Download - The impact of elevated CO2 and global climate change on arbuscular mycorrhizas: a mycocentric approach

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159

Research

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Respiration of the external mycelium in the arbuscular mycorrhizal symbiosis shows strong dependence on

recent photosynthates and acclimation to temperature

A. Heinemeyer

1

, P. Ineson

1

, N. Ostle

2

and A. H. Fitter

3

1

Centre for Terrestrial Carbon Dynamics (CTCD-York), Stockholm Environment Institute (SEI-York Centre), Department of Biology, University of York, PO

Box 373, York YO10 5DD, UK;

2

Centre for Ecology and Hydrology, Lancaster Environment Centre, Library Avenue, Bailrigg LA1 4AP, UK;

3

Department of

Biology, University of York, York YO10 5YW, UK

Summary

• Although arbuscular mycorrhizal (AM) fungi are a major pathway in the globalcarbon cycle, their basic biology and, in particular, their respiratory response totemperature remain obscure.• A pulse label of the stable isotope

13

C was applied to

Plantago lanceolata

, eitheruninoculated or inoculated with the AM fungus

Glomus mosseae

. The extra-radicalmycelium (ERM) of the fungus was allowed to grow into a separate hyphalcompartment excluding roots. We determined the carbon costs of the ERM andtested for a direct temperature effect on its respiration by measuring total carbonand the

13

C:

12

C ratio of respired CO

2

. With a second pulse we tested for acclimationof ERM respiration after 2 wk of soil warming.• Root colonization remained unchanged between the two pulses but warmingthe hyphal compartment increased ERM length.

δ

13

C signals peaked within thefirst 10 h and were higher in mycorrhizal treatments. The concentration of CO

2

inthe gas samples fluctuated diurnally and was highest in the mycorrhizal treatmentsbut was unaffected by temperature. Heating increased ERM respiration only afterthe first pulse and reduced specific ERM respiration rates after the second pulse;however, both pulses strongly depended on radiation flux.• The results indicate a fast ERM acclimation to temperature, and that light is thekey factor controlling carbon allocation to the fungus.

Key words:

arbuscular mycorrhiza (AM), below-ground carbon allocation,

13

C pulselabel, compartment experiment, mycelial growth, mycelial respiration,

Plantagolanceolata

, temperature acclimation of respiration.

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© The Authors (2006). Journal compilation ©

New Phytologist

(2006)

doi

: 10.1111/j.1469-8137.2006.01730.x

Author for correspondence:

Andreas Heinemeyer Tel: +44 1904 43 2991 Fax: +44 1904 432898 Email: [email protected]

Received:

10 January 2006

Accepted:

13 February 2006

Introduction

Elevated CO

2

concentrations in the atmosphere are leading toan increase in global mean temperature, and substantial climaticchanges are expected over the next century (Houghton

et al

.,2001). Elevated CO

2

generally increases plant growth (Ceulemans

et al

., 1999; Norby & Luo, 2004). Temperature effects, andespecially the effects of soil temperature, have been less studiedin the context of global environmental change.

Most experiments on the impacts of rising temperature onecosystems have been conducted under controlled conditionsand have used nonmycorrhizal plants, although most plantslive in association with mycorrhizal fungi (Trappe, 1987)and two-thirds of plants are in symbiosis with arbuscularmycorrhizal (AM) fungi (Fitter & Moyersoen, 1996). AM fungi(Glomeromycota) grow both inside roots and in soil as an extra-radical mycelium (ERM), which directly experiences widevariations in soil environment such as pH, soil moisture and

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temperature. The AM symbiosis appeared contemporaneouslywith land plants (Remy

et al

., 1994) and must therefore haveexperienced major climate changes in the past. However,we still do not understand the basic ecology of this ubiquitoussymbiosis (Fitter

et al

., 2000; Bever

et al

., 2001; Fitter

et al

.,2004). The fungi are obligate symbionts but form a mutualrelationship with the host plant in which the fungus receivesits entire carbon requirement from the host plant but providesthe host with nutrients that are poorly mobile in soil, suchas phosphate, or with other benefits (Newsham

et al

., 1995;Clark & Zeto, 2000). Hence, this symbiosis plays a key rolein linking above- and below-ground carbon cycling (Finlay &Söderström, 1992).

Most of the carbon supply to the fungus seems to be recentlyfixed carbon (Jakobsen & Rosendahl, 1990), and the biochemicalpathways involved are becoming clear (Bago

et al

., 2003),yet we do not know whether plants regulate this process(Graham

et al

., 1997). Changes in the environment thataffect plant photosynthesis, for example reduced lightavailability (Tester

et al

., 1986), affect fungal growthindirectly. Yet the fungus itself might respond to changesin the soil environment directly, independently of host plantresponses; soil temperature is likely to be an important factoraffecting growth and respiration. As fungal carbon supply canconsume up to 20% of net photosynthesis (Smith & Read,1997), any temperature responses of fungal growth and respi-ration might therefore have a significant influence on carboninput and cycling in the soil.

