Extramatrical ectomycorrhizal mycelium c. pdf
Research
Extramatrical ectomycorrhizal mycelium contributes
one-third of microbial biomass and produces, together
with associated roots, half the dissolved organic carbon
in a forest soil
Blackwell Science, Ltd
Mona N. Högberg and Peter Högberg
Department of Forest Ecology, Section of Soil Science, Swedish University of Agricultural Sciences, SE−901 83 Umeå, Sweden
Summary
Author for correspondence:
Mona N. Högberg
Fax: + 46 90 7867750
Email: Mona.Hogberg@sek.slu.se
Received: 11 January 2002
Accepted: 12 February 2002
• A large-scale tree-girdling experiment enabled estimates in the field of the contribution
of extramatrical mycelium of ectomycorrhizal (ECM) fungi to soil microbial biomass
and by ECM roots and fungi to production of dissolved organic carbon (DOC).
• Tree-girdling was made early (EG) or late (LG) during the summer to terminate the
flow of photosynthate to roots and ECM fungi. Determination of microbial C (Cmicr)
and microbial N in root-free organic soil was performed by using the fumigation–
extraction technique; extractable DOC was determined on unfumigated soil.
• Soil Cmicr was 41% lower on LG than on control plots 1 month after LG, whereas
at the same time (that is, 3 months after EG), the Cmicr was 23% lower on EG than
on control plots. Extractable DOC was 45% lower on girdled plots than control plots.
• Our results, which are of particular interest as they were obtained directly in the
field, clearly demonstrate the important contribution by extramatrical ECM
mycelium to soil microbial biomass and by ECM roots to the production of DOC, a
carbon source for other microbes.
Key words: ectomycorrhizal fungi, extractable dissolved organic carbon (DOC),
ectomycorrhizal mycelium, forest soil, fumigation–extraction, microbial N, soil
microbial biomass.
© New Phytologist (2002) 154: 791– 795
Introduction
Biogeochemists, ecologists and soil microbiologists can be
divided into those that neglect and those that recognize the
role of mycorrhizal fungi in ecosystems. A reason for this
division is the problem of assessing the contribution of
mycorrhizal fungi to the soil microbial community, especially
in the field. More precise estimates are available on the carbon
(C) cost of ectomycorrhizal (ECM) symbiosis in laboratory
model systems (Rygiewicz & Anderson, 1994) but only rough
calculations are available for forest ecosystems (Söderström,
1992; Smith & Read, 1997). To date, it has not been possible
to quantify the contribution made by ECM fungi to total
microbial biomass in the soil, despite the need to distinguish
these symbiotic fungi from other mycorrhizal fungi and from
saprotrophic fungi and other decomposers. However, the
phospholipid fatty acid (PLFA) 18 : 2ω6,9 and ergosterol
© New Phytologist (2002) 154: 791– 795 www.newphytologist.com
fungal biomarkers were monitored inside and outside cores
(2.0 dm2) that were root isolated (Wallander et al., 2001).
The root isolation killed ECM fungi inside trenches and
Wallander et al. calculated the ECM biomass by subtracting
the amount of fungi outside, from the values obtained from
inside trenched cores. The ECM biomass, including that of
fungal mantels, was calculated to be approx. 800 kg ha−1.
The amount of ECM mycelium produced in mesh bags
filled with quartz sand, an inert substrate mainly colonized by
mycorrhizal fungi, was approx. 160 kg ha−1. It is unclear if
this estimate applies to the situation in the organic mor-layer,
which is the horizon of greatest biological activity in the
boreal forests.
Ectomycorrhizal fungi use C from their plant hosts, and it
seems unlikely that the fungi ensheathing these plant roots
leak much C to the surrounding soil. However, there is evidently a flora of bacteria on the surfaces of ECM hyphae
791
792 Research
(Garbaye, 1994; Timonen et al., 1998), and several studies
report that ECM fungi and roots produce large amounts of
certain organic acids (Griffiths et al., 1994; Wallander et al.,
1997). The contribution made by ECM roots and ECM
fungi to the production of dissolved organic C (DOC) in the
mor-layer of boreal forests, where concentrations of DOC are
high (van Hees et al., 2000), is not known. This gap in our
knowledge is particularly serious given that DOC affects rates
of weathering of soil minerals (Lundström et al., 2000) and
represents important sources of C for microbes.
