Mineral Weathering And Ectomycorrhizae Of Picea glauca x engelmannii (Moench.) Voss With Emphasis On Piloderma By Kevin R. Glowa B.Sc. 1996. University ofBritish Columbia in association with the Okanagan University College THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE m NATURAL RESOURCES AND ENVIRONMENTAL STUDIES © Kevin R. Glowa, 2000 The University ofNorthem British Columbia March 2000 All rights reserved. This work may not be reproduced in whole or in part, by photocopy or other means, without the permission of the author. Abstract Piloderma is one of the most recognizable ectomycorrhizal fungi in many forest ecosystems in North America. It is a broad host range fungus commonly observed in many economically important coniferous and deciduous trees in central interior British Columbia. Piloderma may benefit these forests through increased soil mineral weathering and nutrient availability. This thesis investigates the role of Piloderma in the weathering of common soil minerals to supply K and/or Mg in the rhizosphere soils of Picea glauca. I also documented the changes in the physical, chemical and mineralogical properties of rhizosphere soils influenced by Pilodermaand other common ectomycorrhizal fungi . In the first study, I investigated the role of Piloderma in weathering and extracting K and/or Mg from biotite, microcline, and chlorite. Specifically, I compared the in vitro growth, morphology, and chemical properties of Piloderma growing within three K and/or Mg-containing minerals that are commonly found in soils of central interior British Columbia. Piloderma cultures were grown for 110 days in medium enriched with K and/or Mg from biotite, microcline, and chlorite, in K and Mg poor media and in medium optimized for proper fungal growth. Chemical analysis indicated that Piloderma more efficiently weathered biotite to obtain K over microcline. The different growth, morphologies, and small amounts ofMg found in all treatments indicated that chlorite and biotite provide a sufficient supply ofMg. Piloderma grown in treatments with insufficient K showed distinct fibrillar growths, hypha! swellings, and hyphae devoid of ornamentation and could be indicative of nutrient deficiency. This study indicated that considerable amounts of K and minor amounts of Mg are essential for !"roper Piloderma growth and, perhaps more that Piloderma could provide sufficient amounts of K from the weathering of biotite. In the second study, the pH, total C and N, cation exchange capacity, and the contents of mica, chlorite, kaolinite, 2:1 type expandable clays, and amorphous materials were compared between two ectomycorrhizosphere soils (soils containing considerable ectomycorrhizal colonization) and non-ectomycorrhizosphere soils (bulk) of Picea glauca x engelmannii (Moench.) Voss) to elucidate the role of ectomycorrhizae on the chemical and mineralogical properties of soils in the field. The two ectomycorrhizosphere soils were 11 characterized by colonization dominated by (1) Piloderma, and (2) Inocy be lacera-like and · Hebeloma-like morphotypes or where Piloderma colonization was <1%. Results showed that pH was one unit lower in ectomycorrhizosphere compared to nonectomycorrhizosphere soils. Total C and N were significantly higher in ectomycorrhizosphere soils where C was three times higher and N was two times higher than non-ectomycorrhizosphere soils. Cation exchange capacity as well as exchangeable K+, and Na+ were higher in ectomycorrhizosphere soils compared to bulk soils where K+ >Na+. Base saturation was significantly lower in non-ectomycorrhizal soils compared to both ectomycorrhizosphere soils. Compared to bulk soils X-ray diffraction did suggest the transformation of mica and chlorite to 2:1 type expandable clays in ectomycorrhizosphere soils. lll TABLE OF CONTENTS Abstract 11 Table of Contents IV List of Tables Vll List ofFigures Vlll Acknowledgements X CHAPTER I INTRODUCTION 1 CHAPTER II REVIEW OF THE RELEVANT LITERATURE 4 2.1 What are mycorrhizae? 4 2.2 Rhizosphere 4 2.3 Fungal dynamics 4 2.4 Biologically induced mineral weathering 5 2.4.1 5 2.5 CHAPTER III What is mineral weathering? 2.4.2 Roots 6 2.4.3 7 Soil microorganisms 2.4.4 Fungi 7 Nutrient allocation 8 THE WEATHERING OF BIOTITE AS A POTASSIUM 11 SOURCE FOR PILODERMA SP. 3.1 Introduction 11 3.2 Meth0ds 13 3.2.1 Isolation of Piloderma 13 3.2.2 Plate preparation and inoculation 14 3.2.3 Growth rate 15 3.3 3.2.4 Morphological and chemical analysis 15 3.2.5 16 Statistical analysis 16 Results 3.3.1 Growth 16 3.3.2 Morphology 17 IV 3.3.3 Chemical composition of ornaments and 18 hyphae between treatments 3.3.4 Chemical composition of ornaments and 18 hyphae within treatments 3.4 Discussion 19 3.4.1 19 Growth of Piloderma 3.4.2 Morphology of ornaments and hyphae 20 3.4.3 Chemical composition of ornaments and 21 hyphae 3.4.4 3.5 Chapter IV Weathering of minerals 21 3.4.5 Concluding remarks 22 Literature cited 23 Chemical and Mineral Composition of Rhizosphere Soils 34 Influenced by Ectomycorrhizae in Hybrid Spruce (Picea glauca x engelmannii (Moench.) Voss) 4.1 Introduction 34 4.2 Methods 35 4.2.1 35 4.3 4.2.2 Sample collection 36 4.2.3 Mycorrhizal assessment 37 4.2.4 Physical and chemical analysis 37 4.2.5 Mineral composition 38 4.2.6 Statistical analysis 39 Results 39 4.3.1 4.4 Description ofstudy area Ectomycorrhizal colonization 39 4.3 .2 Physical and chemical properties 40 4.3.3 40 Mineral composition Discussion 41 4.4.1 Ectomycorrhizal colonization 41 4.4.2 Chemical properties of ECS and N-ECS soils 43 4.4.3 Mineral composition of ECS and N-ECS soils 44 v 4.4.4 4.5 Concluding remarks Literature Cited 46 47 CHAPTER V CONCLUSION AND RECOMMENDATIONS 62 CHAPTER VI LITERATURE CITED 64 VI LIST OF TABLES P. 3.1 Composition ofMMN (Marx 1969) and modified MMN (MMN2) and 28 the relative amounts of K and Mg in each of the stock solutions and dry additives. 3.2. Mean SEM-EDS weight percentages for elemental composition of 29 Piloderma ornamentation and hyphae grown in biotite, chlorite, and microcline enriched nutrient poor (MMN2), nutrient deficient (MMN2), and nutrient rich (MMN) media. (n=3) 4.1 Mean abundance (%) of ectomycorrhizae present in the 55 ectomycorrhizosphere A (ECS-A) and B (ECS-B) samples for white spruce. (n=6) 4.2 Morphological descriptions of the dominant ectomycorrhizae of white 56 spruce in Ae horizon of Luvisol. 4.3 Mean values for physical and chemical properties of two 58 ectomycorrhizosphere (ECS-A and ECS-B) and nonectomycorrhizosphere (N-ECM) soils ofwhite spruce. (n=6) 4.4. Mean contents (g kg" 1) of selected phyllosilicates and oxides in the clay fraction in non-ectomycorrhizosphere (N-ECM) and ectomycorrhizosphere soils of white spruce. (n = 6) Vll 59 LIST OF FIGURES P. 3.1 Mean daily temperature for the 110-day growth period starting at day 7. 30 3.2 a. Petri-lid used to assure that minerals added to the center of plate were 31 not dispersed over entire agar surface. b. A glass rod bent at the end was used to spread mineral over MMN2 agar. c. Prepared growth plate showing mineral circle and mineral edge markers (arrows). d. Piloderma at 16 days on biotite. e. Piloderma at 56 days growth growing past the biotite edge and mineral edge, markers. f. At 110 days growth Piloderma completely grew through plate. Outside of the mineral edge markers samples were taken (tapered arrows) for chemical and morphological analysis. g. Biotite, a K and Mg-rich trioctahedral mica, showing layer structure. h. Chlorite, a Mg rich 2:1:1 layer silicate. i. Microcline, a K-rich feldspar, showing crystalline structure. 3.3 Growth of Piloderma modeled using the logistic growth equation 32 growth (mm)=aM/a+(M-a)e-km(Days>, where a=intercept, M=maximum theoretical growth, k= shape and slope in (a) biotite, (b) chlorite, and (c) microcline enriched nutrient poor (MMN2), (d) nutrient poor (MMN2), and (e) nutrient rich (MMN) growth media for the 11 0 day growth period. Approximate locations of lag (open arrows), exponential growth (closed arrows), and stationary phases (solid arrows) are indicated in the chlorite treatment. (a, M, k superscripted with similar letters a-e are not significantly different)(n=5) 3.4 Scanning electron micrographs of Piloderma lenticular (arrowheads), and verrucose (non-tapering arrows) ornamentation, and hyphae grown in (a) biotite, (b) chlorite, and (c-d) microcline enriched nutrient poor (MMN2), (e-i) nutrient poor (MMN2), and (j-k) nutrient rich (MMN) growth media. c-i. Microcline and nutrient poor (MMN2) treatments showing characteristic fibrillar growth (tapered arrows). g. Swollen hyphal cell characteristic of nutrient poor (MMN2) treatment. Vlll 33 4.1 X-Ray diffraction patterns ofthe clay fraction ofECS-B sample 60 subjected to four pre-treatments showing the relative intensities of the 1.7-, 1.4-, 1.0-, 0.71-, 0.42-, 0.35-, and 0.33-nm reflections. 4.2. X-ray diffraction patterns of Ca-saturated and glycerol-solvated clay fractions ofECS-A, ECS-B, and N-ECS samples showing the relative intensities of the 1. 7- and 1.4-nm reflections. IX 61 ACKNOWLEDGEMENTS I would like to acknowledge the Natural Sciences and Engineering Research Council for financial support and Dr. Joselito Arocena for the additional contracts that kept me financially happy over the duration of my M.Sc. degree. Thanks to Dr. Joselito Arocena who, was my right hand man and supervisor throughout the project. Without his guidance, this project would not have been completed in my lifetime. Special thanks to Dr. Hugues Massicotte, Ms. Linda Tackaberry and Dr. Dave Dick for advice and guidance throughout the project. For laboratory analysis, I would like to thank Caroline von Schilling for the many hours put forth on the SEM-EDS. If it was not for her, I would have went insane. In addition, I would like to thank Dr. D. Dick and Mr. Allan Esler for their help in the central equipment laboratory. I would like to express my appreciation to (1) Riverside Forest Products in Armstrong, B.C, for giving me a wicked, high paying, rewarding weekend student job that kept me glued to the books; (2) Dr. Dan Durall of Okanagan University College for introducing me to the world of ectomycorrhizae; and (3) my parents, Dennis and Myrna, for too many things that I can possibly list. X Chapter I - Introduction Soil supplies plants with necessary elements for proper growth largely from the transformation or weathering of primary minerals enhanced by the activity of microorganisms. Biogeochemical processes in soil weathering occur largely in soil microenvironments where pH (Nye 1981; Marschner and Romheld 1983), cation-exchange equilibria (Chung and Zasoski 1994), organic carbon/acid concentrations (Gardner et al. 1982), metal availability (Sarkar and Wyn Jones 1982), monosaccharide contents (Dormaar 1988), grain-size distribution (Sarkar et al. 1979), moisture, and mineral composition can be different from that ofbulk soil or soil influenced to a lesser extent by the activity of living roots. One important and least understood microenvironment is the area surrounding the rootlets of plants called the rhizosphere. First introduced by L. Hiltner in 1904, rhizosphere was defined as that volume of soil surrounding roots in which growth is stimulated. Currently, the term rhizosphere has been broken down to include the endorhizosphere, rhizoplane, and ectorhizosphere (Sorensen 1997). Endorhizosphere includes the spaces around the mucoid layer, epidermal layer including the root hairs, and the cortical layer. Rhizoplane refers to the root surface, and ectorhizosphere comprises soil that extends a few millimeters from the root surface (Sorensen 1997; Curl and Truelove 1986). The unique morphology of many ectomycorrhizal fungi extends the rhizosphere from 3 mm to several centimeters, or possibly even meters in highly rhizomorphic ectorhizosphere. Due to emanating hyphae and rhizomorphic structures, extended ectorhizosphere zones are termed the "mycorrhizosphere" (Molina and Amaranth us 1990; Harley 1989; Read et a!. 1985; Tinker 1975), and more specifically the ectomycorrhizosphere. Ectomycorrhizae (ECM) are integral parts in healthy forest ecosystems where fungal biomass can exceed that of all microorganisms combined (Brady, 1990). As a result, their biomass represents a significant portion of the nutrient pool, and their activities are the key to providing or limiting access to nutrients for plant growth. In the forests of British Columbia, as much as 90% of tree root tips can be colonized with ectomycorrhizal fungi (Simard et a!. 1997). These root-fungal associations modify many soil processes including mineral weathering and the release of bound nutrients, making them available to plants (Perry et al. 1 1987). Berthelin and Leyval (1982) indirectly demonstrated that the dissolution ofbiotite (K and Mg rich mica) in the rhizosphere of maize (Zea mays L.) was primarily due to endomycorrhizal fungi and bacteria. In 1978, Mojallali and Weed reported that infection of soybean (Glycine max [L.] Merr.) roots by Glomus macrocarpus (an endomycorrhizal fungus) accelerated the process ofK release from mica (phlogopite) in soil. Studies have elucidated that ECM play a direct role in mineral weathering and the release of bound nutrients. Furthermore, weathering of soil minerals is dependent on the species (of microorganism) involved (Berthelin 1983). For example, Cromack, Jr. et al. (1979) reported that Hysterangium crassum on Douglas-fir could supply a high amount of oxalate to extract the Fe and Al from andesite. In vitro studies conducted by Paris et al. ( 1994) found that Pax illus involutus and Rhizopogon luteolus could release the NH4 + ions trapped in the interlayer space of vermiculite. Suillus granulatus and Piloderma croceum were found to connect calcium feldspars and homblends to the tree root in granitic rocks in Sweden (Jongmans et al. 1997). It was suggested that these connections formed by ectomycorrhizal fungi made it possible to directly supply basic cations such asK+, Mg 2+, and Ca2+ from the minerals . Most recently, Wallander and Wickman (1999) studied Paxillus involutus and Suillus variegatus associated with Pinus sylvestris seedlings in mobilizing K from biotite and microcline. Results indicated that seedlings colonized with S. variegatus tended to be more efficient in the uptake ofK from biotite. In addition, microcline-induced growth depression in all seedlings except those colonized by S. variegatus . Although ECM are known to benefit plants by providing essential nutrients, factors governing the association ofECM fungi with certain minerals or ECM-mineral specificity (Arocena et al. 1999) are least understood. If an ectomycorrhizal fungus prefers a specific mineral as source of certain element for its biomass production, then that ECM can be used to supply that element to the trees growing in nutrient-poor soils in B.C and elsewhere. There is a need to understand the biogeochemical processes involved in mineral weathering in hybrid spruce rhizospheres and its relationships to ectomycorrhizae. In addition, little is known about the ectomycorrhizal diversity on Picea glauca x engelmannii. In the future, the knowledge put forth by this study may be used in management practices to protect, rebuild, and sustain healthy Picea glauca x engelmannii stands. The objectives of this thesis were to : 2 1) Elucidate the concept of fungus-mineral relationships and their importance to longterm nutrient supply. Specifically, to investigate from in vitro weathering experiments the preference of Piloderma for biotite, chlorite, and microcline as a source ofK and Mg. I will compare the growth, morphology, and chemical partitioning between hyphae and encrustations on Piloderma grown in the different minerals. 2) Determine the chemical and mineralogical properties of soils influenced by various species of fungi growing on roots of hybrid white spruce, Pice a glauca x engelmannii (Moench.). Specifically, soil pH, total C and N, cation exchange properties, the contents of mica, chlorite, kaolinite, feldspars, 2:1 expanding clays and amorphous minerals will be compared between soils collected from non-ectomycorrhizosphere and two ectomycorrhizosphere soils in the Ae horizon of a Gray Luvisol in the central interior of British Columbia. 