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Ectomycorrhizal fungi in culture respond differently to increased carbon availability

Petra M.A. Fransson, Ian C. Anderson, Ian J. Alexander
DOI: http://dx.doi.org/10.1111/j.1574-6941.2007.00343.x 246-257 First published online: 1 August 2007

Abstract

Carbon (C) availability to ectomycorrhizal fungi is likely to increase at elevated atmospheric CO2. To determine whether there are any broad patterns in species' responses that relate to their ecology, we compared growth, respiration, N uptake and C exudation of 17 fungal isolates in liquid culture. As a surrogate for increased C availability we used three different C:N ratios (10:1, 20:1 and 40:1), moving from conditions of C limitation to conditions of N limitation. Responses were species-specific, and suilloid fungi were the most responsive in terms of growth and respiration. In contrast, a group of eight isolates showed no growth increase above C:N 20:1. This inability to respond was not due to N limitation, although there were marked differences in N uptake between isolates. At higher C availability isolates generally became more efficient in converting C into biomass. Six isolates showed net release of exudates into the culture medium (up to 40% of the C in biomass and respiration). We conclude that the findings were in agreement with field observations, and suggest that pure culture observations can yield ecologically relevant information on how ectomycorrhizal fungi may respond under conditions of elevated CO2.

Keywords
  • elevated CO2
  • nitrogen uptake
  • carbon partitioning
  • functional groups
  • exudation
  • boreal forest

Introduction

Ectomycorrhizal (ECM) fungi are central to carbon (C) and nutrient cycles in northern temperate and boreal forest. Their importance in accessing nitrogen and phosphorus, in many cases by short-circuiting conventional pathways of mineralization, has been clearly demonstrated (Read & Perez-Moreno, 2003). In fulfilling this role, ECM fungi consume between 20% and 30% of current assimilates (Söderström, 2002). Field-based studies show that ‘autotrophic’ below-ground respiration, including roots and associated mycorrhizal fungi, represents 50–65% of total soil respiration (Högberg et al., 2001; Bhupinderpal-Singh et al., 2003; Andersen et al., 2005), and mycorrhizal respiration is considered to represent a high proportion of forest soil respiration (Högberg et al., 2001; Keel et al., 2006). At least 30% of the microbial biomass, and 80% of the fungal biomass, in boreal forest soils comprises the extraradical hyphae of ECM fungi (Wallander et al., 2001, 2003; Högberg & Högberg, 2002). They are therefore an important pathway for the transfer of tree-derived carbon to the soil system. Given this central role in C and nutrient cycles, ECM fungi are potentially an important component of how these forests respond to elevated CO2. Indeed, it is known that elevated atmospheric CO2 increases the supply of fixed C to roots (Norby & Jackson, 2000; Tingey et al., 2000), and that this is likely to feed through to their fungal partners (Alberton et al., 2005), promoting an increase in ECM biomass and colonization and in the extent of extraradical mycelium.

It is estimated that there are between 7000 and 10 000 species of ECM fungi worldwide (Taylor & Alexander, 2005), and individual trees and forests support a functionally and taxonomically diverse ECM fungal community (Agerer, 2001; Horton & Bruns, 2001; Saari et al., 2005). There is evidence that elevated CO2 causes a shift in ECM fungal community composition. Godbold & Berntson (1997) noted an increased abundance of ECM morphotypes with extraradical hyphae and rhizomorphs on Betula papyrifera saplings exposed to elevated CO2 for 24 weeks. Rey & Jarvis (1997) saw a change towards so-called ‘late-successional’ (Deacon & Fleming, 1992) ECM species on B. pendula seedlings exposed to elevated CO2 for 4.5 years, during which time a rhizomorph-producing Leccinum sp. (Müller & Agerer, 1990) became dominant.

In the only field study to date utilizing mature Picea abies in whole-tree chambers, Fransson et al. (2001) also found a shift in community composition, although they were unable to confirm a shift towards morphotypes producing greater amounts of mycelium or rhizomorphs. Thus, the 45% increase in extraradical mycelium noted by Alberton et al. (2005) in their meta-analysis of ECM fungal responses to elevated CO2 might be due to an increase in production of extraradical mycelium by fungi already present on the root system and/or a shift in ECM fungal community composition towards species which inherently produce more.

