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Keywords:

  • chitinase;
  • disturbance;
  • functional similarity;
  • β-glucosidase;
  • mycorrhizosphere;
  • phosphatase;
  • physiological diversity;
  • Wilcoxina

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References
  10. Supporting Information

1. Clearcut logging results in major changes in ectomycorrhizal fungal communities, but whether this results in the loss of key functional traits, such as those associated with nutrient acquisition from soil organic matter, is unknown. Furthermore, little is known about the importance of resource partitioning in structuring ectomycorrhizal fungal communities following disturbance because most research on these communities has focussed on life history strategies. By studying functional traits, such as activities of enzymes involved in the catabolism of organic macromolecules in soil, we can determine whether a physiological potential for resource partitioning exists in pioneer ectomycorrhizal communities and whether severe disturbance affects these important ecosystem services.

2. We used activities of key hydrolytic enzymes in the ectomycorrhizospheres of Douglas-fir seedlings regenerating at clearcut sites as a functional trait to test whether these differed from those at recent wildfire sites or control forests. We sampled the most abundant types of ectomycorrhizas from 16-month-old seedlings from sites exposed to (i) low or (ii) high severity wildfire, (iii) sites that had been clearcut logged in the same year as the fire and (iv) sites that contained control stands of mature Douglas-fir. We expected differences in activities among ectomycorrhizas sampled from different disturbance treatments and among those formed by different fungal species.

3. In spite of large differences in soil chemistry, activities of acid phosphomonoesterase, N-acetylglucosaminidase and β-glucosidase, when averaged among the ectomycorrhizas sampled per site, were not affected by disturbance agent. However, activities varied up to sixfold among mycorrhizospheres of different fungal species on the same seedling. Multivariate analysis also indicated some consistent differences in enzyme profiles among ectomycorrhizas formed by specific fungal species, independent of treatment.

4. The finding that ectomycorrhizal fungal communities exposed to different disturbance agents are functionally similar with respect to the activities of three mycorrhizosphere enzymes supports the conclusion that complementarity exists among ectomycorrhizal fungi. The substantial physiological diversity among ectomycorrhizal fungi at the scale of an individual seedling’s root tips, especially at control mature forests, indicates the potential for resource partitioning within the ectomycorrhizal community and access to a wider range of nutrient sources by each seedling.

5. Functional similarity among ectomycorrhizal fungal communities across a disturbance severity gradient suggests that dry interior Douglas-fir forests are resilient to severe disturbances such as high severity wildfire and clearcutting with forest floor removal. Moreover, our results suggest that current harvesting practices emulate natural disturbances with respect to site-level mycorrhizosphere enzyme activity. The large variation in activity among fungal species, however, indicates that a substantial simplification of the fungal community through other perturbations, as expected with climate change, has the potential to affect ecosystem function.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References
  10. Supporting Information

Ectomycorrhizal (EcM) fungi are dominant members of the soil microbial community in coniferous forests worldwide, where they act as conduits for up to 60% of carbon inputs to soil (Godbold et al. 2006; Högberg & Read 2006). As with other fungi, their mode of nutrition is assimilative, involving the excretion of hydrolytic and oxidative enzymes and subsequent absorption of the breakdown products (Read & Perez-Moreno 2003). The molecules made available contribute to the nutrition not only of the fungal mycelium, but also of symbiotically associated host plants. Historically, this supply of nutrients to host plants was the focus of studies on the physiology of EcM associations (Smith & Read 2008). However, as a result of recent evidence that EcM fungi can form 30% of the microbial biomass in boreal forests (Högberg & Högberg 2002), the role of EcM fungi in decomposition and nutrient cycling is of increasing interest (Schimel & Bennett 2004; Cullings, Ishkhanova & Henson 2008; Talbot, Allison & Treseder 2008). Any disturbance that causes a major change in EcM fungal communities could influence not only subsequent seedling establishment, but also important ecosystem processes.

Although wildfires are natural disturbance agents in most temperate and sub-boreal forests, their extent and severity is predicted to increase in large areas of North America, in concert with longer summers (Hamann & Wang 2006). Under these conditions, stands of ecologically important tree species such as Pseudotsuga menziesii var. glauca (Beissn.) Franco (Interior Douglas-fir) may have difficulty regenerating in their current locations, particularly in drier zones (McKenzie et al. 2004; Griesbauer 2008). As a result EcM fungi may become increasing important both in priming nutrient cycling (Talbot, Allison & Treseder 2008) and facilitating seedling establishment by providing greater access to water (Plamboeck et al. 2007). In recent years, forest managers have aimed to protect biodiversity by mimicking the spatial and temporal distribution of natural disturbances, such as wildfire, at the landscape scale (Weber & Stocks 1998; Peltzer et al. 2000; Lauzon et al. 2006); however, both clearcutting (Jones, Durall & Cairney 2003) and wildfire (e.g. Grogan, Baar & Bruns 2000; Dahlberg 2002; Twieg, Durall & Simard 2007) result in major changes in the species composition of the EcM fungal community. The extent to which EcM fungal species are functionally similar, and the degree of response diversity within this guild of soil microbes (Elmqvist et al. 2003), will determine whether fungal species shifts result in a loss of ecosystem functions that could compromise ecological resilience to more severe disturbance (Folke et al. 2004).

Ectomycorrhizal fungal communities are stratified vertically and horizontally among soil microsites (e.g. Rosling et al. 2003; Lindahl et al. 2007; Tedersoo et al. 2008), with some of this spatial segregation correlated with soil chemistry (Lilleskov et al. 2002a; Parrent, Morris & Vilgalys 2006; Dickie, Richardson & Wiser 2009). Such patterns are suggestive of resource partitioning among fungal species, but could also be a result of species interactions and/or dispersal mechanisms (Peay, Kennedy & Bruns 2008 and references therein). For plants, differences among co-occurring species in physiological or morphological traits related to carbon, water or nutrient acquisition are used as evidence of resource partitioning or niche diversification (e.g. Keeley 1999; Moore 1999; Silvertown 2004; von Felten et al. 2009). For ectomycorrhizal fungi, colonization of different parts of the root system of a tree appear to distinguish niches (Gibson & Deacon 1988); however, few studies, especially ones that are field-based, have tested for differences among EcM fungi in traits related to resource acquisition (e.g. Lilleskov, Hobbie & Fahey 2002b; Jones et al. 2009). Recently McGill et al. (2006) recommended a ‘functional trait approach’ to community ecology to make progress in understanding how the species composition of communities relates to their abiotic environments. Given their importance in fungal nutrition, activities of extracellular enzymes can be considered a ‘functional trait’ for EcM fungi, as defined by McGill et al. (2006). By studying such a trait, it is possible to investigate not only functional diversity and complementarity within EcM fungal communities (Caldwell 2005; Koide, Courty & Garbaye 2007), but also how changes in EcM fungal communities might affect ecosystem function (Peay, Kennedy & Bruns 2008).

