New Phytol. (1999), 143, 589–597
Short-term phosphorus uptake rates in
mycorrhizal and non-mycorrhizal roots of
intact Pinus sylvestris seedlings
JAN V. COLPAERT"*, KATIA K. VAN TICHELEN",
J O Z E F A . V A N A S S C H E "    A N D R E! V A N L A E R E #
" Laboratory of Plant Ecology, Institute of Botany, Katholieke Universiteit Leuven,
Kardinaal Mercierlaan 92, B-3001 Leuven, Belgium
# Laboratory of Developmental Biology, Institute of Botany, Katholieke Universiteit
Leuven, Kardinaal Mercierlaan 92, B-3001 Leuven, Belgium
Received 22 April 1998 ; accepted 9 September 1998

Short-term phosphate uptake rates were measured on intact ectomycorrhizal and non-mycorrhizal Pinus sylvestris
seedlings using a new, non-destructive method. Uptake was quantified in semihydroponics from the depletion of
Pi in a nutrient solution percolating through plant containers. Plants were grown for 1 or 2 months after
inoculation at a low relative nutrient addition rate of 3 % d−" and under P limitation. Four ectomycorrhizal fungi
were studied : Paxillus involutus, Suillus luteus, Suillus bovinus and Thelephora terrestris. The Pi-uptake capacity of
mycorrhizal plants increased sharply in the month after inoculation. The increase was dependent on the
development of the mycobionts. A positive correlation was found between the Pi-uptake rates of the seedlings and
the active fungal biomass in the substrate as measured by the ergosterol assay. The highest Pi-uptake rates were
found in seedlings associated with fungi producing abundant external mycelia. At an external Pi concentration of
10 µM, mycorrhizal seedlings reached uptake rates that were 2.5 (T. terrestris) to 8.7 (P. involutus) times higher
than those of non-mycorrhizal plants. The increased uptake rates did not result in an increased transfer of
nutrients to the plant tissues. Nutrient depletion was ultimately similar between mycorrhizal and non-mycorrhizal
plants in the semihydroponic system. Net Pi absorption followed Michaelis–Menten kinetics : uptake rates
declined with decreasing Pi concentrations in the nutrient solution. This reduction was most pronounced in nonmycorrhizal seedlings and plants colonized by T. terrestris. The results confirm that there is considerable
heterogeneity in affinity for Pi uptake among the different mycobionts. It is concluded that the external mycelia
of ectomycorrhizal fungi strongly influence the Pi-uptake capacity of the pine seedlings, and that some mycobionts
are well equipped to compete with other soil microorganisms for Pi present at low concentrations in soil solution.
Key words : ectomycorrhiza, external mycelium, short-term phosphate uptake, Pinus sylvestris (Scots pine),
Paxillus involutus, Suillus bovinus, Suillus luteus, Thelephora terrestris.

Phosphate ions are mainly supplied to root-absorbing surfaces by diffusion, as mass flow of solution
in soil is unable to supply phosphate ions to roots at
rates that can account for the amount of P absorbed.
Consequently, a local concentration gradient or
depletion zone develops around the roots. The low
bio-availability of P in most soils often limits plant
growth (Marschner, 1995 ; Schachtman et al., 1998).
Additionally, as a result of increasing anthropogenic
*Author for correspondence (fax 32 16 32 1968 ; e-mail
jan.colpaert!bio.kuleuven.ac.be).
N deposition, a number of formerly N-limited forest
ecosystems are now limited by P availability.
The colonization of roots by mycorrhizal fungi can
significantly improve the uptake of P in host plants.
The possible mechanisms underlying the mycorrhizal effect on P uptake are diverse and have been
discussed by Harley (1989), Bolan (1991) and Smith
& Read (1997). Evidence for long-term increased P
inflow rates in ectomycorrhizal plants grown under P
limitation has been reported several times in the last
decade (Bougher et al., 1990 ; Jones et al., 1990,
1991 ; Rousseau et al., 1994 ; Cumming, 1996).
External hyphae of mycorrhizal fungi can absorb P
beyond the root-depletion zones. The subsequent
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590
J. V. Colpaert et al.
hyphal transfer of this P to the plant is faster than the
slow diffusion of Pi towards the roots. The volume of
accessible soil increases when plants are colonized by
mycorrhizal fungi, especially for poorly branched,
coarse-rooted plant species with short root hairs
(Bolan, 1991).
Apart from the more efficient soil exploitation,
differences in absorbing surface area and in kinetics
of Pi influx might affect the Pi uptake efficiency of
mycorrhizal and non-mycorrhizal root systems.
Mycorrhizal roots and external hyphae might have
higher maximum influx rates (Vmax), lower efflux
rates and a higher affinity (lower Km) for Pi uptake
than non-mycorrhizal roots. These features would
result in a more effective P absorption from low
concentrations in the soil solution. They also would
improve the below-ground competitiveness of a host
plant, especially in soils where strong competition
for P exists between microorganisms and plants
(Casper & Jackson, 1997).
These specific physiological aspects of the Pi
uptake process have been poorly investigated on
intact ectomycorrhizal root systems, mainly because
of the P-buffering capacity of normal soils and
difficulties in growing ectomycorrhizal plants in
hydroponic culture systems. In this paper a new
semihydroponic cultivation technique is used to
analyse the short-term Pi uptake process in nonmycorrhizal and mycorrhizal pine seedlings. Four
different mycobionts, having different life strategies
(Deacon & Fleming, 1992), were included in the
study. For the first time, short-term Pi uptake was
studied on intact plant–fungus associations including
the external mycelia. The measurements were nondestructive so that the time course of the Pi-uptake
capacity of individual plants could be followed
during mycorrhizal development.