Elevated CO

2

often increases internal colonization as a resultof an increase in plant growth. Consequently, the amountof ERM in the soil may also increase (Staddon

et al

., 1998).Temperature also increases internal colonization, both indirectly,through increased plant growth (Staddon

et al

., 2002), anddirectly, through the fungal response (Heinemeyer &Fitter, 2004). Yet, to date, hyphal respiration rates havenever been measured directly for AM fungi (Rillig & Allen,1999) and it is not known how much ERM respirationcontributes to the carbon budget of the plant and whetherit shows similar acclimation to temperature changes as doroots (Gifford, 1995; Atkin

et al

., 2000) and soil respiration(Luo

et al

., 2001).Ecological studies increasingly use the stable isotope

13

C to follow a carbon signal through different trophic layers(Ostle

et al

., 2000; Radajewski

et al

., 2000), including AMfungi (Staddon

et al

., 1999a; Miller & Kling, 2000). Wecombined a compartment study with the application of two

13

CO

2

pulse labels during a period of warming the ERM. Weaimed to measure (i) the sensitivity of the respiration ofthe ERM of an AM fungus to temperature and whether itdisplayed acclimation; (ii) how much of the carbonallocated to the fungus from the root carbon pool is recentlyfixed carbon; and (iii) the carbon costs for the host plant ofthe ERM and the dynamics of carbon flux from the roots tothe ERM.

Materials and Methods

Growth conditions

Ten open-top Perspex boxes (23

×

23

×

10 cm) were divided inhalf using Perspex plates. These halves were each further dividedinto two compartments (A and B) with nylon mesh (pore size20 µm; Staniar P/N

°

25T11-20, John Staniar & Co., Manchester,UK), allowing passage of extra-radical hyphae only, not roots,into hyphal compartment B (Fig. 1). The growth mediumin each compartment (1200 g) consisted of a 1 : 1 mixture[volume/volume (v/v)] of coarse builders’ dried silica sand andTerragreen® (an attapulgite clay soil conditioner; TurfproLtd, Staines, UK). Bonemeal was mixed into the medium asa long-term phosphate source (0.25 g l

−

1

soil). After germinationon moist filter paper (Whatman® no. 1) for 2 d (20

°

C in thedark), two ribwort plantain (

Plantago lanceolata

L.) seedlings(Emorsgate Seeds, Norfolk, UK) were planted at the sameposition in each plant compartment A, 3.5 cm from the mesh,and thinned to one seedling after 2 wk. Compartment A wasevenly inoculated (150 g kg

−

1

soil) with fresh inoculum of

Glomusmosseae

((Nicol. & Gerd.) Gerd. & Trappe, isolate UY 316, exP. Bonfante, Torino) consisting of

c.

1 cm pieces of

P. lanceolata

and

Trifolium repens

(L.) roots within the same substrate. Controlpots received the same amount of uninoculated roots of the samespecies in the same substrate, and 150 ml of a filtrate (Whatman®no. 3M) of a further batch of the inoculum. Boxes were placed ina glasshouse for the first 61 d after planting (dap) at 14 : 25

±

3

°

C (day:night), 60–80% relative humidity and a mean photo-synthetically active radiation (PAR) of 450

±

100 µmol m

−

2

s

−

1

over the waveband 400–700 nm at plant level for 16 h d

−

1

.Supplementary light was supplied by six mercury vapour lamps(400 W) giving 200 µmol m

−

2

s

−

1

(400–700 nm) at plant levelduring early morning and evening. PAR was measured twiceweekly throughout the day at plant level using a light meter(SKD210; Skye Instruments Ltd, Powys, UK). In addition, anautomatic weather station (Delta-T Devices Ltd, Cambridge,UK) located next to the glasshouse recorded continuous measure-ments of exterior PAR flux (µmol m

−

2

s

−

1

(400–700 nm)).Soil temperature at 3 cm depth in both compartments of four

boxes was recorded every 10 min (averaged 30-s readings) at1, 3 and 5 cm distance from the mesh with thermistor probes(Grant Instruments Ltd, Shepreth, UK), and both compartmentswere watered daily with deionized water as required. From week 3onwards a half-strength Rorison’s nutrient solution (Hewitt,1966) (containing 1/10 phosphate) was given twice weekly(10 ml) to compartment A, except within 1 cm of the mesh; allcompartments were subsequently watered with deionized waterto remove any nutrient solution from the leaves.

Experimental set-up and soil heating

There were six treatments with three replicates each:

13

C-labelled mycorrhizal and nonmycorrhizal treatments and


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an unlabelled mycorrhizal treatment, all with and without soilheating. Unlabelled mycorrhizal treatments were needed toassess background

13

C contamination during the two pulselabels, to monitor mycorrhizal effects on plant growth, and toprovide a blank for calculating extra-radical mycelium (ERM)

13

C respiration. Two spare boxes were used to determine whensufficient ERM had been produced in the hyphal compart-ment to start treatments. Soil in the hyphal compartmenthad a soil warming cable (Macpenny® Cameron; East RidingHorticulture, Sutton-on-Derwent, UK) inserted with fourvertical bends at a distance of 7.5 cm from the mesh (Fig. 1).In control compartments, the cable was not connected to apower supply. To avoid solar heating of hyphal compartmentsand algal growth, reflecting shields were attached to their outersurface. Soil warming started directly after the first pulse label

(see the next section). Air temperatures in the glasshouse duringthe experimental phase were 12 : 23

±

3

°

C (day:night). Heatingflux across the mesh from the hyphal compartment had nosignificant effect on the plant compartment (less than 0.5

°

Cdirectly behind the mesh) as shown previously (Heinemeyer &Fitter, 2004), but raised soil temperature at the gas sample tubeposition (see the next section) by approx. 6

±

3

°

C. Any soilmoisture differences induced by soil warming were avoided byfrequently watering all compartments to field capacity.