One important aspect to consider in the global change context is whether C storage and mycelial production and turnover rates change under elevated CO2. As several authors have pointed out, more extraradical mycelium offers the potential for both positive feedback on tree response through enhanced nutrient capture, and negative feedback through increased competition for nutrients between fungus and host. Differences in response between ECM fungal species are likely to be important, and the few comparative studies published to date (Gorissen & Kuyper, 2000; Fransson et al., 2005) have indeed shown differences between ECM fungal isolates both in mycelial production and in effects on plant performance. Clearly, screening a large number of ECM fungi in association with their hosts, and measuring species-specific host and fungal responses, would improve our ability to understand and predict effects of elevated CO2 on forest ecosystems. However, this is impractical in the short term, and in this study we investigate whether simpler screening of ECM fungal isolates in asymbiotic culture can yield similar useful information. Previous studies have clearly shown interspecific differences in growth between ECM fungi in response to increased carbon availability in pure culture (Alexander, 1983; Baar et al., 1997; Eaton & Ayres, 2002).

Pure culture studies suffer from two drawbacks. First, not all ECM fungi, including many which are important members of communities in the field, can be grown in asymbiotic culture. Secondly, it is not clear how growth in asymbiotic culture relates to the performance of the fungus in association with its host. Nevertheless, we hypothesized that for a range of culturable ECM fungal species there would be broad patterns in response and that these patterns would relate to what is known about the ecology and morphology of those species in controlled symbiosis or in the field. We believe that where clear differences in pure culture physiology are apparent, these are likely also to be important in the field, although caution should be taken when translating into field conditions.

As a first step we have measured how C supplied to a range of culturable ECM fungi with different ecological strategies is partitioned between growth, respiration and exudation. We used multiple isolates of some species to gauge the extent of within-species variation. As a surrogate for the increased C availability to the fungus resulting from increased C fixation by the host under elevated CO2 we have compared fungal growth, respiration and C exudation at three different C:N ratios, in effect moving from conditions of C limitation to conditions of N limitation. Glucose was supplied as the C source, based on current conceptual models which favour the transfer of hexoses in the symbiosis. Sucrose is assumed to be hydrolysed by cell-wall-bound acid invertase in the apoplast in the plant–fungus interface, and the resulting hexoses are taken up by the fungus (Schaeffer et al., 1995; Nehls et al., 2000). Because of the importance of potential feedbacks to host nitrogen nutrition we also measured fungal N uptake at these different C:N ratios. We attempted to answer the following questions: (1) Do different ECM fungal species respond differently to an increase in the C:N ratio of the growth medium? (2) Do different isolates of the same species respond the same way? (3) Are there broad patterns of response which map onto current ideas about functional groups or ecological strategies of ECM fungi?

Materials and methods

Experimental set-up

Seventeen ECM fungal isolates were used, comprising 13 different ECM species to test for interspecific variation and three isolates each of Laccaria bicolor and Suillus bovinus to test for intraspecific variation (Table 1). The identity of the isolates was confirmed by internal transcribed spacer sequence analysis using standard techniques. Isolates belonging to the genera Suillus and Rhizopogon are closely related and are referred to as ‘suilloid’ species in the results and discussion. Fungi were kept on 25-mL agar plates (pH 5.5) containing Modified Melin Norkrans (MMN) medium (Marx & Bryan, 1975) with the following modifications: 2.5 g L−1 glucose, 10 g L−1 malt extract, 15 g L−1 agar. Plugs of fungal inoculum were cut from each plate with a corer (diameter 4 mm) and placed on new MMN plates for 3–5 days to allow the fungus to resume growth. The plugs were then moved to individual, gas-tight 500-mL Kilner glass jars containing 50 mL liquid Basal Norkrans (BN) medium (Norkrans, 1950) at pH 4.5. The dry weight of five replicate plugs of each isolate was measured after drying at 70°C for 24 h. All fungi were grown at 25°C in the dark at three different C:N ratios, 10:1, 20:1 and 40:1, containing a constant nitrogen content of 0.174 g L−1 (NH4)2SO4 (1.85 mg of NH4+-N per jar), and a carbon content of 1.25, 2.5 and 5 g L−1 glucose, respectively. Five replicates of each isolate and treatment were prepared, and each jar contained a 10-mL scintillation vial with 5 mL 2 M NaOH to trap evolved CO2. The values obtained were not corrected for background levels of CO2, as this was found to be negligible in preliminary experiments; however, this means that absolute respiration rates may be overestimated. We tested for possible effects of NaOH on fungal growth by comparing two isolates (S. bovinus UP63 and Hebeloma velutipes) in the presence of NaOH or an equal volume of H2O. There were no differences.

View this table:
1

Ectomycorrhizal fungal isolates used to study carbon partitioning at three different growth medium C:N ratios

Harvest

After 21 days, fungal biomass was collected on Whatman glass filter paper and dried in an oven at 70°C for 24 h. Biomass dry weight was recorded, and the mean dry weight of the initial inoculum plug was subtracted. Growth media were immediately frozen in plastic bottles, and the scintillation vials were sealed with a tight plastic lid and kept in a cold room at 4°C, to await analysis.