In the current study, our objective was to investigate the effects of disturbance by clearcutting or wildfire on the physiology of P. menziesii var. glauca ectomycorrhizas at several scales. As a relevant functional trait, we selected the activities of three hydrolytic enzymes involved in releasing absorbable forms of carbon, nitrogen and phosphorus from soil organic matter: β-glucosidase, N-acetylglucosaminidase and phosphomonoesterase. β-glucosidase is an enzyme that catalyses the final step in the breakdown of cellulose and other β-1,4 glucans to glucose (Sinsabaugh et al. 2008). We selected this enzyme because it is involved in the breakdown of more labile forms of soil organic carbon (SOC), the fraction of SOC that decreases by the highest proportion after fire (Fernandez, Cabaneiro & Carballas 1997; Knicker 2007). This reduction in labile SOC can result in changes in cellulolytic activities among soil fungi in burned soils (Bastias et al. 2009). N-acetylglucosaminidase acts on chitin and other β-1,4-linked glucosamine polymers (Sinsabaugh 2005), releasing absorbable forms of N. Because N-acetylglucosaminidase is inducible, higher activities can also indicate a switch to fungal utilization of more recalcitrant C sources from more labile ones (Boerner et al. 2008). Phosphomonoesterases break monoester bonds, releasing phosphate (Burns & Dick 2002). The activity of this enzyme is expected to change following wildfire because of heat-induced changes in the availability of phosphorus (Serrasolses, Romanya & Khanna 2008). In ecosystems with acidic soils, such as conifer forests, the activities of these three enzymes tend to be higher, and to vary more, than those of other commonly measured soil enzymes (Sinsabaugh et al. 2008). Given effects of wildfire on the availability of carbon, nitrogen and phosphorus, as well as on EcM fungal species composition, we predicted that activities of the extracellular enzymes would vary, at the community scale, among ectomycorrhizas that developed after clearcutting vs. wildfire. We also hypothesized that functional diversity, expressed as differences in the activities of the three enzymes in the mycorrhizosphere, would be detected among EcM fungal species at the same site. We expected variation among mycorrhizas would be more apparent at sites with living trees, where inoculum of a wider range of EcM fungi would be present, and at sites with intact forest floors, where vertical soil heterogeneity would be greater, thus providing increased opportunities for resource partitioning.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References
  10. Supporting Information

Site descriptions

The study was established over an 850 km2 area in the Cascade Dry, Cool variant of the Interior Douglas-fir biogeoclimatic zone (IDFdk2; Lloyd et al. 1990), along both sides of the North Thompson River Valley near the town of Barriere, British Columbia (51°12′N, 120°07′W). This was where the McLure fire burned 26 000 ha of forest in the summer of 2003 (see Fig. S1). The 16 sites were circum-mesic in soil moisture regime, on Luvisolic and Brunisolic soils (Soil Classification Working Group 1998), and ranged in elevation from 806 to 1268 masl (Table S1). The original canopy at all sites was dominated by P. menziesii var. glauca, with minor components of Pinus contorta var. latifolia Engle. Ex. S. Wats. (lodgepole pine). The understorey at control forests was dominated by Arnica cordifolia, Calamagrostis rubescens, Fragaria virginiana, Linnea borealis, Paxistima myrsenites and Spiraea betulifolia (Stark, Arsenault & Bradfield 2006). Herbaceous vegetation was similar on the clearcut sites, except that F. virginiana was less abundant and Epilobium angustifolium was much more abundant. Sites classified as ‘high severity burns’ had been exposed to a stand-destroying wildfire, with all needles on the trees consumed during the fire. At these sites, it was apparent from the exposed mineral soil that most of the forest floor had been consumed (0·7 ± 0·2 cm depth compared to 4·9 ± 0·6 cm in control mature forests). The remaining burned trees on these sites were salvage logged over the winter and early spring of 2004, before the initiation of our study. Sites classified as ‘low severity burns’ were exposed to surface-level fire. At these sites, at least 80% of the trees survived immediately after the fire. The understorey vegetation was consumed and there was evidence that a good portion of the forest floor was lost during the fire (1·8 ± 0·5 cm remaining). During the first two growing seasons after the fire (2004, 2005), Calamagrostis rubescens, E. angustifolium, Populus tremuloides and Pseudotsuga menziesii seedlings became established on the burned sites (Stark, Arsenault & Bradfield 2006).

Experimental design

Four replicate sites were selected of each of four disturbance treatments: control forests, forests disturbed by low severity burns, forests disturbed by high severity burns, and clearcuts. Each clearcut had two types of plots: those with the forest floor left undisturbed (3·8 ± 0·4 cm depth) and those with the organic horizons removed (referred to as ‘screefed’; 0 cm of forest floor). Using two different factorial arrangements of the treatments, we tested for effects of death and removal of tree stems, and wildfire. Each factorial arrangement included either the screefed or unscreefed clearcut treatment, not both, because these two treatments co-occurred on the same sites and, hence, were not independent (Table 1). The first analysis included the unscreefed plots and evaluated the effects of wildfire and the loss of aboveground tree parts. A significant effect of wildfire in this analysis could be attributed to either to a partial loss of forest floor, or to changes to the mineral soil caused by heating, or both. To parse these two effects we substituted the screefed plots, which lacked forest floor but had not been burned, into the second analysis. A significant interaction between the two factors in second analysis would indicate that loss of forest floor made a significant contribution to the effect of wildfire in this study.

Table 1.   Assignment of treatments in two separate two-factor analyses of variance to examine effects of (i) death and removal of tree stems and (ii) alteration of soil by fire
Analysis 1Trees presentTrees absent
Forest floor present and soil unburnedControl forestClearcut
Forest floor reduced due to burningLow severity burnHigh severity burn
Analysis 2Trees presentTrees absent
Soil unburnedControl forestScreefed clearcut
Soil burnedLow severity burnHigh severity burn

Seedlings

Pseudotsuga menziesii var. glauca seed (seedlot 48523 from the British Columbia Ministry of Forests Tree Seed Centre, Surrey, B.C.) was moist-stratified at 4 °C for 3–6 weeks. From late May to mid June 2004, 1000 seeds were planted 1 cm apart in 5-mm deep depressions in five 1·2 × 1·4 m subplots at each of the control forest and burned sites. At clearcuts, a total of 2000 seeds were planted: 1000 each in screefed and unscreefed subplots.

Soil sampling

The soil was sampled to the base of the A horizon in September 2005. Five subsamples per site were combined and sent to the British Columbia Ministry of Forests, Research Branch Analytical Laboratory in Victoria, B.C. where they were analysed for total C, available P (Bray) and pH (water) according to Kalra & Maynard (1991). Mineralizable N (as NH4 N) was determined using a 2-week anaerobic incubation (Bremner 1996).

Description of ectomycorrhizal tip community

In early October 2005, five seedlings were collected per site (10 per clearcut), one from each subplot, to characterize the EcM fungal community on the root systems (Fig. 1). Only three low severity sites were sampled because one of the sites had been logged in 2004. Root systems were stored at 4 °C until they were examined, up to 4 months later. Every root tip was examined under 400× and/or 1000× magnification. Ectomycorrhizas were grouped into morphotypes based on overall colour and shape of the mycorrhiza as well as the pattern of mantle hyphae and the diameter and appearance of extramatrical hyphae and cystidia (Goodman et al. 1996). Representative samples were frozen for subsequent molecular analysis.

image

Figure 1.  Seedlings of Pseudotsuga menziesii var. glauca growing at a high severity burn site.

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Enzyme assays

In addition to the seedlings collected for community analysis, two seedlings, one from each of two subplots, were excavated at each site (four from each clearcut site – two from each screefed and unscreefed plot, for a total of 40 seedlings), placed on ice and returned to the lab for the enzyme assays. Root systems were washed gently with tap water and then examined under a stereomicroscope. Every mycorrhizal morphotype was assayed, provided that at least five root tips or clusters of that type of mycorrhiza were present on the root system. A maximum of three, and in many cases only one or two, morphotypes fit this criterion on any seedling. Five to seven mycorrhizas, henceforth referred to as an assay group, were sampled per morphotype per seedling. We aimed to assay 21 mycorrhizal tips per seedling, so if only one or two morphotypes were sufficiently abundant on a root system, multiple assay groups of the same morphotype were analysed. For very small root systems only one or two assays groups could be tested, in total, per seedling. Replicate tips of each morphotype were selected from different regions of the root system.

Quantification of activities of β-glucosidase, N-acetylglucosaminidase and phosphomonoesterase was based on the release of 4-methylumbylliferone (MU) from MU-linked substrates following sequential exposure of individual ectomycorrhizal root tips to each substrate using the microplate assay method of Pritsch et al. (2004). Details are provided in Appendix S1. Each tip was assayed for phosphomonoesterase activity for 10 min, followed by N-acetylglucosaminidase for 20 min, and finally β-glucosidase for 20 min. Preliminary experiments showed that these were the minimum times to detect maximum activity and that the order of the incubations did not affect the results. After the final assay, to account for any major differences in size among the mycorrhizal tips, the surface area of each mycorrhiza was determined by using a Scanmaker 8700 (Microtek Lab Inc., Carson, CA, USA) scanner, followed by image analysis using WinRHIZO Basic (v2005b; Regent Instruments Inc., Québec, Canada). Mycorrhizas were frozen at −80 °C in sterile DNA-free water for later DNA analysis.