Pulse label of

δ

13

C and gas sampling technique

Two weeks before the first pulse label, one gas sample tube(diameter 2.1 cm and length 7.5 cm; Universal Container;Sterilin, Stone, UK) was inserted to a depth of 2.0 cm into each

Fig. 1 Experimental set-up during the two 13CO2 labelling periods (for dimensions, see text). The Perspex box was divided into two halves byinserting a 20-µm mesh, which excluded roots but allowed the extra-radical mycelium (ERM) to grow from an inoculated-plant compartmentinto a fungus-only compartment. Each plant was covered by a sealed Perspex chamber with an inflow, an outflow and an inflow disperser fora 13C pulse label application. Fungal respiration with a 13C label was measured over time by taking subsequent gas samples with a syringe froma re-sealable sampling tube positioned in the fungal compartment. Fungal compartments were heated (approx. +6°C at sampling tube position)with a vertically inserted heating cable or were kept at ambient temperature.

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hyphal compartment, 4.5 cm from the mesh (Fig. 1), giving atotal air volume of approx. 19 ml. On the day of the first pulse(61 dap; 20 April 2001), two fitted fans (Turbo-Fan; RoofUnits Group, Dubley, UK) were switched on until the end ofthe experiment, ensuring a constant replacement of internalair with fresh air through a chimney from the top of theglasshouse. During the pulse label, each plant was coveredwith an acrylic chamber (diameter 10 cm; height 19.5 cm;volume 1530 cm

3

(i.e.

π

×

5 cm

2

×

19.5 cm) resting on twohalves of an acrylic dish which were fitted around the stem ofeach plant (Fig. 1), with the joins between chamber and dish,and between dish halves, sealed with petroleum jelly (PhilipHarris, Hyde, UK). Each chamber had an inflow (with a fitteddisperser at the bottom) and an outflow tube at the top. A stableisotope delivery system (SID; Ostle

et al

., 2000) was situated20 m from the glasshouse and 12 lines (13-mm-diametergarden hose) introduced air to the labelling chambers. Eachline was linked to a 360-l gas mix tank with individual flowgauges. The gas was continuously mixed with CO

2

-scrubbedair to approx. 360 ppm CO

2

with 50%

δ

13

CO

2

(Sigma Aldrich,Gillingham, UK) from a gas-tight 2-l bag. The six controlplants received air from outside via two pumps (3 l min

−

1

foreach channel) placed next to the fresh air inflow of the two fans.Outflow tubes were vented externally. All connections weresealed with gas-sealing teflon PTFE tape (B&Q, Eastleigh,UK). Labelling was performed from 12:00 to 15:30 h at aflow rate of approx. 2.5 l min

−

1

, avoiding condensation insidethe chambers. Outside air was scrubbed through the systemfor an additional 2 h before the chambers were removed. Thesecond pulse was applied 2 wk after the first (75 dap; 4 May2001), following the same procedure.

Suba Seal stoppers (Scientific Laboratory Supplies Ltd, Not-tingham, UK) were fitted to the gas sample tubes 4 h beforeany sampling. Gas samples (14 ml) from the tubes were takenwith syringes (25 ml) at 2 h before and 9.5, 21.5, 28.5 and 41 hafter each pulse and used to over-pressure a 10-ml sealedNa-glass Exetainer® (cat #438 W; Labco Ltd, Wycombe, UK).For each sample, a new syringe and needle were used and eachsyringe was purged with 1 ml of the sample air before sampling.Stoppers were removed once the sample had been taken.Analytical determinations of CO

2

concentration and

13

C:

12

Cratios were made at the Natural Environment Research Council,UK (NERC) stable isotope facility (CEH-Merlewood). Fordetails of the

δ13C analysis, see Ostle et al. (2000). Weatherconditions were nearly identical at the times of the two pulses,giving a mean air temperature of 25 ± 2°C and a mean peakdaytime PAR of 500 ± 50 µmol m−2 s−1.

Plant and fungal measurements

One day before the first pulse label for each plant, the maximumwidth and length of all leaves were recorded and later referencedagainst a calibration curve (obtained from scanned leaves ofthe second pulse) to calculate an estimated leaf area, from the

known fresh:dry weight ratio (see the end of this section), inorder to estimate individual shoot biomass for the first pulse.Soon after the last gas sample of each pulse and also shortlybefore the second pulse label, leaf cores (each 0.2 cm2) weretaken from five healthy, fully expanded leaves of each plant,then pooled and weighed both fresh and dry. These shootsamples and a subsample of dried roots were ground to a finehomogenous powder for determination of carbon contentand 13C:12C ratios (see the previous section).

After gas sampling of each pulse, a soil core (diameter 1.9 cm;length 7.5 cm) was taken from the same position in each plantcompartment. Root samples (< 1 mm diameter) from this soilcore were stained and investigated for colonization (Staddon et al.,1998). Roots were cleared in 10% KOH (10 min) and stained(twice) in 0.1% acid fuchsin (35 min), in both cases in a water-bath (85°C). Percentages of total root length colonized (LRC),arbuscules (LRCarb) and vesicles (LRCves) were scored separately.

