Research
Temperature constraints on the growth and functioning of
root organ cultures with arbuscular mycorrhizal fungi
Blackwell Publishing, Ltd.
Mayra E. Gavito1,4, Pål A. Olsson2, Hervé Rouhier2, Almudena Medina-Peñafiel1, Iver Jakobsen3, Albert Bago1 and
Concepción Azcón-Aguilar1
1
Departamento de Microbiología del Suelo y Sistemas Simbióticos, Estación Experimental del Zaidín, CSIC, C/Profesor Albareda 1, E-18008 Granada, Spain;
2
Department of Microbial Ecology, Ecology Building, Lund University, S-223 62, Lund, Sweden; 3Biosystems Department, Risø National Laboratory, PO Box
49, DK-4000 Roskilde, Denmark. 4Present address: Centro de Investigaciones en Ecosistemas, Universidad Nacional Autónoma de México, Apartado Postal
27–3 C. P. 58090, Morelia, Michoacán, Mexico
Summary
Author for correspondence:
Mayra E. Gavito
Tel: +52 56 232777 ext. 32822
Fax: +52 56 232719
E-mail: [email protected]
Received: 7 February 2005
Accepted: 13 April 2005
• In this study we investigated the effects of temperature on fungal growth and
tested whether the differences in fungal growth were related to the effects of
temperature on carbon movement to, or within, the fungus.
• Growth curves and C uptake-transfer-translocation measurements were obtained
for three arbuscular mycorrhizal fungi (AMF) isolates cultured within a 6 –30°C
temperature range. A series of experiments with a model fungal isolate, Glomus
intraradices, was used to examine the effects of temperature on lipid body and 33P
movement, and to investigate the role of acclimation and incubation time.
• Temperature effects on AMF growth were both direct and indirect because,
despite clear independent root and AMF growth responses in some cases, the
uptake and translocation of 13C was also affected within the temperature range
tested. Root C uptake and, to a lesser extent, C translocation in the fungus, were
reduced by low temperatures (< 18°C). Uptake and translocation of 33P by fungal
hyphae were, by contrast, similar between 10 and 25°C.
• We conclude that temperature, between 6 and 18°C, reduces AMF growth, and
that C movement to the fungus is involved in this response.
Key words: arbuscular mycorrhizal fungi (AMF), carbon, growth, mycorrhiza,
phosphorus, temperature, translocation, uptake.
New Phytologist (2005) 168: 179–188
© New Phytologist (2005) doi: 10.1111/j.1469-8137.2005.01481.x
Introduction
The impact of human activity on global climate change has
recently driven our attention to measure and to understand
carbon (C) fluxes between soil and the atmosphere. Ubiquitous
arbuscular mycorrhizal (AM) symbioses, established between
roots of more than 80% of land plants (Smith & Read, 1997)
and biotrophic Glomeromycota fungi, also called arbuscular
mycorrhizal fungi (AMF) (Schüßler et al., 2001), may play an
important role in the movement of C from the atmosphere to
the soil via their host plants. AMF are obligate symbionts that
depend on the host plant C assimilates for life cycle completion.
AMF not only live within the root, but also form an extensive
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hyphal network in the surrounding soil that may rapidly
recycle C released from respiration and the turnover of hyphae
(Staddon et al., 2003a). However, despite their important role
in global C fluxes, and in plant performance and survival in
adverse conditions, it is not possible at present to predict the
response of AMF to a changing climate. Although temperature
is the component of climate change that may have the strongest
direct impact on these fungi, plant performance and the C cycle,
its influence on AMF growth is poorly understood (Fitter
et al., 2000).
Temperature affects the growth of all organisms because
it controls the rates of metabolic reactions. The temperature
range at which most organisms function is between 0 and
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40°C, the lower limit being associated with the transition
phase at freezing and the upper limit being associated with the
increasing influence of catabolism (Gillooly et al., 2001).
Results from the few studies on temperature effects on AMF
showed that mycorrhizal colonization increased in most
studies when temperature increased between 5 and 37°C
(Staddon et al., 2002). Reports indicating adaptation of AMF
isolates to temperatures below 15°C (Hayman, 1974; Daft et al.,
1980) or above 25°C (Schenck & Schröder, 1974; Bowen,
1987; Grey, 1991) are available. Other studies suggest, however,
that AMF growth is limited at low (< 15°C) temperatures
(Daniels-Hetrick & Bloom, 1984; Baon et al., 1994). The
effect of high temperatures (> 35°C) has been studied in much
less detail (Staddon et al., 2002). Unfortunately, none of those
studies followed the growth of both the intraradical and
extraradical phases of the mycorrhizal mycelium.
The extraradical AM mycelium of certain isolates is resistant
to low temperatures and can reinitiate growth after months of
freezing, once it has been formed and established in soil (Addy
et al., 1998). Recent studies demonstrated, however, marked
growth reductions of Scandinavian AMF isolates and native
communities when the fungi had to establish new colonization at temperatures of < 20°C (Gavito et al., 2000, 2003). It
is usually assumed that the intraradical and the extraradical
mycelium develop proportionally, but those studies showed
the extraradical mycelium to be more sensitive to low temperatures than the intraradical mycelium. The former was generally
unable to develop below 15°C, whereas the latter developed
extensively at 5°C. This differential response may have strong
implications for our understanding of the AM symbiosis and
deserves further study.