  
Plant and fungal material
Scots pine (Pinus sylvestris L.) seeds were washed
with distilled water, surface sterilized with H O
# #
(30 %) and sown in a perlite–vermiculite mixture
(vol 2 : 1). The substrate was moistened with a
modified Ingestad nutrient solution (Ingestad &
Ka$ hr, 1985). The optimum macronutrient weight
proportions for P. sylvestris (100 N\15 P\60 K\6
Ca\6 Mg\9 S) were changed to 100 N\9 P\54 K\6
Ca\6 Mg\9 S in order to set P as the growth-limiting
factor.
Thirty-five days after sowing, the seedlings were
inoculated with one of four ectomycorrhizal fungi :
Paxillus involutus (Batsch.) Fr., Suillus luteus (L. :
Fr.) S. F. Gray, Suillus bovinus (L. : Fr.) O. Kuntze
or Thelephora terrestris Ehrh. : Fr. The former three
species were obtained from basidiocarps collected in
1995 in a 20-yr-old Pinus nigra stand in Paal (Belgian
Campine region) ; T. terrestris was isolated in 1994
from spores produced by a basidiocarp found in a
5-yr-old P. sylvestris stand in Lommel (Belgian
Campine region). A sandwich technique was used to
inoculate 10 pine seedlings with each fungus
(Colpaert et al., 1996) – 10 non-mycorrhizal plants
followed the same procedure without addition of
inoculum, and 10 plants were immediately harvested
in order to determine their biomass, f. wt : d. wt ratio
and P content.
Seedlings remained in contact with the inoculum
for 72 h. Plants were subsequently transferred to
plant containers (70 ml Omnifix2 Braun syringes,
27-mm inner diameter) filled with acid-washed,
sieved perlite (1–2 mm particles). Prior to the
transfer, the fresh weight of each seedling was
recorded and adhering inoculum was carefully
removed. During manipulations the root system was
kept moist with nutrient solution sprayed by an
atomizer. The containers were filled with 5 g dry
perlite, corresponding to a volume of 60p2 ml with
a maximum water-holding capacity of 17.0p0.5 ml.
Ten syringes without a plant were also included in
the experiment as blanks. After transplanting the
seedlings, the perlite was immediately watered with
the modified Ingestad nutrient solution. The initial
Pi concentration was 48 µM. Once in the containers,
plants were grown under a strictly controlled
constant P addition rate of 3 % d−" according to the
Ingestad concept (Ingestad & A/ gren, 1995 ; Colpaert
& Verstuyft, 1999). This addition rate was based on
the total amount of P in the seedlings harvested at
inoculation time. After a transition period the
seedlings should reach a relative growth rate (RG) of
only 3 % d−", whereas the maximum RG of nonmycorrhizal Scots pine is 7.5 % d−" (Ingestad &
Ka$ hr, 1985). The mean P content of the seedlings
harvested at inoculation time was used to programme
the nutrient addition rate. Nutrients were added
every 24 h. The perlite surface was covered with
gravel (2–5-mm particles), in order to prevent
growth of algae and to reduce evaporation of water.
All 60 plant containers were hung in holes in a PVC
lid put over a single, large, dark plastic box (60i40
cm). A black plastic foil (5i5 cm) was slipped
around each pine stem to avoid light penetration
along the transparent syringe walls. Below each plant
container, a glass tube (100 ml) was present to check
for percolating water. Except for the blanks without
plants, water percolation was avoided as much as
possible in order to restrict nutrient losses. The daily
nutrient requirements to maintain the RG of 3 % d−"
were supplied in the mean volume of the daily water
requirement of the plants. The conductivity of the
added solution remained below 130 µS cm−". Plants
were grown in a growth cabinet where they were
exposed to an 18 h photoperiod, a photon flux rate of
400 µmol m−# s−", and day\night temperatures of
21\15mC.
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Phosphate uptake by mycorrhizal and non-mycorrhizal pine
Experiment 1
Two plants from each plant–fungus combination
were selected for regular measurements of Pi uptake.
Pi uptake was measured on eight occasions with a
weekly interval : 6, 13, 20, 27, 34, 41, 48 and 55 d
after inoculation. Plants were harvested 56 d after
inoculation.
Experiment 2
The eight remaining plants from each plant–fungus
combination were tested for Pi uptake only 1 d
before they were harvested. Plants from the second
experiment were divided into two groups : half of the
seedlings were harvested 30 d after inoculation, the
other half 30 d later.
Pi uptake measurements
A method for characterizing the relation between
nutrient concentration and flux into roots of intact
plants (Claassen & Barber, 1974) was adapted for
semihydroponics. Net Pi uptake was determined
from the depletion of Pi in a test solution circulating
through the plant containers. The measurements
were performed in the growth cabinets under the
standard growth conditions. The syringes were hung
in dark PVC tubes fixed on a rack. During an
equilibration phase, 100 ml of nutrient solution with
48 µM Pi was dripped by means of multichannel
peristaltic pumps in the containers at a flow rate of
4.2 ml min−" in order to impose equal nutrient
solution concentrations for all plants. During this
phase a second silicon tube, connected to the
draining hole of the container and to the pumps,
sucked away percolating solution. Immediately after
flushing the growth substrate, each container was
integrated in a closed loop of a 130-cm silicon tube
(3 mm inner diameter) connected to the peristaltic
pump. Within 5 min, each test plant received 15 ml
(3 aliquots of 5 ml) of nutrient solution with 48 µM
Pi that was now circulated through the plant
container at a flow rate of 4.2 ml min−". The total
volume of solution in the system was 32p0.5 ml.
Less than 20 % of the container volume was
waterlogged at the start of the measurements. Every
hour 100-µl samples were collected, except during
the first 2 h when samples were taken every 30 min.
