The Inhibitory Effect of Rhododendron maximum L. (Ericaceae) Thickets on Mycorrhizal Colonization of Canopy Tree Seedlings

Transcription

1 The Inhibitory Effect of Rhododendron maximum L. (Ericaceae) Thickets on Mycorrhizal Colonization of Canopy Tree Seedlings John F. Walker Thesis submitted the Faculty of the Virginia Polytechnic Institute and State university in partial fulfillment of the requirements for the degree of Master of Science in Biology Orson K. Miller, Jr., Chairman Khidir W. Hilu Erik T. Nilsen April, 1998 Blacksburg, Virginia Keywords: Ectomycorrhizae, Mycorrhizal colonization, Fungus community, Rhododendron, Tree seedling

2 THE INHIBITORY EFFECT OF RHODODENDRON MAXIMUM L. (ERICACEAE) THICKETS ON MYCORRHIZAL COLONIZATION OF CANOPY TREE SEEDLINGS John F. Walker (ABSTRACT) Thickets of Rhododendron maximum (Rm) in the southern Appalachians impose severe limitations on the regeneration of hardwood and coniferous seedlings. Interactions between Rm thickets and ectomycorrhizal colonization were examined to explain seedling inhibition. Experimental blocks were established in and out of Rm thickets in a mature, mixed hardwood/conifer forest in Macon County, North Carolina. Planted seedlings of red oak (Quercus rubra) and hemlock (Tsuga canadensis) were harvested at the end of the first and second growing seasons. Litter manipulation had no effect on total mycorrhizal colonization. Mycorrhizal colonization and ramification index (# mycorrhizae cm -1 ) were depressed and colonization by Cenococcum geophilum increased in blocks with versus without Rm. After the first year, percent colonization of T. canadensis not in Rm thickets (62 %) was three times higher than in Rm thickets (19%), and the ramification index was increased by more than a factor of four (2.83 versus 0.61). Mycorrhizal colonization levels were correlated with root weight and shoot weight in both hemlock and oak seedlings, but did not explain most of the variation observed. Sporocarps of 69 putatively ectomycorrhizal species were collected on the blocks. Species diversity and overall community structure was similar in and out of Rm thickets. Individual species, i.e. Lactarius speciosus and Russula krombholzii, were significant indicators of forest without Rm thickets. Rhododendron maximum thickets probably affect the process of mycorrhization. The reduced level of mycorrhizal capacity under Rm thickets could be a factor in the increased seedling failure in Rm thickets.

3 ACKNOWLEDGEMENTS First I would like to thank Dr. Orson Miller Jr., who as chairman of my committee was instrumental in developing the design and analysis of this study. His guidance and assistance with the fieldwork, mushroom identifications, and mycorrhizal root counts was indispensable. In addition to being an excellent mentor, Dr. Miller has become an esteemed friend and is always a great pleasure to work with. Secondly I must thank Dr. Erik Nilsen, Dr. Tom Lei, Barry Clinton and Shawn Semones who put in many grueling hours assisting with the plot setup and substrate transfer. Shawn Semones and Dr. Lei were constant companions in the field and provided the majority of the above-ground seedling health data. Shawn Semones, Dr. Nilsen, Dr. Lei, Dr. Miller, and the Statistics Consulting Center of Virginia Polytechnic were all involved in designing the statistical model for the analyses in Chapter 1. The professional guidance, critical input, and teaching of Dr. Nilsen and Dr. Khidir Hilu as members of my committee were greatly appreciated. I would also like to thank Hope Miller for assisting edit this manuscript and in the field. The Biology department of Virginia Polytechnic provided funding in the form of Graduate Teaching Assistantships and Tuition Waivers. A Sigma XI, the research society, grant-in-aid of research helped purchase equipment for additional leachate studies. Funding for Graduate Research Assistantships, and expenses for this project, was provided by the National Research Initiative Competitive Grants Program of the U.S.D.A., grant number I would also like to thank the Coweeta Hydrologic Laboratory for logistical support. The efforts of the Asheville Mushroom Club, who added many records to our sporocarp database during two fall forays at Coweeta, were greatly appreciated. I am grateful for the moral and financial support of my parents, Dr. Jerome and Delette Walker. Finally to Lynette Walker, who became my wife during the course of this program, I owe everything. Acknowledgements iii

4 TABLE OF CONTENTS INTRODUCTION.1 The mycorrhizal symbiosis.2 The ectomycorrhizal class:.4 The ericoid mycorrhizal class:.5 Phosphorus uptake in mycorrhizae:.7 Carbon translocation in mycorrhizae: 8 Interplant mycorrhizal linkages: 9 Thesis objectives: 10 CHAPTER 1. Suppression of ectomycorrhizae on canopy tree seedlings in Rhododendron maximum L. (Ericaceae) thickets in the southern Appalachians 12 Introduction: 13 Materials and Methods: 15 Site Description: 15 Plot layout and Site preparation: 16 Seedling Harvest and Sampling: 16 Statistics: 17 Results: and 1997 hemlock mycorrhizae: and 1997 red oak mycorrhizae: 19 Seedling growth parameters: 23 Correlation of mycorrhizae and seedling health: 27 Discussion: 28 Total mycorrhizal colonization and ramification 28 Colonization by Cenococcum geophilum: 33 Mycorrhizal mycelial interconnections between Rhododendron maximum Table of Contents iv

5 and seedlings: 34 Conclusions: 35 CHAPTER 2. The effects of Rhododendron maximum L. (Ericaceae) thickets, organic layer substrates, and litter materials on an Ectomycorrhizal fungal community in the southern Appalachians 36 Introduction: 37 Sporocarp sampling and community analyses: 37 Sporocarp sampling and ectomycorrhizal colonization: 40 Materials and Methods: 42 Sporocarp Sampling: 42 Analytical methods: 43 Results: 45 General assessment of the ectomycorrhizal fungus community: 45 Ectomycorrhizal fungi in versus out of Rhododendron maximum thickets: 55 Ectomycorrhizal fungi in response to treatments: 55 Discussion: 60 Ectomycorrhizal diversity at the substrate manipulation site: 60 Ectomycorrhizal fungi in versus out of Rhododendron maximum thickets: 62 Ectomycorrhizal fungi in response to treatments: 66 Conclusions 67 SYNTHESIS AND CONCLUSIONS 68 APPENDIX A: Growth of ectomycorrhizal fungi in media containing substances from Rhododendron maximum thicket litter and leaves Methods: 70 Decoction experiments: 70 Leachates: 71 Table of Contents v

6 Results: 72 Ectomycorrhizal growth: 72 Discussion: 72 APPENDIX B: Plot map of the study site 76 REFERENCES 77 VITA 93 Table of Contents vi

7 LIST OF FIGURES Figure 1: Tsuga canadensis total mycorrhizal colonization and colonization by Cenococcum geophilum 20 Figure 2: Tsuga canadensis ramification indices 21 Figure 3: Quercus rubra total mycorrhizal colonization and colonization by Cenococcum geophilum 24 Figure 4: Quercus rubra ramification indices 25 Figure 5: Species area curve 50 Figure 6: Cluster analysis 51 Figure 7: Growth of three species of ectomycorrhizal fungi on media with and without the incorporation of Rhododendron maximum leaf materials 74 Figure 8: Radial growth of ectomycorrhizal fungi on media with or without the addition of Rhododendron maximum or canopy tree leaf materials 75 List of Figures vii

8 LIST OF TABLES Table 1: Seedling mycorrhization in blocks with and without Rhododendron maximum 22 Table 2: Seedling growth in blocks with and without Rhododendron maximum 26 Table 3: Correlation of mycorrhizal colonization and seedling health 28 Table 4: Putative ectomycorrhizal species list for all collections on the substrate manipulation plots Table 5: Count, relative abundance, relative frequency, and indicator values for all putatively ectomycorrhizal fungi collected on the blocks Table 6: Species list; count, percent frequency, and indicator values for putatively ectomycorrhizal fungi on the substrate manipulation plots Table 7: Canopy and subcanopy trees at the study site 59 List of Tables viii

9 INTRODUCTION: The spread of Rhododendron maximum L. in highly productive areas of the southern Appalachians in recent times is well documented (Dobbs, 1995), and poses a substantial problem both economically and ecologically in the region. The inhibitory effect of Rhododendron maximum thickets on tree seedling regeneration in the region has been recognized for decades (see Ch.1 for more background and references). Ecologically, the reduction of canopy regeneration will limit the diversity, and perhaps type, of dominant tree where Rhododendron maximum becomes established. The eventual loss of canopy trees in areas where Rhododendron maximum becomes invasive will limit the potential diversity of the ectomycorrhizal fungus community. Thomson et al. (1993) noted that Rhododendron spp. dominated communities support a limited number of insect species and thus the diversity of bird species is greatly reduced in oak woodlands where rhododendrons have become invasive. Additionally Nilsen et al. (in preparation 1 ) discuss the importance of the current spread of Rhododendron maximum thickets in riparian zones, which potentially impacts the terrestrial aquatic ecosystem interface. The spread of introduced Rhododendron ponticum is a concern in areas of the British Isles, western Scotland, and Ireland, especially on sites with mixed-woodland flora (Thomson et al., 1993). This expansion may have a similar biological foundation as with Rhododendron maximum in the Southern Appalachians. Of the many mechanisms by which Rhododendron maximum could potentially inhibit seedling recruitment, those involving mycorrhizae include: 1) The ericoid mycorrhizae of R. maximum may have an advantage in competition for nutrients with the ectomycorrhizae of canopy tree seedlings, 2) Compounds produced by the ericoid 1 Nilsen ET, Clinton BD, Semones S, Walker JF, Miller OKJr, Lei T (in preparation) Resource availability for canopy tree seedlings of the southern Appalachians in the presence or absence of Rhododendron maximum L. (Ericaceae). 1

10 mycorrhizae of R. maximum may inhibit ectomycorrhizal formation with canopy tree seedlings, 3) The diversity or belowground biomass of ectomycorrhizal fungi, and thus the inoculum potential of ectomycorrhizal fungi, may be reduced in areas densely inhabited by R. maximum, inhibiting mycorrhizal formation with canopy tree seedlings. Ectomycorrhizae have evolved over millions of years in mutualistic symbioses with many of the families of dominant canopy tree species currently present in the southeast (i.e. Pinaceae, Fagaceae, Juglandaceae, and Betulaceae). Over this time a highly specialized interaction has developed between the photosynthetic plant and nutrient foraging fungi. The mycorrhizal symbiosis is highly beneficial to the plant host, and therefore reduction in ectomycorrhizal colonization of canopy tree seedlings could have severe impacts on seedling survival under Rhododendron maximum thickets. The following section is intended review the advantages mycorrhizae confer to plant hosts, discuss the potential for mycorrhizae to influence plant to plant interactions, and provide the reader with a general review of the ecto- and ericoid mycorrhizal symbioses. The mycorrhizal symbiosis: Mycorrhizae, the symbiotic relationships between plants and fungi, occur in representatives of all plant phyla (Harley, 1969) and in 90% of known land plant species (Harley and Smith, 1983). It is well known that mycorrhizal associations are beneficial to the host plants and fungi involved. In most cases neither the phytobionts nor mycobionts can survive in vivo as free living organisms (Hacskaylo, 1967; Harley, 1969; Harley and Smith, 1983). The benefits derived by plants from mycorrhizal associations are generally considered to be a large factor in interspecific plant competition, plant community structure, succession (Allen and Allen, 1990; Molina et al., 1992; Read 1991), nutrient cycling, plant water relations and carbon flux in ecosystems (Vogt et al., 1991). Indeed mycorrhizal fungi are considered to be valuable indicators of forest health (Folke and Knudsen, 1994), and are critical in the regeneration of canopy trees (Amaranthus and 2

11 Perry, 1987). In general the benefits derived by host plants from mycorrhizal fungi have been primarily related to enhanced nutrient acquisition (Read 1991). Additionally, the increased surface area for soil contact in mycorrhizal systems with emanating hyphae is also thought to benefit water relations for the host plants (Parke et al., 1983). Mycorrhizal plants survive dry soil conditions and resist wilting more effectively than non-mycorrhizal plants (Read, 1991; Safer et al., 1971). Increased nutrient uptake and improved water relations of the phytobionts may be synergistic in that moisture stress decreases the uptake of soluble nutrients by plants. This may be offset by the ability of mycorrhizal fungi to maintain nutrient uptake at lower moisture levels than the host plant could tolerate. Mycorrhizae also confer protection to the plant from root pathogens. This relates to the following activities of mycorrhizal fungi: 1) competition for essential nutrients in the rhizosphere, 2) competition for infection sites on and within the root, 3) alteration of host plant physiology, 4) presenting a physical barrier to infection (in sheathing types), 5) inducing changes in the composition of root exudates to select beneficial microflora, 6) producing toxic or inhibitory substances which directly affect pathogens, and 7) compensating for pathogen damage by improving nutrient and water relations. Further benefits to host plants include reduced root respiration rates (Marshall and Perry, 1987), protection from phytotoxic compounds resulting from selective ion absorption (Jana and Nada, 1989, Jones et al., 1988), and increased capacity to cope with environmental extremes (Marx and Krupa, 1978). Mycorrhizal fungi depend on host plants mainly for photosynthate because they typically lack the ability to degrade complex organic carbon residues (Hacskaylo, 1973). Another benefit derived by the mycobiont from host plants is the stability of the environment within the plant root. In the following two sections the classes of mycorrhizae found predominantly with canopy trees (ectomycorrhizae) and Rhododendron maximum (ericoid mycorrhizae) will be reviewed. 3

