Journal of Experimental Botany, Vol. 55, No. 396, pp. 525±534, February 2004
DOI: 10.1093/jxb/erh049
RESEARCH PAPER
Impact of temperature on the arbuscular mycorrhizal (AM)
symbiosis: growth responses of the host plant and its AM
fungal partner
A. Heinemeyer* and A. H. Fitter
Department of Biology, University of York, PO Box 373, York YO10 5YW, UK
Received 22 September 2003; Accepted 21 October 2003
Abstract
The growth response of the hyphae of mycorrhizal
fungi has been determined, both when plant and fungus together and when only the fungus was exposed
to a temperature change. Two host plant species,
Plantago lanceolata and Holcus lanatus, were grown
separately in pots inoculated with the mycorrhizal
fungus Glomus mosseae at 20/18 °C (day/night); half
of the pots were then transferred to 12/10 °C. Plant
and fungal growth were determined at six sequential
destructive harvests. A second experiment investigated the direct effect of temperature on the length
of the extra-radical mycelium (ERM) of three mycorrhizal fungal species. Growth boxes were divided in
two equal compartments by a 20 mm mesh, allowing
only the ERM and not roots to grow into a fungal
compartment, which was either heated (+8 °C) or kept
at ambient temperature. ERM length (LERM) was determined on ®ve sampling dates. Growth of H. lanatus
was little affected by temperature, whereas growth of
P. lanceolata increased with temperature, and both
speci®c leaf area (SLA) and speci®c root length
(SRL) increased independently of plant size.
Percentage of colonized root (LRC) and LERM were
positively correlated with temperature when in symbiosis with P. lanceolata, but differences in LRC were a
function of plant biomass. Colonization was very low
in H. lanatus roots and there was no signi®cant temperature effect. In the fungal compartment LERM
increased over time and was greatest for Glomus
mosseae. Heating the fungal compartment signi®cantly increased LERM in two of the three species but
did not affect LRC. However, it signi®cantly increased
SRL of roots in the plant compartment, suggesting
that the fungus plays a regulatory role in the growth
dynamics of the symbiosis. These temperature
responses have implications for modelling carbon
dynamics under global climate change.
Key words: Acaulospora, arbuscular mycorrhiza, belowground carbon allocation, compartment experiment, extraradical mycelium, global climate change, Glomus, niche
separation, soil warming.
Introduction
Arbuscular mycorrhizal (AM) fungi, all members of the
Glomales (Glomeromycota), form a symbiosis with about
two-thirds of plant species (Fitter and Moyersoen, 1996)
and occur in almost all terrestrial ecosystems (Trappe,
1987; Tinker, 1975). However, only some 150 AM fungal
species have been described so far (Redecker et al., 2000),
in contrast to the over 250 000 known plant species. Yet
typically 20±30 species of AM fungi occur in most plant
communities, so that AM fungal a-diversity seems to be
quite similar to that of plants in the same community
(Fitter, 2001). Niche separation between AM fungal taxa
might therefore be expected (Bever et al., 2001), yet almost
nothing is known about their response to different
environmental conditions, which would be required for
an evaluation of such an hypothesis.
The role of AM fungi in nutrient uptake (Clark and Zeto,
2000) and carbon cycling (Smith and Read, 1997) has been
widely recognized. Potential host plants differ in their
dependency on the AM fungus, most likely due to their
different root system architecture (Newsham et al., 1995;
Hetrick et al., 1991) and thus different ability to acquire
highly insoluble nutrients such as phosphate (Koide,
* To whom correspondence should be addressed. Fax: +44 (0)1904 43 2898. E-mail: [email protected]
Journal of Experimental Botany, Vol. 55, No. 396, ã Society for Experimental Biology 2004; all rights reserved
526 Heinemeyer and Fitter
1991). However, as obligate symbionts, all AM fungi rely
on host plant carbon (Smith and Read, 1997) and
mycorrhizal colonization increases sink strength of the
root (Harris and Paul, 1987; Wright et al., 1998). Any
impact on plant carbon ®xation and growth might have
direct impacts on this carbon allocation to the rootcolonizing AM fungi (Same et al., 1983), with a consequent feedback on the host plant due to changes in fungal
functions such as nutrient uptake or other bene®ts (Fitter
et al., 2000). On the other hand the fungus itself might
respond to any environmental changes directly, independently of any effect on the host plant (Fitter et al., 2000) and,
consequently, might also affect host root morphology
(Hooker et al., 1992).
Global climate is predicted to change dramatically over
the next century (Houghton et al., 2001). The effect of
elevated CO2 on plant growth has been studied in detail,
and current evidence suggests that impacts on AM fungi
are likely to be indirect responses, mediated via changes in
plant growth (Staddon and Fitter, 1998). There is less
certainty about potential temperature impacts on plant
communities, despite a predicted rise in global mean
temperature of about 3 °C over the next century (Houghton
et al., 1995). Due to acclimation responses to temperature,
root growth seems to be more affected by PAR (photosynthetically active radiation) than by temperature
(Aguirrezabal et al., 1994; Fitter et al., 1999). Temperature responses of AM fungi have not been investigated
intensively if compared to studies on elevated CO2.