Measurements were stopped after 8–10 h of circulation. The Pi concentrations were determined
immediately by colorimetry as phosphomolybdate
(Murphy & Riley, 1962). Sample volume was
increased to 500 µl as the Pi concentration approached 5 µM. At the end of the test, the remaining
solution was collected in test tubes, and volume, pH
and conductivity were measured. Plant containers
were disconnected from the tubing and plants were
returned to their original place.
591
In a preliminary experiment the validity and
reliability of the nutrient depletion measurements
were studied using non-mycorrhizal pines and pines
mycorrhizal with T. terrestris or S. bovinus. The
depletion of NH +, NO − and Pi was studied. Uptake
$
%
of these nutrients could be almost completely
inhibited ( 95 %) after addition of the membrane
decoupler CCCP (carbonyl cyanide 3-chlorophenylhydrazone, 10−& M final concentration) to the
circulating nutrient solution (Chalot et al., 1996).
Phosphate was not adsorbed on perlite in detectable
quantities in a 5 µM Pi solution at pH 4.
Uptake of phosphate could be described in terms
of Michaelis–Menten kinetics in the low concentration range (Claassen & Barber, 1974 ; Marschner,
1995). Data were fitted to a depletion curve that was
calculated by integration of the Michaelis–Menten
equation (Dixon & Webb, 1964). Computer-assisted
iterative fitting was performed in the GraphPad
Prism4 program (GraphPad Software, Inc., San
Diego, CA, USA). The net Pi-uptake rate was calculated for individual plants at external Pi concentration of 30, 10 and 5 (only day 59 in Experiment 2)
µM. The uptake rate was calculated from the tangent
lines touching the depletion curve. For plants that
were harvested, specific Pi-uptake rates were expressed per unit root dry weight.
Harvest and analyses
Plant shoots were cut off and the fresh weight of
needles and stems was determined before drying at
85mC. The quartz could easily be removed from the
perlite, and non-absorbed Pi was washed from the
perlite with 200 ml of phosphate-free nutrient
solution. Subsequently the syringes were centrifuged
at 135 g for 30 s in order to remove part of the
solution retained in the perlite. Roots and perlite
were pulled out of the containers, the percentage
colonization of the perlite was estimated, and roots
were separated from the perlite. The fresh weight of
the roots was determined, a subsample of roots (200
mg) was immediately frozen in liquid nitrogen for
ergosterol analyses, and the remaining part of the
roots was dried. The perlite was collected and mixed
well. A subsample of 5 g (wet mass) was frozen in
liquid nitrogen for ergosterol analysis, the rest was
used to determine the wet\dry mass ratio of the
sample.
Duplicate samples of dried plant material (50 mg)
were ashed at 500mC and dissolved in 0.5 M HCl.
Phosphate was determined colorimetrically as phosphomolybdate (Murphy & Riley, 1962).
The ergosterol assay was used to estimate metabolically active fungal biomass in roots (mantle j
Hartig net) and perlite (external mycelia). Ergosterol
is a fungus-specific membrane component, and was
determined by HPLC as described by Nylund &
Wallander (1992) and Colpaert et al. (1997).
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J. V. Colpaert et al.
592

Pi uptake measurements
Plant and fungal growth
All mycorrhizal plants were colonized only by the
inoculated mycobiont. Non-mycorrhizal plants remained fungus-free. At the first harvest (Experiment
2), the whole perlite substrate was colonized by
hyphae, except for plants inoculated with S. bovinus.
Between 60 and 100 % of the substrate volume was
colonized with external mycelium of this species.
Two months after inoculation, large mycorrhizal
clusters were observed on all inoculated plants and
all perlite was invaded by mycelium. Shoot growth
was very regular and no bud formation occurred.
The plants had a mean P concentration in the shoot
of 1.9 mg g−" d. wt at inoculation time. In both
experiments this concentration dropped significantly
and the needles turned slightly reddish, indicating P
deficiency.
Pi concentration (µM)
60
50
40
An example of the results obtained by the depletion
technique is shown in Fig. 1 for a single plant from
each fungus condition, 27 d after inoculation (Experiment 1). The Pi concentration in the solution
circulating through the containers without plants
remained constant during the tests.
The nutrient composition of the circulating solution changed during the Pi uptake tests. At the start
of a test, pH of the Ingestad solution was close to 4.0
and this value decreased to 3.5 after a few hours but
then again slowly rose to 4.0. These changes in pH
were observed both in mycorrhizal and non-mycorrhizal plants. This fluctuation is mainly due to a
preferred uptake of NH + over NO −, when both
%
$
ions are present in the solution, a well known aspect
of N uptake in mycorrhizal and non-mycorrhizal
conifers (Marschner et al., 1991 ; Eltrop &
Marschner, 1996 ; Kronzucker et al., 1996). Over the
pH range observed, phosphate will be present
primarily as the monovalent H PO − species, the
# %
form that is taken up in plants and fungi. The
conductivity of the nutrient solution decreased to
values lower than 25 µS cm−", resulting from an
almost complete depletion of all nutrients.
30
Experiment 1
20
10
0
0
100
200
300
400
500
600
Time (min)
Fig. 1. An example of the Pi depletion in the nutrient
solution circulated through the plant containers. Data
from individual mycorrhizal and non-mycorrhizal Pinus
sylvestris seedlings, 27 d after inoculation (Experiment 1).
The depletion curves for the different plant–fungus
combinations were calculated by integration of the
Michaelis–Menten equation. Closed circle, non-mycorrhizal ; closed triangle, Paxillus involutus ; closed square,
Suillus luteus ; open circle, Suillus bovinus ; open triangle,
Thelephora terrestris ; open square, blank.
The two plants from each plant–fungus combination
had very similar growth rates and Pi-uptake rates.
