophytic' ability which could allow the expansion of underground mycelium in the leaf litter and possibly even the survival of the fungus in the absence of host trees (Laiho, 1970; Harley and Smith, 1983; Lee, 1991). Hutchison (1990a) disagrees, stating that the ectomycorrhizal ability to produce substantial quantities of cellulase, lignase or pectinase in vivo would disrupt the mutually beneficial relationship with host roots and he doubts that ectomycorrhizal basidiomycetes behave also as facultative saprotrophs. On the other hand, Singer (1975) reports potential saprophytic abilities for the ectomycorrhizal fungus Laccaria laccata (Scop, ex Fr.) Cke. and numerous researchers indicate potential parasitic abilities for the purportedly ectomycorrhizal Japanese pine mushroom (T. matsutake) (Ogawa, 1975a and 1975b; Kawai and Ogawa, 1981; Lee et al., 1984 and others). The pyrophilous, ascomycetous, mycorrhizal species Sphaerosporella brunnea, has also been shown to produce cellulases (Egger, 1986) using similar tests to those of Hutchison (1990a). The ability to digest cellulose saprophytically under post-fire conditions could give early successional mycorrhizal fungi a competitive advantage in post-fire situations where few living host root systems may be available for colonization. Some basidiomycetous ectomycorrhizal fungi related to T. magnivelare, including Tricholoma aurantium, have been shown to produce minute amounts of cellulases (Trojanowski et al., 1984) using sensitive 1 4C-labeled tests. Basidiomycetous ectomycorrhizal fungi generally grow slowly in culture (Jakobsen, 1991). T. matsutake grows approximately 1 cm per month on M M N (Ohta, 1990). In the present study T. magnivelare grew approximately 0.6 cm/month on M M N but only about 0.3 cm/month on the test media. 35 Kawai and Abe (1976) and Tominaga (1978) showed independently that only 4 sugar monomers supported significant growth of T. matsutake mycelium. These were glucose, fructose, mannose and maltose. Disaccharides such as sucrose and other polysaccharides such as pectin and cellulose were not utilized by their T. matsutake isolates. Japanese reports have not been consistent for in vitro carbohydrase production by T. matsutake. For example, pectin has been found to support good growth of 1 isolate (IFO 6915) (better in fact than glucose or fructose) (Oyama et al., 1974). Cellulase and lignase production by T. matsutake has not been reported. Indeed, it appeared that T. magnivelare mycelium grew denser colonies on the pectin medium than on the cellulose medium. The cellulose medium appeared to support denser colonies than the l ignin medium. Future studies measuring biomass of mycelium produced in liquid culture containing different complex polymers could quantify these differences. It is possible that soluble sugars were released from the pectin medium during autoclaving and provided the isolates with more sugar allowing for denser colonies to form on the pectin medium than on the lignin or cellulose media. Pinus densiflora roots contain primarily the sugars glucose and fructose (Tominaga. 1978). The mycelia of T. matsutake are reported to colonize the roots of Pinus densiflora (Ogawa, 1977c) and considered to receive nutrients from them (Ohta, 1990). However, it is possible that T. matsutake, commonly accepted as an ectomycorrhizal species, also exists as a root pathogen util izing enzymes to cause necrotic tissues in young root-tips (Lee, 1991 and others). Kawai and Ogawa (1981) reported an initial swelling and darkening of P. densiflora root-tips in response to exposure to T. matsutake mycelium in vitro. Indeed, in the present study anomalous darkened root-tips were found on Pinus contorta roots growing in ectomycorrhizal synthesis trials with isolate 'M' of T. magnivelare. This is discussed further in the Ectomycorrhizal Synthesis chapter of this thesis. However, the lack of ability of T. magnivelare to produce significant amounts of cellulase, lignase and pectinase indicates that it is unlikely to perform well as a litter decomposing saprophyte. As discussed earlier, more sensitive tests could be employed to determine i f the mycelium of the North American pine mushroom produces small quantities of such enzymes. 36 Although the failure to utilize complex carbohydrates alone does not distinguish ectomycorrhizal isolates, and in vitro studies do not necessarily mimic the in vivo abilities of a fungus to produce extracellular enzymes, the negative cellulase, lignase and pectinase results of these tests indicate that the T. magnivelare isolates performed more like ectomycorrhizal than saprophytic fungi. 37 Chapter 3 Ectomycorrhizal Synthesis Trials - In Vitro Inoculation of the Roots of Pinus Contorta, Tsuga Heterophylla and Pseudotsuga Menziesii Seedlings with Tricholoma Magnivelare Mycelium. Introduction There is interest amongst mycologists and foresters to determine the abilities of forest fungi to form ectomycorrhizae. Controlled environment studies, though not mimicking field conditions, aid in determining fungus/host relationships. In vitro synthesis techniques have been employed extensively since Melin, in the 1920's, developed laboratory methods to study mycorrhiza formation. The isolation, maintenance and cultivation of ectomycorrhizal fungi have been reviewed by Molina and Palmer (1982) and subsequently by Heinonen-Tanski and Holopainen (1991). Some of the procedures outlined by these authors were adopted in this research. Ectomycorrhizal synthesis methods have been reviewed by Molina and Palmer (1982) and Fortin et al. (1983) There is a significant volume of literature documenting the in vitro formation of ectomycorrhizae between basidiomycetes and coniferous hosts. Systems used employ both sterile (eg. Wong and Fortin, 1989; Zak, 1975; Nylund and Unestam, 1982) and non-sterile conditions (eg. Unestam and Stenstrom, 1989). More recently sterile, non-sterile and hydroponic systems for ectomycorrhizal synthesis were reviewed by Peterson and Chakravarty (1991). The main synthesis systems described by these authors include test tubes, petri-plates, Erlenmeyer flasks, Mason jars, Leonard jars, growth pouches, plastic pots and root-trainers. The purposes of different systems and the advantages and disadvantages of each is summarized by these authors. The main objective of in vitro mycorrhizal synthesis research is to combine potential mycobiont mycelia with potential host root systems to determine i f mycorrhizae form. The formation of mycorrhizae in vitro can be used to examine early mycobiont/host interactions (Agerer, 1991) and for describing anatomical features of ectomycorrhizae (Massicotte et al., 1987). Laboratory synthesis of ectomycorrhizae is also a useful tool for examining specific aspects of the biology and ecology of this group of forest fungi. 38 Experiments conducted under sterile conditions reduce the risk of erroneous results from potential contaminants including root colonization by other ectomycorrhizal fungi. Isolates of many ectomycorrhizal fungi grow slowly in culture and colonies often have growth rates of less than 1 cm/month on commonly employed agar media (Ohta, 1990). Contaminants, especially saprophytic, ascomycetous molds, can rapidly outcompete or overgrow slow growing ectomycorrhizal isolates in non-sterile systems. By employing substrates autoclaved in Erlenmeyer flasks, sterile conditions can be maintained for extended periods and provide a 3-dimensional medium in which the conifer seedling, root system and potential mycobiont mycelium can expand together. Hence, conical flasks have been used extensively and successfully in synthesis experiments between slow growing isolates of ectomycorrhizal fungi and conifer hosts. For example, Russula aerugina Lindblad.Fr and Sitka spruce (Picea sitchensis Bong. Carr) were shown to form ectomycorrhizae 20 weeks after inoculation with agar plugs taken from slow growing (<1 cm/month) isolates of R. aerugina (Taylor and Alexander, 1989). As well as facilitating long term maintenance of sterility, other advantages of using Erlenmeyer flasks to synthesize ectomycorrhizae include their low initial cost, varying sizes, ease of handling and relatively low space requirements. A wide variety of growth substrates can be employed in Erlenmeyer flask systems. Aside from the 'unnatural' environment created in in vitro synthesis vessels, disdvantages of the Erlenmeyer flask method include reduction of light reaching the seedling (ie. neck of flask is plugged and covered) and the potential build up of carbon dioxide, ethylene, root exudates and fungal toxins. The need to sacrifice seedlings in order to examine the roots and monitor ectomycorrhizal formation may necessitate the production of large numbers of replicate flasks. In nature ectomycorrhizae normally develop 1-3 months after the tree seed germinates (Kendrick, 1985). 'Short' or 'feeder' roots are colonized by existing extramatrical mycelium or hyphae from germinating spores or propagules present in the soil. Colonized roots usually become thickened and often branch in characteristic ways (Agerer, 1987b). 39 Many factors influence the successful establishment of ectomycorrhizae including the effects of strain or isolate (Molina, 1982; Wong et al., 1989), host specificity (Molina, 1979 and 1981; Molina and Trappe, 1982; Malajczuk et al., 1982; Harley, 1985 and 1991; Hutchinson and Piche, 1995) and host succession (tree or stand age) (Sinclair, 1974). Changes in fungal species composition from 'early' to 'late' stage colonizing ectomycorrhizae occur as forests mature (Shaw and Lankey, 1994). Early stage ectomycorrhizal fungi generally colonize seedlings and/or occupy the periphery of expanding root systems of older trees (Gibson and Deacon, 1990). Late stage fungi occur on maturing trees particularly in older parts of the root system (Fleming et al., 1984). Bowen (1994) describes changes in fungal composition dominated initially by the ectomycorrhizal species Hebeloma, Paxillus, Suillus, Thelephora and Laccaria and later by Cortinarius, Russula, Lactarius and Amanita. Read (1991) reviewed early and late stage colonizing mycorrhizae. Inoculum type (Danielson et al., 1983), amount (Mortier et al., 1989; Kropacek et al., 1989; Buschena et al., 1992) and inoculation methodology (Hung and Molina, 1986; Stenstrom, 1989) combine to influence successful ectomycorrhiza synthesis. Slankis (1974) reviewed soil conditions which might influence the formation of ectomycorrhizae. Soil physical characteristics including temperature (Parke et al. 1983), aeration (Stenstrom, 1989) and compaction (Skinner and Bowen, 1974; Chevalier and Poiteau, 1989) affect mycorrhiza development. Substrate nutritional status (Haug, 1989; Gibson and Deacon, 1990; Read, 1991) and pH (Metzler and Oberwinkler, 1989; Kamminga-Van Wijk and Prins, 1989) also influence potential rootlet colonization. It has been observed that late stage ectomycorrhizal fungi are associated with increased levels of soil organic matter (Parke and Linderman, 1983) and that they require higher sugar supplies than early stage colonizers (Shaw and Lankey, 1994). The level of available glucose has been shown to influence colonization in vitro. In petri plate studies, Hutchison and Piche (1995) examined the effects of exogenous glucose on the growth of mycelia and the colonization of potential host seedlings by early and late stage ectomycorrhizal fungi. They found that although added glucose (1 g/L) assisted successful synthesis for late stage colonizers, early stage colonizers were less dependent on exogenous glucose. These 40 authors also found that higher levels of glucose (10 g/L) increased mycelial growth and enhanced mycorrhizal formation by late stage ectomycorrhizal fungi. However, Hutchison and Piche (1995) reported that seedling growth was reduced at higher glucose levels due possibly to the production and release of fungal metabolites which were toxic to plants as a consequence of increased mycelial growth. Conditions in the rhizosphere including the production of growth promoting substances by the host (Strzelczyk and Pokojska, 1989; Gogala, 1989; Horan and Chilvers, 1990) or the mycobiont (Read, 1991; Guttenberger and Hampp, 1992) and interactions with other microorganisms including bacteria (Ohara, 1975; Garbaye and Bowen, 1989; Kropacek and Cudlin, 1989) and other saprophytic or ectomycorrhizal fungi (McAfee and Fortin, 1988) may also be critical in the formation, development and survival of ectomycorrhizae. The in vitro formation of 'mycorrhizae' between Tricholoma matsutake and potential host trees has been studied extensively in Japan. A variety of experiments have been undertaken including non-sterile field studies (eg. Tominaga, 1977a; Kawai and Ogawa 1981; Lee et al., 1984), containerized nursery experiments (eg. Okazawa, 1978; Tominaga, 1977b) and axenic combinations employing Erlenmeyer flasks and liquid inoculum (Yokoyama and Yamada, 1987). Ogawa (1975a) described the blackened, necrotic appearance of T. matsutake colonized root-tips extracted from soil samples collected in the 'shiro' as being similar in appearance to parasitized roots. Subsequently Kawai and Ogawa (1981) were able to synthesize in vitro what they reported as the early stages of T. matsutake colonization (i.e. black, "reacted" zones on fine roots) by inoculating, 2-3 year-old pine seedlings grown in sterilized soil free of "weed mycorrhizae". A build-up of tannins and subsequent darkening of the root epidermal and cortical layers were described by these and other authors (e.g. Lee, 1991) as being similar to the initial stages of a pathogen-plant interaction. Tominaga (1977b) described similar root-tips from seedlings inoculated in pots using isolates of T. matsutake grown in liquid culture. He reported that 'mycorrhizae' developed around the finely branched roots of pine seedlings and that some intercellular penetration of the cortex occurred. 4 1 Yokoyama and Yamada (1987) attempted synthesis between T. matsutake mycelium grown in liquid culture and Pinus densiflora seedlings grown aseptically in Erlenmeyer flasks in an inorganic medium consisting of vermiculite and a nutrient solution. Pine root tissue cultures were also inoculated in nutrient solution. These authors found that the mycelium grew more rapidly and formed denser colonies in the presence of P. densiflora seedling root systems. They also reported the 'mycorrhizae' found on seedlings 18 months after inoculation as appearing similar to 'parasitized' T. matsutake roots described by Ogawa (1975b). Yokoyama and Yamada (1987) also describe the anatomical appearance of intercortical cell invasion by hyphae formed after colonization of root tissue by T. matsutake. Interestingly, intercortical cell invasion by T. matsutake hyphae has also been shown by Ogawa (1975b). This invasion he describes as more similar to parasitic than mycorrhizal in that differentiation into Hartig net did not occur and rootlets became necrotic shortly thereafter. The formation of a well-defined sheath (mantle) or well-developed Hartig net, were not observed on T. matsutake colonized root-tips collected in the field or induced in nursery experiments (Ogawa, 1975b; Tominaga, 1977b; Kawai and Ogawa, 1981; Lee, 1991). Although many researchers indicate that T. matsutake has potentially parasitic capabilities, most continue to refer to it as an ectomycorrhizal fungus. The in vitro formation of ectomycorrhizae by the North American pine mushroom (J. magnivelare) has not yet been documented. Tricholoma magnivelare is purported to exist in ectomycorrhizal relationships with the roots of lodgepole pine, western hemlock and Douglas fir. However, little is known about its biology and ecology and much is inferred from its Japanese relative T. matsutake (the Japanese pine mushroom). The objective of this research was to attempt to synthesize T. magnivelare mycorrhiza and to examine the initial effects of combining its mycelia with the roots of lodgepole pine, western hemlock and Douglas fir seedlings in Erlenmeyer flasks under sterile conditions. Preliminary results are compared with those found for other ectomycorrhizal species, with special reference to research done with the Japanese pine mushroom, T. matsutake. 4 2 Methods and Materials Seedlings Coastal seedlots of Pinus contorta, Dougl., Tsuga heterophylla (Raf.) Sarg., and Pseudotsuga menziesii (Mirb.) Franco, were obtained from the Surrey Seed Orchard, Surrey, B.C. Canada. Seeds were stratified at 4°C for 1 month. Table 3 shows the seed lot numbers, germination rate and source data for each tree species. Table 3. Seed lot numbers and source data for the tree species tested: Douglas f ir (Fd), western hemlock (Hw) and lodgepole pine (PI). Seedlot Species Lab Lat Long Elev. BGC Sub Variant Germ. (m) Z zone 424 Fd 89% 48 49 123 56 610 CWH xm 2 7728 Hw 90% 48 40 124 05 600 CWH vm 1 34887 PI 95% 55 01 127 42 610 ICH g Approximately 100 seeds of each species were surface sterilized using the 30% hydrogen peroxide technique developed by Zak (unpublished) and described by Molina and Palmer (1982). Surface sterilized seeds were transferred aseptically to petri plates containing water agar (15 g/L). These were incubated for 10 days at 20°C in the dark. Plates were examined daily and contaminated seeds culled by aseptically transferring uncontaminated seeds to new agar plates and discarding the remaining contaminated ones. Sterilized seeds were incubated at 20°C for a further 10 days or until germination occurred, resulting in approximately 75 aseptic germinants of each species. 43 Synthesis Vessels Prior to selecting Erlenmeyer flasks for the purposes of this experiment other systems including petri plates (Zak, 1975: Wong and Fortin, 1988), growth pouches (Peterson and Chakravarty, 1991), Mason jars and sealed pots (Xiao, unpublished) were also tested as potential synthesis vessels. The sealed pot consists of a one-quart jar with the bottom removed attached to a 4-inch clay pot with silicone sealant (Figure 5). Petri plates (Tackaberry, pers. comm., 1994) and growth pouches became contaminated during seedling growth and colonization by inoculum. A green alga, bacteria and some molds contaminated the growth pouches within 2 weeks of inoculation (the growth pouch is a non-sterile system). Sealed pots were found to take up substantial space in the incubator. Another problem with this system was that drying of substrate occurred rapidly due to evaporation through the clay pot. It was found that conical flasks were best suited for long term maintenance of sterility and all synthesis attempts discussed hereafter were undertaken in 250 ml Erlenmeyer flasks. Figure 6 shows the synthesis system used. Seventy-five, 250 ml Erlenmeyer flasks containing 100 ml of medium (peat moss:vermiculite:sand in a 1:1:1 ratio) were prepared. Approximately 50 ml of M M N solution (minus the glucose and malt extract) were added to each flask. Flasks were plugged with foam, covered with an aluminum foi l l id, autoclaved at 20 psi for 45 minutes and allowed to cool in a laminar flow hood. Twenty-five germinants of each tree species (one per flask) were transferred aseptically into the center of the flasks and carefully manipulated beneath the substrate surface. The flasks were incubated in a growth chamber at 20°C, and illuminated 16 hours per day at 230 umol m'^s" 1 for 8-10 months to allow seedlings to establish root systems. During this time any visibly contaminated seedlings or flasks were discarded. At approximately 4 week intervals flasks were aseptically aerated (i.e. transferred to a laminar flow hood and uncovered for 30 minutes to permit aeration in a sterile airflow). During aeration approximately 10 ml of sterilized, distilled water was added to those flasks in which the substrate surface appeared dry. During seedling development isolates of T. magnivelare to be used as inoculum were expanded on agar media. 44 Fig. 5. Sealed jar synthesis system tested. 