The mycelium of an AM fungus in effect comprises two
linked mycelia, the internal root-colonizing hyphae and the
extra-radical mycelium (ERM): the ERM is likely to
experience the wider variation in soil parameters such as
pH and nutrient levels, exploring soil pores inaccessible to
roots (Smith and Read, 1997) and nutrient patches in the
upper organic-rich soil horizons (Tibbett, 2000; Hodge
et al., 2001) where there are higher temperature ¯uctuations than in deeper soil horizons. Apart from work on
spore germination (Daniels Hetrick, 1984) and internal
colonization (Jakobsen, 1986), the few studies of temperature response to date were not designed to separate
effects on the fungus from those on the plant (Fitter et al.,
2000). More recently, compartment studies have been used
to investigate differences in nutrient uptake of the ERM
among AM fungal species (Jakobsen et al., 1992). This
approach has not previously been used to detect direct
temperature effects on the ERM.
Successful prediction of impacts of global climate
change on ecosystem functioning must take into account
the AM symbiosis and, in particular, the ERM, which links
above- and below-ground carbon cycling (Smith and Read,
1997) and will be crucial in supplying the possible higher
nutrient demands of the host plant under a changing
climate (Fitter et al., 2000).
In order to determine the responsiveness of AM fungi to
temperature, three hypotheses were tested: (1) a species
more heavily colonized by mycorrhizal fungi (here
Plantago lanceolata) will respond more to temperature
and shading treatments than one less colonized (Holcus
lanatus); (2) when plant and fungus are exposed to
different temperatures, changes in plant growth will
mainly determine any fungal response; (3) warming the
extra-radical mycelium stimulates its growth independently of stimulated plant growth. Two experiments were
carried out: one in pots where plant and fungus experienced the same temperature, was designed to test hypothesis 1; the other was a compartment study, where fungal
temperature was varied independently of the plant and
tested hypotheses 2 and 3.
Materials and methods
Experiment 1
Experimental design: Eighty 10 cm pots were each planted with two
seeds (Emorsgate Seeds, Norfolk, UK) of either Plantago lanceolata
L. or Holcus lanatus L. and kept for 28 and 26 d, respectively, in a
growth chamber (Percival A32L, Percival Scienti®c Inc., Iowa,
USA). Conditions in the growth chamber were 20/18 °C (day/night),
60±80% RH and a mean photosynthetically active photon ¯ux
density (PPFD) of 250 mmol m±2 s±1 at plant level for 16 h d±1. Pots
were thinned to one seedling after 2 weeks. The growth medium
consisted of 380 g (dry weight) of a 1:1 mixture (v/v) of coarse
builders' dried silica sand and terragreenâ (an attapulgite clay soil
conditioner, Turfpro Ltd, Staines, UK). This was evenly inoculated
(40 g kg±1 growth medium) with dried fungal inoculum of Glomus
mosseae (Nicol. & Gerd.) Gerd. & Trappe (isolate UY282),
consisting of c. 1 cm pieces of maize roots in the same medium.
Although this medium does not re¯ect natural soil it enabled a more
uniform nutrient supply and was necessary to measure hyphal length
accurately. The measurement of hyphal length is very dif®cult in
soils with organic matter, since hyphae penetrate and bind together
the particles. Plants were re-randomized weekly inside the chambers
and watered daily with tap water. Half-strength Rorison's nutrient
solution (Hewitt, 1966) containing only 1/20 of the normal
concentration of phosphate was given weekly (2.5 ml per pot)
from day 21. Temperature was recorded every 30 min (averaged
5 min readings) with thermistor probes at 1 cm depth (Grant
Instruments Ltd, Shepreth, UK) and PAR ¯ux measured weekly at
plant level using a light meter (SKD210, Skye Instruments Ltd,
Powys, UK).
There were six sequential destructive harvests and four replicates.
After the ®rst harvest, which was at 26 and 28 d after planting (dap)
for Holcus and Plantago, respectively, half of the pots of each plant
species were transferred to an identical growth chamber under the
same growth conditions, but with a temperature of 12/10 °C (day/
night). P. lanceolata harvests occurred at 28, 52, 59, 66, 73, and 80
dap and H. lanatus at 26, 33, 40, 47, 59, and 82 dap.
