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Keywords:

  • activity profiling;
  • ectomycorrhiza;
  • exoenzyme;
  • fluorescent substrates;
  • functional diversity;
  • microplate assays;
  • soil nutrient mobilization;
  • temperate forest

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions and Perspectives
  7. Acknowledgements
  8. References
  • • 
    Data on the diversity and distribution of enzyme activities in native ectomycorrhizal (ECM) communities are inadequate.
  • • 
    A microplate multiple enzymatic test was developed which makes it possible to measure eight enzyme activities on 14 individual, excised ECM root tips. Hydrolytic and oxidative enzymes are involved in the decomposition of lignocellulose, chitin and phosphorus-containing organic compounds. This test system was used to describe the functional diversity of ECM communities in two forest sites.
  • • 
    This set of tests proved to be accurate and sensitive enough to reveal a high diversity of activity profiles, depending on the fungal symbiont and the soil horizon. Ectomycorrhizas can be classified into specialists and generalists, and appear to complement each other in the same horizon to collectively perform all eight activities studied.
  • • 
    By including a higher number of different assays for more detailed analyses, ECM activity profiling will provide a valuable tool for studying the functional diversity of ECM communities.

Introduction

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

Ectomycorrhizal symbiosis is dominant in the fine absorbing roots of trees in temperate and boreal forests. Many studies have demonstrated that ectomycorrhizas (ECM) – the root tips colonized by the symbiotic fungi – play a major role in the hydro-mineral nutrition of the trees through a variety of mechanisms such as soil exploration and foraging by the emanating hyphae, mineral weathering, mobilization of organic and inorganic phosphorus, iron transport and release of nitrogen from complex organic macromolecules by extracellular enzymes (Smith & Read, 1997). Most of these functions cannot be performed with the same efficiency by the roots alone.

The number of ascomycetes and basidiomycetes forming ECMs is tremendously high, even at the forest stand level. Owing to improved morphotyping techniques (Agerer, 1991) and to fast-developing molecular tools of identification, this species diversity has now been described in a variety of forest types (Horton & Bruns, 1998; Tedersoo et al., 2003; Buée et al., 2004).

However, most of our present knowledge of the functional diversity of ECMs results from experiments on young tree seedlings grown under controlled laboratory conditions very different from those that prevail around the roots of mature trees in forest soils. New methods are therefore needed to describe the functional diversity of ECMs in situ in order to understand the role played by the many fungal partners within ectomycorrhizal communities in complex forest ecosystems. Jany et al. (2003), Buée et al. (2004) and Pritsch et al. (2004) have recently proposed direct methods to assess a range of activities relevant to tree nutrition and metabolic activity on single excised ectomycorrhizal root tips. Their results suggest that each symbiotic fungal species behaves differently in terms of seasonality and ability to perform particular functions when forming ECMs with the roots of forest trees. Jany et al. (2003) compared the [14C] glucose respiration of two ECM species using a microradiorespirometry approach to assess the physiological activity of single ectomycorrhizal tips. They found species-specific differences and a high influence of the respective soil conditions (e.g. drought) on the respiration of these ECM fungi. Buée et al. (2004) studied potential enzymatic activities of phosphatase and dehydrogenase using a microplate system to assess these functional parameters in a 1-yr field survey of field-sampled ECM. Enzyme activities were found to be dependent on species, season, temperature and soil moisture. A newly developed, highly sensitive microplate assay for detection of phosphatase, chitinase, and β-glucosidase activity on single mycorrhizal tips also revealed interspecific differences of field sampled ECM (Pritsch et al., 2004). All these approaches have in common a high enough sensitivity to assess functional parameters on individual ECM tips, allowing the study of field-sampled ECM.

In the present study, we endeavored to improve the efficiency of this approach by addressing a larger number of relevant functions and by designing a set of rapid and sensitive assays using a minimum of root material in order to provide efficient tools for large-scale ecological studies. The eight resulting assays combined on two microplates are derived from those of Buée et al. (2004) and Pritsch et al. (2004) using photometric and fluorometric techniques, respectively, and allow testing for activities related to carbon cycling and the release of phosphorus and nitrogen from organic macromolecules (hydrolase and oxidase enzyme activities). The assays were applied to describe and compare the activity profiles of common ectomycorrhizal types in two central European forest stands.

