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FUNGAL AND BACTERIAL SYMBIOSES AS POTENTIAL BIOLOGICAL MARKERS OF EFFECTS OF ATMOSPHERIC DEPOSITION ON FOREST HEALTH Donald H. Marx USDA Forest Service Southeastern Forest Experiment Station Athens, GA 30602 ABSTRACT Steven R. Shafer USDA Agricultural Research Service NC State University Raleigh, NC 27695-7616 The ecological role and physiological functions of the different microbial symbiotic associations, specifically ectomycorrhizae, endomycorrhizae, and actinorhizae, which occur naturally on roots of forest trees will be discussed. The effects of man-made and natural stress on tree physiological and soil processes which may cause change in (i) microbial symbiont species composition in the root zone; (ii) microbial symbiont species succession in developing stands of trees; and (iii) morphological and physiological attributes of the symbiotic associations will be presented. Essential methodologies needed to study these interacting chemical and biological factors on tree seedlings and on more mature trees will be proposed. Microorganisms are present in great numbers on and near the feeder roots of trees, and they play vital roles in numerous physiological processes. These dynamic processes are mediated by associations of microorganisms participating in saprophytic, pathogenic and symbiotic root activities. The major symbiotic associations on tree roots are mycorrhizae and actinorhizae. MYCORRHIZAE The term mycorrhiza (fungus-root) is used to described a structure that results from a mutually beneficial association between the fine feeder roots of plants and species of highly specialized, root-inhabiting fungi. Mycorrhizae are active, living components of the soil and have some properties like those of roots and some like those of microorganisms. The mycorrhizal fungi derive most if not all of their needed organic nutrition (carbohydrates, vitamins, amino acids) from their symbiotic niche in the primary tissues of roots. Evidence suggests that the mycorrhizal habit evolved as a survival mechanism for both partners of the association, allowing each to survive in the existing environments of low soil fertility, drought, disease, and temperature extremes. Because of this coevolutionary process, mycorrhizae are as common on the root systems of trees and other plants as are chloroplasts in their leaves. In examining plants in a natural environment, the question is not "are the plants mycorrhizal" because they all are, but rather "what type of mycorrhiza is present and what is the degree of mycorrhizal development on the roots?" There are over 4000 publications on mycorrhizae in the plant science literature. 217

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218 Endomycorrhizee This type of mycorrhiza is the most widespread and comprises three groups, Ericaceous mycorrhizae occur on four or five families in the Ericales. Orchidaceous mycorrhizae are a distinct type that occur only in the plant family Orchidaceae. These two groups of endomycorrhizae are not widespread and will not be discussed. Vesicular-arbuscular mycorrhizae (YAM) form the third group of endomycorrhizae. They occur on more plant species than do all other types of mycorrhizae combined and have been observed in roots of over 1000 genera of plants representing some 200 families. It has been estimated (Kendrick and Berch 1985) that over 90% of the 300,000 species of vascular plants in the world form VAM. VAM fungi are ubiquitous in all natural soils throughout the world except where they have been eliminated by man's activities. Inoculum density and fungal species, however, differ in different soils supporting different plants. The fungi forming VAM belong to the class Zygomycetes and the family Endogonaceae, which includes the genera Glomus, Gigaspora, Acaulospora, Sclerocystis, Entrophospora, and Endogone. These fungi have very little host specificity. One VAM fungus may have a plant host range including trees, vines, grasses, legumes, and desert plants. The main characteristic of VAM is the presence of vesicles and/or arbuscules in the primary root cortex. The endodermis, stele, and root meristems are not colonized. Inter- and intracellular hyphae present in the cortex are connected to the external mycelium that spreads and ramifies in soil. Some VAM produce large sporocarps (5-10 mm in diameter) containing many spores, and others form large ( 100-600 um in diameter), single, thick-walled spores on the root surface, in the rhizosphere, or in the root tissues. VAM fungi cannot grow saprophytically in soil and, therefore, can only grow while in symbiotic association with their plant hosts. They may, however, survive for decades in soil as dormant spores without plant associations. VAM increase a plant's uptake of certain nutrients, particularly P. Cu. and Zn. These elements are relatively immobile in soil, and zones of depletion develop near feeder roots. The extramatrical growth of hyphae from VAM fungi can extend beyond the feeder roots and increase the volume of soil from which these elements are absorbed. The additional nutrient absorption due to VAM fungi can result in several-fold growth increases in plants. The degree of plant benefit appears to be related to the plant's P requirement, its ability to absorb nutrients from soil, the amount of available P in soil, and the species of VAM fungus involved. Additions of available P to soil may eliminate the plant's dependence on VAM (Baylis 1970, 1972~. However, it takes from 50 to 100 times the normal P levels found in forest soils to eliminate the VAM dependency of hardwood trees (Kormanik et al., 1982~. There are other significant benefits of VAM to plants. VAM are capable of reducing the effects of various fungal pathogens and suppressing the effects of parasitic nematodes (Schenck 1981~. VAM have also been shown to enhance water uptake, increase tolerance to heavy metals, saline soils and drought, decrease transplant shock, and bind soil into semistable aggregates. Ectomycorrhizee This type of association occurs on about 10% of the world flora. Trees belonging to the Pinaceae (pine, fir, larch, spruce, hemlock), Fagaceae (oak, chestnut, beech), Betulaceae (alder, birch), Salicaceae (poplar, willow), Juglandaceae (hickory, pecan), Myrtaceae (eucalyptus), Ericaceae (Arbutus), and a few others form ectomycorrhizae.