Variation in biomass and P shoot concentration was
lower than 10 % ; variation in root P concentration
and ergosterol concentration was sometimes higher
in mycorrhizal plants but in general differed by no
more than 15 %. The plants from this experiment
had shoot P concentrations from 0.95 mg g−" d. wt in
mycorrhizal plants to 1.25 mg g−" d. wt in nonmycorrhizal plants (Table 1). However, in mycorrhizal roots the P concentration was considerably
higher than in non-mycorrhizal roots and at least
twice as high as in foliage (Table 1). The ergosterol
concentrations were higher in roots colonized with
Table 1. Shoot weights and phosphorus concentrations in mycorrhizal and non-mycorrhizal Pinus sylvestris
seedlings (Experiment 1, n l 2)
P concentration
Ergosterol concentration
Fungus
Shoot
(g d. wt)
Shoot
(mg g−" d. wt)
Root
(mg g−" d. wt)
Root
(mg g−" d. wt)
Perlite
(µg g−" d. wt)
Paxillus involutus
Suillus luteus
Suillus bovinus
Thelephora terrestris
Non-mycorrhizal
0.35
0.40
0.42
0.38
0.43
0.95
0.95
1.03
0.99
1.25
2.06
2.17
2.16
2.89
1.44
1.1
0.6
1.3
1.0
0
32
12
25
7
0
After inoculation plants were grown under P limitation in perlite for 56 d at a relative nutrient addition rate of 3% d−".
Ergosterol concentrations in roots and perlite substrate were used to quantify active fungal biomass.
Printed from the C JO service for personal use only by...
Pi uptake rate per plant (pmol s–1)
Phosphate uptake by mycorrhizal and non-mycorrhizal pine
400
(a)
300
200
Pi uptake rate per plant (pmol s–1)
100
0
0
400
10
20
10
20
30
Time (d )
40
50
60
40
50
60
(b)
300
200
100
0
0
30
Time (d )
Fig. 2. Time course of the Pi-uptake capacities of
mycorrhizal and non-mycorrhizal Pinus sylvestris seedlings
at (a) 10 and (b) 30 µM external Pi. Uptake measurements
were repeated weekly on the same plants over a period of
55 d after inoculation (n l 2, Experiment 1). Closed circle,
non-mycorrhizal ; closed triangle, Paxillus involutus ;
closed square, Suillus luteus ; open circle, Suillus bovinus ;
open triangle, Thelephora terrestris.
P. involutus, S. bovinus and T. terrestris than in S.
luteus roots which were partly senescent (intense
browning, Table 1). High ergosterol concentrations
were found in perlite colonized by P. involutus and
S. bovinus, indicating the existence of a dense
external mycelium ; lower concentrations were recorded in perlite invaded by T. terrestris and S.
luteus (Table 1).
The time course of the mean Pi-uptake capacity at
10 and 30 µM Pi is illustrated in Fig. 2a, b. An
593
exponential increase in the Pi-uptake capacity was
observed for all plants up to 40 d after the start of the
experiment. The relative increase in Pi uptake at
30 µM Pi varied from 5.6 % d−" in non-mycorrhizal
plants up to 7.4, 7.5, 8.4 and 8.5 % d−" in plants
associated with T. terrestris, S. bovinus, S. luteus and
P. involutus, respectively. The striking rise in Piuptake capacity in mycorrhizal plants always coincided with the rapid development of mycorrhizas
and external mycelia, which could be observed
through the transparent walls of the plant containers.
During the last 2 wk of the experiment the increase
in Pi-uptake capacity of most plants slowed down,
except in seedlings colonized by S. bovinus. The
expansion of external mycelia of this species proceeds
more slowly than with the other mycobionts. In the
same period the Pi-uptake capacity of plants associated with S. luteus started to decrease strongly,
probably due to senescing mycelia. In Table 2 the
proportional increase in Pi-uptake capacity in mycorrhizal versus non-mycorrhizal plants is reported
over the whole experimental period. Two weeks
after inoculation, Pi uptake at 30 µM Pi was already
twice as high in the P. involutus-inoculated plants as
in the non-mycorrhizal controls. At an external Pi
concentration of 10 µM, the uptake capacity of the
plants with P. involutus became up to 8.7 times that
of non-mycorrhizal plants. The uptake capacity of
plants inoculated with S. bovinus, S. luteus and T.
terrestris became, respectively, 5.7, 5 and 2.5 that of
non-mycorrhizal plants. At a higher external Pi
concentration, the mycorrhizal effect on the Piuptake capacity was smaller but still very prominent.
Experiment 2
Data on plant and fungal development are shown in
Table 3. The P concentration in the shoots at the last
harvest was slightly lower than in Experiment 1 and
varied from 0.74 mg g−" d. wt, as an average of all
mycorrhizal plants, up to 1.15 mg g−" d. wt in nonmycorrhizal plants. In mycorrhizal roots, P con-
Table 2. Ratio of the Pi uptake rate of mycorrhizal pine seedlings over the uptake rate of the non-mycorrhizal
control plants at 10 and 30 µM external Pi (n l 2, Experiment 1)
Time after inoculation (d)
Pi concentration and fungus
10 µM
Paxillus involutus
Suillus luteus
Siullus bovinus
Thelephora terrestris
30 µM
Paxillus involutus
Suillus luteus
Suillus bovinus
Thelephora terrestris
6
13
20
27
34
41
48
55
–
–
–
–
–
–
–
–
5.7
1.8
1.2
1.6
6.6
3.0
1.6
1.8
6.4
4.2
2.2
2.1
6.6
5.0
2.7
2.5
8.1
4.6
4.1
2.4
8.7
1.8
5.7
1.8
1.3
1.1
1.0
1.2
2.3
1.2
1.1
1.2
3.2
1.4
1.2
1.5
4.1
2.0
1.4
1.8
3.5
2.3
1.5
1.8
3.6
2.5
1.9
2.0
4.1
2.5
2.5
2.2
4.5
1.6
3.2
2.1
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J. V. Colpaert et al.