12 The ectomycorrhizal class: Ectomycorrhizal fungi are associated with all genera of the Pinaceae family, several genera of Betulaceae and Fagaceae, and probably the Juglandaceae (Meyer, 1973; Miller, 1983). The taxa in families which form ectomycorrhizal associations include many of the trees which are important economically and as canopy trees in the Southern Appalachian Mountains and elsewhere. A plethora of families of basidiomycetes contain fungal species which are ectomycorrhizal, and ectomycorrhizal taxa are also found in ascomycete and zygomycete families (see Molina et al., 1992; Miller 1983 & ). More specifically within the basidiomycotina the families Russulaceae, Boletaceae, Amanitaceae, Paxilaceae, Cortinariaceae and Gomphidiaceae are either entirely or predominantly mycorrhizal and among the ascomycetes the tuberales (truffles) are ectomycorrhizal (see Miller, ). Miller (1983) found that as a stand of Pinus monticola D. Don (western white pine) matures the number of putatively ectomycorrhizal fungi increased from five species in 15 year old stands to 78 species in a mature stand. There is also considerable variability between species of pine and the diversity of ectomycorrhizal fungi they support. Changes that have been noted in the composition of communities of ectomycorrhizal fungi at different locations with similar plant communities are thought to be related to variations in climate and edaphic factors. Ectomycorrhizae are characterized by the formation of a sheath of fungal mycelium covering the epidermis of the root. The hyphae of the mycobiont grow through the outer cortex of the root intracellularly, forming the Hartig net. In forming the Hartig net the fungal hyphae branch repeatedly but do not penetrate host cell walls (Marks and Foster, 1973). The proliferation of hyphae between cortical cells creates a large surface for the exchange of nutrients between the mycobiont and phytobiont through the host plant 4

13 plasmalemma and apoplast. Fungal hyphae typically emanate outward from the surfaces of ectomycorrhizae in fans (Brownlee et al., 1983) extending the absorptive surface of the symbiosis through a greater volume of soil than the roots of a non-mycorrhizal plant would typically occupy. This along with the increased surface area for nutrient exchange enhances the host plant s ability to absorb nutrients from the soil. In temperate forest ecosystems ectomycorrhizae are found primarily near the soil surface and in accumulated litter (Harley, 1940; Meyer, 1973). Therefore ectomycorrhizal formation with canopy trees may be inhibited or blocked by the dense mat of ericoid mycorrhizae at the soil surface in Rhododendron maximum slicks. Ectomycorrhizal fungi apparently lack the ability to degrade complex polymers like lignin (Trojanowski, 1984). Thus due to its sclerophyllous nature, Rhododendron maximum litter may be relatively unavailable to ectomycorrhizae compared to ericoid mycorrhizae as a source of nutrients. The ericoid mycorrhizal class: Ericoid mycorrhizae show the highest degree of fungal specificity of all types of mycorrhizae considered here (Harley and Smith, 1983). Known and putative ericoid fungi are limited to a few species of ascomycetes and basidiomycetes. The ascomycete Hymenoscyphus ericae (Read) Korf & Kernan was isolated and described as Pezizella ericae (Read) from ericoid mycorrhizae (Pearson and Read, 1973). Also a basidiomycete species of Clavaria close to C. vermicularis was found to be related to the pelotons (discussed below) of ericoid mycorrhizal fungi in Azalea spp. and Rhododendron spp. using fluorescent antibody techniques (Seviour et al., 1973). The ericoid fungus-rootlets in Rhododendron maximum are comprised of a steele, a single layer of cortex and an epidermis (Dighton and Coleman, 1992). The fungal hyphae do not form a sheath as found in ectomycorrhizae and arbutoid mycorrhizae, nor does the proliferation of a hartig net occur. As reviewed by Peterson and Farquhar (1994), 5

14 following adhesion to the root surface the endophyte forms an appressorium, from which arises a thin, septate penetration hypha. Enzymatic cell wall degradation gives the penetration hyphae access to enter epidermal cells in which coils of hyphae or pelotons are formed. The pelotons are surrounded by the host plasmalemma, which remains intact. Colonized cells become swollen with increased cytoplasmic contents and numbers of organelles (Bonfante-Fasolo et al., 1984). The hyphae of the ericoid mycobionts is described as brown pigmented and septate (Pearson and Read, 1973), and does not emanate from the mycorrhiza to nearly the extent of the other mycorrhizal types discussed above (Read, 1991). Instead, a mat of hair-like mycorrhizae forms between the litter and humus at the soil surface which is finely adapted to maximize contact with the soil for nutrient absorption (Read, 1991). The role of ericoid mycorrhizal fungi in enhancing nutrient uptake is thus attributed to the capacity of the endophytes to produce extracellular enzymes, which mobilize nutrients from the soil pool. These enzymes can degrade structural components of the litter including chitin, lignin and tannins enabling fungal produced protease and phosphatase enzymes to access nitrogen and phosphorous (Read, 1992) which are tied up in the slowly degrading litter of plants such as Rhododendron maximum (Webster and Benfield, 1986). A further consequence of a dense surface mat of ericaceous mycorrhizae is that ectomycorrhizal canopy tree seedlings may face a physical barrier to mineral soil components and compatible fungal inoculum. Mycorrhizal fungi are most effective at acquiring nutrients which have low solubility and/or are present in low concentrations in the soil (Bowen, 1973). In most mycorrhizal types the greatest increase is in uptake of organic phosphorous. Increased absorption has also been demonstrated for N, K, S, Ca, Mg, Fe, Zn, Cl and Rb (Bowen, 1973). Ericoid mycorrhizae primarily provide access to higher amounts of nitrogen, although phosphate uptake is enhanced as well (Read, 1983). This may be especially important in highly mineralized soils with low ph (which have inherently low levels of nitrogen) such as 6

15 found in the sites under study at Coweeta Hydrological Laboratory in North Carolina. Phosphorus uptake in mycorrhizae: Variation in the ability of mycorrhizae to access phosphorus for the phytobiont has been recognized between mycorrhizal classes. Within the different mycorrhizal classes phosphorus uptake also depends on the specific taxa of mycorrhizal fungus involved in the mycorrhiza. This is an area that still needs clarification in the literature. Important variables which affect the ability of a mycorrhiza to absorb phosphorus and translocate it to the host plant include: 1) The amount of internal and external hyphal growth, 2) The magnitude of phosphatase production by the fungus, and 3) The optimum phosphorous level in the soil matrix for assimilation of phosphorous by a specific association (Gianinazzi-Pearson and Gianinazzi, 1986). Enhanced phosphorous relations by mycorrhizae are generally correlated negatively with the availability of phosphorous in the soil, with reduced production of emanating hyphae and then the reduction of infection levels as phosphorous becomes highly available (Gianinazzi-Pearson and Gianinazzi, 1986). Other factors that may be important in this regard are the kinetics of phosphorous exchange between internal hyphae and host cells and the quantity of digestion of internal hyphae by host cells. Lemione et al. (1992) found that acid phosphatase activity of the ericoid mycobiont Hymenoscyphus ericae (Read) Korf & Kernan is limited by high concentrations of phosphorous and high ph in culture media. Dighton and Coleman (1992) found that ectomycorrhizal associations had approximately four times the root surface activity of VAM or ericoids (associated with Rhododendron maximum). Gianinazzi-Pearson and Gianinazzi (1986) reviewed physiological aspects of phosphorous translocation by mycorrhizae. Polyphosphates accumulate in vacuoles of the fungal hyphae, and it is thought that these vacuoles facilitate transfer of polyphosphates 7

16 across an osmotic gradient. Phosphorus concentrations in the hyphae are higher in absorptive regions at the fungus/soil interface and lower at the fungus/plant interface. Translocation across this phosphorus gradient is thought to occur via absorption and release of the polyphosphates from successive vacuoles, with the polyphosphates being transferred between vacuoles through cytoplasmic streaming and mixing of the cytosol. Once the polyphosphates reach the fungus/plant interface, the ATPase on the host plasmalemma may provide a source of ATP for active uptake of phosphorous by the phytobiont in ericoid mycorrhizae and VAM (Gianinazzi-Pearson and Gianinazzi 1986). Carbon translocation in mycorrhizae: The transfer of energy to the mycobionts in mycorrhizal symbioses occurs by the translocation of simple sugars from the plant cells. Harley (1978) provided a review of the subject which lead to the hypothesis that glucose from the host plant is taken up by the fungal hyphae and converted to mannitol. The mannitol was presumed to be relatively unavailable to the host plant, providing a net flux of carbon to the mycobiont. Continued research in this area has confirmed that the mycorrhizal fungus Piloderma croceum Erikss. & Hjortst. does have the ability to convert glucose to mannitol and that mannitol cannot be metabolized by host cells of Pinus sylvestris L. or Fagus orientalis Lypski (Soderhall et al., 1986). Recent evidence has called into doubt the previous view that intracellular hyphae are consumed by the phytobiont in VAM and ericoid mycorrhizae as a means of nutrient transfer. Read (1983) found that the digestion of hyphal coils is proceeded by degeneration of the host cell cytoplasm, thus precluding the potential of digestion as a means of nutrient exchange with the phytobiont. The amount of digestion of intracellular hyphae in both ericoid mycorrhizae and VAM infections also appears to be to limited to provide a substantial avenue for nutrient exchange in these associations (Read, 1983). This creates a significant distinction between ericoid mycorrhizae and orchid 8

17 mycorrhizae. In the latter symbiosis, enzymatic digestion of pelotons is proposed as a mechanism for reversal of the direction of carbon flow to the plant during achlorophytic developmental stages (Harley and smith, 1983). It is therefore unlikely that carbon compounds are translocated from canopy trees to Rhododendron maximum by a digestion mechanism through interplant mycorrhizal linkages. Interplant mycorrhizal linkages: Recent evidence regarding the potential for net carbon transfer between trees by shared mycorrhizal hyphae has enhanced awareness of the importance of mycorrhizae in plant to plant interactions. Many studies have demonstrated the capacity of mycorrhizal hyphae to translocate carbon and nutrients between plants in vitro (Arnebrant et al., 1993; Bethlenfalvay et al., 1991; Brownlee et al. 1983; Finlay and Read, 1986; Francis and Read, 1984; Newman and Eason, 1993; Wittingham and Read, 1982). Francis and Read (1984) suggested a source-sink relationship for carbon translocation in shared hyphae. A review by Newman in 1988, however found no direct evidence of net carbon translocation between plants through interplant mycorrhizal connections. More recent evidence confirms significant net transfer of carbon through ectomycorrhizal mycelial connections among different tree species in situ (Read, 1997; Simard et al., 1997). Simard (1997) found that in the field carbon exchange between Betula papyrifera Marsh and Pseudotsuga menziesii (Mirb.) Franco resulted in average net gain by P. menziesii seedlings of 6% of carbon isotope uptake through photosynthesis. Non-ectomycorrhizal seedlings did not gain carbon, indicating that carbon exchange was through shared mycelia. Additionally, shading of P. menziesii indicated that a source-sink relationship was involved. Perry et al. (1989) envisioned interplant linkages as forming mutualistic guilds in plant communities. Of particular interest to this study, Newman (1988) expressed the potential for interplant linkages to transfer carbon from a canopy tree in full sunlight to a seedling in the understory through a previously established ectomycorrhizal mycelial network, and to mediate plant to plant competition. 9

18 Thesis objectives: Because of the many benefits of mycorrhizae and the perception that Rhododendron maximum may be inhibitory to tree seedling mycorrhization, it was felt that the role of mycorrhizae in the suppression of seedlings under R. maximum thickets should be examined. The hypotheses, which follow, were based on the idea that if the diversity or abundance of ectomycorrhizal fungi is reduced in the presence of Rhododendron maximum, it would indicate that the inoculum potential of areas with R. maximum is probably less than that for areas without R. maximum. If so then the reproduction of canopy tree seedlings may fail in nature in part due to the failure of the tree seedlings to encounter compatible ectomycorrhizal hyphae in the rhizosphere where Rhododendron maximum thickets are present. Furthermore, reduced mycorrhizal colonization of tree seedlings in Rhododendron maximum thickets would probably be associated with reduced growth of the seedlings. In Chapter one the objective was to investigate hypogeous interactions between Rhododendron maximum and canopy tree seedling ectomycorrhizae. We hypothesized that components of the Rhododendron maximum thickets (litter substrates, organic substrates, living R. maximum) may alter colonization levels, change mycobiont abundances on roots, and reduce growth of canopy tree seedlings. In Chapter two the following hypotheses are addressed: 1) the diversity of ectomycorrhizal fungi is different within versus outside Rhododendron maximum thickets, 2) the communities of ectomycorrhizal fungi are structured differently in terms of the relative abundance of their component species in versus out of R. maximum thickets, 3) only certain ectomycorrhizal fungus taxa are distributed differentially with regard to R. maximum thickets, 4) the portion of the ectomycorrhizal fungus community capable of recolonizing manipulated soil substrates is different for substrates from in versus out of R. maximum thickets, and 5) only certain ectomycorrhizal fungi are distributed differently based on their ability to recolonize manipulated soil substrates from in versus out of R. maximum 10

19 thickets. Additionally this study was used to gauge the effect of the disturbance associated with the substrate manipulation (Chapter 1) on the overall ectomycorrhizal community and on individual ectomycobionts on the substrate manipulation plots. In Appendix A the potential for inhibition of ectomycorrhizal fungus growth by Rhododendron maximum is explored in vitro. 11

20 CHAPTER 1 Suppression of Ectomycorrhizae on Canopy Tree Seedlings in Rhododendron maximum L. (Ericaceae) Thickets in the Southern Appalachians 12