45 . Erlenmeyer flask synthesis system 46 Fungal Inoculum Five isolates of T. magnivelare (2-1-1, M, N, P and Q) were obtained using tissue cultures isolated from pine mushroom sporocarps. Isolate 2-1-1 was obtained in 1993 in the Boston Bar area (Berch, pers. comm.). The other isolates were obtained from fresh, young fruitbodies gathered in the Nass Valley, British Columbia in autumn, 1994. Subcultures from these were maintained for 90 days in darkness at 20°C on 20% potato dextrose agar (PDA) prior to inoculations. Figure 7 shows isolate M of T. magnivelare growing on 20% PDA. Isolates were confirmed as T. magnivelare by Dr. Keith Egger at the University of Northern British Columbia using DNA (PCR RFLP) analysis by comparing tissue from sporocarps with tissue from growing colonies. An isolate of Laccaria laccata was maintained under similar conditions on PDA for use as a positive control. Inoculations Each synthesis vessel was inoculated in a laminar flow hood by cutting three approximately 5 mm x 5 mm agar sections from the edges of actively growing colonies and transferring them into the synthesis vessels adjacent to the seedling. The agar pieces were then gently manipulated beneath the substrate surface using a long, sterilized probe. Three to nine replicate flasks of each tree species were inoculated in this manner with each of the five isolates of T. magnivelare. The positive control, Laccaria laccata, was inoculated in a similar manner onto three flasks containing lodgepole pine. For each tree species five flasks were left uninoculated as controls. Ten ml of a 2 g/L solution of dextrose were added to three of the replicate flasks containing the M-isolate of T. magnivelare and one uninoculated control flask for each tree species approximately 5 months post-inoculation to determine i f an increased available sugar concentration might enhance mycelial growth and subsequently induce ectomycorrhiza formation. One replicate of each tree species was examined approximately 2 weeks, 6 weeks and 8 weeks post-glucose addition. A l l flasks were incubated at 20°C and illuminated 16 hours per day at 230 umole m" 2 s" 1 . Flasks were aseptically aerated at approximately 4 week intervals. Approximately 10 ml of sterile water was added to flasks with substrate surfaces that appeared dry after aeration. Inoculated seedlings of 47 Fig. 7. T. magnivelare mycelium employed as inoculum. each tree species and a control were extracted and examined at 1-2 month intervals for 8 months post inoculation. Any contaminated seedlings or flasks were discarded. Laccaria laccata replicates were extracted and examined 3 months post-inoculation. Seedling Examination Twenty-five lodgepole pine, 25 Douglas f ir and 22 Western hemlock seedlings were extracted and examined. Seedling shoots were classified as either surviving (+) or dead (-). Root systems were classified as ++, + or - for well, moderately and poorly developed respectively. Well developed root systems (++) were those with healthy meristems and numerous laterals and fine roots available for potential mycorrhization. Moderate root systems (+) had fewer laterals and fine roots however still had ample rootlets with healthy meristems for potential colonization. Poorly developed root systems (-) had few or no laterals or fine roots available and sometimes showed signs of necrosis (darkening of root-tips, sloughing of cortex). The presence or absence of mycelium in synthesis vessels was initially determined by visually examining and smelling the substrate adhering to the roots and the remaining medium in the synthesis vessels. Cultures of each isolate of T. magnivelare were also re-isolated onto agar plates containing PDA using small amounts of substrate aseptically transferred from flasks containing inoculated pine seedlings (Figure 8). Re-isolated colonies grew into uncontaminated monocultures that were similar in appearance, smell, and microscopic characteristics (i.e. hyaline, undamped hyphae) to the original T. magnivelare colonies isolated directly from sporocarps and used as inoculum. Substrate from uninoculated controls was also plated out to ensure axenic conditions were being maintained in the synthesis systems. Root systems were placed in water-filled petri plates and root-tips examined using a dissecting microscope (10-40X) for indications of mycorrhiza formation. Representative root systems were cleared or cleared and stained with trypan blue (Appendix 1), hand sectioned or squashed whole on slides and examined microscopically (100-1000X) for signs of colonization (mantle and/or Hartig-net development). Semi-permanent slides of representative root-tips were prepared and magnified root-tips were photographed. 49 Results Tables 4, 5 and 6 summarize the results of each synthesis flask for lodgepole pine, western hemlock and Douglas f ir respectively. In each table the seedling number, planting date, inoculation date, fungal isolate used, glucose additions, date examined, seedling shoot and root condition and the presence or absence of mycelium and ectomycorrhizae are shown. Al l seedlings survived until harvested for examination except for two western hemlock (#17 and #22) and a single Douglas f i r (#9). Seedlings of all 3 species performed moderately to poorly in the synthesis vessels. Lodgepole pine and Douglas f ir seedlings were generally larger and less chlorotic than western hemlock seedlings. Regardless of species most root systems were found to be long and coiled around the base of the flasks (Figure 9). Most had at least some lateral and fine roots available for potential fungal colonization. Figure 10 shows an uninoculated control lodgepole pine rootlet from seedling #25 magnified 100X. A l l uninoculated control seedlings remained uncontaminated and uncolonized indicating the system was capable of maintaining long term axenic conditions. No visible mycelium or smell of fungal tissue was present in any of the uninoculated control flasks. Attempts to isolate bacterial or fungal colonies from small amounts of substrate taken from uninoculated control flasks and placed on nutrient agar were negative. Al l lodgepole pine root systems were either moderately or well developed with ample laterals and fine roots present for potential ectomycorrhizal synthesis (Figure 11 and Table 4). The three lodgepole pine seedlings inoculated with the positive control (Laccaria laccata) formed abundant ectomycorrhizae, i.e. swollen, bifurcate root-tips with no root hairs and a distinct mantle or sheath. Figure 12 shows an L. laccata colonized root-tip from lodgepole pine seedling #18 magnified 100X. Microscopic examination of sections of the rootlet magnified 400X show the presence of a mantle (Figure 13) and 1000X to show the Hartig net (Figure 14). During extraction visible mycelium and a distinct smell of pine mushroom mycelium was present on all but two (#7 and #15) of the T. magnivelare inoculated pine seedlings (Table 4). Fungal colonies re-isolated from small amounts of substrate transferred aseptically from flasks onto nutrient agar were similar in macroscopic appearance to original isolates used as inoculum (Figure 8) and 50 Table 4. Summary of Pinus contorta seedling inoculat ion data using f ive isolates of Tricholoma magnivelare ( 2 - 1 - 1 , M , N, P, Q), one isolate of Laccaria laccata (L. l . ) and uninoculated controls. (VM=v is ib le myce l ium; ECM=signs of ectomycorrh iza fo rmat ion ) . Tree # Plant ing Date Inoc Date Isolate Exam Date Shoot Roots V M E C M 1 11 /07 /94 04 /04 /95 2 - 1 - 1 05 /08 /95 + ++ + -2 11 /07 /94 04 /04 /95 2 - 1 - 1 12/10/95 + ++ + -3 11 /07 /94 04 /04 /95 2 - 1 - 1 07 /12 /95 + + + -4 11 /07 /94 04 /04 /95 M 05 /08 /95 + + + . 2 5 27 /07 /94 04 /04 /95 M 29/08 /95 + + + -6 27 /07 /94 04 /04 /95 M 1 15/09/95 + ++ + -7 27 /07 /94 04 /04 /95 M 1 12/10/95 + ++ - -8 27 /07 /94 04 /04 /95 M 1 25/10 /95 + ++ + .2 9 06 /07 /94 04 /04 /95 N 12/07/95 + + + -10 06 /07 /94 04 /04 /95 N 15/09/95 + ++ + -11 06 /07 /94 04 /04 /95 N 07 /12 /95 + ++ + -12 16 /08 /94 28 /03 /95 P 26 /05 /95 + ++ + -13 06 /07 /94 04 /04 /95 P 15/09/95 + ++ + -14 06 /07 /94 04 /04 /95 P 12/10/95 + + + -15 27 /07 /94 28 /03 /95 Q 05 /08 /95 + ++ - -16 27 /07 /94 28 /03 /95 Q 29 /08 /95 + + + -17 27 /07 /94 28 /03 /95 Q 07 /12 /95 + ++ + -18 11 /07 /94 31 /08 /95 L. l . 07 /12 /95 + ++ + + 19 11 /07 /94 31 /08 /95 L. l . 07 /12 /95 + ++ + + 20 11 /07 /94 31 /08 /95 L. l . 07 /12 /95 + ++ + + 21 06 /07 /94 Contro l 26 /05 /95 + ++ - -22 06 /07 /94 Contro l 05 /08 /95 + + - -23 06 /07 /94 Cont ro l 29 /08 /95 + + - -24 06 /07 /94 Contro l 25 /10 /95 + ++ - -25 16/08/94 Contro l 07 /12 /95 + ++ - -1 = glucose added 31/08 /95 2 = s l ight ly swol len, darkened roo t - t ips (Figure 15) 51 Table 5. Summary of Tsuga heterophylla seedling inoculat ion data using f ive isolates of Tricholoma magnivelare ( 2 - 1 - 1 , M , N, P, Q) and uninoculated controls. (VM=v is ib le myce l ium; ECM=signs of ectomycorrh iza fo rmat ion) . Tree # Plant ing Date Inoc Date Isolate Exam Date Shoot Roots V M E C M 1 11/07/94 28 /03 /95 2 - 1 - 1 26 /05 /95 + _2 + -2 11/07/94 28 /03 /95 2 - 1 - 1 05 /08 /95 + ' + + -3 11/07/94 28 /03 /95 2 - 1 - 1 12/10/95 + + + .3 4 11/07/94 28 /03 /95 M 29 /08 /95 + ++ + -5 11/07/94 28 /03 /95 M 07 /12 /95 + + -6 11 /07 /94 28 /03 /95 M 1 15/09/95 + ++ + -7 11/07/94 28 /03 /95 M 1 12/10/95 + + + -8 11/07/94 28 /03 /95 M 1 24/10 /95 + ++ + -9 27 /07 /94 04 /04 /95 N 12/07/95 + + - -10 27 /07 /94 04 /04 /95 N 15/09/95 + + + -11 27 /07 /94 04 /04 /95 N 07 /12 /95 + .2 + -12 27 /07 /94 04 /04 /95 P 12/07/95 + ++ + -13 27 /07 /94 04 /04 /95 P 15/09/95 + ++ + -14 27 /07 /94 04 /04 /95 P 12/10/95 + ++ + -15 27 /07 /94 28 /03 /95 Q 05 /08 /95 + + + -16 27 /07 /94 28 /03 /95 Q 07 /12 /95 + + + -17 27 /07 /94 28 /03 /95 Q 07 /12 /95 - - -18 27 /07 /94 Contro l 05 /08 /95 + ++ - -19 27 /07 /94 Contro l 29 /08 /95 + + - -20 27 /07 /94 Contro l 24 /10 /95 + .2 - -21 27 /07 /94 Cont ro l 07 /12 /95 + ++ - -22 27 /07 /94 Contro l 26 /05 /95 - 2 - -= glucose added 31/08 /95 = necrot ic tissues present = repetat ive r ing pattern on root (Figure 17) 52 Table 6. Summary of Pseudotsuga menziesii seedling inoculat ion data using f ive isolates of Tricholoma magnivelare ( 2 - 1 - 1 , M , N, P, Q) and uninoculated controls. (VM=v is ib le myce l ium; ECM=signs of ectomycorrh iza fo rmat ion) . Tree # Plant ing Date Inoc Date Isolate Examin Date Shoot Roots V M E C M 1 27 /07 /94 28 /03 /95 2 - 1 - 1 05 /08 /95 + ++ + -2 27 /07 /94 28 /03 /95 2 - 1 - 1 29 /08 /95 + + - -3 27 /07 /94 28 /03 /95 2 - 1 - 1 12/10/95 + ++ + -4 16/08/94 28 /03 /95 M 26 /05 /95 + .2 + -5 06 /07 /94 04 /04 /95 M 29 /08 /95 + + + -6 06 /07 /94 04 /04 /95 M 1 15/09/95 + + + -7 06 /07 /94 04 /04 /95 M 1 12/10/95 + .2 + -8 06 /07 /94 04 /04 /95 M 1 24/10 /95 + + + -9 16 /08 /94 28 /03 /95 N 12/07/95 - .2 + -10 06 /07 /94 04 /04 /95 N 15/09/95 + + + -11 06 /07 /94 04 /04 /95 N 07 /12 /95 + + + -12 27 /07 /94 04 /04 /95 P 12/07/95 + ++ + -13 27 /07 /94 04 /04 /95 P 15/09/95 + ++ + -14 27 /07 /94 04 /04 /95 P 12/10/95 + + + -15 06 /07 /94 28 /03 /95 Q 05 /09 /95 + + + -16 06 /07 /94 28 /03 /95 Q 07 /12 /95 + + + -17 06 /07 /94 Cont ro l 26 /05 /95 + .2 - -18 06 /07 /94 Contro l 05 /08 /95 + + - -19 06 /07 /94 Contro l 29 /08 /95 + 2 - -20 06 /07 /94 Contro l 24 /10 /95 + ++ - -21 06 /07 /94 Contro l 07 /12 /95 + ++ - -22 06 /07 /94 04 /04 /95 M 07 /12 /95 + ++ + -23 11 /07 /94 28 /03 /95 M 07 /12 /95 + + + -24 11 /07 /94 28 /03 /95 M 07 /42 /95 + ++ + -25 11 /07 /94 28 /03 /95 M 07 /12 /95 + ++ + -= glucose added 31 /08 /95 = necrot ic tissues present 53 55 Fig. 13. Mantle L. laccata inoculated rootlet (#18) (400X magnification). consisted of hyaline, clampless hyphae as did the original T. magnivelare colonies isolated from sporocarps. No definitive ectomycorrhizal structures were observed on any of the T. magnivelare inoculated seedlings regardless of species. However, two of the lodgepole pine seedlings (#8 with added glucose and #4 without) both inoculated with T. magnivelare (M isolate) showed notable morphological changes on some root apices (Figure 15). Lodgepole pine seedling #4 had many slightly swollen, darkened root-tips which narrowed toward the proximal end of the rootlet. These were similar in appearance to the initial stages of T. matsutake inoculated pine roots described by Kawai and Ogawa (1981) and others (e.g. Lee, 1991) who observed a build-up of tannins and subsequent darkening of the epidermal and cortical cells. For lodgepole pine #4 the root epidermal and cortical cells were however intact (i.e. not sloughed off or separated from the central stele). Some portions of the root system had monopodial (unbranched) root-tips with numerous root hairs. Other portions had bifurcate and slightly swollen, darkened root-tips without root hairs. Microscopic examination of unstained pine #4 rootlets showed no evidence of a mantle or Hartig net present on darkened root-tips. Figure 15 shows a root-tip from lodgepole pine seedling #8 (100X magnification). The root system from lodgepole pine seedling #8 (Figure 16) was cleared and stained with trypan blue. Again most rootlets had darkened swollen root-tips with darkened epidermal and cortical cells. Epidermal and cortical tissues were intact (unsloughed). As with rootlets from lodgepole pine seedling #4, neither a mantle nor Hartig net was observed on root-tip sections prepared from lodgepole pine seedling #8. Table 5 shows that one inoculated western hemlock seedling (#17 + isolate Q) and one uninoculated (control) western hemlock seedling (#22) were dead prior to extraction. These and three other seedlings (#1 + 2-1-1 isolate; #11 + N isolate; and #20 control) had root systems with very few or no healthy rootlets for potential colonization. Necrosis and significant sloughing of blackened cortical tissue occurred on these root systems. Necrosis was most pronounced near the 57 Fig. 15. T. magnivelare inoculated (M-isolate) lodgepole pine #8 rootlet with swollen apex (100X root collar. Most western hemlock root systems were considered either moderately or 'well' developed (Table 5). The healthy western hemlock root systems showed typical red pigmentation and live meristematic and cortical tissue. Tricholoma magnivelare mycelium was detected visibly and by smell in all inoculated western hemlock synthesis flasks except seedling #9. No signs of ectomycorrhizae were seen on any of the western hemlock root systems, however, one seedling (#3 + isolate 2-1-1) had root-tips with a repetitive, brown pigmented ring pattern and no root hairs (Figure 17). No signs of a mantle were seen on this root-tip. Other inoculated western hemlock seedlings had root systems with ample root-tips available for potential colonization. One Douglas fir seedling (#9) was dead when extracted from synthesis vessels. This and four other Douglas f ir seedlings (2 controls; #4 + isolate M; and #7 + isolate M with added glucose) had necrotic root systems with few available laterals or fine feeder roots for potential colonization. The root system of Douglas f ir seedling #4 inoculated with m isolate had mostly necrotic roots. The main root had died and divided into 2 laterals which in turn had blackened necrotic cortical and meristematic tissue. Of the remaining Douglas f ir seedlings nine had 'well' developed and eleven had moderately developed root systems (Table 6). Tricholoma magnivelare mycelium was visible and a pine mushroom smell was detected in all but one (#2) of the inoculated Douglas f ir flasks during extraction. No signs of ectomycorrhizae were observed on any of the Douglas f ir seedlings. Figure 18 shows an uninfected root-tip with abundant root hairs taken from Douglas fir seedling #11 inoculated with T. magnivelare isolate N. The root hairs are somewhat obscured in the photograph by substrate adhering to the surface of the rootlet and the epidermis is detaching due to being squashed by the cover slip. 59 Fig. 17. Hemlock rootlet with repetitive ring pattern (100X magnification from seedling #3 inoculated with isolate 2-1-1. Fig. 18. Douglas fir root-tip from seedling #11 inoculated with isolate N (100X magnification). Note substrate attached and root hairs in background. 60 Discussion Synthesis System and Controls The synthesis system employed worked well for maintaining long-term sterile conditions. The uninoculated controls remained uncontaminated throughout the study and monoxenic conditions were maintained in the majority of the inoculated flasks. Since its inception by Melin in 1922 the Erlenmeyer flask system has been extensively used for in vitro ectomycorrhizal synthesis and has indeed worked successfully in this study for the formation of ectomycorrhizae between the positive control fungus Laccaria laccata and lodgepole pine seedlings. Laccaria laccata is however, an early stage mycorrhizal colonizer forming ectomycorrhizae with a wide range of conifer hosts (Mortier et al., 1989) and, according to Singer (1975), has potentially facultative saprophytic abilities. This fast growing species seems to be able to form abundant mycorrhizae and form them rapidly under a wide range of conditions. The mycelium of L. laccata grows much faster than that of T. magnivelare. In retrospect it would have been more representative had a more closely related Tricholoma species been used as a positive control, preferably one that had previously been shown to form mycorrhizae using the Erlenmeyer flask system. For example, Brunner et al., (1992) successfully synthesized mycorrhizae between Tricholoma vaccinum (Pers.:Fr) and Picea abies (L.) Karst. Tricholoma vaccinum is known to form mycorrhizae with Pinus species (Trappe, 1962) but it is uncertain whether it does so with lodgepole pine, western hemlock or Douglas fir. In order to simplify interpretations, enchance comparisons and allow more conclusions to be drawn, future studies should incorporate other Tricholoma species as positive controls to test the synthesis systems. Seedlings and Root Systems The synthesis system employed allowed the growth and survival of seedlings for more than a year. However, under the restrictive conditions imposed by the Erlenmeyer flasks, seedlings of all three species performed only moderately to poorly relative to field or nursery grown seedlings. In general, lodgepole pine performed better than Douglas fir or western hemlock producing larger shoots and root systems. The poor performance of Douglas fir was not suprising as it generally grows on well 61 drained sites and its roots are sensitive to low oxygen concentrations. Western hemlock can be difficult to grow aseptically as it may need early mycorrhizal colonization in order to survive and grow normally (Xiao pers. comm., 1995). The root systems of most of the seedlings certainly appeared odd having coiled around the bottom of the flasks, sometimes two or three times. 'Well', 'moderately' and 'poorly' developed root systems are subjective terms and in this case apply only to the relative status of roots extracted from the synthesis systems. A l l lodgepole pine control seedling root systems were in relatively good condition compared to Douglas f i r and western hemlock. Each of the latter two species had uninoculated control seedlings with poorly developed and/or necrotic roots indicating the system restricted healthy root system development. From a nursery or field perspective all root systems would probably have been considered reduced. It is likely that increased carbon dioxide concentrations and a build-up of root exudates, ethylene, and possibly fungal toxins from the growing mycelia in inoculated flasks, created a less favourable environment for root development. When autoclaved, peat moss has been shown to release organic acids (phenolics) and other compounds potentially deleterious to developing roots (Petersen and Chakravarty, 1991). This may also have contributed to necrosis of Douglas fir and western hemlock root systems in the flasks. Larger synthesis systems using non-peat moss based media should be explored in future to alleviate unfavourable growing conditions. Mycelial growth The flask synthesis system did support the growth, albeit slowly, of T. magnivelare mycelium. Hyphal strands eminated from the original inoculum plugs and ramified the adjacent substrate. The mycelium did not however fully colonize the growing medium even in flasks to which glucose was added. Upon careful examination only short hyphal strands could be seen emanating into the medium from the original agar plugs of inoculum. Although not nearly as dense as the colonies found on agar media, visible mycelia were observed in the growing medium adjacent to the roots of most of the inoculated seedlings during extraction, at times binding the peat moss, vermiculite, sand mixture into small clumps. When root systems were extracted from inoculated flasks a distinct smell 62 of mushroom, similar to that of the pine mushroom, was present in the growing medium which had adhered to the roots. The pine mushroom odour also lingered in the substrate remaining in the flasks. Evidence that the system supported colonies of pine mushroom mycelium comes mainly from the re-isolation of T. magnivelare mycelium from flasks prior to extracting seedlings root systems. Tricholoma magnivelare mycelia were re-isolated on PDA and grew into colonies similar to the original isolates used as inoculum (i.e. containing dense, hyaline, clampless hyphae). The re-isolated mycelium also smelled like the pine mushroom. In future DNA analysis (RFLP) should be done on re-isolated colonies to confirm their identities. Isolates and Inoculum The origin of the isolate or strain of a fungus can affect the success of synthesis attempts (Wong et al., 1989; Molina, 1981). In the present experiment only five isolates were used, four from the Nass valley and one from Boston Bar. Isolates from throughout the range of T. magnivelare should be tested. At present it is uncertain whether some isolates would perform better than others. A wider range of potential tree hosts should also be tested. The type of inoculum (solid, liquid, slurry, pelletized, etc.) and method of inoculation used in synthesis experiments can also affect the success rate (Visser and Parkinson, 1983; Stenstrom, 1989; Mortier et al., 1989; Peterson and Chakravarty, 1991). Tricholoma magnivelare mycelium grows slowly (less than 1 cm/month on standard media) and the distance that mycelial strands of a fungus wi l l grow through the substrate can markedly impact infection dynamics (Bowen, 1994). In the present study pieces of T. magnivelare colonized agar were carefully placed in close proximity to the root systems of the potential host seedlings after seedlings were well established in the flasks under aseptic conditions. This method allows for limited disruption of the mycobiont colony and host root system. It also provides the fungus with a small amount of substrate containing residual glucose to survive the lag phase and from which to grow into the contaminant-free