Materials and Methods

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

Sites

Ectomycorrhizas were sampled at two field sites, in Germany and France. The German experimental site of Kranzberg is a 50- to 70-yr-old mixed spruce (Picea abies (L.) Karst.) and beech (Fagus sylvatica L.) stand (Pretzsch et al., 1998) located in south-eastern Germany (48°25′ N, 11°40′ E, altitude 490 m). The soil is a gleyic cambisol and the humus form is mull to mull-moder under beech. The litter layer and fermentation horizon (Of) under beech has a thickness of 0.5–2 cm and a pH [H2O] of 4.5. The dark brown horizon (Oh) has a thickness of 1–5 cm and a pH of 3.9. The Ah-horizon has a thickness of 5–15 cm and a pH of 3.7. The forest floor under beech is without any vegetation and covered by litter. The French site is a 100-yr-old oak forest with a continuous canopy and a hornbeam understory (Quercus petraea (Mattuschka) Liebl., Quercus robur Ehrh, Carpinus betulus L.) in north-eastern France (48°75′ N, 6°35′ E, altitude 250 m). The luvic cambisol (pH [H2O] 4.6) has a loamy texture in the A1 horizon (0–5 cm) and top of A2 where fine roots are concentrated. The forest floor is flat with scarce vegetation (oak seedlings, Convallaria majalis L., Deschampsia cespitosa L.).

Sampling

Samples for optimizing the enzymatic tests were collected from April to June 2004. Soil samples (0.5 × 0.5 × 0.2 m deep) were brought to the laboratory in isotherm boxes. Roots were kept in the substrate in a plastic bag at 4°C and assayed within 4 d after sampling. The roots were gently washed and then observed in water with a stereomicroscope (×40). Dominant ECM morphotypes (ectomycorrhizas sharing common morphological traits) were identified according to Agerer's (1987–98) descriptions. At least 14 ECM tips of each morphotype were used for the assays. Three tips of each morphotype were frozen (−80°C) for later molecular identification.

Four abundant ECM (Rhizopogon sp. on pine, Lactarius subdulcis (Bull.) Gray on beech, Tomentella sp. 1 and Lactarius quietus (Fr.) Fr. on oak) were used to develop fluorimetric assays for xylosidase, cellobiohydrolase, glucuronidase and leucine aminopeptidase activities.

To optimize the laccase assay, five ECM morphotypes (L. quietus, Lactarius chrysoreus (Fr.) Fr., Byssocorticium atrovirens (Fr.) Bond. & Sing. ex Sing., Boletus sp. and Entoloma nitidum (Bull.) Fr.) on oak were tested in order to determine the optimal incubation time, type of buffer and pH. To establish the procedure of running several assays on the same ectomycorrhizal tips, two ECM morphotypes (L. quietus and Tomentella sp. 1) were studied in detail.

In August 2004, sampling was simultaneously performed at both sites to study the enzyme activity profiles of the ECM communities. At Champenoux, seven soil cores (0.1 m diameter, 0.2 m deep) were sampled in a 50 m2 plot. At Kranzberg, seven samples (0.5 × 0.5 m, 0.2 m deep) were taken in a 20 m2 area with beech only. Tips of dominant ECM types in each sample and from each horizon were subjected to the eight enzymatic tests which are described in the following sections.

Microplate assays

In order to manipulate ECM tips during the experiments, a newly developed system for microplate assays with sieve strips was used (Pritsch et al., 2004). The sieve strips consisted of a row of eight polymerase chain reaction (PCR) reaction tubes, with their bottom cut off and replaced by a fine Nylon mesh. These sieve strips exactly fit into a row of eight wells in a clear, flat bottomed 96-well plate with a lid (Sarstedt, Inc., Newton, NC, USA) or black 96-well microtitration plate (OptiPlate-96F; PerkinElmer Life Sciences, Villebon-Sur-Yvette, France). This system allows easy and rapid manipulation with minimal damage to the ECM. In our experiments, unbranched, turgescent and living ECM tips (2–4 mm) were excised (Agerer, 1991; Buée et al., 2004; Pritsch et al., 2004) and placed in each tube of a sieve strip. Seven tubes were occupied by one ECM tip whereas the eighth one was left without tip to serve as a control. Following the procedure, which has been described in detail for ECM manipulation during fluorescent enzyme assays (Pritsch et al., 2004), the strips were used in all functional assays to transfer seven tips simultaneously from one row of wells to another in a 96-well microplate, allowing the same tips to incubate, rinse for 3 min, reincubate and rinse until the end of an experimental series.

A number of preliminary experiments were run in order to optimize incubation parameters such as temperature and time (data not shown). Incubation took place on a microplate shaker in a climate chamber at 21°C.

