Directory UMM :Data Elmu:jurnal:T:Tree Physiology:Vol16.1996:
Tree Physiology 16, 787--793
© 1996 Heron Publishing----Victoria, Canada
Carbon and nitrogen allocation in ectomycorrhizal and
non-mycorrhizal Pinus sylvestris L. seedlings
JAN V. COLPAERT,1 ANDRÉ VAN LAERE,1 and JOZEF A. VAN ASSCHE2
1
2
Laboratory of Developmental Biology, Institute of Botany, Katholieke Universiteit Leuven, K. Mercierlaan, 92, B-3001 Leuven, Belgium
Laboratory of Ecology, Institute of Botany, Katholieke Universiteit Leuven, K. Mercierlaan, 92, B-3001 Leuven, Belgium
Received November 29, 1995
Summary We studied carbon and nitrogen allocation in mycorrhizal and non-mycorrhizal Scots pine (Pinus sylvestris L.)
seedlings grown in a semi-hydroponic system with nitrogen as
the growth limiting factor. Three ectomycorrhizal fungi were
compared: one pioneer species (Thelephora terrestris Ehrh.: Fr.)
and two late-stage fungi (Suillus bovinus (L.: Fr.) O. Kuntze,
and Scleroderma citrinum Pers.). By giving all plants in each
treatment the same amount of readily available nitrogen, we
ensured that the external mycelium could not increase the total
nitrogen content of the plants, thereby guaranteeing that any
change in carbon or nitrogen partitioning was a direct effect of
the mycorrhizal infection itself. Carbon and nitrogen partitioning were measured at an early and a late stage of mycorrhizal
development, and at a low and a high N addition rate.
Although mycorrhizal seedlings had a higher net assimilation rate and a higher shoot/root ratio than non-mycorrhizal
seedlings, they had a lower rate of shoot growth. The high
carbon demand of the mycobionts was consistent with the
large biomass of external mycelia and the increased belowground respiration of the mycorrhizal plants. The carbon cost
to the host was similar for pioneer and late-stage fungi. Aboveand belowground partitioning of nitrogen was also affected by
mycorrhizal infection. The external mycelia of Scleroderma
citrinum retained 32% of the nitrogen supplied to the plants,
thus significantly reducing nitrogen assimilation by the host
plants and consequently reducing their growth rate. By contrast, the external mycelia of T. terrestris and Suillus bovinus
retained less nitrogen than the mycelia of Scleroderma
citrinum, hence we attributed the decreased growth rates of
their host plants to a carbon drain rather than a nitrogen
deficiency.
Keywords: carbon allocation, nitrogen partitioning, root respiration, Scleroderma citrinum, shoot/root ratio, Suillus
bovinus, Thelephora terrestris.
Introduction
Most higher plant species in terrrestrial ecosystems have associated mycorrhizal fungi, which have direct access to the
assimilates of their hosts and also serve as carriers for mineral
nutrients to the host (Finlay and Read 1986, Finlay et al. 1988).
These fungi demand large amounts of carbon, and it is this
demand that gives rise to mycorrhizal symbiosis, which, in
turn, is important not only for the growth of both symbionts but
also for the carbon balance of ecosystems. Several studies have
shown that mycorrhizal fungi use a significant fraction of the
net primary production of natural forests (Vogt et al. 1982,
Finlay and Söderström 1992). Laboratory studies suggest that
mycorrhizal symbiosis results in significantly higher belowground carbon allocation than in non-symbiotic conditions.
Recent studies have suggested that mycorrhizae impose a
higher C cost on their hosts under field conditions than under
experimental conditions (Rygiewicz and Andersen 1994,
Tinker et al. 1994).
Ectomycorrhizal fungi can reduce the growth rate of their
host plant (Nylund and Wallander 1989, Dosskey et al. 1990,
Rygiewicz and Andersen 1994). Such yield reductions are
usually attributed to an increase in belowground carbon allocation. We postulate that this cost of symbiosis is partly compensated by an increase in shoot/root ratio in mycorrhizal
plants. We also hypothesize that retention of nitrogen in the
mycobiont might lead to growth repression in the host plant.
Because nitrogen can be a growth limiting factor in forests
(Attiwill and Adams 1993), the diversion of nitrogen for fungal
growth might have consequences for tree growth and nutrient
cycling in natural ecosystems.
We studied growth, nitrogen partitioning, and rates of photosynthesis and respiration in the above- and belowground
parts of mycorrhizal and non-mycorrhizal Scots pine (Pinus
sylvestris L.) seedlings at an early and a late stage of fungal
development. Three ectomycorrhizal fungi were compared: a
pioneer species Thelephora terrestris Ehrh.: Fr., and two latestage fungi, Suillus bovinus (L.: Fr.) O. Kuntze and Scleroderma citrinum Pers. (Fox 1986). We compared a pioneer
fungal species with two late-stage fungal species because there
is evidence that there are important physiological differences
between pioneer and late-stage ectomycorrhizal fungi, and
little is known about the nutrient requirements of the different
fungi. Compared to late-stage fungi, pioneer fungi are charac-
788
COLPAERT, VAN LAERE AND VAN ASSCHE
terized by a more rapid growth, lower energy investment in
biomass and early carpophore development with more germinable propagules (Dighton 1991, Deacon and Fleming
1992). Pioneer fungi also tolerate higher concentrations of
inorganic nutrients than late-stage fungi.
Materials and methods
Plant and fungus material
Half-sib seeds of Pinus sylvestris were surface sterilized in
30% H2O2 for 15 min, sown in a 1/1 (v/v) mix of vermiculite
and perlite and watered with a balanced nutrient solution
(Ingestad et al. 1986). The weight proportions of the macronutrients in the solution were 100 N/60 K/18 P/6 Ca/6 Mg/9 S.
After 10 weeks, plants were selected for uniformity and inoculated with a mycorrhizal fungus or left uninoculated. Three
ectomycorrhizal species were used for inoculation: Thelephora terrestris (24 plants), Suillus bovinus (24 plants) and
Scleroderma citrinum (12 plants). For inoculations, mycorrhizal fungi were grown in 10-cm diameter plastic petri dishes
containing modified Melin-Norkrans agar medium covered
with sterile cellophane sheets. Once the mycelia had covered
most of the cellophane surface, the root system of a selected
seedling was spread over the young mycelia and a thick filter
paper (9 cm in diameter), soaked in Ingestad’s nutrient solution, was used to cover the roots and mycelia. Control plants
were treated in the same way, except that their roots were
placed on Melin-Norkrans agar medium without mycelium.
Three to four days after inoculation, the plants were transplanted to 4-liter containers to allow the growth of extensive
external mycelia.
Growth conditions
After inoculation, two plants were transferred to each 4-liter
container (0.40 × 0.20 × 0.05 m) and 250 g of acid-washed
perlite was added. The perlite was irrigated with 1.45 dm3 of
nutrient solution, resulting in saturation of the substrate to 80%
of its water holding capacity. The perlite in each container was
covered with a dark plastic lid to prevent algal growth. The
containers were weighed at least twice a week so that water use
could be estimated during the experiment. After each weighing, the containers were rearranged on the benches in the
growth room.
Plants were maintained in non-sterile conditions in a growth
chamber in a 16-h photoperiod and a day/night temperature of
22/15 °C. Relative air humidity was at least 70% and photosynthetically active radiation (PAR) was 400 µmol m −2 s −1.
The seedlings were grown at two nutrient addition rates. Initially, nutrients were supplied daily in a single addition but
after 8 weeks two additions per day were necessary.
Treatments
Six plant containers of each fungal treatment received balanced nutrient solution for Scots pine at a low relative addition
rate of 2.6% day −1 (LN treatment). The amount of nitrogen
supplied was based on the nitrogen content of the seedlings
(Ingestad et al. 1986). The nitrogen concentration of the nutrient solution was 20 mg l −1. On the first day, 0.4 mg N was
supplied to each plant and the amount was gradually increased
to 4 mg on Day 90. Distilled water was supplied to compensate
for differences in water use between plants. The initial nitrogen
content of the irrigated perlite in the LN treatment was 20 mg
per container. The perlite substrate was maintained at a pH of
4.0 by the addition of a 1/1 mix of NO −3 and NH −4 . Adsorption
of added N on the perlite was low (< 10%).