3 Chapter II - Literature Review 2.1 What are mycorrhizae? Perhaps the most widespread, and from a nutritional standpoint, more significant associations between plants and microorganisms are those formed between roots and a wide variety of soil fungi (Marschner 1995). A root infected with a fungus is called mycorrhizae, which literally means "fungus root". The significance ofmycorrhizae stems from the fact that in nutrient poor conditions, at least 90% of the 'feeding roots ' of a tree are colonized by ectomycorrhizal fungi (Read 1997) Mycorrhizae represent an intimate symbiotic association between the fine roots of plants and specialized fungi. The known benefits of mycorrhizae to plants include enhanced water and nutrient uptake, protection against pathogens, improved resistance to drought, enlarged root systems, and tolerance to heavy metals (Molina and Amaranthus 1990). More specifically, ectomycorrhizae form intricate, often-colorful, sheaths or mantles around feeder roots of temperate trees and shrubs. The hyphae of ectomycorrhizae penetrate the intercellular or apoplastic space of the root cortex called the "Hartig Net", a zone of nutrient exchange between fungus and host (Foster et al. 1983). 2.2 Rhizosphere The biogeochemical processes in the rhizosphere may modify soil solution chemistry of the root zone leading to changes in soil properties such as pH and redox potential (McBride 1994). Many of the organic compounds found in the ectomycorhizosphere have acidic functional groups, such as carboxyl and phenolic groups (Killham 1994). As a result, pH in rhizosphere soils is 2-3 units lower than in bulk soils. The concentration of protons is directly related to the degree of weathering of many primary minerals (Hinsinger et al. 1993 ). Chung and Zasoski (1994) found that the cation exchange equilibrium was higher in rhizosphere soils compared to that in bulk soils. 2.3 Fungal dynamics Fungal biomass within the humic layer of many soils can be much greater than that found in mineral soil (Harvey et al. 1976). From 69 to 72% of the total mycorrhizal roots 4 have been shown to be located in the organic fraction of mature stands of Abies lasiocarpa (Hook.) Nutt., Abies amabilis (Dougl.) Forbes, and Picea glauca x engelmannii (Moench.) Voss- Abies lasiocarpa (Kimmins and Hawkes 1978; Harvey et al. 1979; Vogt et al. 1981 a). However, Abies concolor (Gord. and Glend.) Lindl. Ex Hildebr. seedlings (white fir) were found to be more productive in mineral soil devoid of organic layers (Alvarez et al. 1979). The increased growth was attributed to different predominant mycorrhizal fungi colonizing their root systems. Abies pindrow (Royle) also grew poorly in thick humic layers (Bakshi 1974). Vogt et al. (1981b) found 80% Abies amabilis (Pacific silver fir) roots colonized by Cenococcum to be located in the humus and A-horizon. In addition, sclerotia (fungal reproductive structures) were predominantly located in the A-horizon. With this in mind, it is clear that fungi dominant not only in humus but may also possibly in mineral soils. The exact role that they play is largely unknown. However, there have been studies to elucidate fungi as integral players in mineral soil processes. 2.4 Biologically induced mineral weathering 2.4.1 What is mineral weathering? Mineral weathering generally occurs whenever water comes into contact with primary mineral (chlorites, micas, feldspars, etc). Generally an irreversible process, soil weathering is a result of cation exchange, hydration, and oxidation (McBride 1994). The mechanisms are influenced by the relative amounts of hydrogen ions in solution (Brady 1990). Protons can penetrate into the octahedral sheets of phyllosilicates and replace Al ions to destabilize the 2:1 layer phyllosilicate and cause increased weathering. Organic acids found in the rhizosphere also increase soil weathering by serv1ng as electron donors to unfilled orbitals of cations and metals which, in tum, facilitates their removal from rhizosphere soil solution (Fanning et a!. 1989). Secondary mineral formation from primary minerals is determined by the relative rates at which the weathering process takes place. The susceptibility of weathering is based on the arrangement of connected silica tetrahedra within its structure (McBride 1994 ). Some primary minerals, such as quartz, have very stable silica arrangements, while 2:1 phyllosilicates (micas) or sheet silicates have structures that are susceptible to chemical weathering. Weathering products (secondary minerals) in the clay fractions of soils are 5 mainly sheet silicates (smectite, vermiculite). The release of particular elements into the soil solution is a direct indicator of the type of mineral present. A soil solution high inK indicates weathering ofK-rich primary minerals. For example, 2:1 layer phyllosilicate mica loses its structural K rapidly by a process of cation exchange with protons or metal ions in the weathering solution (McBride 1994). K-rich feldspars (microcline) are the largest natural reserve ofK in many soils and dictate the K status in many soils (Huang 1989; de Leenheer 1950; Phillippe and White 1952; Jeffries et al. 1956). After quartz and K-feldspars, the micas are the next most extensive group of minerals in many parent materials. Since micas in most soils originate form parent materials and tend to weather to other minerals, they are generally more prevalent in the clay mineralogy of younger, less weathered soils and are less prevalent in more weathered soils (Iacksonetal. 1948, 1952;HseungandJackson 1952;Jackson 1959, 1964). Upon weathering, the micas are known to be an important K source for growing plants. Chlorite, a 2:1:1 layer silicate, can form as an alteration/weathering product of biotite and is present in many soils worldwide. Chlorites, however, are not as abundant as some other clay minerals (micas) but, when present, can supply significant amounts of Mg. The low frequency is mainly due to the low stability of chlorite (Barnhisel and Bertsch 1989). 2.4.2 Roots Biological weathering of minerals (primarily silicates) occurs in the rhizosphere zones of higher plants (Robert and Berthelin 1986). One of the first published studies showed the intensified weathering of interstratified minerals in the rhizosphere of French bean (Phaseolus vulgare L.) (Sarkar et al. 197>}. Berthelin and Leyva! (1982) demonstrated considerable biotite weathering could take place in the rhizosphere of maize plants (Zea mays L.) growing in axenic conditions, i.e., without any rhizosphere microorganisms. They concluded that plant roots themselves could induce silicate weathering. In addition, biological weathering induced by the activities of the roots can occur in a very short period of time. Hinsinger et al. (1993) found that vermiculization oftrioctahedral mica (phlogopite) in the rhizosphere zones ofltalian rye grass (Lolium multiflorum cv. Lemtal) and rape (Brassica napus), occurs in as little as four days. Recently, specific root mineral associations and mineral changes in the rhizosphere of field-grown com were reported by Kodama et a!. 6 ( 1994). April and Keller ( 1990) observed biotite weathering close to the surfaces in some selected forest soils. Most recently, Courchesne and Gobran (1997) studied the mineralogy of bulk and rhizosphere soils to find the effect of roots on mineral weathering in a Podzol supporting Norway spruce (Picea abies (L.) Karst). Not surprisingly, the amounts of amphiboles and expandable phyllosilicates were significantly lower to that found in bulk soils. 2.4.3 Soil microorganisms Except for the work conducted by April and Keller (1990) and Courchesne and Gobran (1997), there has been little work on mineral weathering in naturally occurring forest soils. More importantly, most of the previous studies have been conducted in highly artificial laboratory systems in the absence of microorganisms. Soil weathering processes are not only strongly influenced by roots themselves but by the presence of microorganisms (Berthelin 1988). Specifically, Berthelin (1983) indicated that weathering of soil minerals is dependent on the species of microorganism involved. 2.4.4 Fungi Soil weathering induced by endo and ectomycorrhizal fungi has been documented in laboratory experiments. In 1978, Mojallali and Weed reported that colonization of soybean (Glycine max [L.] Merr.) roots by Glomus macrocarpus accelerated the process ofK release from mica (phlogopite) in soil. Berthelin and Leyval (1982) indirectly demonstrated that the dissolution ofbiotite in the rhizosphere of maize was primarily due to endomycorrhizal fungi and bacteria. This information may have sparked the study by Leyva) and Berthelin (1991) where pine roots (Pinus sylvestris L.) were inoculated with the ectomycorrhizal fungus Laccaria laccata and an acid-producing rhizobacterium (Agrobacterium sp.) to look at the weathering of phlogopite. Results indicated that K losses were greater for the phlogopite particles closely attached to the mycorrhizae. In addition, the mycorrhizal effect was attributed to an increase of exchange surface area, rather than an increase in acidity. Many studies have attributed increased soil weathering to be a result of chemical changes induced by the metabolic products released from mycorrhizal fungi . Robert and Berthelin (1986) concluded that biochemical soil weathering is a result of secretions of living 7 organisms. More specifically, changes in pH (Cumming and Weinstein 1990), metalchelating substances (Lapeyrie 1988), and organic acids (Cromack eta!. 1979; Lapeyrie eta/. 1990; Malajczuk and Cromack 1982) by fungi may play a role in soil weathering. Ochs eta/. (1993) tested the ability of root exudates of mycorrhizal spruce seedlings to dissolve Al oxide and found that they were more efficient weathering agents at pH 4 than the exudates from non-mycorrhizal seedlings or humic materials. Some researchers have suggested that intense chemical weathering could be attributed to calcium oxalate secreted by the hypogeous fungi Hysterangium crassum (Cromack eta!. 1979) and Monotropa mycorrhizae (Snetselaar and Whitney 1990). Oxalate synthesis was also found in the mycorrhizal fungus Paxillus involutus and was able to solubilize phosphate in lime rich soil (Lapeyrie 1988). In contrast, Cromack et al. (1977) reported that fungi accumulate calcium oxalate in their tissues and release oxalic acid and soluble forms of calcium oxalate as by products. Oxalic acid can form stable metal complexes with other metal cations thus influencing soil weathering as well as the release ofP, Fe, and Al hydroxyphosphates from minerals. Early studies have concluded that organic acids produced by fungal mats could increase weathering. Tan et al. (1978) found the mycorrhizal fungus Pisolithus tinctorius to influence soil structure by producing humic compounds. Organic acids were attributed to Fe and P losses from upper soil profiles infected with Hydnellum ferrugineum (Hintikka and Naykki 1967), and Fisher (1972) with Hydnaceae fungal mats. Fisher (1972) also observed greater depletion of Ca, Mg, and Al in the upper part of the A horizon in fungal colonized soil. These early studies have concluded that organic acids produced by fungal mats could have increased weathering. In addition, organic acids secreted by mycorrhizal fungi have been attributed to solubilizing P from rock phosphate or Fe and Al hydroxy phosphates (Bowen 1973). Recently, weatherable minerals have been shown to have minute tubular pores in their structure, formed by organic acids exuded by fungi (Jongmans eta!. 1997). These symbiotic mycorrhizal fungi are believed to translocate the dissolved minerals from these isolated micropores directly to the host plants by their hyphal systems. 2.5 Nutrient allocation Ectomycorrhizal fungi allow roots to explore beyond nutrient depletion zones that 8 develop around them (Sylvia 1998). Roots free of fungal associations soon produce these depletion zones when nutrients are removed from the soil faster than they can be replaced. This is especially true for less mobile ions such as phosphate, copper and zinc. Fungal hyphae can readily go beyond these depletion zones and make essential nutrients available to plants. In addition, because of their narrow diameter, fungal hyphae are particularly efficient in nutrient absorption. Having a smaller diameter prevents depletion zones because the diffusion gradient for a nutrient is inversely proportional to the radius of the absorbing unit (Sylvia 1998). This was also seen with ectomycorrhizae mantle thickness where thinner mantles were more effective in nutrient absorption (Harley and McCready 1952). Leyval and Berthelin (1991) suggested that the role of mycorrhizal fungi is only to increase the absorptive area. The ability to extract nutrients from a much larger soil volume than the absorptive zone of non-mycorrhizal roots is especially important with plants which have a poorly branched root system (Rhodes and Gerdemann 1975). The effects ofmycorrhizae were documented in pine seedlings where Pisolithus arhizus mycorrhizae accounted for less than 20% of the total nutrient absorbing surface mass, and 80% of the absorbing surface area (Rousseau eta!. 1994). In addition to hyphae extending the absorptive surface area of roots, ectomycorrhizae produce hormones that promote branching of feeder roots for certain systems. This not only increases the absorbing root surface but also the contact and exchange zone between fungus and plant (Molina and Amaranthus 1990). In contrast, Wallander eta!. (1997) found ectomycorrhiza-infected pine seedlings (Pinus sylvestris) had smaller root systems than non-mycorrhizal plants. Many of the products released by fungi are a result of cation exchange. Metabolic products are excreted while others are absorbed into the fungal tissue. The fungus Hebeloma crustuliniforme greatly increased ammonium uptake in exchange for H+ in Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco.), Sitka Spruce (Picea sitchensis (Bong.) Carr.), and western Hemlock (Tsuga heterophylla (Raf.) Sarg.)(Rygiewicz et al. 1984). Mycorrhizal fungi are particularly efficient in absorbing less mobile ions such as phosphorus (Bolan 1991; Gianinazzi-Pearson and Gianinazzi 1986; Harley and Smith 1983; Bowen 1973). Pine seedlings (Pinus sylvestris L.) colonized with Suillus variegatus and Paxillus involutus had higher concentrations of P in their shoots compared with non-mycorrhizal seedlings (Wallander et al. 1997). Majstrik (1970) established that for different kinds of 9 ectomycorrhizae, the rates of adsorption of phosphate ions are also different. Fungi, in addition to their absorption capabilities, have been found to accumulate nutrients in their mantles. Ectomycorrhizae may accumulate P in their outer fungal mantle (Harley and Smith 1983; Morrison 1962). It was postulated by Marschner (1995) that Sr, an element with similar uptake mechanisms to that of Ca, accumulates in fungal mantles. Many metals like Pb, Cd, Al, as well as Si are known to accumulate in ectomycorrhizal mantles (Tumau et al. 1994). The accumulation of specific elements could be the specificity of different kinds of mycorrhizal fungi to transport specific elements into plants. An interesting observation by Langlois and Fortin (1978) was that "yellow" mycorrhizae absorbed 3, 10, and 16 times more H 2P04 - than "white", "brown", and "black" mycorrhizae, respectively. However, Wallander et al. (1997) found pine seedlings colonized with Piloderma croceum (a yellow mycorrhizal) fungus had low amounts of Pin their shoots when compared to seedling colonized with white (Suillus variegatus) and light brown (Paxillus involutus) types. 10 1 Chapter III- The Weathering of Biotite as a Potassium source for Piloderma sp. 3.1 Introduction The bright yellow color and wide distribution of some Piloderma species make them easily recognizable mycorrhizal fungi in North America. Being a broad host-range ectomycorrhizal fungus (Molina eta/. 