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Oxalate and ferricrocin exudation by the extramatrical mycelium of an ectomycorrhizal fungus in symbiosis with Pinus sylvestris Blackwell Publishing Ltd

Patrick A. W. van Hees1, Anna Rosling2, Sofia Essén3, Douglas L. Godbold4, David L. Jones4 and Roger D. Finlay2 1

Man-Technology-Environment Research Centre, Department of Natural Sciences, Örebro University, SE-701 82 Örebro, Sweden; 2Department of Forest

Mycology and Pathology, Swedish University of Agricultural Sciences (SLU), Box 7026, SE-750 07 Uppsala, Sweden; 3Department of Natural Sciences, Mid Sweden University, SE-851 70 Sundsvall, Sweden; 4School of Agricultural and Forest Sciences, University of Wales, Bangor, Gwynedd LL57 2UW, UK

Summary Author for correspondence: Patrick A. W. van Hees Tel: +46 (0)19 301339 Fax: +46 (0)19 303169 Email: [email protected] Received: 30 August 2005 Accepted: 22 September 2005

• Accurate estimates of mycelial exudation in time and space are crucial for the assessment of ectomycorrhizal involvement in biogeochemical processes. Knowledge of exudation from mycelia of ectomycorrhizal fungi is still limited, especially for fungi in symbiosis with a host. • Pinus sylvestris seedlings colonized by Hebeloma crustuliniforme were grown in aseptic multicompartment dishes. This novel system enabled identification of exudates originating only from extramatrical mycelium. At harvest, hyphal density and numbers were estimated using microscopic imaging. A fractal geometric approach was adopted for calculation of exudation rates. • The main compounds identified were oxalate and ferricrocin. The exudation rate for oxalate was 19 ± 3 fmol per hyphal tip h−1 (mean ± standard error of the mean) or 488 ± 95 fmol hyphal mm−2 h−1. Ferricrocin rates were approx. 10 000 times lower. The fractal dimension (D) of the mycelia was 1.4 ± 0.1, suggesting an explorative growth. Potassium nutrition was a significant regulatory factor for ferricrocin but not oxalate. • The results suggest that hyphal exudation may alter the chemical conditions of soil microsites and affect mineral dissolution. Calculations also indicated that oxalate exudation may be a significant carbon sink. Key words: ectomycorrhiza, exudation, Hebeloma crustuliniforme, mycelium, oxalate, Pinus sylvestris, siderophores. New Phytologist (2006) 169: 367–378 © The Authors (2005). Journal compilation © New Phytologist (2005) doi: 10.1111/j.1469-8137.2005.01600.x

Introduction Mycorrhizas are mutalistic, symbiotic associations between soil fungi and plant roots. Ectomycorrhizas (EMs) are the most common form of this symbiosis for the majority of boreal forest trees (Smith & Read, 1997). In coniferous boreal forests, >95% of the fine roots are commonly colonized by EM fungi with a high diversity of fungal species (Taylor, 2002). Mycorrhizal colonization has important implications for the forest ecosystem. The crucial impact of EM fungi on tree nutrient uptake, including uptake of major nutrients such as nitrogen (N), phosphorus (P), potassium (K) and magnesium (Mg) as well

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as micronutrients, is well established. The change in tree carbon (C) allocation caused by EM fungi is considerable. It has been estimated that 10–20% of the C transferred below ground by trees is used for growth and maintenance of the mycorrhizal symbiont (Smith & Read, 1997). Active or passive exudation of various organic compounds is another means by which mycorrhizal mycelia may directly and/or indirectly affect both biotic and abiotic processes. This may involve specific compounds such as hydrolytic enzymes, which cleave complex substrates, or cytokinins and indolic compounds that aid EM development ( Jones et al., 2004). However, in quantitative terms it is likely that more simple

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substances such as sugars, low-molecular-weight organic acids (LMWOAs) and peptides dominate the EM exudative flux (Sun et al., 1999). Oxalate is often highlighted as a key exudate as it has been hypothesized to be important in a number of processes involving symbiotic, saprophytic and pathogenic fungi (Gadd, 1999). Our knowledge of root and EM fungal exudates is still limited and several mechanistic as well as methodological questions remain unanswered (Grayston et al., 1996; Jones et al., 2004; van Hees et al., 2005a). However, the experimental data available mostly suggest that EM colonization increases the exudation of LMWOAs, in particular oxalate, into the soil (Ahonen-Jonnarth et al., 2000; Casarin et al., 2003). Siderophores are a group of compounds that have attracted considerable research interest as they are thought to be highly important for the iron (Fe) nutrition of plants, bacteria and fungi. Fungi, including mycorrhizal species, mostly produce the hydroxymate type of siderophores (Haselwandter, 1995). Identification of individual siderophores from EM fungi cultures has shown the presence of ferrichrome and ferricrocin (Haselwandter & Winkelmann, 2002; Holmström et al., 2004). Consequently, siderophore-assisted uptake may be a pathway for Fe supply of both fungus and host. These siderophores have also been detected in solutions of forest soils in the low nM range (Holmström et al., 2004; Essén et al., 2005). A key question is what regulates the production of root and mycorrhizal exudates. This is important especially for exudates such as LMWOAs and siderophores, which through metal complexation [e.g. Fe and zinc (Zn)], mineral dissolution (e.g. K and PO43–) or competition for sorption sites (e.g. PO43–) may increase nutrient availability (Jones, 1998). In this context, ‘mycorrhizal weathering’ and what regulates this process may be highly significant at an ecosystem level (Hoffland et al., 2004). However, it is generally difficult to link nutrient deficiencies to elevated levels of LMWOAs, although the effect of P shortage has been demonstrated for some plants (Jones et al., 2004). The impact of K deficiency on root and mycorrhizal exudation also remains controversial and contradictory observations can be found in the literature (Hoffland et al., 2004). It should, however, be pointed out that, in nonsterile systems, the net effect of exudates in soil could be small because of rapid biodegradation and sorption (van Hees et al., 2003). The C flux resulting from root and mycorrhizal exudation should not be overlooked. In the case of root exudation, c. 5% of the C fixed by the plant may be lost ( Jones et al., 2004), which may correspond to a flux in field soils of 6–9 mol C m−2 yr−1 (van Hees et al., 2005a). In magnitude this flux corresponds to c. 10% of the C lost through soil respiration (van Hees et al., 2005a). Despite many observations that fungi can release large amounts of LMWOAs, the overall quantitative and qualitative impact of EM colonization on exudation needs to be further investigated. Most experiments to date have concerned either nonmycorrhizal seedlings or mycorrhizal fungi grown in pure culture in the absence of plants. In the case of mycorrhizal seedlings,