We explored temperature constraints on the growth
and functioning of the AM symbiosis in the absence of the
confounding effects of shoot growth and photosynthesis. We
conducted a series of studies in monoxenic dual-root organ
cultures with AMF in which roots took up C provided as a fixed
amount of sucrose in the growth medium. AMF are not able
to use this source of C and thus depend on the C provided by
roots in the established symbiosis (Bago et al., 2000). These
experimental systems have been proven useful in physiological
studies of C metabolism because it is otherwise impossible to
untangle the effects of the treatment from the effects of other
microorganisms, soil properties or from host-mediated effects,
on the AMF (Pfeffer et al., 1999). Monoxenic cultures can be
maintained under homogeneous environmental conditions,
providing them with the same amounts and sources of C and
mineral nutrients. They provide the simplest system and the
closest to a pure culture, in which we can study the inherent
capabilities or limitations of AMF isolates in response to
different temperatures.
Growth curves were obtained for three AMF isolates, within
a 6–30°C temperature range, to test if responses to temperature
were similar to those observed in nonsterile pot systems with
whole plants and to establish whether the effects of temperature
New Phytologist (2005) 168: 179–188
on AMF were independent of those on roots. We used 13C
uptake-transfer-translocation curves vs temperature from the
three AMF isolates, and a series of experiments with a model
fungal isolate (Glomus intraradices), to test the hypothesis that
the sensitivity of the extraradical mycelium to low temperatures,
and the difference between intraradical and extraradical development, were a result of reduced uptake or translocation of C to
the extraradical mycelium.
Materials and Methods
Experiment 1. Temperature and root and fungal development.
Three AMF isolates from established monoxenic Ri T-DNAtransformed carrot (Daucus carota L.) root organ cultures (Bécard
& Fortin, 1988) were selected according to origin and culturing
properties. Two fungi were originally isolated from Quebec,
Canada (G. intraradices Schenck & Smith, GINCO/MUCL
43194 and G. cerebriforme McGee, GINCO/MUCL 43208)
and the third (G. proliferum Dalpé & Declerck, GINCO/
MUCL 41827) was isolated from the island of Guadeloupe,
French West Indies, in the Caribbean. All isolates were cultured
subsequently at 24°C, a favourable growth temperature for both
roots and fungi. G. intraradices was obtained with the carrot
root clone line, DC1, and G. cerebriforme and G. proliferum
with line DC2. Cultures were propagated in Petri dishes with
25 ml of minimal (M) nutrient medium containing 0.35%
(w/v) phytagel and 10 g l−1 of sucrose, pH 5.5 (Bécard & Fortin,
1988). A 1.5-cm diameter plug containing roots, spores and
mycelium was transferred to a 1.5-cm diameter hole in the
centre of the plates and maintained at 24°C until abundant
mycelium and spores (3–4 months) were formed. A 1.5-cm
diameter plug from these propagation plates was then transferred to new plates containing the same M medium. Plates
were prepared in excess to provide sufficient plates with homogeneous growth for the experiment. All plates were incubated
at 24°C for 2 wk to allow growth recovery in the inoculum
plug and then transferred to refrigerators or incubators set at
6°, 12°, 18°, 24°, or 30°C. The actual temperatures used in
these treatments were (mean ± standard error, in °C): 6 ± 0.9,
12 ± 0.5, 18 ± 1.4, 24 ± 0.6, and 30 ± 0.9. The plates were
kept for 10 wk (G. intraradices and G. proliferum) or 12 wk
(G. cerebriforme) until the mycelium had filled the plates and
reached the reproductive phase with abundant spore formation. G. cerebriforme cultures were more heterogeneous than
cultures of the other two isolates and therefore incubated
for a further 2 wk. Four to six replicates with the most homogeneous growth were selected for harvest. Prior to harvesting,
root length and hyphal length, whenever possible, were determined by using the gridline intersection method (Tennant,
1975) by turning the plates upside down and recording under
the stereomicroscope. The external mycelium of G. intraradices
forms long hyphae with low branching, which allowed us to
perform hyphal measurements in these cultures at all temperatures tested. The percentage of intersections with branched
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Research
absorbing structures (BAS) (Bago et al., 1998) or spores was also
determined. The mycelium of G. proliferum and G. cerebriforme
formed short, highly branched hyphae with excessive overlapping in different depth planes. This caused difficulty in taking
measurements and, as a result, measurements were taken
from G. proliferum and G. cerebriforme cultured only at the
two lowest temperatures; estimated measurements were made
for cultures grown at the other temperatures. Roots were then
extracted with forceps, shaken in 10-mM sodium citrate to
dissolve the attached phytagel and rinsed with water. Roots
were stained, as described in Phillips & Hayman (1980), to
determine the percentage of mycorrhizal colonization according to the magnified intersections method (McGonigle et al.,
1990). Mycelium was extracted by chopping the solid medium
and shaking for 30 min in 10 mM sodium citrate until dissolved.
Mycelium was collected in a 25-µm mesh sieve, washed with
water, freeze-dried, and weighed.