In a recent large-scale tree-girdling experiment (Högberg
et al., 2001) a 50% loss of soil respiration was interpreted
as a loss of activity by ECM roots and their extramatrical
mycelium. Here, we make use of this unique field experiment to quantify the contribution by the extramatrical
mycelium of ECM fungi to total soil microbial biomass in
the organic mor-layer. We also used this experiment to estimate the production of extractable DOC by ECM roots and
fungi.
Materials and Methods
Field site, experimental design and soil sampling
The forest was a naturally regenerated 45- to 55-year-old
Scots pine (Pinus sylvestris L.) located on a weakly podzolized
sandy silt sediment at Åheden, northern Sweden (64°14′ N,
19°46′ E, 175 m above sea level). The climate is cold with a
mean annual temperature of 1.0°C, and a mean annual
precipitation of 600 mm. There is usually snow cover for
6 months between late October and early May. There was a
sparse understorey of Calluna vulgaris L. and Vaccinium vitisidaea L. The bottom layer consisted of mainly Cladonia
spp. lichens and Pleurozium schreberi moss. The organic morlayer (F + H horizons) was 2 cm thick and had the following characteristics (n = 9, mean ± SD): bulk density 0.16 ±
0.05 g cm−3, C : N ratio 40 ± 5, organic matter content
(weight loss on ignition) 76 ± 5%, pHH20 4.0 ± 0.1, water
content at the time of sampling 193 ± 41% (g g−1 dry wt).
The experiment comprised nine quadratic plots of 900 m2
each (with c. 120 trees each) and was divided into three blocks
(Fig. 1). Girdling was performed in early June 2000 (early
girdling, EG) and in mid-August 2000 (late girdling, LG)
on three plots at a time leaving three plots as control plots.
Girdling had no effects on soil temperature and moisture
(Högberg et al., 2001). Seventy-two days after girdling, the
number of sporocarps and their biomass were reduced by
98.4% and 99.4%, respectively, on the central 100 m2 of EG
plots compared with control plots.
On 12 September 2000, soil from the F and H horizons
was sampled by use of a 0.1-m diameter corer. Sampling was
performed along the border of the central 100 m2 of each
plot. Five composite samples made up from 10 cores each
were taken from each plot.
Fig. 1 Layout of the experiment. Each plot is 30 × 30 m. Treatments
are: C, control; EG, early girdling; LG, late girdling.
Fumigation and determinations of Cmicr and DOC
Soil samples were stored at 4°C overnight. Roots were
thereafter sorted out by hand. After a day at 16°C, microbial
C, Cmicr, and microbial N, Nmicr, were determined by the
fumigation–extraction (FE) method (modified from Brookes,
1985a,b; Vance et al., 1987). Approximately 12 g (w : w)
root-free soil was put into each of 45 50 ml glass beakers,
which were placed in a desiccator (18 dm3 volume). Forty-five
millilitres of ethanol-free CHCl3 (Lichrosolv, Merck no. 2444,
Merck KGaA, Darmstadt, Germany) was used as fumigant
(22°C, 20 h). At the same time as the fumigation process was
started, the nonfumigated soil was shaken (150 rev min−1) for
30 min with 50 ml 0.5 M K2SO4 (mean soil : solution ratio =
1 : 13, w : v) and filtered (Munktell 00H filters (equivalent to
Whatman no. 42), Munktell Filter AB, Grycksbo, Sweden).
The fumigated soil was extracted as described above after
removal of the CHCl3 from the soil by repeated evacuations.
The extracts were kept frozen at −30°C before analysis.