1992), Piloderma should in theory be found on an array of plants, including most ofthe economically important coniferous and deciduous trees of central British Columbia (BC). Piloderma is reportedly found in decaying wood or fragmented litter (Goodman and Trofymow 1996; Nylund 1981; Zak 1976), in acid humus under litter or moss (Agerer 1987 -1998). Earlier reports indicated that Piloderma is confined to organic materials (Goodman and Trofymow 1996), but Arocena eta/. (1999) documented its abundance not only in organic horizons but in Luvisolic Ae horizons in central BC. Piloderma sporocarps are not as conspicuous but taxonomic analysis by Larsen et a/. (1997) indicated that they are present in rotten wood, under moss,' and among roots in the litter layer. Piloderma has been isolated into pure culture and used in synthesis and physiology experiments (Nylund 1981; Nylund and Unestam 1982; Nylund 1987; Massicotte eta/. 1993). Piloderma species, like other ectomycorrhizal fungi, are believed to increase cation uptake of plant roots through increased absorbing surface area contributed by fungal mycelia (Sylvia 1998), chelation, or indirectly by influencing soil microbial activity (Grayston et al. 1996). Studies have elucidated that ECM provide nutrients to the plants through a direct role in mineral weathering and subsequent release ofbound nutrients. Furthermore, weathering of soil minerals is dependent on the species ofmicroorganism involved (Berthelin 1983). For example, Cromack eta/. (1979) reported that Hysteramgium. crassum in Douglas-fir could supply high amounts of oxalate to extract the Fe and Al from andesite. In vitro studies by Paris eta/. (1994) found that Paxillus involutus and Rhizopogon luteolus could release the NH 4+ ions trapped in the interlayer space of vermiculite. In Sweeden, Sui//us granulatus and 1 A version of this manuscript will be submitted to the Can. J. Bot. 11 Piloderma croceum were found to connect calcium feldspars and hornblende to the tree root in granitic rocks (Jongmans et al. 1997). It was suggested that this link may supply cations such asK+, Mg 2+, and Ca2+ from the minerals directly to the roots. Most recently, Wallander and Wickman ( 1999) indicated that Pinus sylvestris seedlings colonized by S. variegatus tended to be more efficient in the uptake ofK from biotite than microcline. In addition, microcline addition induced growth depression in seedlings except those colonized by Paxillus involutus. In North America, most soils are rich in primary minerals such as biotite (mica), microcline (K-Feldspar), and chlorite (2: 1:1 layer silicate). These minerals serve as major sources ofK and Mg, which are considered macronutrients, as they are found at concentrations of 10 and 5 gkg- 1 in dry tissue of most vascular plants (Raven et al. 1999). Kfeldspars (microcline) are the largest natural reserve of K in many soils (Huang 1989). After K-feldspars, micas are the third most extensive group of minerals in soil parent materials and are generally more prevalent in the clay fraction of young, less weathered soils and are less prevalent in more weathered soils (Jackson 1959, 1964). Upon weathering, the micas are an important K source for growing plants. Chlorite, a 2:1:1 layer silicate, can form as an alteration/weathering product ofbiotite. Chlorites, however, are not as abundant asKfeldspars and micas, but when present, can supply significant amounts of Mg. The low content of chlorite in soils is mainly due to the low stability of chlorite (Barnhisel and Bertsch 1989). In the forests of central BC, soil parent materials have undergone relatively little weathering and pedogenesis (Arocena and Sanborn 1999) and are rich in mica, feldspars, and chlorite (Floate 1966; Luttermerding 1992; Kodama 1979). Arocena et al. ( 1999) recently reported mica and chlorite in the eluvial horizons of Gray Luvisol could be as high as 220 and 100 g kg- 1 in the clay fraction, respectively. In other areas in central and northeastern interior BC, Arocena and Sanborn (1999) reported contents of mica and chlorite as high as 650 g kg- 1 and >300 g kg- 1, respectively, in the eluvial horizons. In addition, they found that 1 K-rich feldspars could be as high as 150 g kg- in the sand fraction. Although ECM are known to benefit plants by providing essential nutrients, factors governing the association of ECM with certain minerals (Arocena et al. 1999) are least understood. To date, there have been few studies that have directly explored ECM and their preference in extracting essential elements from certain soil minerals . If an ECM can utilize a 12 specific mineral as source of certain element for its biomass production, then that ECM can be used to supply that element to trees growing in nutrient-poor soils. The objective of this study was to address fungus-mineral associations and its importance to long-term nutrient supply. Specifically, this study investigates through in vitro weathering experiments the "association" of Piloderma with biotite, chlorite, and microcline as a source ofK and Mg. We compared the growth, morphology, and chemical partitioning between hyphae and encrustations on Piloderma grown in the different minerals. 3.2 Methods 3.2.1 Isolation of Piloderma Piloderma colonizing Picea glauca x engelmannii ((Moench.) Voss) root tips were collected near the campus of University ofNorthem British Columbia located in the central interior ofBC, Canada (53° 54' N 123° 49' W). Within 2 hrs of collection, 100-120 healthylooking ectomycorrhizae (ECM) were washed in deionized water, immersed in cold deionized water, stored at 4 °C, and plated in a growth medium. Prior to plating, ECM were surface sterilized by dipping root tips in (i) 85% ethanol (1-2 sec), (ii) 30% H 20 2 for approximately 15±10 sec, and (iii) rinsed three times in sterile deionized water for 50±30 sec (Danielson 1984). After sterilization, three to four ECM were transferred to petri plates (100x15mm) of modified Melin-Norkrans (MMN) (Marx 1969) and potato dextrose agar (PDA) (Difco) with 100 ppm streptomycin, 50 ppm chlorotetracycline, and 2-5 ppm of benomyl (a fungicide to inhibit the growth of Ascomycetes) (Danielson 1984). Plates were incubated at room temperature (20-25 °C) and examined regularly for 2-4 months to avoid bacterial and fungal contamination. When bright yellow fungal growth emanating from the root tips was observed, isolates were transferred to MMN and modified MMN2 media (Table 3 .1.) without antibiotics and fungicides and stored at 4 °C in the dark. These Piloderma cultures were tentatively assumed to be Pilodera fallax since no sporocarp collection was possible. Once cultures grew through one half of the plate, Piloderma pure cultures were stored at 4 oc. All media were autoclaved (liquid cycle) at 121 °C at 103 kPa for 15 minutes. 3.2.2 Plate preparation and inoculation Piloderma was grown in customized MMN2 plates (Figs. 3.2a-c) enriched with (i) 13 biotite (trioctahedral mica), (ii) chlorite (2: 1:1 later silicate), and (iii) microcline (K-feldspar) 3 and in MMN2 plates with no mineral (control). Biotite [K(Mg,Fe 2+)3(Al,Fe +)3Si 3 0 10 (0H,F)z] (Fig. 3.2g) served as a source ofK and Mg, chlorite [(Mg,Fe2+) 5Al(Si 3Al)0 10(0H)s] (Fig. 3.2h) supplied Mg, and microcline [KA1Si 30 8 ] (Fig. 3.2i) supplied K for the growing fungus. To ensure that the growing fungus extracts K and Mg from the minerals, K and Mg contents in MMN2 medium was essentially eliminated (Table 3.1). In addition, Piloderma was grown in MMN media (optimal, nutrient rich) for comparison with mineral-modified MMN2 media and the control. The composition of MMN and MMN2 is given in Table 3.1. To increase the surface area of the minerals, biotite, chlorite, and microcline were ground to silt size (2-53 m) fraction using a coffee grinder (Braun©) for biotite and chlorite and mortar and pestle for chlorite. Ground minerals were dry sieved at 53 m to remove sand (53-2000 m) and larger fragments while the homogeneous mixture of silt sized particles were obtained after the clay-sized particles (<2 m) were removed by successive dispersion/sedimentation technique using the principle of Stoke's Law (Sheldrick and Wang 1993). Approximately 0.05 g of each mineral was placed in 0.2 ml Eppendorf/micro (Gordon Technology) tubes and autoclaved (MDT Castle™ steam sterilizer) with the lid open at 121 °C for 15 min at 103 kPa on the dry cycle. MMN2 and MMN media were autoclaved at 121 °C for 15 minutes at 103 kPa and allowed to cool to 50 °C before pouring into 100x150mm petri-plates. Each plate contained 32 ml ofMMN2 or MMN. To add biotite, chlorite, and microcline to the surface ofMMN2 and to ensure that the mineral would be confined to a 3 em diameter in the center of each plate, a special petri dish lid with a 3 em diameter barrier (Fig. 3.2a.) extended down to the agar surface was used. In addition, the lid was fitted with a paper barrier to prevent air currents from dispersing the mineral outside the 3-cm area. Before the addition of mineral, the lid was sterilized with 85% ethanol (bottom) and allowed to dry. Once dry, the lid was placed on the MMN2 plate (centered on a level petri-swivel) where approximately 0.05 g of sterile mineral was added followed by 4-6 drops of sterile de-ionized water (Fig. 3.2a) to dissipate static electricity and air currents that can easily scatter the mineral. While the MMN2 plate was being spun, a glass rod (0.5x10 em) bent at the end was used to spread the mineral over the mineral circle, a 3-cm diameter area in the center of each plate (Fig. 3 .2b ). If 14 needed, a few drops of sterile water were added to homogeneously spread the mineral over the agar surface. The original (non-modified) petri-lid was then placed on the plate and allowed to dry (1-6 hrs) on a level surface to prevent further dispersion of water-saturated mineral. To ensure that the mineral edge could be located after the fungal growth, sterile 14 kg test fishing line (Berkley™) cut into 1cm lengths was placed around the mineral circle as markers (Figs. 3.2c-f). All plates were examined using a dissecting microscope and plates with mineral outside of the guides were discarded. All plates were then stored at room temperature until inoculation. Five replicate plates containing MMN2 enriched with biotite, chlorite, and microcline, MMN2, and MMN were inoculated with four MMN2 plugs of Piloderma (0.5x0.5 em). All treatments were incubated at room temperature for 110 days (Fig. 3.1). 3.2.3 Growth rate The maximum lateral growth (mm) of Piloderma in each treatment was recorded every two weeks from the inoculation point until the fungus grew to the petri-dish edge in one of the five treatments. The growth of Piloderma in each treatment was analyzed with Statistica™ version 5 (Statsoft Inc. 1995) using non-linear user-specified estimation and the logistic growth model given by: Growth (mm) =aM/a + (M-a)e - kM(Days) (1) Where, a = theoretical growth at time zero, M = maximum theoretical growth, k = shape and slope parameter (Stewart 1991). Growth model parameters were calculated from five replicates in each treatment. 3.2.4 Morphological and chemical analysis Morphological properties and chemical composition of hyphae and ornaments were examined under the microscope after 110 days of growth. Agar plugs (approximately 1 x 0.5 em) colonized with Piloderma was sampled outside of the mineral circle (Fig. 3.2f) from three replicates for each treatment, mounted on aluminum stubs with double sided tape, and freeze-dried for 24 hrs using a Labconco TM freeze dry/shell freeze system. Once dried, samples were Au-sputter-coated for 100 seconds and examined using a Philips XL30 Scanning Electron Microscope equipped with an EDAX™ energy dispersive system (SEM- 15 EDS) for morphology and in situ chemical composition of ornaments and hyphae. Carbon, 0, Na, Mg, Al, Si, K, Ca, and Fe contents (expressed as a weight percent) of ornaments and hyphae were determined from energy dispersive spectrum collected for 200s (20kV) using a standardless EDS technique and correction factors for Z (atomic number), A (absorption), and F (fluorescence). Mean chemical composition for each treatment was calculated by spot analysis often lenticular and verrucose crystals and hyphal wall areas (where ornaments were absent) in each of three replicates in each treatment. 3.2.5 Statistical analysis Analysis of data was conducted by ANOVA using Statistica™ version 5 (Statsoft Inc., 1995). Post hoc comparison of significantly different means was made using planned LSD test statistics. Since sample sizes in each treatment were equal, tests for homogeneity of variances and normal distribution were not conducted. 3.3 Results 3.3.1 Growth Piloderma growth from the inoculation plug was bright yellow (2.5Y 8/8) (Munsell color notation), but sometimes at the initial stages exhibited white color (2.5Y 811) that later turned to bright yellow. With the exception of the MMN2 treatment, all treatments exhibited a cottony-like growth that extended up to 0-5 mm above the agar surface (Figs. 3.2d-f). The MMN2 treatment exhibited less dense, sometimes rhizomorphic, growth and very rarely extended more than 1mm above the agar surface. Piloderma had growth patterns with distinct lag, exponential or accelerated growth. decline, and stationary phases according to the fit (r = 0.99) of growth data to the logistic growth model given in Eq. 1 (Figs. 3.3a-e). With theoretical maximum growth (M) at 36.9, and a k = 0.0013, the biotite treatment had significantly strongest growth among all treatments (Figs. 3 .3a-e ). The biotite treatment exhibited a shorter lag period than all other treatments. Decline and stationary phases were not evident as growth in biotite was still in the exponential phase at 110 days of growth. Chlorite, microcline, and MMN2 treatments had similar growth as shown by similar M values (Figs. 3.3b-d). However, chlorite had a k of 0.0099 that was significantly lower compared to the k in microcline (k = 0.0041) and MMN2 16 (k = 0.0058). Although not significantly different with respect toM, the microcline treatment had slightly more growth than MMN2 and chlorite treatments. Chlorite, microcline, and MMN2 treatments exhibited distinct lag, exponential, decline, and stationary phases. MMN treatment had significantly less growth than biotite treatment but had higher growth compared to chlorite, microcline, and MMN2 treatments (Fig. 3.3e). The MMN treatment had the longest lag phase but similar to biotite, was still in the exponential growth phase at 110 days. 3.3.2 Morphology In all treatments, Piloderma hyphae (2-3 11m wide) were encrusted with verrucose and lenticular ornaments (Figs. 3.4a-f, h-k) . In biotite samples, the verrucose and lenticular ornaments were distinct and ranged in size from 0.2-0.4 11m and 2-7 11m long, respectively (Fig. 3 .4a). The density of verrucose ornaments varied but there usually were 8-15 ornaments in one 11m length ofhyphal surface. Lenticular ornaments occurred sporadically and can be as abundant as 10-14 but usually ranged from 0-4 ornaments for every 10 11m length of hyphae. In chlorite treatment (Fig. 3.4b), verrucose ornaments were less distinct as they were sometimes flattened and had a size and density of 0.2-111m and 2-6 ornamentS per 11m, respectively. Lenticular ornaments were present in similar amounts but were usually 121-lm smaller than those found in biotite. In the chlorite treatment, it was common to observe hyphae that were devoid of ornaments. The microcline and MMN2 treatments had (i) a large portion of hyphae without ornaments (Figs. 3.4c, f, h, i), (ii) lenticular ornaments (4-7 every 10 11m) confined to single hyphae devoid of verrucose ornaments (Figs. 3.4c, e, f), and (iii) fibrillar material and localized hyphal swellings which were not seen in the other treatments (Figs. 3.4c-i). Microcline treatments occasionally had considerable amounts of small (0.050.21-lm) and underdeveloped verrucose ornaments (Fig. 3.4d). In both treatments (microcline and MMN2), the density of verrucose ornaments was usually 2-7 per 11m length of hyphae. In MMN2 treatment, rare, large, bursted swollen hypha! cells containing 3-4% Na were observed (Fig. 3.4g). Hyphae in MMN (nutrient rich) treatments (Figs. 3.4j, k) were encrusted with copious amounts of well-developed large, lenticular ornaments similar in size and density to those observed in biotite treatment. Verrucose ornaments were present in large amounts but were larger (0.5-1.0 11m wide) and denser (10-22/llm) than biotite treatment. 