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219 Some tree genera such as Alnus, Eucalyptus, Casuarina, Cupressus, Juniperus, Tilia, Ulmus, and Arbutus will form both ectomycorrhizae and VAM, depending on soil conditions and tree age. Numerous fungi have been identified as forming ectomycorrhizae. In North America alone it has been estimated that more than 2100 species of fungi form ectomycorrhizae with forest trees. Worldwide, there are over 5000 species of fungi that can form ectomycorrhizae on some 2000 species of woody plants. Among the basidiomycetous fungi, species of Hymenomycetes (mushrooms) in the genera Boletus, Cortinarius, Suill?vs, Russula, Gomphidius, Hebeloma, Tricholoma, Laccaria, and Lactarius and species of the Gasteromycetes (puffballs) in the genera Rhizopogon, Sclerod~erma, and Pisolithus form ectomycorrhizae. Certain orders in the Ascomycetes such as Eurotiales (Cenococcum geophitum), Tuberales (truffles), and Pezizales have species that form ectomycorrhizae on trees. In ectomycorrhizae, intercellular hyphae surround cortical cells forming the Hartig net, and several hyphal layers cover the outside of the feeder root forming the fungus mantle. Ectomycorrhizal colonization normally changes the feeder root morphology and color. They may be unforked, bifurcate, nodular, multi-forked (coralloid), or in other shapes. Their color, which is usually determined by the color of the mycelium of the fungal symbiont, may be jet-black, red, yellow, brown, white, or blends of these colors. Unfortunately, with the exception of the above mentioned fungi, few of the hundreds that exist in any given forest stand can be identified based on characteristics of the ectomycorrhizae. The only sure way of identifying ectomycorrhizal fungi is with their fruit bodies. As with VAM, ectomycorrhizal colonization is limited to the primary cortex and does not spread beyond the endodermis or into meristem tissues of the feeder root. Unlike VAM, however, many ectomycorrhizal fungi can be grown routinely in pure culture. An important aspect of both VAM and ectomycorrhizal fungi is that neither group of fungi can exist saprophytically in nature without a plant-host association. Spores or resistant hyphae may survive long periods in soil without a plant host, but the fungi from these propagules will not grow independent of their plant host as saprophytes. Ectomycorrhizal fungi aid the growth and development of trees. For some trees, such as Pinus, they are indispensable for growth under natural conditions. The obligate requirement of pine for ectomycorrhizae in a natural environment has been clearly shown by numerous workers in tree regeneration trials in former treeless areas and in countries without native ectomycorrhizal trees (Marx 1980, Mikola 1973~. Pine or other obligate tree species can be grown from seed in aseptic culture, hydroponics, or elsewhere without ectomycorrhizae. In these nonmycorrhizal conditions however, the seedlings must be furnished with the factors (high nutrients, water) that are normally supplied by the fungal symbiont and must be kept in a stress-free environment or they will die or not grow normally. Mycorrhizae, especially ectomycorrhizae, appear to be the first line of biological defense against stress for trees. Trees with abundant ectomycorrhizae have a much larger, physiologically active, root-fungus area for nutrient and water absorption than trees with few or no ectomycorrhizae. This increase in surface area comes both from the multi-branching habit of most ectomycorrhizae and from the extensive vegetative growth of hyphae of the fungal symbionts from the ectomycorrhizae into the soil. These extramatrical hyphae function as additional nutrient and water-absorbing entities and assure maximum nutrient capture from the soil by the host. Ectomycorrhizae are able to absorb and accumulate nitrogen, phosphorus, potassium, and calcium in the fungus mantles more rapidly, and for longer periods of time, than nonmycorrhizal feeder roots. Ectomycorrhizae also appear

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220 to increase the tolerance of trees to drought, high soil temperatures, soil toxins (organic and inorganic), and extremes of soil - acidity caused by high levels of sulfur or aluminum. Ectomycorrhizae deter infection of feeder roots by root pathogens, such as species of Pythium or PhytopAthora (Marx and Krupa 1978~. Hormone relationships induced by fungal symbionts cause ectomycorrhizal roots to have greater longevity (length of physiological activity) than nonmycorrhizal roots (Slankis 1973, Ng et al. 1982, Ek et al. 1983~. Not all species of fungi form ectomycorrhizae that have equal benefit to their hosts. Some are more effective than others. Many species of fungi are normally involved in the ectomycorrhizal associations of a forest stand, a single tree species, an individual tree, or even a small segment of lateral root. As many as three species of fungi have been isolated from an individual ectomycorrhiza (Zak and Marx 1964~. Even as a single tree species can have numerous species of fungi capable of forming ectomycorrhizae on its roots at any given time, a single fungus can enter into ectomycorrhizal association with numerous tree species on the same site. A single species of ectomycorrhizal fungus can develop numerous biotypes or clones in a very limited area of a pure stand (Fries 1987~. Some fungi are apparently host-specific; others have broad host ranges and form ectomycorrhizae with members of numerous tree genera in diverse families. Ectendomycorrhizae are intermediate types with features of both ecto- and endomycorrhizae. In comparison to other mycorrhizal types, little research has been done on them. They appear to have limited distribution in forest soils and tree nurseries and are found on roots of normally ectomycorrhizal trees. Little is known of their importance to trees. Factors Affecting Mycorrhizal Development Many factors affect mycorrhizal development. It is necessary, however, to separate those that affect the tree from those that affect the fungal symbionts. Generally, any soil or above-ground condition that influences root growth (i.e., carbon allocation) also influences mycorrhizal development. The first prerequisite to mycorrhizal development is that a susceptible feeder root be preformed by the plant host. Second, there must be viable inoculum of a mycorrhizal fungus present in the rhizoplane to colonize the root. Third, soil chemical, physical, and biological conditions must be favorable for successful symbiotic colonization. The main factors influencing susceptibility of tree roots to mycorrhizal infection appear to be photosynthetic potential and soil fertility. High light intensity and low-to-mocterate soil fertility enhance mycorrhizal development; the other extremes of these conditions (light intensity below 20% of full sunlight and excessively high soil fertility) reduce, or may even prevent, ' mycorrhizal development. However, it normally takes 10 to 50 times the nitrogen and phosphorus normally found in most forest soils to significantly suppress mycorrhizal development of forest trees (Cline et al. in press). Mechanical defoliation that reduces photosynthetic surfaces reduces mycorrhizal development (Last et al. 1979~. Increased photosynthesis due to CO2 enrichment of the atmosphere increases mycorrhizal development (O'Neill et al. 1987~. Light intensity and fertility appear to influence either the biochemical status of feeder roots, such as controlling levels of simple sugars, or the synthesis of new feeder roots, both of which are products of carbon allocation (Ekwebelam and Reid 1983~. Roots growing rapidly because of high soil fertility contain few simple sugars and they are not highly susceptible to symbiotic colonization (Marx et al. 1977~. The supply of photosynthates from the tree host to the fungal symbiont is of paramount importance to the development, function, and maintenance of mycorrhizae.' This supply not only furnishes

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221 the energy and carbon for fungal growth but is intimately connected with nutrient uptake of mycorrhizal roots (France and Reid 1983~. The factors that affect the fungal symbionts directly are those which regulate survival of the fungi in the soil or their growth on roots. Extremes of soil temperatures, pH, moisture, etc., and presence of antagonistic soil microorganisms can affect the survival of symbionts and thereby influence the mycorrhizal fungus inoculum potential of the soil (Marx et al. 1984~. Unfortunately, with the exception of recent work on fungicides (Marx et al. 1986, Marx and Cordell 1987), factors affecting survival of inoculum of specific mycorrhizal fungi in soil have not been studied. Actinorhizae The term actinorhizae refers to the nodules that Frankia spp. form with roots of their host plant. The nodules are lobed, coralloid, infection-induced lateral roots exhibiting restricted apical growth but profuse branching. Actinorhizal plants are fast-growing pioneer plants, mostly trees and shrubs, and are important in the nitrogen economy of temperate forests. Typically, they colonize barren, nutrient-poor soils. Roots of these plants are nodulated by filamentous bacteria in the genus Frankia of the Actinomycetales. Taxonomically, actinorhizal plants are distributed through at least 170 species among 22 genera in ~ plant families (Betulaceae, Casuarinaceae, Coriaraceae, Datiscaceae, Elaeagnaceae, Myricaceae, Rhamnaceae, and Rosaceae). However, not all species in all genera are affected. Like leguminous plants with Rhizobium spp. or Bradyrhizobium spp., actinorhizal plants fix atmospheric N2 into plant-available forms. Actinorhizal plants can fix up to 362 kg N/ha/yr, an amount similar to that fixed by leguminous plant-rhizobial systems. The actinorhizal relationship is beneficial to both organisms. Actinorhizal plants normally form mycorrhizae and actinorhizae at the same time in natural environments. Some species lil~e AInus and Casuarina can form tripartate symbioses, i.e., actinorhizae, VAM, and ectomycorrhizae, on the same tree at the same time. The characteristics of the actinorhizal infection reflect the great degree of interaction between the symbionts. Frankia spp. are identified as thin, usually nonseptate hyphae 0.8 to 1.2 um in diameter, forming intercalary or terminal sporangia bearing spores and vesicles. Some strains have not been observed to sporulate. Some isolates exhibit degrees of host-specificity while others do not. Hyphae of a strain of Frankia that are compatible with the host penetrate a root hair and grow within the root hair cell. Cortical cells in the vicinity begin to divide, even though they are not yet infected. Hyphae then