FEMS Microbiology Ecology 47 (2004) 31^37
www.fems-microbiology.org
Mycelial growth and substrate acidi¢cation of ectomycorrhizal fungi
in response to di¡erent minerals
Anna Rosling , Bjo«rn D. Lindahl, Andy F.S. Taylor, Roger D. Finlay
Department of Forest Mycology and Pathology, SLU, P.O. Box 7026, SE-750 07 Uppsala, Sweden
Received 15 July 2003; received in revised form 22 August 2003; accepted 24 August 2003
First published online 19 September 2003
Abstract
A colorimetric method was developed to permit semi-quantitative measurement of substrate acidification by different ectomycorrhizal
and one saprotrophic fungus growing on media containing one of five different minerals. Overall, substrate acidification differed between
fungal species and the degree of variation in acidification in response to different minerals was highly species-dependent. Mycena galopus
and Cortinarius glaucopus produced the least biomass of all tested species and produced the highest amount of acidification per unit
mycelial density. Substrate acidification by C. glaucopus was inversely related to mycelial density, with particularly high acidification at
low mycelial density on medium enriched with tri-calcium phosphate. Substrate acidification by M. galopus was constant irrespective of
mycelial density and varied only according to mineral treatment, with higher substrate acidification on tri-calcium phosphate compared to
the other minerals.
2 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
Keywords : Cortinarius glaucopus ; Ectomycorrhizal fungus; Substrate acidi¢cation ; Apatite ; Potassium feldspar ; Quartz
1. Introduction
The ability of mycorrhizal fungi and other soil microorganisms to actively weather soil minerals [1,2] suggests
that mineral elements may become available to mycorrhizal plants independently of the cation concentrations in
bulk soil solution. After nitrogen, phosphorus is the
most important macronutrient determining plant growth
in most terrestrial ecosystems [3]. In soils most phosphorus
occurs either in primary minerals, mainly apatite, or in
organic complexes. Low solubility and mobility of either
form results in low phosphorus availability in soil. Biological activity can increase the availability of phosphorus
and other nutrients, both as a result of soil acidi¢cation
due to cation uptake and via the release of weathering
agents such as siderophores and low molecular mass organic acids [4]. In heterotrophic leaching processes, low
molecular mass organic acids play a key role in mineral
weathering as they supply both protons and metal-complexing organic acid anions [5]. Oxalic acid is suggested to
be the organic acid most abundantly released by ectomy-
* Corresponding author. Tel.: +46 (18) 671864; Fax: +46 (18) 673599.
E-mail address : [email protected] (A. Rosling).
corrhizal mycelia [6]. In soil densely colonised by mycorrhizal fungi, Gri⁄ths et al. [7] found a signi¢cant positive
correlation between oxalate and PO33
4 concentrations, suggesting that fungal exudation of organic acids enhanced
weathering and solubility of PO33
in the soil. They con4
cluded that fungi released oxalic acid in excess of that
which was precipitated by Ca2þ and that this caused intensive local weathering that increased the availability of
23
PO33
4 and SO4 . In ¢eld and laboratory studies, e¡ects of
weathering have been detected on both clay minerals and
insoluble phosphates as a result of colonisation by mycorrhizal fungi [6,8]. It was suggested that this was a result of
a more acidic environment caused by oxalate produced by
the mycorrhizal mycelium. Apatite has been demonstrated
to increase growth of phosphorus-de¢cient pine seedlings,
and a larger e¡ect was seen for seedlings growing in association with symbiotic mycorrhizal fungi compared to
non-mycorrhizal controls [9].
Increased production and release of organic acids by
fungi and plants has been observed in response to increased heavy metal concentrations [10] and under condi2þ
tions of PO23
and Kþ de¢ciency [11,12]. We hy4 , Mg
pothesise that weathering by mycorrhizal fungi is an active
process triggered by mineral element de¢ciency in fungi
and the host tree. To investigate these ecologically impor-
0168-6496 / 03 / $22.00 2 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/S0168-6496(03)00222-8
FEMSEC 1583 12-1-04
Cyaan Magenta Geel Zwart
32
A. Rosling et al. / FEMS Microbiology Ecology 47 (2004) 31^37
tant questions additional studies of fungal responses to
de¢ned mineral substrates are necessary. The aim of the
present study was to develop a method to screen patterns
of variation in substrate acidi¢cation by di¡erent fungi
growing on phosphorus-free media amended with di¡erent
minerals. A semi-quantitative colorimetric method was developed to estimate substrate acidi¢cation.
