Research

Arbuscular mycorrhizal fungi respond to the substrate pH

Blackwell Science, Ltd

of their extraradical mycelium by altered growth and root

colonization
Ingrid M. van Aarle, Pål Axel Olsson and Bengt Söderström
Department of Microbial Ecology, Ecology Building, Lund University, SE–223 62 Lund, Sweden

Summary

Author for correspondence: • To test the response of arbuscular mycorrhizal (AM) fungi to a difference in soil
Ingrid M. van Aarle pH, the extraradical mycelium of Scutellospora calospora or Glomus intraradices, in
Tel: + 46 46 2223760 association with Plantago lanceolata, was exposed to two different pH treatments,
Fax: + 46 46 2224158
Email: Ingrid.van_Aarle@mbioekol.lu.se
while the root substrate pH was left unchanged.
• Seedlings of P. lanceolata, colonized by one or other of the fungal symbionts, and
Received: 12 November 2001
nonmycorrhizal controls, were grown in mesh bags placed in pots containing pH-
Accepted: 5 March 2002
buffered sand (pH around 5 or 6). The systems were harvested at approximately
2-wk intervals between 20 and 80 d.
• Both fungi formed more extraradical mycelium at the higher pH. Glomus intra-
radices formed almost no detectable extraradical mycelium at lower pH. The extra-
radical mycelium of S. calospora had higher acid phosphatase activity than that of
G. intraradices. Total AM root colonization decreased for both fungi at the higher
pH, and high pH also reduced arbuscule and vesicle formation in G. intraradices.
• In conclusion, soil pH influences AM root colonization as well as the growth and
phosphatase activities of extraradical mycelium, although the two fungi responded
differently.

Key words: arbuscular mycorrhizal fungi, extraradical mycelium, Glomus intra-

radices, pH, phosphatase activity, root colonization, Scutellospora calospora.

© New Phytologist (2002) 155: 173–182

extraradical mycelium may also be of importance (Jakobsen

Introduction
In nature, there is a large variation in plant growth and
phosphorus uptake in response to colonization by different
arbuscular mycorrhizal (AM) fungi (Ravnskov & Jakobsen,
1995; van der Heijden et al., 1998a,b). The AM fungal
isolates differ in their phosphorus uptake efficiency (Jakobsen
et al., 1992a; Joner & Jakobsen, 1994), and in their supply of
phosphorus to their host ( Jakobsen et al., 1992b; Pearson &
Jakobsen, 1993). Most AM fungi increase the phosphorus
content of plants, although some, such as Scutellospora
calospora, are often rather inefficient in phosphorus transport
to the host plant, and in some plants no, or only a small,
growth response can be seen ( Jakobsen et al., 1992a). These
variations may be the result of differences in fungal root
colonization or physiological characteristics, but the ability of
different AM fungal isolates to produce different amounts of
et al., 1992a; Boddington & Dodd, 1998). The positive
effects of AM fungi on plant growth have often been related
to an increased uptake of less mobile nutrients, especially
phosphorus (Bolan, 1991), from soil regions not accessible to
the root hairs. Whether AM fungal phosphatase activities
contribute significantly to the phosphorus nutrition of AM
plants is still unclear (Joner et al., 2000).
The observed differences in plant response to AM coloni-
zation may not only result from the use of different plant hosts
or fungal isolates, but may also result from variations in envi-
ronmental conditions. For example, three Glomus isolates
exhibited similar efficiencies in mineral acquisition when
grown in acidic soils, but their efficiencies differed when they
were grown in alkaline soil (Clark & Zeto, 1996b). Soil pH is
known to have considerable influence on plant growth in
particular because it influences the mobilization, and thus the