medium. This conservative, non-disruptive approach was chosen because of the slow growth rate of the 63 T. magnivelare mycelium and lack of available information regarding its ability to recover from disturbance, compete with other organisms or grow in the absence of potential host roots. Researchers, using other ectomycorrhizal fungi, have experienced greater success and higher levels of ectomycorrhizal establishment by increasing the level of inoculum used (Hung and Molina, 1986; Buschena et al., 1992) and by thorough mixing of inoculum with the rooting substrate (Kropacek et al., 1989; Stenstrom, 1989). Tricholoma magnivelare mycelium grows extremely slowly in culture and because a limited amount of inoculum of each of the isolates of pine mushroom mycelium was available at the time of inoculation, only a limited number of replicates could be attempted. In future, a larger biomass of mycelium should be grown and varying inoculation techniques attempted such as preparing mycelial slurries and either adding these to synthesis systems or dipping sterile root systems into them prior to transferring seedlings to synthesis vessels. Many other inoculation methods should also be attempted such as placing surface sterilized tree seeds directly on top of T. magnivelare colonies growing in petri plates or the addition to synthesis systems of basidiospores isolated from sporocarps. Kawai and Ogawa (1981) reported that the most important factor for successful inoculation of the Japanese pine mushroom (T. matsutake) is the type of inoculum used. According to these and other authors (e.g. Lee et al., 1984) planting aseptically grown seedlings free of "weed mycorrhizae" directly into the expanding shiro of T. matsutake is the best method (i.e. plant aseptic seedlings directly into colonies of T. matsutake in the field). Planting seedlings free of ectomycorrhizae in pots of soil taken from the shiro is the second best method, but much less effective than field placement. Field methods should also be attempted with T. magnivelare. However, these techniques are not sterile and therefore it may not be possible to avoid the infection of planted root systems by soil microorganisms or colonization by competing ectomycorrhizal fungi. As mentioned in the introduction, early stage ectomycorrhizal fungi generally colonize seedlings and/or occupy the periphery of expanding root systems of older trees (Gibson and Deacon, 1990). Late stage fungi occur on maturing trees particularily in older parts of the root zone (Fleming et al., 1984). Although both early and late stage fungi can infect seedlings in aseptic culture, early stage 64 fungi tend to form ectomycorrhizae more rapidly (Fleming, 1985). Tricholoma magnivelare is probably a late stage colonizer i f it is in fact ectomycorrhizal. However, little is known of the biology and ecological requirements of T. magnivelare. Anecdotal information indicates that in British Columbia T. magnivelare fruit bodies have not been encountered in stands less than 40-50 years old. Pine mushrooms have however been found in second growth stands greater than 40 years of age, indicating that it takes several decades before the underground mycelium (shiro) develops to a stage mature enough to produce sporocarps. Being a late stage colonizer may l imit the ability of T. magnivelare to form ectomycorrhizae with very young seedlings. The slow growth of late stage ectomycorrhizal fungi at least on young roots (Mason et al., 1983) may explain their failure to dominate the rhizosphere of young roots. Their success in later stages may be that they are able to use hormones and other unique polypeptides (Gutenberger and Hampp, 1992) or substances from older root exudates such as auxins, jasmonic acid (Gogala, 1989) or compounds released by organisms associated with litter decomposition (Bowen, 1994; Ho ran and Chilvers, 1990; Garbaye and Bowen, 1989; McFee and Fortin, 1988; and others). Ohara (1975) has indeed found humus derived bacteria, including cellulose and chitin-decomposing microorganisms associated with T. matsutake colonies that differed from microorganisms found outside of the colonies. It is plausible that specific substances or soil microorganisms isolated from T. magnivelare colonies in the field could enhance the success and level of root colonization under controlled conditions. Many other physical and chemical factors have been found to influence successful synthesis of mycorrhizae which may prove important in enhancing T. magnivelare inoculations. These include aeration (Stenstrom, 1989), temperature (Parke et al., 1983) soil physical properties (Skinner and Bowen, 1974; Kropp, 1982) soil pH (Metzler and Oberwinkler, 1989; Kammidga-van Wijk and Prins, 1989) nitrogen and phosphorus concentrations (Ogawa, 1974; Haug, 1989; Gibson and Deacon, 1990), B vitamins (Strelczyk and Pokojska, 1989) and the level of available glucose (Hutchison and Piche, 1995). 65 Hutchison and Piche (1995) examined the effects of glucose additions on the success of in vitro ectomycorrhizae formation by early and late stage colonizers. They reported that early stage ectomycorrhizal fungi appeared to be less dependent than late stage colonizers on exogenous carbohydrate supply for successful formation of ectomycorrhiza. They proposed that additional glucose enhanced mycelial growth and allowed a carbon-balance between the host and late stage mycobiont more conducive to ectomycorrhizae formation. However, the concentration of added glucose was important and high levels (10 g/L) were found to have had detrimental effects on the growth of seedlings, possibly due to toxic fungal metabolites released as a consequence of increased mycelial growth. In the present study, 10 ml of glucose (2 g/L) was added to three T. magnivelare (M-strain) inoculated flasks of each of the tree species approximately 5 months post-inoculation in an attempt to enhance mycorrhizal synthesis. Only seedlings inoculated with the M-strain were tested because the highest amount of inoculum and greatest number of replicates of this strain were available. Although one of the M-isolate inoculated lodgepole pine root systems which exhibited anomylous root-tips contained added glucose (seedling #4) the other one did not (seedling #8). It remains uncertain at present what effect, i f any, the additional glucose had. In an expanded study a greater number of replicates should be tested and different concentrations of glucose added at varying times throughout the study. Anomalous Root-tips Lodgepole pine root systems inoculated with T. magnivelare appeared similar to uninoculated control root systems except for seedlings #4 and #8. This was also true for western hemlock, except for seedling #3 with the repetitive ring pattern. It is possible that the affected roots were a function of the influence of the T. magnivelare mycelium. Japanese researchers have successfully synthesized ectomycorrhizae between T. matsutake and Pinus densiflora (Sieb. et Zucc.) both in vitro (Yokoyama and Yamada, 1987) and in vivo (Tominaga, 1977b; Kawai and Ogawa, 1981; Lee et al., 1984). The root-tip morphotypes found in association with T. matsutake sporocarps have been described by many researchers including Ogawa 66 (1975b), Kawai and Ogawa (1981) and Lee (1991). In summary these authors described charcoal-gray, or brownish to black, elongated, slightly swollen root apices with little or no mantle or Hartig net development (Lee, 1991). Similar types of root-tips have been found beneath T. magnivelare sporocarps in the present study (Chapter 4). These were referred to as 'matchsticks' because of their swollen, dark, elongated root apices, lack of root hairs, and slender necks. As with T. matsutake, only limited evidence of mantles or Hartig nets were observed on T. magnivelare 'matchstick' rootlets examined from field samples. It has been proposed that T. matsutake forms an ectomycorrhiza with some species of Pinus, Picea and Tsuga with parasitic characteristics displayed on the fine roots (Ogawa, 1974). Tricholoma matsutake may have potentially parasitic capabilities. Yokoyama and Yamada (1987) in reporting the synthesis of 'mycorrhizae' between P. densiflora root tissue cultures and T. matsutake described intercortical invasion of pine root tissues by mycelia as being similar to parasitized roots. According to Ogawa (1975b) it is probable that this fungus has a "parasitic character against the seedling or sapling with poor roots". In fact the author (Ogawa) states that he has frequently observed the wilt ing of young pine trees and seedlings in the shiro of T. matsutake. On Tsuga (hemlock) species Ogawa (1977b and 1977d) also reports 'mycorrhizae' with parasitic characteristics, lacking a fungal sheath and Hartig net, similar to those formed on infected Pinus species. It is possible that T. matsutake and indeed T. magnivelare behave parasitically with young or suppressed seedlings. Conversely, it may begin as an ectomycorrhizal fungus but later become parasitic causing root necrosis over time, eventually resulting in the sloughing of epidermal and cortical layers. This potentially parasitic nature may have contributed at least in part to the root system necrosis observed in some of the inoculated flasks. It is possible that the roots of the young seedlings respond to the presence of T. magnivelare as they might a parasitic fungus. This may explain some of the necrosis found particularly on the Douglas f ir and western hemlock inoculated root systems. Dead and dying root systems can at least partially be attributed to the unfavourable conditions imposed by the Erlenmeyer flask system for these species because some of the uninoculated controls also had necrotic root systems. However, all lodgepole pine controls were in 67 relatively good condition with healthy root systems and abundant root hairs. The root-tips observed on lodgepole pine seedlings #4 and #8 did appear to be similar to the early stages of rootlets infected with T. matsutake described by Ogawa (1975b) and others thereafter. For example, Kawai and Ogawa (1981) also reported the results of preliminary in vitro synthesis attempts between isolates of T. matsutake and aseptic P. densiflora seedlings. They too placed mycelium grown on solid media in close proximity to potential host root systems for 8-12 months. These authors described the resultant root infections on 2-3 year old pine seedlings as follows: "In most cases (the) two partners reacted to each other, but the reaction stopped at the incipient stage, leaving black reacted zones on a limited range of fine roots... indicating the marks of a transient mycorrhization." Lee (1991) also reports that T. matsutake colonized root-tips appeared 'slightly parasitized' (i.e. in various stages of decomposition) rather than mycorrhizal. Ogawa (1975b) and Tominaga (1977b) earlier described the necrotic appearance of T. matsutake colonized rootlets similar to parasitically infected roots. Following the establishment of fungal infection of roots by T. matsutake, Ogawa (1975b) describes the initial appearance of brown pigments, tannins or resins in epidermal cells and the cortical cells which eventually darken, turning brownish-black. Lee (1991) also describes the latter stages of T. matsutake infection as the rootlets begin to wither and the root-tips blacken due to what he too believed to be a build-up of tannins in the outer layer of the rootlets. Similar root apices have been described for the related species, T. bakamatsutake, by Terashima (1993). The root-tips found beneath T. magnivelare mushrooms appear similar to those infected with T. matsutake, at least macroscopically (Chapter 4). Whether or not T. magnivelare has parasitic capabilities is as yet uncertain, however, the enzymatic capability tests (Chapter 2) indicate that in culture its mycelium is unable to digest cellulose, l ignin or pectin appreciably as is the case with many ectomycorrhizal fungi (Hutchison, 1990a). Ectomycorrhizal fungi are known to influence seedlings before or in the absence of mycorrhiza formation and altered root systems have been observed prior to mycorrhiza formation (Slankis, 1974; Brown and Sinclair, 1981). This phenomenon may help to explain the anomalous 68 T. magnivelare inoculated lodgepole pine root-tips. Foster and Marks (1966 and 1967) describe tannin build-up in the 'tannin-zone' or 'tannin-layer' of the root cortex of pre-mycorrhizal rootlets of Pinus radiata D. Don. These authors propose that the tannins are a result of polyphenolics (toxic to fungi) produced by the host in order to control mycorrhizal associations thus allowing selective action on soil fungi entering the rhizosphere. It is possible that the mere presence of T. magnivelare mycelium in close proximity affected lodgepole pine root system development. According to Al Abras et al. (1988) the production of phenolic compounds in the outer root cortex can be induced prior to mycorrhiza formation. By examining pre-mycorrhizal root systems monthly, these authors were able to show the deposition of tannins in host root cells prior to direct contact with the mycobiont, presumably, in reaction to the mere presence of the fungus (or its exudates) in the rhizosphere. According to Molina and Trappe (1982), disruption of the cortex by an invading ectomycorrhizal fungus, as indicated by intense safranin staining of cortical cells, was the result of incompatibility between host and mycobiont. This host reaction suggests a type of phenolic defense as displayed during plant-pathogen interactions. Is it possible that having T. magnivelare mycelium in close proximity to the roots initiated a plant-pathogen response? Peterson and Farquhar (1994) proposed that in some instances ectomycorrhizal fungal species appear to become pathogenic, inducing resistance reaction in root cells and deterioration of the root system. According to this author the molecular basis for the switch from mutualistic symbiosis to pathogenesis remains unclear. This switch from an ectomycorrhizal to parasitic mode may explain the degenerated root-tips reported in association with the Japanese pine mushroom (T. matsutake) and a related species, T. bakamatsutake (Terashima, 1993) and may well explain the field collected root-tips found in association with the North American pine mushroom (7". magnivelare) (Chapter 4). It does seen plausible that the darkened, swollen root-tips formed on the lodgepole pine root systems inoculated with the M-isolate were initial responses to the presence of T. magnivelare mycelium. 69 However, because of the preliminary nature of the results many questions remain unanswered. The main question is why were the root-tips found on M-isolate inoculated lodgepole pine seedlings #4 and #8 swollen and pigmented but not the other inoculated lodgepole pines? Is this early necrosis or the deposition of tannins in the epidermal and cortical tissues in reaction to the presence of the fungus? Interestingly, Yokoyama and Yamada (1987) also showed that. T. matsutake formed 'mycorrhizae' on pine but not the other tree species they tested that were found in association with Japanese pine mushrooms. It is possible that Pinus species responded earlier or may have been better able to withstand the potentially negative (parasitic) influence of the T. magnivelare mycelium than Douglas f ir or western hemlock. Lodgepole pine is less susceptible to the some pathogenic fungi such as Armillaria ostoyae (Romagn.) Herink (Morrison, 1981) and Phellinus weirii (Murr.) Gilbn. (Wallis, 1976) than Douglas f ir or western hemlock. The crucial question remains: were the rootlets from the lodgepole pine seedlings #4 and #8 responding to the T. magnivelare mycelium or were they simply anomalous root-tips associated with poor conditions in the Erlenmeyer flasks? I f the latter is true then why were no such root-tips found on the root systems of any of the uninoculated lodgepole pine controls? I f the former is true then what is in fact happening to these root-tips? No evidence of a mantle or Hartig net were found upon microscopic examination of the affected root-tips. This is consistent with the black, "reacted" zones (build-up of tannins in the epidermis and outer cortical cells) observed by Kawai and Ogawa (1981) during initial T. matsutake infection. Although definitive evidence is thus far lacking, these may be the first in vitro T. magnivelare affected root-tips recorded. Further work must be done to confirm that the rootlets were responding to the T. magnivelare mycelium and were not simply anomalies caused by the unnatural conditions imposed by the synthesis system. DNA analysis (PCR-RFLP) should be conducted on the resulting root-tips to determine i f T. magnivelare is associated with them. Future research should also allow a longer time frame for potential colonization to occur and utilize more mature seedlings grown in larger synthesis systems providing conditions more 70 conducive to both mycobiont and potential hosts. A variety of sterile and non-sterile synthesis systems should be tested. A wide range of T. magnivelare isolates should be tested on numerous potential host species. More research is needed regarding strain selection as some may vary with respect to rate of colonization. Various forms of inoculum and inoculation techniques should also be employed as well as the planting of seedlings free of competing mycorrhizae directly into colonies of T. magnivelare in the field. Enhancing pine mushroom production is the objective of a number of research proposals and projects currently underway in British Columbia and the U.S. Pacific Northwest. Further laboratory and field research has been done since this work was completed in 1995, but the results are as yet either unpublished, unavailable due to confidentiality, or the research has been undertaken in private facilities by parties who wish at present to retain the methodology and results for proprietry purposes. Successful synthesis of T. magnivelare ectomycorrhizae has potentially far reaching economic as well as ecological implications for British Columbia. I f in vivo T. magnivelare inoculation techniques for second growth forests are successfully developed and result in enhanced pine mushroom production, the current ecological pressures on first growth forests due to pine mushroom over-harvesting may be significantly alleviated. Pine mushrooms are an extremely valuable commodity in Japan and are quickly becoming a popular culinary addition in North America. The potential for value-added forest management could be immense! 71 Chapter 4 Nass Valley Pine Mushroom Study Area, Establishment of Permanent Sampling Plots, Preliminary Tricholoma Magnivelare Sporocarp Assessments, Shiro Analysis and Examination of the Root-tip Morphotypes Beneath Pine Mushrooms. Literature Review - Mushroom Ecological Studies Many ectomycorrhizal forest macrofungi only become conspicuous when their fruit bodies emerge. Unlike vascular plants it is difficult to define the extent of the organism from visible above ground fruit bodies because the rest of the organism (mycelium) is below ground. Fruiting bodies are ephemeral and the biomass of visible fungus does not necessarily represent the biomass of the whole fungus. To complicate matters fungal populations vary seasonally as well as from year to year. Thus, to estimate spatial and temporal sporocarp distribution, seasonal field sampling, must be done repeatedly, whenever conditions are conducive to fruiting, over a number of years. As host trees mature the fungal community diversifies and the resulting mycorrhizal composition at any given time is a function of many parameters including host species and age, fungal types and inoculum potential, competition, abiotic factors, and soil conditions (Dighton and Mason, 1985). Macrofungal frequency, diversity and composition are the fundamental parameters of ectomycorrhizal community analysis (Villeneuve et al., 1988). Although some mycologists have found the production of sporocarps correlates with below ground mycorrhizal infections (e.g. Deacon, et al., 1983; Natarajan et al., 1992), others have found no such correlation (e.g. Visser and Danielson, 1990; Cripps, 1995). Visser and Danielson (1990) found that, though a species of Suillus fruited abundantly in their plots, its ectomycorrhizal root-tip morphotype was infrequent. Cripps (1995) sampled ectomycorrhizal rootlets associated with trembling aspen (Populus tremuloides Michx.) over four years. She identified ectomycorrhizal root-tip types based on morphology and found that the distribution of species below ground was not reflected in the above ground production of fruit bodies. For example, one member of the genus Paxillus representing 25% of the colonized root-tips in her plots produced only 1 sporocarp in four years! 