Soil for extraction of ERM was taken from directly beneaththe inserted gas-collecting tubes, by using these same tubes totake soil cores (26 cm3). The sample area was then refilledwith the same soil mix. The gas-collecting tubes of each com-partment were then inserted next to this sample area as before.From each soil sample, two subsamples were taken and theirfresh weights obtained, one for oven-drying to calculatemoisture content and one for ERM extraction (Staddon et al.,1999b). ERM length could then be expressed as ERM length(m) per gram dry weight soil.

At 78 dap (7 May 2001), all plants were harvested anddivided into root and shoot. After removal of dead leaves, totalleaf and root fresh weights were recorded. Roots were thenchopped into c. 1-cm pieces and mixed with a glass rod in600 ml of water. Two subsamples were taken at random fromthis pool for measurement of root length and root fresh:dryweight ratio. Shoot dry weight (WS) and root dry weight (WR)were recorded after oven-drying at 65°C for 4 d. Leaf area androot length were measured from scanned images (WinRhizo®;Régent Instruments, Quebec, Canada). Specific leaf area (SLA)and specific root length (SRL) were both calculated. Therelative growth rate (RGR) of shoot dry weight was estimatedas the slope of ln WS over time.

Photosynthesis measurements

Net photosynthesis of P. lanceolata plants was determined bycontinuously measuring the difference between inflow andoutflow CO2 concentrations of each chamber during the first 2 hof each pulse label by taking chamber subsamples via a 6.5-mm-diameter PTFE tube fed back from the outflow lines tothe SID-Infra-red gas analyzer (IRGA) (see Ostle et al., 2000).

Calculation of carbon costs to the plant

We calculated the contribution of ERM growth and respira-tion to plant carbon uptake, with the following assumptions.

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1 Rates of ERM growth were calculated as the differencein ERM length between the start and the end of the 2-wkperiod in both the unheated and the heated hyphal com-partments and were assumed to have been constantduring this period. No allowance was made for hyphalturnover.2 Specific hyphal length was assumed to be 3.6 µg m−1 [givenby Harley & Smith (1983) for another Glomus mosseae isolate]and hyphal carbon content to be 50% (Zhu & Miller, 2003).3 ERM respiration and growth in the plant compartment wereunknown but assumed to be equal to those measured in theambient hyphal compartment for both temperature treatments.4 Total carbon fixed was calculated by assuming a constant16-h mean net photosynthesis for labelled mycorrhizal plantsduring each pulse.

Equations and statistical analysis

Values of 13C content in each gas sample could be calculatedby multiplication of atom% 13Csample (Eqn 3; derived bysubstituting Rsample in Eqn 2 with Eqn 1) by total carbon (µg)(Eqn 4):

Rsample = [(δsample/1000) + 1] × Rref Eqn 1

atom% 13Csample = (Rsample × 100)/(1 + Rsample) Eqn 2

atom% 13Csample = 100 × (δsample + 1000)/(90447.84 + δsample) Eqn 3

Csample = ppmsample × Vsample/VM/1000 × MC/1000 Eqn 4

[Rsample and Rref, 13C:12C ratios of sample and Vienna-Pee Dee

Belemnite (V-PDB) reference (0.0111797; the reference usedfor the ‰13C calculation; see http://deuterium.nist.gov/standards.html); δsample, the δ13C of the sample; ppmsample, theconcentration of CO2 (µl l−1) in the gas sample; Csample, thetotal carbon content of the sample (µg); Vsample, the volumeof the sample tube (0.019 l); VM, the molar volume understandard conditions (22.4 l mol−1); MC, the molar mass ofcarbon (12.01 g mol−1).]

Respiration in each fungal compartment (µg h−1) wascalculated per compartment, firstly as total carbon (ERMresp)(Eqn 5) and secondly as 13C (Eqn 6):

ERMresp = (Cmyc − Ccontrol) × ERM × WFcomp/(ERM × Wsample)/t Eqn 5

13C ERMresp = (13Cmyc − 13Ccontrol) × ERM × WFcomp/(ERM × Wsample)/t Eqn 6

(t = 4 h in all cases.)(Individual gas sample carbon contents (µg) were calculatedfor mycorrhizal (Cmyc) and nonmycorrhizal controls (Ccontrol),

using total Csample (Eqn 4) and 13C content (13Cmyc; 13Ccontrol),

respectively; mean values for nonmycorrhizal controlswere subtracted from those for individual mycorrhizalcompartments. Differences are expressed per hyphal com-partment using substrate weight under the gas sampletube (Wsample = 31.43 g) and in the hyphal compartment(WFcomp = 1200 g) and corresponding ERM length density(m g−1). Substrate weights could be calculated by assuming auniform substrate density of 1.21 g cm−3.)

Total 13C (mg) for shoot and root material could be calculatedby multiplication of 13Cx (mg mg−1) per shoot or root mass(Eqn 7) by total Cx (mg) in individual shoot or root samples(Eqn 8):

13Cx = Wsample × atom% 13Csample/Wsample Eqn 7

Total Cx = Wx × %Csubsample/100 Eqn 8

[atom% 13Csample, the calculated individual atom% 13C(Eqn 3); Wsample (mg) and Wx (mg), sample and total shoot orroot weight, respectively; %Csubsample, the percentage of totalcarbon in the corresponding shoot or root subsample materialused for analysis.]