2. Materials and methods
2.1. Fungal isolates
Fungal isolates that we have tested were seven ectomycorrhizal fungi: Amanita muscaria (L.: Fr.) Hook. (isolate
code UP233), Cortinarius glaucopus (Sch.: Fr.) Fr. (UP21),
Hebeloma crustuliniforme (Bull.) Que¤l. (UP184), Piloderma
byssinum (P. Karst.) Ju«lich (UP185), Piloderma fallax
(Liberta) Stalpers (UP121), Paxillus involutus (Batsch.:
Fr.) Fr. (UP234), and Suillus bovinus (L.: Fr.) Roussel
(UP63), as well as a saprotrophic fungus, Mycena galopus
(Pers.: Fr.) Kumm. (892) as a comparison. Stock cultures
were maintained in darkness at 25‡C, on half-strength
modi¢ed Melin^Norkrans (MMN) medium [13].
2.2. Mineral treatments
Phosphorus-free MMN medium, containing 4.3 g l31
2-(N-morpholino)ethanesulfonic acid sodium salt as a
bu¡er, was prepared using L-alanine as a nitrogen source
to avoid acidi¢cation resulting from ammonium-N uptake
[14]. Using a dispenser, 20 ml of the medium was added to
Table 1
Element composition of the four natural minerals : quartz, potassium
feldspar, apatite and marble
Quartz
SiO2
Al2 O3
CaO
Fe2 O3
K2 O
MgO
MnO
Na2 O
P2 O5
TiO5
LOI
98.10
0.28
6 0.10
0.13
6 0.10
6 0.02
0.00
6 0.06
0.02
0.35
0.30
Ba
Cr
Cu
La
Sr
Y
Zr
23.0
51.4
21.9
6.8
4.6
11.1
360.0
Potassium feldspar
66.70
18.10
0.14
6 0.10
12.00
6 0.02
6 0.003
2.64
0.01
0.00
0.40
729.0
6 10.0
26.7
6.5
83.3
8.6
6 2.0
Apatite
1.67
6 0.04
53.00
0.25
6 0.10
0.75
0.02
0.11
37.40
0.01
0.40
6.9
14.4
25.2
1300.0
279.0
645.0
8.0
2.3. Harvest and measurements
Marble
0.12
0.04
54.70
6 0.10
6 0.10
0.35
0.20
6 0.06
0.01
6 0.002
43.60
32.7
6 10.0
11.8
6 6.0
581.0
6.8
6 2.0
Element oxides and remaining loss on ignition (LOI) are given in percent dry weight. The rare elements, listed at the end (Ba to Zr), are given in mg kg31 .
FEMSEC 1583 12-1-04
9-cm Petri dishes. After solidi¢cation, a 5-ml surface layer
of water agar (15 g agar l31 ) containing 0.25% (w/v) mineral powder (particle size 6 125 Wm) was added. The mineral treatments were TCP (tri-calcium phosphate, BP,
E 341, Merck, Darmstadt, Germany) and four pure minerals: apatite, potassium feldspar, pink calcite marble and
quartz. Quartz was used as a control since it is highly
resistant to weathering. Mineral purity was veri¢ed by
element composition analysis (Table 1) according to
EPA methods 200.7 (ICP-AES) and 200.8 (ICP-QMS)
by Analytica AB (Luleafi, Sweden). After solidi¢cation, a
sterile sheet of nitrogen-free cellophane was placed on the
surface of the medium to enable removal of the fungal
mycelium.