For the second treatment (the HN treatment), control plants
and plants inoculated with T. terrestris or Suillus bovinus
received exactly twice the amount of nutrients as the plants in
the LN treatment (0.8 mg N per plant on Day 1 up to 8 mg on
day 90). The initial nitrogen content of the irrigated perlite in
the HN treatment was 40 mg per container.
Gas exchange measurements
Measurements of net photosynthesis and respiration were conducted on all seedlings before harvest. Carbon dioxide was
measured with an infrared gas analyzer (IRGA) in an open
system. Outside air was allowed to adjust to the ambient
temperature of the growth room. Before measurement, entire
plants were transferred to PVC root cuvettes without disturbing the root substrate. The inner compartment of each cuvette
was exactly the same size as the containers. The bottom and
one side wall (0.2 × 0.4 m) of each inner compartment consisted of a stainless steel grid, with a 1-cm air gap between the
grid and the bottom and side of the root cuvette. Relative
humidity of air entering the root compartment was adjusted to
80--85%. Temperature and relative humidity of air entering
and leaving the root cuvette were measured. Air was circulated
through the root compartment for 4 h before commencing the
CO2 measurements. Measurements were recorded at 10-min
intervals.
Immediately after root respiration was recorded, the air
stream through the root compartment was stopped. To determine photosynthetic rate, a shoot cuvette (3.8 or 8.5 dm3, with
a maximal flow rate through the cuvette of 14 dm3 min −1),
which enclosed both seedlings, was mounted on the root compartment and measurements of CO2 were continued until
steady-state conditions were obtained. Temperature in the
shoot compartment was about 1.0 °C higher than ambient
temperature. Aboveground respiration was determined immediately after the photosynthesis measurements were completed. Steady-state conditions were achieved in the dark after
about 15 min.
Harvest
Plants were harvested at Weeks 5 and 12 after transfer of the
seedlings to the 4-liter containers. Two subsamples of rooting
substrate (approximately 20 g), free of root material, were
transferred to 20 ml of distilled water, stirred for 15 min and
subsequently vacuum-filtered over a 0.45 µm membrane filter.
Conductivity and nitrogen concentration of the filtrate were
determined. The filtered perlite was subsequently washed with
two 20-ml aliquots of distilled H2O, dried at 50 °C, weighed
and stored until used for determination of fungal biomass. We
C AND N ALLOCATION IN MYCORRHIZAL SCOTS PINE
defined the external mycelium as the fungal biomass that was
not associated with roots.
Shoots were separated from roots, dried at 80 °C for at least
4 days and weighed. Carpophores and roots were rinsed free
of perlite on a 1-mm sieve and subsequently treated in the same
way as the shoots. For the calculation of shoot/root ratio and
relative growth rate (RGR), the fungal biomass of the mycelium in the substrate was excluded. The RGR, expressed as %
day −1 was calculated by fitting seedling weight (y) at different
days (x; start day and both harvests) to the equation y = aeRGRx.
Nitrogen determinations
Nitrogen in oven-dried shoots, roots and rooting substrate was
extracted by the Kjeldahl procedure. The NH3 was steam
distilled into H2SO4 (0.05 M) and determined colorimetrically
with Nessler’s reagent. Nitrogen was also determined in the
carpophores of T. terrestris and Scleroderma citrinum formed
during the experiment as well as in a Suillus bovinus mycelium
cultured on a modified Melin-Norkrans medium without malt
extract. Data from the Suillus mycelium were used to calculate
fungal biomass from the N concentration of the washed perlite
substrate (Colpaert et al. 1992). Perlite from the non-mycorrhizal treatment was used to determine the background concentration of the non-mycorrhizal N pool. The N concentration of
the Suillus bovinus mycelium might be slightly overestimated
because the N concentration of the MMN medium (53 mg l −1)
was higher than that of the plant nutrient solution. This error
could result in slight underestimations of fungal biomass,
especially in the LN treatment.
Statistical analyses
Significant differences between means within a single treatment were determined with a one-way ANOVA and the
Tukey’s studentized range test.
Results
Plant and fungal growth
Dry weights of shoots, roots and external mycelia are shown
in Table 1. Decreased shoot growth in the mycorrhizal plants
789
was already apparent 5 weeks after inoculation. Twelve weeks
after inoculation, non-mycorrhizal seedlings had significantly
larger shoots and roots than mycorrhizal seedlings in both
nutrient treatments. Although relative growth rates of mycorrhizal plants decreased during the study, their shoot/root ratios
increased, indicating that mycorrhizal infection suppressed
root growth more than shoot growth (Table 2).
In the LN treatment, plant water use increased exponentially
throughout the experiment (r 2 > 0.98), whereas in the HN
treatment, water use plateaued in the last month of the experiment, indicating that seedlings had entered a more linear
growth phase. The conductivity of the solution in the perlite
was between 20 and 45 µS cm −1 at Week 5 and between 10 and
25 µS cm −1 at Week 12. The amount of nitrogen that could be
washed from the perlite was always less than the amount of
nitrogen added. These observations indicate that plant nutrient
uptake rates were high in both treatments, and there was no
oversupply of nutrients.
By Week 5, T. terrestris had completely colonized the
growth substrate, infecting almost all short roots. Suillus
bovinus and Scleroderma citrinum had colonized 75% of the
growth substrate, infecting 80 and 90% of short roots, respectively. By Week 12, substrate colonization and short root infection were close to 100% in all fungal treatments, indicating that
the nutrient treatments did not affect the colonization rates of
substrate and roots.
Carpophores formed in all containers with T. terrestris and
in two containers with Scleroderma citrinum. The N concentration of the T. terrestris carpophores was 2.5% in the LN
treatment and 2.7% in the HN treatment, whereas the N concentration in Scleroderma citrinum carpophores was 3.8%.
Suillus bovinus formed no carpophores and contained 31 mg
N per g dry weight. When we used these N values to calculate
fungal biomass in the perlite substrate, we found that
Scleroderma citrinum produced the largest external mycelial
biomass, and that T. terrestris (in the LN treatment) formed the
least dense mycelium, 31% of which was made up of small
carpophores. The mycelial biomass of T. terrestis was greater
in the HN treatment than in the LN treatment, and carpophores
accounted for 53% of its biomass.
Table 1. Dry weights of shoots, roots and external mycelia (including carpophores) of mycorrhizal and non-mycorrhizal Pinus sylvestris seedlings
grown at a low or a high nutrient addition rate. Standard errors are shown beside values; for each treatment, means within a column followed by
different letters are significantly different (n = 6, one-way ANOVA, Tukey’s test, α = 0.05).
Shoot (g)
Root (g)
Mycelium (g)
Week 5
Week 12
Week 5
Week 12
Week 5
Week 12
Low N
Control
Thelephora terrestris
Suillus bovinus
Scleroderma citrinum
1.42 ± 0.05 a
1.36 ± 0.03 a
1.34 ± 0.03 a
1.29 ± 0.03 a
5.96 ± 0.15 a
5.31 ± 0.13 b
5.09 ± 0.15 b
4.44 ± 0.10 c
1.50 ± 0.07 a
1.26 ± 0.04 b
1.18 ± 0.05 b
1.11 ± 0.06 b
5.84 ± 0.30 a
4.28 ± 0.40 b
4.07 ± 0.18 b
3.50 ± 0.12 b
0 a
0.18 ± 0.02 b
0.24 ± 0.02 b
0.27 ± 0.02 c
0 a
0.98 ± 0.06 b
0.82 ± 0.07 b
1.18 ± 0.06 c
High N
Control
Thelephora terrestris
Suillus bovinus
2.02 ± 0.04 x
1.78 ± 0.06 y
1.84 ± 0.05 y
9.46 ± 0.39 x
7.04 ± 0.25 y
7.47 ± 0.18 y
1.54 ± 0.10 x
1.33 ± 0.07 x
1.32 ± 0.04 x
7.57 ± 0.49 x
4.82 ± 0.19 y
5.12 ± 0.34 y
0 x
0.24 ± 0.02 y
0.19 ± 0.02 z
0 x
2.27 ± 0.15 y
1.17 ± 0.09 z
790
COLPAERT, VAN LAERE AND VAN ASSCHE
Nitrogen partitioning
Although mycorrhizal plants had a lower dry weight than
non-mycorrhizal plants, the reduction did not result in an
increase in shoot N concentration (Table 3). Plants infected
with Scleroderma citrinum had a lower shoot N concentration
than non-mycorrhizal plants, and this finding was confirmed
by a slight yellowing of the needles. The N concentration of
mycorrhizal roots was significantly higher than that of nonmycorrhizal roots.