Statistical analysis was performed using SPSS v10.0(Norusis, 1999). All data were checked and transformedappropriately to normalize skewed distributions before statis-tical analysis (i.e. log transformation for all but the percentagedata, for which an arcsin transformation was chosen). Datafor CO2 uptake, net photosynthesis, and plant and fungalgrowth were tested for differences between labelling treat-ments with a one-way analysis of variance (ANOVA) withlabelling as the factor. Because unlabelled controls differedfrom labelled treatments only at the first pulse and only inCO2 uptake, all other parameters of unlabelled controls wereno longer treated separately in further analyses. Effects onfungal growth were tested with a repeated measurementdesign of the general linear model (GLM) with time as thewithin-subject and temperature as the between-subject factor.Means of plant growth, plant total 13C, CO2 uptake and netphotosynthesis were tested for differences with a two-wayANOVA, with temperature and mycorrhizal treatment asfactors. Data for δ13C signals, CO2 concentrations in thegas samples, the proportion of second to first pulse label of13C:12C in the gas samples and ERM respiration were testedfor any treatment effects with the repeated measurementdesign (as above) with temperature and mycorrhizal treatmentsas the between-subject factors. For 13C ERM respiration, aone-way ANOVA with temperature as the factor was used todetect significant differences at particular harvests. Totalrespiration was tested with mean PAR received during a 12-h period before the sampling time as a covariate in a repeatedmeasures design (as above) with temperature and label asfactors, omitting the first sample of the first pulse without soilwarming.

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Results

Temperature impact on ERM respiration

Extra-radical mycelium respiration was calculated from twovariables measured in the gas samples, the concentrationof CO2 and the δ13C signal. After the first pulse label, CO2concentration showed diurnal fluctuation (Fig. 2a), beinghighest in the evening and lowest in the morning. CO2concentration was higher in mycorrhizal treatments at bothpulses. The δ13C of labelled treatments did not differ betweentemperature treatments in either pulse but was highest inlabelled mycorrhizal treatments: it increased from a naturalbackground of c. −13.0‰ to c. +1300‰ within the first 10 hand thereafter declined rapidly (Fig. 2b for the first pulse).Significant effects were the same at the second pulse butthe overall δ13C signal in the second pulse was much higher(c. +1900‰) and the difference between heating treatments inthe mycorrhizal compartments was smaller. Control treatmentsin both labelling periods had a δ13C of up to −10.0‰.

In both labelling periods, mean ERM respiration per com-partment was c. 10.0 µg h−1 in ambient treatments butcovaried with PAR received before each gas sampling (Fig. 3).Soil warming had no significant effect on respiration in eitherpulse (Fig. 3). Mean respiration per unit length of extra-radical hyphae (LERM) (data not shown) in the ambienttreatments was 2.4 ng C m−1 h−1; in the soil warming treat-ment it increased slightly after the first pulse to 3.8 ng Cm−1 h−1 but after the second pulse decreased to 1.5 ng Cm−1 h−1 (F1,7 = 19.99, P = 0.003). Respiratory 13C loss percompartment was less than 1.5 µg h−1 and peaked in bothpulses within 10 h after the start of labelling (Fig. 4); valueswere higher and declined more rapidly after the second pulse.Soil warming significantly increased respiration at the secondsampling after the first pulse (F1,4 = 7.76, P = 0.049) (Fig. 4a)but there was no warming effect after the second pulse. Thepercentage of 13C relative to total carbon respired was similarin the two pulses in the ambient treatments and declined from10% initially to less than 3% (Fig. 5). However, the 13C:12Cratio was always (but not significantly) higher in the soil

Fig. 2 (a) Mean CO2 concentrations at different hours of the day and (b) δ13C signals in the gas samples from fungal compartments for the first pulse before and after pulse labelling, during the 41 h period (a.p.l.) ± standard error; data for the second pulse were similar but are not shown. Unlabelled controls were excluded from the analysis and significance values for differences between treatments based on a repeated measures design were as follows: mycorrhizal effect: (a) (first pulse) F1,8 = 11.32, P = 0.010; (b) (second pulse) F1,8 = 9.70, P = 0.014; (a) (first pulse) F1,8 = 30.13, P = 0.001; (b) (2nd pulse) F1,8 = 33.84, P < 0.001; there were no other significant differences for either heating treatment or its interaction with mycorrhizal colonization. A, ambient; H, heated; AC, ambient unlabelled; HC, heated unlabelled; NM, nonmycorrhizal treatments.

Fig. 3 Mean total extra-radical mycelium (ERM) respiration in ambient (open diamonds) and heated (closed diamonds) fungal compartments (as the difference of respiration in mycorrhizal and nonmycorrhizal compartments) during the first (a) and second (b) pulse labels before and after pulse labelling, during the 41 h period (a.p.l.) ± standard error, and corresponding mean photosynthetically active radiation (PAR) (bars) received during a 12-h period before each measurement. There were no significant differences between temperature treatments based on a repeated measures design for either pulse; however, respiration covaried significantly with PAR: (a) F1,7 = 13.81, P = 0.007; (b) F1,7 = 6.71, P = 0.037.


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warming treatment after the second pulse (Fig. 5b), and arepeated measures analysis of the proportion of the respirationratios of 13C:12C of the second normalized by those of thefirst pulse label per temperature treatment (combination ofFig. 5a and b) showed a weakly significant temperature effect(F1,4 = 4.73, P = 0.095).