72 On the other hand, Natarjan et al. (1992) found a correlation between sporocarp production and below ground colonized roots for 10 species of ectomycorrhizal fungi in pine plantations. Shaw and Lankey (1994) undertook fruit body surveys on permanent sampling plots established in even aged scots pine stands. During eight years of sampling these authors found that there was a successional change from early colonizers such as Laccaria laccata and Paxillus involutus through mid-stage ones such as Lactarius and Cortinarius species to later stage ectomycorrhizal colonizers dominated by Amanita muscaria. Thus, although patterns of succession are evident in ectomycorrhizal communities, the relationship of above ground fruit bodies to below ground root-tip colonization remains unclear. Above ground sporocarp production can, however, help locate the underground mycelium of ectomycorrhizal fungi. Indeed, ectomycorrhizal fungi often fruit in nearly the same locations each year. Thus, sporocarp presence, at least qualitatively, indicates the presence below ground of a member of an ectomycorrhizal community that is fruiting at the time of sampling. Sporocarp surveys have been undertaken for many reasons. For example, mushroom counts have been undertaken to determine i f harvesting of chanterelles impacts subsequent mushroom productivity (Norvell et al., 1994). Though results thus far are inconclusive regarding the impact of harvesting on long term chanterelle productivity, Norvell et al. (1994) speculated that factors such as coarse woody debris, canopy openings, soil disturbance, soil compaction, host carbohydrate allocation, and climate affected fruiting patterns. The impacts of varying logging methods (i.e. alternative silviculture systems) on macrofungal distribution and abundance are currently being examined in a research forest in the Sunshine Coast Forest District (Fogarty, 1997, unpublished). One hundred and twenty species of macrofungi were selected for post-treatment sporocarp surveys in a clearcut, shelterwood, extended rotation and an unlogged control. The first year post-treatment results showed a reduction in the number of species fruiting with increased canopy removal and a marked reduction of the mycorrhizal component in the clearcut. However, the preliminary nature of the data allows few conclusions to be drawn regarding 73 the longer term significance of these results. Several years of study are required to determine the demise, maintenance or resurgence of macrofungi due to disturbances. Many other mycologists have examined macrofungal fruit body production in coniferous forests. Villeneuve et al. (1988), by examining the diversity and abundance of fruit bodies of 115 species of ectomycorrhizal fungi, were able to show that differing fungal communities were found in different forest cover types. Host species and age, other associated tree species and vegetation and crown closure are important factors affecting ectomycorrhizal community structure. By comparing sporocarp counts, these authors found that species diversity and frequency was related to the percentage cover of ectomycorrhizal hosts. Ammirati et al. (1987) examined the distribution of 18 species of ectomycorrhizal fungi in a 14 year old Douglas f ir plantation by counting fruit bodies in permanent sampling plots. After only one year of sampling these authors were also unable to infer spatial and temporal fruit body distribution patterns. Patterns of fungal fruiting have been examined in 10 year old stands of Pinus radicata plantations by Malajczuk (1987). By mapping the spatial distribution of sporocarps of 8 species of ectomycorrhizal fungi collected throughout the fruiting season and overlaying stem maps showing tree locations, Malajczuk (1987) found that sporocarps of some species were distributed concentrically around the base of host trees. Dighton (1987) examined ectomycorrhizal fungi in Picea sitchensis (Bong.) Carr. plantations in England. He found members of Laccaria and Paxillus dominant in 1-2 year-old stands; Inocybe and Cortinarius in 2-10 year-old stands; and Lactarius and Russula in 10-20 year-old stands. Subsequently, Marx and Cordell (1988) proposed that either late stage ectomycorrhizal fungi have a preference for older trees or soil conditions in older stands are more conducive to their sporocarp production. Fruit body occurrence was used by Visser and Danielson (1990) to compare diversity of fungal flora in young (<40 year-old) versus mature (>40 year-old) post-fire Pinus banksiana Lamb.(Jack 74 pine) stands in Alberta. They found that the mushroom flora was much less diverse in younger than older stands. Luoma et al.(1990), by collecting sporocarps and determining the dry weight (kg/ha) production of 47 species of hypogeous ectomycorrhizal species, determined that hypogeous sporocarp production was higher in their plots in the spring compared with the fall, in contrast to most epigeous species. This demonstrates the importance of seasonal sampling when studying sporocarp production. Mushroom counts have frequently been used to study the Japanese pine mushroom {Tricholoma matsutake Ito et Ima). For example, the effects of soil moisture on sporocarp production by T. matsutake was studied by Ishikawa and Takeushi (1970). By counting sporocarps they found that irrigation increased fruit body production in T. matsutake shiros. Tominaga (1978) using what was referred to as the 'Hiroshima method' was also able to enhance T. matsutake production by watering underground colonies of mycelium which had had plastic tents placed over them to increase temperature and humidity. Ogawa (1976) examined mushroom production while studying the underground mycelium ('shiro' or 'white castle) of the Japanese pine mushroom. He established 30 x 30 m plots and, by marking fruit body locations with pegs over several years, was able to delineate the extent of underground shiros. Ogawa found that fruit bodies formed mostly in 'fairy ring' patterns, however irregularities occurred where ring expansion, and hence sporocarp production, was inhibited by rocks, tree trunks, microbial antagonists or the lack of host tree rootlets for colonization. The mycelial mat was thickest and whitest where fruit body formation was highest and turned light brown more toward the center of the shiro (where fruiting had occurred in the previous year). According to Ogawa (1976), the central (oldest) part of the shiro "became sick" (necrotic) due to the formation of a temporary impermeable mat of decomposing T. matsutake mycelium. Ogawa (1975a) described the morphological features of the shiro of T. matsutake, its seasonal changes and aging in Japanese pine forests. He described seven zones of the shiro of T. matsutake summarized below: 75 Zone I - mycelial front including newly colonized soil of advancing shiro - advancing mycelia in Zone 1 had not yet formed 'ectomycorrhizal' associations with host root-tips. Zone II - white mycelium, clampless, lack cystida, low density. Mycelial mat of high density with abundant infected roots (branched, blackened, mantleless, coarse surfaced root-tips with no distinguishable or poorly developed Hartig nets microscopically). Zone III - Densest mycelial mat with the most abundant blackened root-tips. Root-tips were darker in colour than in Zone I I . The majority of sporocarp formation occurred here and was dependent on the mass of Zone III mycelium. Zone IV - Initial decomposition of mycelial mat occurred in Zone IV. Most of the colonized root-tips had been dead for almost a year. Various stages of decomposition of the mycelial mat occurred from Zone V to Zone VII with final stages showing complete decomposition of mycorrhiza and mycelium and the formation of an impervious soil layer (presumably due to cell membrane fats accumulating from decomposition of mycelium of T. matsutake). Ogawa (1975b) found that T. matsutake mycelium can maintain some growth at low temperature (0-5°C). Shiro enlargement occurred mainly from spring (April) to late summer (September) whereupon primordia formation and sporocarp development occurred as temperatures dropped and precipitation increased. Sporocarp production generally occurred from mid September to late November. Ogawa et al. (1980), again studying fruit body formation of T. matsutake, determined that the onset of fruiting occurred 3-5 years after shiro initiation into new soil. Lee (1983) in Korea found that T. matsutake sporocarps were more frequent on the S, SW and W side of ridges and in greater numbers within 25 m of the edge of ridges. Ogawa (1977b) also examined a number of shiros on flat ground and on sloped ground and compared mushroom production on 10 x 10 m plots. He reported that flat ground produced more abundant yields than sloped ground. Tominaga et al. (1989) undertook T. matsutake mushroom surveys in the Sichuan district in China and also found fruiting occurred in patterns under conditions of cooling and increased humidity 76 similar to those described earlier by Ogawa (1975b). Fruitings occurred earlier (July-September) on the mountains (3100 m) than at lower elevations (September-November). Ogawa (1975b) also reported that a smaller flush of T. matsutake mushrooms occurred at his sites in spring from late March to early June. He postulated that T. matsutake could produce sporocarps when the mycelium of the shiro had reached "the Zone I I I maturation stage" and the abiotic conditions, especially precipitation and soil temperature, became favourable to fructification. He concluded that the long term collapse of the shiro was caused primarily by the reduction of fine roots in aging pine forests and the increase of antagonistic soil microbes or competing organisms, especially other mycorrhizal fungi with similar or later stage successional niches. Ogawa (1977a) again reported on the morphological features of the shiro describing more completely the vertical distribution of T. matsutake mycelium. In this paper, Ogawa (1977) says that the shiro started to form in 15-20 year-old Pinus densiflora stands and disappeared at about stand ages 40-50. In stands older than 70-80 years, shiros had been out-competed by other mycorrhizal fungi and soil microorganisms. Ohta (1990) estimated the in vivo growth rate of the advancing shiro of T. matsutake at 30 cm per annum. Ogawa (1977c) estimated annual advancement of the mycelial front (Zone I of the shiro) at about 15 cm per year. He also estimated a required volume of 1-2 liters of colonized Zone I I I soil per T. matsutake fruit body formed. Tominaga (1975) also examined the fine roots of P. densiflora colonized by T. matsutake and found darkened root-tips similar to those described by Ogawa (1975a). Tominaga (1975) reported that in young shiros, 2-3 years prior to fruit body production, there were few infected rootlets. One year prior to fruit body production pine rootlets became more abundant and increased numbers of blackened mycorrhizae were formed. In agreement with Ogawa (1975a), Tominaga (1975) found that where T. matsutake fruit bodies were most abundant, the highest percentage of T. matsutake colonized (blackened) rootlets were found. Tominaga (1975) also agrees with Ogawa (1975b) that 1-2 years post fruiting the colonized 77 pine rootlets withered and appeared necrotic, often sloughing the epidermal and cortical layers. This description is similar to Ogawa's (1975a) Zones IV - V I I . The distribution and external morphology of mycorrhizal roots of a relative of T. matsutake called T. bakamatsutake has been studied more recently (Terashima, 1993). He examined rootlets from soil samples taken beneath fruit bodies within shiros. Shiros had been located by mapping fruit bodies from 1987-1990 (Terashima et al., 1993). Terashima (1993) found that the abundance of active (turgid, smooth, dark) T. bakamatsutake colonized root-tips was higher prior to fruiting in June than in December. Actively colonized root-tips were also found to be more abundant toward shiro peripheries, where fruiting was highest. Lee et al. (1986) examined T. matsutake mushroom production in a 55-year-old stand of P. densiflora in Korea. They identified an additional 30 species of ectomycorrhizal macrofungi fruiting in the 55-year-old stand. These authors also examined a 28-year-old stand of P. densiflora that had not yet produced T. matsutake. They identified only 18 species of other ectomycorrhizal fungi in the younger stand and proposed that T. matsutake occurs in older stands with a more diverse fungal community. A greater soil organic matter content, a more developed understorey, and a more conducive rhizosphere were proposed as reasons for appearance of T. matsutake in the older stand. Ito and Ogawa (1979) examined Japanese pine mushroom production over 10 years on 25-year-old stands of P. densiflora in which the understorey had been thinned. They found the number of shiros of T. matsutake increased significantly after 7 years on thinned plots reaching 2.4 times the number found on unthinned (control) plots. The number of potential competitors, both saprophytic and ectomycorrhizal, had decreased on the thinned plots and these authors proposed that their demise resulted in increased T. matsutake production. An earlier report by Iwamura et al. (1966) also found that crown closure reduction by up to 70% actually increased production of T. matsutake mushrooms. To date limited scientific information has been published on the biology of the North American pine mushroom {Tricholoma magnivelare (Peck) Redhead). The ecology of T. magnivelare (then called Armillaria ponderosa) was first examined by Zeller and Togashi (1934) in Washington and 78 Oregon. Later, Kinugawa and Goto (1978) reported that T. magnivelare sporocarps were found in association with stands of western hemlock, Douglas f ir and lodgepole pine in Washington. By mapping sporocarp positions in relation to forest trees near Mt. Rainier, these authors suggested that T. magnivelare fruiting often followed the arcs of tree roots, suggesting a mycorrhizal or parasitic relationship. Villarreal and Perez-Moreno (1989) compiled production data on 3 tonnes of T. magnivelare sporocarps in pine stands near Veracruz, Mexico. Hosford (1994, unpublished) presented preliminary information on T. magnivelare fruit body formation in plots in central Washington. In 1985 Hosford and Ohara (1986, 1990 and 1995) initiated long-term T. magnivelare studies. Some of their results have been summarized in Hosford et al. (1997). Using similar methodologies to those developed by Japanese investigators such as Ogawa, 1975a and b, Ogawa, 1976, Ogawa, 1977a, b, c and d, Ohara and Ogawa, 1982, and Ogawa et al., 1980, they mapped shiros and described the ecosystems including climate, soils, vegetation, fungi and soil microorganisms associated with pine mushroom producing sites. Each autumn sporocarp fruiting locations were marked by colour coded flags. Over a number of years sporocarp production and the relative position of underground mycelia and colonized roots were determined, yielding annual growth maps for shiros. The length of the fruiting season and sporocarp productivity was found to be related directly to rainfall and temperature. Sporocarp production appeared to be initiated when cumulative autumn rainfall reached 140 mm and continued until the accumulation reached 350 mm. Average temperatures were mild (i.e. above 5°C. During drier years (e.g. 1987 when by mid-November only 160 mm of rainfall had accumulated at their sites) mushroom production was lower (Hosford et al., 1997). According to Hosford et al. (1997), mushroom primordia were initiated in the ashy E-horizon and development from primordia to fully mature sporocarps took about 10-20 days depending on soil temperature and moisture. This research is continuing and wi l l expand to include other aspects of T. magnivelare biology and ecology. The extensive commercial harvest of pine mushrooms and recent resource conflicts have led to concern regarding the sustainability of pine mushroom populations and have heightened awareness 79 amongst resource managers and the public for forest fungi. Many more T. magnivelare studies have recently been proposed and/or are currently getting underway in the U.S. Pacific Northwest (Pilz, pers. comm., 1998), and in British Columbia (Wills, pers. comm., 1998). Indeed matsutake research is expanding throughout the world in pine mushroom producing areas because of the high market value of T. matsutake and related species such as the T. bakamatsutake, T. magnivelare and T. caligatum. Examining spatial and temporal patterns of fruiting is essential in understanding the ecology of fungi, however, in order to understand ectomycorrhizal relationships host colonized roots must also be examined (Cudlin, 1991; Visser and Danielson, 1990; Cripps, 1995,). Quantification of ectomycorrhizal communities involves a measure of below ground (colonized root-tips) as well as above ground structures (Harvey et a l , 1979). Meyer (1973) proposed factors affecting the occurrence of ectomycorrhizal fungi in soil including soil type, humus properties and depth, and absolute number of host root-tips. Marx et al. (1991) discussed quantitative assessments of ectomycorrhizae. These authors considered reliable quantitative estimates of root colonization to be the most important measurement in ectomycorrhizal ecology research, especially with respect to inoculation trials. Colonized root systems of host trees can be extracted from soil core samples taken within the shiro for examination and characterization of fungus-root associations. Agerer (1991, 1987a, 1986) outlined the procedures for collection, preparation, description, characterization and preservation of ectomycorrhiza. The identification of ectomycorrhizae has also been reviewed by Ingleby et al. (1990); Roth (1990); Brundrett et al. (1990); Kottke and Oberwinkler (1986); Godbout and Fortin (1984); and Zak (1973). Walker (1986) discussed some of the taxonomic problems associated with ectomycorrhizae identification and outlined points to consider when identifying ectomycorrhizal fungi. Danielson (1984) found that mycelia of most of the ectomycorrhizae isolated in stands of Jack pine were nondescript in culture, rendering in vitro cultural features of limited value in determining the identity of ectomycorrhizal fungi. The absence of conidia in culture has been proposed by Hutchison 80 (1989) as common to all ectomycorrhizal basidiomycetes, but in vitro absence of conidia is also common to other groups of fungi. More useful features used to characterize ectomycorrhizae include colour, reactions to stains and reagents, mantle morphology (inner/outer surfaces), emanating hyphae, cystidia, rhizomorphs, chlamydospores, Hartig net structure, staining of nucleii and septal pore structure (Agerer, 1986). In reviewing the characterization of ectomycorrhizae, Agerer (1991) described 4 groups of features including: Group 1 a) Anatomical -mantle surface view -anatomy of rhizomorphs -structures of emanating hyphae -presence or absence and structure of cystidia b) Morphological -colour sheath -surface texture sheath c) Chemical -reaction to meltzers, KOH and sulfovannillin -autofluorescence Group 2 -soil characteristics -preferences for soil layers (vertical distribution) -distribution horizontally (shiro dimensions) Agerer (1991) suggested that since fruit bodies of a given ectomycorrhiza are often found only in certain soil types it can be inferred that their ectomycorrhizae also prefer particular soil conditions and in some cases may occupy only specific soil horizons. Such ecological features are important for their characterization. 8 1 Group 3 -anatomy of sections (microscopy) -microscopic ultrastructural studies of septal pores and hyphal surface (see also Massicotte et al., 1992) Group 4 -studies of nucleii (e.g. staining, numbers per cell) can be greatly assisted using electron microscopy Agerer (1991) predicted that even with advanced molecular analysis techniques, comprehensive anatomical characterization of ectomycorrhizae wi l l still be needed for decades and outlined procedures for naming and describing an unidentified ectomycorrhiza. Agerer (1987b) initiated a colour atlas of ectomycorrhizae which he describes as a manual for ectomycorrhizae identification and to which he invites submissions. Other general guidelines for characterization and identification of ectomycorrhizae can be found in Mil ler (1982); Wilcox (1982); Molina and Palmer (1982); and Ingelby et al. (1990). Agerer's (1986, 1987a, 1987b, 1991) methodology and techniques have been employed by many researchers to characterize and identify ectomycorrhizae. For example, Mil ler et al. (1991, 1987) used Agerer's (1986) methods to characterize 11 morphologically distinct red alder (Alnus rubra Bong.) ectomycorrhizae. Douglas f ir mycorrhizae in the Netherlands were studied by Jansen and de Vries (1989). These authors were able to distinguish 16 types of mycorrhizae using Agerer's (1986) methods and created a dichotomous key for identification of their root-tip morphotypes from field samples. More detailed structural studies of Douglas f ir tuberculate ectomycorrhizae were done by Massicotte et al. (1992) employing methodology described by Agerer (1986; 1991). Additional methods which assist in the identification and characterization of ectomycorrhizae include: pigment chromotography (Agerer, 1991); isoenzyme analysis (Sen, 1989); immunological approaches (Cleyet-Marel et al., 1989); and restriction fragment length polymorphisms (RFLP's) (separating DNA fragments created using restriction nucleases by gel electrophoresis and examining their patterns) (Rygiewicz et al., 1989). Egger and Sigler (1993) further described molecular 82 techniques using polymerase chain reaction - restriction fragment length polymorphisms (PCR-RFLP). Aldwell and Hall (1987) reviewed serological techniques for identification of mycorrhizal fungi and described fluorescence antibody and enzyme linked immuno sorbent assay (ELISA) techniques. Cudlin (1991) described epifluorescent microscopy for the identification of ectomycorrhizal fungi. More recently Egger (1995) reviewed the molecular analysis of ectomycorrhizal fungal communities. He described techniques for identification of fungal symbionts involving amplification of mycobiont DNA obtained directly from colonized roots. Using fungus-specific primers, followed by restriction endonuclease digestion, Egger (1995) was able to produce taxon-specific restriction fragment patterns. According to Egger (1996, pers. comm.), comparisons of the resulting patterns to those obtained from fungal fruit bodies or reference cultures can confirm with certainty the identification of fungal symbionts. The degree of characterization and analysis of an ectomycorrhiza depends on the objectives of the study. For example, Brunner et al. (1992) painstakingly compared the anatomy and morphology of 3 species of in vitro synthesized mycorrhizae with field collected root-tips of what were putatively the same species. Using Agerer's (1991) techniques, these authors confirmed the in vitro synthesized rootlets were in fact the same species as the field collected roots, and therefore were able to confirm the identity of their isolates used as inoculum. On the other hand, Chu-Chou and Grace (1981, 1983) were able to distinguish 13 types of Douglas f ir mycorrhizae based solely on gross morphology by examining the roots under a dissecting scope at 10-70X. Colour, surface texture of the mantle, attached mycelia and rhizomorphs were used to identify different morphotypes. Thus, for some studies distinguishing root-tip morphotypes macromorphologically can be sufficient. There are still difficulties identifying root-tip populations from soil core samples (Roth, 1990; Danielson, 1984). Although keys to ectomycorrhizal identification are being developed and Agerer's (1987b) Atlas of Ectomycorrhizae is being continually updated, there remain many as yet undescribed species. 