Molecular analysis

DNA was extracted and amplified separately from one to five EcM root tips (mycorrhizas) per enzyme assay group, depending on the variability within the morphotype, and from one to five additional root tips per morphotype per treatment from the seedlings used to describe the EcM community. The entire ITS region of fungal rDNA plus portions of the small subunit and large subunit genes was amplified using primer pairs ITS1f and 4B (White et al. 1990; Gardes & Bruns 1993) or NSI1 and NLC2 (Martin & Rygiewicz 2005), cleaned and sequenced. Each sequence was compared with online databases associated with the National Center for Biological Information (NCBI) and User-friendly Nordic ITS Ectomycorrhiza (UNITE; Kõljalg et al. 2005) using the megablast algorithm of the Basic Local Alignment Search Tool (BLAST; Altschul et al. 1990). We labelled fungi with species names if their ITS sequences matched a named sporocarp or voucher specimen in one of the databases with at least 97% sequence similarity over at least 500 base pairs with an 80% query coverage. Details of extraction, amplification, and processing of sequence data are provided in Appendix S1.

Data analysis

To compare mean enzyme activities among treatment (disturbance) types at the level of the sampled EcM community, activities expressed as MU mm−2 min−1 for each tip were averaged among the five to seven tips per assay group; assay group means from both seedlings per site were then considered subsamples within experimental units, with sites being the experimental units. The GLM procedure in sas v. 9·1 (Cary, NC, USA) was used to analyse the main effects and their interactions, for each response variable separately, with these effects tested against experimental error. Response variables were the mean activity for each enzyme (three variables) and the mean pair-wise ratio of each enzyme to each of the others (three variables). Least square means were used because there were different numbers of assay groups per site, resulting in an unbalanced design. For a few cases where log transformation of data did not achieve anova assumptions, Kruskal–Wallis tests were conducted using the NPAR1WAY procedure.

To examine the potential effects of disturbance treatments on community enzyme activities in a multivariate context, sites were ordinated according to the enzyme activities associated with the sampled tips. The values per tip were averaged for each assay group; then the means of those averages were used as individual site values. Nonmetric multidimensional scaling (NMS) was used in PC-ORD version 4 (MjM Software Design, Gleneden Beach, OR, USA), with Sorensen distance measures. Multi-Response Permutation Procedure (MRPP) was also used to look for potential multivariate (three variables – mean activities of each of the three enzymes) differences among disturbance types (see Appendix S1 for details).

To examine the contributions of long distance exploration types to community-level responses of mycorrhizosphere enzyme activities to disturbance, we repeated both the univariate and multivariate analyses described above on two additional data sets. The first data set excluded the 18 assay groups for which we had no molecular identification. This was to ensure that we had eliminated any assay groups that could have been comprised of long-distance exploration types. From this data set, we then removed any Rhizopogon spp. and Suillus lakei assay groups, the two long-distance exploration types encountered in this study.

Two fungal taxa were found regularly enough to test whether disturbance type affected their associated enzyme activities: Pyronemataceae 1, and Wilcoxina spp. Because these taxa were not found at all sites, activities associated with Pyronemataceae 1 mycorrhizas were compared among low severity burn sites, high severity burn sites and clearcuts, and those of Wilcoxina spp. among control forests, high severity burn sites, and clearcuts. Clearcut data were from either the screefed or unscreefed subplot, depending on occurrence, at each site. Activities of each enzyme, as well as ratios of the natural logs of the activities of pairs of enzymes, were averaged first by assay group, then among all assay groups of these taxa on both seedlings per site.

To test whether ectomycorrhizas formed by different fungi on the same seedling were associated with different enzyme activities, we examined data from all seedlings where at least two identified mycorrhizal types were assayed from the root system. Because inference was at the scale of each seedling, with its unique community of EcM fungal associates, activities of individual root tips were used as replicates (N = 5–7 for each type of mycorrhiza). See Appendix S1 for additional details on these analyses.

Potential physiological relationships among only the most frequently encountered types of ectomycorrhizas, across different sites and treatments, were explored using NMS as described above. Enzyme activities were averaged first across replicate tips for each assay group, then by seedling, for each fungal species that was observed on roots from three or more sites.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References
  10. Supporting Information

Soil chemistry

Wildfire caused major changes to soil chemistry in the upper soil, with no difference between the two severity treatments (one-factor anova, followed by orthogonal contrasts; Table 2). Burning was associated with lower soil carbon and mineralizable nitrogen and higher extractable phosphorus and pH. There was no effect of disturbance on total N (mean = 0·480 ± 0·097%; P = 0·13). Clearcutting alone cause no significant effects on soil chemistry relative to control forests.

Table 2.   Chemical characteristics of surface soils at sites from which Pseudotsuga menziesii var. glauca seedlings were sampled
TreatmentTotal carbon (mg g−1 d wt)Mineralizable nitrogen (μg NH4 g−1 d wt d−1)Bray extractable phosphorus (μg g−1 d wt)pH
  1. Data are presented as means per site ± 1 SEM. N = 2 for low-severity burn sites and 4 for the other treatments. Means within a column followed by different letters were significantly different at P < 0·01 according to planned orthogonal contrasts: (i) unburned vs. burned sites; (ii) control vs. clearcut sites; and (iii) low vs. high severity burn sites.

Control forest201 ± 44 b21·7 ± 6·5 b100 ± 30 a5·9 ± 0·2 a
Low severity burn47 ± 4 a2·2 ± 0·0 a261 ± 7 b7·4 ± 0·0 b
High severity burn64 ± 24 a2·8 ± 1·0 a265 ± 43 b7·0 ± 0·4 b
Unscreefed clearcut146 ± 31 b12·4 ± 4·7 b132 ± 36 a5·3 ± 0·2 a
P from one-factor anova0·040·0030·020·001

Types of ectomycorrhizas sampled

Of the 90 assay groups tested, we were successful in amplifying and sequencing fungal DNA from a single fungus from 72 of these. From these DNA sequences we delineated 29 operational taxonomic units (OTUs) – a proxy for fungal species (O’Brien et al. 2005) (Appendix S1). There was a considerable overlap in the EcM fungi sampled from different disturbance types (Table S2). In total 13 OTUs were sampled from unscreefed clearcut plots, 12 from control forests, 11 from screefed clearcut plots, five from high-severity burn sites and four from low-severity burn sites. Many types of ectomycorrhizas were found in samples from one site only. Ectomycorrhizas formed by only five fungal species were assayed from three or more sites: Wilcoxina rehmii, Cenococcum geophilum, Rhizopogon rudus, Suillus lakei, and a non-Wilcoxina ascomycete from the Pyronemataceae, henceforth referred to as Pyronemataceae 1. The most widely distributed EcM fungi on the assay seedlings were Wilcoxina rehmii, which was found at eight sites representing all treatments, and Pyronemataceae 1, which was found at nine sites representing all treatments except control forests. When more extensive sampling was carried out to characterize the EcM community, these two fungi, grouped together as Pyronemataceous morphotypes, were found to dominate the EcM fungal community on root tips (>80% of EcM root tips) from clearcut or burned sites, but were less abundant on seedlings in control forests (Table 3). Mycorrhizas assayed for extracellular enzyme activities included all major taxa detected in the more extensive survey.

Table 3.   Relative abundance (%) of ectomycorrhizas on Pseudotsuga menziesii var. glauca seedlings germinated and colonized in situ for 16 months at Interior Douglas-fir sites following different kinds of disturbance
 Control forestLow severity burnHigh severity burnClearcutScreefed clearcut
  1. Data are presented as means per site ± 1 SEM. N = 3 for low-severity burn sites and N = 4 for the other treatments.