Adding mineral powders to the water agar a¡ected the
pH of the surface layer. The pH e¡ect of the di¡erent
minerals was estimated by measuring the pH of 0.25%
(w/v) mineral powder suspensions, using a digital pH meter E632 (METROHM, Hesisau, Switzerland). After 30 h,
the pH of the mineral suspensions had stabilised and was
6.5 for TCP, 7.6 for quartz, 7.7 for potassium feldspar, 8.7
for apatite and 9.5 for marble. After solidi¢cation of the
surface layer on the basal medium, the pH of the two
media equilibrated. An overall substrate pH of 5.6^5.7
was measured for all minerals by soaking the content of
a half Petri dish in 1:1 w/v deionised water, and measuring
the pH of the solution after 2 h. We concluded that equilibrium of soluble elements in the agar is reached whereas
di¡erences in physiochemical properties, i.e. chelating capacity of the minerals, in the di¡erent treatments remain.
Each fungal species and mineral combination was replicated six times. Fungal inoculum consisted of single agar
plugs (70 mm) cut from the edge of actively growing mycelium. The experimental cultures were incubated in darkness at 25‡C until harvested.
In order to enable comparison between fungal isolates
with di¡erent growth rates, the plates were examined
weekly and harvested when the fungal mycelia reached a
pre-de¢ned diameter of 45 mm. When half of the mycelia
in a particular treatment combination of species and mineral were su⁄ciently large, all plates in that treatment were
harvested. Occasionally, size variations within treatments
were high and the plates were harvested at di¡erent occasions.
Size and appearance of the mycelium at harvest was
documented by scanning the plates from above and below,
using a £atbed scanner. Average mycelial diameter was
calculated from measurements of the images. The location
of the inoculation plug, the mycelial centre, as well as the
mycelial front was marked on the bottom of each dish.
The cellophane was lifted from each plate and the mycelium was removed and dried at 60‡C for 24 h to measure
mycelial dry weight (DW). The contribution of the agar in
Cyaan Magenta Geel Zwart
A. Rosling et al. / FEMS Microbiology Ecology 47 (2004) 31^37
33
Fig. 1. Estimation of substrate acidi¢cation by growing fungal mycelia using a three-step colorimetric method. a: A fungal mycelium growing out from
a central inoculum plug (C). At harvest, the fungal mycelium was removed and a rectangular slice was cut in the underlying agar, from C through the
mycelial front (M) to the edge of the plate. b: The slice was put on top of a purple pH indicator plate (pH 7). As acidity di¡used from the agar slice,
pH dropped in a zone around the slice, shifting the colour of the indicator plate to yellow at pH below 5.2. Plates were scanned after 24 h. c: Image
analysis produced schematic images of the zones and enabled quanti¢cation of the size of the pH shift zone by counting the number of yellow pixels.
the inoculum plugs to this measurement was found to be
negligible.
2.4. Estimating substrate acidi¢cation using pH indicator
plates
A novel colorimetric method was developed in order to
estimate substrate acidi¢cation as a result of mycelial
growth (Fig. 1). Using a dispenser, 10 ml of pH indicator
medium was added to 9-cm Petri dishes. The indicator
medium contained 15 g l31 agar and 0.1 g l31 each of
the pH indicators bromocresol purple (Sigma-Aldrich)
and alizarin red S (Sigma-Aldrich) [15]. The initial pH
was set at 7. Above pH 6.8, the agar is purple and as
the pH drops the colour gradually changes to orange
and ¢nally to yellow below pH 5.2.
After removal of the cellophane and the fungal mycelium from the growth medium, a rectangular slice of the
underlying agar substrate was cut in transect from the
inoculum plug to the peripheral agar beyond the mycelial
front (Fig. 1). In order to cut identical slices an agar-cutting device was constructed by mounting two razor blades
at a distance of 0.5 cm apart. Two replicate slices were cut
from each plate and the slices were transferred to individual pH indicator plates where they were placed on the
indicator agar with the underside down. Plates were incubated for 24 h at room temperature to allow di¡usion of
acidity from the agar slice into the pH indicator plates
(Fig. 1). Di¡usion of acidity caused a decrease in pH
and a simultaneous colour shift in a zone around the
agar slices from the mineral plates. Zones were detected
by scanning the plates from above against a white background using a £atbed scanner.
able threshold levels in each of the three colour components of the images, red (R), green (G) and blue (B). The
application of threshold levels to images at grey levels of
170 in R, 165 in G and 100 in B produced schematic zone
images in yellow and red on a black background. All zone
images were visually compared to their original plate image to ensure that zones corresponded in size. The number
of yellow pixels in each image was calculated using a customised script (available upon request from the corresponding author) in Director 8 (Macromedia0 , San Francisco, CA, USA).