21 Introduction: Throughout the southern Appalachians Rhododendron maximum L. (Ericaceae) (Rm) forms dense thickets in the understory of mesic north facing slopes, stream banks, and coves. These areas are known to be highly productive and high quality for the production of hardwood lumber. The perception that Rm thickets inhibit canopy tree seedlings began in the 1940 s (Minkler, 1941; Wahlenberg, 1950) and has engendered considerable concern among foresters and plant ecologists. Phillips and Murdy (1985) found that hardwood regeneration and rates of succession were reduced in Rm thickets. Significantly reduced seedling density in gaps with heavy Rm cover was reported by Clinton et al. (1994). Numerous authors have considered Rm to be a problem weed for hardwood production (e.g. Martinez, 1975), and control programs have been investigated (Hooper, 1969; Neary et al., 1984; Romancier, 1971; Wahlenberg and Dolittle, 1950; Yawney, 1962). Currently little is known, however, about the biological basis of this important interaction. Therefore it is clear that research into the mechanisms by which Rm thickets suppress the growth of canopy tree seedlings remains a high priority for forest biologists and foresters alike. There are many possible mechanisms by which Rm might inhibit seedling recruitment. This study is concerned with those involving mycorrhizae. Mycorrhizae may be involved in seedling repression by Rhododendron maximum because they are considered to be important factors in interspecific plant competition, plant community structure, succession (Allen and Allen, 1990; Molina et al., 1992; Read 1991), nutrient cycling, plant water relations and carbon flux in ecosystems (e.g. Vogt et al., 1991). Recent evidence confirming significant net transfer of carbon through ectomycorrhizal mycelial connections among different tree species in situ has enhanced awareness of the importance of mycorrhizae in plant to plant interactions (Read, 1997; 13

22 Simard et al., 1997). Mycorrhizal fungi are considered to be valuable indicators of forest health (Folke and Knudsen, 1994) and are critical in the regeneration of canopy trees (Amaranthus and Perry, 1987). There are several lines of evidence that suggest that mycorrhizae may play an important role in the inhibition of seedling recruitment by Rhododendron maximum. Palmer et al. (1994) investigated the sporocarp production of ectomycorrhizal fungi in southwestern Virginia in a mixed forest with Pinus pungens Lamb., P. rigida Mill. and hardwoods. They found more abundant fruiting over a longer period in areas that experienced a light burn which removed the dense ericaceous ground cover (Vaccinium spp., Rhododendron spp., and Kalmia latifolia L.) than in stands nearby. Similar interactions between ericaceous shrubs and tree seedlings have been the focus of research in the Pacific Northwest. In coastal British Columbia, salal (Gaultheria shallon Pursh), a member of the Ericaceae like Rm, also inhibits the growth and survival of seedlings. Messier (1993) found no difference between levels of mycorrhizal colonization in sites with varying density of salal, and vegetation removal versus control treatments. In Messier s (1993) study containerized seedlings were also not colonized differentially over a range of salal planting densities. Messier (1993) ruled out light as a factor in the salal seedling interaction because the seedlings were taller than the salal. Amaranthus and Perry (1987) examined the effect of the ericaceous plants Pacific madrone (Arbutus menziesii Pursh) and manzanita (Arctostaphylos spp.) on Douglas fir seedling mortality and growth. They found that seedlings grew better in manzanita sites than in open oak and meadow sites, and seedlings in the manzanita formed different proportions of association with dominant mycobionts. Amaranthus and Perry (1989) hypothesized that the dominant morphotype in the manzanita sites was more successful at promoting seedling growth. The ability of ectomycorrhizal fungi to access nutrients and promote seedling growth is known to vary among populations (Antibus et al., 1981; 14

23 Jacobson and Miller, 1992) and genera (Antibus et al., 1981; Marx et al., 1978). Shifts in dominance of mycorrhizal morphotypes could be an important factor in the seedling suppression observed under Rm. The objective of this study was to investigate hypogeous interactions between Rm and canopy tree seedling ectomycorrhizae. We hypothesized that components of the Rhododendron maximum thickets (litter substrates, organic substrates, living R. maximum) may alter colonization levels, change mycobiont abundances, and reduce growth of canopy tree seedlings. Materials and Methods: Site description: The study was conducted in the Southeast Forest Research Station at the Coweeta Hydrologic Laboratory (Coweeta). Coweeta is located in the Blue Ridge Mountain Physiographic Province in Otto, North Carolina ( N, W). General vegetation types at Coweeta have been described as northern hardwood, cove hardwood, oak (with or without Chestnut), or oak - pine communities (Day et al., 1988). High moisture levels and mild temperatures typify the area, which is climatically classified as marine, humid (Swank et al., 1988). Precipitation is distributed equally throughout the season, averaging 180 cm annually (Swank et al., 1988). Field plots were established at an elevation of 1000 m on a north-facing slope that was half covered by thickets of Rm. The site is dominated by mature northern red oaks (Quercus spp.) mixed with a diversity of hardwoods including hickory (Carya glabra L.), red maple (Acer rubrum L.) tulip poplar (Liriodendron tulipifera L.), yellow birch (Betula alleghaniensis L.), hemlock (Tsuga canadensis L.), witch hazel (Hamamelis virginiana L.), and flowering dogwood (Cornus florida L.). 15

24 Plot layout and site preparation: Six ¼ ha blocks were randomly selected, three within (Rm) and three outside (no Rm) dense Rhododendron maximum thickets. Within each block the main treatments were applied to 2 X 2 m subplots with three replicates per treatment. The treatments were: 1) unmanipulated controls, 2) litter and organic substrates from Rm thickets (Rl and Ro respectively), 3) litter and organic substrates from forest without Rm thickets (Fl and Fo respectively), 4) Fl and Ro, and 5) Rl and Fo (Appendix B). Both the surface litter and organic horizon were removed from all but the control plots. Each substrate type was pooled and homogenized prior to being redistributed to randomly designated plots within each block. After the first growing season all Rm leaves which fell on Fl treatments in Rm blocks were removed and equally distributed amongst the Rl treatment plots in blocks with no Rm. Following the substrate manipulation containerized seedlings of hemlock and acorns of northern red oak were planted in the plots. The hemlock seedlings were germinated in a greenhouse in forest soil from the local area. Levels of ectomycorrhizal colonization were less than 5% on the seedlings prior to planting in the field. Seedling harvest and sampling: One seedling of each species was randomly selected and harvested from each of the plots after the first and again after the second growing seasons prior to the onset of senescence. The entire seedling was removed with the surrounding soil and stored at 5 c. The roots were carefully washed free of debris and stored frozen in tap water until they were examined for ectomycorrhizal colonization. Approximately 10 cm total of secondary root (< 2 mm diameter) were examined from 16

25 throughout the length of the root system. These root segments were used for quantification of mycorrhizal colonization (% of living root tips which are ectomycorrhizal) and to calculate the ramification indices (number of mycorrhizae per cm root) (Meyer, 1987). Due to the rocky soil conditions some root tips were damaged during harvest. Therefore the ramification indices may be low for comparison with other studies. Root examination was conducted in large petri plates in water using a dissecting microscope (0.9-4x magnification). Non-mycorrhizal root tips typically demonstrated several to many root hairs, sloughing epidermal cells, lack of a fungal mantle, and were not swollen. These morphological criteria were established by routinely comparing root tip sections lacking a Hartig net at high magnification with a compound microscope. Ectomycorrhizal root tips with a black mantle and typical stiff black rhizomorphs characteristic of the mycobiont Cenococcum geophilum Fr. were recorded separately from all other morphotypes. The identity of C. geophilum mycorrhizae were further confirmed by checking frequently for the characteristic stellate mantle pattern with a compound microscope (Hatch, 1934; Trappe, 1964). No other types of ectomycorrhizae were identified although several different morphotypes were observed on both host species. Stem diameters were measured 1 cm above the soil level. The roots and shoots of the seedlings were dried at 70 c for three days and weighed, except for the roots from the second harvest. The roots from the second harvest are being stored frozen in water to examine for an initial estimate of the distribution of ectomycorrhizal morphotypes within and outside Rm thickets. Statistics: 17

26 Means for all parameters were analyzed using analysis of variance with a generalized linear model with two block types (Rm, no Rm), three replicates of each block type, and three treatment replicates in each block replicate with SAS statistical software (SAS Institute, 1996). Differences for the means between Rm and no Rm blocks were compared using an F test with the type three mean square error term for block replicates with 4,1 degrees of freedom. Differences between means for the control plots between block types were analyzed using T-tests for equally or unequally varying samples with SAS statistical software (SAS institute, 1996). Correlation was performed with the Pearson Product Moment test using SigmaStat statistical software (Jandel Corp., 1995). P values less than.10 were considered statistically significant and P values below 0.01 were considered highly statistically significant for all tests. If all significant differences are lower than P =.05 in a figure, then it is noted in the legend. Variance is presented as ± the standard error of the mean (SEM). Results: 1996 and 1997 hemlock mycorrhizae: The total percent mycorrhizal colonization of one year old Tsuga canadensis seedlings was more than three times higher in blocks without Rm than in blocks with Rm (Tab. 1 & Fig. 1). Ramification indices were approximately 4.5 fold higher in blocks without Rm (Tab. 1 & Fig. 2). Percent colonization by Cenococcum geophilum was higher in blocks with Rm by a factor of 2.3 (Tab. 1 & Fig. 1). Seedlings from the control plots showed similar responses, which were statistically significant for the above three parameters (Figs. 1 & 2). Two year old Tsuga canadensis seedlings in blocks without Rm had higher total colonization levels and ramification indices, and lower levels of colonization by Cenococcum geophilum. Although the same patterns were observed on the control seedlings, they were not statistically significant. On second year seedlings treatments with Rm organic layer substrates had statistically significantly lower percent mycorrhizal colonization by C. geophilum (12.2, ± 3.7) than treatments with no Rm 18

27 organic layer substrates (23.1, ± 5.2) 1996 and 1997 red oak mycorrhizae: One year old Quercus rubra seedlings had lower mycorrhizal colonization and ramification index in blocks with Rm, by 19.5 percentage points and 2.8 respectively (Tab. 1 & Figs. 3 & 4). Percentage colonization by Cenococcum geophilum was nearly equal in blocks with or without Rm. The control seedlings had lower levels of total mycorrhizal colonization and ramification index but had higher percent colonization by C. geophilum in blocks with Rm (Tab.1 & Figs. 3 & 4). After the second growing season Quercus rubra seedlings had 11 percentage points less total mycorrhizal colonization and 1.7 lower ramification index in Rm blocks than in no Rm blocks. Again percentage colonization by C. geophilum was nearly equal. Control seedlings had similar ramification index and total mycorrhizal colonization, but higher C. geophilum colonization in blocks with Rm (Tab. 1 & Figs. 3 & 4). There was a significant increase in percent colonization by C. geophilum in treatments with Rm organic layer substrates (30.20, se 6.0) over treatments with no Rm organic layer substrates (21.83, se 6.2). The ramification index was significantly lower in treatments with Rm litter layer substrates (.611, se.097) than in treatments with no Rm litter layer substrates (.850, se.142). 19

28 % Mycorrhizal Colonization 100 No Rhododendron maximum blocks Total colonization Cenococcum geophilum Rhododendron maximum blocks Total colonization Cenococcum geophilum a a a 70 a a b a b a b b a b a a a 1996 P C P C. Figure 1: Tsuga canadensis total mycorrhizal colonization and colonization by Cenococcum geophilum; 'P.' Pooled mean from all plots including controls, significance analyzed with F-tests. 'C' Control plot mean, significance analyzed with T-tests. Sample sizes are given in Table 1. Means for pairs of bars with different letters are statistically significantly different (P <.05); error bars represent the SEM. 20

29 Ramification index No Rhododendron maximum blocks Rhododendron maximum blocks a a a a 2.5 b a b b P C P C. Figure 2: Tsuga canadensis ramification indices (# mycorrhizal roots/cm secondary root); 'P' Pooled mean from all plots including controls, significance analyzed with F-tests. 'C' Control plots mean, significance analyzed with T-tests. Sample sizes are given in Table 1. Means for pairs of bars with different letters are statistically significantly different (P<.05); error bars represent the SEM. 21

30 Table 1: Seedling mycorrhization in blocks with and without Rm. Means in the same row and in either pooled sample or control cells followed by different letters are statistically significantly different (P <.1); (U) Unequal variance t-test; (E) Equal variance t-test; n (No Rm/Rm). Pooled Samples Controls No Rm Rm No Rm Rm Hemlock 1996 Mean SEM Mean SEM n P Mean SEM Mean SEM n P % Colonization 61.9 a b / a b /9 (U).002 % Cenococcum 4.6 a b / a b /9 (U).03 Ramification index 2.8 a b / a b /9 (U).006 Hemlock 1997 Mean SEM Mean SEM n P Mean SEM Mean SEM n P % Colonization 74.8 a b / /9 (E).22 % Cenococcum / /9 (U).24 Ramification index 3.4 a b / /9 (E).27 Oak 1996 Mean SEM Mean SEM n P Mean SEM Mean SEM n P % Colonization 71.1 a b / /8 (E).30 % Cenococcum / /8 (U).47 Ramification index / /8 (E).21 Oak 1997 Mean SEM Mean SEM n P Mean SEM Mean SEM n P % Colonization 87.1 a b / /7 (U).54 % Cenococcum / /7 (E).28 Ramification index / /7 (E).75 22

31 Seedling growth parameters: One year old Tsuga canadensis seedlings from control plots had a significant reduction in root weight (1.75 mg) and root/shoot ratio (0.12) in blocks with Rm. Shoot height, shoot weight, and shoot diameter were similar in blocks with or without Rm. After the second year there were no significant differences in shoot height, shoot weight, or shoot diameter in blocks with or without Rm (Tab. 2). After the second year shoot weight and shoot height were significantly reduced (12 mg and 0.9 cm respectively) (Tab. 2). First year Quercus rubra seedlings from control plots had significantly reduced root weight (220 mg) and shoot weight (2x) in blocks with Rm. The root/shoot ratio and shoot height were not different in blocks with or without Rm. After two growing seasons the seedlings had a significant reduction in shoot weight (457 mg) and reduced shoot diameter (.4 mm) in blocks with Rm. Shoot height was not affected by the presence or absence of Rm (Tab. 2). 23

32 % Mycorrhizal Colonization No Rhododendron maximum blocks Total colonization Cenococcum geophilum a a a b a Rhododendron maximum blocks Total colonization Cenococcum geophilum a a a b a a a a a a a P C P C. Figure 3: Quercus rubra total mycorrhizal colonization and colonization by Cenococcum geophilum. 'P.' Pooled mean from all plots including controls, significance analyzed with F-tests. 'C' Control plots mean, significance analyzed with T-tests. Sample sizes are given in Table 1. Means for pairs of bars with different letters are statistically significantly different (P<.10); error bars represent the SEM. 24

