The direct response of the external mycelium of arbuscular mycorrhizal fungi to temperature and the implications for nutrient transfer

Soil Biology & Biochemistry 78 (2014) 109e117

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The direct response of the external mycelium of arbuscular mycorrhizal fungi to temperature and the implications for nutrient transfer Gracie Barrett a, b, 1, Colin D. Campbell b, c, Angela Hodge a, * a b c

Department of Biology, University of York, Wentworth Way, York YO10 5DD, UK James Hutton Institute, Craigiebuckler, Aberdeen AB15 8QH, UK Department Soil and Environment, Swedish University of Agricultural Sciences, Box 7014, Uppsala SE-750 07, Sweden

a r t i c l e i n f o

a b s t r a c t

Article history: Received 21 April 2014 Received in revised form 16 July 2014 Accepted 18 July 2014 Available online 11 August 2014

In this study we investigated the direct effects of temperature on the extra-radical mycelium (ERM) of arbuscular mycorrhizal fungi (AMF) and the resulting impact on the host plant nutrition and biomass production. Plantago lanceolata L. plants colonized by Glomus hoi (experiment 1) and either G. hoi or Glomus intraradices (experiment 2) were grown in compartmented microcosm units. AMF hyphae, but not roots, were permitted access to a second compartment containing a 15N:13C dual-labelled organic patch maintained at different temperature treatments. All plants were maintained at ambient temperature. AMF hyphal growth in the patch compartments was relatively insensitive to temperate but results were variable. G. hoi hyphal length density was 5 times higher at ambient (c. 24
C) than cooled (c. 11
C) temperatures but only at the end of the first experiment (105 d after patch addition). In contrast, in the second experiment (86 d after patch addition) AMF hyphal growth was unaffected by temperature in the patch compartment. These differences between experiments are likely due to large variation among replicates in the ERM produced and differences in how the organic patch was applied. In experiment 2, plant biomass and phosphate content differed according to the temperature at which the hyphae of both AMF species grew. Plant biomass was greater when the AMF were grown at c. 18
C than c. 11
C but was no different at c. 21
C. These data show that direct temperature responses by the external hyphae of AMF can independently influence associated host plant growth. However, there were also important differences between the two AMF studied both in the amount of nutrients transferred and the distribution of the nutrients. © 2014 Elsevier Ltd. All rights reserved.

Keywords: Arbuscular mycorrhizal fungi Global climate change Extra-radical mycelium (ERM) Nitrogen Phosphorus Organic material

1. Introduction Most plants form arbuscular mycorrhizal (AM) associations with fungi from the phylum Glomeromycota (Smith and Read, 2008). Whilst uptake of phosphorus (P) is thought to be the most important benefit derived from AM association (Mosse et al., 1973; Smith and Read, 2008), AM fungi (AMF) can also take up inorganic nitrogen (N) from organic material and transfer this to their associated plant host (Hodge et al., 2001; Atul-Nayyar et al., 2009; Leigh et al., 2009). As soil organisms, AMF likely show strong responses to a range of edaphic factors including temperature (Fitter et al., 2000; Tibbett and Cairney, 2007) yet, while the effects of temperature and

* Corresponding author. Tel.: þ44 (0)1904 328562; fax: þ44 (0)1904 328505. E-mail address: [email protected] (A. Hodge). 1 Present address: Royal Horticultural Society, Wisley, Woking GU23 6QB, UK. http://dx.doi.org/10.1016/j.soilbio.2014.07.025 0038-0717/© 2014 Elsevier Ltd. All rights reserved.

other environmental global change factors on plant physiology have been well studied, few plant studies have quantified the impact directly upon the AMF symbiont (see Fitter et al., 2004; Hughes et al., 2008), even though it is known that AMF can alter their host plant responses to, for example, temperature (Atkin et al., 2009). Given the near ubiquity of the AM association, and the fact that they are likely to be increasingly important in future sustainable agricultural systems (Gosling et al., 2006; Rooney et al., 2009; Verbruggen et al., 2010; Fitter et al., 2011) this knowledge gap represents a significant problem when it comes to predicting the way in which plants will respond to predicted temperature rises. Investigating AMF responses to temperature directly, however, is complicated by the fact that as these fungi are obligate biotrophs and so rely entirely on their host plant for their carbon (C) supply, they cannot be grown separately. Therefore, not only will the AMF respond directly to the physiological effects of a change in