2.6. Oxalic acid standard curve
Oxalic acid is suggested to be the organic acid most
abundantly released by ectomycorrhizal mycelia [6]. The
substrate acidi¢cation estimated as described above was
expressed in IM equivalents of oxalic acid. Adding di¡erent amounts of oxalic acid, dissolved in 1 ml of water, on
top of the solidi¢ed, mineral-enriched agar produced standard curves. The oxalic acid solution was spread over the
plates and these were subsequently incubated for 24 h at
room temperature. Final concentrations of oxalic acid in
the plates were 1, 3, 6 and 10 WM. Individual standard
curves were produced for all minerals. Duplicate plates
were set up for each combination of mineral and oxalic
acid concentration. Two additional plates from each mineral treatment served as background controls. Substrate
acidi¢cation from oxalic acid standards was estimated in
the same manner as for experimental plates. These pH
indicator plates were scanned after 5, 18 and 24 h. Substrate acidi¢cation in experimental plates was extrapolated
from the oxalic acid standard curves exempli¢ed in Fig. 2
and expressed in IM equivalents of oxalic acid.
2.5. Image analysis
2.7. Statistical analysis
Images of pH indicator plates were subjected to image
analysis, using Adobe Photoshop 7.0.1 (Adobe Systems,
San Jose, CA, USA), in order to quantify the size of the
pH shift zones. Optimal separation of the pH shift zones
from the background was obtained by determining suit-
FEMSEC 1583 12-1-04
Variation in mycelial density, substrate acidi¢cation and
substrate acidi¢cation per unit mycelial density, between
di¡erent fungal species and minerals was analysed using
two-way analysis of variance (ANOVA) with fungal spe-
Cyaan Magenta Geel Zwart
34
A. Rosling et al. / FEMS Microbiology Ecology 47 (2004) 31^37
cies and minerals as explaining variables. To analyse variations within fungal species, one-way ANOVA was used
with the mineral type as the explaining variable. Di¡erences between individual species or minerals were tested
for signi¢cance using Fisher’s PLSD.
3. Results
3.1. Evaluation of the colorimetric scanning method
The colorimetric scanning method described in this paper enables semi-quantitative estimates of substrate acidi¢cation by fungi (Fig. 1). A strong linear relationship
between substrate acidi¢cation and increasing size of pH
shift zones for oxalic acid standard curves was found in all
mineral treatments, exempli¢ed by potassium feldspar and
TCP in Fig. 2. Oxalic acid standard curves on apatite and
quartz are located close to that of potassium feldspar (Fig.
2). The r2 values were 0.87 for apatite, 0.97 for potassium
feldspar, 0.94 for quartz and 0.90 for TCP.
Di¡usion from the agar slice to the pH indicator plate is
determined by di¡erences in acidity between the two media. Zones of pH shift expand linearly over time as long as
su⁄cient concentration di¡erence remains. Once equilibrium is reached, no distinct zones can be distinguished. The
time needed to reach equilibrium is determined by the
initial acidity of the agar slice. Yellow zones are seen as
long as pH in the zone is 5.2 or lower. Di¡usion eventually
decreases acidity in the centre to pH values above 5.2 and
Fig. 3. Average mycelial density in mg cm32 ( Y S.E.M.) for each of the
eight fungi grown on media enriched with ¢ve di¡erent minerals, quartz
(Q), potassium feldspar (K), apatite (A), TCP (T) and marble (M). Mycelial size and dry weight were determined when at least half of the replicates in each treatment had a mycelial diameter of 4.5 cm. Fungal isolates appear from the left according to decreasing average mycelial
density. Numbers of replicates (n) are given for each mineral in the histogram. Average mycelial densities for minerals within a fungal species
signi¢cantly di¡erent from that of the quartz control are indicated by
an asterisk (P = 0.005).