83 Charles Lefevre, a mycologist at Oregon State University in Corvallis, is currently in the process of describing T. magnivelare colonized root-tips for an Atlas of Ectomycorrhizae (Berch, pers. comm., 1998). Although the 'ectomycorrhiza' of T. magnivelare has yet to be fully described using Agerer's (1991) techniques, other species of Tricholoma have been described. For example, Uhl (1988) used Agerer's (1986) preparation techniques and classification methodology to differentiate ectomycorrhizae formed by Tricholoma flavobrunneum (Fr.) Kummer and Tricholoma aurantum (Paul ex. Fr.). Root colonization by ectomycorrhizal fungi has been examined by many researchers. Egli and Kalin (1991) have devised a 'root window technique' which allows researchers to film root colonization by the mycobiont or trace hyphae from fruit bodies to colonized root-tips in situ. However, the equipment required is expensive, time consuming to set up and requires high maintenance under field conditions. Grand and Harvey (1982) reviewed the quantitative measurement of ectomycorrhizae on plant roots. These authors described sampling, timing, root preparation, morphological features and root-tip counting methods. Root-tip morphotype counts from soil core samples have been used extensively to examine the distribution and abundance of ectomycorrhizal fungi (Harvey et al., 1976, 1979, 1980; Visser and Danielson, 1990; Roth, 1990; Cripps, 1995). Visual estimates, grid estimates, direct counts and mathematical approaches to calculate fungal biomass have been employed (Fogel and Hunt, 1979; Soderstrom, 1979; Vogt et al., 1981; Alexander, 1984; Hunt and Fogel, 1984; and Vogt, 1985). As mentioned earlier some mycologists have found that the production of sporocarps correlates with below ground mycorrhizal infections (Deacon et al., 1983; but others have found no such correlation (Cripps, 1995). Although limited information exists about the ecology of T. magnivelare 'ectomycorrhizae', many factors have been shown to affect the distribution and abundance of ectomycorrhizal root-tips of the Japanese pine mushroom, T. matsutake. The types of ectomycorrhizal root-tips found beneath the 84 Japanese pine mushrooms have been examined and described by many mycologists (Ogawa, 1975b, 1977a; Kawai and Ogawa, 1981; Tominaga, 1975; Lee, 1991 and others). The ecology of the T. matsutake colonies has also been studied extensively. Ogawa (1976) showed that T. matsutake shiros were initiated and concentrated in the top 15 cm of the soil where host fine root-tips were most prolific. Later, Ogawa et al. (1980) described the shiros of T. matsutake as being perennial, starting off as circular colonies or shallow disks of underground mycelium which expanded concentrically producing mushrooms about the 3rd year. These disk shaped colonies subsequently grew into ring-shaped donuts ('fairy rings') after about the 5th year of growth. According to these authors, the 'fairy rings' could reach several meters in diameter and up to 60 cm wide in the ring, 25-30 cm deep and were often broken up in arcs by impediments or by senescence or necrosis of portions of rings as they expanded radially each year. The shiro of T. matsutake has been divided into seven zones as outlined earlier (Ogawa, 1975a) based on mycelial mat, ectomycorrhizal root-tip morphology, and shiro developmental stage. Shiro development over a 5-year period has been summarized by Ogawa (1976): Zone I was a thin zone where the mycelium began to establish as hyphal growth occurred. Mycelia in Zone I I were denser and it is here where mycorrhizal relationships are established with host rootlets. In Zone I I I the mycelial mat (extramatrical hyphae) was thickest. Zone I I I was where the majority of fruit body formation occurred. Zone I I I is also where the highest abundance of blackened, colonized root-tips existed. In Zone IV decomposition of mycelium and withering of rootlets began to occur. More advanced stages of mycorrhizal senescence and decay occurred in Zones V to VI I . Ogawa (1975b) noted that during initial colonization root-tips are coated in fresh, thick, whitish to brownish mycelium. (Zone II). These later become blackened root-tips with reddish-brown bases (Zone I I I and IV). In decomposition Zones V-VI I root tips appeared more blackened, some with a necrotic appearance similar to parasitically infected roots (i.e. sloughed necrotic cortical cells separated from the stele). Ogawa (1977a) described the mycorrhizae formed by T. matsutake on 85 Pinus densiflora roots as elongated, blackened, parasitic, ectotrophic, 'witches broom' type mycorrhiza. Lee (1991) examined the shiros of T. matsutake in a Korean pine forest over 4 years. Lee also reported that the T. matsutake associated root-tips he examined did not appear mycorrhizal, but instead appeared "slightly parasitized". He claims that association with the mycelium of T. matsutake resulted in a net carbon drain to the conifer host. This he based on the chlorotic, thinning crowns noted in stands associated with Korean pine mushrooms. He described the colonized root-tips as dark and necrotic looking. These, Lee (1991) says, may have been root-tips that were in various stages of decomposition consistent with Ogawa's (1977a) findings. Terashima (1993) classified the root-tips found beneath T. bakamatsutake sporocarps into five types showing seven root patterns using the terms defined by Agerer (1987a). He described T. bakamatsutake infected root tips as active i f they are turgid and the external surface is smooth or cottony and white or gray in colour. Tips were inactive i f the external surface was coarse, wrinkled or fissured, and black, and tips were dead i f root-tips looked as i f their epidermal cells had collapsed, leaving only the stele and the unsheathed cortex. Other species of Tricholoma have also been found to form blackened root-tips. Ohara and Ogawa (1982) found "identical" black ectomycorrhiza formed by Tricholoma caligatum (Viv.) Gilbert on Cedrus roots in Algeria to those formed by T. matsutake on Pinus in Japan. Blackened, carbonized-looking ectomycorrhizal root-tips have also been found in other genera. Agerer (1993) described the appearance of Phellodon niger (Fr.: Fr.) Karst colonizing Picea abies (L.) Karst. roots as "carbonizing", somewhat similar in gross morphology to those described for T. matsutake by Kawai and Ogawa (1981) and Lee (1991) and others and for T. magnivelare by Hosford and Ohara (1995). However, Agerer (1993) described a distinctive mantle and Hartig net of P. niger while Ogawa (1975b, 1977a) and Kawai and Ogawa (1981) found only a loose fungal sheath and ill-defined Hartig net on blackened root-tips associated with T. matsutake. 86 Preliminary examination of roots colonized by the North America pine mushroom (T. magnivelare) was done by Zeller and Togashi (1934). They reported tracing "rhizomorphic connections" between basidiocarps and ectotrophic mycorrhizae on rootlets of lodgepole pine near Florence, Oregon. The purportedly mycorrhizal nature of T. magnivelare was later supported by research done by Kinugawa and Goto (1978) who undertook surveys of sporocarp positions in relation to forest trees near Mt. Rainier, Washington. These authors suggested a relationship existed between the location of hemlock roots and the locations of T. magnivelare fruit bodies based on the fact that fruiting often followed arcs of tree roots. Kinugawa and Goto (1978) suggested that T. magnivelare formed ectomycorrhizal associations with a variety of tree species including lodgepole pine, Douglas f ir and western hemlock. Redhead (1992, unpublished) states that although little is known about the ecology of T. magnivelare, it is presumed to be ectomycorrhizal. Hosford (1994, unpublished) described preliminary results of soil surveys and development of the shiro of T. magnivelare in Washington. Root-tip morphology was not described in this article. However, the need for further research in this field was stressed by Hosford (1994). Since then, Hosford and Ohara (1995) and Hosford et al. (1997) have published more comprehensive descriptions of the shiro of T. magnivelare. Using similar methods to those developed for T. matsutake by Ogawa and other Japanese investigators, Hosford and Ohara (1986, 1990 and 1995) described the shiro of T. magnivelare at sites in central Washington. Some of this work is summarized by Hosford et al. (1997). According to these authors, the ectomycorrhizal roots formed by T. magnivelare developed below the ashy E-horizon in the transitional greyish-brown EB-horizon. The shiro was typically the shape of a semicircle, however it was usually broken into arcs. One of the shiros they described had a diameter of about 6 m and expanded at a rate of about 4 cm per year on average. This is more slowly than those described for T. matsutake by Ogawa (1975b) or Ohta (1990) who respectively reported 30 and 15 cm per annum shiro expansion. Hosford et al. (1997) described the physiologically active mycorrhizal zone of T. magnivelare shiros as relatively wide and deep, containing abundant blackish, branched mycorrhizae. According to Hosford et al. (1997), the shiro had a simplified 87 bacterial flora similar to those formed by the Japanese matsutake. The boundary between decomposing mycorrhizae and decayed mycorrhizae could not be distinguished visibly. The ectomycorrhizae they examined were similar to those formed by the Japanese matsutake in that they lacked well-defined Hartig nets. In the present study, root-tip morphotypes extracted from soil samples collected directly beneath North American pine mushrooms were examined and distinctive charcoal coloured, blackened, root-tips similar to those described for the Japanese pine mushroom were found. These were referred to as 'matchsticks' because of their swollen, dark, elongated tips and lack of root hairs, giving the rootlet the appearance of an elongated matchstick. These root-tip morphotypes lacked definitive mantles or well-developed Hartig nets. One of the objectives of the present study was to determine i f the blackened 'matchstick' morphotypes were associated in greater numbers in soil samples collected directly beneath T. magnivelare sporocarps than in soil samples collected outside of the shiro. The objective of the mushroom sampling portion of this study was to establish permanent sampling plots and collect preliminary data on the numbers and grades of T. magnivelare sporocarps produced in the Nass Valley Pine Mushroom Study Area. 88 Introduction Scientific information regarding the biology, ecology, fruit body production and 'ectomycorrhiza' of the North American pine mushroom (Tricholoma magnivelare (Peck) Redhead) is scant (Redhead, 1992; Hosford, 1994 unpublished). Observation of the shiro of the pine mushroom is underway in Western North America (Hosford, 1994 unpublished; Hosford et al., 1997), however to date only a limited amount of ecological information has been published. Spatial and temporal patterns of sporocarp distribution have however been examined for many other ectomycorrhizal fungi (Ammirati et al., 1987; Visser and Danielson, 1990; Luoma et al., 1990). Preliminary ecological studies of the North American pine mushroom (Tricholoma magnivelare (Peck) Redhead) were undertaken by Zeller and Togashi (1934) in Oregon. General descriptions of the size and abundance of pine mushroom fruit bodies are provided. Kinugawa and Goto (1978) undertook surveys of North American pine mushroom positions in relation to forest trees near Mt. Ranier, Washington. They suggested a relationship between T. magnivelare fruit body position and root systems of western hemlock. These authors found that in Washington, T. magnivelare was found in association with stands of Douglas fir, western hemlock and lodgepole pine. Until recently much of the understanding of the sporocarp production and shiro development of T. magnivelare was inferred from research done in Asia with the Japanese pine mushroom (T. matsutake). A summary of some of the T. matsutake ecological research has been presented in the Literature Review. More recently, Hosford and Ohara (1995) have described the ecology of T. magnivelare shiros (colonies of underground mycelium) in central Washington State. Using the basic approach of previous investigators in Japan, they mapped shiros and characterized the topography, climate, soil types, soil moisture and temperature, associated vegetation, sporocarp production, ectomycorrhizae, other fungal species and bacterial populations. Other biotic and abiotic factors were also examined. This project is ongoing and other research avenues are being examined (Hosford et al., 1997; Pilz, 1998 pers. comm.). 89 Tricholoma matsutake fruit body formation and associated 'ectomycorrhizal' root-tips have been studied extensively in Asia (Ogawa, 1975a, 1975b, 1977a; Kawai and Ogawa, 1981; Lee, 1991; and others). Sporocarp counts of T. matsutake have been done to determine the production of fruit body biomass (Lee et al., 1986; Ogawa, 1976) and the extent of the shiro (Ogawa, 1975a; Ogawa et al., 1980). The effects of different treatments on production of T. matsutake sporocarps has also been examined [i.e. thinning the understorey (Ito and Ogawa, 1979); thinning the overstorey (Iwamura et al., 1966); or irrigation and environmental manipulation (Tominaga, 1978)]. Root counts from soil core samples have been used extensively to examine the distribution and abundance of ectomycorrhizal fungi (e.g. Harvey et al., 1978, 1979; Fogel and Hunt, 1979; Vogt, et al., 1981 and 1985; Cripps, 1995). The root-tip morphotypes found in T. matsutake shiros have been described (Ogawa, 1975b; Lee, 1991). Beneath T. matsutake sporocarps charcoal grey or brownish to black, elongated, slightly swollen morphotypes, lacking a distinct Hartig net, predominate (Kawai and Ogawa, 1981; Lee, 1991; and others). Similar types of root-tips have been found directly beneath T. magnivelare sporocarps in the Sunshine Coast Forest District (personal observation, 1994). Similar root-tips have also been reported from Washington state by Hosford and Ohara (1995). According to Hosford et al. (1997) the ectomycorrhizae examined in Washington were also structurally similar to those formed by the Japanese matsutake in that they lacked well defined Hartig nets. The objectives of the pine mushroom ecological studies in the Nass Valley Pine Mushroom Study Area were to: 1. Provide a preliminary description of the study area, particularly the T. magnivelare fruiting sites where permanent sampling plots were placed. 2. Establish 'permanent' pine mushroom sampling plots at three sites in the study area and map the distribution and abundance of fruit bodies in each plot over three sampling periods in autumn, 1994. 3. Undertake preliminary descriptions and comparisons of the types and abundances of seven categories of root-tip morphotypes extracted from soil cores taken inside and outside of pine 90 mushroom shiros with special reference to the blackened, 'matchstick' morphotype found in greater abundance in association with T. magnivelare sporocarps. 4. Collect fresh, young fruit bodies of T. magnivelare for isolation and in vitro cultivation of vegetative mycelium for enzymatic ability tests and ectomycorrhizal synthesis experiments. 9 1 Methods and Materials Study Site Location The 5500 ha pine mushroom study area is located adjacent to the Shumal River drainage which flows southward into the Nass River near New Aiyansh, 80 km north of Terrace, British Columbia (Figures 19 and 20). The study area (Figures 21 and 22) was selected because it has been a renowned pine mushroom producing area over the past decade and is geographically isolated (boat or helicopter access across the Nass River). Pine mushroom harvesting in the study area was restricted to pickers registered in the pine mushroom research project. The study area is in the Interior Cedar-Hemlock moist cold subzone - Hazelton variant (ICHmc2) (Banner et al., 1993). In the Nass Valley near New Aiyansh, the ICH zone occupies the valley floors and lower benches to 600 m. Elevation varies from approximately 150 to 600 m in the study area. A complete description of this subzone can be found in Banner et al.(1993). This subzone encompasses the southern Nass basin and adjacent Skeena and Hazelton mountains. Elevation ranges in the ICHmc2 are from 100-750 m. The soils of the ICH developed mainly from morainal parent materials, but fluvial and colluvial materials are also common. Organic matter decomposition and leaching are important processes in forested sites especially where precipitation is heavy. The zonal soils are Orthic Humo-Ferric Podzols with 5-10 cm thick Hemimor humus forms (Banner et al., 1993). The ICH combines elements of both coastal and interior flora. Fire has had a major influence on vegetation development in the ICH. Stands are serai (historically fire-driven every 90-100 years) especially in the southerly sections of the subzone. It is difficult to find a stand in the ICH that has not been disturbed by fire in the past few centuries (Banner et al., 1993). Major conifer species in the study area include Tsuga heterophylla (Ref.) Sarg. and Pinus contorta (Dougl. ex. Loud.) with Picea sitchensis (Bong.) Carr on wetter sites and near creek draws. 92 Known Pine Mushroom Harvesting Areas in British Columbia Fig. 19. General location of Nass Valley Pine Mushroom Study Area (reprinted with permission from de Geus, 1995b). 93 PINK MUSHROOM PILOT PROJECT INFORMATION SHEET Fig 20 Nass Valley Pine Mushroom Study Area location 94 Fig. 21. Looking south over study area - Shumal River in center, study area to north and east of Shumal drainage. The zonal site series is 01 (hemlock/stepmoss) with mesic to submesic moisture regimes. Feather mosses (step moss, red-stemmed feather moss) and knight's plume carpet the forest floor, but understorey shrubs and the herb layer are relatively sparse. Occasionally there is a well-developed shrub layer of red and black huckleberry, alaska blueberry and false azalea (Banner et al., 1993). On drier sites such as nutrient poor outcrops and ridges where lodgepole pine is dominant, kinikinnick can be found in conjunction with black huckleberry and dwarf blueberry. Devil's club is often found in the creek draws. Wetter site series for the ICHmc2 are also described by Banner et al. (1993). In the dry to mesic site conditions which predominate in the study area, bunchberry, prince's pine, five-leafed bramble and scattered saskatoon berry can be found. A wide variety of macrofungi were found fruiting in the study area including members of the genera Amanita, Boletus, Cantharellus, Clavaria, Clavulina, Cortinarius, Croogomphus, Gomphidius, Hebeloma, Hydnellum, Hydnum, Hygrophorus, Inocybe, Laccaria, Lactarius, Lycoperdon, Lyophyllum, Mycena, Pleurotus, Pluteus, Pseudohydnum, Ramaria, Russula, Sparassis, Suillus, and Tricholoma. Soils In August, 1994, three 25 m x 25 m mushroom sampling plots were established and delineated at approximately 100, 200 and 500 m elevations in areas locally known as the 'Granny', the 'Benches', and 'Boogie Heaven' respectively (Figure 23). Plot layout methodology is further described later in the section entitled Mushroom Counts. Adjacent to each mushroom sampling plot soil pits were dug beneath T. magnivelare sporocarps. Figures 24 and 25 show the Plot 1 soil pit site before and during excavation, respectively. Note the primordia uncovered adjacent to the T. magnivelare sporocarp. Figures 26 and 27 show the upper layers of the Plot 2 soil pits during initial excavation. Note the extensive mycelial mat and exposed roots below the mushroom. Soil horizons were identified in the field and some preliminary soil descriptions were done. A more comprehensive ecological description and classification of the three sampling plots in the Nass Valley Pine Mushroom Study Area was undertaken in autumn, 1995 (Trowbridge and 96 Fig. 23. Nass Valley Pine Mushroom Study Area sampling plots (approximate locations). 