Extracts were analysed for total organic C on a TOC-5000
(Shimadzu Corporation, Kyoto, Japan): the organic C component was combusted to CO2 at 680°C and detected on
an infrared gas analyser. Extractable DOC was determined as
total organic C in extracts from nonfumigated soil. The sum of
organic N and NH4-N in the K2SO4 extracts was determined
as NH4-N at 590 nm by flow injection analysis (FIAstar,
FOSS TECATOR, Höganäs, Sweden) after preincubation
and micro-Kjeldahl digestion (Wyland et al., 1994). The Cmicr
and Nmicr were obtained after correcting for the efficiency of
extraction of microbial biomass C and N, respectively. Cmicr
was calculated as Cmicr = Cf /kEC, where Cf is (organic C
extracted from fumigated soil) − (organic C extracted from
unfumigated soil) and kEC is 0.4. The Nmicr was calculated
as Nmicr = Nf /kEN, where Nf is (organic N extracted from
fumigated soil) − (organic N extracted from unfumigated
soil) and kEN is 0.4. These values of kEC and kEN were from a
similar soil in a Finnish Pinus sylvestris forest of C. vulgaris type
www.newphytologist.com © New Phytologist (2002) 154: 791–795
Research
and were calibrated by microscopic counting (Martikainen &
Palojärvi, 1990). Soil dry weight was determined after drying
at 105°C for 24 h. The organic matter content was determined by loss on ignition (600°C, 4 h) and pH was measured
in water (soil : solution ratio = 1 : 5, v : v).
Statistical analyses
Statistical analyses were performed using SIGMASTAT 2.0
(SPSS Science, Chicago, IL, USA). Effects of treatments and
blocks on Cmicr, Nmicr, microbial C : N and DOC were tested
by two-way ANOVA using the mean values for each plot. If a
significant effect (P < 0.05) was found, Tukey’s post hoc test
was performed to test for significant differences among
treatments and blocks.
Results and Discussion
Microbial C and N contributed 1.6% to soil organic C and
6.6% to soil organic N, respectively, on control plots. These
figures are in agreement with the mean values of 1.8% for C
and 8.5% for N given for a similar Finnish forest soil
(Martikainen & Palojärvi, 1990).
Microbial C was 41% lower on LG plots than on control
plots (P < 0.05), while on EG plots it was 23% lower than on
control plots (difference was nonsignificant) (Fig. 2a). For
Nmicr, there were no differences among treatments (Fig. 2b).
However, the C : N ratios of the soil microbial biomass were
significantly lower (P < 0.05) on both EG and LG than on
control plots (Fig. 2d). In this case, there was also a significant
block effect.
In the girdling experiment, in which up to 56% of soil respiration was lost during the first year after girdling (Högberg
et al., 2001), measured total soil respiratory activity on control plots included that of ECM roots and their fungal sheaths
in addition to that of the root-free soil studied here. In rootfree soil, there should be respiratory activity by the extramatrical ECM mycelium, extramatrical ericoid mycorrhizal
mycelium, saprophytic fungi, bacteria and other soil organisms.
In this study, the extramatrical ECM mycelium is the
Fig. 2 Microbial carbon (a), microbial nitrogen (b), extractable dissolved organic carbon DOC (c) and microbial carbon : nitrogen ratio (d) in
the different treatments: C, control; EG, early girdling; LG, late girdling. Treatments significantly different (P < 0.05) from the control are
indicated by asterisks. Bars are SE bars. Note that the EG and LG treatments are both significantly different from the control in (d); the large
apparent variability is partly caused by a significant block effect in this case.
© New Phytologist (2002) 154: 791– 795 www.newphytologist.com
793
794 Research
major functional component, along with mycorrhizosphere
organisms, that could be negatively affected by the girdling.
Conversely, the activity and growth of the other organisms
could be enhanced because they could use the dying ECM
mycelium as a substrate and/or benefit from a release from
competition for space and nutrients. Thus, based on the average loss of Cmicr in the treatments EG and LG, at least 32%
of the soil microbial biomass was contributed by extramatrical
ECM mycelium. This contribution was calculated to be
equivalent of 145 kg ha−1, corresponding to 58 kg C ha−1 at
a fungal biomass carbon content of 40% by dry weight.