Partitioning of N in above- and belowground biomass is
shown in Figure 1. Mycorrhizal root systems retained a larger
proportion of N than non-mycorrhizal root systems, indicating
that the external mycelia function as an N sink. By Week 12,
mycelia had retained between 15 and 32% of all N assimilated
(Table 4). These values would be higher if the N contained in
the mycorrhizas was also included. Moreover, the recovery of
all N added to the plants was similar or slightly higher in
mycorrhizal plants than in non-mycorrhizal plants, whereas
the amount of N transported to the shoots was considerably
Table 2. Shoot/root ratio and relative growth rate of mycorrhizal and
non-mycorrhizal Pinus sylvestris seedlings grown at a low or a high
nutrient addition rate. Standard errors are shown beside values; for
each treatment, means within a column followed by different letters
are significantly different (n = 6, one-way ANOVA, Tukey’s test,
α = 0.05).
Shoot/root ratio
RGR
Week 5
Week 12
% day −1 r 2
0.95 ± 0.02 a
1.08 ± 0.03 b
1.14 ± 0.03 b
1.16 ± 0.03 b
1.02 ± 0.02 a
1.24 ± 0.03 b
1.25 ± 0.03 b
1.27 ± 0.04 b
2.8
2.6
2.6
2.5
0.97
0.98
0.97
0.97
High N
Control
1.31 ± 0.04 x 1.25 ± 0.03 x 3.2
Thelephora terrestris 1.34 ± 0.03 x 1.46 ± 0.04 y 2.9
Suillus bovinus
1.39 ± 0.04 x 1.46 ± 0.05 y 2.9
0.96
0.93
0.95
Low N
Control
Thelephora terrestris
Suillus bovinus
Scleroderma citrinum
less in mycorrhizal plants than in non-mycorrhizal plants in
both nutrient treatments (Figure 1).
Net photosynthesis and respiration
Plants infected with Suillus bovinus and T. terrestris had
slightly higher assimilation rates than uninfected plants or
plants inoculated with Scleroderma citrinum (Table 5). Although the presence of mycorrhizal fungi had no effect on dark
respiration of shoots, it significantly increased respiration of
roots. The percentage of fixed carbon respired belowground by
mycorrhizal plants was twice that respired by non-mycorrhizal
plants (Tables 5 and 6).
Discussion
A difficulty associated with comparative growth studies of
mycorrhizal and non-mycorrhizal plants is that such studies
only provide a measure of the net benefit of mycorrhizal
infection, including nutritional effects, and thus give no direct
information about carbon allocation to the fungus. We overcame this difficulty by using a semi-hydroponic technique.
This technique greatly reduces the nutritional benefit to the
Table 4. The proportion of assimilated nitrogen in the external mycelia, including carpophores, of mycorrhizal Pinus sylvestris seedlings.
Standard errors are shown beside values; for each treatment, means
within a column followed by different letters are significantly different
(n = 6, one-way ANOVA, Tukey’s test, α = 0.05).
Fungal nitrogen (% of total)
Week 5
Week 12
Low N
Thelephora terrestris
Suillus bovinus
Scleroderma citrinum
13 ± 2 a
16 ± 2 a
26 ± 3 b
19 ± 2 a
18 ± 2 a
32 ± 2 b
High N
Thelephora terrestris
Suillus bovinus
13 ± 2 x
12 ± 1 x
25 ± 2 x
15 ± 1 y
Table 3. Nitrogen concentration in shoots and roots of mycorrhizal and non-mycorrhizal Pinus sylvestris seedlings grown at a low or a high nutrient
addition rate. Standard errors are shown beside values; for each treatment, means within a column followed by different letters are significantly
different (n = 6, one-way ANOVA, Tukey’s test, α = 0.05).
Shoot N (% of dry weight)
Root (% of dry weight)
Week 5
Week 12
Week 5
Week 12
Low N
Control
Thelephora terrestris
Suillus bovinus
Scleroderma citrinum
1.19 ± 0.03 a
1.10 ± 0.03 a
1.12 ± 0.03 a
0.96 ± 0.02 b
1.05 ± 0.04 a
0.95 ± 0.03 a
0.95 ± 0.03 a
0.80 ± 0.02 b
1.31 ± 0.03 a
1.39 ± 0.05 ab
1.46 ± 0.04 b
1.45 ± 0.04 b
1.25 ± 0.03 a
1.43 ± 0.04 b
1.61 ± 0.04 c
1.70 ± 0.05 c
High N
Control
Thelephora terrestris
Suillus bovinus
1.35 ± 0.03 x
1.27 ± 0.02 y
1.30 ± 0.03 xy
1.45 ± 0.04 x
1.38 ± 0.04 x
1.48 ± 0.05 x
1.41 ± 0.03 x
1.64 ± 0.05 y
1.66 ± 0.04 y
1.40 ± 0.05 x
1.88 ± 0.05 y
1.84 ± 0.04 y
C AND N ALLOCATION IN MYCORRHIZAL SCOTS PINE
791
Figure 1. Above- and belowground nitrogen partitioning in Pinus sylvestris
seedlings uninoculated (NM) or inoculated with Thelephora terrestris (T te),
Suillus bovinus (S bo) or Scleroderma
citrinum (S ci). Data were obtained 12
weeks after inoculation. For each plant
part, bars with a different letter are significantly different (n = 6, Tukey’s test,
α = 0.05). LN = low nutrient addition
rate, HN = high nutrient addition rate.
Table 5. Rates of respiration and net assimilation in non-mycorrhizal and mycorrhizal Pinus sylvestris seedlings. Units are: aboveground, nmol
CO2 gDWS−1 s −1; belowground (roots and mycelium), nmol CO2 gDWR−1 s −1. Standard errors are shown beside values; for each treatment, means
within a column followed by different letters are significantly different (n = 6, one-way ANOVA, Tukey’s test, α = 0.05).
Net assimilation rate
Aboveground respiration
Belowground respiration
Week 5
Week 12
Week 5
Week 12
Week 5
Week 12
Low N
Control
Thelephora terrestris
Suillus bovinus
Scleroderma citrinum
20.2 ± 0.9 ab
19.6 ± 0.8 ab
22.1 ± 0.8 a
18.5 ± 1.0 b
14.9 ± 0.5 a
17.4 ± 0.8 ab
17.6 ± 0.8 b
14.7 ± 0.7 a
6.5 ± 0.5 a
6.9 ± 0.6 a
7.0 ± 0.5 a
6.4 ± 0.4 a
6.1 ± 0.5 a
6.3 ± 0.3 a
6.4 ± 0.4 a
6.1 ± 0.4 a
3.6 ± 0.3 a
5.0 ± 0.5 ab
6.5 ± 0.4 b
5.7 ± 0.3 b
2.3 ± 0.2 a
5.1 ± 0.3 b
5.7 ± 0.4 b
4.7 ± 0.3 b
High N
Control
Thelephora terrestris
Suillus bovinus
16.9 ± 1.0 x
17.8 ± 1.0 xy
20.8 ± 0.8 y
14.0 ± 0.7 x
16.1 ± 0.6 x
14.4 ± 1.0 x
6.2 ± 0.6 x
5.9 ± 0.4 x
6.2 ± 0.5 x
5.7 ± 0.4 x
5.3 ± 0.3 x
6.0 ± 0.5 x
4.0 ± 0.3 x
5.5 ± 0.3 y
7.6 ± 0.4 z
2.5 ± 0.2 x
4.8 ± 0.2 y
4.1 ± 0.3 y
plant of the mycorrhizal infection by ensuring that both infected and non-infected plants have good access to the same
nutrients, without affecting the cost of the symbiosis to the host
plant (cf. Kähr and Arveby 1986, Nylund and Wallander 1989,
Kamminga-Van Wijk et al. 1992).