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separation of exudation from the roots and mycelium has typically not been carried out. In addition, there is a need to consider the spatial scale over which these processes (e.g. mineral dissolution) may occur. For a true assessment of C dynamics in the ‘hyphosphere’ it is likely that a µm scale is required, i.e. exudation of individual hyphae has to be considered. The overall aim of this work was to determine exudation rates of LMWOAs and hydroxamate siderophores from the extramatrical mycelium of an EM fungus growing in symbiosis with a host tree seedling. For this purpose, a ‘flat bed’ dish system was developed with collection of exudates in a gel matrix. A fractal geometric approach was employed for calculation of fungal colony properties. Exudation rates are presented on different bases including that of individual hyphal tips. Potassium nutrition was tested as a regulatory factor of the efflux. In addition, a new liquid chromatography–mass spectrometry/mass spectrometry (LC-MS/MS) method for hydroxamate siderophores is presented. The results are discussed in the context of the potential ecological significance of mycorrhizal exudation.

Materials and Methods Axenic mycorrhizal synthesis and preparation of seedlings Seeds of Pinus sylvestris L. were surface-sterilized for 30 min in 33% H2O2 and rinsed in sterilized water. Seeds were germinated on water agar for 7 wk in a climate chamber at 14–16°C during the 16-h photoperiod at a photon flux density (daylight spectrum) of 300 µmol m−2 s−1 and at 6–8°C during the 8-h dark period. Hebeloma crustuliniforme (Bull.) Quel. (isolate code UP184) was maintained in darkness at 25°C on half-strength modified Melin–Norkrans (MMN) medium (Marx, 1969). Fungi were inoculated in large Petri dishes (diameter 12 cm) using a half-strength sugar, full mineral strength MMN, without malt extract. Fungal isolates were grown on top of sterile and moist filter paper (diameter 9 cm) placed at one end of the dish. At the other end of the dish a ventilation hole was made in the lid (approx. 2 cm in diameter), and the hole was covered with sterile filter paper that was attached by tape to the outer surface of the lid. The fungal isolate covered the filter paper after 4 wk of incubation in the dark at 20°C. Nylon mesh bags (45-µm mesh) were sewn with folded edges to give outer-edge dimensions of 3 cm × 5.5 cm, with an opening in the upper shorter end. The mesh bags were sterilized by autoclaving in individual foil packages. Ensuring that the sterility of the entire seedling was maintained, the root system of an individual pine seedling was inserted into each mesh bag. Bags with seedlings were placed on top of the mycelia-covered filter paper inside the Petri dishes. Two bags with seedlings were placed on each plate. To ensure good contact between roots in the bag and mycelia on the paper, all inoculation plugs were removed from the paper. In an attempt to

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increase root–mycelial contact, a piece of moist, sterile filter paper was placed on top of the bags. The sterile ventilation holes in the lids enabled us to grow the mycorrhizal seedlings with the shoot of the seedling inside the Petri dish. The lids were oriented so that the ventilation hole was close to the shoots but did not shade them. The lower half of the dishes was covered in aluminium foil and the dishes were placed in boxes with transparent plastic lids. Air humidity in the box was maintained by keeping moist paper towels in the bottom of the box. The syntheses were incubated for 10 wk in the climate chambers described at the beginning of this section. Experimental systems Nunclon™ eight-compartment dishes (128 mm × 86 mm × 15 mm; Nunc, Roskilde, Denmark) were prepared for seedling introduction by cutting a hole in the wall and lid of the dish in the middle of one of the compartments at the short end of the dish. The hole was cut down to the level of the compartment-dividing walls. Two mineral nutrient stock solutions were prepared, one including K (+K) and the other without K (–K). The final solution contained 195 µM NH4NO3, 44 µM KH2PO4, 50 µM Ca(NO3)2, 75 µM Mg(NO3)2, 25 µM K2SO4, 6 µM KNO3, 5 µM H3BO3, 0.1 µM Na2MoO4, 0.1 µM ZnSO4, 0.1 µ M FeCl3 and 0.1 µM CuSO4. The pH was adjusted to pH 5.0. In the –K solution, K-salts were exchanged for Nasalts. Sterile nutrient agar with 10 ml of stock nutrient solution, 5 g of Gelrite (Merck, Darmstadt, Germany) and 990 ml of MilliQ water (Millipore, Billerica, MA, USA) was prepared and 8 ml was added to each compartment of the multidish using an automatic pipette. After solidification, the entire surfaces of all compartments of the dish were covered by one piece of sterile N-free cellophane film, ensuring that as few as possible air bubbles were trapped under the cellophane. The prepared sterile mycorrhizal seedlings in bags were checked, and well-colonized seedlings were lifted from the synthesis dishes and transferred to the multidishes. The bag approximately covered the two upper compartments on one side of the dish. Pieces of the nutrient agar were cut out and placed on top of the seedling bags to hold them in position. Good contact between the roots and the cellophane surface was ensured when the lid of the dish pressed down on the agar pieces. The shoot exit hole was sealed with sterile lanolin and the whole dish was sealed using parafilm (Fig. 1). In order to maintain the sterility of the entire seedling, the multidishes were organized pair-wise in large square Nunclon™ boxes (24.5 cm × 24.5 cm × 2.5 cm) by attaching the multidishes to the bottom of the box using double-sided tape. A ventilation hole had been cut at one end of the lid as described previously. Sterile, moist pieces of filter paper were placed in each box to maintain air humidity. Each box was sealed using parafilm. For a photograph of the experimental systems, see Fig. 1. The lower half of the boxes was coated