Chemical analysis

Fungal biomass samples were ground using a ball mill and total C and N was determined using a Thermo Finnigan Flash EA elemental analyser (Thermo Electron Corp., Germany). Growth media were analysed for total organic carbon (TOC) and total nitrogen (TN) using a Shimadzu TOC-V CSH with TNM-M module attached (Shimadzu, Germany). Ammonium (NH4-N) content was determined using a TRAACS (Bran+Luebbe, Germany) after suspending samples in 0.5 mL active carbon for 5 min and passing the suspension through a Whatman no. 1 filter paper to decrease interference from organic compounds. Residual glucose in the growth media was determined using an enzymatic reaction (glucose oxidase peroxidase method) described by Southgate (1976), to produce a colour change of a chromophore (o-dianisidine) detectable at 540 nm. The following changes were made: water bath incubation temperature was 37°C, and volumes were adjusted to 300 μL microtitre plate format, using 15 μL sample, 30 μL reagent mix and 150 μL H2SO4 (1+3) to terminate the reaction. Duplicates were analysed for each sample, and microtitre plates were measured using a Spectra max 190 (Molecular Devices, USA).

The total volume of CO2 produced during the experiment was determined by titration of the NaOH in the CO2 traps using an automatic titrator (RTS822, Radiometer, Denmark). Total CO2-C (mg) was calculated. Carbon budgets were carefully calculated based on measured parameters. We took into account biomass C calculated on actual C content in the mycelium (biomass C% varied between 35% and 54%) and corrected for the C added in the inoculum plug for each isolate, respiration losses, glucose-C left in the growth medium and exuded C.

Statistics

A two-way anova was used to test for effects of fungal isolate (17) and C:N ratio (three) of the growth medium, and for interactions between isolate and C:N ratio. The following parameters were tested: biomass, respiration (CO2-C), carbon use efficiency (biomass-C divided by respired C and biomass-C), N uptake (start NH+4-N minus residual NH+4-N), biomass C:N ratios and exudation (TOC minus residual glucose). When significant main effects of fungal isolate were found, individual one-way anova was performed for each isolate to test for C:N treatment effects. Means from each anova were compared using the Tukey–Kramer method at the 5% level of significance.

When the difference between TOC and residual glucose in the growth medium was significantly higher than zero this was taken as exudation of C-based compounds. To test if the mean value for each isolate × C:N ratio combination from the anova analysis was significantly different from zero the following calculation was carried out: Embedded Image where ȳ is the mean value of each isolate × C:N ratio combination, and MSe is the mean square error from the two-way anova. Calculated t-values were compared with the t-distribution at the 5% level of significance, using the degree of freedom for MSe from the anova. Statistical analyses were conducted using Minitab 14.1 and SAS 9.1.3.

Results

The 17 ECM fungal isolates showed large differences in C partitioning. Highly significant overall effects of fungal isolate and C:N treatment, as well as significant differences between isolates in their response to increasing C:N ratio, were found for all tested parameters (Table 2).

View this table:
2

Summary of statistically significant responses of ECM fungal isolates to increased C:N ratio above 10:1

Uptake of glucose and recovery of added carbon

The uptake of glucose from the growth medium varied between 13% and 100%. The mean uptake was 77.3±2.5%, 70.3±3.0% and 44.5±3.4% at C:N ratios of 10:1, 20:1 and 40:1, respectively. The largest glucose uptake was found at a C:N of 10:1 where Laccaria laccata, Paxillus involutus and Rhizopogon roseolus consumed all added glucose, and L. bicolor CRBF581 took up more than 95%. At C:N of 10:1, 10 isolates had a glucose uptake between 75% and 100%, one had <50% (Piloderma byssinum) and none had <25%. A similar uptake pattern was found at C:N 20:1. At C:N 40:1 five isolates had a glucose uptake between 75% and 100% (suilloid isolates) and five had <25% (Amanita muscaria, H. velutipes, L. bicolor S238, Pi. byssinum and Piloderma fallax).

The mean recovery of C at C:N ratio of 10:1, 20:1 and 40:1 was 130.5±3.2, 106.3±1.6 and 98.0±2.9, respectively. Five outliers (L. bicolor S238 and Origine, L. laccata, Pa. involutus and Piceirhiza bicolorata) had a very high recovery at C:N of 10:1 (>160%), possibly explained by C limitation causing the fungi to utilize C in the agar inoculum plug. This amount was small (mean=5 mg) but variable (SE=0.2 mg). A small error in measured parameters, e.g. biomass C content in the inoculum plug, has a large impact on the recovery at low C:N because the total sum of C is low. In addition, respiration was somewhat higher than the calculated theoretical respiration (uptake of glucose from the medium minus incorporation into fungal biomass) for these fungi. One of the outliers (L. laccata) had low recovery at C:N of 40:1 (77%) owing to an underestimation of respiration in all replicates, possibly explained by faulty CO2 traps and/or by leakage through the lids.