Chemicals and solutions for fluorogenic assays

The experimental setup for fluorescence assays was the same as described by Pritsch et al. (2004): incubation plates contained 50 µl incubation buffer, 50 µl H2O and 50 µl substrate per well; rinsing plates contained 50 µl buffer and 100 µl H2O per well; stopping plates were prefilled with 100 µl stopping buffer to which 100 µl of incubated solution was added. For calibration, 50 µl of 4-methylumbelliferone (MU) or 7-amino-4-methylcoumarin (AMC) standards were added to the stopping plate and supplemented with 100 µl of H2O to reach a final volume of 250 µl.

Seven enzyme substrates based on MU and AMC were studied: MU-phosphate (MU-P) for the detection of the acid phosphatase (EC 3.1.3.2), MU-β-d-glucopyranoside (MU-G) for β-glucosidase (EC 3.2.1.3), MU-N-acetyl-β-d-glucosaminide (MU-NAG) for chitinase (EC 3.2.1.14), MU-β-d-glucuronide hydrate (MU-GU) for glucuronidase (EC 3.2.1.31), MU-β-d-xylopyranoside (MU-X) for xylosidase (EC 3.2.1.37), MU-β-d-cellobioside (MU-C) for cellobiohydrolase (EC 3.2.1.91), and l-leucine-AMC (Leu-AMC) for leucine aminopeptidase (EC 3.4.11.1) activities. All chemicals were purchased from Sigma Aldrich Chemicals (Lyon, France).

Stock solutions of each substrate (5 mm) and calibration solutions of AMC and MU (25 mm), were prepared in 2-methoxyethanol (Hoppe, 1983). Substrates were diluted with sterile ultra-pure water (18Ω; Millipore, Billerica, MA, USA) to achieve the desired working concentrations of 0, 50, 100, 200, 300, 400, 500, 600, 700 and 800 µm per well, with each well containing 50 µl of incubation buffer, 50 µl of ultra-pure water and 50 µl of substrate working solution. Stock solutions, diluted substrates and calibration solutions were kept at −20°C in the dark.

Incubation buffers were prepared at a concentration of 150 mm and sterilized. The buffer system used was a mixture of maleic acid and Tris (pH 2–7) which had previously been tested and shown not to interfere with the enzymes under study (data not shown).

Experiments for optimizing substrate concentration were carried out at pH 4.5 at which all enzymes in preliminary experiments showed high, if not maximal, activity.

In order to determine the pH range and the optimum pH of the enzymes, microplates were prepared with increasing pH from 2 to 7 with pH unit steps of 0.5 and a substrate concentration of 500 µm per well.

The stopping buffer used to alkalinize the solution and stop enzyme reactions in one step was Tris 2.5 m pH 10–11. Contrary to the original protocol (Pritsch et al., 2004) ethanol was omitted in the stopping step.

Depending on the substrate, incubation times in black 96-well microtitration plates were 10 min (MU-P), 20 min (MU-NAG and MU-G), 40 min (MU-C) or 60 min (Leu-AMC, MU-X and MU-GU). The incubation times were chosen to allow enzymatic activities of the most active ECM morphotypes to be measured. Measurements were carried out with two different microplate readers: the Victor3 (Wallac Perkin–Elmer Life Sciences, Villebon-Sur-Yvette, France) with an excitation wavelength of 355 nm and an emission wavelength of 460 nm, slit width of 5 nm and the Cary Eclipse Fluorescence Spectrophotometer (Varian, Mulgrave, Australia) with an excitation wavelength of 360 nm and an emission wavelength of 450 nm, slit width of 5 nm.

Each series of experiments included calibration with 0, 100, 200, 300, 400 and 500 pmol MU or AMC per well. The concentrations of released MU or AMC were calculated from the resulting regression lines. Controls related to fluorescence measurements (autofluorescence of mycorrhizas, quenching of the fluorescence, assay without ECM tip) were made according to Pritsch et al. (2004).

Photometric assay for laccase (EC 1.10.3.2) activity

Each well of a clear, flat-bottom 96-well microtitration plate (Sarstedt, Newton, NC, USA) contained 100 µl of Tris maleic acid buffer (pH 4.5) and 50 µl of 2 mm 2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonate (ABTS; Sigma, St Quentin Fallavier, France) solution. After incubating ECM root tips at 21°C for 1 h, 120 µl were transferred to a new plate and the intensity of the green color which had developed was immediately measured at 415–425 nm either with the plate reader Hercules 550 (Bio-Rad, Hercules, CA, USA) or the Spectramax 340 (MWG Biotech, Ebersberg, Germany) (Niku-Paavola et al., 1990). Measured absorptions were converted into enzymatic activities expressed as turnover of ABTS (mol ml−1) OD/(ɛ425× path length (cm)) via the relation ɛ425 = 3.6 × 10−4 m−1 cm−1 and an assumed path length calculated as average liquid height caused by a volume of 120 µl (path length = 0.12 cm3/(π × r2).