Pyronemataceae (including Wilcoxina spp. and Pyronemataceae 1)14 ± 791 ± 898 ± 080 ± 583 ± 3
Cenococcum spp.37 ± 60 ± 01 ± 05 ± 13 ± 2
Boletaceae (including Rhizopogon spp. and Suillus lakeii)19 ± 89 ± 70 ± 01 ± 14 ± 4
Amphinema spp.12 ± 100 ± 00 ± 04 ± 13 ± 2
Russula spp.13 ± 60 ± 00 ± 00 ± 00 ± 0
Melinomyces bicolor0 ± 01 ± 10 ± 11 ± 11 ± 1
Thelephora spp.0 ± 00 ± 00 ± 02 ± 21 ± 1
Tomentella spp.0 ± 00 ± 00 ± 03 ± 20 ± 0

Effects of disturbance type on enzyme activities

There were no significant effects of disturbance from clearcutting or wildfire on the activities of any of the enzymes when activities were averaged across assay groups within a site. In particular, we found no effect that could be attributed to loss of trees, reduction in forest floor thickness, or other effects of burning on remaining mineral soil (P ≥ 0·12 for all main effects and interactions for each enzyme and their ratios in both anovas of Table 1). Furthermore, when all assay groups were included, sites did not group well according to disturbance type in the NMS analysis, although the control forests tended to be located on the left of axis 1 when screefed plots were excluded (Fig. 2a; Table S3) and MRPP tests indicated that disturbance treatments did not differ in extracellular enzyme activities (P ≥ 0·14; Table S4). Removing the 18 unidentified assay groups from the data set did not affect either the univariate (Table S5) or multivariate results (Tables S3 and S4; Fig. 2c,d); however when the six assay groups of Rhizopogon and Suillus mycorrhizas were also removed, the MRPP (P < 0·03), but not the anovas, detected a significant treatment effect (Table S4; Fig. 2e,f). The difference among treatments appeared to be due to a halving of the average phosphatase, together with a lesser reduction in N-acetylglucosaminidase activities, for the low severity fire treatment, whereas mean values for other treatments for all three enzymes changed little with removal of the Rhizopogon and Suillus assay groups (Table S5). This was reflected in the tighter ordination grouping of the low severity sites at the ends of the axes correlated to lower phosphatase when using the most reduced data set (Fig. 2e,f). This reduced data set contained only four assay groups for the low severity burn treatment, all comprised of Pyronemataceous mycorrhizas, compared to 13–15 diverse assay groups for the other treatments (Table S2).

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Figure 2.  Nonmetric multidimensional scaling analysis of all sites using Sorensen distance measures for mean activities of the three enzymes per site, (a) the complete data, including unscreefed clearcut plots; (b) the complete data, including screefed clearcut plots; (c) data set with assay groups of unidentified fungi removed, including unscreefed plots; (d) data set with assay groups of unidentified fungi removed, including screefed plots; (e) data set with assay groups of long distance types and unidentified fungi removed, including unscreefed plots; (f) data set with assay groups of long distance types and unidentified fungi removed, including screefed plots.

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By contrast to the community-level analysis, disturbance type did affect activities associated with the two most frequently sampled types of ectomycorrhizas when examined individually. Mean activities of β-glucosidase associated with Wilcoxina spp. mycorrhizas were higher at control forests than clearcut or severely burned sites (Fig. 3a; F = 14·73, P = 0·0080), whereas phosphatase (F = 0·17, P = 0·84) and N-acetylglucosaminidase (F = 2·75, P = 0·16) activities did not differ. The relative activities of the three enzymes were not affected by disturbance (Kruskal–Wallis P > 0·7 for all three enzyme ratios). Activities of the three enzymes associated with mycorrhizas formed by Pyronemataceae 1, when considered separately, were similar among disturbance types (P > 0·2; Fig. 3b); however, their relative activities varied. Specifically, ratios of phosphatase to β-glucosidase (χ2 = 6·5; P = 0·039) and phosphatase to N-acetylglucosaminidase (χ2 = 7·2; P = 0·027) were lower on Pyronemataceae 1 mycorrhizas in the lower severity burns than in the clearcuts (Fig. 3b).

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Figure 3.  Activities of acid phosphomonoesterase (AP), N-acetylglucosaminidase (NAG) and β-glucosidase (BG) associated with (a) Wilcoxina spp. or (b) Pyronemataceae 1 mycorrhizas on 16-month-old Pseudotsuga menziesii var. glauca seedlings germinated and colonized in situ at Interior Douglas-fir sites following different disturbance types. Bars associated with different letters were significantly different for that enzyme according to a one-factor anova and Bonferroni analysis. Data are presented as means per site ± 1 SEM. N = 3 sites in all cases, except for Wilcoxina from high severity burn sites, where N = 2.

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Differences among mycorrhizal types at the seedling scale

On individual seedlings (average root dry wt 102 ± 18 mg), ectomycorrhizas formed by different fungal species were associated with different levels and types of enzyme activities. Of the seedlings from which we assayed two or more different types of mycorrhizas, 90% exhibited differences in activities or ratios of activities of the three enzymes among mycorrhizas (data not shown). The mycorrhizas on most seedlings differed in N-acetylglucosaminidase (76%) and phosphatase (67%) activities; however, only 38% differed with respect to β-glucosidase activities. For a majority of the seedlings (71%), different ectomycorrhizas were also associated with different relative amounts of the three enzymes. N-acetylglucosaminidase : phosphatase ratios differed in 67%, β-glucosidase : phosphatase ratios in 57% and β-glucosidase : N-acetylglucosaminidase ratios in 52% of seedlings. As examples, Figs 4 and 5 illustrate the results for all seedlings with three types of mycorrhizas. At control forests, activities of all three enzymes varied as much as five- to sixfold among different types of ectomycorrhizas on one seedling (Fig. 4). For some EcM fungal species, mycorrhiza-associated phosphatase activities were six times as high as N-acetylglucosaminidase activities on the same root tips; whereas, for ectomycorrhizas formed by other fungal species on the same seedling, phosphatase activities were half as much as N-acetylglucosaminidase activities (Fig. 4b). Enzyme activities tended to vary less among types of ectomycorrhizas on seedlings sampled from clearcut sites (Fig. 5).

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Figure 4.  Activities of acid phosphomonoesterase (AP), N-acetylglucosaminidase (NAG) and β-glucosidase (BG) associated with Pseudotsuga menziesii var. glauca mycorrhizas from three seedlings (a–c) from control forests. Asterisks indicate significant differences among mycorrhizas formed by different fungi on each seedling for the activity of the enzyme indicated, as detected by Kruskal–Wallis tests. *< 0·05; **< 0·01; ***< 0·001. Data are presented as means per site ± 1 SEM. N = 5–7. Differences were also detected in ratios of activities between pairs of enzymes. Seedling a: BG/AP*, BG/NAG**, NAG/AP***; Seedling b: BG/AP**, BG/NAG nsd, NAG/AP**; Seedling c: BG/AP nsd, BG/NAG**, NAG/AP**.

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Figure 5.  Activities of acid phosphomonoesterase (AP), N-acetyl glucosaminidase (NAG) and β-glucosidase (BG) associated with Pseudotsuga menziesii var. glauca mycorrhizas from four seedlings from unscreefed (a, b, d) or screefed (c) plots on clearcut sites. Asterisks indicate significant differences among mycorrhizas formed by different fungi on each seedling for the activity of the enzyme indicated, as detected by Kruskal–Wallis tests. *< 0·05; **< 0·01; ***< 0·001. Data are presented as means per site ± 1 SEM. N = 5–7. Differences were also detected in ratios of activities between pairs of enzymes. Seedling a: BG/AP nsd, BG/NAG*, NAG/AP**; Seedling b: BG/AP*, BG/NAG nsd, NAG/AP**; Seedling c: BG/AP nsd, BG/NAG*, NAG/AP*; Seedling d: BG/AP**, BG/NAG**, NAG/AP*.

