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- Materials and Methods
In boreal forest ecosystems most of the nutrient uptake by trees is mediated by ectomycorrhizal roots and mycelia (Read, 1991). The fine root density of forest trees is highest in the upper soil horizons (Jackson et al., 1996) and almost all studies of ectomycorrhizal fungal communities therefore restrict soil sampling to the uppermost organic part of the soil profile (Horton & Bruns, 2001). However soils below coniferous forests in the northern hemisphere are commonly podzolised with distinct mineral horizons formed under a thin organic layer (Lundström et al., 2000). The possibility that mycorrhizal fungi may play a direct role in mineral weathering (Landeweert et al., 2001) has resulted in increased interest in mycorrhizal colonisation of mineral substrates but there are still very few studies of interactions of mycorrhizal hyphae with mineral substrates.
In a recent study of the vertical distribution of ectomycorrhizal species in a podzol profile we demonstrated differences in species composition between different horizons in the soil profile (Rosling et al., 2003). A parallel study analysed ectomycorrhizal mycelial distribution in the same profile. The ectomycorrhizal species colonising roots in a horizon were commonly found to produce mycelium in the same horizon (Landeweert et al., 2003). Extensive proliferation of ectomycorrhizal roots and mycelia in response to patches of organic substrate has been observed in microcosm experiments (Unestam, 1991; Bending & Read, 1995; Lindahl et al., 1999; Perez-Moreno & Read, 2000). Newly formed patches are sinks of host-derived carbon (Bending & Read, 1995), and intense mycelial colonisation leads to increased uptake of nutrients from the patches (Bending & Read, 1995; Lindahl et al., 1999; Perez-Moreno & Read, 2000). Some ectomycorrhizal fungi are known to form dense mycelial mats in forest soils, strongly affecting the nutrient availability and weathering rate of the colonised soil (Cromack et al., 1979; Entry et al., 1991; Arocena & Glowa, 2000), however, there are few studies of mycelial biomass allocation in relation to mineral substrate heterogeneity.
The aim of this study was to compare the proliferation of ectomycorrhizal roots and mycelium into different substrates occurring in the mineral horizons of boreal forest podzols. E horizon mineral soil collected from a forest was used in a first experiment, and pure sources of quartz and feldspar were used in a subsequent experiment. The fungi used in the study, Hebeloma crustuliniforme (Bull.) Quél. and Piloderma fallax (Liberta) Stalpers, both form mycelial mats on rock surfaces covered by mosses and occur naturally in the interface between the organic and mineral soil. Mycorrhizal colonisation was visualised and quantified by labelling the seedlings with radioactive carbon dioxide and examining the distribution of labelled assimilates using electronic autoradiography and destructive harvesting followed by liquid scintillation counting.
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- Materials and Methods
Ectomycorrhizal mycelial foraging behaviour and exploitation of litter patches has been studied using microcosm systems (Bending & Read, 1995). Newly formed patches of mycelium colonising the organic substrates were shown to be sinks for host carbon. The direct mobilisation of these substrates by the mycorrhizal fungi themselves could not be conclusively proved but it seems likely that heavy investment of carbon would be necessary for the synthesis of degrading enzymes involved in such a process (Finlay & Read, 1986b; Read, 1991). Selective allocation of carbon to mycelia colonising different mineral substrates has been less well investigated but recent studies (Landeweert et al., 2003; Rosling et al., 2003) have underlined the possible importance of ectomycorrhizal fungi in mineral soil horizons. Half of the mycorrhizal taxa found in a forest podzol were found exclusively in mineral soil horizons and over 60% of the total number of root tips were found in the mineral soil (Rosling et al., 2003).
The highly nonuniform distribution of labelled assimilates in experiment one, with larger amounts of 14C retrieved from the E horizon mineral soil, indicates greater mycelial growth in this substrate. Both H. crustuliniforme and P. fallax form mycelial mats on rock surfaces covered by mosses and P. fallax typically occurs in both the organic and upper mineral horizons of forest soils (Rosling et al., 2003). Mycorrhizal fungi that proliferate in mineral soil may provide an important input of carbon to this habitat, from which the fungi and associated soil organisms could derive energy to influence biogeochemical processes such as weathering (Banfield et al., 1999).