Expression of results

The exoenzymes which play a role in nutrient mobilization are likely to be surface located and slowly released by diffusion or even surface-bound (K. Pritsch, unpublished). This is why all measured activities were calculated per mm2 of projected area of individual ECM tips, determined with the automated image analysis software winrhizo 2003b (Regent Instruments, Inc., Quebec, Canada; Buée et al. 2004); the projected areas are linearly correlated with the surface areas of the ECMs considered as cylinders. The results were finally reported against the incubation time.

For the field study, the potential enzymatic activity of a particular ECM type was expressed as relative activity in the community at each site. The relative activity was calculated as per cent of the average of all studied types of one community, allowing comparison of the activity profiles of each ECM type.

Molecular methods and analyses

The DNA of ECMs from the field study was extracted with the Dneasy Plant Mini Kit (Qiagen SA, Courtaboeuf, France). Polymerase chain reaction amplification was performed on a GeneAmp 9600 thermocycler (Perkin-Elmer Instruments, Norwalk, CT, USA) for the internal transcribed spacer (ITS) region of nuclear ribosomal DNA, using the fungal-specific primer pair ITS1F and ITS4 (Gardes & Bruns, 1993). Successful PCR reactions resulted in a single band on a 0.8% agarose gel (Bioprobe; QBiogene, Illkirch, France) in 1% TBE (Tris buffer-ethylenediaminetetraacetic acid (EDTA)) and stained with ethidium bromide (Bet, 2 µg ml−1; Roche, Rosny-sous-bois, France). The size of the band was estimated with a 1 kb-ladder (Gibco BRL, Cergy Pontoise, France). Amplified products were purified with the Multiscreen-PCR plate system (Millipore) according to the manufacturer's instructions. The DNA concentration was estimated with Low DNA mass Ladder (Invitrogen, Cergy Pontoise, France). Direct DNA sequencing from ECMs was performed on the 8-capillar sequencer CEQ 2000XL (Beckman, Fullerton, CA, USA). Two nanograms of purified template DNA was labelled during a cycle of sequencing reaction with 5 ng CEQ DTCS-Quick Start Kit (Beckman) in a GeneAmp 9600 thermocycler (Perkin–Elmer). All samples were sequenced with the primers ITS1F and ITS4 (Gardes & Bruns, 1993) or with the primers ITS1 and ITS4 (White et al., 1990).

Sequences were corrected with the sequencer 4.1 software. DNA sequences were compared with sequence data in the NCBI database (blast program, http://www.ncbi.nlm.nih.gov/). The relevance of species identifications was assessed according to the geographic origin of the species given by similarity research. Results of sequence analyses and short morphotype descriptions of ECMs included in the field study are given in Table 2.

Table 2.  Description of the 10 ectomycorrhizal (ECM) morphotypes sampled and assayed for enzyme activities in the Champenoux and Kranzberg forest sites in August 2004 and identification of the fungal symbionts
Fungal taxaSiteAgerer"s reference or brief descriptionLength of sequenced ITS (bp)Accession No. of most similar ITS sequence in GenBankIdentitiesE-value
  1. ITS, internal transcribed spacer.

Cortinarius olivaceofuscusCYellow-olive tortuous mycorrhiza with abundant rhizomorphs growing off in flat angles, enclosed air between mantle hyphae652U56050 95%0.0
Lactarius quietus (Fr.) Fr.COrange smooth monopodial–pyramidal mycorrhiza with bent or straight unramified ends687AJ272247 98%0.0
Tomentella sp. 1CBlack, smooth monopodial–pinnate mycorrhiza with straight unramified ends644AY299217 98%0.0
Byssocorticium atrovirens (Fr.) Bond. & Sing. ex Sing.C51 Not sequenced  
Lactarius subdulcis (Bull.) GrayK5 Not sequenced  
Russula fellea (Fr.) Fr.K16853AY061676100%0.0
Cenococcum geophilum Fr.K11 Not sequenced  
Russula nigricans (Bull.) Fr.KPale yellowish brown, smooth monopodial pinnate or unramified mycorrhiza with abundant hyphae859AF418607100%0.0
Xerocomus sp.KSilvery white to olive monopodial–pyramidal mycorrhiza with regular rhizomorphs and air entrapped in the mantle691AY310874 96%0.0
Tomentella sp. 2KBeige to light or grey brown monopodial- pinnate mycorrhiza with smooth surface and short hyalin extramatrical hyphae842AJ534912 91%0.0