invade these cortical cells and begin to form vesicles. A localized thickening, called the primary nodule, develops. A lateral root forms adjacent to the primary nodule, and hyphae grow into the cortical cells from the primary nodule. Thus, while prospective cells for infection in the primary nodule arise from de-differentiation of cortical cells, cells for infection in the "true nodule" that develops from the lateral roots arise from continued production of cells by the apical meristem of the lateral root. The meristem itself is not infected. Once a cortical cell has differentiated behind the meristem and starts to grow, hyphae infect the cell, coil around the nucleus, and fill the cell. As an increasing number of cells in the true nodule become infected and other apical meristems become active, the true nodule takes on a lobed, coralloid appearance. Branched hyphae borne in intracellular clusters near the host cell wall form swollen vesicles at the tips. These vesicles are probably the site of N2 fixation. In alder nodules, each infected host cell contains one cluster of several hundred vesicles in the upper tips of the nodule lobes. Lower parts of the lobes contain

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222 cells with degenerating endophyte, which seems to be digested eventually by the host cells. Spores form in the late stages of the infection cycle, and decaying nodules release spores into the soil. The pattern of cortical cells infected around the stele is host-dependent. The same isolate in roots of different hosts may exhibit a different infection pattern. Nodules last an average of 3 to 4 years but may persist for as long as years. They may be over 3 cm in diameter and each can contain billions of spores. Actinorhizae have great ecological and commercial significance. Many actinorhizal plants (e.g., AInus spp. in the northern hemisphere) are important colonizers of N-poor soils. Leguminous plants and other N-fixation symbioses are absent from large areas in cool climates, such as in Canada and Scandinavia, so a large proportion of soil N in those areas is derived through actinorhizae. Some alder spp. produce 15 to 25 tons of biomass/ha/yr and are among the highest-yielding tree species in the temperate zone. The N2-fixation activities of actinorhizae give rise to the ecological and commercial importance of the relationship. Almost all Frankia spp. can use NH4+, NOB-, and selected amino acids as sources of N and many exhibit growth on N-free cultural media. In nodules, Frankia fixes N2 from the air into NH4+. However, more N2 is fixed than is required by the endophyte, so some NH4+ is exported to the host cell cytoplasm, converted to the amino acid citrulline, and translocated from the nodule to other parts of the plant. Thus, a net accretion of N to the plant and to the site occurs. Most factors affecting mycorrhizal development also affect actinorhizal development. The nodule-forming process seems to be more sensitive than plant growth to soil acidity. Most isolates of Frankia grow best in culture in pH 6.5-7.0 as do most of their tree hosts. Vesicle formation and N2-fixation in culture can be suppressed with increases in the availability of N. and nodulation is also suppressed by increased concentrations of NH4+-N. This latter effect may be attributable to lowered C availablity to the nodules under high-N conditions. As with mycorrhizae, actinorhizae represent a C sink to the host. Nodulation and N2-fixation, therefore, are affected by host photosynthetic rate. The activity of nodules is linked to the supply of photosynthate and is altered by changes in light intensity, foliar area, girdling, mycorrhizal status (P absorption), and C partitioning by the host. Any factor that supresses photosynthesis, stimulates respiration, decreases photosynthetic area, and suppresses C transport to roots affects actinorhizal development and function. For further information on actinorhizae see Akkermans and Roelofsen (1980), Akkermans and Van Dijk (1981), Akkermans et al. (1984) and Stowers (1987~. ECOLOGICAL ASPECTS OF TREE SYMBIOSES Other than the limited information presented above, there is a paucity of research or observational data available on the ecology of endomycorrhizal and actinorhizal associations of trees. The following discussion is, therefore, restricted to ectomycorrhizal associations. Fungal Succession and Forest Development Terrestrial plant communities tend to be dominated by either endomycorrhizal or ectomycorrhizal plants (Moser 1967~. During ecosystem succession to climax forest in most temperate regions, the fungal associates of higher plants change from endomycorrhizal or VAM dominance in herbaceous and scrub communities to ectomycorrhizal dominance associated with trees (Rose 1980~. Tropical plant succession