33 Ramification index No Rhododendron maximum blocks Rhododendron maximum blocks 10 9 a a a a a 8 7 a a 6 a P C P C. Figure 4: Quercus rubra ramification indices (# mycorrhizal roots/cm secondary root); 'P' Pooled mean from all plots including controls, significance analyzed with F-tests. 'C' Control plots mean, significance analyzed with T-tests. Sample sizes are given in Table 1. Means for pairs of bars with different letters are statistically significantly different (P<.05); error bars represent the SEM. 25

34 Table 2: Seedling growth in blocks with and without Rm. Means in the same row and in either pooled sample or control cells followed by different letters are statistically significantly different (P <.1); (U) Unequal variance t-test; (E) Equal variance t- test; n (No Rm/Rm). Pooled Samples Controls No Rm Rm No Rm Rm Hemlock 1996 Mean SEM Mean SEM n P Mean SEM Mean SEM n P Root weight (mg) / a b 0.4 9/9 (E).014 Shoot weight (mg) / /8 (E).66 Root/Shoot 0.41 a b / /8 (E).12 Shoot height (cm) / /9 (E).56 Shoot diameter (mm) / /9 (E).38 Hemlock 1997 Mean SEM Mean SEM n P Mean SEM Mean SEM n P Shoot weight (mg) 34.7 a b / /9 (E).65 Shoot height (cm) 5.5 a b / /9 (E).74 Shoot diameter (mm) / /9 (E).55 Oak 1996 Mean SEM Mean SEM n P Mean SEM Mean SEM n P Root weight (mg) a b / a b /8 (U).022 Shoot weight (mg) a b / a b /9 (E).002 Root/Shoot / /8 (U).51 Shoot height (cm) / /9 (E).38 Oak 1997 Mean SEM Mean SEM n P Mean SEM Mean SEM n P Shoot weight (mg) a b / a b /9 (U).026 Shoot height (cm) / /9 (E).58 Shoot diameter (mm) 3.4 a b / /9 (E).10 26

35 Correlation of mycorrhizae and seedling health: First year Tsuga canadensis seedlings had highly significant correlation between total percent mycorrhizal colonization and both root weight and root/shoot ratio, and significant correlation between total percent colonization and shoot diameter. Root weight and root/shoot ratio were also significantly positively correlated with the ramification index. There was a significant but weak negative correlation for one year old Tsuga canadensis seedlings between percent Cenococcum geophilum colonization and root/shoot ratio (Tab. 3). The two year old Tsuga canadensis seedlings had significant positive correlation for shoot height, shoot weight, or shoot diameter versus both total percent mycorrhizal colonization and ramification index (Tab. 3). One year old Quercus rubra seedlings had weak positive, significant correlation between total percent colonization and both root weight and shoot weight. The ramification index was weakly but significantly positively correlated with shoot weight. In addition the percent colonization by Cenococcum geophilum was weakly but significantly correlated with the root/shoot ratio (Tab. 3). Two-year-old Quercus rubra seedlings had a significant but weak positive correlation between shoot weight and total mycorrhizal colonization (Tab. 3). 27

36 Table 3: Correlation of mycorrhizal colonization and seedling health; * Significant at P <.1; ** Significant at P <.01 Total % Cenococcum % Ramification Colonization Colonization Index Hemlock 1996 r 2 p n r 2 p n r 2 p n Root weight (mg) ** * Shoot weight (mg) Root/Shoot ** * * Shoot height (cm) Shoot diameter (mm) * Hemlock 1997 r 2 p n r 2 p n r 2 p n Shoot weight (mg) ** ** Shoot height (cm) * * Shoot diameter (mm) ** * Oak 1996 r 2 p n r 2 p n r 2 p n Root weight (mg) * Shoot weight (mg) * * Root/Shoot * Shoot height (cm) Oak 1997 r 2 p n r 2 p n r 2 p n Shoot weight (mg) * Shoot height (cm) Shoot diameter (mm) Discussion: Total mycorrhizal colonization and ramification: It is evident from this study that mycorrhizal colonization of tree seedlings is depressed on sites in the southern Appalachians with dense thickets of Rm. Comparisons of mycorrhizal colonization levels between studies are difficult due to differences in site 28

37 preparation, host tree composition, edaphic conditions, and measurement techniques. For example Dahlberg et al. (1997) found that 95 % of all short roots were ectomycorrhizal in an old growth Norway spruce forest. Messier found % colonization on western hemlock (Tsuga heterophylla (Raf.) Sarg.) seedlings, and 99% colonization on Sitka spruce (Picea sitchensis Bong. Carr.) seedlings. Zhou et al. (1997) reported 37.6 % mycorrhizal colonization on two year old northern red oak seedlings in uncut oak and pine stands. The intermediate to high levels of mycorrhizal colonization in this study, especially out of Rhododendron maximum thickets, correspond with our observation of a high ectomycorrhizal sporocarp diversity and abundance in the study area (> 69 species). Mycorrhizal inhibition by Rm could be attributed to many factors. Before elaborating these factors however, it is important to note that the relationship of Rm thickets and tree seedling mycorrhization may be indirect if Rm is associated with a habitat less suitable for mycorrhizal development than areas without Rm present. Also, due to a high rate of mortality of Quercus rubra seedlings in the Rm blocks during the second year of the study, poorly colonized seedlings may have died more frequently than other seedlings. Therefore the level of total percent colonization of oaks may be overestimated in the Rm blocks. If Rm is affecting mycorrhizal colonization of tree seedlings directly, the following factors may be involved: 1) accumulation in the soil of biotoxic compounds produced by Rm or Rm ericoid fungi which reduce mycelial growth, seedling root growth, or mycorrhizal synthesis, 2) reduced light availability to tree seedlings resulting in lower carbohydrate availability for ectomycorrhizal fungi and reduced root development, or 3) competition for resources between the Rm ericoid root system and tree seedling ectomycorrhizal root system. Several studies have examined the relationship between mycorrhizal fungi and allelopathy. Reduced ectomycorrhizal colonization and growth of Douglas fir 29

38 (Pseudotsuga menziesii (Mirb.) Franco) seedlings in response to litter leachates in vitro was reported by Rose et al. (1983). Robinson (1972) found that living heather (Calluna vulgaris (L.) Hull; Ericaceae) produced a compound that reduced the growth of ectomycorrhizal fungi. Lyon and Sharpe (1995) found that Hayscented fern (Dennstaedtia punctilobula (Michx.) Moore) reduced growth and mycorrhizal colonization of northern red oak (Quercus rubra L.) in mini-terracosms. Also with red oak, Hansen and Dixon (1987) reported that inoculation with an ectomycorrhizal fungus (Suillus luteus (L.) Fr.) reduced the allelopathic effect of interrupted fern (Osmunda claytoniana L.) on seedling mortality in a pot study. However, accumulation of biotoxic compounds in the soil is not strongly supported by this study. Although there was a decrease in ramification index in treatments with Rm organic layer substrates on second year oaks, this result could also be attributed to sequestration of nutrients in the sclerophyllus and slowly degrading Rm leaves (Webster & Benfield, 1986) thereby providing less N input to the organic layer. Cenococcum geophilum colonization was species specific (+ on Quercus rubra; - on Tsuga canadensis) in treatments with Rm organic layer substrates after the second year. Substrate removal has been shown to affect seedling mycorrhization in several studies reviewed in Baar (1997). Unfortunately these studies are mainly concerned with removal of sod and thick humus layers in stands of Scot s pine (Pinus sylvestris L.) and are not directly comparable to this study. In addition to the general lack of substrate treatment effects, laboratory assays (leachate media preparations, decoction media, or confrontations experiments) using ectomycorrhizal fungi, Rm substrates and Hymenoscyphus ericeae (an ericoid mycorrhizal fungus) in pure culture have produced no evidence of fungi-toxic activity different from similar substrates from forests without Rm (Nilsen et al., in preparation 2 ; see Appendix A). Nilsen et al. (in preparation 2 ) have, however, found an inhibitory effect of Rm litter on root elongation of two bioassay 2 Nilsen ET, Lei T, Walker JF, Miller OKJr, Semones S, (in preparation) Inhibition of seedling survival under Rhododendron maximum: Could allelopathy be a cause? 30

39 species. Nilsen et al. (in preparation 1 ) found evidence for decreased soil moisture (6%), lower carbon content (20%), and less ammonium (.43 ppm), in blocks with Rm versus blocks with no Rm. Levels of phosphorous are extremely low ( ppm) in both Rm and no Rm blocks. Mycorrhizal colonization levels are often negatively correlated with N availability (e.g. Markkola and Ohtonen, 1988) and therefore depression of N availability should not depress percent mycorrhizal colonization in Rm blocks. Organic substrates in soils are frequently favorable sites for mycorrhizal activity (Harvey et al., 1997; Meyer, 1973; Miller, ; Trappe and Fogel, 1977), so reduced C in Rm blocks could be related to reduced mycorrhizal colonization levels. Due to the extremely low levels of soluble phosphorus ectomycorrhizal colonization may be critical to tree seedlings at an early stage of development. Ectomycorrhizal fungi appear to be able to produce up to four times as much phosphatase as ericoid fungi on Rm at Coweeta (Dighton and Coleman, 1992). Because ectomycorrhizal fungi are extremely well suited to enhance P uptake (Bowen, 1973; Gianinazzi-Pearson & Gianinazzi, 1986; Read, 1983) depressed colonization of tree seedlings may be related to seedling mortality and depressed growth in Rm thickets. Ericoid mycorrhizae primarily provide photobionts access to increased amounts of nitrogen, although phosphate uptake is enhanced as well (Read, 1983). It is possible that Rm ericoid mycorrhizae are better competitors for N than the tree seedling ectomycorrhizae. Light levels are lower throughout the year in blocks with Rm (0.5% full sunlight in versus 5% full sunlight out) (Nilsen et al., in preparation 2 ). The relationship between light levels in the sub-canopy and mycorrhizal colonization of red oak was studied by Zhou et al. (1997), who found that red oak seedlings under intermediate levels of canopy cover (45-75% of PAR) formed more mycorrhizae than cut and full canopy treatments. Zhou (1997) also found that vegetation removal (mostly braken fern (Pteridium aquilinum L.) 31

40 and red maple (Acer rubrum L.) saplings) did not increase mycorrhizal colonization. The relationship between light level and mycorrhizal colonization appears to be species specific. For example Zhou et al. (1997) point out that their figures were different from studies by Bjorkman (1970) (optimum 25-49% of full sunlight; Pinus sylvestris), and Ekwebelam and Reid (1983) (optimum 30.5% of full sunlight; Pinus contorta Dougl.). However in all studies light below a certain level results in less photosynthate available and inhibition of mycorrhizal colonization (Bjorkman, 1970). The light levels both in and out of Rhododendron maximum thickets are well below the optima of all studies known to the authors, yet colonization levels are reasonably high in this study. Obviously under the conditions at the study site mycorrhizal colonization of seedlings can occur even at light levels as low as.5% full sunlight. Many factors other than just light, such as the nutrient status of the soil, affect mycorrhizal colonization. Apparently some ectomycobionts colonize roots of compatible host species regardless of their ability to supply carbohydrate. Ectomycobionts colonizing the seedlings in this study could be getting carbohydrates from canopy trees and losing carbohydrates to the seedlings. Nonetheless, light levels are an order of magnitude lower and could be an important factor in the suppression of mycorrhization in Rm thickets. The study area is situated in a well developed mixed forest with a mature canopy. It is therefore likely that sugar from overlying trees is available to developing seedlings through mycelial interconnections between individual plants with well developed mycorrhizal systems (Simard et al., 1997), both in Rm and no Rm blocks. If lower light levels under Rm reduce mycorrhizal colonization levels, one could speculate that reduced carbohydrate gain by the seedlings from canopy resident individuals could occur. Reduced carbohydrate gain will then likely reduce root tissue available for colonization if light is limiting owing to reallocation of biomass to leaf tissue as predicted by plant growth models (Johnson and Thornley, 1987). Another aspect of resource competition is the dense ericoid mycorrhizal mat produced at 32

41 the mineral organic soil interface, which is likely to impose spatial limitations on the development of ectomycorrhizal root systems in that profile (McConnaughay and Bazzaz, 1992). Furthermore this dense root mat may prohibit seedling roots from easily accessing mycorrhizal innocula in the mineral horizon where it is frequently concentrated (Alvarez et al., 1979; Dahlberg, 1990; Lee, 1981) and is most likely related to the reduction in ramification indices in Rm thickets. Depressed total mycorrhizal colonization was most closely related to root weight for both hemlock and oak in the first year, although at a moderately low level. The intermediate level of correlation between total mycorrhizal colonization and root weight possibly reflects the interrelationships between light intensity, carbon sharing between individuals, nutrient availability, mycorrhizal colonization level, and seedling growth. Healthier root systems make more effective mycorrhizae and effective mycorrhizae can potentially provide resources for larger root systems. Yet more effective mycorrhizae can reduce the need for root tissues for nutrient acquisition, thereby freeing up carbon for photosynthetic tissues. After the second year in both species shoot weight was related to total percent mycorrhizal colonization. Overall colonization levels were lower for hemlock than oaks, an observation which could be related to the slower growth strategy of the more shade tolerant hemlocks, to the fact that the hemlocks were not germinated at the site, or to the larger seed reserves in the oak acorns. Conversely, hemlocks exist as individuals or small clusters on the study site and may suffer greater isolation from compatible mycobionts than the ubiquitous oaks. There is no evidence at the organismal level that indicates an optimum level of overall mycorrhizal colonization exists for a given photobiont. Colonization by Cenococcum geophilum: Cenococcum geophilum is a generalistic ectomycorrhizal ascomycete (Trappe, 1964) which tends to favor periodic drought (Pigott, 1982; Worley & Hacskaylo, 1959). Rose et al. (1983) found that although growth of other ectomycorrhizal fungi was reduced by litter leachates, C. geophilum growth had no response to any leachate preparation. The 33