Inoculation with ectomycorrhizal fungi of Scots pine seedlings growing in a semi-hydroponic system resulted in decreased plant growth. All three mycorrhizal fungi inhibited
root development more than shoot growth. Harley and Smith
(1983) concluded that the shoot/root ratio of mycorrhizal
plants increased because of their enhanced ability to absorb
nutrients, whereas Smith (1980) argued that the mycorrhizalinduced increase in shoot/root ratio was a feed-back response
to an increased nutrient uptake by the mycorrhizae. Hetrick
(1991) observed that increases in shoot/root ratio are greater in
strongly mycotropic plants. Because we used a semi-hydroponic system, the increase in shoot/root ratio of the mycorrhizal plants in our study cannot be attributed to increased nutrient
uptake or increased growth rate of the mycorrhizal plants: we
conclude, therefore, that the increase is a result of the mycorrhizal infection itself. Decreased retention of carbon in the host
roots indicates that the fungus was a stronger sink for carbon
than the host roots. A shift in carbon allocation from root
growth to fungal growth could explain the increase in
Table 6. The proportion of net fixed carbon respired by the root
systems of non-mycorrhizal and mycorrhizal Pinus sylvestris seedlings. Standard errors are shown beside values; for each treatment,
means within a column followed by different letters are significantly
different (n = 6, one-way ANOVA, Tukey’s test, α = 0.05).
Belowground respiration (% NA)1
Week 5
Week 12
Low N
Control
Thelephora terrestris
Suillus bovinus
Scleroderma citrinum
19 ± 2 a
27 ± 2 b
31 ± 2 b
33 ± 3 b
15 ± 1 a
29 ± 2 b
31 ± 2 b
34 ± 2 b
High N
Control
Thelephora terrestris
Suillus bovinus
18 ± 2 x
27 ± 2 y
30 ± 2 y
14 ± 1 x
30 ± 2 y
24 ± 1 y
1
NA = Net assimilation.
shoot/root ratio of the mycorrhizal plants. Miller et al. (1989)
demonstrated that the extramatrical hyphae of ectomycorrhizal
pine seedlings constitute a strong sink for carbon, especially
when a new network of external hyphae was developing. In
792
COLPAERT, VAN LAERE AND VAN ASSCHE
natural ecosystems, roots of young seedlings probably integrate into the existing soil mycelia of late-stage fungi so that
they do not have to sustain an extensive ectomycorrhizal mycelium as would occur on pioneer sites where no active mycelia of late-stage fungi are present. For these reasons, we
expected that the carbon demand of the pioneer fungus T. terrestris would differ from that of the late-stage fungus Suillus
bovinus. However, we found that host plants of both fungi had
similar root respiration rates and similar shoot growth, although the fungi did exhibit different carbon investment strategies. Thelephora terrestris developed a rather sparse substrate
mycelium connected with numerous sporulating carpophores,
whereas Suillus bovinus produced a more slow-growing, dense
substrate mycelium. The pioneer species was more sensitive to
the nutrient regime than the late-stage fungus, and the HN
treatment caused a doubling of its biomass compared with the
LN treatment.
Although it is well known that mycorrhizal fungi have a
large carbon demand, other details of the nutrient requirement
for fungal development are sparse. However, such information
may be important because tree growth is often limited by
nitrogen or phosphorus in natural ecosystems (Attiwill and
Adams 1982). In the field, concentrations of minerals in ectomycorrhizal carpophores are consistently higher than the
concentrations in most plant tissues (Vogt and Edmonds 1980,
Vogt et al. 1982). We found that nitrogen use by the mycorrhizal fungi was extensive. The relatively small biomass of the
external mycelium of the fungi (6--16% of total biomass)
retained a large proportion of the total amount of assimilated
N (12--32%), a proportion that remained high even in the LN
treatment. It is not known how much of this fungal N will
become available to the host plant or how much will be used
by the fungus for cell wall production, metabolism or the
formation of reproductive organs. However, in natural forest
soils, the fungal N pool has a rapid turnover because ectomycorrhizal mycelia are a food source for many organisms (Ek et
al. 1994). Also, only a proportion of the hyphae are metabolically active. Rygiewicz and Andersen (1994) reported that
less than 10% of external hyphae in the roots of 6-month-old
Pinus ponderosa Laws. seedlings were active.
Mycelial biomass production of T. terrestris was positively
correlated with the nutrient status of the culture solution.
Wallander (1995) has postulated that more host carbohydrate
becomes available for production of fungal mycelium and fruit
bodies under N-limiting conditions than under conditions of
adequate N supply. Retention of N for fungal growth is likely
to be species dependent. Abuzinadah and Read (1989) reported
that the mycorrhizal fungus Paxillus involutus (Batsch: Fr) Fr.
retained more N than did the mycorrhizal species Hebeloma
crustuliniforme (Bull.: St. Amans) Quél. and Amanita muscaria (L.: Fr.) Hooker and concluded that fungal retention of
N could account for the decrease in growth and N content of
the birch seedlings infected with P. involutus. We have demonstrated that N retention in the external mycelia of Scleroderma
citrinum was responsible for a significant reduction in N concentration of the host plant shoots. This reduction, in turn,
reduced the relative growth rate of the seedlings (cf. Ingestad
et al. 1986).
Thus, reduced seedling growth in response to ectomycorrhizal infection may be the result of increased belowground
carbon allocation or it may be a consequence of high nutrient
retention by the mycobiont. However, accessibility to nutrient
pools will determine the outcome of nutrient retention by
ectomycorrhizal fungi. Under natural conditions, the external
mycelium of mycorrhizas will likely have better access than
uninfected roots to larger or different nutrient pools (Dighton
1991).
Our data on carbon allocation confirmed earlier observations (Reid et al. 1983, Nylund and Walander 1989, Dosskey
et al. 1990, Finlay and Soderstrom 1992). At Week 5, when
differences in shoot size were small, a slight increase in net
photosynthetic rate was observed in seedlings inoculated with
Suillus bovinus. Plants colonized with Scleroderma citrinum
showed no increase in net assimilation rate; however, this lack
of response might be associated with their low shoot N concentration. Stimulation of net photosynthesis in mycorrhizal seedlings can be explained on the basis that photosynthesis is partly
controlled by sink demand. However, the increase in net photosynthesis in plants infected with Suillus bovinus did not
result in increased shoot growth and failed to compensate for
the cost of symbiosis. Belowground respiration in all mycorrhizal plants was higher than in non-mycorrhizal plants in both
nutrient treatments at both harvests. According to Rygiewicz
and Andersen (1994), the increased respiration rate of mycorrhizal plants is associated with a higher fungal respiration rate,
a colonization respiration for ectomycorrhizal host roots and a
greater proportion of active short roots. The maintenance cost
per unit biomass is larger for hyphae than for roots (Fitter
1991). We conclude that the benefit of mycorrhizas to their
host plants is not necessarily or exclusively a nutritional benefit (Fitter 1991); furthermore, the benefit to the host plant
cannot be measured only in terms of an increase in RGR
(Harley 1989).
Acknowledgments
We thank Ms. A. Wijnants and Mr. R. Laermans for their technical
assistance. Jan Colpaert thanks the Belgian National Fund for Scientific Research (N.F.W.O.) for providing a post-doctoral fellowship.