Plant measurements
At harvest, plants were extracted from the pots, collecting as much
growth medium as possible for fungal measurements (see below),
and were then divided into root and shoot fractions. Total fresh
weight (FW) of leaves and roots and leaf area (AL) were recorded
after cleaning of remaining growth medium. Dead leaves were
removed beforehand, dried, and later added to the total plant weight
(WRS). Roots were chopped into c. 1 cm pieces in a 500 ml beaker
Mycelial growth, mycorrhizas, temperature, and light
®lled with 400 ml water and mixed with a glass rod. Four subsamples
were taken from this pool twice at random, one for measurement of
root length (LR) and subsequently root dry weight (WR) and another
for root length colonization (LRC). Fresh weight of shoots and of the
root subsamples were re-measured, then shoot material and root
subsamples were dried at 70 °C for 3 d and their dry weight (WS and
WR) recorded. Leaf area and LR were calculated using a ¯at bed
scanner (Hewlett Packard, USA) and images were later analysed
with the program Winrhizoâ (ReÂgent Instruments, Quebec, CAN).
Speci®c leaf area (SLA) and speci®c root length (SRL) were both
calculated. Remaining growth medium was kept overnight at 8 °C
for extraction of fungal hyphae the following day.
Fungal measurements
The four root subsamples for mycorrhizal assessment were pooled.
Roots were cleared at room temperature in 20% KOH (2 d), acidi®ed
in HCl (30 min), stained with 0.1% acid fuchsin (3 d) (as in
Kormanik and McGraw (1982) but omitting phenol), and were left in
a destain solution for a further 3 d. Mycorrhizal colonization was
assessed under a compound microscope (Optiphot-2, Nikon Inc.,
Melville, U.S.A.) at 3200 magni®cation using epi¯uorescence
(Merryweather and Fitter, 1991). Scoring followed McGonigle et al.
(1990) with a minimum of 100 intersections. The percentage of
intercepts where hyphae are present is termed root length colonized
(LRC). In addition, intercepts where arbuscules (LRCarb) or vesicles
(LRCves) were visible were scored separately.
From each growth medium sample, three subsamples were taken
and their FW obtained, one for oven-drying to calculate the FW:DW
ratio of the medium and two for extraction of extra-radical mycelium
(ERM). A modi®ed membrane ®lter technique (based primarily on
Abbott et al., 1984; Jakobsen et al., 1992; Sylvia, 1992) was used for
the extraction. The growth medium sample was made up to 500 ml
with tap water, then stirred full speed with a magnetic stirrer (Voss
Instruments Ltd, Maldon, Essex, UK) for 4 min. Before settlement,
200 ml were quickly transferred to a tall beaker stirred at full speed
then at 40% of full speed, allowing larger particles to sediment. After
stirring, two 5 ml samples were quickly taken with a syringe,
centrally, about 1 cm from the surface. Each sample was placed in a
®lter funnel (diameter 19 mm) ®tted with a 0.45 mm mesh nylon
membrane (MSI, Westboro, MA, USA), attached to a vacuum pump
(N740.3FT.18, KNF, Germany). When the ®lter was totally dry, the
funnel was removed and a few drops of the staining solution were
applied to the ®lter. After a short drying period the ®lter was
removed from the ®lter-holder and placed in the dark. After about 1 h
the ®lter was rinsed and dried under vacuum, then mounted on a
labelled slide in destaining solution under a coverslip. Four ®lters
were thus obtained from each pot. Assessment of ERM length
(LERM) per ®lter was carried out by the gridline intercept method
(Miller and Jastrow, 1992) for a minimum of 50 ®elds of view at
3125 magni®cation (Jenamed 2, Zeiss, Leipzig, Germany) using a
10310 grid of 1 cm side length (Graticules Ltd, UK). The hyphal
lengths per ®lter were then converted to hyphal densities (m g±1 DW
growth medium), assuming all hyphae to be mycorrhizal (pots
without inoculum revealed no colonization). For subsequent analysis, the mean value for the four ®lters obtained per plant was used.
Experiment 2
Experimental design: Twelve open-top Perspex boxes (233233
10 cm) were divided into halves using a Perspex plate (3 mm). These
halves were then each further divided into two compartments (A and
B) with nylon mesh (pore size diameter 20 mm, Staniar P/No. 25T1120), allowing passage only of extra-radical hyphae and not roots into
fungal compartment B. The entire box was ®lled with a dried silica
sand and terragreenâ growth medium (as above) and vertically
inserted loops of a soil-warming cable (Macpennyâ Cameron, East
527
Riding Horticulture, Sutton-on-Derwent, UK) were inserted vertically in the fungal compartment B at a 7.5 cm distance from the
mesh. After germination on moist ®lter paper (Whatmanâ No. 1) for
2 d (20 °C in the dark) two P. lanceolata seedlings (supplier as in
Experiment 1) were planted in the middle of each compartment A,
3.5 cm from the mesh, and thinned to one seedling per compartment
after 2 weeks. The 24 compartments A were evenly inoculated (50 g
kg±1 growth medium) with dried fungal inoculum of one of three AM
fungi contrasting in the amount of ERM produced: (a) G. mosseae
((Nicol. & Gerd.) Gerd. & Trappe, isolate UY 282, ex P. Bonfante,
Torino-Trotta, I.), (b) Glomus hoi (Berch & Trappe; isolate UY 110,
Welburn, GB) and (c) Acaulospora sp. (isolate UY 991, Elphin, GB,
ex C Walker, likely to be A. laevis) (all provided by J Merryweather,
University of York, UK) and consisting of c. 1 cm pieces of a
P. lanceolata, Trifolium pratense L., and Sorghum sp. (Sudan grass)
root mixture within the same growth medium). Although the three
AM fungal species originated from various places in Europe, all
species have been cultivated for many years under similar growth
conditions at the University of York collection. Boxes were placed in
walk-in chambers (Southern & Redfern Ltd., Bradford, UK) over the
®rst 3 weeks at 20/17 °C (day/night), 60±80% RH, and a mean PPFD
of 350 mmol m±2 s±1 at plant level for 16 h d±1. Over 125 d on ®ve
occasions growth medium samples with four replicates each (one
from each replicate hyphal compartment) were taken for each fungal
species and temperature treatment combination.