Experiment 2. 13C uptake, transfer and translocation. Twocompartment Petri dishes, inoculated separately with one of
each of the three AMF isolates, were prepared by filling M
medium with sucrose in one compartment (the root compartment) and M medium without sucrose in the other compartment
(the hyphal compartment). A cellophane membrane, which
allows the diffusion of water and nutrients, was placed on
top of the medium on the root compartment of the plate, to
reduce root and hyphal growth into the medium and to speed
up hyphal growth to the hyphal compartment. A 1.5-cm
diameter inoculum plug from a previously established culture
was transferred to the sucrose-amended compartment and
placed on top of the membrane. The membrane successfully
reduced the time required for hyphae to grow into the hyphal
compartment, but it still allowed many hyphae to grow into
the medium at the edge of the plates and in the vicinity of the
roots in the root compartment. These hyphae were measured
to account for short-distance C movement from proximal
hyphae (PH) in the root compartment and large-distance
movement from distant hyphae (DH) in the hyphal compartment. Plates were incubated at 24°C until the hyphal compartment of most was filled with hyphae (2–3 months). Most
cultures were in the absorptive phase with a predominance
of BAS in the extraradical mycelium. Roots crossing the plate
division were periodically removed from the hyphal compartment if they were few and easily removable; otherwise, they
were left in order to maintain the integrity of the mycelium.
Plates with the most homogeneous growth were selected,
and four to six replicates were transferred to an incubator or
refrigerator to acclimate for 48 h at their final temperature
before labeling.
Cultures acclimated at 6, 12, 18 and 24°C, received 0.5 ml
of a filtered-sterilized solution containing 5 mg of [13C]
D -glucose [U- 13C 1 , 99% (w/w) 13 C; Campro Scientific,
Veenendaal, the Netherlands] by pipetting over the root compartment, as described in Olsson et al. (2002), and harvested
© New Phytologist (2005) www.newphytologist.org
after 7 d. Roots were picked with forceps, and mycelium
was collected after dissolving the medium as described in
Experiment 1. Roots, and PH and DH samples, were washed
with water, blotted, freeze-dried and stored at −20°C until
packing for 13C-enrichment analysis.
Approximately 20 µg of mycelium and 100 µg of ground
roots were packed in tin capsules and analysed for 13C
enrichment by using an isotope ratio mass spectrometer
(20–20 Stable Isotope Analyser; PDZ Europa Scientific
Instruments, Crewe, UK) interfaced to a combustion module
(ANCA-NT). The 13C enrichment in roots and mycelium
was calculated by subtracting the natural abundance values
obtained from cultures without label (1.17%) from the excess
atom per cent 13C measured in the labeled cultures.
Experiments with G. intraradices-DC1
Experiment 3. Visual measurements of lipid body fluxes in
extraradical mycelium. Two-compartment Petri dishes were
prepared, as explained in Experiment 2, but a cellophane
membrane on top of the medium covered the entire surface of
the plate, preventing most roots and hyphae from growing
into the medium. The inoculum plug was placed on top of the
membrane in the medium + sucrose compartment. When the
distant mycelium crossed the division and began to spread
over the hyphal compartment (approx. 5 wk after inoculation), the plates were ready for observations. The plates were
acclimatized at either 12 or 24°C for 48 h. Five hours before
microscopical observations, the plates received 1 ml of Nile
red sterile solution to reach a concentration of 0.05 µg ml−1
(Bago et al., 2002). Immediately prior to observation, onethird to one-half of the cellophane membrane in the hyphal
compartment was cut with a scalpel and mounted on a large
slide with 10 mM MES buffer, pH 5.5, and a cover slip. Slides
were maintained at the appropriate temperature prior to a 10min image recording and then returned to their temperature
treatment. In all cases, a cellophane section was never observed
for longer than 30 min because previous tests showed that
cytoplasmic streaming in hyphae, and fluorescence, decreases
markedly after this time. Stained lipid bodies were observed
by using a Leica TCS/NT confocal laser-scanning microscope (Leica Microsystems CMS GmbH, Mannheim,
Germany). Observations were performed with an Ar/Kr laser
(476-nm excitation wavelength) for illumination and a ×63
water-immersion lens (numerical aperture 1.20, free working
distance 220 µm). Confocal images (resolution 512 × 512
pixels) were captured at red wavelength through a long-pass
filter, LP 515. Time series were recorded with 40 single-scan
frames at 1.629-s intervals and saved as video clips. As many
time series as possible were taken from active hyphae for each
section during the 30-min observation period. The number
of time series recorded per plate varied, but a minimum of
20 was measured in each treatment. Measurements were
made on hyphae with cytoplasmic streaming by using the
New Phytologist (2005) 168: 179–188
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quantification features of the Leica Confocal software.
Single lipid bodies were identified in the recorded images and
we measured the distance they moved per unit time, their
diameter, and the hyphal diameter. Because the speed at
which lipid bodies move in cultures is highly variable per se,
the maximum (instead of the average) value was identified from
50 to 150 lipid bodies selected from each section to reduce
the heterogeneity and to increase our chances of detecting a
significant reduction in movement. Maximum values were
averaged across sections to estimate the effect of temperature
on the movement of the lipid bodies in each plate.