Potential changes in microbial respiratory activity, growth
and community composition could have been going on for
3 months in EG plots compared with only 1 month in LG
plots. This difference in time since girdling may help to
explain the more drastic decline in Cmicr in LG plots. Therefore, the 41% loss of Cmicr in the LG treatment may be the
more relevant estimate of ECM biomass. The difference in
biomass between EG and LG plots could also relate to seasonality. For respiration, the highest calculated contribution
by ECM roots and mycelium was found in late summer
(Högberg et al., 2001), which is in line with observations on
C allocation patterns in temperate conifers (Hansen et al.,
1997). This means that the LG treatment was conducted when
the ECM fungal biomass would be expected to be greatest.
Several studies show a seasonal pattern in fungal biomass in
forest soils. High values for fluorescein diacetate (FDA)-active
fungi were found in early spring and autumn (Söderström,
1979; Bååth & Söderström, 1982) and growth of extramatrical
mycelium of ECM fungi was highest in the autumn (Wallander
et al., 2001). At the time of this study, the respiratory activity
was 37–39% lower in EG and LG plots than in control plots,
which suggests a rough correlation between biomass and
respiration.
The lower microbial C : N ratios of 7.1 and 6.2 in the EG
and LG soils, respectively, compared with 8.9 for the control
soil, may reflect a lower abundance of ECM fungi (lower
fungi : bacteria ratio), since the C : N ratio in bacteria is
mostly lower than in fungi (Paul & Clark, 1996). Alternatively, there are no changes in the microbial community with
respect to the abundance of fungi and bacteria. Thus, the
lower C : N ratios are simply the results of the same amounts
of N being associated with smaller amounts of C.
Levels of extractable DOC were 49% and 41% lower
on EG and LG plots, respectively, than on control plots
(Fig. 2c). At the same time, there were no differences in
extractable dissolved organic nitrogen (DON) among treatments (data not shown). This suggests the loss of organic
DOC with a high C : N ratio of 51. It would be of considerable
interest to know the molecular composition of the DOC in
the different treatments, since low molecular weight organic
acids are thought to play a major role in the mineral weathering by ECM fungi (van Breemen et al., 2000) and as DOC
comprises potentially important C sources for other microbes.
We suggest that our estimate of a 32% contribution by
extramatrical ECM mycelium to the total microbial biomass
is a conservative one, primarily because the dead ECM
mycelium could be used as a substrate by other organisms.
The particular strength of our estimates of the contribution of
ECM fungi to microbial biomass and by ECM roots and
fungi to the production of DOC is that they relate to an
organic soil in a field setting, in which the only major manipulation of the studied system is the removal of the C source
of the functional group of interest. Högberg et al. (2001)
demonstrated the vital importance of the flux of current
photosynthates to ECM roots for soil respiratory activity.
Our results show that this flux similarly directly supports
a considerable portion of the soil microbial biomass and is
of utmost importance for the production of DOC.
Acknowledgements
We thank Birgitta Olsson and Bengt Andersson for
conducting the FIA and TOC analysis, respectively. This
study was supported by grants from the Swedish Council for
Forestry and Agricultural Research (SJFR), the EU (project
FORCAST) and the Swedish Natural Sciences Research
Council (NFR).
References
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fungal biomass in a forest soil. Soil Biology and Biochemistry 14:
353–358.
van Breemen N, Finlay R, Lundström U, Jongmans AG, Giesler R,
Olsson M. 2000. Mycorrhizal weathering: a true case of mineral plant
nutrition? Biogeochemistry 49: 53–67.
Brookes PC. 1985a. Chloroform fumigation and the release of soil nitrogen:
the effect of fumigation time and temperature. Soil Biology and
Biochemistry 17: 831–835.
Brookes PC. 1985b. Chloroform fumigation and the release of soil nitrogen:
a rapid direct extraction method to measure microbial biomass nitrogen in
soil: the effect of fumigation time and temperature. Soil Biology and
Biochemistry 17: 837–842.
Garbaye J. 1994. Helper bacteria: a new dimension to the mycorrhizal
symbiosis. Tansley Review, 76. New Phytologist 128: 197–210.