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223 may progress from the nonmycorrhizal state of pioneer plants to an endomycorrhizal status of climax hardwoods, such as those found in rain forest (Ianos 1980~. In ectomycorrhizal fungus succession in forests, it is now recognized that there are distinct early- and late-stage fungi. The pioneering work on this concept was done in Scotland. Dighton and Mason (1985) suggested that early- stage fungi are similar to "r" plant species (Harper 1977), since they spend most of their time in acts of colonization then give way to other species in natural succession. They view late-stage fungi as similar to "K" plant species since the latter are specialists in a resource-limited environment with intense interference from their associates. In aseptic culture (i.e., without competition and other stresses) early- and late-stage fungi form ecto- mycorrhizae on seedlings equally well. However, only the early-stage fungi are able to rapidly colonize seedlings in natural, nonsterile soil that harbors competitors and other stresses. Early-stage fungi may not totally disappear from mature stands, but they are supplanted by more dominant species (Dighton et al. 1986a) or suppressed in reproduction due to canopy characteristics. Last and Fleming (1985) suggest that the differences in root physiology and rhizosphere populations between seedlings and mature trees might be beneficial to volunteer seedlings. Late-stage ectomycorrhizae on the mature trees may transfer carbon to these seedlings through the mycorrhizal bridge (Reid and Woods 1969) and benefit seedling growth in the suboptimal light conditions in closed canopy stands. This carbon transfer to seedlings may also function in clearcuts where ectomycorrhizae are still viably attached to cut stumps and sprouts. As trees become larger internal recycling of nutrients within the tree increases, and demand for nutrients from soil generally decreases (Miller 1979~. The amount of carbon available to support a mycorrhizal fungal symbiont may differ with tree size, with small trees having limited supplies and supporting fungi with limited carbon demands, and larger trees supporting more carbon demanding fungi (Mason et al. 1985, Last et al. 1985~. They concluded that the stage of development of the ecosystem would strongly influence the resources available to mycorrhizal fungi, and that successional stages of fungi occur due to their variable requirements for host carbon. Associated with ectomycorrhizal fungus succession is increased fungal species diversity with increasing stand age and increasing number of host species in the stand (Mason et al. 1982, Last et al. 1984~. There is also evidence that the succession of ectomycorrhizal fungi is influenced by a tree x soil interaction (Last et al. 1984~; seed source, tree vigor and leaf retention (Mason et al. 1982~; and forest fertilization (Hora 1959, Shubin et al. 1977~. Fruit body production by these fungi, which is the main parameter used to observe succession, is also strongly influenced by season of year, rainfall amount and frequency, organic content of soil, root density, and degree of ectomycorrhizal development (Wilkins and Harris 1946~. It is assumed that as ectomycorrhizal development increases so does fruit body production as long as all other factors are equal. The amount of fruit body production is an accurate surrogate for degree of ectomycorrhizal development in bare-root tree nurseries (Marx et al. 1984~. Whether this relationship holds true in complex ecosystems is unknown at this time. Ectomycorrhiz~e and Net Primary Production Ectomycorrhizae are important organs for the accumulation and storage of soil-derived and host carbon-based nutrients. The first are essential for the tree host and the latter for growth of hyphae and production of fruit bodies by the fungi. There are no actual measurements of the quantities and types of plant carbon compounds used

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224 by ectomycorrhizal fungi as a proportion of total photosynthesis. Some estimates, however, have been made in field situations. The amount of photosynthate required to support a healthy and vigorous population of ectomycorrhizal fungi on roots has been estimated in only a few cases. These estimates, based on measured carbon content of fruit bodies and estimated carbon content of the fungal mantle and extramatrical mycelia (turn-over rate), vary from a high of over 50h in Douglas-fir forests in Oregon (Fogel and Hunt 1979) to a more likely 15% for the same tree species (Vogt et al. 1982~. The first report did not verify that all fungal components measured were ectomycorrhizal in origin; the latter report did. Odum and Biever (1984) considered the ectomycorrhizal association a major cybernetic control subsystem in terrestrial plant communities and concluded that it has not been adequately considered in energy flow models. They based this conclusion on measurements of the partitioning of net primary production between 4-year-old loblolly pine and fruit bodies, ectomycorrhizae, and mycelia of specific fungi on severely eroded soils in the Copper Basin of Tennessee. They estimated that from 15 to 25% of net primary production flowed along the ectomycorrhizal pathway of trees on this site. Production of fruit bodies by ectomycorrhizal fungi can be quite large in forest stands. In Sweden, several thousand fruit bodies of over 70 species of ectomycorrhizal fungi were collected during a 3-year period in a 5-ha stand of pure beech (Tyler 1984~. In Scotland, a 5-year average of between 240,000 and 490,000 fruit bodies of ectomycorrhizal fungi/ha were produced yearly under Scots pine (Richardson 1970~. In Georgia, Thacker (1971) collected fruit bodies of ectomycorrhizal fungi for 1 year from areas of about 3 ha in three different forest types. Collected fresh weights were 4812 g of 31 fungal species in a 25-year-old pine plantation, 6560 g of 56 