Experiment 4. Longer-term 13C movement. Cultures were
prepared as explained in Experiment 2 and the plates were
acclimatized and labeled, in the same way, at either 12°C or
24°C. There were at least 12 replicate cultures at each temperature in this experiment and four to six replicates were
harvested 7, 14 and 21 d after labeling to examine 13C
enrichment. Harvesting and sample processing was as
described for Experiment 2.
Experiment 5. 13C movement after temperature switching.
Cultures of G. intraradices-DC1 in two-compartment plates
were prepared as explained above and later acclimatized at
either 12°C or 24°C for 48 h. In this experiment, there were
eight replicates in each temperature treatment. After labeling,
four were kept at the same temperature and four were placed at
the other temperature. There were thus four final temperature
treatments: 11–11°C, 11–24°C, 24–24°C, and 24–11°C,
because the actual mean temperature measured in the 12°C
incubator was 11.1 ± 0.8°C. Plates were harvested 7 d after
labeling and samples were processed, as described for Experiment 2.
Experiment 6. 33P movement from hyphae to roots.
Cultures of G. intraradices-DC1 in two-compartment plates
were prepared, as explained in Experiment 2, and acclimatized
at 10°, 18°, or 25°C for 48 h. The cultures were labeled by
pipetting 0.5 ml of a sterile solution containing 50 KBq of
carrier-free H333PO4 to the hyphal, but not to the root,
compartment. Plates were harvested after 5 d and samples were
extracted as in the previous experiments, oven-dried at 60°C,
weighed, and digested in 5 ml of HClO4:HNO3 (1 : 5; v/v).
Radioactivity was measured in a Packard TR1900 Liquid
Scintillation Analyzer (Packard Instrument Co., Meriden,
CT, USA).
Statistics
All data were analysed by using analysis of variance (ANOVA).
Experiments 1 and 2 had temperature, fungal isolate and
biological tissue as factors. Experiments 3–6, conducted only
with G. intraradices, had the following factors: Experiment 3,
temperature (single factor); Experiment 4, temperature, time,
New Phytologist (2005) 168: 179–188
and biological tissue; Experiment 5, temperature combination
and biological tissue; and Experiment 6, temperature and
biological tissue. All treatments had between four and six replicates.
Data were transformed, if required, to meet assumptions for
ANOVA. Trends in curves were further analysed with polynomial
contrasts to test for linear, quadratic or cubic effects describing
the direction of the effects and significant inflexion points
in the curves (Steel & Torrie, 1980). Analysis of covariance
(ANCOVA) was used to separate the effects of temperature
on root length from those on fungal development, using
root length as a covariate. The analyses were performed by
using STATISTIX7 analytical software (Analytical Software,
Tallahassee, FL, USA).
Results
Experiment 1. Temperature and root and AMF development.
The optimum temperature for root growth in G. intraradicesDC1 and G. proliferum-DC2 cultures was 18°C, and 24°C for
G. cerebriforme-DC2 (Fig. 1). In G. intraradices, temperature
significantly affected the total root length (ANOVA, P < 0.001)
and the colonized root length (ANOVA, P < 0.01). The total root
length increased at temperatures between 6 and 18°C and
decreased at temperatures between 18 and 30°C (quadratic
contrast P < 0.001, cubic P < 0.01), whereas the colonized
root length increased linearly at temperatures from 6 to 30°C
(linear, P < 0.001) (Fig. 1). The total root length in G. proliferumDC2 developed similarly to that of G. intraradices-DC1
(quadratic, P < 0.001), but the colonized root length increased
linearly (P < 0.001) up to 24°C and decreased markedly at
30°C (quadratic, P < 0.001). The DC2 root clone grew poorly
at 30°C and the roots exhibited thickening and a dark red
color, possibly in response to heat stress. Pigmentation was
not observed and the growth reduction was lower in the DC1
root clone. The total root length (P < 0.001) and colonized
root length (P < 0.001) increased linearly from 6 to 24°C in
the three temperature treatments tested for G. cerebriformeDC2. The highest temperature treatment (30°C) increased
the root length colonized by G. intraradices but decreased it in
G. proliferum cultures. This reduction was, however, probably
associated with the growth reduction and pigmentation of
roots of the DC2 clone.
The abundant root growth contrasted with the low fungal
development at 6°C and 12°C. Root colonization in all
isolates at 6°C and 12°C consisted mainly of hyphae with a
few arbuscules located sparsely in the root cortex. Vesicles, and
intraradical spores, in the case of G. intraradices, were seldom
observed at these temperatures. By contrast, at higher temperatures their number was only slightly smaller than that of
arbuscules (data not shown).
Temperature treatments affected the development of extraradical hyphae in cultures with all three AMF isolates (ANOVA,
P < 0.001 Fig. 2). The extraradical mycelium did not develop
beyond the inoculum plug at 6°C or 12°C in the case of
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Fig. 1 Total root length (RL) produced per plate [mean ± standard
error (SE), n = 4 –6] and root length that had been colonized by
the arbuscular mycorrhizal fungus, after 8 wk of treatment, at
different temperatures, in monoxenic cultures with (a) Glomus
intraradices-DC1, (b) G. proliferum-DC2, and after 10 wk in cultures
with (c) G. cerebriforme-DC2. Mean intraradical mycorrhizal
colonization rates (cm d−1) are shown at the top of each bar. Different
letters indicate significant differences among mycorrhizal
colonization rates (Tukey, P < 0.05). Black bar, colonized RL; white
bar, noncolonized RL.