42 ability of ectomycorrhizal fungi to promote seedling growth varies even among populations (Jacobson and Miller, 1992) and C. geophilum may be less effective than other ectomycorrhizal fungi (Marx et al., 1978). Dahlberg (1997) found that the C. geophilum colonization level was 18 percent of the total mycorrhizal colonization in an oligotrophic, Swedish, old-growth, Norway spruce forest. Increased levels of colonization by C. geophilum in Rm thickets may therefore indicate that the mycorrhizal community under Rm is disturbed. A study by Antibus (1980) indicated that C. geophilum occurs four times more frequently on seedlings in oil seeps than in other areas near Barrow, Alaska. Mycorrhizal colonization of tree seedlings by Cenococcum geophilum increased in the presence of Rm thickets in a species-specific pattern. Cenococcum geophilum accounted for 50% of the ectomycorrhizae on Tsuga canadensis seedlings in blocks with Rm thickets. On Quercus rubra seedlings, however, C. geophilum was nearly equally abundant in blocks in and out of Rm thickets in all treatments except for the controls. Apparently the mycorrhizal community or the process of mycorrhization associated with hemlock seedlings was less disturbed than that associated with red oak. Levels of colonization by C. geophilum were generally lower on hemlock than on red oak. This is not surprising, as Malloch and Malloch (1981) also reported species specific colonization levels of C. geophilum (ranging from no colonization on Pinus banksiana Lamb. to 19 of 25 root segments colonized on Abies balsamea (L.) Mill.). Mycorrhizal mycelial interconnections between Rhododendron maximum and seedlings: Dighton and Coleman (1992) reported colonization of Rhododendron maximum by ectomycorrhizae including Cenococcum geophilum, but were unable to quantify their occurrence. Smith et al. (1995) found that Rhododendron macrophyllum D. Don ex G. Don was colonized by ectomycorrhizal fungi rarely when grown in a greenhouse in soils from young Douglas fir forests in the Pacific Northwest USA. Largent (1980) also found 34

43 evidence of ectomycorrhizal colonization of Rhododendron spp. We examined numerous samples of Rhododendron maximum root systems and found no evidence of ectomycorrhizal colonization. Although ectomycorrhizal morphotypes were found tangled amongst the Rhododendron maximum fine roots, upon careful examination they were in no case found to be originating from a R. maximum root. Although low levels of ectomycorrhizal colonization may be present on Rhododendron maximum in the southern Appalachians, ectomycorrhizal mycelial interconnections between R. maximum and canopy tree seedlings are unlikely to be a factor in the carbon budget of the seedlings. Additionally, we are not aware of any indication in the literature for the potential for ericoid mycorrhizal fungi to colonize hardwood or conifer seedlings. Irregardless, carbon flow is likely to be in the direction of the seedling if such connections occur, because the seedlings are heavily shaded under Rhododendron maximum thickets and a source sink relationship is assumed (Read, 1997; Simard et al., 1997). In conclusion Rhododendron maximum thickets are associated with strong mycorrhizal inhibition of canopy tree seedlings in the southern Appalachians. The mycorrhizal inhibition of canopy tree seedlings is probably partially responsible for the reduced seedling growth both above and below ground. Other factors such as the availability of light and the relationship between light quantity and mycorrhizal colonization should be the focus of further investigations. Further understanding of the impact of Rhododendron maximum thickets on ectomycorrhizal fungus/plant interactions will require comparison between ectomycorrhizal sporocarp diversity and ectomycorrhizal root tip diversity between R. maximum thickets and areas without R. maximum. Such a study could provide valuable results for management interests by indicating members of the ectomycorrhizal community which are successful competitors in Rhododendron maximum thickets (Perry et al. 1987). 35

44 CHAPTER 2 The effects of Rhododendron maximum L. (Ericaceae) Thickets, Organic Layer Substrates, and Litter Materials on an Ectomycorrhizal Fungal Community in the Southern Appalachians 36

45 Introduction: The important implications of the documented increase of Rhododendron maximum (Rm) thickets in the southern Appalachians were presented in the Introduction and Chapter 1. The results of this study have shown that total ectomycorrhizal colonization of seedlings is reduced in Rhododendron maximum thickets and that this depression of mycorrhization is correlated with decreased vigor of the seedlings. Concomitant with reduced colonization of seedlings in Rhododendron maximum thickets is a shift in the proportion of ectomycorrhizal morphotypes toward a generalistic, unspecialized, disturbance tolerant ectomycobiont (Cenococcum geophilum Fr.). The hypotheses addressed in this study were 1) the diversity of ectomycorrhizal fungi is different in versus out of Rhododendron maximum thickets, 2) the communities of ectomycorrhizal fungi are structured differently in terms of the relative abundance of their component species in versus out of R. maximum thickets, 3) only certain ectomycorrhizal fungus taxa are distributed differentially with regard to R. maximum thickets, 4) the portion of the ectomycorrhizal fungus community capable of recolonizing manipulated soil substrates is different for substrates from in versus out of R. maximum thickets, and 5) only certain ectomycorrhizal fungi are distributed differently based on their ability to recolonize manipulated soil substrates from in versus out of R. maximum thickets. Additionally this study was used to gauge the effect of the disturbance associated with the substrate manipulation (Chapter 1) on the overall ectomycorrhizal community and on individual ectomycobionts on the substrate manipulation plots. Sporocarp sampling and community analyses: Numerous studies have successfully used abundance and biomass as indicators of the composition of ectomycorrhizal fungal communities (e.g. Bills et al., 1986; Miller, ; Nantel and Neumann, 1992; Palmer et al, 1994; Villeneuve et al., 1989,). The 37

46 results produced by these studies appear to correspond well with plausible explanations. For example Bills et al. (1986) found that the ectomycorrhizal communities were quite different in terms of species composition in nearly pure spruce forests versus heterogeneous hardwood stands nearby. This result was interpreted by Bills et al. (1986) as being related to climatic changes leaving the spruce stands as isolated islands in an area where they once formed large contiguous tracts. Nantel and Neumann (1992) found that the strongest niche dimension of ectomycorrhizal communities was the stand composition but that within a host tree species range the assemblages change in relation to edaphic characteristics. Both the results from Bills et al. (1986) and Nantel and Neumann (1992) fit the hypothesis that the extraordinarily high diversity of ectomycorrhizal fungi evolved as the woody plants radiated across the land surface. As a host tree ranged into new habitats where different edaphic conditions prevailed, and in homogeneous stands of trees in areas with heterogeneous soil environments, ectomycorrhizal fungi speciated to diversify the root soil interface (Pirozynski 1981). This is also evidenced by the associations found with Pseudotsuga menzesii (Douglas fir), which occupies large areas of the Pacific northwest, USA. Pseudotsuga menzesii is a conifer which is associated with an estimated 2000 species of fungi (Trappe, 1977). The relationships between sporocarp abundance, sporocarp biomass, and the status of the fungal thallus below ground are not well known. Many small fruiting bodies, one large fruiting body, or several large fruiting bodies could be produced by the same size mycelium under the same abiotic conditions due to variation in sporocarp morphology, and maternal investment strategies between species, or even between individuals of a species. Sporocarp production in basidiomycetes can also be influenced strongly by environmental factors such as light (Miller, 1967). For some species (Cotter and Bills, 1985; Laiho, 1970; Newell, 1984) and even a complete community (Menge and Grand, 1978) however, there does appear to be an allometric relationship between sporocarp and mycelial biomasses. 38

47 In this context it is important to note that the multivariate statistical methods used to conduct community analyses are based on the occurrence of individuals and not on the fecundity of the individual. Sporocarp sampling for community analysis is akin to counting flowers or fruits fallen on the forest floor, and then trying to use fruit or flower locations to map the locations of individual plants and define plant community structures. For this reason it is not appropriate to exclusively use total abundance of fruiting bodies for the purpose of defining the below ground community of ectomycorrhizal fungi. Bills et al. (1986) used frequency of presence of fungal taxa on small quadrats as a measure of the below ground presence of the fungus. This methodology was based on the suggestion by Pielu (1977) that contiguous quadrats were appropriate for sampling sessile organisms for which sample units were not clearly defined. A compromise is necessary, however, between analyzing presence versus absence and strictly quantitative analyses based on sporocarps. This is due to the following contingencies: 1) The presence of sporocarps in the same location in consecutive years might but does not necessarily designate the occurrence of the same genetic individual, and 2) the size or area of overlap of an individual could be variable to the degree that two separate genetic individuals of the same species could fruit on an individual 2x2 m plot over the course of a two year study. In regard to the latter point, the authors do not know of any studies detailing the level of overlap at the level of populations and genetic individuals. Even if such overlap is rare, the margins of two fairly large individual mycelial systems of the same species could be present in even a small area or at different depths in the soil strata. For this study categorical abundance was used in addition to frequency of occurrence. For abundance based analyses the quantity of sporocarps was converted to categories to deemphasize r-selected species yet allow for the possibility of overlap of separate individuals on a plot. Categorizing the data was also useful as a method for adding some weight to repeated fruiting of a species on a plot, which could possibly be an indication of the existence of separate individuals. Because of the use of categories, however, it is necessary to point out that the abundance values presented in Tabs. 5 & 6 are not 39

48 absolute. Keep in mind, however, that absolute abundances in all fungal community analyses must be interpreted in relation to the contingencies mentioned above, and are never absolute in terms of below ground thalli. Indicator species analysis may turn out to be ideally suited to fungal community analysis because it combines information on both the abundance and frequency of a particular taxon. When a sporocarp is produced, we know that at least part of the individual mycelial unit producing the sporocarp is definitely present at that location. It is for that reason that sporocarp samples provide an invaluable resource when evaluating below ground fungal communities. Additionally, some species such as Russula silvicola Shaffer seem to typically produce isolated individual sporocarps (O.K. Miller and J. Walker, personal observations), a tendency which strengthens their value for community analyses. Sporocarp sampling and ectomycorrhizal colonization: For ectomycorrhizal fungi further information can be gleaned from the identification of morphological types of ectomycorrhizae (e.g. Agerer ; Ingelby et al. 1990). When one uses fungal specific primers to amplify DNA directly from ectomycorrhizal root tips (e.g. Dahlberg et al., 1997: Erland, et al., 1994; Egger 1995;Gardes et al., 1991; Gardes and Bruns, 1993; Henrion et al., 1992; Kraiger et al., 1995; Nylund et al., 1995; Rodgers et al., 1989) it is possible to identify ectomycobionts by comparison with RFLP and sequence databases which already exist for many taxa. One can also identify ectomycorrhizae by comparison with sequence data from sporocarps collected in situ. Both of these methods are time intensive, however, and therefore sporocarp sampling is more tenable for preliminary examination of fungal community structure and for use in large-scale comparisons. Lastly, it is the only way that ectomycorrhizal taxa can be identified and primers synthesized for below ground study. 40

49 The abundance of sporocarps of a taxon in an area is not always directly correlated with the abundance of mycorrhizae of that taxon. Dominant ectomycorrhizal taxa determined by root samples may fruit only occasionally throughout the duration of a long-term study. Fungi, which appear dominant, based on sporocarp production are sometimes poorly represented in samples directly from mycorrhizae. A full discussion of this problem can be found under Ectomycorrhizal diversity at the substrate manipulation site. Identifying ectomycorrhizae from root samples, like sampling sporocarps, provides only a point estimate of the presence of an individual unit of mycelium below ground. In addition, environmental and biotic regulation of the process of mycorrhization is complex, as it is fruit set in fungi. Specialized ectomycobionts present in the root zone may be incompatible with a particular species of seedling, or able to resist colonization with seedlings that are unable to produce sufficient carbohydrate in the mycorrhizosphere to attract the fungus. There is no evidence known to the author suggesting that different thalli cannot colonize the same length of root. Furthermore there is no way to know if sporocarps of a given taxon, found on samples taken a given distance apart, are from the same thallus. For these reasons mycorrhizae present in the root zone, in comparison with sporocarp samples, are not inherently better suited for analysis of fungal community distributions. Sampling of mycorrhizae directly from roots can, however, be our best estimate for gauging the ectomycorrhizal associations of a specific host tree species or age class. Subsamples based on roots of a limited number of photobionts or even aged roots provide a limited window on the overall ectomycorrhizal community in a given area. This is especially true in forest that is composed of mixed hardwoods and conifers of various age classes. It would therefore be desirable to sample the diversity of mycorrhizal morphotypes over broader range of tree associations and age classes for comparison with the sporocarp data. Such a comparison would provide a more accurate depiction of the actual structure of the ectomycorrhizal community in and out of Rhododendron maximum 41

50 thickets than is possible in this study. It would be highly advantageous to estimate both the diversity and abundance of taxa on the roots of seedlings grown in and out of Rhododendron maximum thickets. Examining mycorrhizae from root samples could provide a better indication of the dominant ectomycobionts that are compatible with seedlings in and out of Rhododendron maximum thickets. Shifts in dominant ectomycobionts actually in association with seedlings may be more important than shifts in sporocarp dominance. This is because many of the taxa represented by sporocarps in the thickets may be associated with older subcanopy or canopy stage trees, which do not appear to be affected by Rhododendron maximum thickets (personal observation). Materials and Methods: Sporocarp Sampling: All sporocarps of putative ectomycorrhizal fungi (see Bills, 1985; Molina et al., 1992; Miller 1983 & ) were collected from the substrate manipulation plots (described in Chapter 1) throughout the fruiting season during 1996 and Collections were gathered once a week during all peak fruiting periods. During periods of sparse sporocarp production the plots were checked at least once every two weeks and deteriorated, unidentified, sporocarps were only rarely observed. The number of sporocarps produced on a plot (or in a 4 m 2 area if not collected on a plot) was estimated using the following categories: 1) Rare, 1-2 sporocarps, 2) Infrequent, 3-5 sporocarps, 3) Frequent, 6-10 sporocarps, 4) Common, > 10 sporocarps. All sporocarps were identified in the field or dried and examined microscopically in the laboratory in cases when identification was ambiguous based solely on morphology. Voucher collections, color photos of fresh specimens, and detailed field descriptions are available for taxonomically difficult fungi. 42

