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Keywords:

  • ectomycorrhiza;
  • E horizon mineral soil;
  • electronic autoradiography;
  • Hebeloma crustuliniforme;
  • potassium feldspar;
  • Piloderma fallax;
  • quartz

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • • 
    Ectomycorrhizal fungi occur abundantly in the mineral horizons of forest soils, but their interactions with mineral substrates are largely unknown. We have examined the proliferation of ectomycorrhizal roots and mycelium colonising different mineral substrates.
  • • 
    By exposing the shoots of Pinus sylvestris seedlings to air containing 14CO2, the carbon allocation patterns in intact ectomycorrhizal associations could be monitored using electronic autoradiography.
  • • 
    In plants colonised by either Hebeloma crustuliniforme or Piloderma fallax, a larger fraction of the photosynthetically derived carbon was allocated to a mineral soil substrate compared with a Sphagnum peat. In mycorrhizal seedlings colonised by H. crustuliniforme, carbon allocation was significantly greater to roots and mycelia colonising patches of pure potassium feldspar than to those in patches of quartz.
  • • 
    These results suggest that ectomycorrhizal mycelia may respond to the presence of different mineral substrates by regulating their growth and activity.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

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.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Experimental microcosm systems

Seeds of Pinus sylvestris L. were surface sterilised for 30 min in 33% H2O2, rinsed in sterilised water and germinated on 1.6% water agar for 6–8 wk (14–16°C during the 16 h photoperiod at a photon flux density of 300 mol m−2 s−1 and 6–8°C during the 8 h dark period). Fungal stock cultures of Hebeloma crustuliniforme (Bull.) Quél. (isolate code – UP184) or Piloderma fallax (Liberta) Stalpers (UP121) were maintained in darkness at 25°C on half strength modified Melin-Norkrans (MMN) medium (Marx, 1969). Mycorrhizal seedlings were produced by transferring seedlings and agar inoculum plugs from the fungal stock cultures to Petri dishes with a growth substrate of peat: vermiculite: 1 : 10 MMN mixture (1 : 4 : 2, v : v : v) (Duddridge, 1986; Finlay, 1989). The root systems were well colonised after c. 8 wk, when they were transferred to microcosm systems consisting of a 0.2 × 20 × 20 cm Perspex support covered by sieved (2 mm mesh) Sphagnum peat. The plate was securely attached in a square plastic box (2.5 × 24 × 24 cm). The peat was compressed to a thickness of 2 mm. A hole in the side of each box allowed the shoots to protrude. Each system was covered by a lid and wrapped in aluminium foil to minimise drying out and illumination of the mycorrhizal root system. The systems were incubated vertically in the climate chamber. Systems were watered weekly with deionised water using a Pasteur pipette. The relative humidity of the air in the root compartment chambers was thus kept close to 100%.

Experiment one: Vertically divided microcosms with mineral soil and peat

To compare the extent of mycorrhizal colonisation of E horizon mineral soil and an organic substrate, vertically divided microcosms were set up. In these, half the surface was covered with a 2 mm layer of Sphagnum peat and sieved (2 mm mesh) and the other half was covered with E horizon mineral soil. The E horizon mineral soil was collected from a podzol profile in northern Sweden (Ilvesniemi et al., 2000; Rosling et al., 2003). The pH of the peat was c. 4.2 and the pH of the E horizon mineral soil was c. 3.7. One mycorrhizal pine seedling colonised by either H. crustuliniforme or P. fallax was transferred to each vertically divided microcosm with the root systems equally distributed on both substrates. Five systems were set up for each fungus and incubated as described above. In microcosms, Sphagnum peat is commonly used as a growth substrate for ectomycorrhizal seedlings. The two fungi used in this study are both known to grow well on peat. To illustrate this, reference microcosms with peat as the sole growth substrate were set up for each fungus. Reference systems were not part of the experimental design and serve solely as visual examples of proliferation of the roots and mycorrhizal hyphae of seedlings colonised by the two different ectomycorrhizal fungi when grown in a heterogeneous peat substrate. The growth of the seedlings was followed over 14 wk for H. crustuliniforme and over 19 wk for the more slowly growing P. fallax. Allocation of photosynthetically derived carbon into the different substrates was estimated as described below.