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temperature, but, are also subject to indirect effects driven by the concurrent response of their host plant which may include an altered carbon allocation belowground, which, in turn, will impact upon the growth of the fungal symbiont (Fitter et al., 2000; Heinemeyer and Fitter, 2004). AMF comprise an intra-radical mycelium (IRM) growing within the plant root itself and an extra-radical mycelium (ERM) extending from the root into the surrounding soil. Most studies to date have investigated AMF temperature responses by either exposing a whole colonised plant (Hetrick and Bloom, 1984; Kytoviita and Ruotsalainen, 2007) or just the colonized roots (Smith and Roncadori, 1986; Wang et al., 2002; Ruotsalainen and Kytoviita, 2004) to different temperature regimes. Thus, these studies report the combined effects of temperature on both symbiont partners and not the direct impact upon the fungus itself. Furthermore, the response of the ERM to temperature is seldom considered. In general, internal colonisation increases with temperature between 10
C and 30
C (Smith and Roncadori, 1986; Matsubara and Harada, 1996; Wang et al., 2002) and reduced colonisation is consistently reported below 15
C (Hetrick and Bloom, 1984; Zhang et al., 1995). Moreover, AMF colonisation lead to enhanced plant P capture compared to non-AM plants, but only at temperatures of 15
C and above (Wang et al., 2002; Kytoviita and Ruotsalainen, 2007; Karasawa et al., 2012). Similarly, Ruotsalainen and Kytoviita (2004) reported that AMF colonisation leads to enhanced shoot N content at 17
C but not at 12
C. Several other studies also report that AM plants grow more poorly than non-AM plants at temperatures below c. 15
C but better above c. 15
C (Baon et al., 1994; Liu et al., 2004; but see; Rooney et al., 2011). This suggests that the benefit of being AMF to a host plant is temperature-dependent. Such temperature driven changes in mycorrhizal benefit might reflect either a direct physiological response of the fungus or changes in host C allocation. Since the primary benefit to plants of AMF colonisation is the enhancement of plant nutrition resulting from the ability of the ERM to acquire nutrients from soil, any factor which limits the growth of the ERM might reduce that benefit (e.g. Leigh et al., 2011). The few studies of temperature responses of AMF that have considered the ERM show that its' growth is more limited by lower temperatures than that of roots (Liu et al., 2004; Kytoviita, 2005; but see; Karasawa et al., 2012). Temperatures below 15
C often lead to a reduction (Gavito et al., 2003; Liu et al., 2004; Hawkes et al., 2008) or complete suppression of ERM growth (Gavito et al., 2005) and optimal growth temperatures vary among AMF species (Gavito et al., 2005). To date, only a few studies have measured the direct impact of temperature on ERM growth: Heinemeyer et al. (2006) found that growth of the ERM doubled when warmed by 6
C whilst keeping the host plant at an ambient temperature of c. 12/23
C (night/day), whereas Heinemeyer and Fitter (2004) found only transient effects on growth of the ERM from warming the ERM by 8
C while the host plant remained at c. 12
C. They report however, that the specific root length of the host plant increased, suggesting that direct effects of temperature on AMF growth may have indirect impacts on host plant growth. Karasawa et al. (2012) investigated the impact of a short period of soil chilling on ERM growth, respiration and 13C allocation and found no differences compared to when the soil was not chilled. In contrast, respiration and 13C content of the roots was reduced by chilling. Thus, if, as the majority of these studies suggest, AMF are more temperature sensitive than roots to low soil temperature this would be expected to have large implications for nutrient capture via the fungal symbiont. In this study we conducted two experiments to investigate the direct impacts of temperature on the ability of two AMF species, Glomus intraradices and Glomus hoi, to grow in, and transfer nutrients (N and P) from, an organic nutrient patch. In the first