51 Data on sporocarp production in relation to occurrence of Rhododendron maximum thickets was also gathered from numerous other locations throughout both basins of the Southeast Forest Research Station at the Coweeta Hydrologic Laboratory (Coweeta; described in Chapter 1). The occurrence of Rhododendron maximum was classified as none (>2m distant from R. maximum), near (within 2m of R. maximum), edge (1 m either side of a clearly delineated forest to R. maximum thicket transition), or slick (> 1m within a R. maximum thicket) for all fungi not collected on a plot. The data for this broad range diversity study have not yet been fully analyzed, and are used in this study only as anecdotal evidence for comparison with statistically significant indicator species. All canopy and subcanopy trees were identified on the blocks during April In addition, the presence of all tree taxa with roots potentially growing onto a block was noted at that time. Analytical methods: The species area curve, jackknife estimates, MRPP, and indicator species analyses were generated by PC-ORD Multivariate Analysis of Ecological Data version 3.0 for windows (McCune and Medfford, 1997). Cluster analyses were calculated using Numerical Taxonomy and Multivariate Analysis System version 1.8 (Rohlf, 1994). The subplots of the manipulation experiment (described in Chapter 1) were the sample units, and the frequency class for each species was entered as a categorical variable (coded as 0 = absent 1 = rare, 2 = infrequent, 3 = frequent, 4 = common). Species that produced sporocarps on an individual plot, during both years of the study, were placed in the next higher abundance category. The groups used for both the MRPP and indicator species analyses were presence and absence of Rhododendron maximum thickets, and the treatments applied to the subplots. For the Rhododendron maximum thicket versus no R. maximum comparison scores for all fungi collected in the blocks (not necessarily on a subplot) were used. The treatment 43

52 comparison only included those fungi collected directly on a subplot. MRPP is a test that compares the heterogeneity within a priori groups non-parametrically. Because it is a non-parametric test MRPP is robust against lack of multivariate normality and non-homogeneity of variance, making it well suited for analysis of ecological data (McCune and Medfford, 1997; Biondini et al. 1985). The weighting factor applied to the items in each group was n/sum(n) where n is the number of items in group. This weighting is recommended by Mielke (1984) for use with MRPP and most recent applications of MRPP have followed suit (McCune and Medfford, 1997). Groups containing more similar samples have higher R values, and negative R values are possible when groups are less similar than expected by chance. Statistical significance is based on a test of no difference between groups, and P values represent the chance of a more extreme R value originating randomly (based on a calculated mean within group homogeneity for all possible grouping of the data). The MRPP was calculated using the Sorensen coefficient ( 1-2W/(A+B) ; W = sum of the shared abundances, A & B sums of abundance in individual sample units). The Sorensen coefficient was chosen because it is applicable to ecological data, is robust against heterogeneity in data sets, and can be applied to either presence-absence data or qualitative data (McCune and Medfford, 1997). The topology of the cluster analysis dendrogram was defined by unweighted pair-group method, arithmetic average (UPGMA) using the Bray-Curtis coefficient (Rohlf, 1994). Subsampling (with 500 repetitions) was used to generate the species to area curve. Cluster analyses generated by PC-ORD Multivariate Analysis of Ecological Data version 3.0 for windows (McCune and Medfford, 1997) using Sorensen s distance and UPGMA or nearest neighbor joining gave similar results. Jackknife estimates were developed as a method of estimating species richness in an area more accurately when the area is subsampled and are based on the observation that actual 44

53 number of species in the area will be larger than the number represented in the subsample. Palmer (1990, 1991) discussed the limitations of these methods and noted that they are best suited to application in a restricted area because the estimates are limited to below twice the number of observed species. The first order jackknife (Heltshe and Forrester, 1983; Palmer, 1990) is calculated as S+r1(n-1)/n (S = the number of species observed, r1 = the number of species occurring in one sample unit, n = the number of sample units). The second order jackknife (Burnham and Overton, 1979; Palmer, 1991) is calculated as S+r1(2n-3)/n r2(n-2) 2 /(n(n-1)) (S, r1, and n are as for the first order jackknife; r2 = the number of species occurring on exactly two sample units). Indicator species analysis was performed using Dufrene and Legendre s (1997) method, which is based both on the abundance and frequency of species in a priori groups. Indicator species analysis uses a Monte Carlo technique to test statistical significance based on repeated randomizations (1000 in this study) of the data set. Relative abundance is the abundance of a certain taxon in proportion relative to the abundance of the taxon in all groups. The relative frequency is the percentage of sample units in each group containing a given taxon. The indicator value is a measure of both the relative abundance and reliability of occurrence of the taxon in the group. Higher indicator values for a taxon in a certain group are interpreted as a stronger association of the taxon with that group. The average abundance, percent frequency (proportion of the plots on which the taxon occurred), relative abundance, relative frequency, and indicator values are all relative to the class of sporocarp frequency recorded in the field and as such are not in terms of absolute numbers. Please see the Introduction under Sporocarp sampling and community analysis above for a consideration of the value of absolute numbers of sporocarps for making inferences about community structure. Results: General assessment of the ectomycorrhizal fungus community: 45

54 A list of all putative ectomycorrhizal fungi collected on the substrate manipulation blocks during the two years of this study is given in Table 4. A total of 69 species were collected on the blocks of which 49 species were collected on a subplot (Tabs. 5 & 6). All collections except two were identified to species, one of which (Cortinarius sp. 1 ) possibly has not yet been described. Three taxa were identified to subspecies, two of which were subspecies of Lactarius piperatus (L. ex Fr.) S. F. Gray. The species-area curve (Fig. 5) is climbing at a steady pace still at the maximum area sampled in this study. First and second order jackknife estimates of species richness on the subplots were 77 species and 98 species respectively. The families Russulaceae (Lactarius 10 species, Russula 6 species), Boletaceae (15 species), and Amanitaceae (10 species) were the dominant ectomycorrhizal families on the substrate manipulation blocks (Tab. 5). Dominant ectomycorrhizal species on the subplots in descending order based on percent frequency (percent of sublpots with the taxa present) were Laccaria laccata (25% frequency), Russula silvicola (22% frequency) Boletus affinis (16% frequency), Cantharellus ignicolor (13% frequency), and Clavulinopsis fusiformis (10% frequency) (Tab. 6). Note, however, that the substrate manipulation on these sites possibly affected the distribution of individual fungi on the plots (see Ectomycorrhizal fungi in response to treatments below). 46

55 Table 4: Putative ectomycorrhizal species list for all collections on the Substrate Manipulation Plots Amanita brunnescens Atk. Amanita caesarea (Scop. ex Fr.) Pers. ex Amanita cinnereoconia Atk. croceescens Bas Amanita citrina lavendula (Schaeff) per Roques Amanita flavoconia Atk. Amanita gemmata (Fr.) Gillet Amanita onusta (Howe) Sacc. Amanita pantherina cf velatipes DeCandolle per Fr. Amanita rubescens (Pers. per Fr.) S. F. Gray Amanita virosa (Fr.) Bertillon in DeCandolle Amanita volvata (Pk.) C.G. Lloyd Austroboletus betula (Schw.) E. Horak Austroboletus gracilus (Pk.) Wolfe Boletellus chrysenteroides (Snell) Snell Boletus affinis Pk. Boletus affinis affinis sub. Pk. Boletus affinis maculosus Pk. Boletus bicolor bicolor Pk. Boletus griseus Frost apud Pk. Boletus ornatipes Pk. Boletus pallidus Frost in Pk. Boletus seperans Pk. Boletus subtomentosus Linne: Fr. Bondarzewia berkeleyi (Fr.) Bond & Singer Calostoma cinnabarina Desv. Calvariadelphus pistillaris (Fr.) Donk Camarophyllus borealis (Pk.) Murrill Camarophyllus pratensis (Pers. ex Fr.) Kummer Cantharellus ignicolor Peterson Cantharellus tubaeformis Fr. Clavaria vermicularis Micheli: Fr. Clavariadelphus pistillaris (Fr.) Donk Clavariadelphus truncatus (Quel.) Donk Clavicorona pyxidata (Fr.) Doty Clavulinopsis fusiformis (Fr.) Cor. Coltricia cinnamomea (Pers.) Murrill Coltricia perrrenis (Fr.) Murrill Cortinarius alboviolaceus (Pers. ex Fr.) Fr. Cortinarius bolaris (Pers. ex Fr.) Fr. Cortinarius collinitus Fr. Cortinarius iodes Berk. & Curt. Cortinarius sp. Craterellus cornucopiodes (L. ex Fr.) Pers. 47

56 Table 4 (continued): Putative ectomycorrhizal species list for all collections on the Substrate Manipulation Plots Dermocybe semisanguinea (Fr.) Moser in Gams Elaphomyces sp Entoloma grayanum Pk. Gomphus flocossus (Schw.) Singer Hydnellum ferrugineum (Fr.:Fr.) Karst Hydnellum scabrosum Fr. Hygrophorus eburneus (Bull. ex Fr.) Fr. Inocybe cf. fastigiata (Shaeff.: Fr.) Quel. Inocybe mixtilis Britzelm. Laccaria laccata (Scop. ex Fr.) Berk. & Br Laccaria ochropurpurea (Berk.) Pk. Lactarius allardii Coker Lactarius camphoratus (Bull. ex Fr.) Fr. Lactarius chrysorheus Fr. Lactarius fumosus Pk. Lactarius gerardii Pk. Lactarius griseus Pk. Lactarius helvus Fr. Lactarius peckii (Burl.) Sacc. Lactarius piperatus (L. ex Fr.) S. F. Gray Lactarius piperatus sub. glaucescens Crossl. Lactarius speciosus (Burl.) Sacc. Lactarius volemus (Fr.) Fr. Lactarius zonarius (Bull. ex St.-Am.) Fr. Leccinum rubropunctum (Pk.) Sing. Leccinum scabrum (Bull. ex Fr.) S.F. Gray Leccinum snellii Smith, Theirs & Watling Phellodon melaleucus (Sw. apud F.: Fr. Karst) Phylloporus rhodoxanthus (Schw.) Bres. Pulveroboletus ravanelii (Berk. & Curt.) Murr. Rozites caperata (Fr.) Mich. Russula aeruginea Lindbl. apud Fr. Russula incarnaticeps Murrill Russula krombholzii Shaffer Russula rosea Russula silvicola Shaffer Russula variata Banning & Pk. Russula virescens (Schaeff. ex Zanted) Fr. Scleroderma citrinum Pers. Strobilomyces floccopus (Vahl. ex Fr.) Karst 48

57 Table 4 (continued): Putative ectomycorrhizal species list for all collections on the Substrate Manipulation Plots Thelephora palmata Scop.: ex Fr. Tremellodendron pallidum (Schw.) Burt. Tricholoma davisiae Pk. Tricholoma sejunctum (Sow. ex Fr.) Quel. Tylopilus plumbeoviolaceus (Snell) Snell & Dick Tylopilus rubrobrunneus Mazzer & Smith 49

58 Mean # Species # Plots Figure 5: Species to area - Mean number of ectomycorrhizal fungi (of 500 subsamples) collected on substrate manipulation subplots (2x2 m) over two years 50

59

60 Table 5: Count, number of times the taxon was collected on the block; Relative abundance (Rel. Abun.), proportion of sporocarps produced in the group (No Rm or Rm); Relative Frequency (Rel. Freq.), number of times the taxon occurred in the group; and Indicator Values (Indicator) for all putatively ectomycorrhizal fungi collected on the blocks. Statistical significance is based on a Monte Carlo style test with 1000 randomizations. Rel. Abun. Rel. Freq. Indicator Count NoRm Rm NoRm Rm NoRm Rm P Amanita brunnescens Amanita caesarea Amanita cinnereoconia Amanita citrina lavendula Amanita flavoconia Amanita gemmata Amanita onusta Amanita pantherina Amanita rubescens Amanita virosa Austroboletus betula Austroboletus gracilus Boletellus chrysenteroides Boletus affinis Boletus bicolor bicolor Boletus griseus Boletus ornatipes Boletus pallidus Boletus subtomentosus Camarophyllus borealis Camarophyllus pratensis Cantharellus ignicolor Clavaria vermicularis

61 Table 5: (continued) Count, number of times the taxon was collected on the block; Relative abundance (Rel. Abun.), proportion of sporocarps produced in the group (No Rm or Rm); Relative Frequency (Rel. Freq.), number of times the taxon occurred in the group; and Indicator Values (Indicator) for all putatively ectomycorrhizal fungi collected on the blocks. Statistical significance is based on a Monte Carlo style test with 1000 randomizations. Rel. Abun. Rel. Freq. Indicator Count NoRm Rm NoRm Rm NoRm Rm P Clavariadelphus pistillaris Clavariadelphus truncatus Clavicorona pyxidata Clavulinopsis fusiformis Coltricia cinnamomea Cortinarius alboviolaceus Cortinarius bolaris Cortinarius collinitus Cortinarius iodes Cortinarius sp Craterellus cornucopiodes Elaphomyces sp Entoloma grayanum grayanum Gomphus flocossus Hydnellum ferrugineum Hygrophorus eburneus Inocybe cf. fastigiata Inocybe mixtilis Laccaria laccata Lactarius allardii Lactarius camphoratus Lactarius chrysorheus Lactarius gerardii

62 Table 5: (continued) Count, number of times the taxon was collected on the block; Relative abundance (Rel. Abun.), proportion of sporocarps produced in the group (No Rm or Rm); Relative Frequency (Rel. Freq.), number of times the taxon occurred in the group; and Indicator Values (Indicator) for all putatively ectomycorrhizal fungi collected on the blocks. Statistical significance is based on a Monte Carlo style test with 1000 randomizations. Rel. Abun. Rel. Freq. Indicator Count NoRm Rm NoRm Rm NoRm Rm P Lactarius griseus Lactarius helvus Lactarius piperatus glaucescens Lactarius piperatus piperatus Lactarius speciosus Lactarius volemus Lactarius zonarius Leccinum rubropunctum Phellodon melaleucus Phylloporus rhodoxanthus Pulveroboletus ravanelii Rozites caperata Russula aeruginea Russula incarnaticeps Russula krombholzii Russula rosea Russula silvicola Russula variata Strobilomyces floccopus Tricholoma davisiae Tricholoma sejunctum