with foil to protect the root system of the seedling from light exposure. The boxes were organized so that the shoot part of one box overlaid the foil-covered lower part of the underlying box, thus obtaining a close to horizontal position of the systems which enabled maximum illumination of the seedlings. Systems were grown for 5 wk in a climate room at approx. 15°C and a photon flux density (daylight spectrum) at the levels of the shoots of between 250 and 450 µmol m−2 s−1 during the 16-h photoperiod. The growth of extra-radical mycelia was documented weekly by digital photography. To enable an approximation of the mycelial growth rate during the experiment, the margin of the mycelial front was marked on the lid of the box at weekly intervals. Harvest Multidishes were removed from the boxes and individually documented by digital photography. Numbering the compartments in each multidish (1–8) as well as the edges of all compartments (1–15) enabled systematic documentation of the mycelial cover of individual compartments. Using a scalpel, the cellophane was cut along the compartmentdividing walls. The disjointed sheets of cellophane were lifted from the compartments and placed on a droplet of water in small Petri dishes (4.5 cm diameter). No penetration of hyphae through the cellophane was observed. Each dish was labelled with compartment and corner numbers. Dishes were sealed with parafilm and stored at 4°C until further analysis. The agar in each compartment was lifted out using a small spoon, transferred to individual 50-ml centrifuge tubes and frozen at −25°C until further analysis. Microscope imaging and hyphal length and density determination The pieces of cellophane cut from the individual compartments with mycelium attached were mounted on a microscope glass slide. Slides were observed under an Axiophot Epifluorescence Microscope (Zeiss) at ×400 magnification and images were taken using an Axiocam high-resolution colour digital camera (Zeiss). For each mycelium (i.e. each compartment), images were taken along four transects. The first point on the transect was 0.25 mm behind the mycelial front in order to avoid edge effects. Subsequently, points corresponding to different radii (r) of the mycelium were recorded every 2 mm (estimated from the mycelial front) towards the imagined centre of the colony. At each radius, five independent replicate images were taken. Depending on the mycelium and its position on the cellophane, measured total distances from the mycelial front ranged between 4 and 12 mm [mean ± standard error of the mean (SEM) 7 ± 1 mm; n = 11]. For such details as the total number of replicates, see the section ‘Statistical analysis’. Afterwards, the mycelia were transferred from the cellophane to aluminium (Al) tins, dried at 60°C for 48 h and weighed.

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Fig. 1 The experimental system. Two eightcompartment multidishes (128 mm × 86 mm) with mycorrhizal seedlings are contained in a larger square box (245 mm × 245 mm). A ventilation hole covered with a piece of sterile filter paper was cut (upper middle of the box). Two pieces of sterile moist filter paper were inserted for maintenance of moisture. Note the extramatrical mycelium below the seedling in the right multidish. The image was taken c. 2 wk before harvest.

Hyphal length was estimated using a direct counting, gridline intersect method on printed images (Newman, 1966). The total grid size corresponded to 50 µm × 50 µm and the scaling was determined using a stage micrometer. Lengths were converted to densities by dividing counted length by the area of the grid. These densities (d ) together with corresponding r-values were used for calculations of fractal geometric properties (Eqn 2; see ‘Mathematical approach’ below). The numbers of hyphae crossing the whole length of the middle transect (50 µm) in the main growth direction were recorded and subsequently used in Eqn 3 (see ‘Mathematical approach’ below). The diameter of the hyphae was estimated to be 2.8 ± 0.2 µm (mean ± SEM; n = 200). Analysis Deep-frozen gels were thawed and transferred to a 60-ml plastic syringe. An aliquot (c. 1 ml) of solution was pressed through

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a 0.45-µm filter (Millex HV; Millipore) fitted to the syringe. LMWOAs in the solutions were analysed by capillary electrophoresis (Dahlen et al., 2000). LMWOAs were identified by comparing migration times and spiking with those of known LMWOAs. The recovery of oxalate in the experimental system was tested by applying a small volume to the cellophane (80 µl) of a solution that would yield an increase of 10 µM in the gel compartment (8 ml). A recovery of 75 ± 3% (mean ± SEM; n = 5) was found and concentrations were corrected using this figure. After filtration (0.45 µm), 20 nmol FeCl3 was added to each filtrate (1.5–4 ml). The siderophores were extracted from the filtrate on 2 g of XAD-16 (Amberlite, Fluka, Germany) (Macrellis et al., 2001). The filtrates were applied to the columns and washed in 3 × 5 ml of MilliQ water. The siderophores were eluted in 2 × 5 ml of methanol, dried in vacuo below 40°C and dissolved in 500 or 1000 µl of ammonium formiate


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Table 1 Mobile phase composition during high-performance liquid chromatography (HPLC) analysis Time (min)

Pre-column

Analytical column

Notes

0–10 10–20 20–35 35–60 60–70 70–125

0.5% MeOH 12% ACN 12–39% ACN 39% ACN 0.5% MeOH 0.5% MeOH

12% ACN 12% ACN 12–39% ACN 39% ACN 39% ACN 12% ACN

Column switch after 10 min Siderophores eluted into analytical column Linear gradient Column switch after 60 min Detection stopped after 70 min Regeneration of columns for at least 55 min