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physiology: updating classical views. In: Rennenberg H, Eschrich W,
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Netherlands: Backhuys, 97–108.
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Högberg MN, Nyberg G, Ottosson-Löfvenius M, Read DJ. 2001.
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respiration. Nature 411: 789–792.
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Clark FE, eds. Soil microbiology and biochemistry. San Diego, CA, USA:
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SE, Read DJ, eds. Mycorrhizal symbiosis. London, UK: Academic Press,
409–452.
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horizons of a podzolized pine-forest soil in central Sweden. Soil Biology and
Biochemistry 11: 149–154.
Söderström B. 1992. The ecological potential of the ectomycorrhizal
mycelium. In: Read D, Lewis DH, Fitter AH, Alexander IJ, eds. Mycorrhizas in ecosystems. Cambridge, UK: CAB International, 77– 83.
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© New Phytologist (2002) 154: 791– 795 www.newphytologist.com
795
Extramatrical ectomycorrhizal mycelium contributes
one-third of microbial biomass and produces, together
with associated roots, half the dissolved organic carbon
in a forest soil
Blackwell Science, Ltd
Mona N. Högberg and Peter Högberg
Department of Forest Ecology, Section of Soil Science, Swedish University of Agricultural Sciences, SE−901 83 Umeå, Sweden
Summary
Author for correspondence:
Mona N. Högberg
Fax: + 46 90 7867750
Email: Mona.Hogberg@sek.slu.se
Received: 11 January 2002
Accepted: 12 February 2002
• A large-scale tree-girdling experiment enabled estimates in the field of the contribution
of extramatrical mycelium of ectomycorrhizal (ECM) fungi to soil microbial biomass
and by ECM roots and fungi to production of dissolved organic carbon (DOC).
• Tree-girdling was made early (EG) or late (LG) during the summer to terminate the
flow of photosynthate to roots and ECM fungi. Determination of microbial C (Cmicr)
and microbial N in root-free organic soil was performed by using the fumigation–
extraction technique; extractable DOC was determined on unfumigated soil.
• Soil Cmicr was 41% lower on LG than on control plots 1 month after LG, whereas
at the same time (that is, 3 months after EG), the Cmicr was 23% lower on EG than
on control plots. Extractable DOC was 45% lower on girdled plots than control plots.
• Our results, which are of particular interest as they were obtained directly in the
field, clearly demonstrate the important contribution by extramatrical ECM
mycelium to soil microbial biomass and by ECM roots to the production of DOC, a
carbon source for other microbes.
Key words: ectomycorrhizal fungi, extractable dissolved organic carbon (DOC),
ectomycorrhizal mycelium, forest soil, fumigation–extraction, microbial N, soil
microbial biomass.
© New Phytologist (2002) 154: 791– 795
Introduction
Biogeochemists, ecologists and soil microbiologists can be
divided into those that neglect and those that recognize the
role of mycorrhizal fungi in ecosystems. A reason for this
division is the problem of assessing the contribution of
mycorrhizal fungi to the soil microbial community, especially
in the field. More precise estimates are available on the carbon
(C) cost of ectomycorrhizal (ECM) symbiosis in laboratory
model systems (Rygiewicz & Anderson, 1994) but only rough
calculations are available for forest ecosystems (Söderström,
1992; Smith & Read, 1997). To date, it has not been possible
to quantify the contribution made by ECM fungi to total
microbial biomass in the soil, despite the need to distinguish
these symbiotic fungi from other mycorrhizal fungi and from
saprotrophic fungi and other decomposers. However, the
phospholipid fatty acid (PLFA) 18 : 2ω6,9 and ergosterol
© New Phytologist (2002) 154: 791– 795 www.newphytologist.com
fungal biomarkers were monitored inside and outside cores
(2.0 dm2) that were root isolated (Wallander et al., 2001).
The root isolation killed ECM fungi inside trenches and
Wallander et al. calculated the ECM biomass by subtracting
the amount of fungi outside, from the values obtained from
inside trenched cores. The ECM biomass, including that of
fungal mantels, was calculated to be approx. 800 kg ha−1.