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© 1996 Heron Publishing----Victoria, Canada
Carbon and nitrogen allocation in ectomycorrhizal and
non-mycorrhizal Pinus sylvestris L. seedlings
JAN V. COLPAERT,1 ANDRÉ VAN LAERE,1 and JOZEF A. VAN ASSCHE2
1
2
Laboratory of Developmental Biology, Institute of Botany, Katholieke Universiteit Leuven, K. Mercierlaan, 92, B-3001 Leuven, Belgium
Laboratory of Ecology, Institute of Botany, Katholieke Universiteit Leuven, K. Mercierlaan, 92, B-3001 Leuven, Belgium
Received November 29, 1995
Summary We studied carbon and nitrogen allocation in mycorrhizal and non-mycorrhizal Scots pine (Pinus sylvestris L.)
seedlings grown in a semi-hydroponic system with nitrogen as
the growth limiting factor. Three ectomycorrhizal fungi were
compared: one pioneer species (Thelephora terrestris Ehrh.: Fr.)
and two late-stage fungi (Suillus bovinus (L.: Fr.) O. Kuntze,
and Scleroderma citrinum Pers.). By giving all plants in each
treatment the same amount of readily available nitrogen, we
ensured that the external mycelium could not increase the total
nitrogen content of the plants, thereby guaranteeing that any
change in carbon or nitrogen partitioning was a direct effect of
the mycorrhizal infection itself. Carbon and nitrogen partitioning were measured at an early and a late stage of mycorrhizal
development, and at a low and a high N addition rate.
Although mycorrhizal seedlings had a higher net assimilation rate and a higher shoot/root ratio than non-mycorrhizal
seedlings, they had a lower rate of shoot growth. The high
carbon demand of the mycobionts was consistent with the
large biomass of external mycelia and the increased belowground respiration of the mycorrhizal plants. The carbon cost
to the host was similar for pioneer and late-stage fungi. Aboveand belowground partitioning of nitrogen was also affected by
mycorrhizal infection. The external mycelia of Scleroderma
citrinum retained 32% of the nitrogen supplied to the plants,
thus significantly reducing nitrogen assimilation by the host
plants and consequently reducing their growth rate. By contrast, the external mycelia of T. terrestris and Suillus bovinus
retained less nitrogen than the mycelia of Scleroderma
citrinum, hence we attributed the decreased growth rates of
their host plants to a carbon drain rather than a nitrogen
deficiency.
Keywords: carbon allocation, nitrogen partitioning, root respiration, Scleroderma citrinum, shoot/root ratio, Suillus
bovinus, Thelephora terrestris.
Introduction
Most higher plant species in terrrestrial ecosystems have associated mycorrhizal fungi, which have direct access to the
assimilates of their hosts and also serve as carriers for mineral
nutrients to the host (Finlay and Read 1986, Finlay et al. 1988).
These fungi demand large amounts of carbon, and it is this
demand that gives rise to mycorrhizal symbiosis, which, in
turn, is important not only for the growth of both symbionts but
also for the carbon balance of ecosystems. Several studies have
shown that mycorrhizal fungi use a significant fraction of the
net primary production of natural forests (Vogt et al. 1982,
Finlay and Söderström 1992). Laboratory studies suggest that
mycorrhizal symbiosis results in significantly higher belowground carbon allocation than in non-symbiotic conditions.
Recent studies have suggested that mycorrhizae impose a
higher C cost on their hosts under field conditions than under
experimental conditions (Rygiewicz and Andersen 1994,
Tinker et al. 1994).
Ectomycorrhizal fungi can reduce the growth rate of their
host plant (Nylund and Wallander 1989, Dosskey et al. 1990,
Rygiewicz and Andersen 1994). Such yield reductions are
usually attributed to an increase in belowground carbon allocation. We postulate that this cost of symbiosis is partly compensated by an increase in shoot/root ratio in mycorrhizal
plants. We also hypothesize that retention of nitrogen in the
mycobiont might lead to growth repression in the host plant.
Because nitrogen can be a growth limiting factor in forests
(Attiwill and Adams 1993), the diversion of nitrogen for fungal
growth might have consequences for tree growth and nutrient
cycling in natural ecosystems.
We studied growth, nitrogen partitioning, and rates of photosynthesis and respiration in the above- and belowground
parts of mycorrhizal and non-mycorrhizal Scots pine (Pinus
sylvestris L.) seedlings at an early and a late stage of fungal
development. Three ectomycorrhizal fungi were compared: a
pioneer species Thelephora terrestris Ehrh.: Fr., and two latestage fungi, Suillus bovinus (L.: Fr.) O. Kuntze and Scleroderma citrinum Pers. (Fox 1986). We compared a pioneer
fungal species with two late-stage fungal species because there
is evidence that there are important physiological differences
between pioneer and late-stage ectomycorrhizal fungi, and
little is known about the nutrient requirements of the different
fungi. Compared to late-stage fungi, pioneer fungi are charac-
788
COLPAERT, VAN LAERE AND VAN ASSCHE
terized by a more rapid growth, lower energy investment in
biomass and early carpophore development with more germinable propagules (Dighton 1991, Deacon and Fleming
1992). Pioneer fungi also tolerate higher concentrations of
inorganic nutrients than late-stage fungi.
Materials and methods
Plant and fungus material
Half-sib seeds of Pinus sylvestris were surface sterilized in
30% H2O2 for 15 min, sown in a 1/1 (v/v) mix of vermiculite
and perlite and watered with a balanced nutrient solution
(Ingestad et al. 1986). The weight proportions of the macronutrients in the solution were 100 N/60 K/18 P/6 Ca/6 Mg/9 S.
After 10 weeks, plants were selected for uniformity and inoculated with a mycorrhizal fungus or left uninoculated. Three
ectomycorrhizal species were used for inoculation: Thelephora terrestris (24 plants), Suillus bovinus (24 plants) and
Scleroderma citrinum (12 plants). For inoculations, mycorrhizal fungi were grown in 10-cm diameter plastic petri dishes
containing modified Melin-Norkrans agar medium covered
with sterile cellophane sheets. Once the mycelia had covered
most of the cellophane surface, the root system of a selected
seedling was spread over the young mycelia and a thick filter
paper (9 cm in diameter), soaked in Ingestad’s nutrient solution, was used to cover the roots and mycelia. Control plants
were treated in the same way, except that their roots were
placed on Melin-Norkrans agar medium without mycelium.
Three to four days after inoculation, the plants were transplanted to 4-liter containers to allow the growth of extensive
external mycelia.
Growth conditions
After inoculation, two plants were transferred to each 4-liter
container (0.40 × 0.20 × 0.05 m) and 250 g of acid-washed
perlite was added. The perlite was irrigated with 1.45 dm3 of
nutrient solution, resulting in saturation of the substrate to 80%
of its water holding capacity. The perlite in each container was
covered with a dark plastic lid to prevent algal growth. The
containers were weighed at least twice a week so that water use
could be estimated during the experiment. After each weighing, the containers were rearranged on the benches in the
growth room.
Plants were maintained in non-sterile conditions in a growth
chamber in a 16-h photoperiod and a day/night temperature of
22/15 °C. Relative air humidity was at least 70% and photosynthetically active radiation (PAR) was 400 µmol m −2 s −1.
The seedlings were grown at two nutrient addition rates. Initially, nutrients were supplied daily in a single addition but
after 8 weeks two additions per day were necessary.
Treatments
Six plant containers of each fungal treatment received balanced nutrient solution for Scots pine at a low relative addition
rate of 2.6% day −1 (LN treatment). The amount of nitrogen
supplied was based on the nitrogen content of the seedlings
(Ingestad et al. 1986). The nitrogen concentration of the nutrient solution was 20 mg l −1. On the first day, 0.4 mg N was
supplied to each plant and the amount was gradually increased
to 4 mg on Day 90. Distilled water was supplied to compensate
for differences in water use between plants. The initial nitrogen
content of the irrigated perlite in the LN treatment was 20 mg
per container. The perlite substrate was maintained at a pH of
4.0 by the addition of a 1/1 mix of NO −3 and NH −4 . Adsorption
of added N on the perlite was low (< 10%).