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174 Research

availability, of nutrients (Haynes, 1990; Marschner, 1995).
Also, root colonization and the ability to form extraradical
mycelium were changed when AM fungi were grown with
their host plants in soils of different pH (Abbott & Robson,
1985; Porter et al., 1987). However, in these studies, the AM
fungal extraradical mycelium and the plant roots were both
exposed to the pH treatment.
To test the response of AM fungi specifically to a difference
in soil pH, we allowed the hyphae of S. calospora and Glomus
intraradices, in association with Plantago lanceolata, to grow
out into root-free compartments at different pH values. We
studied not only the amount of extraradical mycelium formed
at different pH values, but also its phosphatase activities.
Since effects of a pH treatment may also be caused indirectly
by the interactions with saprotrophic fungi and bacteria (Olsson
et al., 1998; Ravnskov & Jakobsen, 1999), we also estimated
their biomass. Furthermore, we studied the possible influence
of the different pH treatments on total AM colonization of
the roots and the distribution of internal fungal structures.
Materials and Methods
Experimental set-up
Plantago lanceolata L. was grown in soil-filled mesh bags (root
compartment) placed centrally in a pot and surrounded with
quartz sand (hyphal compartment). Four hundred grams of
acid-washed sand, at a pH adjusted to 5 or 6, were used for
each pot. To establish the lower pH of the sand, it was flushed
with 1.0 l of a 1 mM 2-(N-morpholino)ethanesulphonic acid
(MES) buffer adjusted to pH 4 over a 24-h period. For the
higher pH, a MES buffer adjusted to pH 7 was used. The sand
was air-dried to a water-holding capacity of 60%. The growth
medium used for the root compartment was a mixture of a
moraine clay loam (Jakobsen & Nielsen, 1983) and river sand
(1 : 1), which had been irradiated (10 kGy) to eliminate
indigenous AM fungi. Inoculum (soil–root mixture) of either
S. calospora (Nicol. & Gerd.) Walker & Sanders (BEG 43) or
G. intraradices Schenck & Smith (BEG 87) was mixed into
the growth medium in the proportions 1 : 14. Scutellospora
calospora was originally isolated from a Mediterranean
grassland with a pH of 4.4 in Western Australia and G.
intraradices from a temperate grassland with a pH of 6.0 in
Denmark. One-hundred and fifty grams of the soil/inoculum
mixture were added to the mesh bags with a mesh size of
20 µm. For the non-mycorrhizal controls, 150 g of the soil
without inoculum were used. Nutrients were added to the
growth medium as described by Olsson et al. (1996). Bacterial
inoculum was added as a soil extract from pots with
nonmycorrhizal Cucumis sativus L. plants. The mesh bags
were moistened with distilled water to a water-holding
capacity of 60%.
Seeds of P. lanceolata L. were surface-sterilized for 15 min
in 5% calcium hypochlorite solution, washed with water and
transferred to Petri dishes containing moistened filter paper.
The seeds were pregerminated at room temperature for 7 d in
the dark and subsequently for 4 d in daylight. After germina-
tion, four seedlings were transplanted into each mesh bag,
and the surface of the pots was covered with plastic pellets to
reduce evaporation. The plants were grown in a greenhouse
at an average temperature of 28°C during the day, and 20°C
at night (16-h day, photosynthetic photon flux density of
150 µmol m−2 s−1 and a relative humidity of 60%). The sys-
tem was maintained at a water-holding capacity of 60% by
watering the root compartment with distilled water, and the
hyphal compartment with a 0.5 mM MES buffer, set at either
pH 4 or pH 7, which maintained the pH of the sand at 5 or
6, respectively (Table 1). Sets of four pots of each fungal

Table 1 The pH of the hyphal and root

pH (in H2O; n = 3) compartments at the different harvesting
times
Scutellospora Glomus
Nonmycorrhizal calospora intraradices

Compartment Time (d) Low High Low High Low High

Hyphal 20 5.25 6.17* 4.92 6.08* 4.64 5.98*

32 5.04 5.88* 4.61 5.96* 4.54 5.64*
53 4.93 5.43 4.48 5.53* 4.50 5.53*
67 5.08 5.94* 4.83 5.68* 4.79 5.99*
80 4.96 6.02* 5.19 6.06* 4.73 6.13*

Root 20 7.12 7.07 6.91 6.88 6.92 6.91

32 6.89 6.94 6.62 6.89* 6.74 7.03
53 6.61 6.76 6.30 6.65 6.52 6.85*
67 6.26 6.43 6.00 6.48* 6.13 6.41
80 6.06 6.14 5.86 6.06 5.69 6.11

Means marked with an asterisk are significantly different (P < 0.05) for low- and high pH
treatment (two-tailed t-test with unequal variances). Low, low pH treatment; High, high pH
treatment.