17 3.3.3 Chemical composition of ornaments and hyphae between treatments In all types of ornaments, C was significantly higher in mineral (biotite, chlorite and microcline) treatments compared to both MMN and MMN2 treatments (Table 3.2). However, hyphae had similar amounts of C in chlorite and MMN2 treatments. In ornaments and hyphae, 0 was significantly highest in MMN2 treatments. Carbon and 0 together were the most abundant and were 83-91%, 84-95%, and 86-95% in lenticular ornaments, verrucose ornaments, and hyphal walls, respectively. Generally, the least amount of C and highest amount of 0 was found in lenticular ornaments in all treatments. Even though differences were found between treatments, the Na content was 1-2% in ornaments and hyphae. Lenticular ornaments in chlorite and microcline treatments had the highest amount of N a at 2%. At 0. 7-1%, the Mg content was significantly highest in MMN treatments for ornaments and hyphal walls. With the exception of verrucose ornaments, significantly higher amounts ofMg were found in chlorite treatment compared to MMN2 and microcline treatments. At 0.5%, there was significantly higher amounts ofMg in hyphae from the biotite treatment compared to the MMN2 and microcline treatments (0.3%). Generally, there was higher Mg in biotite and chlorite treatments (0.3-0.5%) compared to MMN2 and microcline treatments (0.2-0.4%). Aland Si in ornaments and hyphae were found at 1 % in all treatments. In the MMN treatment, ornaments and hyphae exhibited 12.5-30 times more K than in the chlorite, microcline and MMN2 treatments (0.2-0.4%). Ornaments and hyphae in biotite treatments had 7.5-15 times K than in the chlorite, microcline and MMN2 treatments. No differences were found in K contents between the chlorite, microcline, and MMN2 treatments. Ca was significantly highest in the MMN treatment at 9%, 4%, and 3% for lenticular ornaments, verrucose ornaments, and hyphal walls, respectively. Verrucose ornaments and hyphae in the biotite treatment had significantly higher Ca than in the chlorite, microcline, and MMN2 treatments. The Fe content in ornaments and hyphae in all treatments was significantly highest in chlorite treatment and ranged from 0.6-2%. 3.3.4 Chemical composition of ornaments and hyphae within treatments Except for the biotite treatment, C was significantly lower in lenticular ornaments than in verrucose ornaments and hyphal walls, while 0, with the exception ofmicrocline, 18 was significantly higher in lenticular ornaments in all treatments (Table 3.2). In the biotite, MMN2, and MMN treatments, Na was lowest in the lenticular ornaments. There were little differences with respect to amounts ofMg, Al, and Si between ornaments and hyphae in all treatments. The microcline, MMN2, and MMN treatments had significantly lower K in lenticular ornaments compared to the amounts in verrucose ornaments and hyphae. In all treatments, Ca was significantly highest (2-4 times) in lenticular ornaments compared to amounts in the verrucose ornaments and hyphal walls. Iron was lowest in lenticular ornaments in the chlorite, microcline, and MMN2 treatments. In general, all elements, except for 0 and Ca, were lowest in lenticular ornaments than in verrucose ornaments and hyphal walls. Verrucose ornaments and hyphae had similar elemental composition in all treatments. 3:4 Discussion 3.4.1 Growth of Piloderma Typical in vitro logistic growth of a fungus is chronologically divided into (i) initial stage where no measurable growth occurs (also called lag phase), (ii) accelerated or exponential growth phase, (iii) a period of declining growth rate, (iv) a stationary phase, and finally (v) death (Griffin 1981). During the 110-day growth period in this study, Piloderma exhibited evident lag and exponential phases in all treatments and the growth data exhibited excellent fit into Eq. 1. Except for the biotite treatment, the lag phase lasted approximately 20 days, a period corresponding to the physiological preparation for the exponential phase, and largely influenced by the nature of nutrients in the medium (Griffin 1981; Machi lis 1957). Differences in lag phases between treatments might indicate differential preference of Piloderma for soil minerals as a source ofK. The unusually long lag phase (approximately 40 days) in the MMN treatment might be related to the sub-optimum nutrient levels for Piloderma during the initial stage of growth. The exponential growth phase was relatively short-lived in the chlorite, microcline, and MMN2 treatments, possibly because of the unavailability ofK, as the exponential phase is strongly affected by available nutrients (Griffin 1981 ). The availability of nutrients, particularly K, might be responsible for the continued exponential growth phase after 110 days in biotite and MMN treatments. The decline in growth as well as the stationary phase observed after 60-80 days in the chlorite, microcline, and MMN2 treatments might be due to low nutrient availability, particularly K 19 and Mg, elements, known to be responsible for enzymatic activity, carbohydrate metabolism, and ionic balance. Although his work was not with ectomycorrhizal fungi, Smith (1924) found that decline in growth, after the exponential phase, is probably caused by limited availability of nutrients to Botrytis. Insufficient nutrients, particularly K+, could hamper biological transport processes such as excretion ofNa+ and H+to avoid toxic levels in hyphae (Griffin 1981, Isaac; 1991). 3.4.2 Morphology of ornaments and hyphae The presence of verrucose and large lenticular crystals on Piloderma hyphae is similar to ornaments reported for other species of fungus (e.g., Cromack et al. 1979; Amott and Webb 1983; Whitney and Amott 1986a, 1986b, 1987; Lapeyrie et al. 1990; Snetselaar and Whitney 1990; Jones et al. 1992; Connolly and Jellison 1994; Arocena et al. 1999). For example, Resinicium bicolor (Connolly and Jellison 1994) and Agaricus bisporus (Whitney and Amott 1986a) have styloid crystals about 5-40 11m in size and elongated rod-like to plate-like crystals, respectively. The well-developed ornaments in the biotite and MMN treatments could be an indicator of healthy Piloderma because of high M values. In contrast, the smaller and less frequent ornaments found in the chlorite, microcline and MMN2 treatments might be attributed to poor nutrient availability as growth in these treatments was characterized by low M values. In addition, lenticular crystals confined to single hyphae devoid of verrucose ornaments in the microcline and MMN treatments might be due to several reasons: (i) partitioning of limited supply of K, (ii) early stage of crystal formation, or (iii) growth of (+) or (-) hyphal strains. The presence of fibrillar and swellings could also be a morphological indicator of nutrient deficiency. In the nutrient poor media (MMN2), burst cells with 3-4% Na might indicate that the transport system for the removal ofNa is no longer functioning. This might be similar to marine fungi that take up K+ and extrude Na+ and H+ preventing the accumulation ofNa+, which might otherwise reach toxic levels (Isaac 1991). The high levels ofNa possibly increased the osmotic pressure and caused the cells to burst. However, it was not determined whether freeze-drying during sample preparation could have caused these cells to burst. 20 3.4.3 Chemical composition of ornaments and hyphae Not surprisingly, C and 0 were most abundant in ornaments and hyphae as they constitute major structural elements of fungi (Griffin 1981 ). The low amount of C in the MMN2 treatment could be due to retarded growth. The high contents of K in ornaments and hyphae for the biotite and MMN treatments suggest that these elements are essential for proper Piloderma growth and required at levels around 1o-3 M as macronutrients (Griffin 1981 ). The low levels of K might have resulted from retarded Piloderma growth in the chlorite, microcline, and MMN2 treatments. It is possible that K could substitute for Mg, when Mg is in short supply. Wallander and Wickman (1999) demonstrated that Pinus sylvestris seedlings, inoculated with Paxillus involutus and Suillus variegatus, could substitute Mg forK in severe K deficient treatments. The low levels of Si, AI, Fe, and Na in ornaments and hyphae could indicate that these elements are required at the micronutrient level at <10- 6 gkg -I . Griffin (1981) indicated that most fungi require Fe as a micronutrient at concentrations of 1o- 6 M. The higher Ca, C and 0 contents in lenticular compared to verrucose ornaments and hypha! walls in all treatments support earlier studies (Cromack et al. 1979; Snetselaar and Whitney 1987; Graustein et al. 1977) that hyphal ornaments are composed ofCa-oxalate. The high C content in ornaments could indicate a carbon to Ca-oxalate complex, or the ornaments being coated by an organic sheath similar to the polysaccharide-rich materials coating Ca-oxalate crystals on Paxillus involutus described by Lapeyrie et al. (1990). If verrucose ornaments are coated with an organic sheath, it is possible that verrucose ornaments are an early-stage predecessor to lenticular crystal formation (Arocena et al. submitted). In addition, verrucose ornaments on Piloderma are known to be rich in corticrocin (Agerer 1987-1998; Brand 1991), an organic molecule that gives Piloderma its color, thus further explaining the lower Ca in verrucose ornaments. The higher amounts of Ca and 0 in the form of Ca-oxalate in lenticular crystals would probably explain the lower Si, AI, Mg, Na, K, and Fe in lenticular crystals compared to verrucose and hyphae. 3.4.4 Weathering of minerals The higher growth rate and contents ofK in ornaments and hyphae of Piloderma grown in biotite compared to the MMN2, chlorite, and microcline treatments may indicate 21 the increased mobilization ofK from biotite. Our finding is consistent with the faster release ofK from a phyllosillicate (mica) than from a tectosilicate (microcline) (Huang 1989; Song and Huang 1988). In 1 M HN03 , the release of K from biotite was 118-190 times greater than from microcline (Huang eta!. 1968). Song and Huang ( 1988) reported K release to be 14-18 times faster in biotite than from microcline in organic-acid solutions. In addition, Huang ( 1989) indicated that the Arrhenius heat of activation forK release from microcline requires 96.24 kJ mor 1 compared to 58.66 kJ mor 1 for biotite. According to the Bowen reaction series, minerals that crystallize at low temperatures (microcline) tend to be more stable and resistant to weathering than minerals that crystallize at high temperatures (biotite) (McBride 1994; Fanning eta/. 1989). Furthermore, K located in the interlayer of biotite is easily accessible to Piloderma as opposed to the K embedded in the framework structure of microcline. In contrast, the low levels of Mg in encrustation and hyphae of Piloderma grown in biotite are probably due to the inability of Piloderma to obtain the Mg bound in the octahedral sheets ofbiotite. Seedlings colonized by Suillus variegatus were more efficient in uptake ofK from biotite compared to that from microcline (Wallander and Wickman 1999). Boyle and Voight ( 1973) attributed the lower amounts of K in seedlings grown in microcline than in seedlings grown in biotite to mineral surface area. Surface area was a likely a factor in this study because biotite is a 2:1 type of phyllosilicate mineral that expands when weathered while microcline is a non-expandable framework silicate. Also, it is possible that K availability from biotite increases at an exponential scale when surface area increases during edge weathering (Scott 1968; Huff 1972; Norrish 1973). The weathering of chlorite released Mg as indicated by the higher amount of Mg in lenticular crystals and hyphae observed in chlorite compared to MMN2 treatments. However, the lack of K in chlorite was the probable reason for the low growth in these treatments as chlorite is easily weathered compared to most clays in acidic environments (Barnhisel and Bertsch 1989). 3.4.5 Concluding remarks Piloderma grown in biotite exhibited higher growth and different morphological characteristics than Piloderma grown in chlorite, microcline and MMN2 treatments. The differences were attributed to the ability of Piloderma to efficiently obtain K from the 22 interlayer ofbiotite. The low levels ofMg, Al, Si, and Fe in all treatments might indicate that these nutrients are necessary micronutrients for optimal Piloderma growth. Generally, verrucose (0.2-1 J..Lm wide at 8-22/)..Lm) and lenticular ornaments (2-7)..Lm long at 0-2/5)..Lm) could be indicators of an environment where the supply of essential elements are sufficient for growth of healthy Piloderma . In addition, fibrillar growths and hypha! swellings could indicate nutrient deficient environments or soils containing little or no biotite. Supporting earlier studies, the high Ca and 0 found in lenticular crystals indicates that they are mainly composed of Ca-oxalate. This study elucidates that Piloderma could be·an important factor in the maintenance of healthy forest ecosystems. Specifically, Piloderma, may be capable of extracting large amounts of essential K from biotite, and supply many, if not all, of the economically important trees in the forests of central British Columbia. In the future, it would be beneficial to study Piloderma in association with conifers to determine the mechanisms by which Piloderma supply plant roots with K extracted from biotite. 3.5 Literature cited Agerer, R. (ed). 1987-1998. Colour Atlas ofEctomycorrhizae. Einhorn-Verlag Eduard Dietenberger GmbH Scwabisch Gmi.ind, Munich, Germany. Amott, H.J. and M.A. Webb. 1983. The structure and formation of calcium oxalate crystal deposits on the hyphae of a wood rot fungus. Scanning Electron Microsc. 4:17 471758. Arocena, J.M., K.R. Glowa, H.B. Massicotte and L. Lavkulich. 1999. Chemical and mineral composition of ectomycorhizosphere soils of subalpine fir (Abies lasiocarpa (Hook.) Nutt.) in the Ae horizon of a Luvisol. Can J. Soil Sci . 79 :25-35. Arocena, J.M . and P. Sanborn. 1999. Mineralogy and genesis of selected soils and their implications for forest management in central and northeastern British Columbia. Can. J. Soil Sci. 79:571-592. Barnhisel, R.I. and P.M. Bertsch. 1989. Chlorites and hydroxy-interlayered vermiculites and smectite. In J.B. Dixon and S.B. Weed (eds) Minerals in soil environments. Soil. Sci . Soc. Am, Inc. 23 Berthelin, J. 1983 . Microbial weathering processes. pp. 223-262. In Krumbein, W.E. (ed) Microbial geochemistry. Blackwell Scientific Publ., Oxford, UK. Boyle, J.R. and G.K. Voight. 1973. Biological weathering of silicate minerals. Implications for tree nutrition and soil genesis. Plant Soil. 38:191-201. Brand, F. 1991. Ektomykorrhizen au Fagus sylvatica- characterisierung und identifizierung, okologische kennzeischnung und unsterile kutivierung. Libri Botanica 2:1-229. Connolly, J.H. and J. Jellison. 1994. Calcium translocation, calcium oxalate accumulation, and hyphal sheath morphology in the white-rot fungus Resinicium bicolor. Can. J. Bot. 73:927-936. Cromack, Jr., K.P. Sollins, W.C. Grausten, K. Speidel, A.W. Todd, G. Spycher, C.Y. Li and R. L. Todd. 1979. Calcium oxalate accumulation and soil weathering in mats of the hypogeous fungus Hysterangium crassum. Soil Biol Biochem. 11:463-468. Danielson, R. M. 1984. Ectomycorrhizal association of Jack pine stands in northern Alberta. Can. J. Bot. 62:932-939. Fanning, D.S., Z.K. Vissarion and A.E. Mohamaed. 1989. Micas. In J.B. Dixon and S.B. Weed ( eds) Minerals in Soil Environments. Soil. Sci. Soc. Am, Inc. Floate, M.J. 1966. A chemical, physical and mineralogical study of soils developed on glacial lacustrine clays in north central British Columbia. Can. J. Soil Sci. 46:227-236. Goodman, D.M. and J.A. Trofymow. 