Results and Discussion

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

The aim of the first part of our work was to develop a set of sensitive enzyme assays for functional profiling related to enzymatic activities of individual ECM tips. For each single assay, the optimal incubation time, substrate concentration and pH were derived from existing protocols. The detailed results are given in the following section.

Optimal concentration

Substrate saturation has already been discussed for chitinase, phosphatase and β-glucosidase by Pritsch et al. (2004). Saturation was reached at 500 µm of substrate per well for chitinase and β-glucosidase, whereas it was above 800 µm for acid phosphatase.

Cellobiohydrolase activity (Fig. 1a) reached an optimum at a substrate concentration of 400 µm per well and did not increase further at higher concentrations. For glucuronidase activity (Fig. 1b), the saturating substrate concentration was between 400 µm and 500 µm per well. With a concentration greater than 500 µm, we observed a small decrease of the activity. The four ECM morphotypes had similar xylosidase activities and reached saturation between 400 µm and 600 µm per well (Fig. 1c). Above 600 µm per well, the activity decreased. Leucine aminopeptidase activity showed an optimum at a substrate concentration of 300–400 µm for Tomentella sp. 1, L. quietus and Rhizopogon sp., and 400–500 µm for L. subdulcis (Fig. 1d). The activity decreased for all four ECM morphotypes at concentrations greater than 500 µm per well. For laccase, the substrate concentration was found to be optimal at 2 mm for L. quietus, Cortinarius anomalus (Fr. Fr.) Fr. and Tomentella sp. (data not shown).

image

Figure 1. Enzyme activities at increasing substrate concentrations (µmol MU or AMC per well) expressed as 4-methylumbelliferone (MU) or 7-amino-4-methylcoumarine (AMC) released per unit of time and per ectomycorrhizal (ECM) tip projected area (pmol mm−2 min−1) for individual excised ECM root tips. Bars represent SE (n = 7). Closed circles on solid line, Lactarius quietus; open circles on solid line, Lactarius subdulcis; closed circles on broken line, Tomentella sp. 1; open circles on broken line, Rhizopogon sp.

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Optimal pH

Optimal pH (4.5) has been discussed for chitinase, phosphatase and β-glucosidase activity by Pritsch et al. (2004).

The highest cellobiohydrolase activity was measured at acidic pH (< 4.5) for L. subdulcis (45 pmol mm−2 min−1) before a sharp decrease of activity until pH 7 (Fig. 2a). For the three other species, the activity was constant from pH 2 to pH 4 and highest from pH 4.5 to pH 5 (30 pmol mm−2 min−1 for L. quietus and Rhizopogon sp.; 15 pmol mm−2 min−1 for Tomentella sp. 1). Above pH 5, the activity was slightly reduced. The optimal pH was 4.5 for all species.

image

Figure 2. Enzyme activities at increasing pH expressed as 4-methylumbelliferone (MU) or 7-amino-4-methylcoumarin (AMC) released per unit of time per ectomycorrhizal (ECM) tip projected area (pmol mm−2 min−1) for individual excised ECM root tips. Bars represent SE (n = 7). Closed circles on solid line, Lactarius quietus; open circles on solid line, Lactarius subdulcis; closed circles on broken line, Tomentella sp. 1; open circles on broken line, Rhizopogon sp.

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For glucuronidase activity, the optimal pH-range differed depending on the species: pH 3–4.5 for L. subdulcis and Rhizopogon sp., pH 4.5–5.5 for L. quietus and pH 3.5–5 for Tomentella sp. 1 (Fig. 2b).

Lactarius subdulcis showed highest xylosidase activity around pH 4–4.5, whereas it was around pH 4.5–5 for L. quietus (Fig. 2c). The pH optima were pH 4–5 for Rhizopogon sp. and pH 4.5–5.5 for Tomentella sp. 1.