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(nonmycorrhizal, S. calospora or G. intraradices) at each pH
(low or high) treatment were randomly distributed over
plastic trays with transparent covers to prevent contamination
between treatments. These trays, containing in total 90 pots,
were distributed at random in the glasshouse. Pots and growth
propagators were regularly redistributed.
At 20, 32, 53, 67 and 80 d after transplantation, three ran-
domly taken pots from each treatment were harvested. Some
subsamples of the sand and soil were frozen in order to be able
to determine the fatty acid content later. Other subsamples
were immediately used to determine the pH (in H2O; 25 g
fresh weight substrate in 50 ml distilled water, shaken at 200
rotations min−1 for 2 h, followed by 15 min of sedimenta-
tion). A third set of subsamples was used to determine the acid
phosphatase (ACP) and alkaline phosphatase (ALP) activities.
The remaining sand was used to collect the extraradical
mycelium by wet sieving (van Aarle et al., 2001). If no hyphae
could be detected by eye, the decanted liquid was checked
under a dissecting microscope for hyphae, and these were
collected with forceps. The fresh weight of the hyphae was
measured, after which it was used to study the ACP and
ALP activity. Plants were collected and washed clean of soil.
Dry weights were determined for shoots and for subsamples
of roots. The total root dry weight was estimated by calcula-
tions based on the fresh weight proportion. Another sample
of the roots was used to study the possible ALP activity,
while a third sample was used to assess the mycorrhizal
colonization.
Phosphatase activity measurements
Alkaline and acid phosphatase activities of the extraradical
mycelium and phosphatase activity of mycorrhizal roots were
visualized with enzyme-labelled fluorescence substrate (ELF,
Molecular Probes, Eugene, USA), which is a fluorogenic
phosphatase substrate (van Aarle et al., 2001). The overall
phosphatase activity of the mycorrhizal mycelium was assessed
with a light microscope according to a relative scale ranging
from no activity (denoted 0) to high activity (denoted 5) in all
of the mycorrhizal hyphae. At each harvest, roots of one
replicate of each fungal treatment and each pH were hand-
sectioned and stained with alkaline ELF buffer solution to
visualize phosphatase-active intraradical hyphae.
To measure ACP and ALP activity of the sand and soil, a
modified procedure based on that of Tabatabai & Bremner
(1969) was used. Phosphatase activities were determined
spectrophotometrically using p-nitrophenyl phosphate as
substrate (Sigma Chemical Co., St. Louis, MO, USA). Five
grams of substrate were added to 40 ml of distilled water
and shaken at 200 revolutions min−1 for 15 min. Aliquots of
the suspension, 20 µl for ALP and 40 µl for ACP activity
determination, were incubated in 100 µl p-nitrophenyl
phosphate solution (4 mg p-nitrophenyl phosphate ml−1 in
deionized water) and 100 µl buffer solution. For the ALP
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Research 175
determination alkaline buffer solution was used (1.5 mol 2-
amino-2-methyl-1-propanol l −1, pH 10.3; Sigma), and for
the ACP determination citrate buffer (90 mM citrate and
10 mM chloride, pH 4.8; Sigma). Adding 2 ml 0.05 M
NaOH to the ALP solution or 1 ml 0.1 M NaOH to the ACP
solution after 2 h stopped the reaction. Substrate suspensions
of each of the fungal treatments were deactivated by heating
to boiling three times in a microwave oven and these were
used as substrate blanks to determine background levels of the
substrate. The addition of distilled water instead of the
substrate solution to the buffer solution produced blanks to
check the background of the p-nitrophenyl phosphate
substrate. The absorbance was measured at 420 nm after filtra-
tion through a 0.45-µm hydrophilic PTFE filter (Lida,
Werner-Glas & Instrument AB, Kungsängen, Sweden). Enzyme
activities (expressed as U g−1 dry wt of substrate) were calculated
after correction using the blanks. One unit of phosphatase
activity, under the specified conditions, was defined as
the amount of enzyme activity that liberated 1 µmol of
p-nitrophenol in 1 h.
Fatty acid analysis as a biomass indicator
Lipids were extracted from sand (10 g fresh weight) and soil
(5 g fresh weight) samples by vortexing for 15 s with a one-phase
chloroform–methanol–citrate buffer (Olsson et al., 1997).
Lipid extracts were separated from soil pellets after centrifugation
at 3000 g. The lipids were fractionated into neutral lipids,
glycolipids and phospholipids on prepacked silica columns
(100 mg sorbent mass, Varian, Harbor City, USA) by eluting
with 1.5 ml chloroform, 6 ml acetone and 1.5 ml methanol,
respectively. The fatty acid residues of the neutral lipids and
phospholipids were transformed into free fatty acid methyl
esters, which were identified and quantified using gas
chromatography, according to the method described by
Frostegård et al. (1993).
The nomenclature of the fatty acids follows that used by
Tunlid & White (1992). Signature phospholipid fatty acids
indicate the biomass of specific groups of microorganisms
(Tunlid & White, 1992). Phospholipid fatty acids 16:1ω5,
18:1ω7c, 20:4 and 20:5 were analysed as possible indicators
of AM fungal biomass (Olsson & Johansen, 2000). These
phospholipid fatty acids also occur in varying amounts in
other soil microorganisms (Olsson, 1999). Neutral lipid fatty
acids 16:1ω7c, 16:1ω5, 18:1ω7c, 20:4 and 20:5 were also
considered since they are common in storage lipids of AM
fungi (Graham et al., 1995; Olsson & Johansen, 2000) and
provide sensitive indicators of AM fungi (Olsson, 1999). The
neutral lipid fatty acid 16:1ω7c may constitute a large part of
the neutral lipid fatty acids of G. intraradices, while the reason
for not including phospholipid fatty acid 16:1ω7c is that it
has been shown not to be an important part of AM fungal
phospholipid fatty acids ( Johansen et al., 1996). Phospholipid
and neutral lipid fatty acid 18:2ω6,9 was used as an indicator