1996. Pilodermafallax (Libert) Stalpers +Pseudotsuga menziesii (Mirb) Franco, CDEl. In: Goodman D.M., Durall D., Trofymow, J.A. and S.M. Berch (eds). 1996. Concise Descriptions ofNorth American Ectomycorrhizae. Mycologue Publications, and Canada-B.C. Forest Resource Development Agreement, CanadiaP Forest Service, Victoria, B.C. pp. CDE1.1-CDE1.4 Graustein, W.C., K. Cromack Jr. and P. Sollins. 1977. Calcium oxalate occurrence in soils and effects on nutrient and geochemical cycles. Science 198 :1252-1254. Grayston, S.J., D. Vaughn and D. Jones. 1996. Rhizosphere carbon flows in trees, in comparison with annual plants: the importance of root exudation and its impact on microbial activity and nutrient availability. Applied Soil Ecology 5:29-56. Griffin, D.H. 1981. Fungal physiology. pp 131-167. John Wiley and Sons, Inc. Huang, P.M. 1989. Feldspars, Olivines, Pyroxenes, and Amphiboles. pp. 975-1050. In J.B Dixon and S.B . Weed (eds) Minerals in Soil Environments. Soil. Sci. Soc. Am, Inc. 24 Huang, P.M., L.S . Crosson and D.A. Renni . 1968. Chemical dynamics ofK release from K minerals common in soils. Trans. Int. Congr. Soil Sci., 9th 1968 (Adelaide, Australia) II:705-712. Huff, W.D. 1972. Morphological effects on illite as a result of potassium depletion. Clays Clay Miner. 20:295-301. Isaac, S. 1992. Fungal-plant interactions. pp. 41-42. University Printing House, Cambridge. Jackson, M.L. 1959. Frequency distribution of clay minerals in major great soil groups as related to the factors of soil formation. Clays Clay Miner. 35:111-112. Jackson, M.L. 1964. Chemical compostion of soils. pp. 71-141. In F.E. Bear (ed) Chemistry of the soil. Reinhold Publishing Corp., New York. Jones D, W.J. McHardy, M.J. Wilson and D. Vaughn. 1992. Scanning electron microscopy of calcium oxalate on mantle hyphae of hybrid larch roots from a farm forestry experimental site. Micron and Micros. Acta 23:315-317. Jongmans, A. , N. Van Breemen, U. Lundstrom, P.A.W. van Hees, R.D. Finlay, M. Srinivasan, T. Unestam, R. Giesler, P.A. Melkerud and M. Olsson. 1997. Rock-eating fungi. Nature 389:682-683. Kodama, H. 1979. Clay minerals in Canadian soils. Their origin, distribution and alteration. Can. J. Soil Sci. 59:37-58. Lapeyrie, F., C. Picatto, J. Gerard and J. Dexheimer. 1990. T.E.M. study of intracellular and extracellular calcium oxalate accumulation by ectomycorrhizal fungi in pure culture or in association with Eucalyptus seedlings. Symbiosis 9:163-166. Larsen, M.J., J.E. Smith and D. McKay. 1997. On Piloderma bicolor and the closely related P. byssinum, P. croceum, and P. fallax. Mycotaxon 63:1-8. Luttermerding, H.A. 1992. Vertisolic soils field tour, British Columbia portion. Integrated Mgt Branch, B.C. Ministry of Environment, Lands and Parks, Victoria, B.C. Machilis, L. 1957. Factors affecting the lag phase of growth of the filamentous fungus, Allomyces macrogynus . Amer. J. Bot. 44: 113-119. Marx, D.H. 1969. Antagonism ofmycorrhizal fungi to root pathogenic fungi and soil bacteria. Phytopathology 59:153-163 . Massicotte, H.B., L.H. Melville, R. Molina and R.L. Peterson. 1993. Structure and histochemistry of mycorrhizae synthesized between Arbutus menziesii (Ericaeae) and 25 two basiodiomycetes, Pisolithus tinctorius (Pisolithaceae) and Piloderma bicolor (Corticiaceae). Mycorrhiza 3:1-11. McBride, M.B. 1994. Environmental Chemistry of Soils. Oxford University Press, Inc. Molina, R., H.B. Massicotte and J.M. Trappe. 1992. Specificity phenomena in mycorrhiza; symbioses: community-ecological consequences and practical implications. pp. 357423. In: M FAllen (ed). Mycorrhizal functioning, an integrative plant-fungal process. Routledge, Chapman and Hall, New York. Norrish, K. 1973. Factors in the weathering of mica to vermiculite. pp. 417-432. In J.M. Serratosa (ed) 1972 Proc. Int. Clay Conf., Div. de Ciencias, Madrid. Nylund, J.E. 1981. The formation of ectomycorrhizae in conifers: structural and physiological studies with special reference to the mycobiont, Piloderma croceum Erikss. & Hjorts. PhD Thesis., Uppsala University, Sweden. Nylund, J.E. 1987. The ectomycorrhizal infection zone and its relation to acid polysaccharides of cortical cell walls. New Phytol. 106:505-516. Nylund, J.E. and T. Unestam. 1982. Structure and physiology of ectomycorrhizae. I. The process ofmycorrhiza formation in Norway spruce in vitro. New Phytol 91:65-79. Paris, F., P. Bonnaud, J. Ranger and F. Lapeyrie. 1994. Alteration d 'un phyllosilicate par des champignons ectomycorhiziens in vitro. Acta Bot. Gallica 141:529-532. Raven, P.H., R.F. Evert and S.E. Eichhorn. 1999. Biology of plants (6th edition). p. 728.W.H. Freeman and Company/Worth Publishers. Scott, A.D. 1968. Effect of particle size on interlayer potassium exchange in mica. Trans. 9th Int. Congr. Soil Sci., , Adelaide, Aust. 11:649-660. Sheldrick, B.H. and C. Wang. 1993. Particle size analysis. pp. 499-512. In Carter, M.R. (ed) Soil sampling and methods of analysis. Lewis Pub I., Boca Raton, Florida. Smith, J.H. 1924. On the early growth rate of the individual fungal hyphae. New phytol. 37:341-343. Snetselaar, K.M and K.D. Whitney. 1990. Fungal calcium oxalate in mycorrhizae of Monotropa uniflora . Can. J. Bot. 68:533-543 . Song, S.K and P.M. Huang. 1988. Dynamics ofK release from K-bearing minerals as influenced by oxalic and citric acids. Soil. Sci. Soc. Am. J. 52 :383-390. Statsoft Inc. 1995 . Statistica for windows (computer program manual). Tulsa, Oklahoma. 26 Stewart, J. 1991. Calculus. pp. 485-486. Wadsworth, Inc. Sylvia, D.M. 1998. Mycorrhizal symbioses. pp. 408-426. In D.M. Sylvia, J.J. Fuhrmann, P. G. Hartel and D.A. Zuberer (eds) Principles and applications of soil microbiology. Prentice Hall, New Jersey. Wallander, H. and T. Wickman. 1999. Biotite and microcline as a K source in ectomycorrhizal and non-ectomycorrhizal Pinus sylvestris seedlings. Mycorrhiza 9:25-32. Whitney, K.D. and H.J. Amott. 1986a. Calcium oxalate crystals and basidiocarp dehiscence in Geastrum saccatum (Gasteromycetes). Mycologia 78:649-656. Whitney, K.D. and H.J. Amott. 1986b. Morphology and development of calcium oxalate in Gilbertella persicaria (Mucorales). Mycologia 78:42-51 Whitney, K.D. and H.J. Arnott. 1987. Calcium oxalate crystal morphology and development in Agaricus bisporus. Mycologia 79 :180-187. Zak, B. 1976. Pure culture synthesis ofPacific madrone ectomycorrhizae. Mycologia 68: 362-369. 27 Table 3.1. Composition of Melin Modified Norkrans (MMN, Marx 1969) and modified MMN (MMN2) dry additives and the relative amounts ofK and Mg in each of the stock solutions. Stock A1 Stock A2 Stock A3 10 0.5 0.5 Stock B Stock c Stock D Stock E Malt Dextrose (g) AgarAgar H20 (ml) Contents g/1 CaCh KH 2P04 NaCl (NH4)2HP04 MgS0 4 Fe Citrate Citric Acid Thiamine Hydrochloride K Mg 0.251 ppm 143.7 ppm <1 ppm 5 3 50ml 50 ml 0.1 <1 ppm Trace ppb 14.79 ppm Trace ppm from Fe citrate Trace ppb 50 ml 50ml 50 rnl 50ml 3 ml 3rnl 1 ml 1 ml Media MMN MMN2 10 10 1 not present, ppm-parts per million, ppb-parts per billion 28 2712 ppm 577.8 ppm 3g 146.2 ppm 14.7 ppm 10 g 13 g 12 12 796 896 Table 3.2. Mean weight percentages for elemental composition of Piloderma ornaments and hyphae grown in biotite, chlorite, and microcline enriched nutrient poor (MMN2), nutrient deficient (MMN2), and nutrient rich (MMN) media determined by SEM-EDS. (n=3) Treatment Lenticular Biotite Chlorite Microcline MMN2 MMN Verrucose Biotite Chlorite Microcline MMN2 MMN Hyphal walls Biotite Chlorite Microcline MMN2 MMN c 0 Na Mg AI Si K Ca Fe 47.5\ (4.0) 1 49.2\ (3.7) 49.4\ (5 .3l 42.7 X (3 .3) 42.9b, (4.1) 40.7a\ (3.4) 41 . (3 .8) 41.0\ . (5.2) 46.3\ (2.4) 39.8\ (3.4) 1.2\ (0.50) 2.0b (0 .72) 2.2be (0.53) 1.3\ (0.44) 1.1\ (0.79) 0.29\ (0.17) 0.46b (0.26) 0.25a (0 .12) 0.32a (0 .22) 0.7 1\ (0.30) o.3oab (0.18) 0.34b (0.21) 0.24\ (0.12) 0.34b (0 .20) o.25ab, (0.12) 0.91 , (0.30) 0.89 (0.27) 0.83, (0.40) 0.81 (0 .33) 0.92, (0.27) 2.7" (2 .0) 0.22\ (0.17) 0.26bx (0.15) 0.18bx (0.14) 3.8\ (0.92) 5.6". (2.3) 3.8bx (1.5) 4 .3\ (2.5) 7.1 dx (2 .5) 9.4\ (2 .0) 0.94a (0.64) 1.33\ (0.44) 0.5 8\ (0 .25) 0.91 \ (0.40) 1.1\ (0.40) 54.4\ (4.0) 54.6\ (2.8) 54.1 "y (4.9) 51.7\ (3 .6) 46.9\ (3.3) 35 .6\ (3.7) 38.8b"y (3.0) 39.4\ (5.6) 42.7 dy (4 .1) 37 .2a\ (3 .5) 1.7\ (0.52) 1.9ab (0.65) 2.3b (0 .66) 1.8\ (0.43) 2.0a\ (1.1) 0.53"yz (0 .34) 0.45a (0.19) 0.25b 1. 1ab"y (0.27) l.Oab (0.33) 1.2ey (0 .72) 0.91 a (0.36) 1.2b"y (0.47) 3.1a (2.2) 0.25\ (0.17) 0.41\ (0.29) 0.40by (0 .17) 5.4\ (1.12) 2.0\, ( 1.4) b 1.0 y (0.56) 1. 1bed Y (0.65) 0.63 by (0.26) 4.3\ (2 .1) 1. 1ab 0.40a (0.19) 1.4\ (0.56) 0.39 (0 .25) 0.43 (0.21) 0.36xy (0 .29) 0.36 (0 .18) 0.36y (0.19) (0.84) 1.5\y (0.51) 0.80by (0 .3 0) 1.1 adyz (0.36) 1.3"edy (0.43) 53.9\ (3 .7) 53 .7a\ (4.1) 55 .1\ (4.6l 51.9 y (2.6) 48.8\ (3.5) 36.6\ (4.6) 38.7\ (4.4) 38.2\x 1.6\ (0.60) 2.3be (0.87) 2.4e (0.63) 1.5"y, (0.63) 1.9a\ (1.1) 0.46a\ (0.24) 0.47a (0.37) 0.3 1e (0.192 0.33e b (0.19) 1.2ey (0.34) 0.34 (0.18) 0.45 (0.28) 0.41 y (0.36) 0.32 (0.16) 0.35 xy (0.21) 1. 1abed xy 3.3a ( 1.8) 0.24bxy (0.18) 0.40\ (0.24) 0.37bz (0.22) 5.7\ ( 1.5) 1.7a, (1.1) 1.2 by (0.87) 1.06by (0.88) 0.73 by (0.41) 3.1"y ( 1.4) 1. 1ab (0.60) 1.8\ (0.67) 0 .93a y (0.42) 1.14abz (0.32) 1.2bxy (0.40) rb. (5.5l 42.7 y (3.1) 36.6\ (3.5) (0.13~ (0.26~ 0.99a (0 .29) 1.2dy (0.73) 0.92e (0.29) 1.1bedxy (0 .25) Values in parentheses are standard deviations. For the chemical differences of ornaments and hyphae between the different treatments, means superscripted by the same letter (a-e) in each column are not significantly different. (p>0.05) For the chemical differences between ornaments and hyphae in similar mineral treatments, means subscripted by the same letter (x-z) in each column are not significantly different. (p>0.05) 29 23.8 23.6 23.4 ..-. 23.2 p 'a;' 23.0 ..... ::J 1§ 22.8 ~ E 22.6 ~ 22.4 22.2 22.0 0 ~ 20 ~~ 40 ~ ~ 60 ~ ~~ 80 ~ ~ 100 ~~ 120 Days Growth Fig. 3.1. Mean daily temperature for the 110-day growth period starting at day 7. 30 31 Figs. 3.2 a. Petri-lid used to assure that minerals added to the center of plate were not dispersed over entire agar surface. b. A glass rod bent at the end was used to spread mineral over MMN2 agar. c. Prepared growth plate showing mineral circle and mineral edge markers (arrows). d. Piloderma at 16 days on biotite. e. Piloderma at 56 days growth growing past the biotite edge and mineral edge, markers. f. At 110 days growth Piloderma completely grew through plate. Outside of the mineral edge markers samples were taken (tapered arrows) for chemical and morphological analysis. g. Biotite, a K and Mg rich trioctahedral mica, showing layer structure. h. Chlorite, a Mg rich 2:1:1 layer silicate. i. Microcline, a K-rich feldspar, showing crystalline structure. 35 35 31 I ~ (!) 31 Biotite + MMN2 27 I 0 a• 1.o2•. M• M .a•. k•.oo13• 23 r=0.99 19 s: l 15 0 11 40 DAYS 60 80 b 20 40 DAYS 60 80 100 35 31 31 Mlcrocllne + MMN2 27 I a=0.38b, M=15.8bcd, k=0.0041.,. 23 MMN2 (Control) 27 a=0.42b, M=12.4bd, k=0.0058cd 23 ~ 15 19 19 (!) 15 r=0.99 11 11 7 7 c 3 I r=0.99 100 35 -1 15 3 20 ~ \ 19 a 3 (!) a=0.40b, M=11 .1b, k=0.0099b 23 11 7 I Chlorite + MMN2 27 0 20 40 DAYS 60 80 d 3 -1 100 0 20 40 DAYS 60 80 100 35 31 I l (!) MMN (optimal, nutrient rich) 27 a=0.23b, M=25.6 8 , k=.0024•• 23 19 15 r=0.99 11 7 e 3 -1 0 20 40 60 80 100 DAYS Fig 3.3a.Growth of Piloderma modeled using the logistic growth equation Growth (mm)=aM/a+(Ma)e-kM(Days), where a=intercept, M=maximum theoretical growth, k= shape and slope in (a) biotite, (b) chlorite, and (c) microcline enriched nutrient poor (MMN2), (d) nutrient poor (MMN2), and (e) nutrient rich (MMN) growth media for the 11 0 day growth period. Approximate locations of lag (open arrows), exponential growth (closed arrows), and stationary phases (solid arrows) are indicated in the chlorite treatment. (a, M, k superscripted with similar letters a-e are not significantly different)(n=5) 32 33 Figs. 3.4 a-k. Scanning electron micrographs of Piloderma lenticular (arrowheads), and verrucose (non-tapering arrows) ornamentation, on hyphae grown in (a) biotite, (b) chlorite, and (c-d) microcline enriched nutrient poor (MMN2), (e-i) nutrient poor (MMN2), and (j-k) nutrient rich (MMN) growth media. c-i. Microcline and nutrient poor (MMN2) treatments showing characteristic fibrillar growth (tapered arrows). g. Swollen hyphal cell characteristic of nutrient poor (MMN2) treatment. 1 Chapter IV- Chemical and Mineral Composition of Rhizosphere Soils Influenced by Ectomycorrhizae in Hybrid Spruce (Picea glauca x engelmannii (Moench.) Voss) 4.0 Introduction Biogeochemical processes in soil mineral weathering occur largely in soil microenvironments influenced by microorganisms where pH (Nye 1981; Marschner and Romheld 1983), cation-exchange equilibria (Chung and Zasoski 1994), organic carbon/acid concentrations (Gardner et al. 1982), metal availability (Sarkar and Wyn Jones 1982), monosaccharide contents (Dormaar 1988), grain-size distribution (Sarkar et al. 1979), moisture, and mineral composition can be different from that of bulk soils. Soil processes can vary considerably with respect to different microenvironments because of the diversity in biological associations. Berthelin (1983) suggests microorganismal and tree specificity with respect to the influence of organisms on mineral weathering. An example is in ectomycorrhizospheres or the soil in the immediate vicinity of ectomycorrhizal associations (Perry et al. 1987). Due to emanating hyphae and rhizomorphic structures, extended ectorhizosphere zones are termed the "mycorrhizosphere" (Molina and Amaranthus 1990; Harley 1989; Read et al. 1985; Tinker 1975), and more specifically the ectomycorrhizosphere. A study by Arocena et al. (1999), found that pH and cation exchange capacity in Abies lasiocarpa ectomycorrhizospheres (ECS) influenced by Piloderma (ECS-1) were different from ECS of Mycelium radicis atrovirens and cottony yellow brown types or where Piloderma infection was <2% (ECS-2). Total carbon and nitrogen contents were sigmficantly different, and usually followed the order ECS-1 Mg 2+>K+>Na+. The amount of exchangeable K+ and Na+ in ECS soils was significantly higher than that in N-ECS soils. The exchangeable Ca 2+ was lowest inNECS soils at 2.36 cmolc kg- 1 and was significantly lower than ECS-B (3.34 cmolc kg- 1) but not different to ECS-A (2.65 cmolc kg- 1) . The base saturation(%) was significantly different between ECS and N-ECS soils at 67 and 88.2%, respectively. 4.3.3 Mineral composition Based on x-ray diffraction (XRD) analysis, the following phyllosilicates in the clay fraction of all samples were identified: mica, chlorite, 2: I expandable clays, kaolinite and quartz (Fig. 4.1). The XRD peaks at 1.0 and 0.5 nm indicated that muscovite was the species of mica in the samples. The amount of mica was lowest in ECS soils compared to N-ECS samples and followed the order: ECS-AECS-B> N-ECS (Arocena et al. 1999). In addition, root exudates of Pinus sylvestris were found to be higher in ECS soils than in non-ectomycorrhizosphere soils (Grayston et al. 1996). Because N is also a necessary component of microbial biomass (Arocena et al. 1999), 43 the relative contents of total N between ECS and N-ECS soils paralleled total C. There was no difference in the C and N contents in ECS-A and ECS-B soils (Table 4.3). However, higher total C and N in ECS-A compared to ECS-B was reported in subalpine fir ectomycorrhizospheres and may have been associated with high biomass per unit of root weight in ECS-A samples (Arocena et al. 1999). The C/N ratios differed significantly between ECS and N-ECS soils (Table 4.3) and were similar to those results found in subalpine fir where ECS-A had the highest C/N ratio and followed the order: ECS-A>ECSB>N-ECS (Arocena et al. 1999). In all samples, the C/N ratio were close to the threshold of 20: 1 indicating higher mineralization rates compared to fresh organic matter with C/N ratio between 40:1-100:1 (Myrold 1998). Since clay contents in N-ECS and ECS samples were similar, the higher CEC in ECS so"ils compared to N-ECS soils could be attributed to higher amounts of organic matter due to different degrees ofECM colonization as suggested by Arocena et al. (1999) (Table 4.3). As organic carbon increases, the CEC will also increase due to the negative charge associated with organic matter. Total carbon and CEC were highest in ECS soils and paralleled the findings by Arocena et al. (1999) in A. lasiocarpa. The slight difference in CEC between ECS-A and ECS-B could be attributed to the C content ofECS soils as well as the pH because organic matter has pH-dependent charge. The lower pH found in ECS-A soils could have caused slightly lower CEC compared to ECS-B but the difference in CEC was more likely attributed to the increased amounts of organic matter in ECS-B soils (Table 4.3). ECS soils with higher amounts of 2:1 type clays compared to N-ECS soils could also explain the high CEC . 