Impacts on fungal growth

Soil moisture content did not differ amongst pulse labels ortreatments (data not shown). In nonmycorrhizal treatmentsthere was no colonization of roots and LERM was < 0.04 mg−1. In mycorrhizal treatments, LERM increased between thetwo pulses but when the hyphal compartment was heatedthe increase in LERM was greater, and was 75% higher inheated than unheated compartments at the second sampling(Table 1). In contrast, total (LRC) and arbuscular (LRCarb)root colonizations did not change between the two pulses andwere unaffected by soil warming (Table 1).

Carbon budget and carbon costs for the host plant

ERM length density increased during the 2-wk period by 0.60and 2.77 m g−1 for ambient and heated treatments, respectively(Table 1). These values equate to a minimum hyphal biomassproduction in the entire box of 0.19 and 0.54 mg C d−1,

assuming constant ERM growth and no death in eithertreatment during the 2-wk period. For the ambient treatmentin the first pulse and for both temperature treatments in thesecond pulse, mean ERM respiration was c. 10.0 µg C h−1 percompartment (Fig. 3) providing an additional carbon demandfor the entire box of 0.48 mg C d−1. For the heated treatmentin the first pulse, the figures were 13.6 µg C h−1 per compart-ment and 0.57 mg C d−1 per box. Therefore, total ERM carbondemand was constant at 0.67 mg C d−1 for the ambient treatmentin both labelling periods but decreased in heated treatmentsfrom 1.11 at the first to 1.02 mg C d−1 at the second pulse. Totalcarbon demand by the ERM corresponded to less than 1% of netphotosynthesis (126 and 181 mg C d−1 for the first and secondpulses, respectively), assuming constant mean net photo-synthesis of mycorrhizal plants of 6.6 and 8.4 µmol m−2 s−1

over a 16-h light period, respectively (data not shown).

Plant 13C:12C analyses

The mean natural abundance of 13C was 4.6 mg 13C g−1 inshoot and root material of all control plants. Four days afterthe first pulse, the 13C content of shoots was enriched by anaverage of 2.8 mg 13C g−1 (data not shown) but there wereno differences among mycorrhizal or heating treatments.At the second harvest, after the second pulse, labelled plantscontained significantly more 13C than unlabelled controls in

Fig. 4 Mean extra-radical mycelium (ERM) respiration of 13C in ambient (open diamonds) and heated (closed diamonds) fungal compartments (as the difference of 13C respired from mycorrhizal and nonmycorrhizal compartments) during the first (a) and second (b) pulse labels before and after pulse labelling, during the 41 h period (a.p.l.) ± standard error. There were no significant differences between temperature treatments based on a repeated measures design including only the last four measurements in either pulse; however, there was a temperature effect only during the first pulse, at 9.5 h a.p.l., as indicated by two-way analysis of variance: (a) F1,4 = 7.76, P = 0.050.

Fig. 5 Mean ratio of 13C to total carbon respired from ambient (open diamonds) and heated (closed diamonds) fungal compartments during the last four measurements after pulse labelling (a.p.l.) for the first (a) and second (b) pulse labels ± standard error. There were no significant differences between temperature treatments based on a repeated measures design in either pulse: (b) F1,4 = 2.16, P = 0.215. Note that the prelabelling ratios (solitary open and closed data points) for each pulse are also given, but these were not used in the statistical analysis.


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both shoots (30.02 mg vs 11.7 mg; F1,14 = 7.00, P = 0.019) androots (18.9 mg vs 10.2 mg; F1,14 = 11.73, P = 0.004; Fig. 6b);further, 13C biomass content in shoots (12.1 mg) and roots(10.0 mg) was unaffected by temperature in unlabelledplants (Fig. 6b). At the second pulse, labelled mycorrhizal andnonmycorrhizal plants had similar 13C shoot contents bothbefore (30.3 mg) and after (27.2 mg) the gas sampling

period. Consequently, in all treatments, the shoot 13C contentdeclined similarly, by an average of 3.1 mg over 2 d (Fig. 6a).However, in labelled plants (Fig. 6b), mycorrhizal roots had35% less 13C content than the roots of nonmycorrhizal plants.Further, mycorrhizal roots had the lowest 13C (14 mg)under ambient temperature, although there was no significantinteraction with soil warming (P = 0.176).

Table 1 Effects of soil warming on (a) length of colonized roots (LRC) and extra-radical mycelium (ERM) length density and (b) plant growth for the first and second labelling periods

(a)

(b)

13C-labelled mycorrhizal treatment Repeated measures ANOVA

Ambient Heated Heating Pulse Heating × Pulse

First pulseLRC (%) 52.9 54.1LRCarb (%) 39.0 40.3ERM (m g−1) 3.19 3.79 ns ns ns

Second pulse ns ns nsLRC (%) 51.6 53.0 ns 7.49* 13.17**LRCarb (%) 38.3 37.9ERM (m g−1) 3.79 6.56

13C-labelled mycorrhizal treatment Nonmycorrhizal treatment Two-way ANOVA

Ambient Heated Ambient Heated Mycorrhizal

Second pulseWS (g) 2.64 2.54 2.57 2.35 nsSLA (cm2 g−1) 103.7 106.3 92.2 93.4 6.78*WR (g) 1.95 2.16 2.58 2.20 nsSRL (m g−1) 135.0 119.5 140.2 148.7 ns

(a) Significant F-ratios in the repeated measures design for the mean values of LRC, arbuscular LRC (LRCarb) and ERM length density (m per g dry weight of soil) in labelled mycorrhizal treatments for ambient and heated treatments are presented for the first and second pulse labels; neither nonmycorrhizal nor unlabelled treatment means differed significantly from these means. (b) Significant F-ratios for the two-way analysis of variance (ANOVA) for shoot dry weight (WS), specific leaf area (SLA), root dry weight (WR) and specific root length (SRL) at the final harvest (second pulse only) are presented for labelled mycorrhizal and nonmycorrhizal treatments, either ambient or heated, respectively; mean values of unlabelled plants did not differ from these means. There were no significant heating or interaction effects on plant growth in the second pulse.ns, not significant; *P < 0.05; **P < 0.01.