63 Ectomycorrhizal fungi in versus out of Rhododendron maximum thickets: Canopy and sub-canopy trees present on the blocks are listed in Table 7. Block type (with or without Rhododendron maximum thickets) did not define any clusters of plots in the cluster analysis (Fig. 6). MRPP did not detect grouping at the block type level either, with R =.006 (P <.02, R is the chance corrected within group agreement). Individual fungal taxa which were statistically significant indicators of block type were Austroboletus betula (indicators norm = 1, Rm = 14; P<.05), Lactarius speciosus (indicators norm = 24, Rm = 1; P<.01), and Russula krombholzii (indicators norm = 13, Rm = 0; P<.05). Other species which showed potential as indicators were Amanita brunnescens (indicators norm = 0, Rm = 7; P<.382), Amanita rubescens (indicators norm = 0, Rm = 8; P <.15), Cantharellus ignicolor (indicators norm 4, Rm 16, P<.15), Russula silvicola (indicators norm = 7, Rm = 24; P <.15), and Russula variata (indicators norm = 8, Rm = 1; P <.25). Twenty-five species of putatively ectomycorrhizal fungi were collected only on blocks with Rhododendron maximum thickets. Out of those 25 species, 19 species were collected only once. There were 19 species of putatively ectomycorrhizal fungi on only blocks without Rhododendron maximum thickets, and of those species 14 were collected only once. Ectomycorrhizal fungi in response to treatments: As was the case for effects at the block type level, the ectomycorrhizal fungi collected on the plots did not show a pattern in the cluster analysis dendrogram (Fig. 6). Within group homogeneity for the MRPP using treatments as groups was also low (R =.026, P <<.01). The following fungi were statistically significantly indicative of treatment type: 55

64 1) Amanita flavoconia (indicators FF = 0, RR = 0, RF = 17, FR = 0, Control = 0; P <.05), 2) Austroboletus betula (indicators FF = 1, RR = 0, RF = 0, FR = 0, Control = 26; P <.01), 3) Hydnellum ferrugineum (indicators FF = 0, RR = 0, RF = 0, FR = 0, Control = 15; P <.06), 4) Russula variata (indicators FF = 0, RR = 2, RF = 18, FR = 0, Control = 0; P <.06), and 5) Strobilomyces floccopus (indicators FF = 0, RR = 0, RF = 17, FR = 0, Control = 0; P <.04). Leccinum rubropunctum (indicators FF = 0, RR = 1, RF = 0, FR = 0, Control = 13; P <.15) was also a potential indicator favoring control plots, but was only collected three times on the plots and was not statistically significantly indicative. 56

65 Table 6: Species List; Count, number of plots the taxon was collected on; Percent frequency (% Freq.) the proportion of plots the taxon was collected on; and indicator values for putatively ectomycorrhizal fungi on the substrate manipulation plots. Statistical significance is based on a Monte Carlo style test with 1000 randomizations. FR, plots with no Rm litter and with Rm organic layer substrates, etc. Count % Freq. FF RR RF FR CON P Amanita brunnescens Amanita flavoconia Amanita gemmata Amanita onusta Amanita pantherina velatipes Amanita rubescens Amanita virosa Austroboletus betula Austroboletus gracilus Boletellus chrysenteroides Boletus affinis Boletus bicolor bicolor Boletus griseus Boletus ornatipes Boletus pallidus Camarophyllus borealis Cantharellus ignicolor Clavaria vermicularis Clavariadelphus pistillaris Clavariadelphus truncatus Clavulinopsis fusiformis Coltricia cinnamomea Cortinarius collinitus Cortinarius sp,

66 Table 6: Species List; Count, number of plots the taxon was collected on; Percent frequency (% Freq.) the proportion of plots the taxon was collected on; and indicator values for putatively ectomycorrhizal fungi on the substrate manipulation plots. Statistical significance is based on a Monte Carlo style test with 1000 randomizations. FR, plots with no Rm litter and with Rm organic layer substrates, etc. Count % Freq. FF RR RF FR CON P Entoloma grayanum grayanum Gomphus flocossus Hydnellum ferrugineum Inocybe cf. fastigiata Inocybe mixtilis Laccaria laccata Lactarius allardii Lactarius camphoratus Lactarius gerardii Lactarius piperatus glaucescens Lactarius piperatus piperatus Lactarius speciosus Lactarius volemus Lactarius zonarius Leccinum rubropunctum Phellodon melaleucus Phylloporus rhodoxanthus Pulveroboletus ravanelii Russula aeruginea Russula krombholzii Russula rosea Russula silvicola Russula variata Strobilomyces floccopus Tricholoma davisiae Tylopilus plumbeoviolaceus

67 Table 7: Canopy and subcanopy trees at the study site by block (block #, F = no R. maximum, R = R. maximum; see appendix B) 1F 2F 3R 4R 5F 6R Red Maple Aceraceae VAM C C C C C C (Acer rubrum L.) Black Locust Leguminoseae VAM C R R C R C (Robinia pseudoacacia L.) Northern Red Oak Fagaceae ECTO C C R C R R (Quercus rubra L.) Sourwood Ericaceae ER, ECTO? S C C S R C (Oxydendron arboreum DC.) Chestnut Oak Fagaceae ECTO C C C C C C (Quercus montana Willd.) Striped Maple Aceraceae VAM S R R S S R (Acer pennsylvanicum L.) Fraser Magnolia Magnoliaceae VAM? S R S C R R (Magnolia fraseri Walt) Black Birch Betulaceae ECTO S R R R C C (Betula lenta L.) Hickory Juglandaceae ECTO R C R R R C (Carya spp.) Black Gum Nyssaceae VAM R C S C C R (Nyssa sylvatica Marsh) Black Oak Fagaceae ECTO R C R R R R (Quercus ellipsoidalis E.J. Hill) Hemlock Pinaceae ECTO R S R R R R (Tsuga canadensis Carr.) Black Cherry Rosaceae VAM 1 R R R R R S (Prunus serotina Ehrl.) C = present in the canopy, S = present in the subcanopy only, R = only potentially present within the root zone (within 50 m), A = absent ECTO = Ectomycorrhizal, VAM = Vessicular - arbuscular mycorrhizal, ER = Ericoid mycorrhizal 59

68 Discussion: Ectomycorrhizal diversity at the substrate manipulation site The estimates of overall diversity on the blocks and on the subplots are probably lower than the actual number of species present at the site. Sporocarp sampling is not an exhaustive survey of fungal taxa in an area because firstly some taxa fruit only rarely and go undetected during a two-year study such as this. Secondly, some ectomycorrhizal taxa such as Piloderma croceum Erikss. & Hjortst. form laminar fruiting bodies below the litter which are not visible without disturbing the treatments of a study such as this. Thirdly other taxa such as Cenococcum geophilum Fr. are not associated with a known perfect stage. And lastly many taxa produce strictly hypogeous fruiting bodies and are rarely visible above the soil surface. Gardes and Bruns (1996) compared the distribution patterns of ectomycorrhizal fungus sporocarps and ectomycorrhizae over four years in a western California Pinus murcata D. Don stand. They found that Suillus pungens Thiers & Smith fruited commonly but produced mycorrhizae only rarely. Other taxa were equally represented above and below ground, and others such as Russula amoenolens Romagnesi and two thelephoroid fungi were dominant mycorrhizae that were rarely collected aboveground. Similarly, in a six year study of a 100 year old Picea abies (L) Karst stand Dahlberg et al. (1997) found that 12 fungus species which accounted for only 30 percent of the mycorrhizal biomass and abundance produced 74 percent of the sporocarp biomass, and that mostly or entirely hypogeous taxa produced half of the abundance of mycorrhizae. The species - area curve (Fig. 5) indicates that subsampling of the area is an additional factor in the underestimation of species richness. As more area is sampled the curve continues to climb. Bills et al. (1986) suggested 100 contiguous 2x2 m plots 60

69 as a guideline for subsampling a limited area, a suggestion followed in the study by (Nantel and Neumann, 1992). However, the level of access to the plots required to apply the treatments for this study precluded using contiguous plots. The species area curve (Fig. 1) has an inflection point after the addition of approximately 40 2x2 m plots where the slope tapers off appreciably. A sample size of 40 or more contiguous or nested plots may therefore be appropriate for studies seeking to compare ectomycorrhizal communities across a broad geographic, edaphic, or host ranges and requiring sampling at a large number of locations. The higher estimates of species diversity derived by the first and second order jackknife may account for the affect of subsampling. However, our inability to detect the presence of certain taxa and the lack of sporocarp production by certain taxa is not accounted for in the jackknife estimates. Because this study was conducted in a limited tree assemblage (albeit a heterogeneous one) with reasonably constant edaphic characteristics the jackknife estimates can only be interpreted as potentially representative of stands with similar canopy and subcanopy composition, similar abiotic soil conditions and similar soil microflora. Ectomycorrhizal communities vary in relation to stand age (Dighton et al, 1986; Marks and Foster, 1967; Mason et al., 1982; Miller, 1983), the host tree composition of a stand (e.g. Bills et al., 1996; Hallingback, 1994, Villeneuve, 1989), and edaphic characteristics within the range of a particular assemblage (Nantel & Neumann, 1992). Throughout the range of habitats where Rhododendron maximum thickets are present at Coweeta we have recorded approximately 250 species of ectomycorrhizal fungi. The results of this study derived solely from fungi fruiting at the substrate manipulation sites therefore are not necessarily indicative of other ectomycorrhizal communities in the region where Rhododendron maximum thickets occur. A comparison of ectomycorrhizal diversity in and out of Rhododendron maximum thickets over a broader range of the conditions mentioned above, and over a broader 61

70 geographic range, would therefore be highly desirable. Vogt et al (1992) provides references indicating that the species composition of an area can be determined only through long term sampling if based on sporocarps, the time required ranging from 3-8 years in mesic climates. Therefore in addition to the need for sporocarp samples from a broader range of habitats, a longer term study will be necessary to accurately compare the ectomycorrhizal fungus communities in versus out of Rhododendron maximum thickets. Ectomycorrhizal fungi in versus out of Rhododendron maximum thickets: For the purposes of this study the composition of the root zone was treated as uniform in terms of canopy and subcanopy trees (Tab. 7). All of the blocks for this study are located in close proximity to each other across a hillside with a consistent aspect and slope. It is likely that roots extend between the blocks. In addition most of the trees found on a particular block also occur in the forest surrounding all the other blocks. The lack of differences in the composition of the ectomycorrhizal fungus community in and out of Rhododendron maximum thickets, with both MRPP and cluster analysis, is probably related to the presence of mature ectomycorrhizal host trees in the overstory above R. maximum thickets at the study site. The portion of the fungus community compatible with older age class trees may not be affected by the presence of Rhododendron maximum thickets. Lack of grouping could also potentially be related to any combination of the following factors: 1) confounding effects from disturbance of the subplots by treatment application, 2) confounding effects from the treatments themselves, 3) the limited area sampled, 4) the variable nature of the effect of Rhododendron maximum thickets on individual ectomycobiont species, and 5) the failure to detect potentially dominant ectomycorrhizal fungi which fruit rarely or never above ground. Although this is a short-term study of only two years, additional 62

71 collections of taxa which fruit more periodically are unlikely to provide resolution at the block level because of the extremely low R value from the MRPP. With a larger scale sampling scheme and using undisturbed contiguous plots a pattern of association between Rhododendron maximum thickets and ectomycorrhizal community structure may become evident. It is known that the total percent mycorrhizal colonization is reduced and level of colonization by a particular ectomycobiont (Cenococcum geophilum) is increased in Rhododendron maximum thickets at these sites (see Chapter 1). We could hypothesize, therefore, that if the ectomycorrhizal community is not different in and out of Rhododendron maximum thickets, then the failure of the seedlings to form as many mycorrhizae in R. maximum thickets is potentially due to a factor affecting the process of mycorrhization, and not due to a dirth of potential fungal associates. And, as mentioned in Chapter 1, the seedlings may be cut off from potential ectomycorrhizal innocula by the presence of the dense Rhododendron maximum root mats in the thickets. Alternatively the effect of Rhododendron maximum thickets on total colonization may be due to inhibition of the portion of the ectomycorrhizal community compatible with the two species of seedlings assayed (hemlock and red oak). We have no evidence to support this last point. Although there was no apparent change in overall community composition, individual ectomycorrhizal fungi were correlated more strongly with either forest type (Rm or no Rm) based on indicator values. The number of fungal taxa fruiting only in Rhododendron maximum thickets (25) was similar to the number of taxa fruiting only out of the thickets (19). The large number of rare species with 100% average abundance either in or out of Rhododendron maximum thickets at the study site indicates the need for a broader level examination of the diversity of ectomycobionts in versus out of R. maximum thickets. Additional observations of these taxa will be necessary if they are to be informative for indicator analyses. 63

72 Skewed but not absolutely differential habitat preferences such as those for the indicator values of Cantharellus ignicolor, Russula silvicola, and Lactarius speciosus (Tab. 5) should be expected even if a taxon is better adapted to one habitat than the other (in versus out of Rhododendron maximum thickets for this study). This observation is based on distribution patterns known for lichens and decomposers, in which substrate adaptation is frequently recognized as a higher percent occurrence (and not absolute exclusion) on one substrate versus the other. The following observations on the value of the statistically significant indicator species from the study site were based on a visual observation of all of the sporocarp collection records from Coweeta. Austroboletus betula (a strong indicator of Rhododendron maximum thickets based on the plot study) was found on one occasion fruiting abundantly in a large arc near the study site, in forest without R. maximum thickets. In a riparian habitat Austroboletus betula was collected once at the edge of a Rhododendron maximum thicket and on another occasion again in forest without R. maximum thickets. The fact that the taxon is strongly related with Rhododendron maximum thickets on the study site may thus be an artifact due to the size of the sample area. The manner by which Austroboletus betula fruited in the open forest suggested the presence of a large semicircular thallus producing over a half dozen large fruiting bodies along the thallus margin. On the study sites Austroboletus betula, which occurred in high relative abundance and relative frequency on one block (4R), may have been represented by a single large individual on that block. If this is the case then sporocarp sampling has probably overestimated the importance of this taxon as an indicator of R. maximum thickets. Unlike Austroboletus betula, Lactarius speciosus occurred on every block except 4R. Fruiting of Lactarius speciosus was most frequent and abundant, however, on block 2F. This pattern of fruiting of Lactarius speciosus on the plots may tax our 64