Recovery and utilization of added nitrogen

The mean recovery of N was 101.8±1.4%. The amount of N incorporated into biomass ranged from 10% to 100%. The mean values were 57.4±2.6%, 73.6±2.6% and 90.4±3.5% at C:N of 10:1, 20:1 and 40:1, respectively. The highest incorporation of N was found at a C:N of 40:1 (Cortinarius glaucopus, Laccaria spp. and Pic. bicolorata). The lowest values were found at C:N of 10:1, with seven isolates (H. velutipes, L. bicolor S238, Pi. byssinum and Suillus spp.) incorporating <50%.

Growth response to increased C:N ratio (Fig. 1)

1

Biomass production (mg) of 17 ECM fungal isolates grown in liquid culture for 21 days at C:N ratios of 10:1 (white), 20:1 (grey) and 40:1 (black). Isolates are ordered according to growth at a C:N of 40:1 as a percentage of growth at 10:1. Bars show SEs, and different letters indicate differences between C:N treatments within isolates tested by one-way anova and the Tukey–Kramer method at the 5% level. n=5 for all isolate × C:N ratio combinations with the following exceptions: n=4, L. bicolor CRBF581, L. bicolor Origine, S. bovinus BL and S. variegatus all C:N ratios; n=4, R. roseolus C:N 40:1 and S. bovinus UP63 C:N 20:1; n=3, Pic. bicolorata C:N 20:1 and S. bovinus UP65 all C:N ratios.

There were significant overall effects of fungal isolate (F=55.9, P<0.001) and C:N treatment (F=401.9, P<0.001) on biomass production, as well as significant differences between isolates in their response to increasing C:N ratio (F=13.7, P<0.001). Biomass at a C:N of 20:1 was 96–180% of that at C:N of 10:1 while biomass at a C:N of 40:1 was 89–342% of that at a C:N of 10:1. Eight isolates (Co. glaucopus, L. bicolor CRBF581, L. laccata, Pa. involutus, R. roseolus and S. bovinus spp.) showed a significant growth increase at a C:N of 20:1 compared with at 10:1 and a further significant increase at a C:N of 40:1. Seven isolates (A. muscaria, Cenococcum geophilum, H. velutipes, L. bicolor S238, Pic. bicolorata, Pi. byssinum and Suillus variegatus) showed no significant growth response above a C:N of 20:1. The growth of one of these (A. muscaria) was reduced at C:N of 40:1 compared with at 20:1. The growth of Pi. fallax did not respond to increasing C:N ratio. There was a strong negative relationship between biomass at C:N of 10:1 and the increase in biomass at 40:1 (R=−0.538, P=0.026). In Fig. 1, and in all subsequent bar charts, the isolates are arranged from left to right in order of increasing growth response to higher C availability calculated as the ratio of biomass at C:N of 40:1 to biomass at 10:1. From this arrangement it is clear that, as a group, the suilloid fungi were the most responsive to increased C:N ratio.

Respiration (Fig. 2)

2

Total respiration (mg C) for 17 ECM fungal isolates grown at three different growth medium C:N ratios over 21 days. Isolates are ordered according to growth at C:N of 40:1 as a percentage of growth at 10:1. C:N 10:1 (white), 20:1 (grey) and 40:1 (black), bars show SEs, and different letters indicate differences between C:N treatments tested within an isolate by one-way ANOVA and the Tukey–Kramer method at the 5% level. n=5 for all isolate × C:N ratio combinations with the following exceptions: n=4, L. bicolor CRBF581, L. bicolor Origine, S. variegatus and S. bovinus BL all C:N ratios; n=4, R. roseolus C:N 40:1 and S. bovinus UP63 C:N 20:1; n=3, Pic. bicolorata C:N 20:1 and S. bovinus UP65 all C:N ratios.