The amount of ECM mycelium produced in mesh bags
filled with quartz sand, an inert substrate mainly colonized by
mycorrhizal fungi, was approx. 160 kg ha−1. It is unclear if
this estimate applies to the situation in the organic mor-layer,
which is the horizon of greatest biological activity in the
boreal forests.
Ectomycorrhizal fungi use C from their plant hosts, and it
seems unlikely that the fungi ensheathing these plant roots
leak much C to the surrounding soil. However, there is evidently a flora of bacteria on the surfaces of ECM hyphae
791
792 Research
(Garbaye, 1994; Timonen et al., 1998), and several studies
report that ECM fungi and roots produce large amounts of
certain organic acids (Griffiths et al., 1994; Wallander et al.,
1997). The contribution made by ECM roots and ECM
fungi to the production of dissolved organic C (DOC) in the
mor-layer of boreal forests, where concentrations of DOC are
high (van Hees et al., 2000), is not known. This gap in our
knowledge is particularly serious given that DOC affects rates
of weathering of soil minerals (Lundström et al., 2000) and
represents important sources of C for microbes.
In a recent large-scale tree-girdling experiment (Högberg
et al., 2001) a 50% loss of soil respiration was interpreted
as a loss of activity by ECM roots and their extramatrical
mycelium. Here, we make use of this unique field experiment to quantify the contribution by the extramatrical
mycelium of ECM fungi to total soil microbial biomass in
the organic mor-layer. We also used this experiment to estimate the production of extractable DOC by ECM roots and
fungi.
Materials and Methods
Field site, experimental design and soil sampling
The forest was a naturally regenerated 45- to 55-year-old
Scots pine (Pinus sylvestris L.) located on a weakly podzolized
sandy silt sediment at Åheden, northern Sweden (64°14′ N,
19°46′ E, 175 m above sea level). The climate is cold with a
mean annual temperature of 1.0°C, and a mean annual
precipitation of 600 mm. There is usually snow cover for
6 months between late October and early May. There was a
sparse understorey of Calluna vulgaris L. and Vaccinium vitisidaea L. The bottom layer consisted of mainly Cladonia
spp. lichens and Pleurozium schreberi moss. The organic morlayer (F + H horizons) was 2 cm thick and had the following characteristics (n = 9, mean ± SD): bulk density 0.16 ±
0.05 g cm−3, C : N ratio 40 ± 5, organic matter content
(weight loss on ignition) 76 ± 5%, pHH20 4.0 ± 0.1, water
content at the time of sampling 193 ± 41% (g g−1 dry wt).
The experiment comprised nine quadratic plots of 900 m2
each (with c. 120 trees each) and was divided into three blocks
(Fig. 1). Girdling was performed in early June 2000 (early
girdling, EG) and in mid-August 2000 (late girdling, LG)
on three plots at a time leaving three plots as control plots.
Girdling had no effects on soil temperature and moisture
(Högberg et al., 2001). Seventy-two days after girdling, the
number of sporocarps and their biomass were reduced by
98.4% and 99.4%, respectively, on the central 100 m2 of EG
plots compared with control plots.
On 12 September 2000, soil from the F and H horizons
was sampled by use of a 0.1-m diameter corer. Sampling was
performed along the border of the central 100 m2 of each
plot. Five composite samples made up from 10 cores each
were taken from each plot.
Fig. 1 Layout of the experiment. Each plot is 30 × 30 m. Treatments
are: C, control; EG, early girdling; LG, late girdling.