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The irregular distribution of EcM fungal species among seedlings meant that we could not easily test whether differences in enzyme activities associated with their mycorrhizas were consistent across seedlings, sites and treatments. Therefore we used NMS to compare activities associated with types of ectomycorrhizas found at three or more sites (Fig. 6). While NMS indicated significance of each of one-, two-, and three-dimensional structures (= 0·002, 0·032 and 0·028 respectively for the Monte Carlo test), the relative reduction in stress of a three-dimensional solution beyond a two-dimensional one was negligible. Hence, a two-dimensional ordination was used. Phosphatase [Pearson’s correlation coefficient (r) = −0·651] and N-acetylglucosaminidase (= −0·470) were negatively correlated and β-glucosidase was positively correlated (= 0·399) to axis 1. Phosphatase (= 0·741) and N-acetylglucosaminidase (= 0·350) were positively correlated, and β-glucosidase was negatively correlated (= −0·142), to axis 2.

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Figure 6.  Nonmetric multidimensional scaling analysis of the enzyme profiles of all ectomycorrhizal types found at three or more sites using relative Sorensen distance measure. Control forest– green symbols; low severity burn – yellow; high severity burn – red; unscreefed clearcut – blue and screefed clearcut – black. Each data point represents the mean activity for each type of ectomycorrhiza on one seedling. At three sites, the same type of mycorrhiza was found on both sampled seedlings. Pairs of data points from two seedlings at one site have the same horizontal or vertical strikethrough lines. In all other cases, the data points represent mycorrhizas of that fungus from only one seedling per site. Note that two square data points, one red, one black, partially overlap in the center of the figures. R2 = 0·67 for axis 1 and 0·32 for axis 2.

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The NMS analysis illustrated that some differences in enzyme profiles among mycorrhizal types were consistent enough to be detected across treatments. In particular, enzyme profiles of mycorrhizas formed by Suillus lakeii and Rhizopogon rudus, two closely related species, grouped together on the left side of axis 1, whereas mycorrhizas formed by Pyronemataceae 1 were further right on axis 1. The two Suillus/Rhizopogon samples from control forest sites were furthest to the left. Mycorrhizas of Pyronemataceae 1 were not encountered in sufficient numbers to assay at control forest sites, so some of the difference in enzyme profile could be due to site differences. However, samples from both groups were found at screefed clearcut and low severity burn sites, and these points were clearly separated.

The NMS analysis also showed that ectomycorrhizas formed by some fungal species had a wider range of enzyme profiles than others. The enzyme profiles of Wilcoxina rehmii and Cenococcum geophilum mycorrhizas showed a wider scatter than those of other species, and did not group by disturbance type. The scatter was especially wide for W. rehmii and C. geophilum mycorrhizas from different control forests. Activities of W. rehmii mycorrhizas from control forests varied considerably along both axes 1 and 2; C. geophilum activities from forested sites showed substantial variation along axis 2 only.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References
  10. Supporting Information

Disturbance treatments and community-level enzyme activites

In contrast to our hypothesis, we did not find major differences in the activities of three extracellular enzymes among ectomycorrhizal communities that developed after clearcutting vs. wildfire. We had predicted that activities would vary with disturbance type for two reasons. First, because of the relative availability of different inoculum sources (i.e. spores vs hyphae associated with live ectomycorrhizas), we expected different species of EcM fungi to colonize seedlings in sites with living trees (low severity burn and control forests) and in sites with thicker forest floors (control forests and unscreefed clearcut plots) than in more disturbed sites (Jones, Durall & Cairney 2003). Our detailed examination of the EcM fungal community on root tips confirmed that both clearcutting and wildfire had influenced the EcM fungal species colonizing the roots and, as a result, different types of ectomycorrhizas were present in our enzyme assays from different treatments. For example, Pyronemataceae 1 was not found on assay seedlings from control forests, and Cenococcum spp. and Thelephora spp. mycorrhizas were not sufficiently abundant on burned sites to be sampled from assay seedlings from those treatments. We expected to see a change in overall EcM fungal community enzyme profiles as a result of these changes in the relative abundance of different EcM fungal species in the communities. Lab-based studies have demonstrated that EcM fungal species differ considerably in their ability to use different forms of mineral nutrients (e.g. Finlay, Frostegård & Sonnerfeldt 1992). Furthermore, earlier work in mature beech, oak and spruce forests found that mycorrhizas associated with different EcM fungi had different enzyme profiles (Courty et al. 2005; Buée et al. 2007; Rineau & Garbaye 2009).

The second reason for expecting differences in mycorrhiza-associated enzyme activities among disturbance treatments is that the physiology of individual EcM fungal species may vary depending upon abiotic factors in their environment. Changes in soil structure and chemistry alter the excretion of enzymes by EcM fungi, roots and rhizosphere microflora (Boerner et al. 2008; Rineau & Garbaye 2009). For example, acid phosphatase activities of roots and fungi are typically higher in environments with low orthophosphate (Shieh, Wodzinsk & Ware 1969; Gilbert et al. 1999). Chitin has been demonstrated to be an important N source for ericoid mycorrhizal fungi in organic soils (Kerley & Read 1995), and Buée et al. (2007) found higher N-acetylglucosaminidase activities associated with ectomycorrhizas in decayed wood microsites in a mature oak forest that had been heavily colonized by saprotrophic fungi. Activities of β-glucosidase, which cleaves cellobiose, are often correlated with soil organic matter (Chang, Chung & Tsai 2007). However, disturbance effects were not observed at the scale of the sampled ectomycorrhizal community in our study, in spite of increases in extractable phosphorus, together with decreases in carbon content and depth of forest floor, at burned sites.

Our results appear to represent an example of functional complementarity among EcM fungi across treatments, resulting in similarity in overall enzyme activities among EcM fungal communities on these young seedlings, a situation that has also been reported for saprophytic soil fungi (Setala & McLean 2004; Deacon et al. 2006). Other researchers have found complementarity for mycorrhizosphere enzyme activities within EcM communities (Buée, Vairelles & Garbaye 2005; Courty et al. 2005), but our finding that this can result in similar overall enzyme profiles between very different EcM communities appears to be unique. Other explanations for the absence of a relationship between community-scale enzyme profiles and soil chemistry include that (i) influences from factors such as seedling phenology (Courty, Breda & Garbaye 2007) outweigh that of soil chemistry, (ii) mycorrhizas respond to finer scale differences in soil chemistry than are detectable with conventional soil sampling (Schimel & Bennett 2004) or (iii) responses in the mycorrhizosphere to changes in soil chemistry are smaller than those of extramatrical hyphae. The latter situation would be expected to be most obvious for long-distance exploration-type mycorrhizas (LDETs) (Agerer 2001). Therefore, we tested whether inclusion of Suillus lakei and Rhizopogon spp., the two long distance mycorrhizas found in the current study, dampened our ability to detect differences in community-level enzyme profiles among disturbance types. When both the LDETs and all unidentified mycorrhizas were removed from the data set, multivariate analyses detected differences among treatments, whereas this was not the case when only the unidentified mycorrhizas were removed. This may indicate that Suillus and Rhizopogon mycorrhizas are filling an important complementary role within the community; however by removing both the unidentified and LDETs from the data set, the number of assay groups in the low severity burn treatments were reduced to a much smaller and less diverse set than the other treatments. Because it was a reduction in phosphatase activities at the low severity burn sites with removal of the LDETs that seemed to drive the significant result in this analysis compared to those on the more complete data sets, we must interpret these results with caution.

Although we found little evidence that the EcM fungal community responded uniformly or collectively to changes in environment as did Buée, Vairelles & Garbaye (2005), there was evidence that enzyme activities varied as predicted when ectomycorrhizas formed by the same fungal species or group of species were considered. For example, phosphatase activities associated with Pyronemataceae 1 mycorrhizas were lower, relative to the other two extracellular enzymes, at the high-phosphorus burned sites than clearcut sites. β-glucosidase associated with Wilcoxina spp. mycorrhizas was higher at control forests, with their higher carbon contents, than at burned sites. The placement of Suillus and Rhizopogon mycorrhizas in the NMS analysis indicates that, as expected, they produced more phosphatase at control forests than at burned sites. This is consistent with Courty et al. (2005) and Buée et al. (2007), who found that enzyme profiles of mycorrhizas formed by the same fungus could vary among sites, implying that mycorrhizal fungi have a degree of physiological plasticity. Such plasticity is important for ecosystem resilience after severe disturbance. The finding that enzyme profiles of individual types of ectomycorrhizal changed as expected, but those of the overall sampled community did not, provides additional support for the idea that EcM fungal species within a community are functionally complementary.