176 Research
of the biomass of saprotrophic fungi, and 10 bacteria-specific
phospholipid fatty acids (i15:0, a15:0, i16:0, 10Me16:0, i17:0,
a17:0, cy17:0, 10Me17:0, 10Me18:0 and cy19:0) were
used as indicators of bacterial biomass (Frostegård & Bååth,
1996).
Colonization assessment
Root samples were stained with Trypan blue according
to a modification of the procedure of Phillips & Hayman
(1970). Total AM fungal root colonization, arbuscular and
vesicular colonization were determined as the percentage
of root length colonized with the magnified intersections
method, as described by McGonigle et al. (1990). Root
length colonized by fine endophytes (Thippayarugs et al.,
1999) was quantified independently of the AM fungal
colonization.
Statistical analysis
Averages, standard errors and t-tests were calculated using
Microsoft Excel 2000. Data on dry weight, fatty acid
contents, phosphatase activities in the substrate and
percentage colonization were subjected to ANOVA, while the
Fig. 1 Fatty acids indicating arbuscular mycorrhizal fungal biomass (n = 3) in the hyphal compartment (a–c) and in the root compartment
(d–i). (a–f) Neutral lipid fatty acids 16:1ω5 and 16:1ω7c; (g–i) phospholipid fatty acid 16:1ω5. Open symbols, low pH treatment; closed symbols,
high pH treatment. Values are mean ± SE; where no error bars can be seen, the SE is smaller than the symbol. The pH treatments were compared
using two-way ANOVA (pH × time) for 20–80 d.
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visual estimates of phosphatase activity of extraradical
mycelium were subjected to Paired sign and Kruskal–
Wallis tests, using STATVIEW 5.0.1 (SAS Institute Inc., Cary,
NC, USA). The time factor of the ANOVA is not reported
in most cases since its significance is obvious already from
the graphs.
Results
Substrate pH
The pH of the hyphal compartment was significantly
different between the pH treatments for all, except for the
nonmycorrhizal plants at 53 d (Table 1). The pH of the root
compartment showed only occasional significant differences
between the pH treatments and, at the last harvest, no
differences were found between the pH treatments. In none
of the treatments could a difference in pH (two-tailed t-test
with unequal variances) between the first and the last harvest
be detected for the hyphal compartment, whereas the pH
of the root compartment was significantly lower (two-tailed
t-test with unequal variances) at the last harvest from both
pH treatments. The experimental system thus seems to have
worked according to our intentions.
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Growth of the extraradical mycelium
The AM fungal signature neutral lipid fatty acid 16:1ω5 was
detected in both the hyphal and the root compartment for
both the S. calospora and G. intraradices treatments, but not
the nonmycorrhizal samples (Fig. 1a–f ). Neutral lipid fatty
acid 16:1ω7 was detected in the hyphal compartment only
for the G. intraradices treatment. These neutral lipid fatty
acids increased significantly over time and in the hyphal
compartment a significantly greater increase was observed at
the higher pH than at the lower pH. The amounts of neutral
lipid fatty acids were higher in the root compartment than
in the hyphal compartment, and in the root compartment
significantly more of the neutral lipid fatty acids were found
with G. intraradices treatment than with S. calospora or
nonmycorrhizal treatments. The nonmycorrhizal treatments
did not show any changes in neutral lipid fatty acids over time.
No mycelium was found by wet sieving at the first two
harvests, while at the third harvest (53 d), mycelium could be
collected from the sand of the hyphal compartment from both
the S. calospora and G. intraradices treatments (except for the low
pH treatment of G. intraradices). There was large variation in
the amount of extraradical mycelium between replicates, but
generally the amount of extraradical mycelium increased over
time. No mycelium was recovered from the nonmycorrhizal
treatment. The pH treatment did not influence the amount of
extraradical mycelium formed in the root compartment while
it did affect the amount in the hyphal compartment.
Fig. 2 Total (a,b), arbuscular (c,d) and
vesicular (e,f) colonization (n = 3) by
Scutellospora calospora (a,c) and Glomus
intraradices (b,d,f). No colonization of the
roots was seen in the nonmycorrhizal
controls. Open symbols, low pH treatment;
closed symbols, high pH treatment. Values
are mean ± SE; where no error bars can be
seen, the SE is smaller than the symbol. The
pH treatments were compared using two-
way ANOVA (pH × time) for 53–80 d, because
in these cases mycelium had grown out into
the hyphal compartment.
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Research 177
The content of the AM fungal signature phospholipid fatty
acid (16:1ω5) in the root compartment (Fig. 1g–i) was
roughly similar for S. calospora and G. intraradices treatments
and slightly, but significantly, higher than in the nonmycor-
rhizal treatment. The background level of phospholipid fatty
acid 16:1ω5 in the nonmycorrhizal treatment can be attrib-
uted to bacteria (Olsson, 1999). With S. calospora, the low
pH-treated system exhibited a significantly higher content of
phospholipid fatty acid 16:1ω5 than the high pH treatment.
In the hyphal compartment the phospholipid fatty acid
16:1ω5 could not be detected. Other possible AM fungal sig-
nature fatty acids (18:1ω7c, 20:4 and 20:5) were not higher
in S. calospora and G. intraradices treatments than in nonmy-
corrhizal treatment at the first four harvests. These were there-
fore not used as indicators of extraradical mycelium growth in
this study.
Arbuscular mycorrhizal fungal root colonization
To examine the influence of pH on mycorrhizal colonization
only the data from the last three harvests, where mycelium was
present in the hyphal compartment, were included in the
two-way ANOVA. Total root colonization by S. calospora was
significantly higher in the low pH treatments from 53 to 80 d
(Fig. 2a). Root colonization by S. calospora at 80 d was low
but still increasing, and at the last harvest an average of 23%
and 18% of the total root length had been colonized for
low- and high pH treatment, respectively. The highest root