Temperature was recorded in the growth medium for both
compartments (in four boxes per temperature treatment) at similar
distances from the mesh every 10 min. (averaged 30 s readings) with
thermistor probes (as in Experiment 1) and PPFD measured weekly
at plant level. Compartments A and B were watered daily with tap
water. From week three onwards a half-strength Rorison's nutrient
solution (Hewitt, 1966) was given weekly (12.5 ml) to compartment
A (containing 1/20 phosphate) and B (containing 1/10 phosphate),
except within 1 cm of the mesh. For this procedure plant
compartment A was always watered ®rst; all compartments were
subsequently watered with tap water to remove nutrient solution
from the leaves.
The ®rst sampling after 20 dap represents the beginning of the
treatment and over the following 15 weeks the temperature in the
growth chamber was maintained at 12/10 °C (day/night). Over this
period one end of the fungal compartment B was either kept at
ambient temperature or heated (giving each four replicates per
treatment) to approximately 8 °C above ambient temperature at
sampling position for each fungal species. Heating fungal compartment B did not affect the temperature of the plant compartment A
(Fig. 1). Any moisture differences between heated and unheated
compartments B were limited by manually watering all the
compartments at least three times per day.
Plant measurements
On the last sampling day, plants were extracted from compartment A
and divided into root and shoot fractions. Analysis followed as
described for Experiment 1.
Fungal measurements
On each sampling day a growth medium core (9 cm depth 3 1.5 cm
in diameter) was taken from compartment B, but in a random
location along an arc 8.5 cm from each shoot base, at approximately
4.5 cm distance from the mesh. Samples were taken at identical
positions in compartments B and used for ERM extraction (as
described for Experiment 1); the sample area was then re®lled with
the same growth medium, gently compressed and watered with
nutrient solution. Root samples were stained and investigated for
LRC as described in Experiment 1. In this study both clearing
(10 min) and staining (35 min) were conducted in a waterbath
528 Heinemeyer and Fitter
Fig. 1. Experimental design for the compartment experiment. The entire box was divided into two equal halves by a 20 mm mesh (M). Plant
position (PP), sample area position (SP), and heating cable position (HC) are indicated with arrows. Data are means (bars indicate minima and
maxima) of the medians of the four replicates per temperature treatment (open symbols: ambient; ®lled symbols: heated) measured in the growth
medium at different distances from the mesh in either plant compartment (A) or fungal compartment (B) after heating was switched on (different
distances between thermistor probes of ambient and heated treatments were due to number of probes available and box position determining cable
length). The lines shown are best ®t polynomials (dashed line: ambient, R2=0.87; solid line: heated, R2=0.94).
(80 °C). To improve staining results, samples were acidi®ed and
stained twice. The percentage of LRC and also LRCarb and LRCves
were scored (see above).
Statistical analysis
Analysis was performed in SPSS v6.1 (Norusis, 1994). All data were
checked and transformed appropriately (e.g. arcsine transformation
for percentage values and mostly log or square root transformation
for other parameters, however, data were presented as back
transformed data in most tables and ®gures) to normalize skewed
distributions before statistical analysis and data were also checked
for equality of variance (Levene's test in SPSS); no cases were
encountered where data could not be made suitable for ANOVA. In
Experiments 1 and 2 plant growth data were all analysed with a twoway ANOVA (temperature and harvest or temperature and fungal
species as factors); species differences in Experiment 2 were also
named by Bonferroni's multiple comparisons test. The F-ratio
method was used to test for signi®cant difference of ®tted regression
lines (Sokal and Rohlf, 1981) if data were plotted against plant
biomass. Data for ERM growth in Experiment 2 were tested for any
treatment effects with the repeated measurement design of the
general linear model (GLM), with species and temperature as
between-subject factors. These data were also tested with a one-way
ANOVA, with treatment as the only factor, in order to detect
signi®cant treatment effects for individual species at particular
harvests. Since the ERM of Acaulospora sp. was very low (<0.2 m
g±1 DW growth medium), apart from the last sampling time, these
data were excluded from the GLM-analysis. Furthermore, in one
compartment of each Glomus species and in two different treatments
of Acaulospora sp. roots had grown through the mesh into the fungal
compartment B; these data were also excluded from further analysis.