For the second treatment (the HN treatment), control plants
and plants inoculated with T. terrestris or Suillus bovinus
received exactly twice the amount of nutrients as the plants in
the LN treatment (0.8 mg N per plant on Day 1 up to 8 mg on
day 90). The initial nitrogen content of the irrigated perlite in
the HN treatment was 40 mg per container.
Gas exchange measurements
Measurements of net photosynthesis and respiration were conducted on all seedlings before harvest. Carbon dioxide was
measured with an infrared gas analyzer (IRGA) in an open
system. Outside air was allowed to adjust to the ambient
temperature of the growth room. Before measurement, entire
plants were transferred to PVC root cuvettes without disturbing the root substrate. The inner compartment of each cuvette
was exactly the same size as the containers. The bottom and
one side wall (0.2 × 0.4 m) of each inner compartment consisted of a stainless steel grid, with a 1-cm air gap between the
grid and the bottom and side of the root cuvette. Relative
humidity of air entering the root compartment was adjusted to
80--85%. Temperature and relative humidity of air entering
and leaving the root cuvette were measured. Air was circulated
through the root compartment for 4 h before commencing the
CO2 measurements. Measurements were recorded at 10-min
intervals.
Immediately after root respiration was recorded, the air
stream through the root compartment was stopped. To determine photosynthetic rate, a shoot cuvette (3.8 or 8.5 dm3, with
a maximal flow rate through the cuvette of 14 dm3 min −1),
which enclosed both seedlings, was mounted on the root compartment and measurements of CO2 were continued until
steady-state conditions were obtained. Temperature in the
shoot compartment was about 1.0 °C higher than ambient
temperature. Aboveground respiration was determined immediately after the photosynthesis measurements were completed. Steady-state conditions were achieved in the dark after
about 15 min.
Harvest
Plants were harvested at Weeks 5 and 12 after transfer of the
seedlings to the 4-liter containers. Two subsamples of rooting
substrate (approximately 20 g), free of root material, were
transferred to 20 ml of distilled water, stirred for 15 min and
subsequently vacuum-filtered over a 0.45 µm membrane filter.
Conductivity and nitrogen concentration of the filtrate were
determined. The filtered perlite was subsequently washed with
two 20-ml aliquots of distilled H2O, dried at 50 °C, weighed
and stored until used for determination of fungal biomass. We
C AND N ALLOCATION IN MYCORRHIZAL SCOTS PINE
defined the external mycelium as the fungal biomass that was
not associated with roots.
Shoots were separated from roots, dried at 80 °C for at least
4 days and weighed. Carpophores and roots were rinsed free
of perlite on a 1-mm sieve and subsequently treated in the same
way as the shoots. For the calculation of shoot/root ratio and
relative growth rate (RGR), the fungal biomass of the mycelium in the substrate was excluded. The RGR, expressed as %
day −1 was calculated by fitting seedling weight (y) at different
days (x; start day and both harvests) to the equation y = aeRGRx.
Nitrogen determinations
Nitrogen in oven-dried shoots, roots and rooting substrate was
extracted by the Kjeldahl procedure. The NH3 was steam
distilled into H2SO4 (0.05 M) and determined colorimetrically
with Nessler’s reagent. Nitrogen was also determined in the
carpophores of T. terrestris and Scleroderma citrinum formed
during the experiment as well as in a Suillus bovinus mycelium
cultured on a modified Melin-Norkrans medium without malt
extract. Data from the Suillus mycelium were used to calculate
fungal biomass from the N concentration of the washed perlite
substrate (Colpaert et al. 1992). Perlite from the non-mycorrhizal treatment was used to determine the background concentration of the non-mycorrhizal N pool. The N concentration of
the Suillus bovinus mycelium might be slightly overestimated
because the N concentration of the MMN medium (53 mg l −1)
was higher than that of the plant nutrient solution. This error
could result in slight underestimations of fungal biomass,
especially in the LN treatment.
Statistical analyses
Significant differences between means within a single treatment were determined with a one-way ANOVA and the
Tukey’s studentized range test.
Results
Plant and fungal growth
Dry weights of shoots, roots and external mycelia are shown
in Table 1. Decreased shoot growth in the mycorrhizal plants
789
was already apparent 5 weeks after inoculation. Twelve weeks
after inoculation, non-mycorrhizal seedlings had significantly
larger shoots and roots than mycorrhizal seedlings in both
nutrient treatments. Although relative growth rates of mycorrhizal plants decreased during the study, their shoot/root ratios
increased, indicating that mycorrhizal infection suppressed
root growth more than shoot growth (Table 2).
In the LN treatment, plant water use increased exponentially
throughout the experiment (r 2 > 0.98), whereas in the HN
treatment, water use plateaued in the last month of the experiment, indicating that seedlings had entered a more linear
growth phase. The conductivity of the solution in the perlite
was between 20 and 45 µS cm −1 at Week 5 and between 10 and
25 µS cm −1 at Week 12. The amount of nitrogen that could be
washed from the perlite was always less than the amount of
nitrogen added. These observations indicate that plant nutrient
uptake rates were high in both treatments, and there was no
oversupply of nutrients.
By Week 5, T. terrestris had completely colonized the
growth substrate, infecting almost all short roots. Suillus
bovinus and Scleroderma citrinum had colonized 75% of the
growth substrate, infecting 80 and 90% of short roots, respectively. By Week 12, substrate colonization and short root infection were close to 100% in all fungal treatments, indicating that
the nutrient treatments did not affect the colonization rates of
substrate and roots.
Carpophores formed in all containers with T. terrestris and
in two containers with Scleroderma citrinum. The N concentration of the T. terrestris carpophores was 2.5% in the LN
treatment and 2.7% in the HN treatment, whereas the N concentration in Scleroderma citrinum carpophores was 3.8%.
Suillus bovinus formed no carpophores and contained 31 mg
N per g dry weight. When we used these N values to calculate
fungal biomass in the perlite substrate, we found that
Scleroderma citrinum produced the largest external mycelial
biomass, and that T. terrestris (in the LN treatment) formed the
least dense mycelium, 31% of which was made up of small
carpophores. The mycelial biomass of T. terrestis was greater
in the HN treatment than in the LN treatment, and carpophores
accounted for 53% of its biomass.
Table 1. Dry weights of shoots, roots and external mycelia (including carpophores) of mycorrhizal and non-mycorrhizal Pinus sylvestris seedlings
grown at a low or a high nutrient addition rate. Standard errors are shown beside values; for each treatment, means within a column followed by
different letters are significantly different (n = 6, one-way ANOVA, Tukey’s test, α = 0.05).
Shoot (g)
Root (g)
Mycelium (g)
Week 5
Week 12
Week 5
Week 12
Week 5
Week 12
Low N
Control
Thelephora terrestris
Suillus bovinus
Scleroderma citrinum
1.42 ± 0.05 a
1.36 ± 0.03 a
1.34 ± 0.03 a
1.29 ± 0.03 a
5.96 ± 0.15 a
5.31 ± 0.13 b
5.09 ± 0.15 b
4.44 ± 0.10 c
1.50 ± 0.07 a
1.26 ± 0.04 b
1.18 ± 0.05 b
1.11 ± 0.06 b
5.84 ± 0.30 a
4.28 ± 0.40 b
4.07 ± 0.18 b
3.50 ± 0.12 b
0 a
0.18 ± 0.02 b
0.24 ± 0.02 b
0.27 ± 0.02 c
0 a
0.98 ± 0.06 b
0.82 ± 0.07 b
1.18 ± 0.06 c
High N
Control
Thelephora terrestris
Suillus bovinus
2.02 ± 0.04 x
1.78 ± 0.06 y
1.84 ± 0.05 y
9.46 ± 0.39 x
7.04 ± 0.25 y
7.47 ± 0.18 y
1.54 ± 0.10 x
1.33 ± 0.07 x
1.32 ± 0.04 x
7.57 ± 0.49 x
4.82 ± 0.19 y
5.12 ± 0.34 y
0 x
0.24 ± 0.02 y
0.19 ± 0.02 z
0 x
2.27 ± 0.15 y
1.17 ± 0.09 z
790
COLPAERT, VAN LAERE AND VAN ASSCHE
Nitrogen partitioning
Although mycorrhizal plants had a lower dry weight than
non-mycorrhizal plants, the reduction did not result in an
increase in shoot N concentration (Table 3). Plants infected
with Scleroderma citrinum had a lower shoot N concentration
than non-mycorrhizal plants, and this finding was confirmed
by a slight yellowing of the needles. The N concentration of
mycorrhizal roots was significantly higher than that of nonmycorrhizal roots.