G. intraradices, but did in G. proliferum and G. cerebriforme
(Fig. 2). Overall, the dry weight of extraradical hyphae increased
linearly, with temperature, up to 24°C for the three isolates
(P < 0.001). The mean extraradical hyphal growth rate data
showed that the three isolates had very different growth rates
at the same temperature (Fig. 2). For G. intraradices there was a
steady increase in extraradical mycelium mass up to 30°C, but
growth of G. proliferum showed three phases: little development at 6°C or 12°C; an increase between 12 and 24°C; and
a decrease between 24 and 30°C (cubic contrast P < 0.001).
The extraradical mycelium developed best at 30°C in
G. intraradices, whereas the optimal temperature for G. proliferum
and G. cerebriforme was 24°C. However, it was not possible to
determine the optimal temperatures for G. proliferum and
G. cerebriforme with the same certainty as for G. intraradices
owing to missing plates and to the negative effect of high
temperature on the root clone, DC2. Covariance analysis
© New Phytologist (2005) www.newphytologist.org
Fig. 2 Dry weight of extraradical hyphae produced per plate
[mean ± standard error (SE), n = 4 –6] by the arbuscular mycorrhizal
fungus after 8 wk of treatment, at different temperatures,
in monoxenic cultures with (a) Glomus intraradices-DC1,
(b) G. proliferum-DC2, and after 10 wk in cultures with
(c) G. cerebriforme-DC2. Mean hyphal growth rates (mg d−1) are
shown at the top of each bar. Different letters indicate significant
differences among hyphal growth rates (Tukey, P < 0.05).
showed that the effect of temperature on the development
of AMF isolates was independent of its effects on root length
(F-test, P = 0.38).
The extraradical mycelium in cultures with G. intraradices
allowed quantitative microscopical examination to be performed
at all temperatures, but the extraradical mycelium of G. proliferum
and G. cerebriforme was too highly branched to allow reliable
counting at temperatures of > 12°C (Table 1). The hyphal length
in G. intradices increased significantly (ANOVA, P < 0.001) and
linearly (P < 0.001) with temperature. There were no significant
differences in hyphal length at 6°C and 12°C in G. proliferum,
but there was a significant (ANOVA, P < 0.001) increase from 6 to
12°C in G. cerebriforme. The temperature treatments also produced a clear change in fungal morphology and phenology
(Table 1). In G. intraradices, the development of BAS was affected
by temperature (ANOVA, P < 0.001), being very low at 6°C and
12°C, peaking at 18°C and 24°C and dropping markedly at 30°C
(cubic contrast, P < 0.001). Spore formation increased mostly linearly with temperature (ANOVA, P < 0.001; linear, P < 0.001).
The two isolates growing at 6 and 12°C formed a few single
radial runner hyphae that crossed the plates from the inoculum
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Data are expressed as mean (± standard error) of total hyphal length, hyphal length with spores or with branched absorbing structures (BAS). The abundant and profuse branching of mycelium
in G. proliferum and G. cerebriforme cultures prevented quantitative measurements; a subjective visual evaluation was used for these two isolates. ND, not determined.
0.11 (0.14)
0.71 (0.99)
ND
Spores > BAS
ND
0.003 (0.003)
0.23 (0.23)
ND
Spores > BAS
ND
0.15 (0.09)
0.04 (0.06)
BAS > spores
BAS = spores
Spores > BAS
1.19 (1.04)
3.43 (5.73)
Countless
Countless
Countless
0.05 (0.04)
0.15 (0.08)
0.51 (0.07)
0.44 (0.26)
0 (0)
0.39 (0.37)
0.41 (0.48)
6.74 (2.47)
9.78 (4.60)
28.1 (11.6)
6°C
12°C
18°C
24°C
30°C
0.19 (0.17)
0.09 (0.04)
2.95 (2.09)
2.78 (1.82)
6.11 (1.94)
0.02 (0.03)
0.03 (0.06)
BAS > spores
BAS = spores
Spores > BAS
0.72 (0.31)
5.77 (2.42)
ND
Countless
ND
With spores
Total
With BAS
With spores
Total
With BAS
With spores
Total
Temperature
Glomus cerebriforme
Glomus proliferum
Glomus intraradices
Table 1 Influence of temperature treatments on growth and phenology of the external mycelium of arbuscular mycorrhizal fungi (AMF) in root organ cultures
With BAS
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Fig. 3 Experiment 1. 13C-enrichment in roots (R, squares), proximal
extraradical hyphae (PH, triangles), and distant extraradical hyphae
(DH, circles), as excess atomic per cent, after subtraction of the
natural abundance of 13C (1.171). Mean ± standard error (SE) (n = 4–
6) values were measured in monoxenic cultures with (a) Glomus
intraradices-DC1, (b) G. proliferum-DC2 and (c) G. cerebriformeDC2, 7 d after giving a 13C-sucrose pulse to the root compartment
and incubating at temperatures between 6°C and 24°C.
plug to the wall without forming a network, in contrast to that
observed when all isolates were growing at temperatures of
> 12°C. BAS and spore clusters were formed directly on the
runner hyphae at low temperatures, whereas at temperatures
of > 12°C, BAS and spores were formed mainly on fine,
highly branched hyphae. Spores and BAS were scarce at
temperatures of < 18°C and increased markedly at 24°C.