species in a 70-year-old pine stand, and 9991 g of 74 species in a 70-year-old pine-oak stand. This report is the only one in the literature suggesting that fungal succession and increased species diversity may occur in aging stands in the southern U.S. ECTOMYCORRHIZAE AND ATMOSPHERIC DEPOSITION Several controlled-exposure studies in recent years have shown that ambient or above-ambient levels of atmospheric deposition can affect ectomycorrhizal development on seedlings. Direct exposure of specific ectomycorrhizae of loblolly pine to Of and SO: caused a significant reduction in their respiration, but respiration in nonmycorrhizal roots was reduced more. Certain ectomycorrhizae were tolerant of On, while others were more tolerant of SO2 (Carney et al. 1 97S, Garrett et al. 1982~. Shafer et al., ( 1985) found that simulated rain treatments of pH 4.0 and 3.2 inhibited ectomycorrhizal development of loblolly pine seedlings, but that rain treatment of pH 2.4 stimulated development. Mahoney et al., (1985) found that root growth of loblolly pine was more heavily impacted by O3 and SO2 in nonmycorrhizal than in mycorrhizal seedlings. Their results suggested that ectomycorrhizae altered the pollution effects on root and shoot growth resulting in more root growth and elimination of the effect of pollutants. Reich et al. (1985, 1986) found that seedlings of white pine and northern red oak produced more short roots and higher percentages of ectomycorrhizae after exposure to O~, and that SO2 and acid rain treatments decreased both root parameters. One could predict that any significant change in carbon allocation patterns to roots caused by air pollution woulcl affect root growth and, subsequently, mycorrhizal development (Reich et al. 1987, Winner et al. 1987~. Also, any significant change in soil chemistry causing increased levels of available metals (A1, Zn, Pb, etc.) in solutions could cause dysfunction in one or both partners in the symbiotic association (McCreight and

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225 Schroeder 1982, Jones et al. 1986, Wasserman et al. 1987~. Recently, Dixon and Buschena (1988) showed that growth of nonmycorrhizal pine seedlings was reduced and that ectomycorrhize protected the pine seedlings from toxicity caused by Cd, Cu. Ni, Pb, and Zn. In Poland, Kowalski ( 1987) found distinct qualitative and quantitative changes in ectomycorrhizae of various tree species planted on a prepared and fertilized site damaged by industrial emission (SO2, Zn, and Pb). The percentage of living ectomycorrhizae decreased with increasing pollution levels, but ectomycorrhizae were prevalent on even the most pollution-damaged living trees. He concluded that reduced photosynthetic activity, strongly altered soil chemistry, and high concentrations of heavy metals were responsible for ectomycorrhizal depression. In the Netherlands, Termorshuizen and Schaffers (1987) found three times more ectomycorrhizae, 20 times more fruit bodies, and much greater fungus diversity in 5 to 1 0-year-old Pinus sylvestris than in 50- to 80-year-old trees. This observation appears to conflict with the succession and diversity concept discussed earlier. However, in the older stand, these fungus parameters were negatively correlated with concentrations of SO2, O3, and NH3. They were positively correlated with the mean number of needleyears of these older trees. There were no differences in soil chemistry between the two stands of trees. They concluded that the lack of fungal species diversity on the old trees could cause a decreased resistance to both man-made and natural stress. In Scotland, Dighton et al., ( 1 986b) applied 1500 mm/yr of pH 3.0 rain for 5 years to P. sylvestris trees and found a reduced abundance of ectomycorrhizae and short roots. No abnormality in morphology was found in any ectomycorrhizae. They concluded that increased levels of Al and Mn in the acid treated soils reduced root growth, which caused a reduction in ectomycorrhizae. Numerous other researchers in other parts of Europe have reported similar depressions in ectomycorrhizae on trees with various degrees of foliar symptoms of air pollution damage (Blaschke 1981, 1982, Schlechte 1986~. BETTER METHODS ARE NEEDED TO STUDY SYMBIOSES Basically, we know only that unhealthy trees seem to have depressed root symbiotic development compared to healthy trees. Biologically, this suppression is predictable. The major problem is separating cause from effect -- the old chicken-and-egg dilemma. At this time, we do not know what normal or abnormal populations of mycorrhizae or actinorhizae are for any tree species, of any age, on any site, anywhere in the world. How many species of symbionts are normal? What proportion of the short roots should be symbiotically colonized? Is microbial succession from young to older trees a worldwide phenomenon? Does it occur in actinorhizal and VAM tree systems? Is succession caused directly by changing root physiology, soil characteristics, or rhizosphere populations? If so, could atmospheric deposition alter succession? Or is succession happening simply because it has had time to happen? Can normal succession be interrupted by any form of plant stress? For ectomycorrhizae, is the presence of late-stage fungi the result of physiological change in aging trees? Or is it simply due to the fact that late-stage fungi do not proliferate or