4.4.3 Mineral composition of ECS and N-ECS soils The lower amounts of mica and chlorite in ECS soil compared to N-ECS soil may indicate different rates of mineral weathering between ECS and N-ECS soils. This is consistent with the findings of Arocena et al. (1999) where there were significantly lower amounts of mica and chlorite in ECS samples compared to N-ECS of A. lasiocarpa. These findings also corroborate other studies (e.g., April and Keller 1990; Hinsinger et a!. 1992; Kodama et a!. 1994; Courchesne and Go bran 1997). The slightly higher ratios of the sum of 2:1 expandable clays over mica, and chlorite in ECS soils compared to N-ECS (Table 4.4) 44 may indicate a transformation of mica and chlorite to 2:1 expandable clays as suggested earlier by Arocena et a!. ( 1999). The high rate of mica and chlorite weathering to 2: 1 expandable clays was also indicated by the higher amounts of exchangeable K+ and Mg 2+ in ECS compared to N-ECS soils. The higher amounts of exchangeable K+ and Mg 2+ could stem from the breakdown of muscovite and chlorite, respectively. These results are consistent with those of Arocena et al. (1999) and suggest that common ECM (e.g. Piloderma spp., Cenococcum geophilum, MRA) associated with A. lasiocarpa rhizospheres are inducing similar effects on mineral weathering in ECS of hybrid white spruce in central interior BC. Although early reports on high rates of mica and chlorite weathering were mostly for the rhizosphere of agricultural crops, the weathering mechanisms may also have been operative in the rhizosphere of trees even though they involved different groups of fungi (vesicular arbuscular mycorrhizae & ectomycorrhizae) (Arocena et al. 1999). In rhizosphere zones, both physical and chemical processes increase rates of mineral weathering. The pressures exerted by growing roots and associated fungal hyphae could mechanically alter minerals by causing realignment, bending, and fracturing (April and Keller 1990). In addition, Robert and Berthelin (1986) showed micrographs ofhyphae probing between mica flakes to extract essential K+. In many studies, root-induced release of interlayer K+ was the main reason for the high rates of chemical weathering in rhizosphere zones (Hinsinger et al. 1991; Hinsinger et al. 1992; Hinsinger and Jaillard 1993; Kodama et al. 1994). The formation ofvermiculite (a weathering product of mica) is usually caused by the release ofK+ from mica (Fanning et al. 1989) and can take place within a few days (Hinsinger et al. 1992). Primarily, whenever the uptake ofK+ by plant roots was greater than the release ofK+ from the mica, the release ofK+ from mica was enhanced (Arocena et al. 1999). The formation of vermiculite from phlogopite (a mica species) in the rhizosphere was found when the K+ concentration fell below 80 11mol dm- 3 (Hinsinger and Jaillard 1993). The complete dissolution of the mica lattice could elucidate another pathway of mica weathering in the rhizosphere (Hinsinger and Jaillard 1993). Higher rates of mineral weathering, as seen by the lower amounts of mica, chlorite, and kaolinite in ECS samples, could be attributed to the larger amounts of organic acids in rhizosphere zones. There was a significant increase in the amount of carbon in ECS samples compared to N-ECS soils (Table 4.2). As suggested by Arocena et al. (1999), the increase in 45 carbon could have been a result of organic acids. Organic acids have been found at higher levels in rhizosphere soil than in bulk soil (Grierson 1992; Griffiths et al. 1994). In addition, concentrations of organic acids may be high in the micro-environments around fungal hyphae (Ochs eta!. 1993; Drever and Vance 1994). Griffiths et al. (1994) indicated that Gautieria monticola, an hypogeous ectomycorrhizal fungus which produces large amounts of oxalic acid, weathers soil minerals. Both citric and oxalic acid are involved in the dissolving of feldspars (Manley and Evans 1986). Organic acids (e.g., oxalic, citric) produced by ECM in rhizosphere soils could initiate the removal of the hydroxide sheet from chlorite (Arocena et al. 1999). Direct removal ofMg2+ from the hydroxide sheet by the action of fungal hyphae such as those from Piloderma, is also a possible pathway in the weathering of chlorite (Arocena et al. 1999). Oxalate produced in ECM is an active agent of mineral weathering because of its high complexing capability (Robert and Berthelin 1986; Lap eyrie 1988). For example, the ECM system of Hysterangium erassum in Douglas-fir produced high amounts of oxalate to extract the Fe and Al from andesite (Cromack et al. 1979). Similarly, Watteau and Berthelin (1994) studied the mycorrhizal fungus Suillus granulatus and found that the fungus produced malic and citric acids which released iron from biotite. In this study, ECSB samples had approximately 11% Suillus spp. and could have had an influence in ECS-B samples. In a related study (data not shown), we found that verrucose and globular ornaments in ECM appear to be crystals of metal-oxalate complexes. 4.4.4 Concluding remarks This study elucidates that soil properties such as total C, total N, pH, cation exchange capacity, and exchangeable cations are different in ectomycorrhizosphere soils (ECS) compared to non-ectomycorrhizosphere soils (N-ECS). Total C and N were higher in ECS soils than N-ECS soils. Soil pH was ten times lower in ECS soils and followed the order ECS-AMg 2+>K+>Na+. The high cation exchange capacity in ECS soils was most likely the result of high total organic matter, which has a high amount of pH-dependent charge. No differences in the chemical composition were found between Piloderma-dominated (ECS-A) soils and soils that were dominated by Inocy be-like, 46 Amphinema byssoides-like, Hebeloma-like, and Suillus-like (ECS-B) fungi . This may suggest that there is no difference in biological activities of various ECM morphotypes in hybrid white spruce. Results of our chemical analyses of ECS soils paralleled those found in A. lasiocarpa (Arocena et al. 1999) and could suggest that the ECM are exhibiting the same processes interspecifically. Although there was no significant difference in the amounts of mica, chlorite, kaolinite, and 2:1 expandable clays between ECS and N-ECS soils, these trends do support the findings by earlier workers (Arocena et al. 1999; Kodama et al. 1994; Hinsinger and Jaillard 1993; Hinsinger et al. 1991; Hinsinger et al. 1992; April and Keller 1990). Mica, chlorite, kaolinite, and FeOOH (goethite) were lower in ECS soils compared to N-ECS soils. The ratios of vermiculite plus smectite over mica and chlorite indicate that weathering of mica and chlorite may have occurred in ECS soils. This may be especially true in ECS-A where the vermiculite and smectite were higher compared to ECS-B and N-ECS . Weathering of the minerals may have been a result of organic acids released by mycorrhizal fungi as organic carbon contents were significantly higher in ECS soils than N-ECS soils. No differences were seen in the rate of mica and chlorite weathering between the two ECS samples tested. Unforeseen problems associated with this project may have included the local variations in soil make-up (sand, silt and, clay) which can occur within a few meters and the difficulty to locate and collect Piloderma ectomycorrhizospheres (ECS-A) on spruce roots. In order to successfully show the differences between the two ECS soils, one must be sure that all samples used come from soil with the same physical properties (sand, silt, clay). In the future, studies will be needed to address the efficiency of each individual ECM morphotype in extracting specific exchangeable cations from specific minerals. This has significant implications for tree nutrition as soils with high CEC and high amounts of exchangeable cations are considered fertile soils, able to supply trees with essential nutrients. 4.5 Literature cited Agerer, R. (ed) 1987-1998. Colour Atlas ofEctomycorrhizae. Einhorn-Verlag Eduard Dietenberger GmbH Scwabisch Gmtind, Munich, Germany. Alexiades, C.A. and M.L. Jackson. 1965 . Quantitative determination ofvermiculite in soils. 47 Soil Sci. Soc. Amer. Proc. 29:522-527. Amebrant, K., H. Ek, R.D. Finlay and B. Soderstrom. 1993 . Nitrogen translocation between Alnus glutinosa (L.) Gaertn. seedlings inoculated with Frankia sp. and Pinus contorta Doug. Ex Loud seedlings connected by a common ectomycorrhizal mycelium. New Phytol. 130:231-242. April, R. and D. Keller. 1990. Mineralogy ofthe rhizosphere in forest soils ofthe eastern United States. Biogeochemistry 9:1-18. Arocena, J.M., K.R. Glowa, H.B. Massicotte and L. Lavkulich. 1999. Chemical and mineral composition of ectomycorrhizosphere soils of subalpine fir (Abies lasiocarpa (Hook.) Nutt.) in the Ae horizon of a Luvisol. Can J. Soil. Sci. 79:25-35. Arocena, J.M. and K.R. Glowa. 1999. Mineral weathering in ectomycorrhizospheres of subalpine fir (Abies lasiocarpa (Hook.) Nutt.) as revealed by soil solution composition. Forest Ecol. and Manage. In press. Berthelin, J. 1983. Microbial weathering processes. pp. 223-262. In Krumbein, W.E. (ed) Microbial geochemistry. Blackwell Scientific Publ., Oxford, UK. Bethlenfalvay, G.J., M.G. Reyes-Solis, S.B. Camel and R. Ferrera-Cerrato. 1991. Nutrient transfer between root zones of soybean and maize plants connected to a common mycorrhizal mycelium. Physiologia Plantarum. 82:423-432. Brand, F. 1991. Piloderma croceum. In: Agerer, R. (ed) Color Atlas ofEctomycorrhizae, plate 62. Einhorn-Verlag, Schwiibisch Gmiind. Brownlee, C., J.A. Duddridge, A. Malibari and D.J. Read. 1983 . The structure and function of ectomycorrhizal roots with special reference to their role in forming interplant connections and providing pathways for assimilate and water transport. Plant Soil 71 : 433-443 . Courchesne, F. and G.R. Gobran. 1997. Mineralogical variations ofbulk and rhizosphere soils from a Norway spruce stand. Soil. Sci. Soc. Am. J. 61 :1245-1249. Chung, J.-B . and R.J. Zasoski. 1994. Ammonium-K and ammonium-Ca exchange equilibria in bulk and rhizosphere soil. Soil Sci. Soc. Am. J. 58 :1368-1375. Cromack, Jr., K.P . Sollins, W. C. Grausten, K. Speidel, A. W. Todd, G. Spycher, C. Y. Li and R. L. Todd. 1979. Calcium oxalate accumulation and soil weathering in mats of the hypogeous fungus Hysterangium crassum . Soil Biol. Biochem. 11:463-468. 48 Curl, E.A. and B. Truelove. 1986. The rhizosphere. Springer-Verlag. Berlin. Dawson, A.B. 1989. Soils ofthe Prince George-McLeod Lake Area. Report no. 23 . British Columbia Soil Survey. Victoria, B.C. Derbsch, H. and J.A. Schmitt. 1987. Atlas der Pilze des Saarlandes 2. Nachweise, Okologie, Vorkommen und Beschreibungen. Nat. Landsch. Sonderb. 3:1-816. Dormaar, J.F. 1988. Effect of plant roots on chemical and biochemical properties of surrounding discrete soil zones. Can. J. Soil Sci. 68:233-242. Drever, J.L. and G.F. Vance. 1994. Role of organic acids in mineral weathering processes. pp. 138-161. In: Pittman ED, Lewman Ed (eds) Organic acids in geological processes. Springer, Berlin Heidelberg. Fanning, D.S., V.S. Keramidas and M.A. El-Desoky. 1989. Micas. pp. 467-525 . In J.B . Dixon and S.B. Weed (eds) Minerals in soil environments. 2nd ed. SSSA Book series no. 2. Madison, USA. Favre, J. 1960. Catalogue descriptif des champignons superieurs de la zone subalpine du Pare national Suisse. Erg. Wiss. Unters. Schweiz. National. 6: 321-610. Finlay, R. and D.J. Read. 1986. The structure and function ofthe vegetative mycelium of ectomycorrhizal plants. I. Translocation of 14C-labeled carbon between plants interconnected by a common mycelium. New Phytol. 103: 143-156. Fitter, A.H. and J. Garbaye. 1994. Interactions between mycorrhizal fungi and other soil organisms. Plant Soil 159: 123-132. Fogel, R. and G. Hunt. 1983. Contribution ofmycorrhizae and soil fungi to nutrient cycling in a Douglas-fir ecosystem. Can. J. For. Res. 12: 219-232. Francis, R. and D. J. Read. 1984. Direct transfer of carbon between plants connected by vesicular-arbuscular mycorrhizal mycelium. Nature 307: 53-56. Gardner, W.K. , D.G. Parbery and D.A. Barber. 1982. The acquisition of phosphorus by Lupinus a/bus L:I. Some characteristics of the soil/root interface. Plant Soil 68:19-32. Goodman, D.D., D.M. Durall, J.A. Trofymow and S.M. Berch. 1996. A manual of concise descriptions ofNorth American Ectomycorrhizae. BC Ministry of Forests, Victoria, B.C. Mycologue Publications, and Canada-B .C. Forest Resource Development Agreement, Canadian Forest Service, Victoria, B.C. Grayston, S.J., D. Vaughn and D. Jones. 1996. Rhizosphere carbon flows in trees, in 49 comparison with annual plants: the importance of root exudation and its impact on microbial activity and nutrient availability. Applied Soil Ecology 5: 29-56. Grierson, P.F. 1992. Organic acids in the rhizosphere of Banksia integrifolia L.F. Plant Soil 144: 259-265. Griffiths, R.P., J.E. Baham and B.A. Caldwell. 1994. Soil solution chemistry of ectomycorrhizal mats in forest soil. Soil Bioi. Biochem. 25: 331-37. Harley, J.L. 1989. The significance ofmycorrhizae. Mycol. Res. 92:129-139. Hamiman, S.M.K. and D.M. Durall. 1996a. Cenococcum geophilum Fr.+ Picea engelmannii (Parry) Engelm. p. CDE 10.3-10.4. in Goodman, D.D., D.M. Durall, J.A. Trofymow., and S.M. Berch (ed) A manual of concise descriptions of north American ectomycorrhizae. British Columbia Ministry of Forests. Victoria. Hamiman, S.M.K. and D.M. Durall. 1996b. Amphinema byssoides-like + Picea engelmannii (Parry) Engelm. P. CDE 6.1-6.4. in Goodman, D.D., D.M. Durall, J.A. Trofymow., and S.M. Berch (ed) A manual of concise descriptions of north American ectomycorrhizae. British Columbia Ministry of Forests. Victoria. ectomycorrhizae. British Columbia Ministry of Forests. Victoria. Hinsinger, P. and B. Jaillard. 1993. Root-induced release ofinterlayer potassium and vermiculitization of phlogopite as related to potassium depletion in the rhizosphere of ryegrass. J. Soil Sci. 44: 525-534. Hinsinger, P., B. Jaillard and J.E. Duffey. 1992. Rapid weathering of a trioctahedral mica by the roots ofryegrass. Soil Sci. Soc. Am. J. 56: 977-982. Hinsinger, P., J.E. Dufey and B. Jaillard. 1991. Biological weathering of micas in the rhizosphere as related to potassium absorption bv plants. p. 98-105. In McMichael, B.L. and H. Persson (ed) Plant roots and their environment. Elsevier Science Publishers. Amsterdam. Ingleby, K., P.A. Mason, F.T. Last and L.V. Fleming. 1990. Identification of ectomycorrhizae. Institute of Terrestrial Ecology. Research Publication 5. Jongmans, A., N. Van Breemen, U. Lundstrom, P. A. W. van Hees, R.D. Finlay, M. Srinivasan, T. Unestam, R. Giesler, P.A. Melkerud and M. Olsson. 1997. Rock-eating fungi. Nature 389: 682-683. Kalra, P. and D.G. Maynard. 1991. Methods manual for forest soil and plant analysis. 50 Information Report NOR-X-319. Forestry Canada, Edmonton, AB. Knapp, A. 1951. Die europaischen Hypogaen-Gattungen und ihre Gattungstypen. Schweiz. Z. Pilzkd. 29: 4. Kodama, H., S. Nelson, A.F. Yang and N. Kohyama. 1994. Mineralogy ofrhizospheric and non-rhizospheric soils in corn fields. Clays and Clay Miner. 42: 755-763. Kreisel, H(ed). 1987. Pilzflora der Deutschen Demokratischen Republik. Fischer, Jena. Lap eyrie, F. 1988. Oxalate synthesis from soil bicarbonate by the mycorrhizal fungus Paxillus involutus. Plant and Soil110:3-8. Leyval, C. and J. Berthelin. 