Respiration differed significantly between isolates (F=41.8, P<0.001) and C:N ratios (F=30.6, P<0.001), and the response to increasing C:N ratio also differed significantly between isolates (F=13.0, P<0.001). Seven isolates (A. muscaria, Pic. bicolorata, Pi. byssinum, R. roseolus, S. bovinus UP63 and BL, S. variegatus) showed significantly increased respiration at one or both of the higher C:N ratios and a further two isolates (Co. glaucopus, S. bovinus UP65) showed a clear trend (nonsignificant) for this response. Three isolates (L. bicolor CRBF581, L. bicolor Origine, Pa. involutus) showed no response to C:N ratio. Five isolates (Ce. geophilum, H. velutipes, L. bicolor S238, L. laccata and Pi. fallax) showed a significant decrease in respiration at one or both of the higher C:N ratios. In the case of H. velutipes, L. laccata, L. bicolor S238 and Pi. fallax respiration at C:N of 40:1 was <33% of that at 10:1. The suilloid fungi, which were the most responsive in terms of biomass, were also the most responsive in terms of respiration (Fig. 3). Piloderma fallax, which had lower respiration at higher C:N also had lower biomass, but the other four isolates (Ce. geophilum, H. velutipes, L. bicolor S328, L. laccata), which had lower respiration, had higher biomass.

3

The increase in respiration (mg C) and biomass (mg C) in growth media at a C:N of 40:1 as a percentage of that at 10:1 for 17 ECM fungal isolates. A. mus., A. muscaria; C. geo., Ce. geophilum; C. gla., Co. glaucopus; H. vel., H. velutipes; L. bic., L. bicolor; L. lac., L. laccata; P. inv., Pa. involutus; P. bic., Pic. bicolorata; P. bys., Pi. byssinum; P. fal., Pi. fallax; R. ros., R. roseolus; S. bov., S. bovinus; S. var., S. variegatus.

Exudation (data not shown)

For six isolates (Ce. geophilum, L. laccata, Pi. fallax, R. roseolus, S. bovinus UP65 and S. variegatus) the difference between TOC and residual glucose was significantly higher than zero for at least one C:N ratio, i.e. exudation was taking place. Rhizopogon roseolus showed a significant increase in exudation at C:N of 20:1 compared with at 10:1 and a further significant increase at 40:1. Piloderma fallax showed significant differences between all three C:N treatments, with the highest exudation at C:N of 20:1 and no exudation at 10:1. The remaining four isolates (Ce. geophilum, L. laccata, S. bovinus UP65 and S. variegatus) showed a significant increase in exudation at 40:1 compared with one or both of the lower C:N ratios. The amount of carbon exuded was about 5% of the total C in biomass and respiration in most cases, but in L. laccata at C:N of 20:1 and 40:1, Pi. fallax at C:N of 20:1 and 40:1, S. variegatus at C:N of 40:1 and S. bovinus UP65 at C:N of 40:1 exudation-C was more than 40% of the C in biomass and respiration.

C-use efficiency (data not shown)

There were significant overall effects of fungal isolate (F=30.5, P<0.001) and C:N treatment (F=174.29, P<0.001) on C-use efficiency (biomass-C divided by respired C and biomass-C), as well as significant differences between isolates in their response to increasing C:N ratio (F=20.2, P<0.001). C-use efficiency ranged from 16% to 85%. At higher C availability isolates generally became more efficient in converting C into biomass. Six isolates (H. velutipes, L. bicolor S238, Pa. involutus, Pi. byssinum, S. bovinus BL and UP63) at C:N of 10:1 and two isolates (R. roseolus, S. bovinus UP63) at C:N of 20:1 had a C-use efficiency below 25%. Three isolates at C:N of 40:1 (H. velutipes, L. bicolor S238 and Pi. fallax) had an efficiency over 75%. Five isolates (Ce. geophilum, H. velutipes, L. laccata, L. bicolor S238 and Pa. involutus) showed a significant increase in C-use efficiency at both of the higher C:N treatments. Another seven isolates showed an increased efficiency at one of the higher C:N ratios.

Nitrogen uptake (Figs 4 and 5) and pH (data not shown)

4

NH+4-N uptake of 17 ECM fungal isolates in liquid culture for 21 days at C:N ratios of 10:1 (white), 20:1 (grey) and 40:1 (black). Bars show SEs, and different letters indicate differences between C:N treatments within isolates tested by one-way anova and the Tukey–Kramer method at the 5% level. n=5 for all isolate × C:N ratio combinations with the following exceptions: n=4, L. bicolor CRBF581, L. bicolor Origine, S. variegatus and S. bovinus BL all C:N ratios; n=4, R. roseolus C:N 40:1 and S. bovinus UP63 C:N 20:1; n=3, Pic. bicolorata C:N 20:1 and S. bovinus UP65 all C:N ratios.

5

Relationship between nitrogen uptake (% of supplied NH+4-N) and biomass production (mg) at a C:N ratio of 10:1 (white) and 40:1 (black). Those isolates with >75% or <50% available nitrogen uptake at C:N ratio of 10:1 are shown (see text).