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Fig. 6 (a) Mean decline in 13C of shoots of Plantago lanceolata over a 2-d period after the second pulse label and (b) 13C content of roots at the final harvest ± standard error. Unlabelled controls were excluded from the analysis and significance values for differences between treatments based on a two-way analysis of variance were as follows: (a) no significant differences; (b) mycorrhizal: F1,8 = 3.76, P = 0.089; there were no other significant differences for either treatment or their interaction. A, ambient; H, heated; AC, ambient unlabelled; HC, heated unlabelled; NM, nonmycorrhizal treatments.


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Impacts on plant growth and photosynthesis

Plant growth was unaffected by warming of the hyphal compart-ment. Mycorrhizal and nonmycorrhizal plants had similar shootand root dry weights (Table 1, Fig. 7) and shoot RGR (0.059d−1) but mycorrhizal plants had significantly greater leaf areaand SLA.

The mean plant CO2 uptake during the 3.5-h period in thefirst pulse was higher for outside air controls (1.63 ± 0.10 mmolCO2) than in labelled treatments (1.20 ± 0.07 mmol CO2)(F1,16 = 14.13, P = 0.002). During the second pulse, mycor-rhizal plants had a 20% higher mean CO2 uptake (1.85 ± 0.06mmol CO2) in 3.5 h than nonmycorrhizal plants (F1,14 = 6.34,P = 0.025). However, the mean value of net photosynthesis,6.1 and 8.0 µmol m−2 s−1 for the first and second pulses,respectively, did not differ between pulses or treatments.

Discussion

We report here the first quantitative estimate of the respira-tion of the ERM of an AM fungus and its response to soilwarming, excluding background respiration. However, thereis also surprisingly little information on ectomycorrhizalrespiration. Ettema et al. (1999) and Hedlund & Augustsson(1995) reported basidiomycete (which are generally thicker asthey tend to form rhizomorphs) hyphal respiration of 40 µg Cg−1 d−1 and 0.055 µg C m−1 h−1, respectively, in comparisonwith which our findings of 0.2 µg C g−1 d−1 and 0.003 µg Cm−1 h−1 seem to be very low. However, their results were obtainedfrom field soil with the addition of glucose and antibiotics orin axenic culture, respectively.

We used gas sample tubes, which enabled us to calculatefungal compartment ERM respiration, and demonstratedan initial increase in ERM respiration under soil warming(Figs 3a and 4a), which disappeared after 2 wk of warming.This response resembles the acclimation to temperatureshown by roots (Atkin et al., 2000). However, in a warmerenvironment, longevity of hyphae might decline as reportedfor roots (Fitter, 1996; Norby & Jackson, 2000), leading toaccumulation of dead hyphae, which were not measured in

this study; the significantly lower respiration rate per unithyphal length (LERM) after 2 wk of soil warming is consistentwith the accumulation of dead hyphae, whereas the increasedratio of 13C to total carbon respired (Fig. 5b) is not, as itindicates more rapid carbon allocation to the ERM. Bothobservations could be explained by an increase in younger,and therefore thinner but also more active, mycelium undersoil warming; such an increase is likely to have occurred.Nonetheless, there was a residue of 13C label in the ERM bythe time the second label was applied. Although values in13C-labelled treatments had returned to near starting values(c. +13 and +30‰ for nonmycorrhizal and mycorrhizaltreatments, respectively), the enrichment in the second pulsewas very high (c. +1900‰), and consequently any residueeffect was negligible.

Soil warming increased ERM growth in the hyphal com-partment after 2 d and by nearly twofold after 2 wk (Table 1),as previously found (Heinemeyer & Fitter, 2004); yet therewas no difference in LRC or LR and hence total LRC, suggestingthat internal mycelium length does not determine growthof ERM as suggested by Tinker (1975) and Staddon et al.(2004). However, it is still debatable whether an increasedERM mass enters a slow turnover carbon pool in soils (Treseder& Allen, 2000; Rillig et al., 2001; Staddon et al., 2003) andit is known that the ERM length varies considerably amongspecies (Smith et al., 2004; Munkvold et al., 2004). Wesuggest that as temperature rises ERM mass will become agreater carbon sink than soil respiration, which seems toacclimate, as shown in prairie and forest studies (Grace &Rayment, 2000; Luo et al., 2001); further, even if productivityis expected to increase in a warmer climate (Parton et al.,1995), rates of decomposition of soil organic matter seem tobe unaffected by warmer conditions (Giardina & Ryan, 2000).