the zone ceases to expand and eventually disappears. Estimating substrate acidi¢cation based on the size of the pH
shift zone is only relevant as long as the time of zone
detection is kept constant and zones are still expanding
linearly at this time. Substrate acidity was su⁄cient for
linear expansion of the pH shift zone over 24 h for all
oxalic acid concentrations tested, in apatite, potassium
feldspar and quartz. The bu¡ering e¡ect of TCP decreased
acidi¢cation in this treatment and pH shift zones were
only detected at oxalic acid concentrations of 3 WM and
above. Marble had an even higher bu¡ering capacity and
was not analysed further.
3.2. Mycelial density
Fig. 2. Standard curves for 1, 3, 6 and 10 WM oxalic acid applied to
plates containing four di¡erent minerals. Increasing concentrations resulted in an increased size of the yellow pH shift zone. Linear regressions for apatite, potassium feldspar and quartz overlapped and are represented by the curve for potassium feldspar (K). The high bu¡ering
capacity of TCP (T) shifted this curve to the right in the diagram.
FEMSEC 1583 12-1-04
Mycelial density (mg cm32 ) was used as a measure of
biomass in order to enable comparison of fungal species
with di¡erent mycelial growth rates. Occasionally plates in
the same treatment were harvested at di¡erent times due
to the large variation in mycelial expansion rate. In these
cases, the growth rate (mg day31 ) within treatments was
checked to ensure that plates were comparable. All plates
analysed for substrate acidi¢cation had comparable mycelial sizes.
The mycelial density was signi¢cantly a¡ected by both
mineral type (P 6 0.0001) and fungal species (P 6 0.0001),
and there was also a signi¢cant interaction between the
two (P 6 0.0001). The average mycelial density was signi¢cantly higher on marble than the other four minerals
(P = 0.012) and the lowest densities were found on potassium feldspar. Averaged over all species, the phosphorus
Cyaan Magenta Geel Zwart
A. Rosling et al. / FEMS Microbiology Ecology 47 (2004) 31^37
35
celial growth on di¡erent mineral-enriched substrates is
expressed as acidi¢cation equivalent to given oxalic acid
concentrations (Fig. 4a). Using the current method, four
fungal isolates, C. glaucopus, M. galopus, P. involutus and
S. bovinus, caused measurable substrate acidi¢cation on
quartz, potassium feldspar, apatite and TCP. Mycelial
growth of A. muscaria and H. crustuliniforme did not
cause detectable substrate acidi¢cation in any of the mineral treatments whereas the two Piloderma species P. byssinum and P. fallax caused detectable substrate acidi¢cation in all mineral treatments except TCP. In the TCP
treatment the size of the pH shift zones for the two Piloderma species was close to the detection limit, at acidi¢cation equivalent of 3 WM oxalic acid. Substrate acidi¢cation
could thus not be accurately estimated and these measurements were excluded from further analysis.
Substrate acidi¢cation was signi¢cantly a¡ected by both
mineral type (P 6 0.0001) and fungal species (P 6 0.0001)
with a signi¢cant interaction (P 6 0.0001). Averaged over
all species, signi¢cantly higher acidi¢cation was detected
on TCP compared to quartz, potassium feldspar and apatite (P 6 0.0001). Averaged over all mineral treatments,
mycelial growth of S. bovinus and P. involutus caused
most substrate acidi¢cation while C. galopus caused the
least (Fig. 4a).