There were very marked differences in N uptake between isolates (F=21.8, P<0.001) and between C:N treatments (F=82.5, P<0.001), and significant differences between isolates in their response to increasing C:N ratio (F=6.5, P<0.001; Fig. 4). Thirteen isolates took up significantly more NH+4-N at one or both of the higher C:N ratios. Nine isolates (Co. glaucopus, L. bicolor CRBF581, L. laccata, Pa. involutus, Pic. bicolorata, R. roseolus and S. bovinus spp.) took up significantly more NH+4-N at C:N of 20:1 and 40:1 than at 10:1. Uptake from the growth medium varied between 18% and 100%, and generally increased with increasing biomass production (Fig. 5). The lowest uptakes were observed in the 10:1 treatment (mean value 65.1±2.3%). Laccaria bicolor S238 had a notably lower N uptake (41–74%) than the other Laccaria isolates in all C:N treatments. Both Piloderma species and Ce. geophilum had an NH+4-N uptake between 41% and 68%, irrespective of C:N treatment.

At a C:N of 10 : 1 there was a weak but significant relationship between growth and N uptake (Fig. 5), but very great differences in the amount of N taken up by different isolates. A group of isolates (L. laccata, L. bicolor Origine, L. bicolor CRBF581 and Pa. involutus) took up over 75% of available N, while others (L. bicolor S238, Pi. byssinum, S. bovinus BL, S. bovinus UP63 and S. variegatus) took up <50%. At a C:N of 40 : 1, 10 isolates took up over 90% of the NH+4-N; uptake in the other six was linearly related to their ability to grow at that C:N ratio.

pH values were reduced from the initial 4.5 down to between 2.3 and 3.7. Mean values were pH 2.9±0.02, 2.7±0.02 and 2.7±0.03 for C:N ratios of 10:1, 20:1 and 40:1, respectively, and the reduction in pH normally became larger with increasing biomass production/increasing nitrogen uptake.

C:N ratios in mycelium (data not shown)

The C:N ratios in the mycelium were 10.9±0.3, 11.4±0.5 and 12.0±0.3 at C:N of 10:1, 20:1 and 40:1, respectively. There were significant overall effects of fungal isolate (F=8.4, P<0.001) and C:N treatment (F=3.4, P=0.036) on C:N ratio, as well as significant differences between isolates in their response to increasing C:N ratio (F=5.5, P<0.001). One isolate, Pic. bicolorata, differed significantly from the majority of other isolates in that its C:N ratio was very high (14.3±0.5–16.1±0.7).

Discussion

There were large and significant differences in growth, C partitioning and N uptake between ECM fungal isolates in response to increasing C:N ratio in the growth medium. With a few exceptions (see above), all the C and N added to the closed culture system was accounted for.

Growth

There were marked differences between isolates both in their ability to grow at low C:N ratio (10:1) and in the magnitude of their response to an increase in C availability. The physiological reason for the difference between isolates is unclear, but failure to respond to increasing C availability cannot be due to N limitation, as those isolates which grew slowly or failed to respond did not take up all the N in the culture medium. It is possible that some other mineral element was limiting growth, but this seems unlikely as these are generally considered to be provided in excess in culture media. It is possible that temporal differences in growth response are important; however, we only had a single harvest.

All but one of the isolates were able to respond positively in pure culture to increased C availability. Alberton et al. (2005) concluded from their meta-analysis of field and pot experiments that an increase in C availability to ECM fungi resulting from elevated CO2 leads to an increase in the amount of fungal biomass associated with the root systems, most importantly as extraradical mycelium. Our pure culture data are in line with this conclusion. Increased extraradical mycelium increases the potential for a positive feedback of elevated CO2 on nutrient capture.

In general, those isolates which produced most biomass at low C:N ratio were not the most responsive to increased C:N. We could predict that the ‘responsive’ isolates might become relatively more abundant on the root systems of plants grown at elevated CO2, replacing those which perform relatively better at lower C availability. In the experiment reported here the four most responsive isolates were suilloid, suggesting that ability to respond to increased C availability might be a feature of this fungal clade. An important feature of these fungi is that in symbiosis they are capable of producing abundant extraradical mycelium and extensive rhizomorph systems (Agerer, 2001). This is also true of Pa. involutus, which was the sixth most responsive isolate. These results are in line with the observations of Godbold & Berntson (1997) and Rey & Jarvis (1997) who noted an increased abundance of ECM morphotypes with abundant extraradical mycelium on Betula spp. seedlings exposed to elevated CO2. Further support for the idea that suilloid fungi have a high carbon demand comes from the field experiment of Kuikka et al. (2003) who found that the colonization frequency of S. bovinus tended to decrease in response to defoliation of Pinus sylvestris. In contrast, the one fungus (Pi. fallax) in our trial which failed to respond to an increase in C availability also declined in mycorrhizal root tip abundance in Norway spruce forest after elevated CO2 treatment (Fransson et al., 2001). Taken together these parallels between observations in pure culture and the outcomes of field and pot trials give us confidence that pure culture observations are ecologically relevant. However, in this context it is important to note that not all the isolates capable of producing extensive extraradical hyphae and rhizomorphs in symbiosis (e.g. A. muscaria, Pi. fallax) responded to higher C:N ratio, and some which did respond (e.g. L. bicolor) do not produce rhizomorphs. Thus, while some of the most responsive fungi are rhizomorph producers, this feature in isolation could not be used to predict community changes under enhanced CO2.