1991. Weathering of mica by roots and rhizospheric microorganisms of pine. Soil Sci. Soc. Am. J. 55: 1009-1016. Manley, E.P. and L.J. Evans. 1986. Dissolution of feldspars by low-molecular-weight aliphatic and aromatic acids. Soil Sci. 141: 106-112. Marschner, H. and V. Romheld. 1983. In vivo measurement of root-induced pH changes at the soil-root interface: effects of plant species and nitrogen source. Z. Planzenphysiol. Bodenkd. 111 :241-251. Marschner, H. , Romheld, V. and I. Cackman. 1987. Root-induced changes of nutrient availability in the rhizosphere. J. Plant Nut. 10: 1175-1184. Massicotte, H.B., Molina, R., Luoma, D.L. and J.E. Smith. 1994. Biology of the ectomycorrhizal genus, Rhizopogon. II. Patterns ofhost-fungus specificity following spore inoculation of diverse hosts grown in monoculture and dual culture. New Phytol. 126: 677-90. McKeague, J.A. 1967. An evaluation of O.lM pyrophosphate and citrate-dithionite in comparison with oxalate as extractant of the accumulation products in podzols and some other soils. Can. J. Soil Sci . 47 : 95-99. Meidinger, D. and J. Pojar. 1991. Ecosystems ofBritish Columbia. BC Min. afForests Report Ser. No. 6. Victoria, BC. Molina, R. and M. Amaranthus. 1990. Rhizosphere biology: Ecological linkages between soil processes, plant growth, and community dynamics. pp. 51-58 . In A.E Harvey and L.F Neuenschwander (eds). Proceedings: Management and productivity ofwestern montane forest soils. Boise, ID, April 10-12, 1990. Intermountain Research Station, University ofldaho, Boise, ID. 51 Molina, R., H. Massicotte and J.M. Trappe. 1992. Specificity phenomena in mycorrhizal symbioses: Community-ecological consequences and practical implications. p. 357421. In M.F. Allen (ed) Mycorrhizal Functioning - An integrative plant-fungal process. Chapman and Hall, New York. Myrold, D. D. 1998. Transformations of nitrogen. p. 259-294. In D.M. Sylvia, J.J. Fuhrmann, P.G. Hartel. and D.A. Zuberer (eds) Principles and applications of soil microbiology. Prentice Hall, New Jersey. Newman, E.l. and W.R. Eason. 1993. Rates of phosphorus transfer within and between ryegrass (Lolium perenne) plants. Funct. Ecol. 7: 242-48. Nye, P.H. 1981. Changes of pH across the rhizosphere induced by roots. Plant Soil61 :7-26. Ochs, M. , I. Brunner, W. Stumm and B. Cosovic. 1993. Effect of root exudates and humic substances on weathering kinetics. Water, Air, Soil Pollut. 68: 213-229. Paris, F., P. Bonnaud, J. Ranger and F. Lapeyrie. 1994. Alteration d'un phyllosilicate par des champignons ectomycorhiziens in vitro. Acta Bot. Gallica 141: 529-532. Perry, D.A., R. Molina and M.P. Amaranthus. 1987. Mycorrhizae, mycorrhizospheres, and reforestation: current knowledge and research needs. Can. J. For. Res. 17: 929-940. Rauscher, T. and G. Chevalier. 1995. Tuber melanosporum. In: Agerer, R. (ed)- Color Atlas of Ectomycorrhizae, plate 87. Einhorn-Verlag, Schwiibisch Gmiind. Read, D.J., R. Francis and R.D. Finlay. 1985. Mycorrhizal mycelia and nutrient cycling in plant communities. pp. 193-217. In Fitter A.H. (ed) Ecological Interactions in Soil: Plants, Microbes and Animals. British Ecological Society Special Publication No. 4. Ricek, E.W. 1989. Die Pilzflora des Attergaus, Hausruck-und Kobernausserwaldes. Abh. Zool.-Bot. Ges. 6sterr. 23 . Robert, M. and J. Berthelin. 1986. Role ofbiological weathering and biochemical factors in soil mineral weathering. p 453-495 . In Huang, P.M. and M. Schnitzer (eds) Interactions of soil minerals with natural organics and microbes. SSSA Spec. Pub I. No. 17., Soil Sci. Soc. Am., Madison, WI. Rygiewicz, P.T. and C.S. Bledsoe. 1984. Mycorrhizal effects ofpotassium fluxes by northwest coniferous seedlings. Plant Physiol. 76: 918-923 . Rygiewicz, P.T., C.S. Bledsoe and R.J. Zasoki. 1984. Effects ofmycorrhizae and solution pH on eN) ammonium uptake by coniferous seedlings. Can. J. For. Res. 14: 8855 52 892. Sarkar, A.N., D.A. Jenkins and R.G. Wyn Jones. 1979. Modifications to mechanical and mineralogical composition of soil within the rhizosphere. pp 125-136. In J.L. Harvey and R. Scott-Russell (eds). The soil-root interface. Academic Press. London. Sarkar, A.N. and R.G. Wyn Jones. 1982. Effects ofrhizosphere pH on the availability and uptake ofFe, Mn, and Zn. Plant Soil66:361-372. Schmid-Heckel, H. 1988. Pilze in den Berchtesgadener Alpen. Forsch. Ber. Berchtesg. 15: 1136. Sheldrick, B.H. and C. Wang. 1993. Particle size analysis. p. 499-512. In Carter, M.R. (ed) Soil sampling and methods of analysis. Lewis Publ., Boca Raton, Florida. Simard W.S. , R. Molina, J.E. Smith, D.A. Perry and M.D. Jones. 1997. Shared compatibility of ectomycorrhizae on Pseudotsuga menziesii and Betula papyrifera seedlings grown in mixture in soils from southern British Columbia. Can. J. For. Res . 27:331-342. Soderstrom, B. and D.J. Read. 1987. Respiratory activity of intact and excised ectomycorrhizal mycelial systems growing in unsterile soil. Soil Biol. Biochem. 19:231-236. Statsoft Inc. 1995. Statistica for windows (computer program manual). Tulsa, Oklahoma. Theisen, A.A. and M.E. Harward. 1962. A paste method for preparation of slides for clay mineral identification by X-ray diffraction. Soil Sci. Soc. Amer. Proc. 26:90-91. Tinker, P.B. 1975. Soil chemistry of phosphorus and mycorrhizal effects on plant growth. pp. 353-371. In F.E. Sanders, B. Mosse and P.B. Tinker (eds) Endomycorrhizae. Academic press, London. Trappe, J.M. 1964. Mycorrhizal hosts and distribution of Cenococcum graniform e. Lloydia 27 : 100-106. Warren, C.J. and M. J. Dudas. 1992. Acidification adjacent to an elemental sulfur stockpile: I. Mineral weathering. Can. J. Soil Sci. 72: 113-126. Watteau, F. and J. Berthelin. 1994. Microbial dissolution of iron and aluminum from soil minerals : efficiency and specificity ofhydroxamate siderophores compared to aliphatic acids. Eur J. Soil Biol. 30: 1-9. Wittingham, J. and D.J. Read. 1982. Vesicular-arbuscular mycorrhizae in natural vegetation 53 systems III. Nutrient transfer between plants with mycorrhizal connections. New Phytol. 90: 277-84. 54 Table 4.1. Mean abundance (%) of ectomycorrhizae present in the ectomycorrhizosphere A (ECS-A) and B (ECS-B) samples for hybrid white spruce. (n=6) Amphinema byssoides-like ECS-A 0.10(0.24f Cenococcum geophilum Fr. 5.43(1.78) Hebeloma-like Hysterangium-like Inocy be-like 0.40(0.98) Ectomycorrhizae Brown, finely grainy 1 Brown, finely grainy 2 _Y E-strain 1 0.17(0.40) ITE.6-like Lactarius-like Leccinum-like My celium radicis atrovirens (MRA) Piloderma Russula-like 1 Russula-like 2 Suillus-like 0.20(0.31) 0.65(0.69) 93 .0(1.74) 0.10(0.24) Tan brown, finely grainy Tomentella-like 1 Tomentella-like 2 Tuber-like 1 Tuber-like 2 Tuber-like 3 Number of root tips counted per sample Percentage of mycorrhizal tips per sample Percentage of dead tips per sample Percentage of non-mycorrhizal tips per sample z Values in parentheses are standard deviations Y represents absence of ectomycorrhizae 55 883(94.7) 54.9(9.37) 44.9(9.72) 0.17(0.41) ECS-B 18.7(20.33) 0.24(0.58) 0.57(1.17) 9.73(5 .01) 0.05(0.12) 13.3(9.07) 1.64(3.59) 29.6(24.93) 3.64(3.60) 0.16(0.40) 0.20(0.48) 4.26(5 .06) 0.73(0.96) 0.30(0.75) 0.50(0.80) 11.3(20.35) 0.08(0.18) 0.95(1.43) 0.16(0.40) 1.09(1.30) 1.03(1.36) 1.81(4.33) 914(61.0) 48.9(7.46) 49.0(8.82) 2.18(3 .38) Table 4.2. Morphological descriptions of the dominant ectomycorrhizae of hybrid white spruce in Ae horizon of Luvisol. Ectomycorrhizae Amphinema byssoides-like Branching pattern, surface texture, lustre, color Irregularly branched to monopodia! pinnate, felty to cottony, matte to reflective, white-dull yellow/brown Cenococcum geophilum Fr. Single to monopodia! pinnate (rare), finely to coarsely grainy, shiny, black Hebeloma-like Monopodia! pinnate, cottony, matte to reflective, white to slightly brown/white Hysterangium-like Monopodia! pinnate, wooly, reflective, white lnocybe-like Not branched, smooth to finely grainy, matte to shiny, tan brown Mantle characteristics, outer mantle (OM)- inner mantle (IM) All cells turning distinctly yellow in 10% KOH, mantle thickness variable; OM felt-net prosenchyma, hyphae smooth to finely verrucose, 2.5-4 !-lil1 wide, large clamped septa; IM net prosenchyma to synenchyma, hyphae smooth, 2.5-5 !-lil1 wide, septa not clamped Mantle 5-25 !-lil1 thick; OM net synenchyma, hyphae 4-7 f..lm wide, with distinct "stellar" pattern ; IM net synenchyma, hyphae 1-4 f..lm Mantle 5-30 flm thick; OM net prosenchyma, hyphae 3-6 flm, common clamped septa, common enlarged junctions; IM net synenchyma, hyphae 26 flm , no septa Mantle 10-25 flm thick; OM felt-net prosenchyma, hyphae 2.5-4 flm thick, druse-like ornamentation, common clamped septa; IM net prosenchyma to net synenchyma, hyphae 2-4 1-1m , no ornaments, common undamped septa Mantle 10-20 flm thick; OM felt-net prosenchyma, hyphae 0.5-3 flm wide, oil-like bodies, common septa, swollen/ enlarged junctions and multiseptate hyphae ; IM net synenchyma, hyphae 1-2 flm , oil like bodies, common seEta 56 Emanating hyphae (EH), mycelial strand (MS), C:rstidia (C) EH abundant, 2.5-3.5 !-lil1 wide, white-yellow to dull yellow/brown, smooth to verrucose, clamped septa, H-shaped anastomoses with clamp, frequently branched; MS abundant, loose to smoothundifferentiated, hyphae as perEH EH common, straight, thick walls, 3-6 flm wide, septate, smooth, dark brown-black; MS not observed EH common, straight, 2-6 !-lil1 wide, white, verrucose medium ornamentation, common clamped septa, H-shaped anastomoses without clamp, hyphae stain blue with toluidine blue; MS loose-smoothundifferentiated, hyphae as per EH ; No C observed EH rare, 2.5-4 flm wide, large crystalline ornamentation, common clamped septa; MS as per EH; No C observed EH uncommon ; MS and C not observed ITE.6-Iike Single, smooth, shiny , brown to tan brown Mycelium radicis atrovirens (MRA) Single, finely grainy to slightly felty , shiny, black/dark brown, root apex sometimes hyaline Piloderma Irregular systems, coarsely felty, matte, bright yellow Suillus-like Single to monopodia! pinnate, cottony, reflective, white to dull white to golden yellow Tuber-like I Single monopodia! pinnate, felty, matte, cream yellow/gray Tuber-like 2 Single to monopodia! pinnate, felty , matte, cream yellow/gray Tuber-like 3 Single to monopodia! pinnate, felty, smooth , matte, tan brown Mantle 8-15 IJlil thick ; OM net synenchyma, hyphae 1-3 1-1m wide, distinct hypha) cells that stain pink to bright pink in toluidine blue (that give rise to emanating hyphae); IM net synenchyma, hyphae 1.5-3 1-1m wide Mantle I O-I5 IJlil thick; OM net prosenchyma with typically inflated outer hypha! cells, hyphae I-5 IJlil wide; IM net synenchyma, hyphae 2.5-3 IJlil wide Mantle 10-40 IJlil thick; OM felt prosenchyma, finely verrucose to crystalline, hyphae 2.5-3 IJlil wide, septate; IM net prosenchyma, hyphae 1.5-2.5 IJlil wide Mantle 10-40 IJlil thick; OM net prosenchyma, medium to large crystalline/needlelike ornaments, hyphae 2-4 1-1m wide, septate; IM net synenchyma, hyphae 2-3 1-1m Mantle I5-30 1-1m thick; OM non-interlocking to interlocking irregular to regular synenchyma, hyphae 3-42 1-1m wide; IM net synenchyma, cells 2-9 1-1m wide Mantle I5-30 1-1m thick; OM non-interlocking to interlocking irregular synenchyma, hyphae 5-17 !-liD wide ; IM net synenchym a, cells 3-4 1-1m wide Mantle I5-30 1-1m thick ; OM interlocking irregular synenchyma, hyphae 3-6 1-1m wide, possibly oil like bodies; IM net synenchyma, cells 1-3 [;!ID wide 57 EH rare to common, hyphae 3-4 !-liD, common crystalline to verrucose, common clamped septa, anastomoses contact without clamp; MS and C not observed EH common, 2-3 !-liD wide, finely verrucose, septate, brown/black, no clamp ; MS and C not observed EH abundant, 3 IJlil wide, bright yellow, verrucose, septate, H-shaped anastomoses with septa, no clamps; MS numerous , strand hyphae as per EH EH abundant, 2-4 IJlil wide, none to rare medium to large crystalline, needlelike ornaments, septate, Hshaped anastomoses without clamp, no clamps; MS numerous, smooth undifferentiated, strand hyphae as per EH EH rare to common, 3-6 1-1m wide, clamped septa; NoMS and C EH and MS absent; C common, bristle-like to awl shaped, 40-300 1-1m long, apex width 2-3 !-liD, medial width 2-5 1-1m wide, basal width 4-6 1-1m , wall thickness 1-1.5 1-1m EH and MS absent ; C rare to common, bristle-like to awl shaped, 20-100 IJlil long, apex width 1-2 IJID , medial width 1-21-lm wide, basal width 2-4 [;!ID Table 4.3. Mean values for physical and chemical properties of two ectomycorrhizosphere (ECS-A and ECS-B) and non-ectomycorrhizosphere (N-ECM) soils ofwhite spruce. (n=6) Soil Properties Particle Size Dist. (g kg-1) Sand Silt Clay ECS-A ECS-B N-ECM 550 (112)z 382 (92) 68 (21) 529 (80) 398 (65) 73 (16) 482 (92) 450 (73) 68 (20) pH Total Carbon (g kg-1 soil) Total Nitrogen(g kg-1 soil) CIN ratio CEC (cmolc kg- 1 soil) Ex. Cations (cmolc kg-1 soil) Ca Mg K Na 4.11a (0.13) 19.7a (4.29) 0.85a (0.14) 23 .1a (2.21) 5.87a (1.10) 4.26a (0.15) 21.3a (6.44) 0.87a (0.17) 24.4a (5 .63) 6.61a (0.83) 5.lb (0.17) 6.05b (1.32) 0.39b (0.06) 15 .4b (1.99) 3.90b (0.61) 2.65ab (0.67) 0.84 (0.24) 0.29a (0.06) 0.12a (0.03) 3.34a (0. 76) 1.11 (0.27) 0.32a (0.04) 0.12a (0.02) 2.36b (0.69) 0.78 (0.34) 0.17b (0.04) 0.07b (0.01) Base saturation (%) 67.la (11.6) 67.8a (7.9) 88.2b (16.3) zy alues in parentheses are standard deviations. a-c Across each row, means followed by the same letter are not significantly different. 58 Table 4.4. Mean contents (g kg·') of selected phyllosilicates and oxides in clay fraction in non-ectomycorrhizosphere (N-ECM) and ectomycorrhizosphere soils ofwhite spruce. (n = 6) Minerals Mica Chlorite KaoliniteY Amor. Ah03 Amor. Fe203 Amor. Si02 FeOOH# Vt+Sm (Vt+Sm)/Mi (Vt+Sm)/Ch ECS-A ECS-B N-ECM 177.5 (12.6)z 99.4 (25 .9) 43.7 (25.4) 2.18 (0.57) 3.49 (0.27) 1.01 (0.18) 14.9a (1.70) 104.6 (10.3) 0.56 (0.04) 1.13 (0.37) 181.6 (18.1) 102.9 (32.5) 40.7 (32.2) 2.48 (0.55) 4.63 (0.96) 1.00 (0.10) 18.1b (2.42) 94.2 (7.00) 0.55 (0.08) 1.07 (0.42) 194.3 (23.4) 103.2 (30.9) 69.5 (67.3) 3.09 (0.71) 3.91 (1.28) 0.86 (0.14) 17.8ab (0.54) 94.6 (21. 7) 0.48 (0.09) 1.03 (0.47) Vt =Vermiculite, Sm =Smectite, Mi=Mica; Ch =Chlorite;# FeOOH (goethite)- assumed as the dominant crystalline Fe oxide Across each row, means superscripted with similar letters are not significantly different. z values in parentheses are standard deviations. Yn=4. 59 Ectomycorrhizosphere B (ECS-B) 1.41 0 .709 1.41 0 .334 Ca. 54% RH 0 .709 K -54% RH 0.426 0 .355 0 .334 1.01 K • 550°C 0.709 0 .426 0 .355 0 .319 0 .334 1.79 0 5 1.41 10 Ca ·Glycerol 0 .709 15 20 25 30 35 40 45 Fig. 4.1. X-Ray diffraction patterns of the clay fraction ofECS-B sample subjected to four pre-treatments showing the relative intensities ofthe 1.7-, 1.4-, 1.0-, .71 -, .42-, .35-, and .33nm reflections. 60 1.79 Ca-Sal. and Glycerol Solvated ECS-A 10 1 .79 12 14 1S 20 22 20 22 20 22 ECS-8 Ca - Giycerol 1.41 10 12 10 1 . 41 1 .79 \ N - ECM · Ca-Giycerol 10 0 .709 12 14 16 1a Fe Ka • 2 e Fig. 4.2. X-Ray diffraction patterns of Ca-saturated and glycerol-solvated clay fractions of ECS-A, ECS-B, and N-ECS samples showing the relative intensities ofthe 1.7- and 1.4-nm reflections. 