Leucine aminopeptidase activity was constant for all four species from pH 2 to pH 5.5 (Fig. 2d). In this pH range, the maximal activity was shown by Tomentella sp. 1 (40 pmol mm−2 min−1). The activity increased twofold from pH 5.5 to pH 6 (Tomentella sp. 1 and Rhizopogon sp.) or from pH 5.5 to pH 6.5 (L. quietus and L. subdulcis). The activity was then constant to pH 7 for all species. For laccase, a pH optimum of 4.5–5 and lower has been described by Eggert et al. (1996), Bourbonnais et al. (1997) and Kanunfre & Zancan (1998).

Optimal incubation time

The enzyme activities recorded by these methods may result not only from the ectomycorrhizal fungus but also from the whole ectomycorrhizal complex, including root tissues and other associated microorganisms such as bacteria and free-living microfungi. In the case of field sampled ECMs, all these components belong to a natural mycorrhizosphere and neither can nor should be separated. To avoid proliferation of bacteria during the assays, which would lead to an overestimate of the bacterial contribution to enzyme activities, reaction times must be kept as short as possible. The fluorescence method has already been shown to allow the detection of enzymes such as acid phosphatase, chitinase and β-glucosidase after incubation times as short as 5, 10 and 20 min, respectively (Pritsch et al., 2004). For phosphatase detection, the fluorescence method therefore allowed a six- to over ten-fold reduction of incubation times (30–60 min) compared with the previously used photometric method using pNPP (Bartlett & Lewis, 1973; Tibett et al., 1998; Buée et al., 2004). In the present study, enzymes with lower activities could also be easily detected after 40 min (cellobiohydrolase) or 60 min incubation (xylosidase, glucuronidase and leucine aminopeptidase).

Laccase activity remained constant over 2 h for all morphotypes studied: L. quietus, L. chrysorrheus, B. atrovirens, Boletus sp. and E. nitidum (data not shown). Differences in laccase activities between the five ECM morphotypes appeared even before 30 min of incubation but were more distinct after 1 h, the incubation time which was chosen for routine assays. Controls with pasteurized mycorrhizal tips (30 min, 80°C) showed that oxidation of ABTS was restricted to living mycorrhizas.

Optimized protocol for subsequent assays on the same ectomycorrhizal tips

Assaying for different activities in succession on the same root tips was studied with two ECM morphotypes (L. quietus and Tomentella sp. 1). Since it has been previously demonstrated that there is no reduction in enzyme activities during longer incubation intervals (Pritsch et al., 2004), a number of combinations and orders for incubation with different substrates were tested. All MU substrates were put on one plate, to avoid interference with the two other substrates (ABTS and AMC). For MU-substrates, no interference of substrates was observed, resulting in the following optimal procedure: 14 tips of each ECM morphotype divided into two subsamples of seven tips each were assayed; the first subsample was used for all MU-substrates (e.g. chitinase, phosphatase, β-glucosidase, glucuronidase, xylosidase and cellobiohydrolase activities, in any order) and the second subsample was used to test for leucine aminopeptidase and laccase activity (in that order).

The final design for multiple activity tests with eight different substrates on two subsamples of seven mycorrhizal tips each (incubation time, pH, type and concentration of substrate) is summarized in Table 1.

Table 1.  Optimized experimental conditions (incubation time, pH and substrate concentration) for multiple enzyme activity tests with eight different substrates on two subsamples of seven mycorrhizal tips each
SubsampleTests in orderSubstrateIncubation time (min)pHConcentration (µm)
No. 17 ECM tipsGlucuronidase (glr)MU-β-d-glucuronide hydrate604.5500
Xylosidase (xyl)MU-β-d-xylopyranoside604.5500
Cellobiohydrolase (cel)MU-β-d-cellobioside404.5400
β-Glucosidase (gls)MU-β-d-glucopyranoside204.5500
No. 27 ECM tipsChitinase (nag)MU-N-acetyl-β-d-glucosaminide204.5500
Acid phosphatase (pho)MU-phosphate104.5800
Leucine aminopeptidase (leu)l-leucine-AMC606.5400
Laccases (lac)ABTS604.5  1.105

At this point, it is important to stress that the necessity to extract ECMs from soils causes a loss of extramatrical mycelium (emanating hyphae and strands). It is known that extramatrical mycelium plays a major role in nutrient mobilization and uptake by ECMs (Agerer, 2001; Landeweert et al., 2003) and that there might be specific differentiations of mycelia depending on species and nutrient pools in soils (Agerer, 2001). Therefore, it has to be kept in mind that the interpretation of the results relies on activities measured in the close vicinity of ECMs deprived from emanating hyphae and strands because of extraction from the soil. Whether these activities correlate with the corresponding activities of whole ECMs, including their complete extramatrical mycelium, or whether parts of mycelia remote from the roots display functions other than those close to roots, are challenging questions for further studies. Despite the acknowledged importance of the extramatrical mycelium, there is still a lack of methods allowing study of functional aspects of field-grown mycelia in a specific way (Leake et al., 2004). Such studies need major methodological developments, which were beyond the scope of this exploratory work.