Fig. 4. Substrate acidi¢cation after fungal growth on four minerals,
quartz (Q), potassium feldspar (K), apatite (A) and TCP (T) estimated
equivalent to WM concentrations of oxalic acid. For the two Piloderma
isolates, substrate acidi¢cation was below the detection limit of 3 WM
oxalic acid in treatment T. For minerals within each fungal species,
mean values of substrate acidi¢cation and substrate acidi¢cation per
unit mycelial density di¡ering signi¢cantly from that of the quartz control are indicated by asterisks (P = 0.005). a: Mean substrate acidi¢cation expressed equivalent to WM oxalic acid ( Y S.E.M.) in each treatment. b: Substrate acidi¢cation per unit mycelial density in di¡erent
treatments. The mean for each treatment is given as equivalents of
oxalic acid WM (mg cm32 )31 ( Y S.E.M.).
content of the apatite (Table 1) and TCP did not signi¢cantly stimulate growth. However, P. fallax, H. crustuliniforme and M. galopus grew more densely on apatite compared to quartz, and P. fallax and M. galopus grew more
densely on TCP. S. bovinus and A. muscaria grew less
densely on TCP than on quartz. S. bovinus also grew
less densely on apatite and potassium feldspar. A. muscaria, P. involutus and M. galopus grew more densely on
marble than on quartz. On average, for all mineral treatments, the mycelial density di¡ered as follows : Piloderma
byssinum s P. fallax s S. bovinus = A. muscaria s P. involutus s M. galopus = H. crustuliniforme = C. glaucopus
(Fig. 3).
3.3. Estimated substrate acidi¢cation after mycelial growth
The substrate acidi¢cation produced as a result of my-
FEMSEC 1583 12-1-04
3.4. The relationship between substrate acidi¢cation and
mycelial density
When expressed per unit mycelial density, substrate
acidi¢cation was also signi¢cantly a¡ected by both mineral
type (P 6 0.0001) and fungal species (P 6 0.0001) and
there was a signi¢cant interaction between the two
(P 6 0.0001). Averaged over all species, substrate acidi¢cation per unit mycelial density was also signi¢cantly higher on TCP (P 6 0.0001). This di¡erence was mainly due to
a drastic increase in relative acidi¢cation by C. glaucopus
on TCP. Due to sparse mycelial growth of C. glaucopus
Fig. 5. Relationships between substrate acidi¢cation, expressed as IM
equivalents of oxalic acid (WM), and mycelial density (mg cm32 ) for
C. glaucopus and M. galopus illustrated by plotting all replicates for
substrates containing quartz (Q), potassium feldspar (K), apatite (A) and
TCP (T). Left: C. glaucopus demonstrates a general trend for all minerals of increasing substrate acidi¢cation at lower mycelial densities.
Right : In M. galopus substrate acidi¢cation appears to be independent
of mycelial density. Higher acidi¢cation is detected in treatment T.
Cyaan Magenta Geel Zwart
36
A. Rosling et al. / FEMS Microbiology Ecology 47 (2004) 31^37
and M. galopus, relative substrate acidi¢cation was signi¢cantly higher in these species compared to P. involutus and
S. bovinus that produced more biomass (P 6 0.0001). In
M. galopus and P. involutus acidi¢cation per unit mycelial
density was signi¢cantly lower on apatite compared to
quartz. In S. bovinus acidi¢cation per unit mycelial density
was signi¢cantly higher on potassium feldspar and apatite
compared to quartz (Fig. 4b).
For C. glaucopus an overall trend, for all minerals, of
higher substrate acidi¢cation at low mycelial densities was
found (Fig. 5, left). In contrast, M. galopus did not demonstrate any relationship between substrate acidi¢cation
and mycelial density on any mineral treatment (Fig. 5,
right).
4. Discussion
In studies of fungal weathering the displacement method
is commonly used in which clearing zones in mineral-enriched agar plates are detected after fungal growth [16].
Although straightforward in its design this technique has
two major disadvantages. No quantitative measurements
of weathering capacity are obtained, and substrate discoloration as a result of fungal growth makes detection of the
clearing zone di⁄cult for many species. The current colorimetric method overcomes both shortcomings and provides a useful semi-quantitative method to estimate substrate acidi¢cation. Apart from substrate acidi¢cation,
fungal weathering of minerals, like all biologically mediated weathering, is a complex process involving dissolution, selective transport and re-crystallisation [17]. In order
to achieve a better understanding of the fungal-mediated
weathering of di¡erent minerals, future studies may combine the current colorimetric method with crystallographic
studies of primary and secondary mineral particles in the
surface ¢lm.