Fumigation and determinations of Cmicr and DOC
Soil samples were stored at 4°C overnight. Roots were
thereafter sorted out by hand. After a day at 16°C, microbial
C, Cmicr, and microbial N, Nmicr, were determined by the
fumigation–extraction (FE) method (modified from Brookes,
1985a,b; Vance et al., 1987). Approximately 12 g (w : w)
root-free soil was put into each of 45 50 ml glass beakers,
which were placed in a desiccator (18 dm3 volume). Forty-five
millilitres of ethanol-free CHCl3 (Lichrosolv, Merck no. 2444,
Merck KGaA, Darmstadt, Germany) was used as fumigant
(22°C, 20 h). At the same time as the fumigation process was
started, the nonfumigated soil was shaken (150 rev min−1) for
30 min with 50 ml 0.5 M K2SO4 (mean soil : solution ratio =
1 : 13, w : v) and filtered (Munktell 00H filters (equivalent to
Whatman no. 42), Munktell Filter AB, Grycksbo, Sweden).
The fumigated soil was extracted as described above after
removal of the CHCl3 from the soil by repeated evacuations.
The extracts were kept frozen at −30°C before analysis.
Extracts were analysed for total organic C on a TOC-5000
(Shimadzu Corporation, Kyoto, Japan): the organic C component was combusted to CO2 at 680°C and detected on
an infrared gas analyser. Extractable DOC was determined as
total organic C in extracts from nonfumigated soil. The sum of
organic N and NH4-N in the K2SO4 extracts was determined
as NH4-N at 590 nm by flow injection analysis (FIAstar,
FOSS TECATOR, Höganäs, Sweden) after preincubation
and micro-Kjeldahl digestion (Wyland et al., 1994). The Cmicr
and Nmicr were obtained after correcting for the efficiency of
extraction of microbial biomass C and N, respectively. Cmicr
was calculated as Cmicr = Cf /kEC, where Cf is (organic C
extracted from fumigated soil) − (organic C extracted from
unfumigated soil) and kEC is 0.4. The Nmicr was calculated
as Nmicr = Nf /kEN, where Nf is (organic N extracted from
fumigated soil) − (organic N extracted from unfumigated
soil) and kEN is 0.4. These values of kEC and kEN were from a
similar soil in a Finnish Pinus sylvestris forest of C. vulgaris type
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Research
and were calibrated by microscopic counting (Martikainen &
Palojärvi, 1990). Soil dry weight was determined after drying
at 105°C for 24 h. The organic matter content was determined by loss on ignition (600°C, 4 h) and pH was measured
in water (soil : solution ratio = 1 : 5, v : v).
Statistical analyses
Statistical analyses were performed using SIGMASTAT 2.0
(SPSS Science, Chicago, IL, USA). Effects of treatments and
blocks on Cmicr, Nmicr, microbial C : N and DOC were tested
by two-way ANOVA using the mean values for each plot. If a
significant effect (P < 0.05) was found, Tukey’s post hoc test
was performed to test for significant differences among
treatments and blocks.
Results and Discussion
Microbial C and N contributed 1.6% to soil organic C and
6.6% to soil organic N, respectively, on control plots. These
figures are in agreement with the mean values of 1.8% for C
and 8.5% for N given for a similar Finnish forest soil
(Martikainen & Palojärvi, 1990).
Microbial C was 41% lower on LG plots than on control
plots (P < 0.05), while on EG plots it was 23% lower than on
control plots (difference was nonsignificant) (Fig. 2a). For
Nmicr, there were no differences among treatments (Fig. 2b).
However, the C : N ratios of the soil microbial biomass were
significantly lower (P < 0.05) on both EG and LG than on
control plots (Fig. 2d). In this case, there was also a significant
block effect.
In the girdling experiment, in which up to 56% of soil respiration was lost during the first year after girdling (Högberg
et al., 2001), measured total soil respiratory activity on control plots included that of ECM roots and their fungal sheaths
in addition to that of the root-free soil studied here. In rootfree soil, there should be respiratory activity by the extramatrical ECM mycelium, extramatrical ericoid mycorrhizal
mycelium, saprophytic fungi, bacteria and other soil organisms.
In this study, the extramatrical ECM mycelium is the
Fig. 2 Microbial carbon (a), microbial nitrogen (b), extractable dissolved organic carbon DOC (c) and microbial carbon : nitrogen ratio (d) in
the different treatments: C, control; EG, early girdling; LG, late girdling. Treatments significantly different (P < 0.05) from the control are
indicated by asterisks. Bars are SE bars. Note that the EG and LG treatments are both significantly different from the control in (d); the large
apparent variability is partly caused by a significant block effect in this case.