The contents of methanol (MeOH) and acetonitrile (ACN) in ammonium formiate buffer, pH 4.0, 11 mM, are shown.

buffer (pH 4.0, 11 mM). The four hydroxamate siderophores were separated with on-line purification and preconcentration by column-switching capillary high-performance liquid chromatography (HPLC) and selectively detected by electrospray ionization mass spectrometry (ESI-MS/MS), based on the method described by Essén et al. (2005) modified by gradient elution that enabled analysis of ferrioxamine B. The instrumental set-up of Essén et al. (2005) was used with the addition of one Shimadzu LC-10ADvp pump (Shimadzu, Osaka, Japan). The injection volume was 70 µl. The mobile phase compositions described in Table 1 were obtained by mixing two mobile phases with 4 and 50% acetonitrile, respectively, at a total flow rate of 10 µl min−1. The flow of the methanol containing the mobile phase was 10 µl min−1. The ferric siderophores were detected by multiple reaction monitoring (MRM) of the most intense fragments of the proton adducts, i.e. m/z (mass/change) 741.3→569.4 for ferrichrome, 771.3→599.4 for ferricrocin, 801.3→629.4 for ferrichrysin and 641.3→414.4 for ferrioxamine B, on a triple quadrupole mass spectrometer (Sciex API 3000; Applied Biosystems, Streetsville, Ontario, Canada) operated in positive electrospray ionization mode with MS/MS parameter settings optimized by direct infusion at 10 µl min−1. The method is linear in the range 1–10 nM (R 2 = 0.995, n = 10). The chromatographic concentration limits of detection (3 × background noise) were 0.36 and 0.33 nM for ferrichrome and ferricrocin, respectively. The XAD-16 sample clean-up procedure provided a further preconcentration of 3–6× as a result of sample volume reduction. The XAD-16 extraction recovery was 80 ± 3% in the concentration range 1–10 nM. Mathematical approach The aim of the calculations was to express hyphal exudation on the basis of time and various measures such as number of hyphal tips, unit length, unit surface area and biomass. In order to achieve this, equations were derived for calculation of the cumulative sum of tips or hyphal length (or, alternatively, area or biomass) that had contributed to the exudation during the duration of the experiment. The number of tips as well as total hyphal length (area /biomass) will increase over time and,

to calculate this, the fractal geometric approach of Ritz & Crawford (1990) was adopted, which describes the total hyphal length as a function of the radius of the colony: L(r) = Kr D

Eqn 1

[L, total length of the hyphae from the centre of the colony (r = 0) to the radius r; K, a constant; D, the fractal dimension.] L divided by πr 2 gives the average hyphal density of the whole colony. Consequently, a D-value of 1–2 implies a lower density of the mycelium at larger r (explorative growth) while D = 2 gives an even density over the colony. D in the range 2–3 suggests an exploitative growth. The radius at harvest of the mycelium of each compartment was estimated from digital images. Invariably these radii indicated a colony centre in the lower part of the bags with seedlings. For each colony, the set of densities (d; mm mm−2) obtained at counting (see Microscope imaging, hyphal length and density determination) was plotted against corresponding r-values (note that here d is measured at specific r-values, not averaged for the whole colony as would have been obtained by division of Eqn 1 by πr 2) in the form: ln d = a ln(r) + ln K ′

Eqn 2

(a, the slope of fitted linear equation corresponding to D − 2; ln K ′, a constant.) All ln (d ) vs ln (r) plots showed significant correlations (P < 0.05) and R 2 values ranging between 0.47 and 1.00 (nplots = 11; 9 with R 2 > 0.75) were obtained. In three plots the largest r-value closest to the mycelial front was omitted as these values deviated strongly from the linear relationship, probably as a result of a breakdown of the relationship at the front (Ritz & Crawford, 1990). A relationship between the measured hyphal densities and the number of hyphae mm−1 ( fhyphae) of the circumference at the radius r was empirically derived on basis of the microscope images (see Microscope imaging, hyphal length and density determination): f hyphae = 0.85 × d(R 2 = 0.78, n = 100)

Eqn 3

The total number of hyphae (N ) crossing the circumference of a colony with radius r was estimated according to:


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Fig. 2 Drawing of part of a multidish (see Fig. 1) for illustration of the approach used for calculation of the fraction of the area between the radius at the compartment wall (r1) and the radius at the mycelial front (r2) covered by the mycelium. This fraction was used for estimation of L × tadj and N × tadj [see text; L, total length of the hyphae from the centre of the colony (r = 0) to the radius r; N, the total number of hyphae (N) crossing the circumference of a colony with radius r; tadj, XXX] and subsequently hyphal exudation (Eqns 8 and 9). θ, angle of mycelium covering the compartment (see text); 1, compartment walls; 2, mycelial front; 3, centre of fungal colony; 4, bag containing seedling.