73 assumption of similar floral composition of the root zone across blocks. Block 2F is the only block with Hemlock stems present (as a subcanopy resident, Tab. 7). Additionally the sporocarps of Lactarius speciosus on other block were all produced on treatments with either litter or organic layer substrates from blocks with no Rhododendron maximum thickets. It could be suggested, therefore, that if Lactarius speciosus is an ectomycobiont strictly compatible with hemlock, it may have existed only on block 2F prior to the substrate manipulation in association with the hemlock (and not due to a lack of Rhododendron maximum thickets). The patterns of fruiting observed over the course of the study could then be explained by the distribution of Lactarius speciosus inoculum among all the blocks with the pooled forest substrates. Subsequent development of limited associations with hemlock, potentially occurring with less density in the root zones of blocks other than 2F, could then possibly explain the fruiting pattern of Lactarius speciosus at the sites. Unfortunately, Lactarius speciosus was not collected in areas outside the study sites during the course of this study, so no additional observational data are available to support the hypothesis that L. speciosus is poorly adapted for the conditions in Rhododendron maximum thickets (in contrast to the specificity redistribution hypothesis). Russula krombholzii was collected on every block with no Rhododendron maximum and on none of the blocks with R. maximum, and may thus have the best potential for being indicative of forest habitats without R. maximum thickets. Russula krombholzii, as with Lactarius speciosus, was only collected on plots with treatments including substrates from forest without Rhododendron maximum thickets. To our knowledge Russula krombholzii has not been collected on any site in a Rhododendron maximum thicket. Many collections of Russula ssp. from outside the study area still need to be identified however, therefore we cannot be sure that Russula krombholzii is a good indicator of forest without Rhododendron maximum thickets. 65

74 Ectomycorrhizal fungi in response to treatments: As was true for the effect of block type, treatment effects on the distribution of ectomycorrhizal fungi were species specific, but did not relate to overall ectomycorrhizal community structure. Since there were few differences in mycorrhizal colonization levels by treatment type (see Chapter 1), the lack of grouping of ectomycorrhizal sporocarps by treatment type is not surprising. Confounding factors as mentioned for effects by block type could also be a factor here. The homogenization of the substrates was a fairly severe disturbance to the mycelium in the substrates, and the fungi fruiting on the plots had to grow from severed hyphal fragments or other propagules if they originated in the substrates. There is no way to estimate how frequently fungi fruiting on the plots originated from the underlying mineral horizon which was left intact in all treatments, or how frequently they originated from the walkways between the plots. In terms of the ability of ectomycorrhizal fungi to recolonize the substrates, in general the substrates from Rhododendron maximum thickets were not different from those from areas with no R. maximum thickets. The fact that several species of ectomycorrhizal fungi (i.e. Amanita flavoconia, Russula variata, and Strobilomyces floccopus) were statistically significantly indicative (P <.10) of the Rhododendron maximum thicket litter, no R. maximum thicket organic layer substrate combination is peculiar. Austroboletus betula, Hydnellum ferrugineum, and Leccinum rubropunctum all indicated lack of tolerance to disturbance and were most abundant on the controls. Many other species (e.g. Boletus affinis, Boletus pallidus, and Clavulinopsis fusiformis) apparently were disturbance tolerant and did not show a preference to a particular treatment. The difference between the number of species on the subplots versus the total number on the blocks (19 species) could be explained by the increase in sample area. This also 66

75 tends to indicate diversity of ectomycorrhizal fungi on the subplots was not severely depressed by the disturbance to the plots during the substrate manipulation. If this is the case then the validity of ectomycorrhizal counts following plot manipulations is strengthened. Brundrett and Abbott (1995) found that some bait plants (Mirbelia dilatata R. but not Eucalyptus calophylla Lindley) formed somewhat fewer mycorrhizae in treatments where hyphal networks in soil cores were disrupted by breaking up the core. They also reported a lot of variation between mycorrhizal colonization levels related to the amount of organic matter, and the amount of hyphae in the soil. To summarize the results of this study in respect to the hypotheses presented in the introduction, we have found that 1) the diversity of ectomycorrhizal fungi is not apparently different in versus out of Rhododendron maximum thickets, 2) the communities of ectomycorrhizal fungi are not apparently structured differently in versus out of R. maximum thickets in terms of the relative abundance of their component species, 3) only certain ectomycorrhizal fungus taxa are distributed differentially with regard to R. maximum thickets, 4) the portion of the ectomycorrhizal fungus community capable of recolonizing manipulated soil substrates is not different for substrates from in versus out of R. maximum thickets, and 5) only certain ectomycorrhizal fungi are distributed differently based on their ability to recolonize manipulated soil substrates from in versus out of R. maximum thickets. Although the amount of disturbance associated with the substrate manipulations affected the upper soil profiles, the ectomycorrhizal fungal community apparently recolonized the substrates equitably. Despite the fact that a few species of ectomycorrhizal fungi did indicate an intolerance to disturbance, species richness probably was not severely depressed at the study site due to the substrate manipulation. 67

76 SYNTHESIS AND CONCLUSIONS In conclusion Rhododendron maximum thickets are associated with strong mycorrhizal inhibition of canopy tree seedlings in the southern Appalachians. For example on one-year old hemlock seedlings mycorrhizal colonization was three fold lower in Rm thickets than outside Rm thickets. Cenococcum geophilum (a disturbance related ectomycorrhizal fungus) occurred more frequently on seedlings in Rm thickets than outside Rm thickets. For example on one-year old hemlocks percentage colonization by C. geophilum was twice as high within Rm thickets than outside Rm thickets. The mycorrhizal inhibition of canopy tree seedlings was correlated with reduced seedling growth both above and below ground, but explained only about one third of the variation. The differences in mycorrhizal colonization found on seedlings within versus outside of Rm thickets were not reflected in the distribution of ectomycorrhizal fungus sporocarps. Overall diversity and abundance of ectomycorrhizal fungal sporocarps were similar within and outside of Rm thickets. Only individual taxa were found to be more frequent and abundant in forest with or without Rm thickets. Many of the ectomycorrhizal fungi in Rm thickets are associated with mature canopy trees, which occur above the thickets at the study site, however, and may not be compatible with tree seedlings. Because the mycorrhizal colonization of seedlings is depressed, but sporocarp dominance and diversity is similar within versus outside Rm thickets, it is thought that Rm thickets affect the process of mycorrhization. In addition, fungitoxicity of Rm litter or organic layer substrates does not appear to be a factor related to mycorrhizal colonization of seedlings. Other factors such as the availability of light and the relationship between light quantity and mycorrhizal colonization should be 68

77 the focus of further investigations of the inhibition of seedlings by Rhododendron maximum thickets. 69

78 APPENDIX A Growth of ectomycorrhizal fungi in media containing substances from Rhododendron maximum thicket litter and leaves Methods: Decoction experiments: The decoction experiments were conducted using solid Hagem s media as modified by Van Cotter (in Cripps and Miller, 1995) with and without the addition of 16 g/l of chopped leaf materials. Leaf materials used were Rhododendron maximum L., hemlock (Tsuga canadensis (L.) Carr.), red oak (Quercus rubra L.), birch (Betula lenta L.), and cherry (Prunus serotina Ehrh.). The leaves were cut into fine pieces and incorporated in the media which was then autoclaved for 18 minutes at 250 C. The warm media was agitated to evenly distribute the leaf material and immediately poured into petri plates (10 cm diameter). All leaf materials were collected during July and were stored frozen until used. After cooling the decoction media plates were inoculated with one of three ectomycorrhizal fungi. In the first experiment (Fig. 7) Cenococcum geophilum Fr. (VTCC 1409), Pisolithus tinctorius Pers.(VTCC 3303), and Suillus pictus (Pk.) Smith and Thiers (VTCC 3326) were used. In the second experiment (Fig. 8) only Cenococcum geophilum (VTCC 1409) and Pisolithus tinctorius (VTCC 3303) were used. For both experiments the fungi were grown for one month at 20 C on solid Hagem s media as modified by Van Cotter (in Cripps and Miller, 1995). From these plates uniform squares of mycelium and agar were cut from near the margin of the fungus culture and used to center inoculate the decoction media plates. Immediately after inoculation two lines were drawn on the lid of the plate. Measurements of radial 70

79 growth were taken along the predetermined lines from the edge of the inoculum. For the first experiment (Fig. 7) measurements were taken over a period from days at approximately one week intervals. The radial growth in the second experiment (Fig. 8) was measured only once, 4 weeks after inoculation. For both experiments the plates were incubated at 18 C. Differences between mean growth on control plates versus decoction media plates were analyzed with T-tests for each fungus on each measurement date for the first experiment (Fig.5). In the second experiment (Fig. 8) a two-way analysis of variance (one treatment was the fungus inoculum and the other treatment was the type of leaf material added) was used. The sample sizes were 10 replicates for the first experiment (Fig. 7) and 7 replicates for the second experiment (Fig. 8). Leachates: Litter and organic layer substrates were collected from three sites in Rhododendron maximum thickets and pooled. The substrates were chilled for transportation to the lab and immediately after arrival were soaked (at 50 g/l for the organic substrates and 20 g/l for the litter substrates) in tap water for 24 hours at room temperature in the dark. After this period the leachates were drained through cheesecloth and first filtered through Whatman #1 filter paper then through a Seitz filter with Hercules Sterilizing Filter Sheets (grade ST3; size L6). The sterile leachates were stored at 3 C for three days prior to use. The leachates were combined one to one with autoclaved (250 C; 18 min.) double strength solid Hagem s media as modified by Van Cotter (in Cripps and Miller, 1995) after it cooled to approximately 50 C. The leachate medium was then poured onto petri plates (10 cm diameter) and allowed to cool. Straight Hagem s media as modified by Van Cotter (in Cripps and Miller, 1995) was used for the control plates. Ten replicate plates of each substrate type were center inoculated with P. tinctorius (VTCC 3303) and incubated as described above in the decoction section and observed after four weeks. Means for 71

80 radial growth were not analyzed statistically because there were no visually apparent differences between treatments. Voucher cultures for all fungi are being maintained in the Virginia Tech Culture Collection. Results: Ectomycorrhizal growth: Two of the three species of ectomycobionts (Suillus pictus and Cenococcum geophilum) showed no affect of Rhododendron maximum leaf decoctions on their growth rate. However, the growth of P. tinctorius was highly statistically significantly (P <.01) reduced by more than 65% (Fig. 7). In order to determine if the inhibition of P. tinctorius is a specific response to Rhododendron maximum leaf extracts the decoction experiment was repeated with leaves from 4 species. The growth of the mycorrhizal fungi was statistically significantly (P<.05) inhibited by all leaf types except Rhododendron maximum. Litter from birch, red oak, and cherry inhibited growth of P. tinctorius over 50% more than that of Rhododendron maximum. The radial growth of P. tinctorius was not inhibited by leachates from Rhododendron maximum litter or organic layer substrates. Discussion: Does leaf material from Rhododendron maximum inhibit ectomycobiont growth? Among the ectomycorrhizal species examined, only Pisolithus tinctorius growth was inhibited by Rhododendron maximum leaf material. However, this inhibition was less than that from leaves of other species. Therefore it is not likely that the leaf litter on the forest floor under Rhododendron maximum will be any more inhibitory to ectomycorrhizal growth than leaf litter from forest without R. maximum present. 72

81 The lack of any Rhododendron maximum organic or litter leachate effect on ectomycorrhizal fungus growth suggests that the inhibition observed in the decoction experiments is either an artifact due to the intense heat of autoclaving or related specifically to fresh leaf materials. However since a toxic compound would have to accumulate in the litter or organic horizon to affect the growth of ectomycorrhizal fungi in the field, it is unlikely that suppression of ectomycorrhizal colonization and reduced seedling growth (See Chapter 1) in Rhododendron maximum thickets is due to an activity of toxins from R. maximum. 73

82 Radial Growth (mm) P.t. control P.t. decoction S.p. control S.p. decoction C.g. control C.g. decoction Time (days) Figure 7: Growth of three species of ectomycorrhizal fungi on media with (decoction) and without (control) the incorporation of Rhododendron maximum leaf materials. Each point is a mean of ten plates per treatment and errors represent the standard error of the mean. Means for P.t. were highly statistically significantly different (P<.01) on all sample dates. P.t.=Pisolithus tinctorius, C.g.=Cenococcum geophilum, S.p.=Suillus pictus 74

83 Radial Growth (mm) P.t. C.g. c c a b ab ab 0-5 T.c. P.s. B.l. Q.r. R.m. Con. Figure 8: Radial growth of the ectomycorrhizal fungi Pisolithus tinctorius and Cenococcum geophilum on media with and without the incorporation of Rhododendron maximum or canopy tree leaf materials. Each point is a mean of 7 plates containing a decoction media with leaves of one of five species or control media. Error bars refer to the standard error of the mean, and treatments followed by different letters are statistically significantly different (P<.05); significance tested using two-way ANOVA. T.c.=Tsuga canadensis, P.s.=Prunus serotina, B.l.=Betula lutea, Q.r.=Quercus rubra, R.m.=Rhododendron maximum Con.=control; abbreviations for fungi are as for Figure 7. 75

84 APPENDIX B Plot map of the study site at Coweeta; distance among main plots is not to scale 76