Experiment two: Peat microcosms with pure mineral patches

In order to examine the possible differential colonisation of different pure mineral substrates by ectomycorrhizal roots and mycelium, patches of pure minerals were introduced at the margin of the growing mycelia in peat microcosm systems. Quartz and potassium feldspar were major constituents of the E horizon material used in experiment one, constituting 50% (quartz) and 20% (potassium feldspar) of the total mineral fraction (Melkerud et al., 2000). Pure mineral pieces of potassium feldspar or quartz were obtained from the Swedish Geological Survey and ground in a ball mill type MM2 (Retsch, Haan, Germany) and sieved to collect fractions with a particle diameter between 0.02 and 2 mm. After soaking 1 g of mineral grains in 1 ml of distilled water for 72 h the pH of the liquid was measured to determine the pH of the minerals. The pH was within the range 4.3–4.5 for both minerals. Minerals were washed in 70% ethanol and sterilised at 120°C for 2 h. Plastic dishes were constructed from square weighing boats (2 × 2 cm) by cutting the edges down to a height of 2 mm. The dishes were lined with 1 g of dry peat and soaked with 1 g of distilled water. In each dish, 1 g of either ground quartz or potassium feldspar was sprinkled on top of the peat. Four dishes (two of each mineral) were introduced at the margin of the growing mycelia in five 7-month-old peat microcosms containing pine seedlings colonised by H. crustuliniforme. Squares of peat were removed and replaced with the mineral dishes so that the mineral sprinkled surface of the dishes was level with the surrounding peat. Over the 15 wk duration of the experiment, the root and mycelial growth was documented weekly by photography. Allocation of photosynthetically derived carbon into the mineral dishes was estimated as described below.

14CO2-labelling, electronic autoradiography and destructive 14C analysis

Plants in experiments one and two were labelled by supplying 14CO2 to the shoots (Finlay & Read, 1986a). The vertically divided microcosms (experiment one) were continuously labelled over 1 wk by incubating all the boxes in a 60 × 40 × 30 cm plastic box with a 14CO2 enriched atmosphere. CO2 was removed from the box by a constant replacement of air in the box with CO2-free air pumped through soda lime. The CO2 level within the box was then replenished by circulating air from within the box through a flask containing phosphoric acid to which a 14C-enriched Na2CO3 solution was added using a peristaltic pump. The pump was regulated by an infrared gas analysis (IRGA) CO2 system (ADC BioScientific Ltd, Herts, UK) to maintain a carbon dioxide concentration of between 340 and 370 ppm. The specific activity of the added Na2CO3 was 370 MBq mol−1. The box maintained a temperature of 19°C and the photon flux density at the level of the plant shoot was 200 mol m−2 s−1. Pure mineral patch systems (experiment two) were pulse labelled by supplying 0.74 MBq 14CO2 to the shoot of each seedling following the method of Finlay & Read, 1986a.

The distribution of radioactivity in the systems was detected nondestructively using a Packard Instant Imager electronic autoradiography system (Packard Instrument Co. Meriden, CT, USA) as described by Lindahl et al. (1999). To acquire an autoradiographic image, radioactive disintegration events were recorded for 15 min with a spatial resolution of 0.5 mm. Autoradiographic scanning was performed immediately after 1 wk of continuous labelling in experiment one and 24, 48 and 72 h after the pulse labelling of experiment two.

Following autoradiographic scanning, further re-distribution of 14C was prevented by rapid air-drying at 60°C until further processing for destructive measurements. Roots were extracted from each substrate. After mixing, two to four subsamples were taken from the root-free colonised substrates. Root material was divided into subsamples and from each shoot two subsamples of needles and one from the stem were taken. The total weight of the content of the mineral dishes in experiment two was recorded before subsamples were taken for destructive analysis of 14C content. All samples were combusted in a Packard sample oxidizer, model 307 (Packard Instrument Co., Meriden, CT, USA) and the radioactivity was measured in a TriCarb 2100 TR scintillation counter (Packard Instrument Co., Meriden, CT, USA).

Statistical analysis

Data were analysed using a mixed model (Littell et al., 1996) to account for the dependence caused by the fact that the same plant was used as an experimental unit for the contrasting substrates used in the two different experiments. On the basis of preliminary analyses, data were ln-transformed before analysis to reduce heteroscedasticity. For experiment one, substrate (E or P), species and the species*substrate interaction were used as the fixed part of the model, while the plant (each replicate) was regarded as a random factor. For experiment two, the plant (each replicate) and the plant*mineral interaction were regarded as random factors.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Experiment one: Carbon allocation in vertically divided microcosms

In microcosms containing peat only, both roots and mycelia of H. crustuliniforme and P. fallax colonised seedlings grew vigorously (Fig. 1a,b). In vertically divided microcosms, roots and mycelia of mycorrhizal pine seedlings grew vigorously in E horizon mineral soil whereas the growth on peat was feeble. This was the case for both fungal species (Fig. 1c,d). Labelling the seedlings with 14CO2 for 1 wk enabled us to detect the incorporation of photosynthetically derived carbon into roots and mycelium during this period. Electronic autoradiographic analysis of the patterns of 14C distribution in the two substrates showed that, for both mycorrhizal species, more 14C had accumulated in the E horizon mineral soil than in the peat substrate (Fig. 1e,f).