594
Table 3. Data on the shoot weight and phosphorus concentration in mycorrizal and non-mycorrhizal Pinus
sylvestris seedlings from Experiment 2 (meanspSE, n l 4)
P concentration
Shoot
(g d. wt)
Harvest and fungus
Harvest 1 (t l 30 d)
Paxillus involutus
Suillus luteus
Suillus bovinus
Thelephora terrestris
Non-mycorrhizal
Harvest 2 (t l 60 d)
Paxillus involutus
Suillus luteus
Suillus bovinus
Thelephora terrestris
Non-mycorrhizal
Ergosterol concentration
Shoot
(mg g−" d. wt)
Root
(mg g−" d. wt)
Root
(mg g−" d. wt)
Perlite
(µg g−" d. wt)
0.14p0.01
0.16p0.01
0.16p0.01
0.15p0.01
0.15p0.01
a*
a
a
a
a
1.00p0.02
0.90p0.04
1.09p0.03
0.99p0.05
1.13p0.03
a
a
b
a,b
b
1.30p0.07
1.32p0.08
1.22p0.10
1.43p0.11
1.19p0.11
a,b
a,b
a
b
a
1.0p0.1 c
0.6p0.1 b
0.4p0.1 b
0.4p0.1 b
0a
22p3 c
9p1 b
7p1 b
7p1 b
0a
0.32p0.02
0.36p0.02
0.36p0.01
0.33p0.02
0.38p0.02
m
m,n
m,n
m,n
n
0.79p0.06
0.72p0.04
0.75p0.04
0.71p0.05
1.15p0.07
m
m
m
m
n
1.67p0.10
1.97p0.12
1.87p0.08
2.09p0.11
1.26p0.04
n
n,p
n,p
p
m
1.3p0.1
1.0p0.1
1.1p0.1
0.9p0.1
0
38p2 q
31p2 q
21p2 p
12p2 n
0m
p
n
n,p
n
m
1.2
45
Ergosterol (µg g–1 perlite)
Pi uptake rate (nmol s–1 g–1 d. wt root)
1.2
Pi uptake rate (nmol s-1 g-1 d. wt root)
After inoculation, plants were grown in semihydroponics for 30 or 60 d. Nutrient addition rate was 3% d−" with P as
growth-limiting element. Ergosterol concentrations in roots and perlite substrate were used to quantify active fungal
biomass.
*Means with the same letter within each harvest are not significantly different (one-way ANOVA, Tukey’s test, α l
0.05).
(a)
1.0
0.8
0.6
72
0.4
71
0.2
0.0
44
53
Paxillus
involutus
Suillus
luteus
Suillus
bovinus
75
58
0
50
100
150
200
250
300
Pi uptake rate per plant (pmol
350
400
s–1)
Fig. 4. The relationship between the mean Pi-uptake rate
of the different pine–fungus combinations and their
respective ergosterol concentration in the substrate. Plants
from Experiments 1 (n l 2) and 2 (both harvests, n l 4).
Closed circle, non-mycorrhizal ; closed triangle, Paxillus
involutus ; closed square, Suillus luteus ; open circle,
Suillus bovinus ; open triangle, Thelephora terrestris. y l
0.125xk4.61 ; r# l 0.92.
1.0
0.6
15
0
42
Thelephora
Non terrestris mycorrhizal
(b)
0.8
30
79
64
0.4
63
0.2
47
0.0 Paxillus
involutus
Suillus
luteus
Suillus
bovinus
46
30
42 25
Thelephora
Non terrestris mycorrhizal
Fig. 3. Specific Pi-uptake rates of mycorrhizal and nonmycorrhizal plants after (a) 29 and (b) 59 d (Experiment 2).
Pi uptake was calculated for external Pi concentrations of 5
(white), 10 (grey) and 30 (stippled) µM. Numbers in the
columns represent the percentage of Pi-uptake capacity
that is retained in comparison to the uptake at 30 µM Pi
(100 %). Bars represent standard errors (n l 4).
centration was again considerably higher than in
non-mycorrhizal roots (Table 3). Shoots of plants
colonized with P. involutus were significantly smaller
than those of non-mycorrhizal plants. The ergosterol
determinations show the rapid development of P.
involutus mycorrhizas with an abundant external
mycelium (Table 3). The other fungi grew somewhat
more slowly (Suillus sp.) or did not form such an
extensive external mycelium (T. terrestris). Senescence of S. luteus mycelium was not obvious in this
experiment. Ergosterol data from the last harvest
confirm this observation (Tables 1, 3).
The specific Pi-uptake capacities of the plants 1
and 2 months after inoculation are shown in Fig. 3a,
b. Large heterogeneity was found in the net Piuptake rates of the plants. At the first harvest, 30 d
after inoculation, all mycorrhizal plants had higher
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Phosphate uptake by mycorrhizal and non-mycorrhizal pine
specific Pi-uptake rates than the control plants.
Between both harvests the specific Pi-uptake rate of
non-mycorrhizal plants decreased, whereas it remained unchanged or increased in mycorrhizal
seedlings. The Pi-uptake rate decreased when the
external Pi concentration decreased from 30 to 10 or
5 µM (Fig. 3). At 10 and 5 µM Pi non-mycorrhizal
plants retained only 42 and 25 % of the Pi uptake
measured at 30 µM Pi. The reduction in uptake
rate was much smaller in plants colonized with
P. involutus or S. luteus.
A positive correlation was found between the
mean Pi-uptake rates of the plants from both
experiments and the ergosterol concentration in the
perlite, irrespective of fungal species and harvest day
(Fig. 4).