Peat is commonly used as a growth substrate for mycorrhizal seedlings in flat bed microcosms and was also selected as the organic substrate in comparison with the E horizon mineral soil in the vertically divided microcosms. As both H. crustuliniforme and P. fallax grow well in peat when no optional substrate is provided it can be concluded that E horizon mineral soil is the preferred growth substrate for H. crustuliniforme and P. fallax when both substrates are available. As mycelial development on peat alone was vigorous for both species, the poor performance on peat in the vertically divided systems is likely to depend on resource reallocation towards roots and mycelia in the mineral substrate. This growth pattern emphasizes the spatial integration of the mycelium into one interconnected unit (Read, 1992), where the performance of one part of the mycelium is affected by the local conditions in other parts, as previously demonstrated for wood decaying fungi (Dowson et al., 1989).
In experiment one, 70–80% of the labelled carbon in the below ground compartment was allocated to the E horizon mineral soil. This greater growth in the E horizon substrate may be a response to the availability of elements such as potassium, magnesium and phosphorus within the mineral soil. There are, however, several other possible reasons for this nonuniform distribution of activity. The pH of the two substrates is slightly different with a pH of 4.2 in the peat and of 3.7 in the E horizon mineral soil. The two substrates have different bulk densities and thus different water holding capacities, however, both substrates were watered regularly in our experiment. The podzol E horizon mineral soil contains visible organic material and is not a pure mineral substrate. Dead hyphal fragments or other sources of organic nutrients may have influenced the mycelial growth. These differences complicate interpretation of the observed differences in mycelial growth. For these reasons the subsequent experiment (experiment two) was based on a comparison of two identical substrates, differing only with respect to which of two defined minerals was added. In this experiment, there was significantly higher allocation of labelled carbon to roots and mycelium associated with potassium feldspar patches than to quartz patches. The cause of this difference is not clear from the results of the present study, but the pH, particle size and moisture of the two substrates were similar. Potassium feldspar (Huang, 1989) is easier to weather than quartz (Drees et al., 1989) and is a possible source of mineral nutrient elements, mainly potassium, but also magnesium and sodium. Selective mycelial exploitation of organic patches (Bending & Read, 1995) was accompanied by mobilisation and removal of elements from the patches (Lindahl et al., 1999; Perez-Moreno & Read, 2000). It was not possible to demonstrate element depletion in the present study since pure mineral sources were used, however, other studies suggest that mycorrhizal interactions with different minerals do occur. Species-specific substrate acidification responses by fungi to different minerals have been demonstrated in vitro (Rosling et al., 2004) and mycorrhizal pine seedlings colonised by Suillus variegatus and Rhizopogon roseolus have been shown to increase their production of oxalic acid in response to elevated aluminium concentrations (Ahonen-Jonnarth et al., 2000). Wallander et al. (2002) demonstrated mobilisation and accumulation of phosphorus and potassium by ectomycorrhizal rhizomorphs colonising apatite-amended sand in mesh bags buried in forest humus, suggesting that ectomycorrhizal fungi may be important in accumulating and possibly transporting these elements to forest trees. Laboratory experiments with tree seedlings also suggest that ectomycorrhizal fungi may play a role in mobilising phosphorus from apatite and increasing phosphorus content and biomass production of their host plants (Wallander, 2000).
The amount of labelled carbon in the extraradical mycelium in experiment one was 50–60% of the total amount found in the system. This figure is much higher than that estimated in earlier studies (Leake et al., 2001) and in unpublished studies of our own using pulse labelling. In those experiments typically 2–12% of the recovered activity was present in the extramatrical mycelium. In experiment one the label was supplied to the mycorrhizal systems continuously over a 1-wk period and it is possible that respiratory losses of 14C from the plant (Högberg et al., 2002) could have been higher than those from the mycelium during this period. This would result in proportionally more of the label being retained in the fungal mycelium after a long incubation period and would explain the discrepancy between this experiment and other studies measuring the allocation of 14C a short time after pulse feeding.
Molecular methods now enable us to study the spatial and temporal distribution of different ectomycorrhizal fungi with increasing taxonomic resolution but our knowledge of the functional interactions of different species with different substrates is still extremely limited. There is increasing evidence that many ectomycorrhizal fungi may play an important role in mobilising organic polymers of nitrogen and phosphorus, increasing their availability to their host plants (Read & Perez-Moreno, 2003). Mineral substrates have been less well studied but recent evidence suggests that some taxa associate exclusively with mineral soil horizons (Dickie et al., 2002; Landeweert et al., 2003; Rosling et al., 2003). There is also an accumulating body of evidence to suggest that interactions between mycorrhizal fungi and mineral particles may influence mineral nutrient availability and plant growth (Wallander, 2000). The present study demonstrates differential allocation of carbon to mycelium colonising different mineral substrates and highlights the need for more detailed studies of the interactions of mycorrhizal mycelia with minerals.