N(r) = fhyphae × 2πr = 0.85 × d × 2πr = 1.7πK ′r a+1

Eqn 4

where d is replaced with the unlogarithmic form of Eqn 2. The total length of hyphae between the radii r1 and r2 (Lr1→r2 ; mm; for r1 and r 2 see Fig. 2) is derived by integration of Eqn 4 with respect to r: Lr →r (r ) = 1

2

r2

r2

r1

r1

N (r ) =

1.7πK ′  × r a +2    a +2 

Eqn 5

This equation is the same expression as Eqn 1 but relates L to the observed hyphal densities at individual r-values for each colony. In order to calculate hyphal exudation rates per hyphal tip (where the number of tips is estimated by N ) or unit length (or area/biomass, all using L) and unit time (t) (e.g. molexudate per tip h−1 and molexudate mm−1 h−1) the cumulative L × t or N × t were estimated. These correspond to the cumulative product of hyphal length or tips multiplied by time for the duration of exudation. This was achieved by summing products of discrete L- or N-values at step-wise increasing r-values (1 mm) and the inverted radial growth rate representing the time factor: L ×t =

ri =r2

∑ Lr + 0.5 × rs

ri =r1

i

Eqn 6

and N ×t =

ri =r2

∑ N r + 0.5 × rs

ri =r1

i

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Eqn 7

(rs, the inverted radial growth rate (h mm−1) for the colony.) However, only a part of the colony was contained within the compartment where exudates had been collected in the gel. The area of the compartment covered by the colony was estimated by a graphical approximation where r2 was the radius of the hyphal front and r1 was the minimum radius where the compartment wall crossed the colony (Fig. 2). The part of the hypothetically circular colony contained within each compartment was estimated by subtracting the area at r2 from that of r1 and measuring the angle (θ) of the part of the circle covered (Fig. 2), giving an area with the appearance of a ‘slice of doughnut’. The small parts of this area not included within the compartment walls were graphically assessed and subtracted. The maximum error of this approach was estimated to 5%. Calculations for L, N, L × t and N × t between r1 and r2 were subsequently adjusted using this approach for mycelium area approximation (referred to as L × tadj and N × tadj). Finally, exudation per hyphal tip or unit length/area/biomass and time was computed: E tip =

C exudate × V gel nexudate = N × t adj N × t adj

Eqn 8

and EL =

C exudate × V gel nexudate = L × t adj L × t adj

Eqn 9

[Etip (mol per tip h−1) and E L (mol mm−1 h−1), exudation rates; Cexudate, the concentration (mol dm−3; M) of the exudates in the gel; Vgel, the volume of the gel (8 cm−3).] Area-based numbers (EA; mol mm−2 h−1) were calculated from E L using the average hyphal diameter (2.8 µm). Estimates on a dry weight (d. wt) biomass (Ew; mol g−1 h−1) basis were converted from E L using the quotient between measured dry weight biomass (wmeas) and calculated total length, wmeas L−1 (g mm−1). For these three estimates (E L, EA and Ew) it was assumed that exudation was uniform along the hyphae and constant over time. For comparison between measured and calculated dry weight biomasses the equation of Paul & Clark (1996) was used: w calc = πrh2LeS c

Eqn 10

[wcalc, the calculated dry weight biomass; rh, the hyphal radius (1.4 µm); L, total hyphal length; e, hyphal density (1.3 g cm−3); Sc, the content of solids (0.3).] Statistical analysis In total, seven multidishes with K supplied (+K) and seven without K (–K) were incubated. At harvest, sufficient mycelium had grown in eight multidishes (five +K and three –K), and in four of these dishes mycelium was found in two


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compartments. Statistical analysis showed that the variance within multidishes was as great as the variance between them, with the exception of specific growth rate (rs), and so each compartment was treated as a replicate. This yielded n = 6 for +K and n = 6 for –K. Statistical significance testing of experimental factors (+K/–K and multidish; P < 0.05, unless otherwise stated) was performed on log-transformed values using analysis of variance (GLM-ANOVA; MINITAB 1.3; Minitab Inc., State College, PA, USA). Differences between individual means were calculated with Student’s t-test. Outliers were detected using Grubb’s test. Calculations based on the analytical error for oxalate determination indicated that elevations > 0.65 µM above the background concentration were significant. All averages unless indicated are presented as mean ± standard error of the mean (SEM).

Results

deviation between transects of individual mycelia at the same r was 46 mm mm−2. The growth rate (rs) did not vary between the K treatments but there were significant differences between individual multidishes. The average week-to-week variation in rs (inverted growth rate) equalled 8 h mm−1. Almost no significant differences between the variables were observed because of either very similar average values or large variation. The biomass (wmeas) of the mycelia recovered from the cellophane of individual compartments ranged from 30 to 550 µg, with higher average values seen for the –K treatment, partly because of greater density and partly because of greater surface coverage. Calculated total hyphal lengths (L; Eqn 5) for the compartments ranged from 11 to 190 m (60 ± 17 m; mean ± SEM; n = 12). Using Eqn 10 this yielded wcalc in the range 30–470 µg (160 ± 40 µg; mean ± SEM; n = 12). Plotting of the two biomass variables (not shown) against each other indicated a relationship of wmeas = 1.14 ± 0.17 wcalc (R 2 = 0.92).

Colony properties The mycelia on the excised cellophane pieces were generally characterized by sparingly distributed, well-separated hyphae. No cords or rhizomorphs were observed. However, parts of the mycelia adjacent to the root tips were not studied. Maintained sterility of the systems was verified at harvest as no bacteria were found and all hyphae had regular clamps, indicating that they belonged to the inoculated EM fungus. The D-values (fractal dimension) derived from Eqn 2 for the mycelia suggested explorative growth with little difference between the +K and –K treatments (Table 2). No significant difference was seen for the K ′ value despite a much higher average for –K because of the large variance. The greater K ′ value was also consistent with the higher average hyphal density (d ) observed in the –K treatment. The within-colony variation in hyphal density estimated from the pooled standard

Exudation of LMWOAs and siderophores Oxalate was the only LMWOA that was consistently detected in the gelrite-filled compartments at harvest. Occasionally malonate and acetate were identified at concentrations lower than that of oxalate; however, citrate could not be detected (not shown). Background concentrations of oxalate in the gel were 8.8 ± 0.3 µM and average increases at harvest were 4.3 ± 0.6 µM. Among the hydroxymate-type siderophores studied, ferricrocin could be identified in six samples, of which two also contained ferrichrome. The levels of siderophores were c. 10 000 times lower than for oxalate, with an average concentration of 0.29 ± 0.02 nM. Ferrichrome concentrations were in the same range (< 0.19 nM). With one exception, all background concentrations of siderophores were below the limit of detection.