experiment G. hoi alone was screened for its ability to transfer nutrients under cooled versus ambient temperature conditions. Microcosms in which AMF ERM but not plant roots were permitted access to a compartment containing an organic patch of 15N:13C dual-labelled grass shoots were used. The ‘no AMF access’ treatment was included to determine if N movement via mass flow or diffusion was an important N transfer pathway under these experimental conditions. As the latter was found not to be the case in the second experiment we omitted these ‘no AMF access’ controls, thus allowing two AMF species and a greater number of temperature treatments to be examined. In the second experiment, G. intraradices was also included because its growth has been reported to be severely repressed at temperatures below 15
C (Smith and Roncadori, 1986; Liu et al., 2004; Gavito et al., 2005) while G. hoi was found to grow and transfer nutrients to its host plant even at temperatures of 10e12
C (Barrett et al., 2011). In both experiments the temperature of the patch compartment was cooled to varying degrees whilst the host plant in the adjacent compartment always remained at ambient temperature. We tested the following hypotheses: (i) that AMF promote nutrient cycling by capturing nutrients from decomposing organic matter and (ii) that AMF effectively capture nutrients even when temperature is reduced. We further hypothesised that (iii) growth of G. intraradices hyphae would be more adversely affected than G. hoi, as G. intraradices is frequently reported to be temperature sensitive (Gavito et al., 2005; but see; Lekberg et al., 2011). 2. Materials and methods 2.1. Experimental set-up Two microcosm experiments were carried out in successive years; experiment 1 (Expt 1) began on 25th May 2007, experiment 2 (Expt 2) on 23rd November 2008. In Expt 1 the growth and N capture ability of the AM fungus G. hoi was compared at ambient (c. 24
C) or a cooled (c. 11
C) temperature treatment. In Expt 2, the growth and nutrient (N and P) capture ability of two AM fungi, G. hoi (isolate number UY 110, University of York) and G. intraradices (isolate BB-E, Biorhize, Dijon, France), were compared at 4 different temperature treatments; c. 11, 14, 18 or 21
C. In both experiments the plant compartment was maintained at the ambient temperature in the glasshouse. In Expt 1, destructive harvests took place at 30 and 105 d after patch addition. In Expt 2, at 36 d a nondestructive harvest took place followed by a full destructive harvest at 86 d. These sampling and harvests time points were broadly based on previous work examining temperature impacts on AMF development in similar experimental systems (e.g. Barrett et al., 2011; Karasawa et al., 2012). Although changes to the nomenclature of many AMF species have recently been proposed (e.g. Krüger et al., 2012; Redecker et al., 2013), here we retain the previous name of ‘G, intraradices’ given the phylogenetic position of the particular isolate used in this study is uncertain. AMF cultures of G. hoi and G. intraradices were established three months prior to the start of experiments, in pots with Plantago lanceolata in a sand:Terragreen mix (see below) with 0.25 g l1 bone meal (Vitax, Leicestershire, UK), a complex P and N source to encourage AMF development. Either, 50 g fresh weight of G. hoi (Expt 1 and 2) or G. intraradices (Expt 2 only) inoculum comprising root pieces and growth medium was added to the plant compartments of the experimental microcosms. 2.2. Microcosm set-up Microcosm units (adapted from Hodge and Fitter, 2010) were made by joining two open top plastic boxes (each

G. Barrett et al. / Soil Biology & Biochemistry 78 (2014) 109e117

14.5  14.5  15 cm), to form two compartments. The first compartment (plant compartment) was separated from the second (patch compartment) by a double thickness nylon mesh barrier. In Expt 1, half (n ¼ 16) of the microcosms contained a 20 mm nylon mesh membrane which allowed the passage of AMF hyphae but not roots (Fig. 1a). The other half contained a 0.45 mm mesh (Anachem, Bedfordshire, UK) which allowed diffusion of compounds between compartments but prevented the passage of both roots and AMF hyphae. In Expt 2, all 32 units contained a 20 mm mesh barrier thus permitting AMF hyphal growth into the patch compartment. Microcosms were filled with an autoclaved (121
C; 30 min) sand:Terragreen® (an attapulgite clay soil conditioner; OIL-DRI Ltd, Cambridgeshire, UK) mixture (1:1 v/v) together with an AMF inoculum (as above) and 0.3 g of bone meal (Vitax, Leicestershire, UK). P. lanceolata L. seeds (Emorsgate Wild Seeds, Nottingham, UK) were surface sterilised in 100% bleach for 5 min, rinsed in deionised water and pre-germinated on moist filter paper (3 d at

111

20
C in the dark). Three germinated seeds were then transferred to each plant compartment and microcosms were transferred to a glasshouse. Seedlings were thinned to one per unit after 2 weeks. Average, maximum and minimum temperatures in the glasshouse were recorded daily over the course of both experiments and were 21.4 ± 4.9
C (maximum, 34.3
C; minimum, 14.3
C; Expt 1) and 19.1 ± 2.6
C (maximum, 24.1
C; minimum, 14.9
C; Expt 2). Photosynthetically active radiation (PAR) levels were recorded weekly at midday and averaged 220 ± 70 mmol m2 s1 (Expt 1) and 205 ± 45 mmol m2 s1 (Expt 2). In Expt 2 natural light was supplemented with 400 W halogen bulbs in the mornings and evenings to extend the photoperiod to 16 h. In both experiments, the moisture content of the growth medium in both compartments was maintained by checking daily with a soil moisture probe (ThetaProbe type ML2x, Cambridge, UK) and adjusting to 0.081 m3 m3 e 0.104 m3 m3 with de-ionized water as required. Two weeks after planting, plants were fed with 50 ml of low N (2.5 mmol l1 of NH4NO3) and P (0.034 mmol l1 of NaH2PO4.2H2O) nutrient solution (as Leigh et al., 2009) twice weekly. Organic patches and temperature treatments were started 30 d after seedling addition to the microcosm units (see below). 2.3. Temperature treatments During the set-up of the patch compartments 115 cm lengths of polyvinyl chloride PVC tubing (4 mm ID, Nalgene, Roskilde, Denmark) were coiled inside the compartment space (Fig. 1), this was then attached to a purpose built cooling system. A coolant solution (anti-freeze and tap water; 1:1 v/v) at ± 2.5
C was pumped through the coils of a line of 4 microcosm units then returned to the reservoir. Consequently, the average temperature of the patch compartment was reduced relative to that of the glasshouse. During set-up, the patch compartment of all microcosm units was wrapped in a layer of double-thickness bubble wrap for insulation. Polystyrene inserts (11.5  11.5 cm, 2.5 cm thickness) were inserted each side of the dividing membrane between the two compartments (Fig. 1a) to prevent the adjacent plant compartment from being cooled (Table 1). In Expt 1, four lines of four microcosm units were set-up, producing 16 replicates for the cooled patch compartment treatment (8 with 20 mm and 8 with 0.45 mm mesh). A further 16 microcosm units were set-up exactly as above but were not connected to the