97 Fig. 24. Plot 1 soil pit site prior to excavation. Fig. 26. Plot 2 soil pit during excavation showing mycelial mat in upper horizons. Fig. 27. Plot 2 soil pit during excavation showing exposed mycelium and roots below the mushroom. 99 Macadam, 1996). A copy of this report complete with site attributes, vegetation communities, soil descriptions, profiles, and chemical analysis for the three pine mushroom study plots is available from the British Columbia Ministry of Forests, Research Branch in Victoria. Below is a summary of their findings. The study site was confirmed to be located in the moist, cold Hazelton variant of the Interior Cedar-Hemlock biogeoclimactic zone (ICHmc2) in the Hw-stepmoss site series, sub-mesic phase (01(l)b) (Appendix 2). Trowbridge and Macadam (1996) list trees, shrubs, herbs, mosses and lichens recorded from each plot. Both lodgepole pine and western hemlock were present in the main or subcanopy of each plot. Lodgepole pine was the dominant overstorey species in Plots 1 and 2 and western hemlock in Plot 3. The tree stratum was fairly dense with percent cover averaging approximately 75% (Figure 28 and Appendix 3). In all plots, the shrub and herb strata were very low in diversity and had low percent cover values (7% and 4% respectively). Black and red huckleberry, princes pine and rattlesnake plantain were present in all plots. Red-stemmed feather moss, stepmoss and pipe cleaner moss were also present in all plots with an overall percent cover value of nearly 90% (Trowbridge and Macadam, 1996) (Appendix 3). The soils were coarse textured and formed on glacio-fluvial terraces (Plots 1 and 2) or morainal t i l l (Plot 3). A l l plots had a strongly developed S bryophytic layer, with litter material intermixed. Under this layer were typical Fm horizons making up the Hemimor humus forms. Fungal mats of abundant white and grey mycelium were sometimes present at the forest floor - mineral soil interface. Ae horizons were typically present but not always continuous. Subsurface mineral horizons were classified as Bm and podzolic Bf soils, and were confirmed as Orthic Humo-Ferric Podzols (Plots 1 and 2) and Eluviated Dystric Brunisols (Plot 3). The mineral soil horizons were dominantly sandy loam to sand in texture. Coarse fragment content in the subsoil was high as is typical for these parent materials, ranging from 30-80% by volume. Soil development was not considered deep as C horizons were designated at approximately 60 cm at each plot (Trowbridge and Macadam, 1996) (Appendix 4). 100 Fig. 28. Canopy showing lodgepole pine and western hemlock. Fig. 29. Portion of plot 1 site showing T. magnivelare sporocarps. 101 For each horizon soil analyses were done including: pH; total carbon and nitrogen; available phosphorus; cation exchange capacity and exchangable calcium, magnesium, and potassium; and extractable iron and aluminium. Results of these tests are summarized in the report by Trowbridge and Macadam (1996) and shown in Appendix 4. Mushroom Counts Methodology In August, 1994 three T. magnivelare sampling plots measuring 25 m x 25 m were placed over areas of ground where pine mushrooms were fruiting at the time of plot establishment. Figure 29 shows a portion of plot site #1 with T. magnivelare fruit bodies. Plots were laid out with the origin at the southwest corner. Sporocarps were located and mapped using the distance in centimeters north and east of the origin. Three samplings (mushroom counts) were undertaken in autumn, 1994, in each plot (September 10-12, September 28 and October 11). Mushroom locations were marked with plastic pegs. A different coloured plastic peg was used for each sampling. Data were collected on the number, quality or grade (Appendix 5) and location of each sporocarp in each plot. Primordia less than one centimeter in diameter were not counted but were left intact where encountered during sampling. Sporocarp maps were drawn showing the locations of mushrooms in each plot on each sampling. For clarity, the locations of tightly clustered sporocarps were shifted slightly (+/- 10 cm) on the sporocarp maps to allow data points which were obscured due to overlap to be visible on the figures. Although data on the numbers and grades of mushrooms in Plot 2 on the third sampling were collected, the locations of sporocarps in Plot 2 on the third sampling are not shown on the sporocarp map. During the third sampling in Plot 2 sporocarps were located, picked and graded and markers were placed in the seven mushroom fruiting sites identified that day. However, researchers were forced to leave the study area prior to measuring the distances and mapping sporocarps in Plot 2 in order to catch a boat across the swollen Nass River which was flooding due to heavy rains and becoming hazardous. Sporocarp location data were to be measured by a local forest technician who was to return to the Plot 2 after the river had subsided and measure the markers. This information 102 has not been received. Other data (numbers, grades) collected on these sporocarps are included in the tabulated Results. Unfortunately, some indiscriminate commercial harvesting of pine mushrooms occurred in the plots between samplings. It is also possible that some mushrooms may have been removed by animals. Deer, elk, moose, bear and even squirrels purportedly consume pine mushrooms (Mills, 1998, pers. comm.). Attempts were made to locate evidence of sporocarp removal (small holes remaining after the removal of a mushroom and recently replaced moss and litter covering holes) left by harvesters. Holes were considered to have been due to pine mushroom fruit body removal i f a mycelial mat with distinctive pine mushroom odour was present in the hole. Sometimes T. magnivelare primordia still remained adjacent to where sporocarps had been removed, facilitating the identification of holes as being caused by the removal of a T. magnivelare sporocarp. As with sporocarps, holes putatively left by pine mushroom pickers were counted and their locations marked and mapped. Mushroom sampling data was tabulated and plot maps were examined for possible shiro characteristics - 'fairy rings', 'arcs' or apparent groupings of pine mushrooms. Fresh, young fruiting bodies of T. magnivelare were collected adjacent to plots for laboratory analysis and tissue cultures. Root Core Sampling In October, 1994 soil core samples measuring 10 x 10 x 10 cm were collected with a hand trowel adjacent to each mushroom sampling plot. At each site two soil core samples were collected - one from directly beneath a T. magnivelare sporocarp ('in the shiro') and another from outside the shiro (>3 m away from any visible T. magnivelare sporocarps ('not in the shiro'). The 6 cores were placed in separate plastic bags, shipped to the University of British Columbia and frozen. Prior to examination, a 6 x 6 x 7 cm subsample was cut from the center of each original core using a sharp blade. This subsample included the moss, litter layer and organic matter, the Ae horizon where present, and some mineral soil beneath the Ae horizon. Subsamples were placed in water and soaked overnight. 103 Sample preparation The following day all visible roots were extracted from the water bath and placed in a 2 mm sieve overlying a 0.6 mm sieve and thoroughly washed with a gentle stream of tap water. Wood, mineral and humus debris was gently crumbled away from root tissue during sieving. Using a fine forceps, all visible roots were removed from the sieves and placed in water in 90 mm petri plates for examination. With the aid of a dissecting scope residual roots missed during preliminary sorting were found, extracted with forceps and combined with roots from preliminary sorting for examination. Figure 30 shows a portion of the root mat from preliminary sorting. Root-tip Morphological Descriptions Prior to root-tip counting, preliminary descriptions for seven distinct types of root-tip, herein called morphotypes, were done by S. Berch and F. Fogarty at Glyn Road Research Station, Victoria, B.C. from subsamples collected for this purpose. The seven categories of morphotypes found were referred to as: 1) uncolonized, 2) Cenococcum geophilum, 3)'Cenococcum-hke', 4) 'matchstick', 5) white over tan, 6) golden brown and 7) multiples. The morphotypes were categorized using ectomycorrhizal description forms based on some of Agerer's (1991) characterization criteria. Some micromorphological features were examined when describing each of the morphotypes. However, root-tip morphotype counts from soil core samples were primarily based on macromorphological features easily distinguishable at 10-50X magnification under a dissecting microscope. More comprehensive descriptions of root-tip morphotypes 2-6 are given in the Results and in Appendix 6. Root-tip Morphotype Counts For the sieved samples all root-tip morphotypes were counted with the aid of a dissecting microscope (10X-50X). Roots matted together or embedded in the mycelial mat were gently teased apart to aid examination (Figure 31). No attempt was made to distinguish live from inactive or dead root-tips. A l l root-tips counted were placed in one of the seven categories. Root-tips were considered ectomycorrhizal i f they were ensheathed by a mantle of fungal hyphae and/or possessed a Hartig net when examined microscopically. Supplemental microscopic examinations were conducted to 104 Fig. 30. Root mat during preliminary sorting showing rootlets embedded in dense, white mycelium (8X magnification). Fig. 31. Root-tips extracted from mycelial mat for examination (12X magnification). 105 complement macroscopic examinations to ensure consistency in counting. Each root-tip was counted separately even i f it was part of a complex, branching structure. Examination of Primordia-Rootlet Connections and Mycelium. Initial attempts were also made to carefully follow hyphae from the base of primordia to blackened root-tips by gently washing the organic matter away from soil cores collected directly underneath primordia for this purpose. Primordia were embedded in the mycelial mat/root mat, so direct hyphal connections were not discernible between primordia and rootlets. The mycelial mat was so dense in the organic and upper mineral horizons at the time of sampling that it obscured tracing mycelia directly from primordia to potentially colonized rootlets. Figure 32 shows a primordium with rootlets embedded in its base after rinsing away the organic matter. Mycelium from the base of the primordium was examined microscopically and found to consist of a mass of septate, branched, hyaline hyphae with no apparent clamp connections. Mycelium taken from a rootlet attached to the base of the primordium also had hyaline hyphae with slightly swollen, 'ball-joint-like' septae and what appeared to be septal constrictions (Figure 33). Anastomoses, or hyphal branching in the form of 'H'-shapes (Figure 34), were apparent in numerous locations in the mycelium on prepared slides. Data Analysis The number of each morphotype in each sample was multiplied by four to yield approximate morphotype numbers per litre for each sample. The estimated numbers of each morphotype/litre in each sample were tabulated (Table 8). For convenience of analysis, data were transposed so that the rows are columns (Appendix 7) in order to compare the numbers of the seven root-tip morphotype categories 'in shiro' and 'not in shiro' from the three different plot locations. In Appendix 7 'Loc' refers to location of the sample: l= ' in shiro' and 2='not in shiro'. The 'in shiro' and 'not in shiro' counts were made in close proximity adjacent to each plot and were therefore treated as a pair (i.e. there are three pairs of measurements for each morphotype - one 'in shiro' and one 'not in shiro' count for each plot). The numbers of each morphotype 'in shiro' and 'not in shiros' were compared. 106 L • • •: 1 l 1 S i . • r • a •'illliilli;. 1 H m% ** Fig. 32. Tricholoma magnivelare primordium with rootlets embedded in its base (12X magnification). Fig. 33. Hyphae with slightly swollen 'ball-joint-like' septae in mycelium taken from base of T. magnivelare primordium. 107 Fig. 34. Hyphal branching in the form of 'H-shapes' (anastamoses) in mycelium taken from the base of T. magnivelare primordia. 108 Three tests of the null hypothesis (that there was no difference between the expected number of root-tip morphotypes beneath and adjacent to T. magnivelare sporocarps) were carried out: (i) a simple t-test of the difference (between 'in shiro' and 'not in shiro'), (ii) a sign test of the difference and (iii) a signed-rank test of the difference. 109 Results Mushroom Counts The locations of T. magnivelare sporocarps and holes presumed to be the result of sporocarp removal on three samplings are mapped for Plots 1, 2 and 3 in Figures 35, 36 and 37 respectively. Sporocarps and holes were distributed in a manner in which no definitive 'fairy rings' or arcs of underground mycelium (shiro) could be confirmed. Plausible groupings of sporocarps can however be inferred from the data. Figure 35 shows that most of T. magnivelare sporocarps were collected in the second sampling in the mid-south of plot 1. Four discernible clusters (of >3 sporocarps) were centered at approximately: 300 cm N/1250 cm E; 400 cm N/750 cm E; 650 cm N/1200 cm E; and 1150 cm N/1400 cm E. Figure 36 shows the distribution of T. magnivelare sporocarps in Plot 2. Again the majority of sporocarps were collected on the second sampling. There are two plausible clusters of mushrooms in the northwest at 1500 cm N/800 cm E and at 2100 cm N/1000 cm E. Figure 37 shows the distribution of T. magnivelare sporocarps in Plot 3. The second sampling also yielded the majority of sporocarps. Two clusters occurred in the midwest portion at 1100 cm N/950 cm E; 1300 cm N/800 cm E; and one in the mideast at 1500 cm N/2000 cm E. Individual T. magnivelare sporocarps were found scattered throughout the plots. A total of 94 sporocarps and holes presumed to have contained sporocarps were measured in the three sampling plots. Table 7 shows numbers and grades of sporocarp in each plot for each sampling. The majority of sporocarps collected (31) were graded as #l's. Most of these were collected during the 2nd sampling (22) and fewest in the 3rd sampling (1). Plots 1 and 2 produced more grade #1 sporocarps (10 and 15 respectively) than Plot 3 (only 6). No grade #2 sporocarps were collected in any plots on any samplings, and only 1 grade #3, 4 grade #4, and 8 grade #5 sporocarps were collected. Twenty-three (23) grade #6 sporocarps (usually containing insect larvae) were found during the 3 sampling periods. Twenty-seven (27) holes which appeared to have been caused by pickers removing T. magnivelare sporocarps were encountered. These were referred to as grade #7 in Table 7. 110 F i g u r e 35. N a s s V a l l e y P l o t #1: L o c a t i o n o f T. m a g n i v e l a r e 2500 s p o r o c a r p s o n three s a m p l i n g d a t e s in fa l l , 1994. • Sampling 1 • Sampling 2 • Sampling 3 2000 1500 • • ? North 1000 500 • • B • • • a • a* B 1 D • • • 0 i i i i i 1 l 1 I I 0 500 1000 1500 2000 2500 East (cm) 111 F i g u r e 36. N a s s V a l l e y P l o t #2: L o c a t i o n o f T. m a g n i v e l a r e s p o r o c a r p s o n three s a m p l i n g d a t e s in fa l l , 1994. 2500 2000 B • Sampling 1 • Sampling 2 1500 E u sz t o 1000 500 • • • • 500 1000 1500 2000 E a s t ( c m ) 2500 112 F i g u r e 37. N a s s V a l l e y P l o t #3: L o c a t i o n o f T. m a g n i v e l a r e s p o r o c a r p s o n three s a m p l i n g d a t e s in fa l l , 1994. 2500 • a 2000 D • • • Sampling 1 • Sampling 2 • Sampling 3 • th (cm) 1500 • & ft o • 1000 500 • a • • • • 0 i i i i i i i i 0 500 1000 1500 2000 2500 East (cm) 113 Table 7. Numbers and grades3 of Tricholoma magnivelare sporocarps in Plots 1, 2 and 3 from three samplings in September and October, 1995. T. magnivelare sporocarp grade. 7 b Plot Samp. 1 2 3 4 5 6 Total 1 5 6 2 13 1 2 10 3 2 3 18 3 - 2 - 1 3 34 1 1 8 9 2 2 9 - 4 1 14 3 C - 1 2 2 1 1 7 30 1 2 6 8 3 2 3 2 8 4 17 3 1 . . . . i 2 1 5 30 Total 31 0 1 4 8 23 ' 27 94 a Appendix 5 shows the pine mushroom grading system employed. D holes presumed caused from picking of T. magnivelare sporocarps. c sporocarp locations not mapped for third sampling in Plot 2. 114 Root-Tip Morphological Descriptions Preliminary descriptions of the root-tip morphotypes 2, 3, 4, 5 and 6 are given in Appendix 6. In summary, the seven categories of root-tip morphotypes were as follows: 1. uncolonized - light to dark brown root-tips with root hairs and no evidence of swelling or fungal colonization macroscopically. No evidence of a mantle or Hartig net microscopically. 2. Cenococcum geophilum - jet black mycorrhiza with black bristle-like, eminating hyphae. Dark, stellate mantle pattern evident microscopically. 3. ' Cenococcum-Wte' - black mycorrhiza but no bristles visible and no stellate mantle pattern microscopically. 4. 'matchstick' - charcoal-black, slightly swollen, elongated root-tip with a narrow 'neck' (ie. a slight constriction between the swollen, colonized apex and uncolonized, narrower proximal portion of the rootlet). The darkened surface often appeared covered in thin layer or 'dusting' of whitish-grey mycelium (Figure 38). Generally no mantle or distinct Hartig net were visible microscopically. However, what appeared to be a loose mycelial 'sheath' and palmate invaginations ('finger-like' hyphal projections) (poorly developed Hartig net?) were observed on epidermal and outer cortical cells from longitudinal sections of one of the colonized rootlets at 1000X magnification oil immersion (Figure 39). 5. white mycelium over tan root tip - slightly swollen, light cinnamon-coloured root-tip covered in dense sheath of white mycelium (Figure 40). Relatively thick mantle (two layers with patches of looser, thick extramatrical hyphae) and distinguishable Hartig net present with abundant palmate hyphal invaginations. 6. golden brown - no mantle but 'finger-like' hyphal projections ('Hartig net') seen microscopically. 7. multiples - 2 or more of the above types usually C. geophilum and one of the other types distally or proximally on the same root-tip. Morphotype #4 ('matchstick') (Figures 41 and 42) most resembles the charcoal grey to black, elongated, slightly swollen root-tips described by other researachers in association with T. matsutake 115 Fig. 38. Root-tip morphotype #4 ('matchstick') showing proximal constriction and darkened surface with whitish-gray remnant mycelium (32X magnification). Fig. 39. Longitudinal section through morphotype #4 rootlet showing loose mycelial sheath (remnant 'mantle'?) and palmate invaginations (poorly developed 'Hartig net') on epidermal and outer cortical cells (1000X magnification). 116 Fig. 40. Root-tip morphotype #5 (white/tan) (8X magnification). Fig. 41. Morphotype #4 root-tip. Whole mount unsquashed (120X magnification). 117 Fig. 42. Morphotype #4 root-tip. Whole mount squashed (120X magnification). 118 in Asia and with T. magnivelare in North America. Morphotype #5 is similar to rootlets purportedly in the early stages of T. magnivelare colonization. Root-tip Morphotype Counts Table 8 shows the estimated numbers of each root-tip morphotype per litre of soil. Of the morphotypes only Cenococcum geophilum was identified to species. The total number of root-tips per litre varied from 11,600 (plot 3) to 15,792 (plot 1). The largest variation between the 'in shiro' and 'outside shiro' samples occurred with morphotype #4 ('matchstick'). A greater number of 'matchstick' type root-tips were found beneath T. magnivelare fruit bodies than away from T. magnivelare fruit bodies in all three plots. The opposite was true for C. geophilum and 'Cenococcum-like' root-tip morphotypes in that fewer were found in shiros (Table 8). In general there were a greater number of each of root-tip morphotypes #5 (white/tan), #6 (golden brown) and #7 (multiples) found in the shiro (Table 8). The results of the t-test for each root-tip type are summarized in Appendix 7. DIFF1-DIFF7 refer to differences in numbers between root-tip morphotypes 1-7 found directly beneath pine mushrooms (Loc 1) and >3 m away from pine mushrooms (Loc 2) within each plot. Small p-values (e.g. p <0.05) imply that there was a statistically significant difference between the beneath pine mushroom and away from pine mushroom mean values assuming the sample measurements for the three plots were independent and their distributions were normal. The t-test results suggests that there was a significant difference (p=0.0083) 'in shiro' and 'out of shiro' for root-tip #4 (the 'matchstick' morphotype). There is also a possibility that root-tip #2 (C. geophilum) would have shown a significant difference given a larger sample size (p=0.0601). The t-test results showed no significant differences for the 'in shiro' and 'out of shiro' counts for any of the other root-tip morphotypes. The nonparametric tests (i.e. the sign test and the sign-ranked test), which are less powerful but do not require normality (Zar, 1974), showed no significant differences for any of the root-tip morphotypes including root-tip morphotype #4. 