reproduce as rapidly, do not produce spores in a manner that ensures rapid dissemination, do not produce as many spores as do early-stage fungi and, therefore, take longer to appear in the system? Are the late-stage fungi more or less susceptible to dysfunctions in physiology of roots or foliage? Do exposure experiments on tree seedlings with early-stage ectomycorrhizae contribute meaningfully to our understanding of the reaction of late-stage/mature trees to the same exposure. Since we cannot recognize "normal", how are we to recognize "abnormal" populations of symbiotic associations? The answer is very simple -- we can't! Answers to these simple questions exist only for a few very isolated situations that may not apply elsewhere. The biggest problem is that reliable and standardized methods to study symbiotic relationships in the field do not exist (Schenck 1982~. Only in recent

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226 years have we been able to develop reasonably standardized methods for mycorrhizal research on small tree seedlings with specific ectomycorrhizal fungi in a rather controlled environment, the tree nursery. Cline et al. (in press) discussed briefly the inherent problems in mycorrhizal assessments and concluded that these assessments must be coupled to the root growth rate, or to existing root biomass. Percent short root colonization is a widely used and reasonably reliable assessment parameter for seedlings of comparabl sizes, but it has serious limitations elsewhere. It is important to know the total number of short roots that are available for potential colonization. A percent of short roots colonized tells us little because one seedling may have 4000 short roots, and another 400. A visual estimation of 50% infection tells little unless the 1 0-fold difference in total number of short roots is also known. Number of mycorrhizae/cm of lateral root has little value unless total number and length of lateral roots are also known. The diverse morphology of ectomycorrhizae also creates assessment problems. One mature ectomycorrhiza of Pisolithus tinctorius may have more surface area and more fungus tissue (more carbon demand) than 20 ectomycorrhizae formed by Cenococcum geophilum on the same seedling. How do we accurately measure the amount of fungal biomass? Gravimetric (France et al. 1985) and catalytic potential (Iyer 1978) methods have been developed but neither method measures lateral root length or short roots. Growth rate of lateral roots may have profound effects on mycorrhizal assessments. For example, mycorrhizal status may appear to decrease in a situation that stimulates lateral root growth. In the presence of a lateral root growth stimulator, mycorrhizal infection may lag behind lateral root extension and short root proliferation, thereby, suggesting a depression in mycorrhizae. Examples of conditions or agents that stimulate lateral root growth include fertilization (especially with phosphorus), warming of soils in spring, and water becoming available after prolonged deficit. Conversely, mycorrhizal status may appear to increase in the presence of a lateral root growth inhibitor. If root growth slows or stops, the growth rate of the fungi and roots may be more closely matched or the mycorrhizae may develop faster than new short roots can be produced. The result is a net increase in numbers of mycorrhizae over time and a higher percentage of short roots colonized without a significant increase in total numbers of short roots. A few inhibitors of lateral root growth are rapid defoliation (storm damage), moderate soil water deficits, soils getting colder in fall, soil compaction, and depleted available soil phosphorus. Thus, the time of root sampling for mycorrhizal assessments can influence greatly the perception of mycorrhizal status. Very few methods have been developed to study survival of inoculum of microbial symbionts in soil. Depression of symbiotic associations could be caused by depressed survival of their inocula in soil. None of these symbionts can be isolated directly from soil but, in the case of ectomycorrhizal and actinorhizal symbionts, they can be reisolated from roots with considerable effort. Reisolation results, which vary from 1 to 10% success, however, do not relate directly to survival potential of the specific microorganisms. Techniques have been developed to study the effects of various soil treatments on survival of vegetative inoculum of Pisolithus tinctorius in nursery soil (Marx et al. 1986, Marx and Cordell 1987~. This procedure uses nonmycorrhizal roots of pine seedlings as a fast "trap" of viable inoculum and is reasonably quantitative. This technique could be modified to test survival of other propagules of these microorganisms, such as spores and sclerotia. A problem remains, however, because of the apparent physiological differences between early-stage (like P. tinctorius) and late-stage fungi. With this technique, can seedling roots be used as a "trap" for inocula of late-stage fungi? Can nonmycorrhizal seedlings be transplanted into variously treated soil collected