ECM activity profiling in the two forests stands

Ten ECM morphotypes (Table 2), identified at the genus (3) or species (7) level, sampled in the Champenoux and Kranzberg sites in August 2004, have been subjected to the set of eight enzymatic tests (Table 1). The results confirmed the low variability of enzyme activities among the seven ECM tips within a sample, as already found when optimizing the tests (Figs 1 and 2). Exceptionally, higher variability was only observed, when absolute values were low, as in the case of glucuronidase activity (Fig. 3). By contrast, enzyme activities may vary widely among different samples of the same ECM, taken a few meters apart in the same soil horizon (intersample variation). In the example of Tomentella sp. 1 in the A1 horizon at the Champenoux site (Fig. 3), sample 2 markedly differed from the three other samples by higher leucine-aminopeptidase activity and low laccase activity. This result indicates that enzymatic activities may be strongly influenced by the specific conditions at each sampling point. Thus, in further studies, the choice of a sampling strategy will need to take into account the fact that the activity profile of a given ECM morphotype is very homogenous at the centimeter scale, but highly variable at the meter scale.

image

Figure 3. Enzymatic activity profiles of Tomentella sp. 1 sampled in Champenoux A1 horizon in four different places a few meters apart in August 2004. The polar graphs have been drawn from the relative activities (solid line polygon) calculated per cent of the mean value of the four samples. The two dotted line polygons are ± SE (n = 7) among the seven ectomycorrhizal (ECM) tips used in the laboratory test to measure each enzyme activity. Abbreviations for enzymes: pho, acid phosphatase; nag, N-acetyl-glucosaminidase; gls, β-glucosidase; cel, cellobiohydrolase; xyl, xylosidase; lac, laccase; glr, glucuronidase; leu, leucine aminopeptidase.

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At both sites, the 10 ECM types displayed remarkably different mean activity profiles (Fig. 4a,b). Two main factors are likely to be responsible for these different activity profiles: (1) species-specific enzyme expression, as already reported by Gramss et al. (1998) for mycelia in pure culture, Agerer et al. (2000) for sporocarps and Buée et al. (2004) for ectomycorrhizas; and (2) influence of local soil conditions (e.g. horizontal and vertical soil heterogeneity).

image

Figure 4. Enzymatic activity profiles of the 10 ectomycorrhizal (ECM) types sampled in different soil horizons in the Champenoux and Kranzberg forest sites in August 2004. The polar graphs have been drawn from the relative activities calculated as per cent of the mean value of all ECM types studied in the same community (site). Each graph was built with the mean value of the one to four replicates (root samples taken a few meters apart) within each species–horizon combination. The number of replicates (N) is given on top of each graph. Abbreviations for enzymes: pho, acid phosphatase; nag, N-acetyl-glucosaminidase; gls, β-glucosidase; cel, cellobiohydrolase; xyl, xylosidase; lac, laccase; glr, glucuronidase; leu, leucine aminopeptidase. (a) Champenoux site with two horizons: A1, top row; A2, bottom row. (b) Kranzberg site with three horizons: Of, top row; Oh, medium row; Ah, bottom row.

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At the Champenoux site (Fig. 4a), Tomentella sp. 1 had very low activities for all enzymes, while the other ECMs appeared specialized in at least one function (Fig. 4a; B. atrovirens. laccase; C. olivaceofuscus, xylosidase; L. quietus, glucuronidase, xylosidase and leucine aminopeptidase). For the last three ECMs, shifts in their activity profiles were observed with depth. The observed increase of laccase activity of B. atrovirens in the deeper horizon (A2) points to the fact that laccase activity is involved in breaking down recalcitrant phenolic molecules, as found in humic substances, resulting in the release of entrapped nitrogen and phosphorus (Criquet et al., 1999). Therefore, higher laccase activity may indicate a possible mechanism of mobilization of these nutrients by B. atrovirens. The activity profiles of C. olivaceofuscus and L. quietus were also different in the deeper A2 horizon. While the former had higher β-glucosidase, L. quietus lost its high β-glucosidase and cellobiohydrolase relative activities compared with the A1 horizon, but had high leucine aminopeptidase values and acquired high relative activities for phosphatase and chitinase. These shifts in activities may reflect the need for a better ability to mobilize organic phosphorus and to gain access to nitrogen from proteins and chitin in the deeper horizon.