Substrate acidi¢cation is the result of several simultaneously ongoing processes. Proton and carbon dioxide release are consequences of biological activity of roots and
micro-organisms and result in local acidi¢cation of the
substrate [4]. Production and exudation of low molecular
weight organic acids, oxalic acid in particular, is common
in fungi, but factors inducing production and subsequent
release vary for di¡erent groups of fungi [5]. In the current
study we also found large variation between species in
substrate acidi¢cation per unit mycelial density. We can
conclude that substrate acidi¢cation is not simply proportional to the fungal biomass, but largely dependent on
species-speci¢c responses to the environment.
Fungal production and subsequent exudation of oxalic
acid have di¡erent functions in the physiology and ecology
of di¡erent groups of fungi [18]. Oxalic acid production in
Aspergillus niger is largely dependent on the pH of the
substrate, with production drastically decreasing at substrate pH below 3 [19]. The types of carbon and nitrogen
FEMSEC 1583 12-1-04
sources available to fungi may also in£uence the production of oxalic acid [18]. Increased production by fungi has
been found in response to excess carbohydrate availability
[5] and the inverted growth^acidi¢cation relationship for
C. glaucopus (Fig. 5) described in this study can be explained in terms of limited growth, resulting in excess carbon being exuded into the substrate [5]. Addition of TCP
to the agar ¢lm may create an environment with a high
calcium concentration that the fungi have to tolerate in
order to grow. This environmental stress may be alleviated
by precipitation of calcium oxalate outside the cells [20].
Phosphorus de¢ciency in plants is associated with changes
in the carbon metabolism, and among other reactions, the
production and exudation of organic acids are increased
[21]. Signi¢cantly increased oxalic acid exudation has been
demonstrated for P. involutus under magnesium and potassium de¢ciency compared to non-de¢cient conditions
[12]. De¢ciency responses may, however, be di⁄cult to
separate from general stress responses. In the present
study, fungi were probably not phosphorus-de¢cient and
substrate acidi¢cation patterns are largely a result of the
fungal response to the element composition of the mineralenriched agar ¢lm.
Exudation in fungal mycelia is localised mainly at the
growing hyphal tips, where excess water and metabolic byproducts are deposited outside the hyphae [22]. Under
¢eld conditions exudates condition the micro-environment
in the fungus^soil interface. It has been suggested that a
large fraction of the exuded metabolites is re-absorbed by
the fungus [23] or consumed by the rich microbial communities commonly found in association with mycorrhizal
mycelia in soil [24]. The factors determining net substrate
acidi¢cation in soil are still poorly understood because of
high spatial and temporal complexity. A comprehensive
understanding of the reactions of organic acids in soil is
also still lacking [25]. Several studies have demonstrated a
strong relationship between low molecular weight organic
acids and weathering, but detected ¢eld concentrations of
organic acids in the bulk soil solution are too low to a¡ect
the weathering rate of feldspar [26]. Field measurements,
however, fail to estimate local concentrations in the acidic
extracellular gel at the micro-organism^mineral interface
[17]. The ¢ndings of the present study suggest that there
are signi¢cant species^mineral interactions regulating local
substrate acidi¢cation. Such interactions may further increase the discrepancy between measurements in bulk soil
and the actual acidity in the micro-habitat of fungal hyphae involved in weathering of minerals.
Acknowledgements
We gratefully acknowledge ¢nancial support from The
Swedish Research Council for Environment, Agricultural
Sciences and Spatial Planning (FORMAS). We also acknowledge the great assistance of Ola Rosling at Gap-
Cyaan Magenta Geel Zwart
A. Rosling et al. / FEMS Microbiology Ecology 47 (2004) 31^37
minder (www.gapminder.com) for writing the script that
enabled easy quanti¢cation of pixels of di¡erent colours.
Thanks to Petra Fransson and Hans Rosling for critical
reading of the manuscript and providing constructive ideas
for its improvement.
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