Partitioning of N in above- and belowground biomass is
shown in Figure 1. Mycorrhizal root systems retained a larger
proportion of N than non-mycorrhizal root systems, indicating
that the external mycelia function as an N sink. By Week 12,
mycelia had retained between 15 and 32% of all N assimilated
(Table 4). These values would be higher if the N contained in
the mycorrhizas was also included. Moreover, the recovery of
all N added to the plants was similar or slightly higher in
mycorrhizal plants than in non-mycorrhizal plants, whereas
the amount of N transported to the shoots was considerably
Table 2. Shoot/root ratio and relative growth rate of mycorrhizal and
non-mycorrhizal Pinus sylvestris seedlings grown at a low or a high
nutrient addition rate. Standard errors are shown beside values; for
each treatment, means within a column followed by different letters
are significantly different (n = 6, one-way ANOVA, Tukey’s test,
α = 0.05).
Shoot/root ratio
RGR
Week 5
Week 12
% day −1 r 2
0.95 ± 0.02 a
1.08 ± 0.03 b
1.14 ± 0.03 b
1.16 ± 0.03 b
1.02 ± 0.02 a
1.24 ± 0.03 b
1.25 ± 0.03 b
1.27 ± 0.04 b
2.8
2.6
2.6
2.5
0.97
0.98
0.97
0.97
High N
Control
1.31 ± 0.04 x 1.25 ± 0.03 x 3.2
Thelephora terrestris 1.34 ± 0.03 x 1.46 ± 0.04 y 2.9
Suillus bovinus
1.39 ± 0.04 x 1.46 ± 0.05 y 2.9
0.96
0.93
0.95
Low N
Control
Thelephora terrestris
Suillus bovinus
Scleroderma citrinum
less in mycorrhizal plants than in non-mycorrhizal plants in
both nutrient treatments (Figure 1).
Net photosynthesis and respiration
Plants infected with Suillus bovinus and T. terrestris had
slightly higher assimilation rates than uninfected plants or
plants inoculated with Scleroderma citrinum (Table 5). Although the presence of mycorrhizal fungi had no effect on dark
respiration of shoots, it significantly increased respiration of
roots. The percentage of fixed carbon respired belowground by
mycorrhizal plants was twice that respired by non-mycorrhizal
plants (Tables 5 and 6).
Discussion
A difficulty associated with comparative growth studies of
mycorrhizal and non-mycorrhizal plants is that such studies
only provide a measure of the net benefit of mycorrhizal
infection, including nutritional effects, and thus give no direct
information about carbon allocation to the fungus. We overcame this difficulty by using a semi-hydroponic technique.
This technique greatly reduces the nutritional benefit to the
Table 4. The proportion of assimilated nitrogen in the external mycelia, including carpophores, of mycorrhizal Pinus sylvestris seedlings.
Standard errors are shown beside values; for each treatment, means
within a column followed by different letters are significantly different
(n = 6, one-way ANOVA, Tukey’s test, α = 0.05).
Fungal nitrogen (% of total)
Week 5
Week 12
Low N
Thelephora terrestris
Suillus bovinus
Scleroderma citrinum
13 ± 2 a
16 ± 2 a
26 ± 3 b
19 ± 2 a
18 ± 2 a
32 ± 2 b
High N
Thelephora terrestris
Suillus bovinus
13 ± 2 x
12 ± 1 x
25 ± 2 x
15 ± 1 y
Table 3. Nitrogen concentration in shoots and roots of mycorrhizal and non-mycorrhizal Pinus sylvestris seedlings grown at a low or a high nutrient
addition rate. Standard errors are shown beside values; for each treatment, means within a column followed by different letters are significantly
different (n = 6, one-way ANOVA, Tukey’s test, α = 0.05).
Shoot N (% of dry weight)
Root (% of dry weight)
Week 5
Week 12
Week 5
Week 12
Low N
Control
Thelephora terrestris
Suillus bovinus
Scleroderma citrinum
1.19 ± 0.03 a
1.10 ± 0.03 a
1.12 ± 0.03 a
0.96 ± 0.02 b
1.05 ± 0.04 a
0.95 ± 0.03 a
0.95 ± 0.03 a
0.80 ± 0.02 b
1.31 ± 0.03 a
1.39 ± 0.05 ab
1.46 ± 0.04 b
1.45 ± 0.04 b
1.25 ± 0.03 a
1.43 ± 0.04 b
1.61 ± 0.04 c
1.70 ± 0.05 c
High N
Control
Thelephora terrestris
Suillus bovinus
1.35 ± 0.03 x
1.27 ± 0.02 y
1.30 ± 0.03 xy
1.45 ± 0.04 x
1.38 ± 0.04 x
1.48 ± 0.05 x
1.41 ± 0.03 x
1.64 ± 0.05 y
1.66 ± 0.04 y
1.40 ± 0.05 x
1.88 ± 0.05 y
1.84 ± 0.04 y
C AND N ALLOCATION IN MYCORRHIZAL SCOTS PINE
791
Figure 1. Above- and belowground nitrogen partitioning in Pinus sylvestris
seedlings uninoculated (NM) or inoculated with Thelephora terrestris (T te),
Suillus bovinus (S bo) or Scleroderma
citrinum (S ci). Data were obtained 12
weeks after inoculation. For each plant
part, bars with a different letter are significantly different (n = 6, Tukey’s test,
α = 0.05). LN = low nutrient addition
rate, HN = high nutrient addition rate.
Table 5. Rates of respiration and net assimilation in non-mycorrhizal and mycorrhizal Pinus sylvestris seedlings. Units are: aboveground, nmol
CO2 gDWS−1 s −1; belowground (roots and mycelium), nmol CO2 gDWR−1 s −1. Standard errors are shown beside values; for each treatment, means
within a column followed by different letters are significantly different (n = 6, one-way ANOVA, Tukey’s test, α = 0.05).
Net assimilation rate
Aboveground respiration
Belowground respiration
Week 5
Week 12
Week 5
Week 12
Week 5
Week 12
Low N
Control
Thelephora terrestris
Suillus bovinus
Scleroderma citrinum
20.2 ± 0.9 ab
19.6 ± 0.8 ab
22.1 ± 0.8 a
18.5 ± 1.0 b
14.9 ± 0.5 a
17.4 ± 0.8 ab
17.6 ± 0.8 b
14.7 ± 0.7 a
6.5 ± 0.5 a
6.9 ± 0.6 a
7.0 ± 0.5 a
6.4 ± 0.4 a
6.1 ± 0.5 a
6.3 ± 0.3 a
6.4 ± 0.4 a
6.1 ± 0.4 a
3.6 ± 0.3 a
5.0 ± 0.5 ab
6.5 ± 0.4 b
5.7 ± 0.3 b
2.3 ± 0.2 a
5.1 ± 0.3 b
5.7 ± 0.4 b
4.7 ± 0.3 b
High N
Control
Thelephora terrestris
Suillus bovinus
16.9 ± 1.0 x
17.8 ± 1.0 xy
20.8 ± 0.8 y
14.0 ± 0.7 x
16.1 ± 0.6 x
14.4 ± 1.0 x
6.2 ± 0.6 x
5.9 ± 0.4 x
6.2 ± 0.5 x
5.7 ± 0.4 x
5.3 ± 0.3 x
6.0 ± 0.5 x
4.0 ± 0.3 x
5.5 ± 0.3 y
7.6 ± 0.4 z
2.5 ± 0.2 x
4.8 ± 0.2 y
4.1 ± 0.3 y
plant of the mycorrhizal infection by ensuring that both infected and non-infected plants have good access to the same
nutrients, without affecting the cost of the symbiosis to the host
plant (cf. Kähr and Arveby 1986, Nylund and Wallander 1989,
Kamminga-Van Wijk et al. 1992).