Respiration

Respiration was significantly affected by altered C availability, and the response differed between isolates. In general those isolates with the highest biomass response, including the suilloid isolates, also had the highest positive respiratory response. Forest soil respiration is known to increase in response to elevated CO2 (King et al., 2004; Heath et al., 2005), and mycorrhizal respiration has been suggested to constitute a high proportion of forest soil respiration (Högberg et al., 2001; Keel et al., 2006). The responses seen in pure culture are consistent with these field observations. Direct measurements of respiration have seldom been performed in intact plant–ECM fungal systems owing to methodological difficulties. Despite the difficulties of separating extraradical mycelium from mycorrhizal roots, attempts have been made to estimate fungal respiration, arriving at values of 20–30% of below-ground respiration (Söderström & Read, 1987; Rygiewicz & Andersen, 1994).

The balance between growth, respiration and turnover determines the contribution that increased C flow to ECM fungi under elevated CO2 has on soil C store (Langley & Hungate, 2003). We are not able to estimate mycelial turnover rates from our experiment, and nothing is known about turnover rates of ECM fungal mycelia in the field, although Staddon et al. (2003) found that some of the extraradical mycelia of arbuscular mycorrhizal fungi could turn over in a matter of days. However, we did find large differences between isolates in carbon-use efficiency, i.e. the balance between growth and respiration. For most, but not all, isolates the proportion of absorbed glucose converted into biomass was higher at higher C:N ratios, indicating the potential for positive feedback through the mycelia of ECM fungi to the soil C store at elevated CO2. However, because of the differences between isolates, the extent of this will depend on the possible changes to ECM fungal community structure discussed above.

Three isolates (two L. bicolor isolates and Pa. involutus) did not increase respiration at higher C:N ratios although they increased biomass production. For five isolates (Ce. geophilum, H. velutipes, L. bicolor S238, L. laccata and Pi. fallax), respiration at high C availability was only one-third of that at low C availability. The reasons for this unexpected response are unknown.

Nitrogen uptake

There was great variation between isolates in their ability to absorb N from the culture medium, and this appeared in part to correspond with what is known about the ecology of the species in question in the field. N uptake was generally related to growth, with many isolates increasing uptake at higher C:N ratios. This was particularly marked for the S. bovinus isolates, which had relatively low N uptake at C:N of 10:1 but complete utilization at C:N of 40:1. However, there was a distinct group of isolates (L. laccata, L. bicolor CRBF581, L. bicolor Origine and Pa. involutus) which took up over 80% of the available N even at the lowest C:N ratio. These ECM fungi are regarded as ‘nitrophilic’ species. For example, Pa. involutus ectomycorrhizas increased in abundance with increasing N availability along a gradient of N deposition in Alaska (Lilleskov et al., 2002), and sporocarp production of L. bicolor and Pa. involutus increased after N fertilization (Brandrud, 1995). It has been suggested that there may be a close relationship between C availability and N transfer in the symbiosis. Under high C availability the large C flux towards the fungal compartment ensures the assimilation of organic N, which is then released to the host plant. In contrast, under conditions of C depletion synthesis of organic N might be strongly down-regulated and ammonium would instead be transferred to the plant (Chalot et al., 2006). In contrast to the ‘nitrophilic’ species, N uptake by Pi. fallax and Pi. byssinum in culture was relatively low and these species only took up 40–65% of the available N even at a C:N ratio of 40:1. Lilleskov et al. (2002) and Toljander et al. (2006) found that mycorrhizas of Piloderma spp. declined in abundance along gradients of increasing N availability in the field, and both Kårén (1997) and Fransson et al. (2000) found that Pi. fallax mycorrhizas declined after N fertilization.

The observed reduction in pH resulting from uptake of ammonium from the growth medium was expected, and was in general negatively correlated with increasing biomass production/N uptake. Species such as Laccaria sp. and Pa. involutus had very similar and low pH values irrespective of C:N treatment, since most or all of the added ammonium was taken up from the growth medium even at low C:N ratio. All suilloid species showed larger pH reductions with increasing growth medium C:N ratio, i.e. increasing biomass production.