© New Phytologist (2002) 154: 791– 795 www.newphytologist.com
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major functional component, along with mycorrhizosphere
organisms, that could be negatively affected by the girdling.
Conversely, the activity and growth of the other organisms
could be enhanced because they could use the dying ECM
mycelium as a substrate and/or benefit from a release from
competition for space and nutrients. Thus, based on the average loss of Cmicr in the treatments EG and LG, at least 32%
of the soil microbial biomass was contributed by extramatrical
ECM mycelium. This contribution was calculated to be
equivalent of 145 kg ha−1, corresponding to 58 kg C ha−1 at
a fungal biomass carbon content of 40% by dry weight.
Potential changes in microbial respiratory activity, growth
and community composition could have been going on for
3 months in EG plots compared with only 1 month in LG
plots. This difference in time since girdling may help to
explain the more drastic decline in Cmicr in LG plots. Therefore, the 41% loss of Cmicr in the LG treatment may be the
more relevant estimate of ECM biomass. The difference in
biomass between EG and LG plots could also relate to seasonality. For respiration, the highest calculated contribution
by ECM roots and mycelium was found in late summer
(Högberg et al., 2001), which is in line with observations on
C allocation patterns in temperate conifers (Hansen et al.,
1997). This means that the LG treatment was conducted when
the ECM fungal biomass would be expected to be greatest.
Several studies show a seasonal pattern in fungal biomass in
forest soils. High values for fluorescein diacetate (FDA)-active
fungi were found in early spring and autumn (Söderström,
1979; Bååth & Söderström, 1982) and growth of extramatrical
mycelium of ECM fungi was highest in the autumn (Wallander
et al., 2001). At the time of this study, the respiratory activity
was 37–39% lower in EG and LG plots than in control plots,
which suggests a rough correlation between biomass and
respiration.
The lower microbial C : N ratios of 7.1 and 6.2 in the EG
and LG soils, respectively, compared with 8.9 for the control
soil, may reflect a lower abundance of ECM fungi (lower
fungi : bacteria ratio), since the C : N ratio in bacteria is
mostly lower than in fungi (Paul & Clark, 1996). Alternatively, there are no changes in the microbial community with
respect to the abundance of fungi and bacteria. Thus, the
lower C : N ratios are simply the results of the same amounts
of N being associated with smaller amounts of C.
Levels of extractable DOC were 49% and 41% lower
on EG and LG plots, respectively, than on control plots
(Fig. 2c). At the same time, there were no differences in
extractable dissolved organic nitrogen (DON) among treatments (data not shown). This suggests the loss of organic
DOC with a high C : N ratio of 51. It would be of considerable
interest to know the molecular composition of the DOC in
the different treatments, since low molecular weight organic
acids are thought to play a major role in the mineral weathering by ECM fungi (van Breemen et al., 2000) and as DOC
comprises potentially important C sources for other microbes.
We suggest that our estimate of a 32% contribution by
extramatrical ECM mycelium to the total microbial biomass
is a conservative one, primarily because the dead ECM
mycelium could be used as a substrate by other organisms.
The particular strength of our estimates of the contribution of
ECM fungi to microbial biomass and by ECM roots and
fungi to the production of DOC is that they relate to an
organic soil in a field setting, in which the only major manipulation of the studied system is the removal of the C source
of the functional group of interest. Högberg et al. (2001)
demonstrated the vital importance of the flux of current
photosynthates to ECM roots for soil respiratory activity.
Our results show that this flux similarly directly supports
a considerable portion of the soil microbial biomass and is
of utmost importance for the production of DOC.
Acknowledgements
We thank Birgitta Olsson and Bengt Andersson for
conducting the FIA and TOC analysis, respectively. This
study was supported by grants from the Swedish Council for
Forestry and Agricultural Research (SJFR), the EU (project
FORCAST) and the Swedish Natural Sciences Research
Council (NFR).
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