We also determined ERM carbon costs for the host plant,which were very similar for the two pulse periods and were< 1% of net photosynthesis, similar to the value of 0.8% givenby Jakobsen & Rosendahl (1990). However, we were onlyable to estimate the ERM in the plant compartment and didnot account for root internal colonization with dense coloniza-tion of thicker hyphae and storage vesicles; total fungal carbon

Fig. 7 Effects of mycorrhizal and temperature treatments on (a) leaf area (AL), (b) root length (LR) and (c) plant root and shoot biomass (WRS) of Plantago lanceolata at the final harvest ± standard error. Significance values for differences between mycorrhizal treatments based on a two-way analysis of variance were as follows: (a) F1,14 = 5.33, P = 0.036; (b) F1,14 = 4.00, P = 0.065; there were no other significant differences for either treatment or their interaction. A, ambient; H, heated; NM, nonmycorrhizal treatments.


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costs might then increase to over 5%, as reported by Snellgroveet al. (1982), Koch & Johnson (1984) and Cooper (1984).Further, we had to assume a similar hyphal mass for ourG. mossea strain to that used by Harley & Smith (1983),which is somewhat uncertain. In our study, the carbon costfor ERM respiration was slightly higher than for growth, asestimated by Harris & Paul (1987). Further, although theratio of 13C:12C respired (Fig. 5) was the same in ambient andheated treatments in the first pulse, it increased slightly undersoil warming in the second pulse. This shows (i) that there wasrapid carbon transport to the ERM in < 10 h, (ii) that onlyc. 10% of the carbon respired came from recently fixedcarbon, and (iii) that there was faster carbon allocation insidethe ERM under higher temperature, possibly as a result ofincreased production of young hyphae.

The finding that control plants did not show any 13C enrich-ment demonstrates that we successfully prevented contamina-tion of unlabelled plants via 13C leakage from labellingchambers; roots of labelled plants increased 13C content byc. 18 mg (Fig. 6b), sufficient to follow a 13C signal into thehyphal compartment. Mycorrhizal plants had less labelledcarbon than nonmycorrhizal plants in the roots, but not inthe shoots. Although the difference in root 13C was onlymarginally significant (P = 0.089), it may demonstrate alloca-tion of fixed carbon from mycorrhizal roots to the AM fungus(Jakobsen & Rosendahl, 1990) without affecting the shootcarbon pool. However, warming of the ERM did not result ina further decrease.

Diurnal differences in measured CO2 concentrations (Fig. 2a)reflected different ERM activities, corresponding to thecarbon fixation of the plants: the concentration of root sucrose,which is believed to be one of the main forms of carbon trans-ported to the fungus (Bago et al., 2003), increases duringthe day, consistent with peak activity of the AM fungus duringand shortly after the photoperiod. Further, as ERM respirationclearly depended on short-term PAR effects, our findingscould be explained both by a transfer mechanism based onleakage of carbon compounds into the apoplast followed byfungal capture, and by a transfer mechanism with greaterregulation by the plant. There are two technical problemsin measuring hyphal respiration as influenced by the plantcompartment. First, mycorrhizal roots may have higherrespiration rates than nonmycorrhizal roots (Solaiman &Saito, 1997); because we assumed that the 24% greater rootlength in the nonmycorrhizal plants (Fig. 7b) should havecounteracted such an effect, we might have underestimatedERM respiration. Secondly, δ13C signals in the first pulsewere highly variable, as is often the case (Figs 2b and 4a),which made it difficult to test statistically for differences.The variation might reflect either individual differences inthe growth of the hyphal front or differences in ERM activityamong replicates.

Plants showed typical mycorrhizal responses – increasedleaf area, increased SLA and reduced root length (Harris &

Paul, 1987; Gavito et al., 2001) – and mean net photosynthesisranging between 4.5 and 8.8 µmol m−2 s−1 was very similar todata for this species obtained by Staddon et al. (1999a) undersaturated light. Hyphal length densities were between3 and 7 m g−1, within the range reported for glomaleanfungi in other compartment experiments ( Jakobsen et al.,1992; Hodge et al., 2001) but lower than values obtained inpot experiments or field data. The latter sometimes exceed100 m g−1 (Sylvia, 1990), although much of this may beeither dead (Sylvia, 1988) or nonmycorrhizal (Sylvia, 1986)material.

Photosynthesis is expected to increase under highertemperature and elevated CO2 (Ceulemans et al., 1999) ifwater supply is not limited. Plants will then need an increasednutrient supply to maintain higher growth rates (Olesniewicz& Thomas, 1999). The greater amount of ERM in the warmerenvironment found here will increase the effectiveness withwhich the host plant explores soil and if, as suggested byStaddon & Fitter (1998) for elevated CO2, more carbon wereavailable to the fungus, the positive growth response of theERM of AM fungi might well supply this increased nutrientdemand in a warmer climate.

In this study, we have obtained evidence for faster carbonallocation to and increased respiration of the ERM under highersoil temperature, but also rapid acclimation and an immediateresponse to changes in available carbon from a previouslymixed root carbon pool. The positive growth response ofthe ERM to higher temperature together with acclimationof its respiration might lead to a significant increase in carbonaccumulation in soils when the climate becomes warmer.However, light seems to be the overall controlling factor incarbon allocation to the fungus. This study underlines theimportant role of the AM fungal symbionts in global carboncycling and suggests that a more mycocentric view in ecologicalstudies should be considered in future climate modelling.

Acknowledgements

The authors are very grateful to C. Abbott, who lookedafter the plants. We would also like to thank P. Wilson, whoprovided us with the AM inoculum. This work was partlyfunded by the NERC (UK), the Evangelische Studienwerk(Germany) and the University of York.

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