Table 2 Variables describing colony properties at harvest for extramatrical Hebeloma crustuliniforme mycelium Mathematical constants

Colony growth

Treatment

n

D

K′

n

Radius (r) (mm)

Rate (rs ) (h mm−1)

Density (d) (mm mm−2)

Biomass (w) (µg)

+K –K Overall ANOVA

3 6 9

1.46 ± 0.05a 1.43 ± 0.16a 1.44 ± 0.10

3500 ± 900a 10800 ± 2500a 8400 ± 1800

5 6 12

18 ± 2a 20 ± 2a 19 ± 1

41 ± 5a 44 ± 2a 42 ± 3 M

199 ± 28a 334 ± 18b 272 ± 25 K

102 ± 20a 209 ± 88a 186 ± 50

Values are mean ± standard error of the mean. Means followed by different superscript letters are significantly different (see Statistical analysis). Mathematical constants are from Eqn 2 (see Materials and Methods), where D (= a + 2) is the fractal dimension and K′ is a constant. Two D values < 1 were omitted as values < 1 are not theoretically possible (would indicate a decreasing number of hyphae with r). The colony growth variables are the estimated colony radius at harvest, the inverted growth rate (measured weekly over 3 –4 wk) and the average hyphal density and biomass of the measured part of the colonies at harvest. The treatments were potassium present in the gel (+K) or omitted from it (–K). Statistical testing [analysis of variance (ANOVA)] was performed for the factors potassium supply in the gel (+K/–K; K) and individual multidishes (M).


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Values are mean ± standard error of the mean. Means followed by different superscript letters are significantly different (see Statistical analysis). In the cases of length, area and biomass, uniform exudation along the hypha is assumed. Treatments were potassium present in the gel (+K) or omitted from it (–K). Statistical testing (analysis of variance) of the factors potassium supply in the gel (+K/–K; K) and individual multidishes (M) did not reveal any differences.

0.24 ± 0.07a 0.03 ± 0.01a 0.16 ± 0.05 61 ± 16a 6 ± 0b 39 ± 11 2.2 ± 0.7a 1.9 ± 0.8a 2.0 ± 0.5 5 6 11 +K –K Overall

642 ± 142a 333 ± 117a 488 ± 95

n Treatment

EA (fmol mm−2 h−1)

Ew (pmol µg−1 h−1)

n

4 2 6

1.79 ± 0.36a 0.36 ± 0.02b 1.22 ± 0.31 5.6 ± 1.3a 2.9 ± 1.0a 4.3 ± 0.8 23 ± 5a 14 ± 3a 19 ± 3

0.53 ± 0.14a 0.06 ± 0.01a 0.34 ± 0.10

Ew (fmol µg−1 h−1) EA (amol mm−2 h−1) Etip (amol per tip h−1) EL (fmol mm−1 h−1) Etip (fmol per tip h−1)

EL (amol mm−1 h−1) Ferrocrocin exudation Oxalate exudation

Table 3 Oxalate and ferrocrocin exudation by extramatrical Hebeloma crustuliniforme mycelium calculated on a tip (Etip), length (EL), area (EA) and biomass dry weight (Ew) basis

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In the case of oxalate, the highest average exudation rates were seen for the +K treatment regardless of the unit employed (Table 3). However, no significant differences were found between the treatments or between the multidishes. Ferricrocin exudation followed the same pattern as oxalate, and for Etip the +K average was statistically higher. The ratio between Etip and E L values for oxalate and ferricrocin (Table 3) suggested that exudation at a tip would correspond to exudation along 5.1 ± 1.5 and 3.6 ± 1.4 mm of a hypha, respectively.

Discussion Colony growth and experimental approach The fractal nature of fungal colonies has been demonstrated in a growing number of reports. Fractals are, in a wide sense, geometric shapes with structural features repeated across all scales, which implies that the structure at one point is correlated to that at any other (Ritz & Crawford, 1999). In the case of mycelia, basically two scales are considered, that of the hyphae and that of the colony as a whole. In its basic form, when the distribution of the colony can be described by one fractal dimension (D), referred to as self-similar fractals (Ritz & Crawford, 1999), the colony length (or mass) can be described by Eqn 1. An average D-value of 1.44 indicated an explorative foraging strategy with a lower hyphal density towards the mycelial front resulting in a larger total surface coverage. This D-value is at the lower end of the range but is comparable to those obtained in experiments with fungi in agar cultures with Trichoderma viride (1.4–2.0; Ritz & Crawford, 1990, 1999). Experiments with the EM fungi Paxillus involutus and Suillus bovinus in symbiosis with P. sylvestris showed D-values ranging between 1.5 and 1.8 in nonsterile soil (Donnelly et al., 2004). The cited studies demonstrate that a multitude of factors may influence the fractal dimension, such as species, individual strain and age of the colony. Older colonies tend to have higher D-values (Ritz & Crawford, 1990; Donnelly et al., 2004). The availability of nutrients, potentially both C and inorganic elements (e.g. N), may also be of importance, and greater fractal dimensions are typically seen for systems with higher supply (Baar et al., 1997). Moreover, in a soil environment, mycorrhizal mycelia may develop a more exploitative foraging strategy in response to nutrient-rich patches (Donnelly et al., 2004). However, as demonstrated by Baar et al. (1997), a high D-value does not necessarily imply a large biomass. This finding was supported by our results, in which no correlation between D and the hyphal density (d ) was found (not shown). The comparison between calculated biomass (wcalc) and measured weight (wmeas) provided a means of indirectly checking the accuracy of the calculated number of hyphae (N ), total hyphal length (L) and area estimation. There was a close relationship between wcalc and wmeas, although the wcalc