Experiment 2. 13C uptake-transfer-translocation studies.
Overall, 13C enrichment increased linearly (P < 0.001) with
temperature in cultures of the three AMF isolates (Fig. 3). 13C
enrichment in roots, PH and DH was generally similar and
indicated that C was transferred and translocated to PH and
DH in proportion to the amount that had been taken up.
G. intraradices was the only AMF in which 13C enrichment
increased significantly in DH at 18°C compared to PH.
G. intraradices-DC1 showed the highest 13C-enrichment
values of the three monoxenic AM cultures, at 18°C and at
24°C.
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Table 2 Confocal laser microscopy visual measurements of
translocation of fluorescent lipid bodies in the external mycelium of
Glomus intraradices at two temperatures
Maximum flux
Mean hyphal diameter
Mean volume of lipid
bodies in movement
12°C
24°C
3.94 (0.36) µ s−1
8.28 (0.49) µ
0.98 (0.03) µ3
3.43 (0.28) µ s−1
8.65 (0.86) µ
1.04 (0.07) µ3
Data are expressed as the mean (± standard error) of the maximum
speed of translocation of lipid bodies, the average diameter of hyphae
with movement of lipid bodies and the average volume of the lipid
bodies in plates maintained at 12 and at 24°C (n = 4 plates, at least
50 measurements averaged per plate).
Experiment 3. Visual measurements of lipid body fluxes in
the extraradical mycelium. There was no evidence that lipid
bodies moved more slowly at 12°C than at 24°C, that the
movement of lipid bodies occurred in different types of
hyphae, or that there was any difference in lipid body size
as a result of temperature treatment (Table 2). Visual
screening indicated, in general, that lipid body movement was
similar in G. intraradices-DC1 cultures at 12°C and at 24°C
because there were no significant differences between
temperature in any of the variables measured.
Experiment 4. Longer-term 13C movement. When we
examined whether 13C enrichment in roots would be different
with a longer translocation time, we found that 13C
enrichment in roots increased with time at both 12°C and
24°C (ANOVA, P < 0.05; Fig. 4) and linearly with incubation
time (P < 0.01). The increase in 13C enrichment in hyphae
from 12 to 24°C was, on the other hand, marginally
significant (ANOVA, P = 0.05) and the enrichment did not
change significantly with time at both temperatures. There
was a nonsignificant trend for an increase in 13C enrichment in
hyphae with increasing time at 24°C that was not observed at
12°C. The difference in root and hyphal response indicated that
C translocation from intraradical mycelium to distant external
hyphae did not continue after the first 7-d period, and this
seemed to be more pronounced at 12°C than at 24°C.
Experiment 5. 13C movement after temperature switching.
The effect of temperature combination treatment on 13C enrichment was significant (P < 0.01), but there were no significant
differences between root and fungal tissue and no factor
interactions (Fig. 5). Switching cultures from high to low and
from low to high temperatures revealed that temperature had
to be low before and after the pulse in order to yield a
significant decrease in 13C enrichment in root or fungal tissue
(Fig. 5). When the cultures treated at 24°C were restored after
the chilling period, C was taken up, transferred and translocated
as in the cultures that had not been cooled down. Transfer was
© New Phytologist (2005) www.newphytologist.org
Fig. 4 Experiment 2. 13C-enrichment in roots (R) and extraradical
hyphae (H), as excess atomic per cent, after subtraction of the natural
abundance of 13C (1.171). Mean ± standard error (SE) (n = 4 –5)
values were measured in monoxenic cultures with Glomus
intraradices-DC1, 7 d (white bar), 14 d (black bar) and 21 d (striped
bar) after giving a 13C-sucrose pulse to the root compartment and
incubating at 12°C or 24°C.
Fig. 5 Experiment 3. 13C-enrichment in roots and extraradical
hyphae, as excess atomic per cent, after subtraction of the natural
abundance of 13C (1.171). Mean ± standard error (SE) (n = 4) values
were measured in monoxenic cultures with Glomus intraradices-DC1
acclimated to 11 or 24°C after giving a 13C-sucrose pulse to the root
compartment and incubating at either 11 or 24°C for 7 d. Different
letters indicate significant differences among treatment combinations
in 13C-enrichment in roots (white bar) and extraradical hyphae (black
bar) (Tukey, P < 0.05).
not separated from translocation in this experiment because
we did not separate PH and DH.
Experiment 6. 33P movement from hyphae to roots. Total
33P activity was significantly higher (ANOVA, P < 0.05) in roots
than in hyphae (Fig. 6). However, total 33P uptake and
translocation to the intraradical mycelium were similar within
the 10–25°C temperature range. It was not possible to separate
33P transfer from fungus to plant from translocation because
the measurement was made in roots with the fungus.