Inoculation with ectomycorrhizal fungi of Scots pine seedlings growing in a semi-hydroponic system resulted in decreased plant growth. All three mycorrhizal fungi inhibited
root development more than shoot growth. Harley and Smith
(1983) concluded that the shoot/root ratio of mycorrhizal
plants increased because of their enhanced ability to absorb
nutrients, whereas Smith (1980) argued that the mycorrhizalinduced increase in shoot/root ratio was a feed-back response
to an increased nutrient uptake by the mycorrhizae. Hetrick
(1991) observed that increases in shoot/root ratio are greater in
strongly mycotropic plants. Because we used a semi-hydroponic system, the increase in shoot/root ratio of the mycorrhizal plants in our study cannot be attributed to increased nutrient
uptake or increased growth rate of the mycorrhizal plants: we
conclude, therefore, that the increase is a result of the mycorrhizal infection itself. Decreased retention of carbon in the host
roots indicates that the fungus was a stronger sink for carbon
than the host roots. A shift in carbon allocation from root
growth to fungal growth could explain the increase in
Table 6. The proportion of net fixed carbon respired by the root
systems of non-mycorrhizal and mycorrhizal Pinus sylvestris seedlings. Standard errors are shown beside values; for each treatment,
means within a column followed by different letters are significantly
different (n = 6, one-way ANOVA, Tukey’s test, α = 0.05).
Belowground respiration (% NA)1
Week 5
Week 12
Low N
Control
Thelephora terrestris
Suillus bovinus
Scleroderma citrinum
19 ± 2 a
27 ± 2 b
31 ± 2 b
33 ± 3 b
15 ± 1 a
29 ± 2 b
31 ± 2 b
34 ± 2 b
High N
Control
Thelephora terrestris
Suillus bovinus
18 ± 2 x
27 ± 2 y
30 ± 2 y
14 ± 1 x
30 ± 2 y
24 ± 1 y
1
NA = Net assimilation.
shoot/root ratio of the mycorrhizal plants. Miller et al. (1989)
demonstrated that the extramatrical hyphae of ectomycorrhizal
pine seedlings constitute a strong sink for carbon, especially
when a new network of external hyphae was developing. In
792
COLPAERT, VAN LAERE AND VAN ASSCHE
natural ecosystems, roots of young seedlings probably integrate into the existing soil mycelia of late-stage fungi so that
they do not have to sustain an extensive ectomycorrhizal mycelium as would occur on pioneer sites where no active mycelia of late-stage fungi are present. For these reasons, we
expected that the carbon demand of the pioneer fungus T. terrestris would differ from that of the late-stage fungus Suillus
bovinus. However, we found that host plants of both fungi had
similar root respiration rates and similar shoot growth, although the fungi did exhibit different carbon investment strategies. Thelephora terrestris developed a rather sparse substrate
mycelium connected with numerous sporulating carpophores,
whereas Suillus bovinus produced a more slow-growing, dense
substrate mycelium. The pioneer species was more sensitive to
the nutrient regime than the late-stage fungus, and the HN
treatment caused a doubling of its biomass compared with the
LN treatment.
Although it is well known that mycorrhizal fungi have a
large carbon demand, other details of the nutrient requirement
for fungal development are sparse. However, such information
may be important because tree growth is often limited by
nitrogen or phosphorus in natural ecosystems (Attiwill and
Adams 1982). In the field, concentrations of minerals in ectomycorrhizal carpophores are consistently higher than the
concentrations in most plant tissues (Vogt and Edmonds 1980,
Vogt et al. 1982). We found that nitrogen use by the mycorrhizal fungi was extensive. The relatively small biomass of the
external mycelium of the fungi (6--16% of total biomass)
retained a large proportion of the total amount of assimilated
N (12--32%), a proportion that remained high even in the LN
treatment. It is not known how much of this fungal N will
become available to the host plant or how much will be used
by the fungus for cell wall production, metabolism or the
formation of reproductive organs. However, in natural forest
soils, the fungal N pool has a rapid turnover because ectomycorrhizal mycelia are a food source for many organisms (Ek et
al. 1994). Also, only a proportion of the hyphae are metabolically active. Rygiewicz and Andersen (1994) reported that
less than 10% of external hyphae in the roots of 6-month-old
Pinus ponderosa Laws. seedlings were active.
Mycelial biomass production of T. terrestris was positively
correlated with the nutrient status of the culture solution.
Wallander (1995) has postulated that more host carbohydrate
becomes available for production of fungal mycelium and fruit
bodies under N-limiting conditions than under conditions of
adequate N supply. Retention of N for fungal growth is likely
to be species dependent. Abuzinadah and Read (1989) reported
that the mycorrhizal fungus Paxillus involutus (Batsch: Fr) Fr.
retained more N than did the mycorrhizal species Hebeloma
crustuliniforme (Bull.: St. Amans) Quél. and Amanita muscaria (L.: Fr.) Hooker and concluded that fungal retention of
N could account for the decrease in growth and N content of
the birch seedlings infected with P. involutus. We have demonstrated that N retention in the external mycelia of Scleroderma
citrinum was responsible for a significant reduction in N concentration of the host plant shoots. This reduction, in turn,
reduced the relative growth rate of the seedlings (cf. Ingestad
et al. 1986).
Thus, reduced seedling growth in response to ectomycorrhizal infection may be the result of increased belowground
carbon allocation or it may be a consequence of high nutrient
retention by the mycobiont. However, accessibility to nutrient
pools will determine the outcome of nutrient retention by
ectomycorrhizal fungi. Under natural conditions, the external
mycelium of mycorrhizas will likely have better access than
uninfected roots to larger or different nutrient pools (Dighton
1991).
Our data on carbon allocation confirmed earlier observations (Reid et al. 1983, Nylund and Walander 1989, Dosskey
et al. 1990, Finlay and Soderstrom 1992). At Week 5, when
differences in shoot size were small, a slight increase in net
photosynthetic rate was observed in seedlings inoculated with
Suillus bovinus. Plants colonized with Scleroderma citrinum
showed no increase in net assimilation rate; however, this lack
of response might be associated with their low shoot N concentration. Stimulation of net photosynthesis in mycorrhizal seedlings can be explained on the basis that photosynthesis is partly
controlled by sink demand. However, the increase in net photosynthesis in plants infected with Suillus bovinus did not
result in increased shoot growth and failed to compensate for
the cost of symbiosis. Belowground respiration in all mycorrhizal plants was higher than in non-mycorrhizal plants in both
nutrient treatments at both harvests. According to Rygiewicz
and Andersen (1994), the increased respiration rate of mycorrhizal plants is associated with a higher fungal respiration rate,
a colonization respiration for ectomycorrhizal host roots and a
greater proportion of active short roots. The maintenance cost
per unit biomass is larger for hyphae than for roots (Fitter
1991). We conclude that the benefit of mycorrhizas to their
host plants is not necessarily or exclusively a nutritional benefit (Fitter 1991); furthermore, the benefit to the host plant
cannot be measured only in terms of an increase in RGR
(Harley 1989).
Acknowledgments
We thank Ms. A. Wijnants and Mr. R. Laermans for their technical
assistance. Jan Colpaert thanks the Belgian National Fund for Scientific Research (N.F.W.O.) for providing a post-doctoral fellowship.
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