Mycelial C:N ratios in the present study were lower than the C:N ratio (20.2±0.8) of ECM mycelia collected from Norway spruce forest by Wallander et al. (2003). This is not altogether surprising as the mycelium in our experiment was much younger than that measured by Wallander and colleagues, and the ratio of cytoplasm to cell wall was probably higher. In addition, Wallander's hyphae were growing in sand-filled mesh bags where N availability was low.

Exudation

Exudation of carbon-based compounds was detected for six of the 17 isolates. Because determination was partly based on a colorimetric assay of residual glucose there was potential interference from pigments released into the culture medium. For example, Pa. involutus is known to produce oxalic acid (Yamaji et al., 2005), but released pigment in our experiment, and we did not detect exudation for this isolate. It is possible therefore that our data underestimate the extent of exudation by ECM fungi. Where exudation was detected it amounted to between 5% and 40% of the C in biomass and respiration, and tended to increase with increasing C:N ratio. This is a significant component of the C budget for these fungi. ECM fungi exude a range of different compounds, including low-molecular-weight organic acids, amino acids, sugars and siderophores (Arvieu et al., 2003; Holmström et al., 2004; van Hees et al., 2005). However, the rates and dynamics of mycorrhizal exudation are still largely unknown (van Hees et al., 2005). In natural substrates, exudation may directly (through affecting solubility and mobility) or indirectly (through stimulating microbial activity) increase nutrient availability to roots and mycorrhizas (Jongmans et al., 1997; Casarin et al., 2004; Jones et al., 2004). Hence, exudation has important implications for C-cycles, and is an additional mechanism by which positive feedback on nutrient capture might occur at elevated CO2.

Differences between isolates

Because intraspecific variation in pure culture physiology of ECM fungi has been widely reported (Anderson et al., 1999; Guidot et al., 2005), we compared three isolates of each of two fungal species (S. bovinus and L. bicolor). The three isolates of S. bovinus responded in a similar way with respect to growth and N uptake to increasing C:N ratio, but varied somewhat in respiration and particularly (UP65) in exudation at high C availability. The L. bicolor isolates also responded in a broadly similar fashion in terms of growth, but one (S238) had markedly reduced capacity for NH+4 uptake and declined in respiration at the highest C:N ratio. The distinctive behaviour of this isolate is of particular interest as it was obtained from the same sporocarp collection used to provide the monocaryotic strain S238N-H82, whose genome is currently being sequenced (Martin et al., 2004).

Conclusions

In this work we have demonstrated that different ECM fungal isolates respond differently in growth, respiration, exudation and N uptake to an increase in the C:N ratio of the growth medium, and that for many of the isolates the responses map onto what is known about the ecology of the species concerned. This suggests that, despite artificial culture conditions at a temperature much higher than would be experienced by ECM fungi in the field, the pure culture comparative approach adopted here is useful in defining functional groups of ECM fungi and in predicting how ECM fungi are likely to respond to elevated CO2. Clearly, absolute values for, for example, respiration obtained here cannot be scaled up to field conditions. An important drawback of this approach at present is that fungi in two families, Russulaceae and Thelephoraceae, which are known to be the symbionts in a high proportion of mycorrhizas in the field (Horton & Bruns, 2001), are not, thus far, easily grown in pure culture. This problem is compounded because members of Russulaceae overwhelmingly form so-called ‘smooth’ mycorrhizal exploration types (Agerer, 2001), i.e. with little or no extraradical mycelium, and it is critical to know how these species respond to elevated CO2.

Alberton et al. (2005) showed that plants colonized by a mixed ECM community had a much larger response to elevated CO2 than plants colonized by a single species. Our data on fungal growth and N uptake confirm that species-specific responses to increased C availability have the potential both to drive ECM community changes in response to elevated CO2 and to determine the nature of the feedbacks on nutrient supply to the host. In addition, fungal-species-specific responses to elevated CO2 demonstrated in a study by Gorissen & Kuyper (2000) suggest that S. bovinus has a higher demand for C than L. bicolor, but that L. bicolor has a higher demand for N. Our data are consistent with this interpretation. In this respect dual-culture experiments where hosts are grown with a range of contrasting ECM symbionts are a promising way forward.

Acknowledgements

We thank Trude Vrålstad, Francis Martin, Björn Lindahl and Andy Taylor for fungal isolates, and Duncan White and Pamela Parkin for technical support. Funding was provided by Carl Trygger's foundation, Sweden, and the UK Natural Environment Research Council. I.C.A. receives funding from the Scottish Executive Environment and Rural Affairs Department.

Footnotes

  • Editor: Christophe Tebbe

References

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