Discussion
This study provides the first conclusive evidence for: (1) the
negative direct effects of low (< 15°C) temperatures in the
development of three isolates of AMF and of a high (> 24°C)
New Phytologist (2005) 168: 179–188
185
186 Research
Fig. 6 Experiment 4. Total 33P activity in roots (black bar) and
extraradical hyphae (white bar) measured per plate in monoxenic
cultures with Glomus intraradices-DC1, after adding 33P to the
hyphal compartment and incubating for 5 d at 10°, 18°, and 25°.
Data are expressed as mean ± standard error (SE) (n = 4–5).
temperature on one of the isolates; (2) the existence of both
direct and host-mediated effects in the response of AMF to
temperature changes, even in the absence of photosynthesis and
shoot growth differences; and (3) a reduction in C movement
to distant extraradical hyphae as one of the mechanisms
involved in the response of AMF to varying temperatures.
One of the most important confounding factors in pot or
field experiments is the effect of temperature on the host plant
because growth and photosynthesis, both critical for C
availability to the fungal partner, can be strongly affected by
temperature changes (Morison & Lawlor, 1999). Root organ
cultures had the potential to minimize these confounding
factors, but our results indicated that the amount of C for
the fungus was affected by temperature, even in the absence
of shoot growth and photosynthesis. Strictly direct effects on
AMF may be estimated if temperature changes do not alter
substantially the amount of plant C available for fungal development. Temperatures below 10°C are known to reduce ion and
water uptake in numerous plants, but low temperature effects
on C uptake by transformed roots had not been investigated.
This information was not considered relevant in most other
studies conducted, to date, with this system. The sensitivity of
the root clone DC2 to a 30°C temperature was also unknown.
Although the use of the monoxenic system did not allow us to
measure direct and indirect effects completely independently
from each other, it still permitted a more reliable quantification of both host and fungal growth and a first approximation
to the involvement of C nutrition in the response of AMF to
temperature manipulations. There was, for example, strong
evidence for the direct effects of temperature on AMF growth,
based on the clear difference in temperature optima for the
partners and the lack of covariance between fungal growth and
root growth. Heinemeyer & Fitter (2004) recently published
results from the first study on the direct effect of temperature
on both phases of the mycorrhizal mycelium. They concluded
that localized warming, from 12 to 20°C, significantly increased
the length of the extraradical mycelium of AMF but, if temper-
New Phytologist (2005) 168: 179–188
ature effects on root growth or root colonization were accounted
for, the effect was not significant.
The results supported our hypothesis that C movement
was involved in the response of AMF to temperature changes,
because of the differences measured in root-C uptake and C
translocation in the fungus. The fact that C movement to the
fungus was affected by temperature, whereas root P uptake
and P translocation by the fungus were not, suggested that the
negative effect of low temperatures did not extend to the
movement of other nutrients. The lack of temperature effects
on P uptake, translocation and transfer between 10 and 25°C
is consistent with results from another study conducted in
that temperature range (Wang et al., 2002). The negative
effects of low temperatures on the fluidity of membranes
and translocation of solutes in fungi has been documented
(Jennings, 1995), and one might have expected a reduction in
the movement of both C and P. C movement had never been
studied in relation to temperature in AMF. This is the first
evidence suggesting that C availability is involved and that
the movement of nutrients within the mycelium is not an
important component of the response of AMF to varying
temperature. However, as C normally arrives to AMF via shoot
photosynthesis and not via root C uptake, this should be
further investigated with whole plants.
Carbon was transferred to the AMF mycelium, in most cases
proportionally to the amount taken up by the roots, indicating that transfer and translocation had not been substantially
reduced. Additionally, temperature manipulations after C uptake
had taken place affected only slightly the amount of C reaching the DH. The reduction in C movement to DH at temperatures of < 18°C was, nevertheless, too small to explain
growth reductions in some cases. The shortage of C might
have explained most of the growth reduction at 6°C, but not
at 12°C, because the amount of C entering the fungus at
12°C was clearly higher than at 6°C, whereas growth was still
very depressed. Fungal growth limitation below 18°C might
be intrinsic, at least partially, to the fungus or may be related
to other unknown processes.
The shapes of the growth curves for roots and the three fungal isolates, and 13C uptake-transfer-translocation curves
showed that: (1) root growth was much more tolerant to temperatures below 18°C than fungal growth; (2) fungal growth
was very limited at temperatures of < 18°C, despite the good
development of roots and a clear increase in C availability
between 6 and 18°C; and (3) the fungal isolates had different
growth rates, but there was no evidence suggesting that the
Canadian isolates were more tolerant to temperatures below
18°C than the tropical isolate. Moreover, the tropical isolate
grew better at temperatures of < 18°C than one of the Canadian isolates. The negative effect of temperatures below 18°C
on extraradical AMF development from previous studies in
pots (Gavito et al., 2000, 2003 and Gavito et al., unpublished),
was thus confirmed in the monoxenic system, with no indication, so far, for adaptation of AMF to these temperatures. The
www.newphytologist.org © New Phytologist (2005)
Research
establishment of single isolates in pot cultures and monoxenic
plates requires that growing conditions are mild, otherwise
growth is very poor. There was thus a possible bias owing
to maintenance of the isolates at mild temperatures for
some generations, but this was unavoidable. There is evidence
that subculturing for a few generations does not affect the
response of AMF to temperature. In Gavito et al. (2003),
both the entire (nonsubcultured) AMF community of a
Danish soil and a single (subcultured) native isolate failed
equally to produce extraradical mycelium at a soil temperature
of 10°C.