Functional diversity among ectomycorrhizal fungi

We detected clear differences in enzyme profiles among ectomycorrhizas formed by different fungal species on the same seedling and, as predicted, differences were greater at control forests. Previous studies have detected differences in enzyme profiles among ectomycorrhizas formed by different fungi at the plot scale (Buée, Vairelles & Garbaye 2005; Courty et al. 2005; Buée et al. 2007; Rineau & Garbaye 2009), but not at the scale of a single host plant. Although a diverse assemblage of EcM fungi increases seedling growth only under some conditions and not others (Jonsson et al. 2001; Baxter & Dighton 2005), differential contribution to nutrient release from soil organic matter could be one mechanism responsible. Such complementary may be especially important at low fungal species richness (Setala & McLean 2004), such as occurs on very young seedlings. It would also contribute to resilience in ecosystem response to disturbance, which often causes nutrient depletion.

Our study design, with sites distributed over 850 km2, provided an opportunity to recognize consistent physiological differences among EcM fungi that were independent of treatment or site. Few studies have attempted to compare the physiology of EcM fungal species at this scale (e.g. Lilleskov et al. 2002a). Instead, the vast majority of studies have been conducted at single sites or plots to minimize variability. In the current study, while some taxa exhibited a large degree of phenotypic plasticity, others appeared to have more consistent enzyme profiles. For example, mycorrhizas formed by Suillus lakeii and Rhizopogon rudus exhibited profiles that were clearly distinct from mycorrhizas of Pyronemataceae 1, indicating that these may be taxonomically based differences.

Although enzyme assays on mycorrhizas excised in the field can be criticized because both carbon supply from the host and extramatrical hyphae extending into the soil are severed, these measurements have the power to provide important information about relevant physiological differences among EcM fungal species and their associated microflora (Buée, Vairelles & Garbaye 2005; Courty et al. 2005; Buée et al. 2007; Rineau & Garbaye 2009). The enzymes that we investigated here are extracellular, surface-bound enzymes and, therefore, do not depend on a continuous supply of carbon from the host (Burns & Dick 2002). Hence the activities measured represent the suite of enzymes present on surfaces at the time of sampling. Furthermore, there was no evidence that differential hydrophobicity of the mantles among mycorrhizal types influenced our results. The mantles of many LDETs are hydrophobic (Agerer 2006), yet none of the root tips in our assay were buoyant in the assay solutions and enzyme activities associated with the LDET mycorrhizas were within the range of activities of other types of mycorrhizas, if not slightly higher.

It is important to continue to develop techniques whereby activities of extramatrical hyphae can be studied because these are major sites of nutrient absorption (Bending & Read 1995; Timonen & Sen 1998). Nevertheless, some localized volumes of soil may contain mycorrhizal root tips without extensive extramatrical hyphae of the corresponding fungus (Genney, Anderson & Alexander 2006). In such regions, enzymes associated with the mycorrhizosphere may well serve important roles in nutrient acquisition. This is true for mycorrhizas of all exploration types. Even classic long-distance exploration types, whose rhizomorphs and mycelial fans extend considerable distances from the root, such as those formed by Sullius spp. and Rhizopogon spp. (Agerer 2001, 2006), have considerable quantities of hyphae close to the mycorrhizal tip (V. Ward, B. Twieg, personal observations). These hyphae are in an ideal location to absorb nutrients released by extracellular enzymes in the mycorrhizosphere. In summary, we feel that the mycorrhizal root tip assay, which provides a fine-scale snapshot of one type of activity in the mycorrhizosphere of naturally colonized roots, when combined with information from other approaches, will make a significant contribution to our understanding of the location, scale and importance of biodiversity to mineralization processes in soil.

Conclusion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References
  10. Supporting Information

Three conclusions emerge from this study. First, the degree of divergence in enzyme activities among EcM fungi depends upon the scale at which it is considered. For a single seedling, associating with several EcM fungal species, rather than one, will likely increase the diversity of enzyme activity associated with its root system. On the other hand, there is sufficient functional complementarity among ectomycorrhizas formed by different fungal species that, at a plot or stand level, enzyme profiles may not differ substantially, even when the fungal species composition does. Similar results were recently found by Bledsoe and collaborators (C. S. Bledsoe, pers. comm.): uptake of 15N-ammonium varied widely among EcM morphotypes within a soil core, but the range of uptake rates was similar among cores. Second, our finding that functional diversity with respect to degradation of organic matter occurs even among early-colonizing fungi means that there is potential for resource partitioning after major disturbances. Third, there appears to be sufficient response diversity (see Elmqvist et al. 2003) for ectomycorrhiza-associated hydrolytic enzyme activity that these dry Douglas-fir ecosystems can be considered resilient to disturbance by clearcutting or high severity wildfire. The species shifts in EcM fungal communities commonly observed following these disturbances do not appear to result in disruptions to this important ecosystem service or contribute to the low regenerative capacity of dry interior Douglas-fir forests.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References
  10. Supporting Information