119 1* •a a 5S cu S e ft vi I ^ 1 ST" o c o o CN VO r o in o VO CN CN O c o vo ON CO vo f N O o VO I s o u JS v> S VO o CN c o CN 00 r-<N CN oo o 00 CO cu s o cu — cu VI Si ft s CU •-o •a cu « cu o CU ca B fN a o o 00 o VO ON 00 o o 00 CN Os VO CO CO 00 00 Ov CO o 00 CN 00 oo CO f N OS VO CN 00 CN o CO CN •r, o CN CN CN O CN •s & .9 g .2- & - i « O 2 I "S 1 1 CU S( JB cu US cu Si 2 •-cu ft CU Xl S s e oo s E o S3 93 CO A 2 « .2 1 at X 60 © O CQ S vi a © o JS vi 8 O Os O CU ft >. H cu N "3 "3 «u s s CN s a CN fie CN CO CO O 00 o VO M it CU cu S 1-O CU cu >. e CU -«-* 'JS vo Os B I B cu "© ©JO 00 VO CN O o 00- 00 CN OS CN CN o CO r-- 00 CN O o 1—1 I T ) <—1 f N s S CN Os VO O H Discussion Mushroom Counts Plots encompassing shiros of T. magnivelare have been established at three sites in the Nass Valley Pine Mushroom Study Area. Primary fruiting portions of T. magnivelare shiros were located in Plot 1 in the mid-south. In Plot 2 sporocarps were clustered toward the middle and in the northwest portion of the plot. In Plot 3 sporocarps were concentrated in the mid portion of the plot however mushrooms were scattered throughout the plot. Neither the direction of shiro advancement nor the extent, biomass or distribution of underground mycelium can be quantitatively inferred from this data. However, fruiting portions of the shiros of T. magnivelare are located in each of the mushroom sampling plots which wi l l allow longer term measurements to be done. As stated in the introduction to this thesis the original intention of the Nass pine mushroom ecological study was for mushroom sampling to occur over several years with the cooperation of both the Nisga'a Tribal Council and the Terrace District Ministry of Forests. It is hoped that longer term sporocarp production data wi l l continue to be collected at the study site because a single season of limited data is insufficient for either mushroom productivity, shiro or ectomycorrhizal analysis. The first year mushroom production results are preliminary, limited data is available, and it is too early to assess the long-term implications or draw meaningful conclusions. To gain an understanding of the fruiting patterns and developing colonies of macrofungi takes years of continued field and laboratory assessment. Five to ten years of frequent sampling may be required to generate meaningful data (Ammirati et al., 1987). Spatial and temporal patterns of sporocarp formation must be carefully studied in each of the plots over several years. Further sampling should be done in the plots paying special attention to the locations of host trees, host root systems, microtopography and potential impediments to shiro expansion. Locations of canopy openings, coarse woody debris and climatic conditions should also be monitored for these and other factors can also affect shiro development and sporocarp production. Seasonal fluctuations should be determined by examining a number of parameters such as sporocarp production and the horizontal and vertical distribution of mycelium and colonized rootlets over time (Parke, 1985). 1 2 1 Unfortunately, in the present study mushroom research plots were disturbed by commercial harvesters. Overall, for the three plots during three sampling periods, holes represented approximately 30 percent of the data points. In Plots 2 and 3 on the first sampling there were fewer actual sporocarps counted than indicative holes presumed left by the removal of sporocarps by commercial harvesters. There may have been even more mushrooms removed by harvesters which went undetected as resulting holes are often covered with leaf litter or moss post harvest. Further quantitative analysis was not attempted because there were too many holes in the data. In order to avoid indiscriminate commercial mushroom harvesting, research plots must be located in areas where access is either limited or controllable. Optimally in an area where commercial pickers are well enough informed not to disturb mushroom research sampling plots but rather to actively participate in cooperative data collection efforts. The impetus to harvest pine mushrooms is great. The high market value of pine mushrooms, combined with the 'goldrush' mentality of some mushroom harvesters may mean it wi l l take some time before research requirements are satisfied. In the meantime, frequent sampling and researcher presence throughout the season are the best ways to alleviate such problems at research sites accesible to the public. According to Hosford et al. (1997) sporocarp production data should be collected every 3 days throughout the fruiting season. Under optimal conditions mushrooms fruiting from the shiros in the plots would appear in the form of a 'fairy ring' - or disc-shaped colony expanding from the. original inoculum source (basidiospores or vegetative mycelium) (Ogawa, 1976). Over time the shiro is broken into arcs when impediments such as tree stems, rocks, suboptimal soil conditions, lack of host root-tips or competitors are encountered (Ogawa et al.,1981). By mapping sporocarps, host trees and root systems, as well as impediments to colony expansion, arcs of the expanding shiro could probably be delineated in each of the plots. Spororcarp production on arcs of actively expanding underground mycelium of the Japanese pine mushroom (7". matsutake) has been described by Ogawa (1975a, 1976, 1977a and others) and preliminary work has been done with T. magnivelare (Kinugawa and Goto, 1978; Hosford and Ohara, 1990; Hosford, 1994, unpublished; Hosford et al., 1997; Pilz, pers. comm., 1998; and the 122 present study). Much more T. magnivelare ecological research has been proposed or is underway in the U.S. Pacific Northwest (Pilz, pers. comm., 1998) as well as in British Columbia (Wills, pers. comm., 1998; Olivoto, pers comm. 1998). Large knowledge gaps still exist and enhanced, integrated biological and ecological research is needed to better understand pine mushroom productivity. Root-tip Morphotype Counts Prior to discussing the results of the root-tip assessments it must be noted that, as with the sporocarp counts, the first year root-tip morphotype analyses are preliminary. According to Parke (1985) a measurement of root-tip colonization from a single point in time is inadequate for assessing mycorrhizal colonization. With limited data available, it is too early to assess the long-term implications or draw meaningful conclusions regarding the distribution and abundance of the different root-tip morphotypes. A greater number of samples from a larger number of plots over a longer period of time need to be examined in order to generate sufficient information for analyses. The abundance of ectomycorrhizal root-tips isolated from soil core samples has however been examined over several years by many authors (e.g. Cripps, 1995; Vogt, 1985). The abundance of below ground ectomycorrhiza does not neccessarily represent the abundance of sporocarps produced (Cripps, 1995; Parke, 1985). Although quantitative assessment of root-tips may not necessarily correlate with abundance of fruit bodies, one would expect some associated colonized root-tips to be found beneath sporocarps of a given ectomycorrhizal fungus (Agerer, 1993). Indeed Ogawa (1975a), Tominaga (1975) and Lee (1991) all found that the blackened, carbonized ectomycorrhizae were most abundant beneath sporocarps of T. matsutake. The root-tip morphotype associated most abundantly with T. magnivelare sporocarps in the Nass Valley study plots in October, 1994 has been determined. Greater numbers of charcoal grey to black, 'matchstick' (morphotype #4) root-tips were found directly underneath pine mushroom fruit bodies. Whether the beneath sporocarp 'matchstick' numbers are significantly greater than the away from sporocarp numbers is uncertain at present. A l l three statistical tests employed assume that the counts for the plots were independent. The t-test also assumes that the differences came from a normally distributed population of differences. The two non-parametric tests (i.e. the sign test and 123 the sign-ranked test) do not assume a normal distribution. The assumptions could not be tested because of the small sample size, however the tests used are robust and showed significant differences for root-tip morphotype #4. However, the presence of large numbers of 'matchstick' morphotypes beneath T. magnivelare sporocarps in the Nass Valley is relevant. This, combined with descriptions of similar blackened root-tips found in soil directly beneath T. magnivelare sporocarps in Washington (Hosford et al., 1997) and in Asia beneath sporocarps of related species such as T. matsutake (Ogawa, 1975b) and T. bakamatsutake (Terashima, 1993), suggests that these were in fact T. magnivelare colonized root-tips. Microscopically, the absence of a well-defined mantle or well-developed Hartig net in root-tip morphotype #4 is also consistent with Hosford et al. (1997) description of the dark (blackish) T. magnivelare rootlets in Washington State as well as Ogawa's (1975b) and Lees's (1991) findings for T. matsutake colonized rootlets in Asia. Although in lower numbers, putatively T. magnivelare colonized 'matchstick' morphotypes were also found greater than 3 m away from visible pine mushroom sporocarps (i.e. 'outside' of the shiro) (Table 8). These may have been the result of root-tip colonization by dispersed, successfully germinated basidiospores. Alternatively, they could be part of the shiro at an earlier or later stage of development. Hosford et al. (1997) reported T. magnivelare colonies in Washington State measuring up to 6 meters in diameter Further core sampling should be undertaken to examine the shiro of T. magnivelare. By comparing rootlets in core samples taken seasonally, at increasing distances away from T. magnivelare sporocarps and at different depths, the size and depth of the colony, rootlet morphology, seasonal fluctuations in morphotype populations and other shiro characteristics could be examined. More detailed studies of shiro zonation similar to those done with T. matsutake in Japan and Korea wi l l assist in better understanding the below ground ecology of T. magnivelare. Identification of the other associated root-tip morphotypes to species combined with seasonal collection and analysis of root-tips over several years wi l l also help further describe the shiro of T. magnivelare. 124 These and other associated research projects are currently underway in the U.S. Pacific Northwest and preliminary results have recently been published (Hosford and Ohara, 1995; Hosford et al., 1997). Microbial interactions in the shiro of T. magnivelare should also be examined. A better understanding of the influence of soil microorganisms including soil bacteria, fauna and other fungi on the development of the under ground mycelia of T. magnivelare is needed. Hosford et al. (1997) indicated that studies of soil microflora associated with T. magnivelare are being initiated in Washington. The preliminary root-tip results are not conclusive evidence that morphotype #4 was associated with T. magnivelare. The limited number of samples assessed represent only a single point in time. Fluctuations over time in the number of rootlets available for colonization (Parke, 1985), the potential change in the number of root-tips colonized by a particular mycobiont (Cripps, 1995) or the number of active or inactive rootlets (Harvey et al., 1980) were not taken into account. As well, only Cenococcum geophilum was identified to species and the identity of the other morphotypes is at present uncertain. More definitive evidence could have been generated from molecular analysis of mycobiont DNA to identify which of the root-tips observed were in fact colonized by T. magnivelare. Egger (1996, pers. comm.), using polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) analysis (a technique for enzymatic amplification of DNA sequences from minute quantities of DNA (Mullis and Faloona, 1987)) has shown that tissue cultures isolated from T. magnivelare sporocarps produced virtually identical restriction digestion patterns to tissues taken directly from T. magnivelare sporocarps (Chapter 2 ). Since DNA sequences specific to the fungus are amplified directly, PCR-RFLP analysis can be applied to any tissue including cultures, fruit bodies or mycorrhizal roots (Egger, 1995; Egger, 1996, pers. comm.). According to Egger (1996, pers. comm.) PCR-RFLP analysis of excised 'matchstick' root-tips would determine conclusively whether or not 'matchstick' (morphotype #4) is in fact the 125 ectomycorrhizal habit of T. magnivelare. Excised root-tips of each morphotype should be subjected to DNA analysis in an attempt to ascertain their identities. Molecular analysis of DNA from 'matchstick' and the other root-tip morphotypes (e.g. white/tan #5) should be performed to determine i f they represented a different species or various stages of T. magnivelare rootlet colonization. The white/tan root-tips (morphotype #5) were similar, at least in terms of gross morphology, to the tanoak (Lithocarpus densiflorus (Hook and Arn.) Rehd.) ectomycorrhizae collected beneath a pine mushroom (Hosford, et al., 1997 - Appendix 6). A relatively thick mantle and an easily distinguishable Hartig net were observed on morphotype #5 but the microscopic characteristics were not reported from the tanoak ectomycorrhiza. Morphotype #5 also looked macromorphologically similar to the ectomycorrhiza of Russula xerampelina (Shaet. Seer.) Fr. on Picea abies (L.) Karst. rootlets as shown in the Altas of Ectomycorrhizae (Agerer, 1987b). It is likely that either the morphotype morphology varies at different stages in development (Terishima, 1993) or there was more than one type of ectomycorrhizal fungus that appeared as white mycelium over tan root-tips. Lee (1991) reported pale brown uncolonized T. matsutake rootlets. Terashima (1993) described a number of root-tip morphotypes found beneath T. bakamatsutake including a sheathless form with a poorly developed Hartig net. Morphotype #6 had what appeared to be a Hartig net (palmate hyphal invaginations on cortical cells) but no signs of a mantle. It is uncertain at present whether this was an earlier stage of T. magnivelare colonization or a different species with 'sheathless' ectomycorrhizae similar to those described by Al Abras et al. (1988). DNA analysis is again necessary to test these possibilities. Tricholoma magnivelare rootlets described by Hosford et al. (1997) lacked well-developed Hartig nets similar to those described by Japanese researchers for T. matsutake. There are indications that T. magnivelare may behave more like a parasitic fungus than a mycorrhizal one. Hypothetically, in the mycelial mat, T. magnivelare mycelium could colonize host rootlets initially forming a loose 'sheath' of mycelium on the surface. The overpowering of host defence systems may allow some intercortical colonization forming what appears similar to a Hartig net around cortical cells. Subsequently, rootlets release phenolics (toxic to fungi) and/or produce tannins in response to the 126 fungus which, combined with necrosis of the epidermal and cortical cells, turn the rootlet surface black. Subsequent senescence and sloughing of the mycelium would leave the rootlet surface black, coarse and fissured. This of course is unsubstantiated and many other possible infection scenarios could be proposed. According to Hosford et al. (1997) the dense mycelial mat of T. magnivelare suggests that it may be capable of aggressively weathering mineral soil through organic acid exudates. Many ectomycorrhizal fungi have some saprobic ability (Singer, 1975; Hosford et al., 1997). However, the mycelium of T. magnivelare does not appear to have the ability to appreciably degrade the major wood components cellulose, l ignin or pectin (Chapter 2). As discussed in the literature review, Ogawa (1975a) described seven distinct zones of the shiro of T. matsutake. Zone I I I is where fruit bodies formed and where the blackened T. matsutake root-tip morphotype was most abundant. Ogawa (1975a) and subsequently Kawai and Ogawa (1981) reported that the type of relationship formed between T. matsutake and the rootlets of Pinus densiflora resembled that of a parasitic fungus (i.e. appeared necrotic with darkened cortical cells). Ogawa (1975b) also described the initial appearance of brown pigments like tannins or resins in epidermal and cortical rootlet cells. The cortical cells turned brownish-black, eventually sloughing of outer layers followed established fungal infection. Lee (1991) agreed describing a dichotomously branched "witches broom" of necrotic, parasitized-looking pine rootlets associated with T. matsutake in Korea. He observed that in the latter stages mycorrhizae begin to wither and the roots blacken due to what he also believed to be the formation of tannins. Lee (1991) cited poor performance of host trees in T. matsutake producing areas to support the theory that T. matsutake may act parasitically. Symptoms similar to a type of root disease were reported on pine seedlings inoculated with T. matsutake (Tominaga, 1978). Indeed seedlings inoculated with T. magnivelare in the present study (Chapter 3) also performed poorly and signs of necrosis occurred on some of the western hemlock and Douglas f ir root systems. It is uncertain whether root necrosis was solely a function of the restrictive growing conditions produced by the Erlenmeyer flasks in which the seedlings were grown or partially due to a reaction to the 127 T. magnivelare mycelium because the control seedlings also performed poorly. Some lodgepole pine seedlings showed anomalous (slightly swollen, darkened) rootlets which may have been initial responses to the presence of the fungus in the synthesis system. It is too early to be certain and more research is needed. Whether mycelium of the North American pine mushroom (T. magnivelare) is capable of parasitic, saprophytic, or ectomycorrhizal growth has been further discussed in Chapter 2 (Enzyme tests) and Chapter 3 (Ectomycorrhizal Synthesis Trials) of this thesis. The importance of T. magnivelare to the B.C. economy combined with the lack of scientific information regarding its biology and ecology justify further research in this province. A long-term, integrated approach using sporocarp mapping, detailed examination of the development of the shiro and ectomycorrhiza identification using molecular analysis of DNA should be initiated. Study sites should be established in each of the major pine mushroom producing areas in the province in differing forest cover types. Tricholoma magnivelare colonies and potential host trees within study sites should be located and mapped to assist in determing the extent of potentially colonized host root systems. Other parameters such as the presence of obstacles to shiro development and the production of fruit bodies over several years should be studied. Associated plants, fungi, soil microflora and potential 'indicator' species should be determined. Research plots should be placed in areas where commercial harvesters cooperate in research efforts or areas which are inaccessible to commercial harvesters (e.g. helicopter access only). Thus far, the pine mushroom studies in the Nass Valley have served to give preliminary descriptions of the vegetation and soils associated with T. magnivelare fruiting sites in the study area. Preliminary methodology has been developed for the establishment of permanent sampling plots and the collection of T. magnivelare sporocarp production data. Some of the potential problems and limitations associated with this type of field research have been identified. The examination of root-tip morphotypes from soil collected directly beneath pine mushrooms has provided preliminary descriptions of the various types of root-tips found. A greater abundance of the charcoal grey to blackened ('matchstick') morphotype combined with descriptions given by other 128 researchers for the Japanese pine mushroom (T, matsutake) and those recently for T. magnivelare in Washington has helped to identify the colonized root-tips associated with T. magnivelare sporocarps. DNA analysis could confirm that these were in fact, T. magnivelare colonized rootlets. 