178 Research
Table 2 Phosphatase activity of the extraradical mycelium of two
arbuscular mycorrhizal fungi, Scutellospora calospora and Glomus
intraradices
S. calospora G. intraradices
Low High Low
(n = 9) (n = 8)
ALP 2.8 2.1 nd
(2–3) (1–3)
ACP 3.3 3.1 nd
(3– 4) (1– 4)
Alkaline (ALP) and acid (ACP) phosphatase activities, from 53 to
80 d, were estimated after staining with the enzyme-labelled
fluorescence substrate; 0 = no activity to 5 = high activity. Average
values are given with the minimum and maximum values in
parentheses. Low, low pH treatment; High, high pH treatment;
nd, not determined since no hyphae were found in the hyphal
compartment.
colonization by G. intraradices was at 67 d, there being 84%
and 79%, respectively, for low pH and high pH treatment
(Fig. 2b). Total colonization was significantly higher for the
low pH treatment between 53 d and 80 d. Roots colonized by
G. intraradices were also colonized by a fine endophyte
(Thippayarugs et al., 1999). The fine endophyte colonization,
estimated independently from the colonization by G.
intraradices, appeared first after 20 d, and increased after that
linearly over time. The increase was similar for both pH treat-
ments, that is 58% and 44% of the root length for low (R 2 =
0.94) and high (R 2 = 0.84) pH treatment at 80 d, respectively.
Neither the roots colonized by S. calospora nor nonmycorrhizal
roots showed any colonization by fine endophytes.
Arbuscular colonization by S. calospora occurred in 9% of
the total root length for both of the pH treatments at 80 d
(Fig. 2c). This fungus does not form any vesicles. Glomus
intraradices showed significantly higher root lengths with
arbuscules and vesicles for the low pH treatment (Fig. 2d,f ).
Root length with arbuscules and vesicles reached the maxi-
mum estimated value at 53 d. The maximum root length
showing arbuscular formation by G. intraradices was 40% of
total root length for both pH treatments and for vesicles the
values were 20% and 16% for the low pH and high pH treat-
ments, respectively.
Phosphatase activities
The phosphatase activities of the extraradical mycelium, as
visualized by the ELF substrate, were, except for a few
extremes, constant for all samples within each treatment. In
Table 2 the data from each fungus at each pH treatment were
averaged and the minimum and maximum are also given.
Both ACP (P < 0.001) and ALP (P = 0.15) activities were
higher for the extraradical mycelium of S. calospora than G.
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intraradices. The S. calospora extraradical mycelium seemed to
have higher phosphatase activity at low pH than at high pH
and the ACP activity was higher than the ALP activity
(P = 0.016). This contrasts with G. intraradices where ALP
seemed to have the higher activity (P = 0.22). For both pH
High treatments the phosphatase activity of the intraradical
(n = 9) mycelium of both species (n = 1), as determined by staining
with alkaline ELF substrate solution, increased over time and
1.7
(0 – 4) reached a maximum at 67 d. Overall, the roots colonized by
G. intraradices showed more phosphatase active intraradical
0.8 mycelium than roots colonized by S. calospora, and maximum
(0 –1)
values estimated were 2 and 4 (according to a relative scale)
respectively.
In general, the phosphatase activities of the substrates in the
hyphal and root compartment were for both fungi similar to
those of the nonmycorrhizal samples (results not shown).
Phosphatase activities were occasionally found in the hyphal
compartment, and the highest values detected were 2.2 U g−1
for ALP, and 1.4 U g−1 for ACP. In the root compartment ACP
activity was normally higher than in the hyphal compartment,
where the highest ACP activity detected was 12.3 U g−1,
while the highest ALP activity was only 6.1 U g−1.
Growth of plants, bacteria and saprotrophic fungi
Neither the plants colonized by S. calospora or G. intraradices,
nor the nonmycorrhizal plants showed any significant
difference in total plant or shoot dry weight between the high
pH and the low pH treatment (Table 3). The S. calospora-
colonized plants had a significantly higher root dry weight at
the higher pH, but Glomus intraradices-colonized roots were
significantly heavier only at the last harvest in the high pH
treatment. Shoot, root and total plant dry weight of G.
intraradices-colonized plants were significantly lower than
those of plants colonized by S. calospora or nonmycorrhizal
plants (three-way ANOVA).
The bacterial biomass, as indicated by bacteria-specific
phospholipid fatty acids, increased significantly over time for
all fungal treatments in the hyphal compartment (two-way
ANOVA, Fig. 3). Both G. intraradices and S. calospora seemed to
depress bacterial colonization of the hyphal compartment
at higher pH (Fig. 3). The pH treatment had no effect on
bacterial biomass in the non-mycorrhizal treatment (Fig. 3,
Table 4). For the root compartment only the S. calospora
treatment showed a difference between the pH treatments:
amounts of bacteria-specific phospholipid fatty acids were
greater at the lower pH (two-way ANOVA, Table 4). In the root
compartment a significantly higher bacterial biomass was
found for S. calospora- than for G. intraradices-treated pots.
The bacterial biomass of the S. calospora treatment signifi-
cantly increased in the root compartment over time. Both S.
calospora and G. intraradices treatments showed significantly
higher amounts of bacteria-specific phospholipid fatty acids
in the root compartment than the nonmycorrhizal treatment
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Research 179