This research was funding by a Special Research Opportunity Grant (MJ, DD, SS) and a Discovery Grant (MJ) from the Natural Sciences and Engineering Research Council (NSERC) of Canada. Anne Bernhardt, Ben Chester, Julie Deslippe, Shufu Dong, Alanna Leverrier, and Adam McCaffrey provided excellent field assistance. We are also grateful to Jean Roach, Duane Hennig, Brent Olsen, Norm Fennell, Kelly Low, and Carmen Smith for their assistance with site selection and road access, Bill Clark for excellent technical assistance, and Pierre-Emmanuel Courty for helpful discussions and advice.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References
  10. Supporting Information
  • Agerer, R. (2001) Exploration types of ectomycorrhizae. A proposal to classify ectomycorrhizal mycelial systems according to their patterns of differentiation and putative ecological importance. Mycorrhiza, 11, 107114.
  • Agerer, R. (2006) Fungal relationships and structural identity of their ectomycorrhizae. Mycological Progress, 5, 67107.
  • Altschul, S.F., Gish, W., Miller, W. & Wootton, J.C. (1990) Basic local alignment search tool. Journal of Molecular Biology, 215, 403410.
  • Bastias, B.A., Anderson, I.C., Ignacio Rangel-Castro, J., Parkin, P.I., Prosser, J.I. & Cairney, J.W.G. (2009) Influence of repeated prescribed burning on incorporation of 13C from cellulose by forest soil fungi as determined by RNA stable isotope probing. Soil Biology & Biochemistry, 41, 467472.
  • Baxter, J.W. & Dighton, J. (2005) Phosphorus source alters host plant response to ectomycorrhizal diversity. Mycorrhiza, 15, 513523.
  • Bending, G.D. & Read, D.J. (1995) The structure and function of the vegetative mycelium of ectomycorrhizal plants V. Foraging behaviour and translocation of nutrients from exploited litter. New Phytologist, 130, 401409.
  • Boerner, R.E.J., Giai, C., Huang, J. & Miesel, J.R. (2008) Initial effects of fire and mechanical thinning on soil enzyme activity and nitrogen transformations in eight North American forest ecosystems. Soil Biology & Biochemistry, 40, 30763085.
  • Bremner, J.M. (1996) Nitrogen availability indexes. Methods of Soil Analysis, Part 2, (ed. C.A.Black), pp. 13241345, American Society of Agronomy, Madison.
  • Buée, M., Vairelles, D. & Garbaye, J. (2005) Year-round monitoring of diversity and potential metabolic activity of the ectomycorrhizal community in a beech (Fagus silvatica) forest subjected to two thinning regimes. Mycorrhiza, 15, 235245.
  • Buée, M., Courty, P.E., Mignot, D. & Garbaye, J. (2007) Soil niche effect on species diversity and catabolic activities in an ectomycorrhizal fungal community. Soil Biology & Biochemistry, 39, 19471955.
  • Burns, G.B. & Dick, R.P. (2002) Enzymes in the Environment; Activity, Ecology and Applications. Marcel Dekker, New York.
  • Caldwell, B.A. (2005) Enzyme activities as a component of soil biodiversity: a review. Pedobiologia, 49, 637644.
  • Chang, E.H., Chung, R.S. & Tsai, Y.H. (2007) Effect of different application rates of organic fertilizer on soil enzyme activity and microbial population. Soil Science & Plant Nutrition, 53, 132140.
  • Courty, P.E., Breda, N. & Garbaye, J. (2007) Relation between oak tree phenology and the secretion of organic matter degrading enzymes by Lactarius quietus ectomycorrhizas before and during bud break. Soil Biology & Biochemistry, 39, 16551663.
  • Courty, P.E., Pritsch, K., Schloter, M., Hartmann, A. & Garbaye, J. (2005) Activity profiling of ectomycorrhiza communities in two forest soils using multiple enzymatic tests. New Phytologist, 167, 309319.
  • Cullings, K., Ishkhanova, G. & Henson, J. (2008) Defoliation effects on enzyme activities of the ectomycorrhizal fungus Suillus granulatus in a Pinus contorta (lodgepole pine) stand in Yellowstone National Park. Oecologia, 158, 7783.
  • Dahlberg, A. (2002) Effects of fire on ectomycorrhizal fungi in Fennoscandian boreal forests. Silva Fennica, 36, 6980.
  • Deacon, L.J., Pryce-Miller, E.J., Frankland, J.C., Bainbridge, B.W., Moore, P.D. & Robinson, C.H. (2006) Diversity and function of decomposer fungi from a grassland soil. Soil Biology & Biochemistry, 38, 720.
  • Dickie, I.A., Richardson, S.J. & Wiser, S.K. (2009) Ectomycorrhizal fungal communities and soil chemistry in harvested and unharvested temperate Nothofagus rainforests. Canadian Journal of Forest Research, 39, 10691079.
  • Elmqvist, T., Folke, C., Nystrom, M., Peterson, M., Bengtsson, J., Walker, B. & Norberg, J. (2003) Response diversity, ecosystem change, and resilience. Frontiers in Ecology & the Environment, 1, 488494.
  • von Felten, S., Hector, A., Buchmann, N., Niklaus, P.A., Schmid, B. & Scherer-Lorenzen, M. (2009) Belowground nitrogen partitioning in experimental grassland plant communities of varying species richness. Ecology, 90, 13891399.
  • Fernandez, I., Cabaneiro, A. & Carballas, T. (1997) Organic matter changes immediately after a wildfire in an Atlantic forest soil and comparison with laboratory soil heating. Soil Biology & Biochemistry, 29, 111.
  • Finlay, R.D., Frostegård, A. & Sonnerfeldt, A.M. (1992) Utilization of organic and inorganic nitrogen-sources by ectomycorrhizal fungi in pure culture and in symbiosis with Pinus contorta Dougl ex Loud. New Phytologist, 120, 105115.
  • Folke, C., Carpenter, S., Walker, B., Scheffer, M., Elmqvist, T., Gunderson, L. & Holling, C.S. (2004) Regime shifts, resilience, and biodiversity in ecosystem management. Annual Review of Ecology Evolution & Systematics, 35, 557581.
  • Gardes, M. & Bruns, T.D. (1993) ITS primers with enhanced specificity for basidiomycetes—application to the identification of mycorrhizae and rusts. Molecular Ecology, 2, 113118.
  • Genney, D.R., Anderson, I.C. & Alexander, I.J. (2006) Fine-scale distribution of pine ectomycorrhizas and their extramatrical mycelium. New Phytologist, 170, 381390.
  • Gibson, F. & Deacon, J.W. (1988) Experimental study of establishment of ectomycorrhizas in different regions of birch root systems. Transactions of the British Mycological Society, 91, 239251.
  • Gilbert, G.A., Knight, J.D., Vance, C.P. & Allan, D.L. (1999) Acid phosphatase activity in phosphorus-deficient white lupin roots. Plant, Cell & Environment, 22, 801810.
  • Godbold, D.L., Hoosbeek, M.R., Lukac, M., Cotrufo, M.F., Janssens, I.A., Ceulemans, R., Polle, A., Velthorst, E.J., Scarascia-Mugnozza, G., De Angelis, P., Miglietta, F. & Peressotti, A. (2006) Mycorrhizal hyphal turnover as a dominant process for carbon input into soil organic matter. Plant and Soil, 281, 1524.
  • Goodman, D.M., Durall, D.M., Trofymow, J.A. & Berch, S.M. (eds) (1996) A Manual of Concise Descriptions of North American Ectomycorrhizae, Mycologue Publications and Canada-B.C. Forest Resource Development Agreement, Canadian Forest Service, Victoria, BC, Canada.
  • Griesbauer, H. (2008) Regional, Ecological and Temporal Patterns in Douglas-fir Climate-growth Relationships in the British Columbia Interior. MSc thesis, University of Northern British Columbia, Prince George.
  • Grogan, P., Baar, J. & Bruns, T.D. (2000) Below-ground ectomycorrhizal community structure in a recently burned bishop pine forest. Journal of Ecology, 88, 10511062.
  • Hamann, A. & Wang, T.L. (2006) Potential effects of climate change on ecosystem and tree species distribution in British Columbia. Ecology, 87, 27732786.
  • Högberg, M.N. & Högberg, P. (2002) Extramatrical ectomycorrhizal mycelium contributes one-third of microbial biomass and produces, together with associated roots, half the dissolved organic carbon in a forest soil. New Phytologist, 154, 791795.
  • Högberg, P. & Read, D.J. (2006) Towards a more plant physiological perspective on soil ecology. Trends in Ecology & Evolution, 21, 548554.
  • Jones, M.D., Durall, D.M. & Cairney, J.W.G. (2003) Ectomycorrhizal fungal communities in young forest stands regenerating after clearcut logging. New Phytologist, 157, 399422.
  • Jones, M.D., Grenon, F., Peat, H., Fitzgerald, M., Holt, L., Philip, L.J. & Bradley, R. (2009) Differences in 15N uptake amongst spruce seedlings colonized by three pioneer ectomycorrhizal fungi in the field. Fungal Ecology, 2, 110120.
  • Jonsson, L.M., Nilsson, M.C., Wardle, D.A. & Zackrisson, O. (2001) Context dependent effects of ectomycorrhizal species richness on tree seedling productivity. Oikos, 93, 353364.