The temperature treatments altered mycelium growth,
growth pattern, and phenology of intraradical and extraradical colonization in all isolates. Temperatures of < 18°C
resulted in reduced growth, the formation of a loosely connected and weak network, and a delay in the production of
absorptive and reserve/propagation structures. These results
suggest that growth at temperatures of < 18°C was not only
diminished, but more susceptible to damage. Mycelium structure could be easily disrupted, with few reserve and propagation
structures that could reinitiate colonization or new growth
after injury. High temperatures (30°C) reduced mycelium
growth of G. proliferum, perhaps as a consequence of its negative effect on the growth of root clone DC2. G. intraradices,
on the other hand, grew best at 30°C, despite a reduction
in root growth. G. intraradices and G. cerebriforme could still
form a good network with abundant structures at 30°C, following the phenology scheme observed at mild temperatures
(18°C and 24°C). The negative effect of high temperature
(30°C) supported results from field experiments (Monz et al.,
1994; Rillig et al., 2002; Staddon et al., 2003b; Heinemeyer
et al., 2004) showing that warming might be detrimental
for fungal development in some regions or when the soil
is warmed above the temperature optima for AMF.
The studies published, to date, with different AMF, hosts
and growth systems, suggest that AMF have a narrower range
(> 18°C and < 30°C) of temperature for good development
than previously believed, although they may grow between 5
and 37°C, as reported by Staddon et al. (2002). Studies by
D’az-Raviña et al. (1994), with soil bacteria, by Bååth (2001),
with soil fungi, and by Pietikäinen et al. (2005), with both soil
bacteria and fungi, showed similarly that growth curves of
these two groups of soil microbes, in response to temperature,
were not bell-shaped, but rather steep-peak shaped. Temperature optima for both fungi and bacteria in Scandinavian
forest soils were between 25 and 30°C, with drastic growth
increases between 0 and 25°C, and marked reductions above
30°C. Interestingly, the temperature optima for soil fungi and
bacteria found in those studies and in Domsch et al. (1980),
for soil fungi from temperate climate soils, correspond to high
temperature values that rarely occur in temperate soils.
Arbuscular mycorrhizal fungi are a special case because of
their obligate symbiotic nature, and it is therefore expected
that temperatures for AMF growth should coincide, at least in
© New Phytologist (2005) www.newphytologist.org
part, with the temperatures for root growth. Plants seem to
have more tolerance to low temperatures than soil microbes,
as exemplified by the higher sensitivity of soil organic matter
decomposers than of the net primary productivity to low
temperatures (Kirschbaum, 1995). This may explain why the
lower limit of the temperature optima for AMF is around 18°C,
slightly below the lower limit measured for other soil fungi.
When the cultures treated at 24°C were restored after a
chilling period at 11°C, C was taken up, transferred and
translocated as if the cultures had not been cooled down. This
suggests that the physiological activity recovered rapidly to the
same level as that in plates maintained at a constant temperature of 24°C. There was, however, some evidence for reduced
root C uptake and reduced translocation to distant hyphae at
low temperatures when we allowed a 2-wk longer incubation
time. This suggests a lag response of some processes after continuous exposure to low temperatures. Measuring the movement of C from shoot to roots, and to AMF in less artificial
systems, is required to elucidate whether the arrival of C from
the host plant to AMF is significantly diminished at very low
or very high temperatures.
We conclude that the effects of temperature on fungal
growth were partly direct and partly a consequence of changes
in the availability of plant C for AMF. Our results have implications for the current understanding of the development and
functioning of AMF in natural environments at present or in
the context of a rapidly changing climate. Evidence for severe
growth constraints at very low or very high temperatures is
increasingly relevant in this scenario and does not support the
general belief that AMF are highly plastic and tolerant to variable environmental conditions. We provide evidence that the
ability to grow at temperatures of < 20°C may be rare, even in
isolates from temperate and cold environments. This suggests
that AMF are probably temperature-limited in temperate and
cold environments and that their development is probably
confined to short periods in the summer, and in the warmer,
upper soil layer. Most plants do not grow, or grow only very
slowly, at temperatures below 10°C and C is probably very
limiting for fungal growth. Moreover, new roots produced in
the growing season in natural systems probably become colonized by previously established mycelium that has the potential to reconnect and reinitiate growth, so that no substantial
AMF new growth is needed to have a mycelial network. Low
temperature-negative effects on AMF may represent a bigger
problem in ploughed agricultural fields where mycelial
networks are continuously disrupted so that new networks
have to be re-established.
Acknowledgements
We thank GINCO (Glomales in vitro collection) and
Stéphane Declerck for kindly providing us with monoxenic
cultures of Glomus proliferum and Glomus cerebriforme for
our experiments. This work was supported by Marie Curie
New Phytologist (2005) 168: 179–188
187
188 Research
Fellowships of the European Commission for Mayra E. Gavito
in Spain and Almudena Medina-Peñafiel in Denmark, and
a Carl Trygger Foundation (Sweden) postdoctoral fellowship
for Mayra E. Gavito.
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