Table 3 The average shoot and root dry

weight of Plantago lanceolata, measured at Plant dry wt (g; n = 3)
the different harvesting times
Scutellospora Glomus
Nonmycorrhizal calospora intraradices

Time (d) Low High Low High Low High

Shoot 20 0.052 0.072 0.061 0.054 0.054 0.047

32 0.185 0.190 0.154 0.162 0.127 0.118
53 0.394 0.420 0.377 0.355 0.305 0.251
67 0.820 0.869 0.778 0.781 0.705 0.718
80 1.082 1.341 1.278 1.390 1.256 1.319

P-value pH 0.094 0.56 0.94

Time < 0.001 < 0.001 < 0.001
pH × time 0.22 0.69 0.37

Root 20 0.018 0.033 0.023 0.027 0.035 0.031

32 0.075 0.062 0.066 0.093 0.077 0.055
53 0.333 0.207 0.239 0.229 0.201 0.161
67 0.428 0.412 0.384 0.443 0.307 0.291
80 0.874 0.763 0.652 0.900 0.505 0.691

P-value pH 0.089 0.022 0.16

Time < 0.001 < 0.001 < 0.001
pH × time 0.42 0.034 < 0.001

Low, low pH treatment; High, high pH treatment.

Fig. 3 Bacterial phospholipid fatty acid content (n = 3) in the hyphal compartment of the nonmycorrhizal (a), Scutellospora calospora (b) and
Glomus intraradices (c) treatments. Open symbols, low pH treatment; closed symbols, high pH treatment. Values are mean ± SE; where no error
bars can be seen, the SE is smaller than the symbol. The pH treatments were compared using two-way ANOVA (pH × time) for 20 – 80 d.