  • Kalra, Y.P. & Maynard, D.G. (1991) Methods Manual for Forest Soil and Plant Analysis. Report NOR–X–319. Forestry Canada, Edmonton.
  • Keeley, J.E. (1999) Photosynthetic pathway diversity in a seasonal pool community. Functional Ecology, 13, 106118.
  • Kerley, S.J. & Read, D.J. (1995) The biology of mycorrhiza in the Ericaceae, XV. Chitin degradation by Hymenoscyphus ericae and transfer of chitin-nitrogen to the host plant. New Phytologist, 131, 369375.
  • Knicker, H. (2007) How does fire affect the nature and stability of soil organic nitrogen and carbon? A review. Biogeochemistry, 85, 91118.
  • Koide, R.T., Courty, P.E. & Garbaye, J. (2007) Research perspectives on functional diversity in ectomycorrhizal fungi. New Phytologist, 174, 240243.
  • Kõljalg, U., Larsson, K.-H., Abarenkov, K., Nilsson, R.H., Alexander, I.J., Eberhardt, U., Erland, S., Høiland, K., Kjøller, R., Larsson, E., Pennanen, T., Sen, R., Taylor, A.F.S., Tedersoo, L., Vrålstad, T. & Ursing, B.M. (2005) UNITE: a database providing Web-based methods for the molecular identification of ectomycorrhizal fungi. New Phytologist, 166, 10631068.
  • Lauzon, E., Bergeron, Y., Gauthier, S. & Kneeshaw, D. (2006) Fire Cycles and Forest Management: An Alternative Approach for Management of the Canadian Boreal Forest. Sustainable Forest Management Network, Edmonton.
  • Lilleskov, E.A., Hobbie, E.A. & Fahey, T.J. (2002b) Ectomycorrhizal fungal taxa differing in response to nitrogen deposition also differ in pure culture organic nitrogen use and natural abundance of nitrogen isotopes. New Phytologist, 154, 219231.
  • Lilleskov, E.A., Fahey, T.J., Horton, T.R. & Lovett, G.M. (2002a) Belowground ectomycorrhizal fungal community change over a nitrogen deposition gradient in Alaska. Ecology, 83, 104115.
  • Lindahl, B.D., Ihrmark, K., Boberg, J., Trumbore, S.E., Högberg, P., Stenlid, J. & Finlay, R.D. (2007) Spatial separation of litter decomposition and mycorrhizal nitrogen uptake in a boreal forest. New Phytologist, 173, 611620.
  • Lloyd, D., Angove, K., Hope, G. & Thompson, C. (1990) A Guide to Site Identification and Interpretation for the Kamloops Forest Region. Land Management Handbook Number 23. BC Ministry of Forests, Kamloops.
  • Martin, K.J. & Rygiewicz, R.T. (2005) Fungal-specific PCR primers developed for analysis of the ITS region of environmental DNA extracts. BMC Microbiology, 5, 28.
  • McGill, B.J., Enquist, B.J., Weiher, E. & Westoby, M. (2006) Rebuilding community ecology from functional traits. Trends in Ecology & Evolution, 21, 178185.
  • McKenzie, D., Gedalof, Z., Peterson, D.L. & Mote, P. (2004) Climatic change, wildfire, and conservation. Conservation Biology, 18, 890902.
  • Moore, P.D. (1999) Mixed metabolism in plant pools. Nature, 399, 109110.
  • O’Brien, H.E., Parrent, J.L., Jackson, J.A., Moncalvo, J.-M. & Vilgalys, R. (2005) Fungal community analysis by large-scale sequencing of environmental samples. Applied and Environmental Microbiology, 71, 55445550.
  • Parrent, I.L., Morris, W.F. & Vilgalys, R. (2006) CO2-enrichment and nutrient availability alter ectomycorrhizal fungal communities. Ecology, 87, 22782287.
  • Peay, K.G., Kennedy, P.G. & Bruns, T.D. (2008) Fungal community ecology: a hybrid beast with a molecular master. Bioscience, 58, 799810.
  • Peltzer, D.A., Bast, M.L., Wilson, S.D. & Gerry, A.K. (2000) Plant diversity and tree responses following contrasting disturbances in boreal forest. Forest Ecology & Management, 127, 191203.
  • Plamboeck, A.H., Dawson, T.E., Egerton-Warburton, L.M., North, M., Bruns, T.D. & Querejeta, J.I. (2007) Water transfer via ectomycorrhizal fungal hyphae to conifer seedlings. Mycorrhiza, 17, 439447.
  • Pritsch, K., Raidl, S., Marksteiner, E., Agerer, R., Blaschke, H., Schloter, M. & Hartmann, A. (2004) A rapid and highly sensitive method for measuring enzyme activities in single mycorrhizal tips using 4-methylumbelliferone-labelled fluorogenic substrates in a microplate system. Journal of Microbiological Methods, 58, 233241.
  • Read, D.J. & Perez-Moreno, J. (2003) Mycorrhizas and nutrient cycling in ecosystems – a journey towards relevance? New Phytologist, 157, 475492.
  • Rineau, F. & Garbaye, J. (2009) Does forest liming impact the enzymatic profiles of ectomycorrhizal communities through specialized fungal symbionts? Mycorrhiza, 19, 493500.
  • Rosling, A., Landeweert, R., Lindahl, B.D., Larsson, K.-H., Kuyper, T.W., Taylor, A.F.S. & Finlay, R.D. (2003) Vertical distribution of ectomycorrhizal fungal taxa in a podzol soil profile. New Phytologist, 159, 775783.
  • Schimel, J.P. & Bennett, J. (2004) Nitrogen mineralization: challenges of a changing paradigm. Ecology, 85, 581602.
  • Serrasolses, I., Romanya, J. & Khanna, P.K. (2008) Effects of heating and autoclaving on sorption and desorption of phosphorus in some forest soils. Biology & Fertility of Soils, 44, 10631072.
  • Setala, H. & McLean, M.A. (2004) Decomposition rate of organic substrates in relation to the species diversity of soil saprophytic fungi. Oecologia, 139, 98107.
  • Shieh, T.R., Wodzinsk, R.J. & Ware, J.H. (1969) Regulation of the formation of acid phosphatases by inorganic phosphate in Aspergillus ficuum. Journal of Bacteriology, 100, 11611165.
  • Silvertown, J. (2004) Plant coexistence and the niche. Trends in Ecology and Evolution, 19, 605611.
  • Sinsabaugh, R.L. (2005) Fungal enzymes at the community scale. The Fungal Community, 3rd edn (eds J.Dighton, P.Oudermans & J.White), pp. 237247, CRC Press, New York.
  • Sinsabaugh, R.L., Lauber, C.L., Weintraub, M.N., Ahmed, B., Allison, S.D., Crenshaw, C., Contosta, A.R., Cusack, D., Frey, S., Gallo, M.E., Gartner, T.B., Hobbie, S.E., Holland, K., Keeler, B.L., Powers, J.S., Stursova, M., Takacs-Vesbach, C., Waldrop, M.P., Wallenstein, M.D., Zak, D.R. & Zeglin, L.H. (2008) Stoichiometry of soil enzyme activity at global scale. Ecology Letters, 11, 12521264.
  • Smith, S.E. & Read, D.J. (2008) Mycorrhizal Symbiosis, 3rd edn. Academic Press, New York.
  • Soil Classification Working Group (1998) The Canadian System of Soil Classification. Agriculture and Agri-Food Canada Publication 1646 (Revised). NRC Research Press, Ottawa.
  • Stark, K.E., Arsenault, A. & Bradfield, G.E. (2006) Soil seed banks and plant community assembly following disturbance by fire and logging in interior Douglas-fir forests of south-central British Columbia. Canadian Journal of Botany, 84, 15481560.
  • Talbot, J.M., Allison, S.D. & Treseder, K.K. (2008) Decomposers in disguise: mycorrhizal fungi as regulators of soil C dynamics in ecosystems under global change. Functional Ecology, 22, 955963.
  • Tedersoo, L., Suvi, T., Jairus, T. & Kõljalg, U. (2008) Forest microsite effects on community composition of ectomycorrhizal fungi on seedlings of Picea abies and Betula pendula. Environmental Microbiology, 10, 11891201.
  • Timonen, S. & Sen, R. (1998) Heterogeneity of fungal and plant enzyme expression in intact Scots pine–Suillus bovinus and –Paxillus involutus mycorrhizospheres developed in natural forest humus. New Phytologist, 138, 355366.
  • Twieg, B.D., Durall, D.M. & Simard, S.W. (2007) Ectomycorrhizal fungal succession in mixed temperate forests. New Phytologist, 176, 437447.
  • Weber, M.G. & Stocks, B.J. (1998) Forest fires and sustainability in the boreal forest of Canada. Ambio, 27, 545550.
  • White, T.J., Bruns, T., Lee, S. & Taylor, J. (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR Protocols: A Guide to Methods and Applications (eds M.A.Innis, D.H.Gelfand, J.J.Sninsky & T.J.White), pp. 315322, Academic Press, New York.

Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References
  10. Supporting Information

Fig. S1. Map of sites.

Table S1. Physical characteristics of sites.

Table S2. Fungi associated with the mycorrhizal root tips used in the enzyme assays.

Table S3. NMS ordination results for the mean activities per tip for three multivariate data sets, including or excluding long-distance exploration types.

Table S4. Summary of MRPP results for the mean activities per tip for three multivariate data sets, including or excluding long-distance exploration types.

Table S5. Activities per tip for the three univariate data sets, including or excluding long-distance exploration types, analysed according to the designs outlined in Table 1.

Appendix S1. Additional details for the Materials and methods section.

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