Results
Temperature impact in experiment 1
Growth of H. lanatus was only slightly affected by
temperature over the experimental period of 56 d. Only
leaf area (AL) and root length (LR) were signi®cantly
higher at 20 °C than at 12 °C (Table 1), although the mean
plant dry weight (WSR) was 40% greater in the 20 °C
treatment. Length of colonized root (LRC) and of extraradical mycelium (LERM) were both very low, did not
differ between temperature treatments, and declined after
initially peaking between 33 and 40 dap (<10% and <0.8 m
g±1, data not shown) to less than 2% and 0.5 m g±1 at the
last harvest (Table 1); the root length colonized by
arbuscules (LRCarb), was always very low (Fig. 2A).
Total LRC was nearly three times higher in the 20 °C
treatment (Table 1).
Growth of P. lanceolata was greater at the higher
temperature after 52 d (Table 1). Similar differences were
observed for AL and LR (Table 1). Colonization of roots
was 25±30% by the end of the experiment. Both LRC and
LERM increased over time and total LRC was nearly three
times and LERM almost twice as large under the higher
temperature (Table 1). The percentage LRCarb increased
over time to about 25% (Fig. 2A) and was signi®cantly
higher in the 20 °C than in the 12 °C treatment (P <0.05).
Percentage LRCves also showed a signi®cant treatment
effect during an initial peak between 28 and 52 dap with
values of 2% and 5% for the 12 °C and 20 °C treatments,
respectively, but thereafter declined to about 1% in both
treatments (Fig. 2B). There were signi®cant differences in
the morphology of P. lanceolata as measured by increased
SLA and SRL (Table 1) and these were direct responses to
increased temperature. Even though both SRL and SLA
showed a pronounced ontogenetic drift, the effect of
temperature was independent of plant biomass as shown by
analysis of covariance. There was a signi®cant difference
in the ®tted regression lines of both SRL and SLA on ln
biomass between the temperature treatments (Fig. 3). The
same analysis revealed that LERM was greater at 20 °C than
at 12 °C at a given plant biomass (Fig. 4A), but there was
Mycelial growth, mycorrhizas, temperature, and light
529
Table 1. Temperature effects on plant growth and colonization of roots (LRC) and extra-radical mycelium length (LERM) of
Glomus mosseae grown in symbiosis with either Holcus lanatus or Plantago lanceolata in the pot experiment
Data are the means 6 SE at the last harvest for plant biomass (WRS), leaf area (AL), speci®c leaf area (SLA), root length (LR) and speci®c root
length (SRL), LRC, total LRC, LERM, and LERM per pot per unit LR (LERM/LR) or unit total LRC (LERM/total LRC) for either at 12 °C or 20 °C
grown H. lanatus or P. lanceolata plants, respectively. Signi®cances between temperature treatments are based on a two-way ANOVA. n.s., not
signi®cant, *P <0.05, **P <0.01, ***P <0.001.
WRS (g)
AL (cm2)
2
±1
SLA (cm g )
LR (m)
SRL (m g±1)
Temperature
Plant growth (n=4)
°C
H. lanatus
12
20
12
20
12
20
12
20
12
20
0.960.3 n.s.
1.360.3
45.360.3*
82.9611.2
200.1613.0 n.s.
220.5627.2
162.4646.5*
216.4627.0
323.6612.1 n.s
319.6615.9
Fungal growth (Glomus mosseae) (n=4)
P. lanceolata
*
0.460.0
0.560.1
19.261.7**
37.964.5
105.562.7*
171.321.3
23.663.2**
49.469.6
0.4611.6**
0.568.4
H. lanatus
Total LRC (m)
LRC (%)
±1
LERM (m g
soil)
LERM/LR (m m±1)
LERM/total LRC (m m±1)
P. lanceolata
*
1.360.6
3.661.3
1.060.4 n.s.
1.560.5
0.460.1 n.s
0.560.0
1.160.4 n.s.
0.960.1
81.8610 n.s.
74.5617.8
5.560.5*
14.563.9
24.463.5
29.564.6
1.160.2
2.160.1***
18.262.9 n.s
17.663.0
80.9618.6 n.s.