129 General Conclusions Tricholoma magnivelare is the most economically important wild edible mushroom species in British Columbia, yet it is not well understood ecologically. Research is currently underway to elucidate its trophic character and ecological role in forest ecosystems. Enhanced scientific information wil l assist in developing long-term pine mushroom management strategies in the province. Although preliminary, the results of this research support claims that T. magnivelare has characteristics of an ectomycorrhizal fungus. The limited ability of its mycelium to appreciably degrade cellulose, lignin and pectin in vitro is consistent with other known ectomycorrhizal fungi. More strains should be tested and more sensitive tests applied to determine i f it does in fact have limited saprobic abilities. Initial results of the ectomycorrhizal synthesis studies indicate that lodgepole pine rootlets may be affected in the presence of T. magnivelare mycelium. However, it is uncertain at present whether the affected root-tips were a host response to the presence of the pine mushroom mycelium, the early stage of ectomycorrhizal colonization or simply a function of the adverse growing conditions in the synthesis system. The response appears similar in some respects to the initial stages of a host-parasite relationship. Longer term studies employing a wider range of isolates and hosts in a variety of synthesis systems are needed to clarify this. Mushroom sampling plots have been established in the Nass Valley Pine Mushroom Study Area. Sporocarp measurement data are preliminary and more information is required to determine i f the fruiting pattern is consistent with other known ectomycorrhizal fungi. However, some of the methodology and protocols necessary to examine this have been developed and potential research problems identified. The presence in soil directly beneath basidiocarps of abundant colonized root-tips similar in morphology to those described for related species (T. matsutake and T. bakamatsutake) in Asia, as well as for T. magnivelare in the Pacific Northwest, support the theory of a host-mycobiont relationship. The identity of the root-tip morphotypes should be confirmed using DNA analysis (PCR-RFLP). Knowledge of which morphotypes are in fact T. magnivelare colonized wil l allow better monitoring and assessment of host-mycobiont interactions and shiro development. 130 Whether this host-mycobiont relationship is strictly mycorrhizal or somewhat parasitic remains unclear at present. Tricholoma magnivelare may have more than one trophic character, as is suspected for the Japanese pine mushroom (T. matsutake). The dense mycelial mat in the organic layers and upper mineral soil may even have some limited saprobic ability. Regardless of its ecological role more comprehensive pine mushroom inventories are required. Productivity levels need to be determined in different pine mushroom ecosystems. 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Pure culture synthesis of bearberry mycorrhizae. Can. J. Bot. 54: 1297-1305. Zar, J.H. 1974. Biostatistical Analysis. Prentice-Hall, Englewood Cliffs, New Jersey. 620 p. Zeller, S.M. and Togashi, K. 1934. The American and Japanese Matsu-takes. Mycologia 26: 544-558. 149 Appendix 1. Clearing and staining procedures used for root systems from synthesis flasks. STAINING PROCEDURE 1. Heat roots in 10% KOH at 90°C (don't let boil) for approx. 1 hour (w/v). 2. Wash roots with tapwater (at least 3 times). 3. Bleaching roots: bleach for 1-2 hrs. Prepare just before use: 30 ml of 10% H 2 0 2 or 3 ml of 30% H 2 0 2 + 3 ml N H 4 O H + 567 ml (or 587 ml) dH 2 Q Total Volume 600 ml Let them get to light yellow but don't leave too long; wash 3X in tap water. 4. Last wash add a few drops of lactic acid in distilled water. 5. Let sit in acidified water for at least 10 minutes. 6. Stain using lactic acid:glycerol:distilled water in a 1:1:1 proportion and 0 .1% trypan blue stain (w/v). 7. Heat with staining solution at 90°C for at least 30 minutes or leave sit overnight. 8. Destain using lactic acid:glycerol:distilled water in a 1:1:1 proportion. 9. Examine under the microscope. 150 Appendix 2 Summary of silc attributes for the three pine mushroom plots. Attribute Plot 1 Plot 2 Plot 3 Biogeoclimatic zone/subzone/variant lCHmc2 Moist Cold, Hazelton variant ICHmc2 Moist Cold, Hazelton variant ICHmc2 Moist Cold, Hazelton variant Site series 01(l)b Hw - Step moss, Submesic phase 01(l)b Hw - Step moss, Submesic phase 01(l)b Hw - Step moss, Submesic phase Map sheet (1:50 000) 103 P/3 103 P/6 103 P/6 Latitude (approx.) 55° 14.8' •<• 55° 15.5' 55° 15.5' Longitude (approx.) 129° 9' 129° 9.9' 129° 11 r Elevation (m) 110 195 516 Slope (%) 3 13 19 Aspect (°) 110 160 136 Landforra Glaciofluvial terrace Glaciofluvial terrace Morainal blanket Soil classification Orthic Humo-Ferric Podzol Orthic Humo-Ferric Podzol Eluviated Dystric Brunisol Humus form classification Hemimor Hemimor Hemimor Soil texture Loamy sand/sand Loamy sand/sand Sandy loam/loamy sand Course fragment content 30-80% 22-55% 20-45% Soil moisture/nutrient regimes 2/B 2+/B 3/B (from: Trowbridge and Macadam, 1996) 151 Appendix .1. Summary' of vegetation data for the three pine mushroom study plots. Species Stratum Plot 1 Plot 2 Plot 3 Mean Constancy' Trees: Percent cover Pinus contorta A 25 20 15 20 1.00 B 25 10 18 0.67 C 0.00 Tsuga heterophylla A 10 35 23 0.67 B 40 60 20 40 1.00 C 3 15 9 0.67 Total tree stratum: Al l 65 80 80 75 1.00 Shrubs: Alnus rubra B <1 <1 0.33 Menziesia ferruginea B 1 <1 1 0.67 Paxistima myrsinites B 2 2 0.33 Rub us parviflorus B 1 T 1 0.33 Sorbus scopulina . B <1 T <1 0.67 Vaccinium membranaceum1 B 2 4 4 3 1.00 Vaccinium parvifolium B 1 1.5 3 2 1.00 Total shrub stratum: B 6 7 8 7 1.00 Herbs: Chimaphila urtibellata C <1 1 2 2 1.00 Clintonia uniflora C <1 <1 0.33 Cornus canadensis C 1 1 1 0.67 Goodyera oblongifolia C <1 1 2 2 1.00 Linnaea boreal is C 1 1 0.33 Orthilia secunda C 1 1 0.33 Pyrola asarifolia C <1 <1 <1 0.67 Total herb stratum: C 1 6 6 4 1.00 Moss and lichens Cladina spp. D 1 1 0.33 Dicranum ssp. D 5 5 0.33 Hylocomium splendens D 15 20 30 22 LOO Peltigera spp. D <1 <1 0.33 Pleurozium schreberi D 65 > 3C ) 25 41 1.00 PliUium crista-castrensis D < 1 4 0.67 Rhylidiadelphus triquetrus D 1( ) 1C 0.33 Rhytidiopsis robusta D ( > K ) 3i t IS 1.00 Total moss and lichen stratum: D 9' 1 T. 5 9! > 85 > 1.00 ' Constancy refers to the proportion of plots in which the species were found. 'The i" membranaceum looked somewhat like V. alaskaensc in that leaf margins were not consistently fine-toothed, however other I", alaskaense characteristics were lacking. (from: Trowbridge and Macadam. 1996) 152 Appendix 4 Soil profile descriptions Tor the three pine mushroom study plots. Plot 1 soil profile description (20.8m @ 52° from NW corner): Horizon S/L Fml Fm2 Bf (Ae) Bm BC Depth (cm) (2) 4-1 1-0, 0-18 (pocket) 18-38 38-62 62+ P R O F I L E DIAG RAM S / L Description Bryophyte layer dominated by Pleurozium schrebeh and Hylocomium splendens, intermixed with individual particles of newly accreted litter (leaves, needles, twigs, and cones). Compact-matted; slightly decomposed fibric material; abundant fine and medium roots. Matted and tenacious; abundant gray and white mycelia resembling an Ae horizon in color; abundant charcoal at interface. Strong brown (7.5 YR 4/6, moist); loamy sand; 30% coarse fragments; very weak subangular blocky to single grain; plentiful to abundant fine, medium and coarse roots; clear, wavy boundary. Light brownish gray (10YR 6/2, moist); loamy sand; 30% coarse fragments; very weak subangular blocky to single grain; few fine and medium roots; abrupt, broken boundary. Dark yellowish brown (10YR 4/5, moist); sand; 40% coarse fragments; very weak subangular blocky to single grain; plentiful medium, few fine and coarse roots; clear, smooth boundary. Dark to dark yellowish brown (10YR 3.5/3, moist); sand, 80% coarse fragments (60% stones); single grain, few fine roots; gradual smooth boundary. Dark grayish brown (2.5Y 4/2, moist); 50+% coarse fragments; single grain, very few occasional fine roots; also in turbated area left side, mid-profile. Horizon Total C C/N CEC exch. Ca exch. K exch. Mg Total N MinN Avail. P ext. Fe ext. Al pH % Ratio Meq./IOOG % ppm % (CaCl2) Fml&2 39.87 53.16 42.69 24.97 2.66 5.45 0.75 128.6 69.4 0.241 0.235 3.79 Bf 1.26 14.85 8.57 0.07 0.07 0.02 0.08 1.6 64.2 0.283 0.385 4.53 Bm 1.15 15.63 7.94 0.15 0.05 0.01 0.07 0.8 85.6 0.112 0.238 4.87 BC 0.73 10.85 6.71 0.13 0.05 0 0.07 0.8 94.9 0.102 0.22 5.08 C 0.6 9.35 5.25 0.16 0.03 0.02 0.06 07 98.8 0 131 0.265 5.08 (from: Trowbridge and Macadam. 1996) 153 Appendix 4. Soil profile descriptions for the three pine mushroom study plots (con't). Plot 2 soil profile description (20.8m @ 46° from the NW plot comer): Horizon S/L Fml Fm2 Ae Bfl Bf2 BC Depth (cm) (2) 4-3 3-0 0-1 1-14 14-32 32-54 54-60+ Description Bryophyte layer dominated by Pleurozium schreberi and Hylocomium splendens, intermixed with individual particles of newly accreted litter (leaves, needles, twigs, and cones). Compact-matted and tenacious; slightly decomposed fibric material; abundant fine and medium roots. Matted, moderately decomposed; abundant gray and white mycelia with few yellow near decaying wood; common, medium charcoal at interface, 10% decaying wood. Light brownish gray (10YR 6/2, dry); loamy sand; 25% coarse fragments; very weak to moderate subangular blocky, to single grain; abundant gray and white mycelia contributing to color; few fine and medium roots; abrupt, smooth boundary. P R O F I L E D I A G R A M S / L Q BC O Strong brown (7.5YR 4/6, moist); loamy sand; 30% coarse fragments; very weak subangular blocky to single grain; small patches of gray and white mycelia; plentiful to abundant fine, medium and coarse roots; clear, smooth boundary. Brown to dark brown (10YR 4/3, moist); sand; 40% coarse fragments; very weak to moderate subangular blocky, to single grain; plentiful medium, few fine and coarse roots; clear, smooth boundary. Light olive brown (2.5YR 5/4, moist); sand; 50% coarse fragments; single grain, very few fine roots; gradual smooth boundary. Dark grayish brown (2.5Y 4/2, moist); 55% coarse fragments; single grain. Horizon Total C C/N CEC exch. Ca exch. K exch. Mg Total N MinN Avail. P ext. Fe ext. Al PH % Ratio Meq./IOOG % ppm °/ (CaCl2) Fml&2 42.66 93.17 42.19 3.47 3.06 2.09 0.46 7.7 121.6 0.275 0.276 3.25 Bfl 1.2 12.05 10.16 0 0.19 0.01 0.1 4 110.8 0.42 0.602 3.95 Bf2 0.37 '4.95 5.65 0.02 0.07 0.01 0.08 0.9 50.7 0.136 0.276 4.72 BC 0.35 5.02 5.15 0.02 0.06 0 0.07 0.6 44.3 0.105 0.221 4.94 C 0.33 5.22 4.89 0.03 0.04 0 0.06 0.2 30.5 0.085 0.18 4.84 (from: Trowbridge and Macadam, 1996) 154 Appendix 4. Soil profile descriptions for the three pine mushroom study plots (con't). Plot 3 soil profile description (20.7m @ 41° from NW plot comer): P R O F I L E D I A G R A M Horizon Depth (cm) Description S/L (2) Bryophyte layer dominated by Rhytidiopsis robusla, Hylocomium splendens, and Pleurozium schreberi, intermixed with individual particles of newly accreted litter (leaves, needles, twigs, and cones). Fm 0-3 Compact-matted and tenacious, slightly to moderately decomposed fibric material, abundant fine and medium roots; abundant gray and white mycelia, no charcoal present. Ae 0-5 Light brownish gray (10YR 6/2, moist); sandy loam; 20% coarse fragments; weak fine subangular blocky to single grain; abundant fine and medium roots; very hydrophobic due to mycelia; abrupt, smooth to broken (discontinuous) boundary. Bfj 5-17 Brown to.strong brown (7.5YR 5/5, moist); loamy sand to sand; 35% coarse fragments; weak, fine subangular blocky to single grain; plentiful to abundant fine, medium and coarse roots; clear, wavy boundary. Bm 17-32 Dark yellowish brown (10YR 4/6, moist); loamy sand to sand; 40% coarse fragments; weak fine subangular blocky to single grain; abundant fine and medium roots; clear, wavy boundary. BC 32-53 Brown (10YR 5/3, moist); loamy sand; 45% coarse fragments (60% stones); weak fine subangular blocky; few fine roots; gradual smooth boundary. C 53-60+ Yellowish brown (10YR 5/4, moist); loamy sand; 45% coarse fragments; single grain, very few fine roots. Horizon Total C C/N CEC exch. Ca exch. K exch. Mg Total N Miri N Avail. P ext. Fe ext. Al pH % Ratio Meq./IOOG % ppm % (CaCI2) Fm 51.33 53.34 45.85 10.47 4.6 4.41 0.96 24.3 122.6 0.261 0.154 3.74 Ae 3.09 39.35 18.09 0.32 0.27 0.5 0.08 1.1 2.0 0.21 0.153 3.35 Bfj 1.01 13.36 9.18 0.04 0.19 0.06 0.08 0.8 23.7 0.211 0.382 4.67 Bm 0.69 9.8 6.83 0.06 0.11 • 0.05 0.07 11 43.9 0.114 0.236 5.03 BC 0.31 6.76 4.69 0.09 0.06 .0.05 0.05 0.6 56.5 0.118 0.218 5.17 C 0.31 4.71 4.5 0.09 0.07 0.11 0.07 0.8 41.7 0.144 0.205 4.81 (from: Trowbridge and Macadam. 1996) 155 Appendix 5. Pine mushroom grading system. Grade 1 "Button"; veil completely intact. Grade 3 Less than 50 per cent veil intact. Grade 5 Mature mushroom with flat cap. veil Grade 2 More than 50 per cent veil intact. Grade 4 Vei l completely broken; cap edges rounded. Grade 6 Over-mature mushroom; may be wormy. (from: de Geus. 1995b) 156 Appendix 6. MORPHOTYPE 2 DESCRIPTION. Ectomycorrhiza type: Fungus species: Plant species: Collect information: #2 Cenococcum geophilum Tsuga heterophylla, Pinus contorta Plot #1 Boogie Heaven. September/94. Beneath T. magnivelare sporocarp. Date: Described by: Most distinctive characteristic(s): Habit: Colour: Rhizomorphs or strands: Emanating hyphae: Abundance: Diameter: Clamps: Septation: Ornamentation: Cystidia: Mantle: Thickness: Surface texture: Outermost layer: Tissue type: Colour: Hyphal diameter: Hartig net: Other descriptions: February 1/95 S.M. Berch and F.W. Fogarty Jet black mycorrhizae with bristle-like emanating hypha or hairs. Stellate pattern of mantle (synenchymous). Unbranched (monopodial) mantle, smooth to woolly. Black mantle. Absent Present, wide/bristly often bent or broken. Abundant 4-5 urn None seen. Septate Pigmented wall. Absent Black, pigmented, stellate pattern. 3-5 um Smooth with bristle-like hyphae. Synenchymous - stellate pattern. Dark brown. 3-5 um Hyaline (hyphal) invaginations (1-3 um wide). Roth (1990). 157 Appendix 6 (con't). MORPHOTYPE 3 DESCRIPTION. Ectomycorrhiza type: Fungus species: Plant species: Collect information: #3 'Cenococcum-like' Tsuga heterophylla, Pinus contorta Plot #1 Boogie Heaven. September/94. Beneath T. magnivelare sporocarp. Date: Described by: Most distinctive characteristic(s): Habit: Colour: Rhizomorphs or strands: Emanating hyphae: Abundance: Diameter: Clamps: Septation: Ornamentation: Cystidia: Mantle: Thickness: Surface texture: February 1/95 S.M. Berch and F.W. Fogarty Black mantle, no bristles and no stellate pattern microscopically. Unbranched tips. Black. Absent. Present. Common. 3-5 um None seen. Septate. Pigmented wall. Absent Pigmented. 3-5 um Smooth. Hartig net: Other descriptions: Palmate invaginations (approx. 2-5 um wide). Berch et al. (1984) discuss a 'cenococcum-like' anamorph common in many ectomycorrhizal communities. 158 Appendix 6 (con't). MORPHOTYPE 4 DESCRIPTION. Ectomycorrhiza type: Fungus species: Plant species: Collect information: Date: Described by: Most distinctive characteristic(s): Habit: Colour: Rhizomorphs or strands: Abundance: Hyphal diameter: Clamps: Septation: Emanating hyphae: Cystidia: #4 Unknown (potential T. magnivelare) Tsuga heterophylla, Pinus contorta Plot #1 Boogie Heaven. September/94. Beneath T. magnivelare sporocarp. February 1/95 S.M. Berch and F.W. Fogarty Charcoal grey to black, slightly swollen, elongated root-tip with a slight constriction between the swollen, colonized apical portion and narrower proximal uncolonized portion of the rootlet (Figures 38 and 41). Unbranched. Charcoal grey to black. None seen however rootlets were embedded in a dense mycelial mat beneath sporocarps. Mycelium from the base of a primordium was examined microscopically and found to consist of a mass of septate, branched hyaline hyphae with no apparent clamp connections (Figure 33). Mycelium taken from the mycelial mat in which rootlets were embedded and appearing to be attached to the base of the primordium also had hyaline hyphae with slightly swollen, 'ball-joint-like' septae with what appeared to be septal constrictions (Figure 33). Hyphal branching in the form of 'H'-shapes (anastamoses) were apparent at numerous locations in the mycelium (Figure 34). Mycelial mat dense in root mat. 2-4 um None seen [H-structures (anastomoses) (Figure 34)]. Septate, swollen look like 'ball-joints' (Figure 33). None seen. Absent 159 Appendix 6 (con't). MORPHOTYPE 4 (CON'T) Mantle: External surface of rootlets coarse, fissured and charcoal grey to black. A fine dusting of remnant mycelium was observed on the surface of most rootlets. No definitive mantle or compact fungal hyphae found in the form of a sheath on the surface of black root-tips. However, what appeared to be a thin, loose mycelial sheath was observed on longitudinal root-tip section (1000X oil immersion) at transition zone from the mycelial layer covering rootlets to the darkened, brown thickened zone behind the root-tip (Figure 39). The brown, thickened zone (Figure 41) could be caused by build-up of tannins or pigments in the epidermis or disrupted, necrotic cortical cells. Surface texture: Outermost layer: Tissue type: Colour: Hartig net: Photographs and illustrations: Other descriptions: Coarse, fissured. 'Dusting' of remnant mycelium. Coarse on some could be disrupted necrotic epidermal and cortical tissue. Charcoal gray to black. No definitive Hartig net was discernible however longitudinal section through root-tips showed what appeared to be palmate hyphal invaginations (finger-like projections surrounding epidermal and outer cortical cells) (Figure 39 - 1000X oil immersion). (Figures 31, 38 and 41). Similar to Ogawa's (1975a) and Kawai and Ogawa's (1981) descriptions of Tricholoma matsutake 'ectomycorrhizae' and Hosford et al. (1997) description of purportedly T. magnivelare colonized rootlets. 160 Appendix 6 (con't). MORPHOTYPE 5 DESCRIPTION. Ectomycorrhiza type: Fungus species: Plant species: Collect information: Date: Described by: Most distinctive characteristic(s): Habit: Colour: Rhizomorphs or strands: Abundance: Diameter: Consistency: Surface texture: Hyphal diameter: Clamps: Septation: Ornamentation: Emanating hyphae: Abundance: Diameter: Clamps: Septation: Ornamentation: Cystidia: Mantle: Thickness: Surface texture: #5 Unknown (potential T.magnivelare - early stages of colonization). Tsuga heterophylla, Pinus contorta Plot #1 Boogie Heaven. September/94. Beneath T. magnivelare sporocarp. February 1/95 S.M. Berch and F.W. Fogarty White mycelium overlying a tan (light, cinnamon coloured) rootlet. Occasional branching. White mycelium on tan-coloured root. Strands common. Approx. 8.5 um. Dense near root, denser at distance from root. Approx. 1.8 um No Yes No Common, hyaline. Common. 2 um None seen. Septate No. Absent Present. Relatively thick (or thin with patches of extramatrical hyphae). Hairy-cottony. 161 Appendix 6 (con't). MORPHOTYPE 5 DESCRIPTION (CON'T). Outermost layer: Tissue type: Colour: Hyphal diameter: Innermost layer: Tissue type: Colour: Hyphal diameter: Loose (patches of organized prosenchymous tissue?). Hyaline. <2 um Thin, (densly packed). Hyaline. 1-2 um (variable). Hartig net: Present and distinguishable (abundant palmate hyphal invaginations). Photographs and illustrations: Similar in gross morphology to root-tips photographed by Hosford et al. (1997) - plate 7 and Agerer (1987) - plate 2. Other description: Gross morphology similar to the tanoak (Lithocarpus densiflorus (Hook and Arn.) Rdhd) ectomycorrhizae collected directly beneath a T. magnivelare sporocarp (Hosford et al., 1997 - plate 7). However, not similar microscopically to those described for T. magnivelare by Hosford and Ohara (1995) in that morphotype #5 had a well-defined mantle and Hartig net. Morphotype #5 was also similar in gross morphological appearance to Russula xerampelina (Shaeff. Seer.) Fr. on Picea abies (L.) Karst. rootlets as depicted by Agerer, 1987 (atlas - plate 2). Morphotype #5 may in fact represent more than one type of fungus both of which appear similar in gross morphology (i.e. white mycelium/tan rootlet). 162 Appendix 6 (con't). MORPHOTYPE 6 DESCRIPTION. Ectomycorrhiza type: Fungus species: Plant species: Collect information: #6 . Unknown. Potential T. magnivelare? Tsuga heterophylla, Pinus contorta Plot #1 Boogie Heaven. September/94. Beneath T. magnivelare sporocarp. Date: Described by: Most distinctive characteristic(s): Habit: Colour: Rhizomorphs or strands: Emanating hyphae: Cystidia: Mantle: Hartig net: Other Description: February 1/95 S.M. Berch and F. W. Fogarty Golden brown rootlet with no visible mantle but 'Hartig net' or finger-like hyphal projections microscopically. Tips unbranched and appearing non-mycorrhizal. Golden brown. Absent Absent Absent Absent Finger-like projections (palmate hyphal invaginations) approx. 1-3 um wide, no clamps seen. Similar to 'sheathless' ectomycorrhizae described by Abras et al. (1988) with no mantle present but obvious Hartig net. Terashima (1993) described a 'sheathless' ectomycorrhizae with a poorly developed Hartig net assiciated with T. bakamatsutake. 163 Appendix 7. Results of t-tests, sign, and sign-ranked tests of root-tip data. Root-tip data arranged for statistical analysis. Locations directly beneath pine mushrooms (Loc 1) or >3 m away from pine mushrooms (Loc 2). Plot Loc Typel Type2 Type3 Type4 Type5 Type6 Type7 Total 1 1 1792 1840 1120 5248 1024 1008 2432 14464 1 2 1904 7472 3312 480 560 496 1568 15792 2 1 896 2336 792 3988 280 952 3028 12272 2 2 1108 5440 3804 512 336 388 2464 14052 3 1 1476 2044 2324 4744 748 172 1872 13380 3 2 1200 4536 2604 296 304 3966 2264 11600 Results of t-tests. DIFF1-7 refers to differences between numbers directly beneath pine mushrooms (Loc 1) and those >3 m away from pine mushrooms (Loc 2) for root-tip morphotypes 1-7. Root-tip variable Mean Std. Dev. T Prob>/T/ DIFF1 -15.3333 257.016 -0.103332 0.9271 DIFF2 -3742.67 1664.58 -3.894372 0.0601 DIFF3 -1828.00 1401.90 -2.258495 0.1524 DIFF4 4230.67 672.8605 10.89041 0.0083 DIFF5 284.000 294.6183 1.669625 0.2369 DIFF6 284.000 440.7085 1.116162 0.3805 DIFF7 345.333 655.9308 0.911887 0.4581 Results of sign tests and signed-rank tests. Root-tip variable M(Sign) Prob>=/M/ S(Sign Rank) Prob>=/S DIFF1 -0.5 1.00 0 1.00 DIFF2 -1.5 0.25 -3 0.25 DIFF3 -1.5 0.25 -3 0.25 DIFF4 1.5 0.25 3 0.25 DIFF5 0.5 1.00 2 0.50 DIFF6 0.5 1.00 2 0.50 DIFF7 0.5 1.00 2 0.50 Note: M(Sign) and S(Sign Rank) are test statistics for the sign test and signed-rank test respectively. 164 

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