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tended to slightly underestimate the wmeas. Besides any unknown systematic errors, the deviations should be regarded in the light of the fairly large variation in the data set. For example, an average hyphal diameter of 3.0 µm instead of 2.8 µm would have yielded a 1 : 1 correlation. Hyphal exudation This study provides evidence for, and quantification of, exudation by the extramatrical mycelium of an EM fungus growing in symbiosis with a host tree. The combination of P. sylvestris and H. crustuliniforme was chosen because of the favourable growth of this fungus in the experimental system compared to other tested fungi (not shown). Efflux by mycelia has been strongly suggested in a number of studies (e.g. Ahonen-Jonnarth et al., 2000; Casarin et al., 2003), although separation of exudates from roots, mycorrhizal root tips and extramatrical mycelium is difficult to achieve. The experimental set-up employed here ensured that the measured exuded compounds originated from the extra-radical mycelium only. Another question is where the zone of active exudation along the hypha is located, and if this can vary with species, age of the mycelium, growth phase, morphology, type of exudates, etc. Work by Sun et al. (1999) demonstrated for Suillus bovinus that exudation of a variety of organic compounds, including oxalate, can occur at the apical zone of the hypha. However, the possibility of release of substances from the proximal part of the mycelium was not specifically studied. As conclusive evidence is lacking, calculations presented here are based upon one of either two assumptions, that exudation occurs only at the hyphal tips, or alternatively that there is uniform release along the hypha. The –K treatment tended to show lower rates of oxalate exudation in comparison with the +K treatments, although the observation was not statistically significant. However, there was a negative relationship (not shown), regardless of K status, between hyphal density and exudation rate, where exudation rate increased as hyphal density decreased. This was especially obvious above 200 mm mm−2. A similar observation was made by Rosling et al. (2004a) for Cortinarius glaucopus, which caused higher substrate acidification at lower densities. The effect appears to be species dependent as no such effect was seen for Mycena galopus. Besides metabolic regulation of the efflux rate with the density or possibly the radius of the colony, an alternative explanation is re-uptake of oxalate by the hyphae. While oxalate does not seem to be re-absorbed by mycelia, in contrast to other substrates (e.g. glucose and mannitol; Sun et al., 1999), more investigations are required to clarify this. Oxalate is the most common LMWOA associated with EM exudation, but in previous studies citrate and malate have also been identified in exudates (Marschner, 1994; AhonenJonnarth et al., 2000). The average Ew rate for oxalate obtained in this work is directly comparable to literature values for other EM fungi. These rates, typically derived from culture

experiments with glucose as an external C source, may range between 0.003 and 8.2 pmol µg−1 h−1 (Casarin et al., 2003; Rosling et al., 2004a). In contrast, oxalate exudation for nonmycorrhizal P. sylvestris roots has been estimated at 0.024 ± 0.007 pmol µg−1 h−1 (mean ± SEM) and that for mycorrhizal roots at 0.10 ± 0.04 pmol µg−1 h−1 (Ahonen-Jonnarth et al., 2000). This implies that on a biomass basis H. crustuliniforme produced 85 times more oxalate than nonmycorrhizal tree roots. However, when the larger surface area of hyphae (36 500 cm2 g−1; rh = 1.4 µm) vs that of roots (250 cm2 g−1; rroot = 0.4 mm) was considered and exudation rates were expressed on an area basis, this difference almost disappeared. A number of factors may influence the EM exudation of oxalate. Paris et al. (1996) demonstrated an increase of oxalate efflux, in particular under combined K and Mg deficiency but also for single deficiency of K or Mg in some treatments. However, in the present study, no effect of K omission was observed. It must be pointed out that various fungi may respond differently to both their own nutrient status and that of their host plant, and further systematic studies are warranted. Furthermore, the general C and energy supply may be important, as shown in work with saprophytes ( Jacobs et al., 2002), but again conclusive studies are largely lacking. In soil, the spatial and temporal variation can be expected to be considerable. For example, increased growth and exudation in nutrient (e.g. P) ‘hot spots’ as well as responses to toxic metals (Al and heavy metals) have been suggested (AhonenJonnarth et al., 2000; Donnelly et al., 2004). Another aspect worth considering is whether fungi are capable of reacting to different mineral substrates, as indicated by Arvieu et al. (2003) and Rosling et al. (2004a). The multidish system developed is suitable for this kind of experiment, and such manipulations should be within the scope of future studies. In soil environments, the importance of the sink strength for exudates should not be overlooked. In the case of roots, microbial removal of LMWOAs from soil is believed to enhance exudation substantially ( Jones et al., 2004), but very little is known if this applies to fungi. In soil, adsorption of exudates to the solid phase or formation of Ca-oxalate(s) (Arvieu et al., 2003; Rosling et al., 2004a) could play a similar role. The effect of the background level of oxalate in the current experiment can be discussed in the same terms, although it should be pointed out that it was directly comparable to measured field soil concentrations ( Jones, 1998; van Hees et al., 2005a). This strategy of Fe acquisition is well established for roots (Jones et al., 2004), and evidence suggests that EM fungi have similar mechanisms to those of plants, although the complexing agents are different (Leyval & Reid, 1991). However, uptake of Fe by mycorrhizal roots was not as efficient as that by nonmycorrhizal ones (Leyval & Reid, 1991). In the present study, a low Fe concentration was supplied (100 nM), as knowledge of how EM fungi react to a complete lack or very low levels of Fe is still limited. Although the results must be viewed in the light of the Fe status of this particular experiment,