image

Figure 1. Ectomycorrhizal seedlings of Pinus sylvestris growing in flatbed microcosms with peat (P), colonised by either Hebeloma crustuliniforme (a) or by Piloderma fallax (b). In vertically divided microcosms with E horizon mineral soil (E) on one side and P on the other, both fungi (c, d) demonstrated more vigorous growth in E horizon mineral soil compared with peat. When the systems were 14-wk-old (H. crustuliniforme (c)) or 19-wk-old (P. fallax (d)), shoots were exposed to CO2 (340–370 ppm) containing 370 MBq mol−1 14CO2. After 1 wk, distribution of radioactivity as CPM (counts per min) in the systems was measured nondestructively using electronic autoradiography. Electronic autoradiographic scan images overlaying schematic views of the vertically divided microcosms for H. crustuliniforme (e) and P. fallax (f) demonstrate heavy allocation of current assimilates to root tips and the growing mycelial front. The colour bar indicates the scale of detected activity levels in CPM.

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Destructive sampling of the microcosms followed by sample oxidation and liquid scintillation spectroscopy confirmed the visual patterns of 14C distribution. In seedlings colonised with either H. crustuliniforme or P. fallax, about 80% of the accumulated 14C was retrieved from the below ground compartment. In systems with H. crustuliniforme, c. 60% of the total amount of 14C in each system was incorporated into the extraradical mycelium (i.e. in the root-free, colonised substrate). There was significantly more activity detected in the E horizon material compared with the peat substrate. This was true for activity in mycelia (P < 0.0001) and roots (P < 0.0001) separately as well as for the total activity (P < 0.0001) in the different substrates. There were no significant differences between the two species. In systems with P. fallax the corresponding figure was 50%. Seventy to 80 % of the 14C in the below ground compartment was detected in the E horizon mineral soil (Fig. 2a,b). For both fungi, the total amount of 14C allocated to extraradical mycelium in E horizon mineral soil (E) was significantly higher than that allocated to peat (P). Carbon allocation to roots followed the same pattern but the difference was only statistically significant for P. fallax (Fig. 2b).

image

Figure 2. Activity measured as disintegrations per min (DPM) from the destructive harvest of vertically divided microcosms. Mean activity is presented as 100 000 DPM (± SE) in the two contrasting substrates, E horizon mineral soil (E) and peat (P), showing distribution between mycelia and roots as well as the total activity, for (a) Hebeloma crustuliniforme and (b) Piloderma fallax, n = 5.

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Experiment two: Carbon allocation in peat microcosms with pure mineral patches

In peat microcosms with pine seedlings colonised by H. crustuliniforme, root and mycelial proliferation was more intense in patches of pure potassium feldspar compared with patches of pure quartz (Fig. 3a). Pulse labelling with 14CO2 and autoradiographic scanning of the systems after 72 h enabled the allocation of newly assimilated carbon to be visualised (Fig. 3b). Most of the 14C that was transported to the root system was allocated to mycorrhizal roots and the extending mycelial fronts (Fig. 3b). Significantly more 14C (P = 0.021) was allocated to dishes with potassium feldspar compared with dishes with quartz (Fig. 4).

image

Figure 3. Flatbed peat microcosms containing Pinus sylvestris seedlings colonised by Hebeloma crustuliniforme and pure mineral patches of either potassium feldspar (K) or quartz (Q). Fifteen weeks after introducing mineral patches at the growing mycelial front (a), the shoots were pulse labelled with 0.74 Mbq 14CO2. The distribution of radioactivity in the systems was detected by nondestructive electronic autoradiography. Electronic autoradiographic scan images overlaying schematic views of the mineral patches in the microcosms (b) demonstrate that greater amounts of labelled carbon are allocated to root tips and mycelia associated with patches of potassium feldspar compared to patches of quartz. The colour bar indicates the scale of detected activity levels in counts per min (CPM).

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image

Figure 4. Mean radioactivity, measured in thousands of disintegrations per min (DPM ± SE) per gram, in patches of the two contrasting mineral substrates, quartz and potassium feldspar (K–feldspar) colonised by roots and mycelia of Hebeloma crustuliniforme colonising 14CO2-labelled ectomycorrhizal Pinus sylvestris seedlings (n = 5).

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

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.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We gratefully acknowledge financial support from The Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (FORMAS). We are also indebted to Sven Snäll at SGU (Swedish Geological Survey, Uppsala, Sweden) for providing minerals and grinding equipment and for sharing his great experience in the field of mineral weathering. We thank Ulf Olsson from the Department of Biometry & Informatics, SLU for help with the statistical analyses. We also thank Petra Fransson and Hans Rosling for critical reading of the manuscript and providing constructive ideas for its improvement.

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  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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