Many investigators have determined ion uptake in
intact non-mycorrhizal plants in short-term experiments by monitoring nutrient depletion in solutionculture systems (Claassen & Barber, 1974 ; Jungk et
al., 1990 ; Marschner, 1995). However, most plants
under field conditions are associated with mycorrhizal fungi that can have a significant impact on the
acquisition of minerals by the host plant. Growing
ectomycorrhizal plants in hydroponics is difficult,
especially with mycobionts with hydrophobic mycelia. The semihydroponic system with perlite is a
valuable alternative as both hydrophilic and hydrophobic fungi grow well in this substrate, at least
when waterlogging and permanent flooding with
nutrient solution are avoided (Colpaert & Verstuyft,
1999).
In the present work, both plants and fungi grew
very well in the perlite. In a few weeks a dense
external mycelium developed in the mycorrhizal
conditions. Wallander & Nylund (1992) previously
found a stimulating effect of P starvation on the
development of the external mycelium of ectomycorrhizal fungi associated with pine. Plant shoots
had low P concentrations (Tables 1, 3). The P
concentration in shoots of P. sylvestris seedlings can
vary from 0.6 to 5 mg g−" d. wt (Ingestad, 1979 ;
Wallander & Nylund, 1992 ; Colpaert et al., 1997).
Except for S. luteus, plants and fungi behaved in a
similar way in both experiments. The more prominent senescence of S. luteus mycelia in Experiment 1
than in Experiment 2 could have been caused by the
repeated Pi uptake measurements in Experiment 1.
The hydrophobic Suillus species often do not grow
very well in semihydroponic systems in which the
substrate is regularly irrigated (Wallander & Nylund,
1992).
Phosphate-uptake rates were expressed per unit
root dry weight. Although it would be better to
measure the nutrient-absorbing surface area of the
root systems, root dry weights can be measured
595
easily and very accurately for a large number of
plants. A major problem is that we do not know the
proportion of the root and hyphal surfaces that are
effectively involved in nutrient absorption. Uptake
rates of phosphate were calculated from the
Michaelis–Menten equation assuming a single
mechanism of uptake. However, both in plants and
higher fungi at least two independently operating
uptake systems may be active (Beever & Burns,
1980 ; Schachtman et al., 1998). Over the external
concentration range used here, the high-affinity
system for Pi uptake contributes most to the uptake.
The Km value of the high-affinity Pi uptake systems
in plant roots, filamentous fungi and excised mycorrhizas seems to be in the 1–10 µM Pi concentration
range. From the present experiment we cannot
determine the relative contribution of the pine and
the mycorrhizal fungi to the Pi uptake, as both
organisms were growing in the same compartment.
Nevertheless, it is possible that in seedlings colonized
by hydrophobic fungi producing impermeable outer
sheath layers (Suillus sp.), almost all Pi will be first
absorbed by the mycobiont. Translocation of P to
the root cortex can then occur only through the
fungal symplast (Ashford et al., 1989). This statement is not valid for fungi with hydrophilic mycorrhizas (e.g. T. terrestris) in which apoplastic movement of solutes through the mantle remains possible.
A marked increase in the net Pi-absorption
capacity of the pine root systems was observed with
all mycorrhizal fungi studied (Table 2, Fig. 3). The
time course of this increase was clearly fungusdependent (Fig. 2), and illustrates that differences in
establishment of the mycobionts can affect physiological responses of mycorrhizal plants during an
experiment. However, since the actual development
of the different fungi during an experiment is difficult
to evaluate, sequential harvests are recommended
(Jones et al., 1991). The weekly non-destructive
determination of the uptake rate of the growthlimiting nutrient in our experiment allowed us to
follow the development of the fungi even more
closely without a need for weekly harvests.
The constant percolation of nutrient solution
during the uptake analyses counteracts the development of depletion zones around roots and hyphae.
Therefore the high Pi-uptake rates of the mycorrhizal root systems cannot be ascribed to an improved nutrient scavenging of the substrate by the
fungi. The high Pi uptake in the mycorrhizal
conditions strongly suggests that a much larger
number of Pi transporters are operating in the
mycorrhizal root systems than in the non-mycorrhizal roots. The Pi-uptake rate of the seedlings was
positively correlated with the ergosterol concentration in the perlite, independently of the fungal
species involved or the developmental stage of the
mycobiont (Fig. 4). This indicates that the increase
of absorptive surface area provided by the mycelium
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596
J. V. Colpaert et al.
outside the root plays a substantial role in the Pi
uptake of the plants. Ergosterol is a major sterol in
fungal membranes, and not only correlates with
active fungal biomass but probably also with total
surface area of external mycelia involved in Pi
absorption. A possible causal relationship between
the long-term P influx in pines and willows and the
absorbing surface area of the external mycelia of
their ectomycorrhizal associates was also found by
Jones et al. (1990) and Rousseau et al. (1994). Ion
influx rates in cells are determined not only by the
quantity of available transporters, but also by
qualitative aspects of the uptake systems. Lower
efflux rates or more efficient transporters in hyphal
membranes may contribute to the improved net Pi
uptake in the mycorrhizal plants, especially at the
lower Pi concentration. A comparison of the nutrient
uptake rates at decreasing external Pi concentrations
indicates that at least some mycobionts have a higher
affinity for Pi uptake than non-mycorrhizal roots
(Fig. 3). This suggests that mycelia of ectomycorrhizal fungi might be more effective than roots in
competing with free-living microorganisms for recently mineralized or solubilized P.
The increased Pi-uptake capacity of the mycorrhizal plants did not result in improved growth of
the host plants, because all plants received the same
amount of readily available P. Even non-mycorrhizal
plants with a low Pi-uptake capacity eventually
absorbed most of the ions present in the perlite
solution. The fact that mycorrhizal plants could not
benefit from their higher Pi-absorbing capacity
resulted in similar growth rates between mycorrhizal
and non-mycorrhizal plants. In semihydroponic
systems, the partial retention of P in rapidly
expanding fungal mycelia can result in a lower
transfer of P into shoots of mycorrhizal pines, in
particular under P limitation (Tables 1, 3 ;
Cumming, 1996 ; Colpaert & Verstuyft, 1999).