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concentrations of siderophores both in cultures and in soil solutions are commonly in the low nM range, as also observed here. Further work is required to elucidate the molecular, physiological and environmental factors that regulate the release of siderophores from EM when associated with the host. The reason why K-status affected ferricrocin exudation is not apparent, and the results also need to be viewed in the light of the relatively few replicates. Environmental impact of hyphal exudation Mycorrhizal exudates have been implicated in a number of biogeochemical cycles in soil (Gadd, 1999; Jones et al., 2004; Finlay & Rosling, 2005). Despite this, quantitative data concerning exudation rates and the concentrations that this efflux can give rise to in the mycorhizosphere and hyphosphere are still largely lacking. Fungal weathering assisted by LMWOAs is a topic that has been intensively debated (Sverdrup et al., 2002), in particular with regard to the so-called ‘micropores’ found in mineral grains of podzol E horizons. These are thought to be created by fungi, a hypothesis supported by the occasional occurrence of hyphae in the pores (e.g. Hoffland et al., 2004). Although these pores have been estimated to account for only a small fraction of total feldspar weathering (Smits et al., 2005), a key question is whether concentrations can reach such levels that substantial weathering can take place. For example, if a H. crustuliniforme hypha was contained in a water-filled micropore (internal diameter 5 µm, length 50 µm) and exudation at the tip was assumed, already after 1 h of exudation an oxalate concentration of 30 mM in the void of the tunnel could be achieved. However, this concentration would be critically dependent upon the diffusion out of the pore, as surrounding bulk soil solution concentrations would typically be 10 000 times lower. The corresponding figure for ferricrocin would be 1.5 µM or c. 1000 times more than the surrounding soil solution. In the case of oxalate (L2– below), the exudation commonly coincides with an enhanced H+ efflux (e.g. Casarin et al., 2003), and if a 1 : 1 or 2 : 1 H+ : L2– ratio is assumed the resulting pH within the tunnel would be 2.9 or 1.7, respectively. These pH values can be compared with those encountered at the tips of wooddecomposing fungi (pH 2.5; Gadd, 1999). However, it is still unclear if this acidification is a result of an efflux of protonated acids or a H+-efflux caused by an up-regulation and/or coordination with H+-ATPase activity, as suggested for roots ( Jones et al., 2004). Extrapolation of the dissolution rates for the feldspar andesine presented by Stillings et al. (1996) would then, for the three exudation scenarios 0 : 1, 1 : 1 and 2 : 1 H+ : L2–, give rates of 1.2 × 10−14, 1.4 × 10−14 and 2.1 × 10−14 mol andesine cm−2 s−1, respectively. In the first two cases ligand-promoted dissolution would dominate while in the last dissolution would be largely pH driven. In comparison, the dissolution rate in water of pH 4, typical for soil solutions, would be 7.1 × 10−16 mol cm−2 s−1 or 15–30 times lower.

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Although the values should be interpreted with great care, they do indicate that EM hyphae may create microsites of intense weathering. The effect on mineral dissolution by ferricrocin is difficult to assess directly. Research performed for goethite indicates that hydroxymate types of siderophores may induce a significant dissolution despite very low concentrations and may also enhance other ligand- or H+-promoted weathering (Kraemer, 2004). This could suggest a significant contribution despite the much lower concentrations expected than for oxalate. Another question is the amount of C that can possibly be transferred to the soil through hyphal exudation. Using the ergosterol values and estimated fraction for EM fungi of total fungal biomass (60%) for forest soils given by Bååth et al. (2004) and a conversion factor from Stahl & Parkin (1996; c. 10 µg ergosterol mg−1 active fungal biomass), an active EM biomass of 10 (O horizon) and 0.3 mg g−1 soil d. wt (mineral soil 0–30 cm) or c. 185 g m−2 can be estimated. Corresponding production rates for oxalate would range from 0.3 ± 0.1 (> 10 cm depth) to 20 ± 5 (O horizon) nmol oxalate g−1 h−1. These figures are within the range of < 0.01 to 50 nmol g−1 h−1 that has been modelled for coniferous forests depending upon soil type and depth (van Hees et al., 2005b). Similarly, if the values were scaled up for a ‘typical’ soil profile (van Hees et al., 2005a), the resulting C flux would be 6.5 mol C m−2 yr−1. This figure is directly comparable to the calculated flux of 6.8 mol C m−2 yr−1 based on soil solution concentrations, and would also be significant in relation to typical estimates of soil C respiration (60–75 mol C m−2 yr−1; van Hees et al., 2005a). It must, however, be pointed out that all these calculated numbers will include EM biomass contained in root tips as well, and not only the extramatrical mycelium. However, the latter may comprise 80% of the total EM biomass (Wallander et al., 2001). The exudation of oxalate can also be viewed in the light of the internal C allocation of the fungus. Employing values presented by Rygiewicz & Andersen (1994) for H. crustuliniforme in symbiosis with P. ponderosa, oxalate efflux (Table 3) could comprise 2–4% of the C received by the fungus or c. 0.2% of the C fixed by the host. Root exudation may correspond to c. 10% of the total C allocated below ground (Grayston et al., 1996), and the calculated numbers compare favourably. Although oxalate is just one of a wide range of compounds possibly exuded by hyphae (Sun et al., 1999), we expect it to be a major constituent (Gadd, 1999). All the calculations presented in this paper are subject to possible inherent inaccuracies and should be interpreted with care. The values refer to one fungus and exudation rates may vary greatly, both qualitatively and quantitatively, between fungal species. In addition, as discussed, in the soil environment a multitude of biotic, abiotic, spatial and temporal factors are likely to affect exudation. Nevertheless, the figures obtained support the idea that hyphal exudation, acting on a microspatial scale, has a significant impact on processes operating at the whole soil and probably the ecosystem level.


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Acknowledgements PvH, AR and RF thank the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (FORMAS) for financial support. PvH would also like to thank the Swedish Research Council (VR) for support.

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