71.2621.4
P = 0.086
Fig. 2. Effects of temperature on mean percentage of root colonization of (A) arbuscules (LRCarb) or (B) vesicles (LRCves) in either Plantago
lanceolata (diamonds) or Holcus lanatus (triangles) roots at the six harvests (days after planting, dap) 6SE (standard error, n=4). Temperature
treatments were either 12 °C (open symbols) or 20 °C (®lled symbols) and started after the ®rst harvest. Signi®cances between treatments based
on a two-way ANOVA differed only for P. lanceolata and were as following: (A) harvest: F5,36=3.14, P=0.019; temperature: F1,36=4.42, P=0.043;
(B) harvest: F5,36=5.96, P <0.001; temperature: F1,36=5.91, P=0.020; there were no other signi®cant differences for either treatment or interaction
terms.
no temperature effect on LERM if it was expressed per unit
LR or LRC (Table 1). Even though %LRC did not differ
signi®cantly between treatments (P=0.086) (Table 1), the
large difference in total LRC was still evident when plotted
as a function of plant biomass (Fig. 4B), with similar-sized
plants having greater total LRC at 20 °C than at 12 °C,
indicating a direct response to increased temperature.
Effect of temperature in the compartment experiment
2
Heating the fungal compartment B did not affect growth of
P. lanceolata, although it did alter root morphology: SRL
was higher for plants grown with hyphae connected to a
heated fungal compartment (Table 2). However, plant
growth did respond to the fungal species. Overall plants
had the highest biomass if grown with Acaulospora sp.
(Table 2).
Percentage LRC and %LRCarb declined in the order G.
mosseae >G. hoi >Acaulospora sp., but heating the fungal
compartment B had no effect on any measure of
colonization in the root compartment A (Table 2). LERM
increased progressively during the experiment (Fig. 5)
with a lag phase prior to colonization of the fungal
compartment B, that differed in duration among the
species, followed by a phase of more or less exponential
increase. At all times hyphal length differed between
species (GLM, P <0.001) and in the order G. mosseae
>G. hoi >Acaulospora sp. Further, LERM was signi®cantly
530 Heinemeyer and Fitter
Fig. 3. Effects of temperature on (A) speci®c root length (SRL) or (B) speci®c leaf area (SLA) of Plantago lanceolata at all harvests against ln
plant biomass. Temperature treatments were either 12 °C (open symbols and dashed line) or 20 °C (®lled symbols and line). The lines shown are
best ®t regression lines with (A) y (12 °C)= ±83.8x+8.4, r2=86%; y (20 °C)= ±54.2x+96.6, r2=66%; (B) y (12 °C)= ±93.7x+20.3, r2=93%;
y (20°C)= ±72.5x+102.6, r2=93%. Signi®cances between temperature treatments based on the F-ratio method (Potvin et al., 1990) comparing the
two ®tted lines were as following: (A) F2,44=25.30, P <0.001; (B) F2,44=8.28, P <0.001.
Fig. 4. Effects of temperature on (A) extra-radical mycelium (ERM) length g±1 growth medium or (B) total length of colonized roots (total LRC)
of Glomus mosseae grown in symbiosis with Plantago lanceolata at all harvests against (A) ln plant biomass and (B) plant biomass. Temperature
treatments were either 12 °C (open symbols and dashed line) or 20 °C (®lled symbols and line). The lines shown are best ®t regression lines with
(A) y (12 °C)=0.29x+1.45, r2=42%; y (20 °C)=0.45x+2.19, r2=64%; (B) y (12 °C)=11.39x+0.49, r2=60%; y (20 °C)=23.29x±0.13, r2=65%.
Signi®cances between temperature treatments based on the F-ratio method (Potvin et al., 1990) comparing the two ®tted lines were as following:
(A) F2,44=6.87, P <0.001; (B) F2,44=6.83, P <0.001.
(GLM, P <0.001) greater in the heated fungal compartments B (Fig. 5), but for each species this difference was
transient: the effect of heating was signi®cant for G.
mosseae and G. hoi at harvest three and four (Fig. 5). On
the last sampling day, however, there was no longer a
temperature effect on the ERM of either Glomus spp.,
whereas it became more evident (though not yet signi®cant) for Acaulospora sp. (Fig. 5).
Discussion
Higher temperature enhanced the growth of both the
internal and external mycelium of AM fungi in P.
lanceolata. Enhanced growth responses of AM fungi to
higher temperature have been reported earlier (Furlan and
Fortin, 1973; Graham and Leonard, 1982); however, the
few experiments either exposed the symbiosis to the
different temperatures right from the germination stage,
which leads to differences in spore germination (Daniels
Hetrick, 1984) and growth of infection units (Tinker,
1975), or were single harvest experiments. Single harvest
experiments must be interpreted with great caution
because they may involve comparisons of individuals
that have grown at different rates and therefore are at
different developmental stages (Staddon et al., 1998,
1999). Treatments were applied for shorter periods in this
Mycelial growth, mycorrhizas, temperature, and light
531
Table 2. Treatment effects on plant growth and colonization of roots (LRC) by Glomus mosseae (GM), G. hoi (GH) and
Acaulospora sp. (A sp.) grown in symbiosis with Plantago lanceolata in the compartment experiment
Data are the means 6 SE at the last harvest for plant biomass (WRS), speci®c root length (SRL), total LRC, LRC, arbuscular LRC (LRCarb), and
vesicular LRC (LRCves) for P. lanceolata plants at either ambient (A) or heated (+8 °C, H) temperature grown in symbiosis with one of the three
AM fungi. Signi®cances between temperature treatments are based on a two-way ANOVA; there were no signi®cant interactions. Note that
number of replicates (n=4) was reduced to n=3 in ambient treatments of GH and A sp. and in heated treatments of GM and A sp. n.s., not
signi®cant, *P <0.05, **P <0.01, ***P <0.001; different letters indicate signi®cant differences between species according to multiple comparison
by Bonferroni.