The ECM types at the Kranzberg site also showed different enzyme activity profiles within each horizon, and the same ECM expressed different enzyme profiles in different horizons (Fig. 4b). Especially high activities of the ECMs at Kranzberg were found for Tomentella sp. 2 (cellobiohydrolase and β-glucosidase), Xerocomus sp. (chitinase and phosphatase), R. nigricans and C. geophilum (laccase, glucuronidase and xylosidase), L. subdulcis (laccase and leucine aminopeptidase).

In the case of Tomentella sp. 2 (Fig. 4b), a shift in distinct activities (leucine aminopeptidase increase in Oh, and chitinase increase in Ah) was observed in different horizons, displaying two ways of N-mobilization from N-containing biopolymers (proteins and chitin). Xerocomus sp. ECMs showed increased intensity from Of to Ah horizons but not different expression patterns of potential enzyme activities (Fig. 4b). By contrast, R. fellea showed rather different profiles in the Of horizon with high leucine aminopeptidase activity and a lack of almost all activities (especially phosphatase and glucuronidase) found in the deeper horizons Oh and Ah (Fig. 4b). L. subdulcis, an abundant contact type (Agerer, 2001) in the litter layer, may use its proteolytic capacity to release amino acids from fresh litter while simultaneously profiting from its high laccase activity, which may enable this fungus to cope with high amounts of phenolic compounds in weakly degraded beech litter. In the Ah horizon, C. geophilum and R. nigricans displayed very similar profiles with high laccase, glucuronidase and xylosidase activities, following probably the same strategy in nutrient mobilization, as discussed for B. atrovirens at Champenoux.

At the stand level, the observed high functional diversity reflects a clear complementarity of the different ECM types. For the Champenoux site, the four types inhabiting the A1 horizon, or the three types in the A2 horizon, when considered together, were performing the eight enzymatic activities with values of c. 100% of the mean values in the community. Exceptions are phosphatase and chitinase activities which are slightly under-represented in the A1 horizon. At Kranzberg, similarly, the mean of all types in one horizon is around or above average of the total community with two clear exceptions: a very low glucuronidase activity in the litter layer and a total lack of leucine aminopeptidase activity in the Ah horizon. This suggests that all activities are not constitutively produced, but induced under specific soil conditions.

Although each ECM type appears to be more or less specialized in a small number of activities among the eight we measured here, activity profiles are clearly influenced by the location and also by the horizon from which the samples were taken. However, the occurrence of different ecotypes belonging to the same species but adapted to different soil horizons cannot be ruled out. Such short distance genetic and functional variability has not been reported, but Jany et al. (2002, 2003) showed that C. geophilum displayed a high diversity in ribotypes and respiration potential activity at the forest stand scale. To be elucidated, this point would necessitate more sophisticated molecular methods discriminating at the genet level.

Conclusions and Perspectives

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

The direct in situ approach of the functional diversity of ECM communities, first proposed by Buée et al. (2004) and Pritsch et al. (2004), provides a very valuable tool for describing and understanding the role of individual ECM populations within a complex community. Sensitive and rapid tests are needed to process a large number of samples for large-scale ecological studies. This has been achieved here by using fluorogenic substrates and combined tests on the same root tips. However, in order to realistically describe the actual functioning of the ECM community, the quantification of as many putative functions as possible is required. The perspective for future research is therefore to design new tests concerning not only extracellular enzymatic activities but also other mechanisms related to nutrient mobilization and uptake, such as iron chelation or mineral weathering. This work was the first step in developing ectomycorrhiza activity profiling (EMAP) for studying the functional community structure of this key symbiotic complex and to provide new indicators of stability or disturbance in forest ecosystems.

Acknowledgements

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

We thank Shilpi Sharma for language corrections and helpful comments on the manuscript. The PhD scholarship of the first author was funded by grants of the French Ministry of Research and of the Lorraine region. Part of this research work has been supported by the programme Biological Invasions of the French Ministry of Ecology and Sustainable Development. K.P. was funded by the German Research Community in the frame of SFB607 (http://www.sfb607.de). P.-E.C. and K.P. received grants from the PROCOPE program funded by German Academic Exchange Service (DAAD) and the French Ministry of Foreign Affairs. The authors are also grateful to the Office National des Forêts for permitting sampling and measuring in the Champenoux State Forest.

References

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