Phosphate uptake in plant and fungal cells is under
biochemical control, a control that is probably
mediated by the phosphorus status of the cells
(Beever & Burns, 1980 ; Schachtman et al., 1998). In
mycorrhizal root systems, phosphate absorption
might be regulated by the intracellular P concentration of both mycobionts and root cells. We
assume that the P status of the mycelia is probably
most important for the Pi absorption from the
solution, whereas the P concentration of the plant
tissues is more likely to affect the transfer of Pi at the
fungus–plant interface. In Pisolithus tinctorius Pi
absorption and efflux is regulated by the cellular P
content (Cairney & Smith, 1992, 1993). To optimize
the Pi-absorption capacity in the external mycelia,
the intracellular Pi concentration in the hyphae is
kept low by incorporation of Pi in polyphosphate and
by transfer of P to the mycorrhizas.
Compared to other studies, the specific Pi-uptake
rates found in our experiments were high. Harley &
McCready (1952) report a Pi-uptake rate of 0.12 and
0.3 nmol g−" d. wt s−" for excised Fagus mycorrhizas
in a 6.3 and 32 µM Pi solution, respectively, at pH
5.5. These values are in the same order as those
recorded for pine roots colonized with T. terrestris
which was the least efficient mycobiont in Pi uptake
in the present study. This again stresses the
importance of the external mycelium for the enlargement of the nutrient-absorbing surface. It is
also important to notice that we expressed uptake
rate on the basis of total root weight of the seedlings,
whereas Harley & McCready (1952) divided by the
weight of the mycorrhizas as unit of reference.
Cumming (1996) studied $#P uptake in pine seedlings
transferred from sand to a hydroponic culture. In
this way, the external mycelia were not included in
the tests. Mycorrhizal colonization nevertheless
enhanced Pi uptake in P-limited pine seedlings 1.3,
2.6 and 3.3-fold in roots colonized with Laccaria
bicolor, P. involutus and Pisolithus tinctorius, respectively, compared with non-mycorrhizal roots.
Jones et al. (1990, 1991) obtained a mean P inflow
rate of 0.2 nmol g−" s−" over a 50 d period in
Salix viminalis plants colonized with T. terrestris,
compared to only 0.1 nmol g−" s−" for nonmycorrhizal willow.
The differences in growth and Pi uptake among
the different mycobionts studied may be related to
different life strategies. Paxillus involutus had the
most rapid and strongest effect on the Pi absorption
of the seedlings. Cairney & Smith (1993) studied Pi
uptake of in vitro cultures of Pisolithus tinctorius,
Hebeloma crustuliniforme and P. involutus. They also
found that Pi absorption in P. involutus was three to
four times higher than in the other ectomycorrhizal
fungi. P. involutus grew very rapidly in our experiment but it retained a lot of the assimilated P in
its own tissue. Paxillus involutus is suggested to be a
common stress-tolerant ruderal species in pioneer
and disturbed habitats (Deacon & Fleming, 1992) ; it
is a generalist with a very broad spectrum of host
plants, has little soil preference, and seems rather
insensitive to environmental pollution such as soil
acidification and nitrogen accumulation in forest soil
(Arnolds & Jansen, 1992). The two Suillus spp. are
normally associated with Pinus spp. and are sensitive
to nitrogen deposition (Arnolds & Jansen, 1992).
Both isolates were collected from the same P. nigra
stand on a poor, sandy soil. Suillus luteus behaves
more as a pioneer fungus that follows the periphery
of the expanding pine root systems. Suillus bovinus
was clearly the slowest-growing species in our
experiment, and it certainly has more features of Kselected organisms than the other fungi studied
(Deacon & Fleming, 1992). Its Pi-uptake rate did not
match that of P. involutus but there is evidence that
S. bovinus has better access to organic P compounds
than P. involutus (Timonen & Sen, 1998). The
smallest effect on the Pi uptake was found in plants
Printed from the C JO service for personal use only by...
Phosphate uptake by mycorrhizal and non-mycorrhizal pine
associated with T. terrestris. This fungus does not
form a dense external mycelium, rapidly colonizes
the substrate, and seems to be less efficient in Pi
uptake at low external Pi concentrations. It is a
typical ruderal colonizer of pine seedlings and
probably has low competitive abilities.
              
The authors are very grateful to the technical staff of the
Institute of Botany for the development of the new
instruments used in the Pi-uptake experiments. We are
also grateful to the Research Fund K. U. Leuven for the
financial support (OT project 97\24).

Arnolds E, Jansen E. 1992. New evidence for changes in the
macromycete flora of the Netherlands. Nova Hedwigia 55 :
325–351.
Ashford AE, Allaway WG, Peterson CA, Cairney JWG. 1989.
Nutrient transfer and the fungus–root interface. Australian
Journal of Plant Physiology 16 : 85–97.
Beever RE, Burns DJW. 1980. Phosphorus uptake, storage and
utilization by fungi. Advances in Botanical Research 8 : 127–219.
Bolan NS. 1991. A critical review on the role of mycorrhizal fungi
in the uptake of phosphorus by plants. Plant and Soil 134 :
189–207.
Bougher NL, Grove TS, Malajczuk N. 1990. Growth and
phosphorus acquisition of karri (Eucalyptus diversicolor F.
Muell.) seedlings inoculated with ectomycorrhizal fungi in
relation to phosphorus supply. New Phytologist 114 : 77–85.
Cairney JWG, Smith SE. 1992. Influence of intracellular
phosphorus concentration on phosphate absorption by the
ectomycorrhizal basidiomycete Pisolithus tinctorius. Mycological
Research 96 : 673–676.