Temperature
WRS (g)
SRL (m g±1)
Total LRC (m)
LRC(%)
LRCarb (%)
LRCves (%)
A
H
A
H
A
H
A
H
A
H
A
H
Plantago lanceolata
Species
Warming
GM
GH
A sp.
d.f.=2
P-value
d.f. = 1
P-value
6.760.6 b
7.161.1
90.867.8
101.6611.1
137.9618.0 a
151.569.2
41.165.0 a
39.567.1
21.862.8 a
16.962.6
0.960.4 a
3.361.6
6.862.0 b
6.061.1
90.769.4
103.465.1
42.6624.2 b
80.769.9
10.464.2 b
20.162.4
6.063.7 b
13.461.2
0.060.0 b
0.060.0
11.061.0 a
9.260.7
80.3610.1
103.364.2
27.768.3 b
47.762.3
5.261.3 b
6.062.0
3.261.0 b
3.661.5
0.760.3 a
0.960.5
*
n.s
n.s.
*
**
n.s.
***
n.s.
**
n.s.
**
n.s.
Fig. 5. Temperature effects on log transformed data of the extraradical mycelium (ERM) length g±1 growth medium of Glomus
mosseae (GM, diamonds), G. hoi (GH, triangles), and Acaulospora sp.
(circles) grown in symbiosis with Plantago lanceolata in either
ambient (open symbols) or heated (+8 °C, ®lled symbols) fungal
compartments over 126 d after planting (dap). Signi®cances between
temperature treatments based on a one-way ANOVA at a particular
harvest differed as following: (GM) harvest 3: F1,5=17.67, P=0.008;
harvest 4: F1,5=8.99, P=0.030; (GH) harvest 3: F1,5=6.60, P=0.050;
harvest 4: F1,5=8.64, P=0.032. There was no signi®cant difference for
the Acaulospora sp. at any one harvest.
study and the effects were followed over several harvests,
so that it was possible to allow for such developmental
effects.
When both plant and fungus were warmed, an enhanced
growth response of the ERM to temperature was demonstrated that was independent of plant biomass (Fig. 4A),
but not of LR (Table 1), because of a temperature-induced
increase in SRL. In other words, the ratio of LERM to root
length remained constant, even though variation was
considerable; this is in agreement with model ®ndings by
Fitter (1991) that there is an optimal number of entry
points per unit LR at low phosphate concentrations.
Although the ratio was greater in P. lanceolata than in
H. lanatus, the much lower colonization intensity in H.
lanatus, which was expected for this fast growing, ®nerooted species under the relatively low irradiance provided, resulted in LERM per unit root length colonized
(LRC) being very similar at 71±82 m m±1, within the range
reported by Sanders et al. (1977) for another Glomus sp.
The constancy of this ratio suggests regulation of the
amount of ERM attached to a given amount of internal
fungal biomass, and might indicate that the host plant
controls carbon allocation to the fungal partner (Graham
et al., 1997).
Although the pot experiment could not determine
whether the ERM responded directly to temperature,
independently of the host plant, the compartment experiment clearly showed such a direct temperature response of
the ERM. The length of ERM produced differed among
AM species (Fig. 5) as reported in other studies (Sylvia,
1992; Jakobsen et al., 1992), but at about 6 m g±1 growth
medium was in the range of other studies (Boddington
et al., 1999) and differences re¯ected different degrees of
internal colonization (Table 2). Further, sampling 4.5 cm
from the mesh might have underestimated the overall
amount of ERM of the less colonized species as the rate of
development of the front of mycelium growth also differs
among AM fungal species (Dodd, 1994). Differences in
532 Heinemeyer and Fitter
LERM might re¯ect different functions (Dodd, 1994; Dodd
et al., 2000) and might also be an important reason for
possible niche separation of AM fungi based on speciesspeci®c differences in ERM production (Fitter et al.,
2000). It may be signi®cant that the most responsive (and
fastest-growing) isolate, G. mosseae, came originally from
Italy, where it would have experienced a greater mean soil
temperature.