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227 from mature stands and form mycorrhizae with late-stage fungi? Another ecological aspect of symbiotic associations, especially ectomycorrhizae, which has been completely ignored is what can happen to the airborne spores of these fungi while in flight. During fruit body production by these fungi, thousands of spores/m3 of air/day (LeTacon et al., 1987) are airborne for various periods of time. What effects do gaseous pollutants, such as Of and S02, have on their viability during this spore-flight period? What effect does acidic rain have during spore washout from the air? There is no easy way to assess accurately root symbiotic associations on tree seedlings, on small trees, or in natural soil. Reliable and reproducible methods to qualitatively and quantitatively assess root symbioses must be developed if we are ever to understand their reaction to man-made and natural stresses in either experimental or real-world situations. When we realize that the "abnormal" environment of today may be the "normal" environment of tomorrow, we must not delay in developing these methods. CONCLUSION Common biological sense indicates that root symbiotic associations are responsive to effects of atmospheric deposition on forests. Unfortunately, at this time, base-line data does not exist as to the symbiotic status of healthy forests. Techniques to assess quantitatively and qualitatively symbiotic associations on forests containing trees of different age and species, do not currently exist and must be developed before this information can be produced. Because of these limitations, it is doubtful that symbiotic associations can be used today as potential biological markers of the effects of atmospheric deposition on forest health. SUMMARY 1. Root symbioses (mycorrhizae or actinorhizae) are as common on roots of plants in forest ecosystems as are chloroplasts in leaves of plants in the ecosystems. 2. A forest ecosystem, a stand of mixed tree species, a plantation of trees, a single tree, or a small segment of lateral root on a tree can have many types of symbioses with multiple microbial species. 3. Susceptible short roots on lateral roots must be formed by the tree host before root colonization by microbial symbionts can occur. 4. Viable inoculum of the symbiotic microorganisms must be present near susceptible roots before symbiotic colonization can occur. 5. Edaphic factors can strongly influence root colonization by affecting root growth and, probably, inoculum survival and viability of the microbial symbionts. 6. Vitality of root symbiotic associations is strongly dependent on tree host physiology, mainly photosynthesis. Reduced photosynthesis equals reduced root symbioses. 7. Ectomycorrhizal fungal succession apparently occurs during the development of forest stands. Increasing age of stands and increasing number of tree species in the stand increase ectomycorrhizal fungus species diversity.

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228 S. Current methods to measure root symbionts or their function have severe limitations, especially for ecological studies, and better methods must be developed. REFERENCES Akkermans, A.D.L., D. Baker, K. Huss-Danell, J.D. Tjepkema. (eds.~. 1984. Frankia Symbioses. Developments in Plant and Soil Sciences, Vol. 12. Martinus Nijhoff/Dr W. Junk Publ., The Hague. 258 pp. Akkermans, A.D.L., W. Roelofsen. 1980. Symbiotic nitrogen fixation by actinomycetes in AInus-type root nodules. Chap. 12 in Stewart, W.D.P.; Gallon, J.R. Nitrogen Fixation. Academic Press, London. 451 pp. Akkermans, A.D.L., C. van Dijk. 1981. Non-leguminous root-nodule symbioses with actinomycetes and Rhizobium. Pp. 57- 103 in Broughton, W.J. (ed.~. Nitrogen Fixation. Vol. 1: Ecology. Clarendon Press, Oxford. 306 pp. Baylis, G.T.S. 1970. Root hairs and phycomycetous mycorrhizas in phosphate- deficient soil. Plant Soil 33:713-716. Baylis, G.T.S. 1972. Minimum levels of phosphorus for nonmycorrhizal plants.Plant Soil 36:233-234. Blaschke, H. 1981. Schadbild und Atiologie des Tannensterbens. II. Mycorrhizastatus und pathogene Vorgange im Feinwurzelbereich als Symptome des Tannessterbens. Eur. J. For. Pathol. 11 :375-379. Blaschke, H. 1982. Schadbild und Atiologie des Tannensterbens. III. Das Vorkommen einer Phytophthora-Faule an Feinwurzeln der Weisstanne (Abies alba Mill.~. Eur. J. For. Pathol. 12:232-238. Carney, J.L., H.E. Garrett, H.G. Hedrick. 1978. Influence of air pollutantgases on oxygen uptake of pine roots with selected ectomycorrhizae. Phytopathology 68:1160-1163. Cline, M.L., R.J. Stephans, and D.H. Marx. (In press). Influence of atmospherically deposited nitrogen on mycorrhizae: A critical literature review. Ann. Rev. Physiology. Dighton, J., P.A. Mason. 1985. Mycorrhizal dynamics during forest treedevelopment. Pp. 117- 139 in Moore, D.; Casselton, L.A.; Wood, D.A.; Frankland, J.C. (eds.~. Developmental Biology of Higher Fungi. Cambridge Univ. Press, Cambridge. Dighton, J., J.M. Poskitt, D.M. Howard. 1986a. Changes in occurrence of basidiomycete fruit bodies during forest stand development: With specific reference to mycorrhizal species. Trans. Br. Mycol. Soc. 87:163-171. Dighton, J., R.A. Skeffington, K.A. Brown. 1986b. The effects of sulphuric acid (pH 3) on roots and mycorrhizas of Pin?~s sylvestris. Pp. 739-743 in Gianinazzi-Pearson, V., S. Gianinazzi. (eds.~. Physiological and Genetical Aspects of Mycorrhizae, C~RS-INRA, Dijon, France. Dixon, R.K., C.A. Buschena. 1988. Response of ectomycorrhizal Pinusbanksiana and Picea glauca to heavy metals in soil. Plant and Soil 105:265-271.

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