61 Chapter V - Conclusion and Recommendation In vitro and field studies strongly suggest that Piloderma plays a large role in weathering of soil minerals to provide essential nutrients for the growth of Picea glauca x engelmannii.Piloderma grown in biotite exhibited higher biomass and different morphological characteristics than Piloderma grown in chlorite, microcline and MMN2 treatments. The vigorous growth in biotite medium was attributed to the ability of Piloderma to efficiently obtain K from the interlayer of biotite compared to Kin the framework structure of microcline. The lower density of lenticular ornaments in nutrient-poor media compared to biotite and MMN media could be used as indicators for unhealthy environments. Another possible indicator forK-deficient growing medium could be the frequent fibrillar growths and hypha! swellings in Piloderma observed in nutrient-poor MMN2 medium. In the field study, the chemical and mineralogical properties of ectomycorrhizosphere soils were different to that of bulk soils and suggest that different ECM systems might be involved in mineral weathering. The high exchangeable Kin ECS-A and ECS B soils may indicate that Piloderma and other ECM may be involved in weathering of mica and the release of interlayer K. Contents of mica, chlorite, kaolinite, and FeOOH were lower in ectomycorrhizosphere soils compared to bulk soils, and these findings corroborate earlier reports (Arocena et al. 1999; Kodama et al. 1994; Hinsinger and Jaillard 1993; Hinsinger et al. 1991; Hinsinger et al. 1992; April and Keller 1990). Vermiculite and smectite (secondary weathering products of mica) were higher in ECS-A (Piloderma-dominated) soils compared to ECS-B (<1% Piloderma colonization) soils, and supporting the results from in vitro studies suggesting the ability of Piloderma to induce accelerated rate of biotite weathering. The findings from this thesis illustrate the importance of ectomycorrhizal ~ ~ in soil formation, particularly the transformation of common soil minerals. One of the possible applications of the knowledge that Piloderma can weather biotite more efficiently as source of potassium could be the need for Piloderma inoculation of coniferous seedling prior to planting in a potassium-deficient environment. Although our in vitro studies strongly suggest more efficient weathering of biotite over microcline and chlorite by Piloderma, the field situation might have different outcomes due to the influence oftree roots, competition from other fungi, and environmental factors such as moisture content and temperature. The presence of organic ligands in soil water and root exudates might also influence fungal- 62 mineral preference. In the future, studies are needed to establish the exclusive role of Piloderma in the ectomycorrhizosphere of spruce. This could be done through controlled experiment (e.g., greenhouse) where spruce trees growing in biotite, chlorite and microcline will be exclusively infected with Piloderma. Because of the wide diversity of ectomycorrhizae in forest ecosystems, in vitro and greenhouse studies will also be needed to elucidate specific roles of other common ECM, particularly Cenococcum geophilum Fr., Amphinema byssoides-like, and MRA that are known to colonize economically important trees in British Columbia. Also, it will be important to investigate the mineralogy of minerals used in this study to elucidate the role of ECM in the formation of vermiculite and smectite in ECS soils observed in the field study. 63 Chapter VI - Literature Cited Alvarez, I.F., D.L. Rowney and F.W. Cobb Jr. 1979. Mycorrhizae and growth ofwhite fir seedlings in mineral soil with and without organic layers in a California forest. Can J. For. Res. 9:311-315. Arocena, J.M., K.R. Glowa, H.B. Massic9tte and L. Lavkulich. 1999. Chemical and mineral composition of ectomycorhizosphere soils of subalpine fir (Abies lasiocarpa (Hook.) Nutt.) in the Ae horizon of a Luvisol. Can J. Soil Sci. 79:25-35 April, R. and D. Keller. 1990. Mineralogy of the rhizosphere and forest soils ofthe eastern United States. Biogeochemistry 9:1-18. Bakshi, B.K. 1974. Mycorrhizae and its role in forestry . Forest Research Institute and Colleges. Dehra Dun. India. Project Report No. P.L. 480. Barnhisel, R.I and P.M. Bertsch. 1989. Chlorites and hydroxy-interlayered vermiculites and smectite. pp. 729-788 in J.B Dixon and S.B. Weed (eds) Minerals in Soil Environments. 2"d ed. Soil. Sci. Soc. Am Book series no. 2. Madison, WI. Berthelin, J. 1983. Microbial weathering processes. pp. 223-262. In Krumbein, W.E. (ed) Microbial geochemistry. Blackwell Scientific Publ., Oxford, UK. Berthelin, J. 1988. Microbial weathering processes in natural environments. p. 375. In Lerman A. and M. Meybeck (eds) Physical and Chemical Weathering in Geochemical Cycles. Kluwer Academic Publishers, Dordrecht. Berthelin, J and C. Leyva!. 1982. Ability of symbiotic and nonsymbiotic rhizosphere microflora of maize (Zea mays) to weather micas and to promote plant growth and plant nutrition. Plant and Soil 68, 369-377. Bolan, N.S. 1991. A critical review on the role ofmycorrhizal fungi in the uptake of phosphorus by plants. Plant and Soil 134:189-207. Bowen, G. D. 1973. Mineral nutrition ofectomycorrhizae. pp. 151-205. In Marks, G.C. and T.T. Kozlowski (eds) Ectomycorrhizae. Academic press, NY. Brady, N.C. 1990. Origin, nature, and classification of parent materials. pp 23-46. In The Nature and Properties of Soils. Macmillan Publishing Company, NY. Chung, J.B . and R.I. Zasoski. 1994. Ammonium-K and ammonium-Ca exchange equilibria in bulk and rhizosphere soil. Soil Sci. Soc. Am. J. 58:1368-1375. 64 Courchesne, F. and G.R. Gobran. 1997. Mineralogical variations of bulk and rhizosphere soils from a Norway Spruce stand. Soil Sci. Soc. Am. J. 61:1245-1249. Cromack, K. Jr., P. Sollins, W.C. Graustein, K. Speidel, A.W. Todd., G. Spycher, C.Y. Li and R.L. Todd. 1979. Ca oxalate accumulation and soil weathering in mats of the hypogeous fungus Hysterangium crassum . Soil Biol. Biochem. 11 :463-68. Cromack, K. Jr., P. Sollins, R.L. Todd, R. Fogel, A.W. Todd, W.M. Fender, M.E. Crossley and D.A. Crossley. 1977. The role of oxalic acid and bicarbonate inCa cycling by fungi and bacteria. Some possible implications for soil animals. Ecological Bulletin (Stockholm) 25:246-252. Cumming, J.R. and L.H. Weinstein. 1990. Utilization of AlP0 4 as a phosphorus source by ectomycorrhizal Pinus rigida seedlings. New Phytol. 116:99-106. Curl, E.A. and B. Truelove. 1986. The Rhizosphere. p. 288. New York: Springer-Verlag. de Leenheer. L. 1950. Mineralogical characterization of the sand-fraction in soil profiles. Trans. Int. Congr. Soil Sci.2:84-89. Dormaar, J.F. 1988. Effect of plant roots on chemical and biochemical properties of surrounding discrete soil zones. Can. J. Soil Sci. 68:233-242. Fanning, D.S., V.Z Keramidas and M.A. El-Desoky. 1989. Micas. pp. 467-525 . In J.B Dixon and S.B . Weed (eds) Minerals in soil environments. Soil Sci. Amer. Book Series#l. Soil Sci. Amer., Madison, WI. Fisher, F. 1972. Spodosol development and nutrient distribution under Hydnaceae fungal mats. Soil Sci. Soc. Amer. Proc. 36:492-95. Foster, R.C., A.D. Rovira and T.W. Cook. 1983. Ultrastructure ofthe root-soil interface. The American Phytopathological Society. St. Paul, MN. Gardner, W.K., D.G. Parbery and D.A. Barber. 1982. The acquisition of phosphorus by Lupinus a/bus L: I. Some characteristics of the soil/root interface. Plant Soil 68 : 1932. Gianinazzi-Pearson, V. and S. Gianinazzi. 1986. The physiology of improved phosphate nutrition in mycorrhizal plants. pp. 101-109. In Gianinazzi-Pearson, V. and S. Gianinazzi (eds) Physiological and Genetical aspects ofmycorrhizae. Proc. 1st European Symposium on Mycorrhizae, Dijon, 1-5 July 1985. INRA, Paris Harley, J.L. 1989. The significance ofmycorrhizae. Mycol. Res. 92 :129-139. 65 Harley, J.L. and C.C. McCready. 1952. The uptake ofphosphate by excised mycorrhizal roots ofbeech. II. Distribution of phosphate between the host and fungus. New Phytol. 51:56-64. Harley, J.L. and S.E. Smith. 1983. Mycorrhizal symbiosis. Academic Press, London. Harvey, A.E., M.J. Larsen and M.F. Jurgensen. 1976. Distribution of ectomycorrhizae in mature Douglas-fir /larch forest soil in western Montana. For. Sci. 22:393-398 . Harvey, A.E., M.J. Larsen and M.F. Jurgensen. 1979. Comparative distribution of ectomycorrhizae in soils of three western Montana forest habitat types. For. Sci. 25:350-358. Hinsinger, P., F. Elsass, B. Jailard and M. Robert. 1993. Root-induced release ofinterlayer potassium and vermiculization of phlogopite as related to potassium depletion in the rhizosphere ofryegrass. J. Soil. Sci. 44:525-534. Hinsinger, P., J.E. Dufey and B. Jaillard. 1991. Biological weathering of micas in the rhizosphere as related to potassium absorption by plants. pp. 98-105. In McMichael, B.L. and H. Persson (ed) Plant roots and their environment. Elsevier Science Publishers. Amsterdam. Hinsinger, P., B. Jailard and J.E. Duffey. 1992. Rapid weathering oftrioctahedral mica by roots ofryegrass. Soil Sci. Soc. Amer. J. 56:977-982. Hintikka, V. and 0. Naykki. 1967. Notes on the effects ofthe fungus Hydnellumferrugineum (Fr.). Karst. on forest soil and vegetation. Communicationes Instituti Forestalis Fenniae 62:1-22. Hseung, Y. and M .L. Jackson. 1952. Mineral composition ofthe clay fraction: III. Of some main soil groups of China. Soil. Sci. Soc. Am. Proc. 16:294-297. Huang, P.M. 1989. Feldspars, Olivines, Pyroxenes, and Amphiboles. pp. 975-1050. In J.B Dixon and S.B. Weed (eds) Minerals in Soil Environments. Soil. Sci. Soc. Am, Inc. Jackson, M.L. 1959. Frequency distribution of clay minerals in major great soil groups as related to the factors of soil formation. Clays Clay Miner. 35:111-112. Jackson, M.L.1964. Chemical composition of soils. pp. 71-141. In F .E. Bear (ed) Chemistry of the soil. Reinhold Publishing Corp., New York. Jackson, M.L., Y. Hseung, E.J. Evans and R.C. VandenHeuvel. 1952. Weathering of claysize minerals in soils and sediments. II. Chemical weathering of layer silicates. Soi 1 66 Sci. Soc. Am. Proc. 16:3-6. Jackson, M.L., S.A. Tyler, A.L. Bourbeau and R.P. Pennington. 1948. Weathering sequence of clay-sized minerals in soils and sediments. I. Fundamental generalizations. J. Phys. Colloid Chern. 52:1237-1260. Jeffries, C.D., E. Grissinger and L. Johnson. 1956. Distribution of important soil minerals in Pennsylvania soils. Soil Sci. Soc. Am. Proc. 20:400-403. Jongmans, A.G., N. Breeman, U. Lundstrom, P.A.W van Hees, R.D. Finlay, M. Srinivasan, T. Unestam, R. Giesler, P.A Melkerud and M. Olsson. 1997. Rock eating fungi. Nature 389:682-3. Killham, K. 1994. Soil Ecology. Cambridge University Press, Cambridge, England. Kimmins, J.P. and B.C. Hawkes. 1978. Distribution and chemistry of fine roots in a white spruce-subalpine fir stand in British Columbia: implications for management. Can. J. For. Res. 8:265-279. Kodama, H., S. Nelson, A.F. Yang and N. Kohyama. 1994. Mineralogy ofrhizospheric and non-rhizospheric soils in corn fields. Clays Clay Miner. 42:755-763. Lapeyrie, F., J. Raager and D. Vairelles. 1990. Phosphate-solubilizing activity of ectomycorrhizal fungi in vitro. Can. J. Bot. 69:342-346. Lapeyrie, F. 1988. Oxalate synthesis from soil bicarbonate by the mycorrhizal fungus Paxillus involutus. Plant and Soil110:3-8 Leyval, C. and J. Berthelin. 1991 . Weathering of a mica by roots and rhizospheric microorganisms of pine. Soil Sci. Soc. Amer. J. 55: 1006-1 009. Ma1ajczuk, N. and K. Cromack. 1982. Accumulation ofCa Oxalate in the mantle of ectomycorrhizal roots of Pinus radiata and Eucalyptus marginata. New Phytol. 92:527-531. Majstrik, V. 1970. The uptake of 32 P by different kinds of ectotrophic mycorrhizae of Pinus. New Phytol. 69:295-298 . Marschner, H. 1995. Mineral nutrition of higher plants. London:acedemic press. Marschner, H. and V. Rornheld. 1983. In vivo measurement ofroot-induced pH changes at the soil-root interface: Effects of plant species and nitrogen source. Z. Planzenphysiol. Bodenkd. 111 :241-251. McBride, M.B. 1994. Environmental chemistry of soils. Oxford University Press, Inc, N.Y. 67 McCalla, T.M. 1946. Influence of some microbial groups on stabilizing soil structure against falling water drops. Soil Sci. Soc. Am. Proc. 11:260-263 Mojallali, M. and S.B. Weed. 1978. Weathering of micas by mycorrhizal soybeen plants. Soil Sci. Soc. Am. J. 42:367-372 Molina, R. and M. Amaranthus. 1990. Rhizosphere biology: Ecological linkages between soil processes, plant growth, and community dynamics. pp. 51-58. In A.E Harvey and L.F Neuenschwander (eds). Proceedings: Management and productivity of western montane forest soils. Boise, ID, April 10-12, 1990. Intermountain Research Station, University ofldaho, Boise, ID. Morrison, T.M: 1962. Absorption of phosphorus from soils by mycorrhizal plants. New Phytol. 61 :10-20. Nye, P .H. 19.81. Changes of pH across the rhizosphere induced by roots. Plant Soil 61 :726. Ochs, M., I. Brunner, W. Stumm and B. Cosovic. 1993. Effects of root exudate and humic substances on weathering kinetics. Water Air Soil Poll. 68:213-229. Paris, F., P. Bonnaud, J. Ranger and F. Lapeyrie. 1994. Alteration d' un phyllosilicate par des champignons ectomycorhiziens in vitro. Acta Bot. Gallica 141:529-532. Perry, D.A., Molina, R. and M.P. Amaranthus. 1987. Mycorrhizae, mycorrhizospheres, and reforestation: current knowledge and research needs. Can. J. For. Res. 17:929-940. Phillippe, M.M. and J.L. White. 1952. Acid soluble potassium and microcline content of the silt fractions of 12 Indiana soils. Soil Sci. Soc. Am. Proc. 16:371-380. Read, D. 1997. The ties that bind. Nature. 388:517-518. Read, D.J., R. Francis and R.D. Finlay. 1985. Mycorrhizal mycelia and nutrient cycling in plant communities. pp. 193-217. In Fitter A.H. (ed) Ecological Interactions in Soil: Plants, Microbes and Animals. British Ecological Society Special Publication No. 4. Rhodes, L.H. and J.W. Gerdeman. 1975. Phosphate uptake zones ofmycorrhizal and nonmycorrhizal onion. New Phytol. 75:555-61. Robert, M. and J. Berthelin. 1986. Role of biological and biochemical factors in soil mineral weathering. pp. 453-95. In P.M. Huang and M. Schnitzer (eds) Interactions of soil minerals with natural organics and microbes. SSSA Spec. Publ. 17. ASA, CSSA, and SSSA. Madison, WI. 68 Rousseau, J.V.D., D.M. Sylvia and A.J. Fox. 1994. Contribution of ectomycorrhizae to the potential nutrient-absorbing surface of pine. New Phytol. 128:639-644. Rygiewicz, P.T., C.S. Bledsoe and R.J. Zasoki. 1984. Effects ofmycorrhizae and solution pH on eN) ammonium uptake by coniferous seedlings. Can. J. For. Res. 14:885-892. 5 Sarkar, A.N., D.A. Jenkins and R.G. Wyn Jones. 1979. Modifications to mechanical and mineralogical composition of soil within the rhizosphere. pp. 125-136. In J.L. Harvey and R. Scott-Russell (eds). The soil-root interface. Academic Press. London. Sarkar, A.N. and R.G. Wyn Jones. 1982. Effects ofrhizosphere pH on the availability and uptake ofFe, Mn, and Zn. Plant Soil66:361-372. Simard W.S., R. Molina., J.E. Smith., D.A. Perry and M.D. Jones. 1997. Shared compatibility of ectomycorrhizae on Pseudotsuga menziesii and Betula papyrifera seedlings grown in mixture in soils from southern British Columbia Can. J. For. Res . 27:331-342. Snetselaar, K.M and K.D. Whitney. 1990. Fungal Ca oxalate in mycorrhizae of Monotropa uniflora. Can. J. Bot. 68:533-543. Sorensen, J. 1997. The rhizosphere as a habitat for soil microorganisms. pp. 63-127. In J.D . Elasas, J. Trevors and E.M.H. Wellington (eds) Modem Soil Microbiology. Marcel Dekker, INC. Sylvia, D.M. 1998. Mycorrhizal symbioses. pp. 408-426. In D.M. Sylvia, J.J. Fuhrmann, P. G. Hartel and D.A. Zuberer (eds) Principles and applications of soil microbiology. Prentice Hall, New Jersey. Tan, K.H., P. Sihanonth and R.L. Todd. 1978. Formation ofhumic acid-like compounds by the ectomycorrhizal fungus Pisolithus tinctorius. Soil Sci. Soc. Amer. J. 42:906-908 . Tinker, P.B. 1975. Soil chemistry of phosphorus and mycorrhizal effects on plant growth. pp. 353-371. In F.E. Sanders., B. Mosse and P.B. Tinker (eds) Ectomycorrhizae. Academic press, London. Tumau, K., I. Kottke and J. Dexheimer. 1994. Paxillus involutus/Pinus sylvestris mycorrhizae from heavily polluted forest II. Ultrastructural and cytochemical observations. Bot. Acta. 107:73-80. Vogt, K.A. , R.L. Edmonds and C.C. Grier. 1981a. Seasonal changes in biomass and vertical distribution of mycorrhizal and fibrous-textured conifer fine roots in 23 and 180-year- 69 old subalpine Abies amabilis stands. Can. J. For. Res. 11 :223-229. Vogt, K.A., L. Robert and C.C. Grier. 1981 b. Dynamics of ectomycorrhizae in Abies amabilis stands: The role of Cenococcum graniforme. Holarct. Ecol. 4:167-173. Wallander, H. and T. Wickman. 1999. Biotite and microcline as a K source in ectomycorrhizal and non-ectomycorrhizal Pinus sylvestris seedlings. Mycorrhiza 9:25-32. Wallander, H., T. Wickman and G. Jacks. 1997. Apatite asaP source in mycorrhizal and non-mycorrhizal Pinus sylvestris seedlings. Plant and Soil. 196:123-131. 70