Cairney JWG, Smith SE. 1993. Efflux of phosphate from the
ectomycorrhizal basidiomycete Pisolithus tinctorius : general
characteristics and the influence of intracellular phosphorus
concentration. Mycological Research 97 : 1261–1266.
Casper BB, Jackson RB. 1997. Plant competition underground.
Annual Review of Ecology and Systematics 28 : 545–570.
Chalot M, Brun A, Finlay RD, So$ derstro$ m B. 1996. Kinetics,
energetics and specificity of a general amino acid transporter
from the ectomycorrhizal fungus Paxillus involutus. Microbiology 142 : 1749–1756.
Claassen N, Barber SA. 1974. A method for characterizing the
relation between nutrient concentration and flux into roots of
intact plants. Plant Physiology 54 : 564–568.
Colpaert JV, Verstuyft I. 1999. The Ingestad concept in
ectomycorrhizal research : possibilities and limitations. Physiologia Plantarum 105 : 233–238.
Colpaert JV, Van Laere A, Van Assche JA. 1996. Carbon and
nitrogen allocation between symbionts in ectomycorrhizal and
non-mycorrhizal Pinus sylvestris L. seedlings. Tree Physiology
16 : 787–793.
Colpaert JV, Van Laere A, Van Tichelen KK, Van Assche JA.
1997. The use of inositol hexaphosphate as a phosphorus source
by mycorrhizal and non-mycorrhizal Scots pine (Pinus sylvestris). Functional Ecology 11 : 407–415.
Cumming JR. 1996. Phosphate-limitation physiology in ecto-
597
mycorrhizal pitch pine (Pinus rigida) seedlings. Tree Physiology
16 : 977–983.
Deacon JW, Fleming LV. 1992. Interactions of ectomycorrhizal
fungi. In : Allen MJ, ed. Mycorrhizal functioning. An integrative
plant–fungal process. New York, USA\London, UK : Chapman
& Hall, 249–300.
Dixon M, Webb EC. 1964. Enzymes, 2nd edn. London, UK :
Longman, Green & Co.
Eltrop L, Marschner H. 1996. Growth and mineral nutrition of
non-mycorrhizal and mycorrhizal Norway spruce (Picea abies)
seedlings grown in semi-hydroponic sand culture. I. Growth
and mineral nutrient uptake in plants supplied with different
forms of nitrogen. New Phytologist 133 : 469–478.
Harley JL. 1989. The significance of mycorrhiza. Mycological
Research 92 : 129–139.
Harley JL, McCready CC. 1952. The uptake of phosphate by
excised mycorrhizal roots of the beech. III. The effect of the
fungal sheath on the availability of phosphate to the core. New
Phytologist 51 : 342–348.
Ingestad T. 1979. Mineral nutrient requirements of Pinus
silvestris and Picea abies seedlings. Physiologia Plantarum 45 :
373–380.
Ingestad T, A/ gren GI. 1995. Plant nutrition and growth : basic
principles. Plant and Soil 168/169 : 15–20.
Ingestad T, Ka$ hr M. 1985. Nutrition and growth of coniferous
seedlings at varied relative nitrogen addition rate. Physiologia
Plantarum 65 : 109–116.
Jones MD, Durall DM, Tinker PB. 1990. Phosphorus relationships and production of extramatrical hyphae by two types of
willow ectomycorrhizas at different soil phosphorus levels. New
Phytologist 115 : 259–267.
Jones MD, Durall DM, Tinker PB. 1991. Fluxes of carbon and
phosphorus between symbionts in willow ectomycorrhizas and
their changes with time. New Phytologist 119 : 99–106.
Jungk A, Asher CJ, Edwards DG, Meyer D. 1990. Influence of
phosphate status on phosphate uptake kinetics of maize (Zea
mays) and soybean (Glycine max). Plant and Soil 124 : 175–182.
Kronzucker HJ, Siddiqi MY, Glass ADM. 1996. Kinetics of
NH + influx in spruce. Plant Physiology 110 : 773–779.
%
Marschner H. 1995. Mineral nutrition of higher plants, 2nd edn.
London, UK : Academic Press.
Marschner H, Ha$ ussling M, George E. 1991. Ammonium and
nitrate uptake rates and rhizosphere pH in non-mycorrhizal
roots of Norway spruce [Picea abies (L.) Karst.]. Trees 5 : 14–21.
Murphy J, Riley JP. 1962. A modified single solution method for
the determination of phosphate in natural waters. Analytica
Chimica Acta 27 : 31–36.
Nylund J-E, Wallander H. 1992. Ergosterol analysis as a means
of quantifying mycorrhizal biomass. In : Norris JR, Read DJ,
Varma AK, eds. Methods in microbiology, Vol. 24. London,
UK : Academic Press, 77–88.
Rousseau JVD, Sylvia DM, Fox AJ. 1994. Contribution of
ectomycorrhiza to the potential nutrient-absorbing surface of
pine. New Phytologist 128 : 639–644.
Schachtman DP, Reid RJ, Ayling SM. 1998. Phosphorus
uptake by plants : from soil to cell. Plant Physiology 116 :
447–453.
Smith SE, Read DJ. 1997. Mycorrhizal symbiosis, 2nd edn.
London, UK : Harcourt Brace & Company.
Timonen S, Sen R. 1998. Heterogeneity of fungal and plant
enzyme expression in intact Scots pine–Suillus bovinus and
–Paxillus involutus mycorrhizospheres developed in natural
forest humus. New Phytologist 138 : 355–366.
Wallander H, Nylund J-E. 1992. Effects of excess nitrogen and
phosphorus starvation on the extramatrical mycelium of
ectomycorrhizas of Pinus sylvestris L. New Phytologist 120 :
495–503.
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