Only P. lanceolata had a clear growth response to
temperature in the pot experiment (Table 1); both SLA and
SRL declined over time, but also increased under higher
temperature. These temperature-induced changes were
independent of plant biomass which suggests that they may
have been mediated by a direct fungal response to
temperature. Biomass of P. lanceolata was very different
between the pot and the compartment experiment, possibly
due to differences in length of the growing periods or pot
sizes. However, in both cases SRL was greater if connected
to a heated mycelium. A reduction in SRL due to AM
colonization has been reported (Torrisi et al., 1999;
Atkinson et al., 1994), but the ®nding that changing the
environment of an AM fungus can affect plant morphology
with no change in the plant's environment is novel. This
result suggests that the causation is fungus to plant:
enhanced fungal growth causes changes in plant growth
that stabilizes colonization density.
The different LRC patterns in the pot experiment
re¯ected the expected different degrees of mycorrhizal
colonization in the two host plants (cf. Gange and West,
1994; Abbott et al., 1977; Fitter, 1996; Eissenstat et al.,
2000). Colonization was lower than in some studies, but
not untypical. The higher LRC together with an increase in
LERM in the warmer environment might increase the
competitiveness of P. lanceolata due to increased phosphate uptake (Smith et al., 2000) or enhancement of other
bene®ts (Dodd et al., 2000) compared with H. lanatus,
which often co-occurs with P. lanceolata in the ®eld.
These results support Tinker's view (Tinker, 1975) that the
total LRC is ecologically more important than its percentage, although to test such an hypothesis would require the
comparison of a greater number of plant species.
The positive growth response of the AM symbiosis to a
warmer environment might have important implications
for nutrient uptake and carbon cycling. Although the
temperature difference in this study (8 °C) was large,
temperature changes approaching this might occur in
topsoils if global mean temperature rises by 3 °C over the
next century (Houghton et al., 1995). Host plant photosynthesis is expected to increase under the predicted
changes (Saxe et al., 2001; Grace and Rayment, 2000), and
plants will therefore need an increased nutrient supply to
maintain higher growth rates (Ceulemans et al., 1999),
which could possibly be supplied by the response of the
ERM. However, viability of the ERM was not measured
and its turnover time may be as short as 5±6 d (Staddon
et al., 2003). Differences in viable LERM might have been
smaller.
All three AM fungal species in the compartment
experiment showed an enhanced ERM production in the
warmer environment, yet the response time differed
according to the overall amount of ERM and signi®cant
differences for two of the three species tested were
transient (Fig. 5). The transience of the temperature effects
in G. mosseae and G. hoi might have been due to the
mycelial front passing the sampling points at different
rates. The increased ERM growth might also lead to a
higher carbon input to the soil as AM hyphae have high
concentrations of both chitin (and chitosan) and excrete
large amounts of glomalin (Grif®n, 1994; Wright and
Upadhyaya, 1996), both very stable in soil (Rillig et al.,
2001). Increased ERM growth might therefore lead to
accumulation of these compounds, possibly increasing
carbon sequestration (Fitter et al., 2000).
Both increased temperature and elevated CO2 (Treseder
and Allen, 2000) may increase external mycelium growth.
The interaction between temperature and elevated CO2
might be rather complex as little is known about either
plant growth responses in the natural environment or
effects on other soil microorganisms, which are closely
linked to the ERM (Mar Vazquez et al., 2000), for
example, grazing by collembola on the ERM (Warnock
et al., 1982; Klironomos et al., 1999; Gange, 2000).
Despite frequent watering, possible moisture differences in
fungal compartments which could have resulted in osmotic
effects (i.e. a nutrient gradient towards the heating cable
position) cannot be ruled out completely; possibly affecting ERM growth into the fungal compartment. Although
controlled conditions can never re¯ect natural conditions,
these ®ndings clearly indicate the need for future experiments examining any temperature impacts under ®eld
conditions; molecular techniques in combination with
staining techniques might offer a very valuable tool for this
approach (Redecker, 2000).
In conclusion, ®rst, when plant and fungus were both
subject to varying temperatures, the impact on the internal
and external part of the AM fungus was related to different
biomass or root growth dynamics, respectively; and
second, when only the ERM was subject to a different
temperature, increased ERM growth was independent of
plant growth but increased SRL. This implies that both
plant and fungus can regulate the symbiosis, resulting in
stable colonization densities. These ®ndings underline the
important role of the AM fungal symbionts in global
carbon cycling, but leave unanswered key questions about
the responsiveness of different mycorrhizal symbioses to
temperature. Land plants in nearly all ecosystems around
the world interact closely with AM fungi and a more
mycocentric point of view is needed in the development of
predictions of impacts of a rising global temperature on
ecosystem functioning.
Mycelial growth, mycorrhizas, temperature, and light
Acknowledgements
The authors are grateful to JW Merryweather and J Denison, who
provided us with the AM inoculum, and to C Abbott, who looked
after the plants. This work was partly funded by the NERC (National
Environmental Research Council), DAAD (German Academic
Exchange Service), and the University of York. The ®rst author
now works for the Centre for Terrestrial Carbon Dynamics (CTCD).
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