(three-way ANOVA), and a considerable increase was observed
in the last harvest.
Low amounts of saprotrophic fungal biomass, as indicated
by neutral lipid and phospholipid fatty acid 18:2ω6,9, were
detected in the hyphal compartment. Saprotrophic fungi were
only occasionally detected in the hyphal compartment by
microscopic observations. In the root compartment the bio-
mass of saprotrophic fungi increased significantly over time
for all fungal treatments (see Table 4 for values at 80 d). For
S. calospora, it was initially relatively constant but at the final
harvest a marked increase was observed.
Discussion
This study demonstrated an effect of pH on AM fungal
hyphae even though the roots of the host plant were not
exposed to the same pH. In earlier studies where the effect of
pH on AM hyphal development, root colonization and plant
growth has been examined, both symbionts were subjected to
the same pH (Abbott & Robson, 1985; Clark & Zeto,
1996a). By using mesh bags we created a hyphal
compartment that excluded the host roots. Here, we have
shown that the pH of a substrate colonized by the AM fungal
mycelium influences not only the growth of the mycelium but
also the AM colonization of the host plant roots.
The increased extent of root length with vesicles and arbus-
cules in G. intraradices at the lower pH could indicate a stress
response, especially since no hyphae were found in the hyphal
compartment (Figs 1a–c and 2d,f ). Siqueira et al. (1984)
found no correlation between total root colonization and pH
of the soil, and they suggested that the effect of the pH might
be greater on the fungus than on the host plant. Other studies

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180 Research

Table 4 Fatty acids indicating bacterial and saprotrophic fungi (SE in parentheses) at 80 d in hyphal and root compartments

Fatty acids (nmol g−1 dry wt)

Saprotrophic fungi
Bacterial Phospholipid Neutral lipid Phospholipid

Compartment Low High Low High Low High

Hyphal Non-mycorrhizal 0.29 0.23 0.05 0.06 0.09 0.04

(0.11) (0.01) (0.00) (0.00) (0.04) (0.00)
Scutellospora calospora 0.37 0.25 0.11 0.06 –a 0.01
(0.01) (0.01) (0.05) (0.00) (0.01)
Glomus intraradices 0.30 0.24 0.07 0.09 – 0.04
(0.01) (0.01) (0.01) (0.00) (0.00)
Root Non-mycorrhizal 8.68 8.99 1.01 1.26 2.28 3.25
(0.69) (0.42) (0.20) (0.12) (0.56) (0.55)
S. calospora 19.67 16.01 1.66 1.39 5.25 4.49
(1.21) (0.18) (0.53) (0.27) (1.05) (1.19)
G. intraradices 13.99 12.15 1.56 1.49 3.23 3.14
(2.98) (1.86) (0.20) (0.29) (0.87) (0.67)

Low, low pH treatment; High, high pH treatment. aNo saprotrophic fungal fatty acid could be detected.

(Medeiros et al., 1994; Clark & Zeto, 1996a) indicate that
AM fungal colonization can be strongly affected by pH per se,
although the effects on different fungal isolates differed. In
this study the amount of extraradical mycelium in the hyphal
compartment did not appear to be related to the fungal colo-
nization of the roots. However, a large amount of AM fungal
neutral lipid fatty acids in the root compartment for G. intra-
radices was correlated with high AM fungal colonization, and
for S. calospora both neutral lipid fatty acid content in the root
compartment and colonization were low (Figs 1e,f and 2).
The pH of the root compartment declined strongly during
the experimental period, while only a small effect of the pH
treatment on soil pH was detectable (Table 1). The decreased
soil pH at the low pH treatment probably contributed to
a higher root colonization and AM fungal biomass of S.
calospora in the root compartment (Figs 1h and 2a). This may
look like a contradiction to the result in the hyphal compart-
ment (Fig. 1b), but the actual pH value was close to 6 both in
the root compartment at the low pH treatment and in the
hyphal compartment at the high pH treatment (Table 1).
This indicates that S. calospora has a pH optimum of c. 6. The
pH treatment influenced root growth only in AM colonized
plants, indicating that this effect was mediated by the AM
fungal responses to pH.
The lower pH of the growth substrate of the extraradical
mycelium inhibited mycelial growth and possibly spore for-
mation in both species (Fig. 1b,c). This agrees with Abbott &
Robson (1985), who showed that the spread of extraradical
mycelium by a Glomus isolate was strongly inhibited at low
soil pH. It is likely that low growth of extraradical mycelium
at lower pH was caused by aversion to the substrate. Acidic
soils are known to have a fungistatic effect on spores of Glomus
mosseae (Siqueira et al., 1984). The hyphae of S. calospora in
our experiment did not appear to be severely stressed at this
lower pH, since their ALP activity was not reduced (Table 2).
In other studies it was shown that stress caused by simulated
acid rain (pH 3–4) reduced the ALP activity of AM fungal
extraradical mycelium in a range of substrates (Vosatka &
Dodd, 1998; Malcová et al., 1999). Local responses of AM
fungal extraradical mycelium have been found, mainly as
proliferation in patches rich in organic material (Mosse, 1959;
St John et al., 1983; Ravnskov et al., 1999). This stresses the
importance of using experimental systems that allow separa-
tion of extraradical mycelium and roots when studying effects
on extraradical mycelium, since in nature mycorrhizal hyphae
can reach patches not available to plant roots.
In the present study, the pH of the hyphal compartment
was buffered with a MES buffer. MES has been shown to
stimulate extraradical mycelium growth, but only signifi-
cantly so at concentrations 100 times higher than in our study
(Vilariño et al., 1997). In our study, similar amounts of MES
were added for all treatments and so it is unlikely to be the
cause of differences in extraradical mycelium biomass. The
addition of MES to the hyphal compartment, containing
acid-washed sand largely deprived of nutrients, should not
influence the nutrient uptake. It has been observed to affect
nutrient uptake in plants (Medeiros et al., 1993), although at
a concentration four times higher than here, and plants were
grown in a nutrient solution. Since the decrease in pH of the
root compartment over time was greater than the difference in
the pH levels employed (Table 1), we conclude that the acid-
ifying capacity of the roots had a stronger influence on the pH
of the root compartment than the effect of diffusion of MES
from the hyphal compartment to the root compartment.
Our results show that the ELF substrate can be used both
to detect and estimate the extent of extraradical mycelium

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with active phosphatases (Table 2). Most studies on phos-
phatases of AM fungal extraradical mycelium have focused on
ALP activity. Our results indicate that ACP activity of the
extraradical mycelium contributed significantly to total phos-
phatase activity, in agreement with recent findings by Joner
& Johansen (2000). Thus, ACP activity should also be con-
sidered in phosphatase studies, especially since recent obser-
vations have shown the vacuoles of AM fungi to be acidic
(Ezawa et al., 2001). Our results (Table 2) indicate that phos-
phatase activity of the extraradical mycelium can be highest
at a substrate pH quite different from the pH for optimal
enzyme activity. In most cases no significant phosphatase
activity could be detected in the substrate of the hyphal com-
partment. However, where phosphatase activity was detected
in the substrate, the values were of the same order as those
observed by Joner & Jakobsen (1995) and Joner et al. (1995).
In the root compartment, the observed ACP activity in the
substrate probably did not originate from the AM fungi, since
similar values were found for all fungal treatments. This
supports the view of Joner et al. (2000) that AM fungal phos-
phatases are not actively released into the soil.
Interaction studies between AM fungi and saprotrophic
microorganisms have often reported conflicting results (Ames
et al., 1984; Posta et al., 1994; Olsson et al., 1996). As shown
in Fig. 3, temporal differences may be one factor explaining
such discrepancies. In an earlier study, saprotrophic fungi,
detected by phospholipid fatty acid 18:2ω6,9, were inhibited
by AM fungal extraradical mycelium in root-free dune sand
(Olsson et al., 1998). This could not be confirmed here since
the biomass of saprotrophic fungi was too variable (Table 4).
The two fungi showed differences in their ability to colo-
nize the roots and to produce extraradical mycelium biomass
(Figs 1 and 2, respectively). These differences persisted
throughout the experiment and can not be explained only by
a difference in infective propagule density of the inoculum.
Since AM fungal signature neutral lipid fatty acids have been
shown to correlate with the number of spores formed by the
extraradical mycelium (Olsson et al., 1997), the high neutral
lipid fatty acid values indicate that sporulation was higher in
G. intraradices. High amounts of AM fungal neutral lipid fatty
acids in the extraradical mycelium indicate that high amounts
of neutral lipids, mainly triacylglycerides, have been trans-
ported from the intraradical mycelium (Bago et al., 2000).
This carbon cost to the plants may have caused the lower
weight of G. intraradices-colonized plants compared with
S. calospora-colonized and nonmycorrhizal plants. The differ-
ences in responses of the AM fungal isolates support previous
reports showing the importance of studying more than one
fungal isolate (van der Heijden et al., 1998a,b; Smith et al.,
2000; Ezawa et al., 2001).
In conclusion, the results show that the growth of AM fun-
gal mycelia is directly influenced by the pH of the substrate.
This is further evidence that AM fungi are organisms that
respond to a heterogeneous environment. We have also shown
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Research 181
that the way in which AM fungi species respond to pH may
vary between species. This study also reveals a possible
connection between the response of the extraradical mycelium
and the colonization strategy within the host root. Finally, the
results support the view that the external phosphatases of AM
fungi are of little importance.
Acknowledgements
The financial support of the Swedish Council for Forestry and
Agricultural Research is gratefully acknowledged. We thank
Professor Anne Ashford for critically reading the manuscript.
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