Table 1 Mean, maximum and minimum temperatures of the patch compartment and adjacent mean plant compartment temperature over the course of the two experiments. In experiment 1 there were two temperature treatments (cooled and ambient). In experiment 2 there were 4 temperature treatments each position number refers to the distance of the microcosm unit from the coolant reservoir, position 1 being nearest the coolant reservoir, and therefore the coolest, and position 4 the furthest from the coolant reservoir and thus the warmest. Temperature treatment

Fig. 1. Experimental set-up for the temperature treatments for the two experiments (Expt 1 and 2). a) Microcosm set-up in Expt 1. The patch compartment was cooled by pumping a coolant solution through a PVC tubing coil. Polystyrene panels either side of the membrane insulated the plant compartment from the temperature changes in the adjacent patch compartment. Half the microcosm units contained a 0.45 mm mesh between the compartments which prevented both root and arbuscular mycorrhizal fungi (AMF) hyphal access to the second (patch) compartment. This allowed measurement of N movement via mass flow or diffusion from the patch compartment to the plant in the absence of AMF hyphae. b) Generation of the four patch compartment temperature treatments in Expt 2. A chilled coolant solution was pumped around the patch compartments of four microcosm boxes. Each pump was connected to two lines of four microcosm boxes. Numbers 1e4 indicate the position of the units from the coolant reservoir with position 1 being the coolest and position 4 the warmest (see Table 2). The arrows indicate the direction of the water flow around the system.

Experiment 1 Patch Compartment Mean (
C) Maximum (
C) Minimum (
C) Plant Compartment Mean (
C)

Cooled

Ambient

10.8 ± 0.2 17.0 7.4

23.7 ± 0.3 27.6 18.6

22.2 ± 0.2

22.3 ± 0.3

Experiment 2 Patch Compartment Mean (
C) Maximum (
C) Minimum (
C) Plant Compartment Mean (
C)

Position 1

Position 2

Position 3

Position 4

11.2 ± 0.1 14.1 9.4

13.5 ± 0.2 16.3 11.5

17.6 ± 0.5 20.4 15.7

21.1 ± 0.2 24.4 19.4

22.1 ± 0.1

22.2 ± 0.2

22.2 ± 0.2

22.1 ± 0.1

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cooling system (ambient patch compartment treatment). Temperature readings from the cooled patch compartments, a random selection of 10 ambient patch compartments and 5 plant compartments from each treatment, were recorded daily at midday from thermometers at patch depth (c. 5 cm; Table 1). Cooled units were randomly rotated within each line weekly, to minimise any effects of temperature increase. Ambient units were placed alongside the cooled ones and rotated in the same way to prevent environmental gradients influencing growth. Four treatments were established: 1) AMF hyphae (AMF) were permitted access to a cooled (C) patch compartment (i.e. C þ AMF). 2) AMF hyphae were excluded from a cooled patch compartment (i.e. C  AMF). 3) AMF hyphae were permitted access to an ambient (A) patch compartment (i.e. A þ AMF). 4) AMF hyphae were excluded from an ambient patch compartment (i.e. A  AMF). In Expt 2, the outlet of each pump was fitted with a polypropylene Y-s connector (inner diameter 4e5 mm, VWR international Ltd, Lutterworth UK). This allowed each pump to be connected to two lines of four microcosms, doubling the replicate number (n ¼ 32). The set-up was modified (see Fig. 1b) to create four temperature treatments in which there was an increasing mean patch compartment temperature from the first box (position 1) nearest the coolant reservoir to the fourth box (position 4) furthest from the coolant reservoir. This was achieved by modifying the length of the tubing coils and by using variable lengths of PVC tubing to connect microcosm units so that the coolant became progressively warmer as it travelled along the line. Loops between positions 2 to 3 and 3 to 4 on the line were passed through warm water baths (35
C) to enhance the temperature change (Fig. 1b). Thermometers were used to monitor the temperature of all patch compartments and two plant compartments for each of the four positions. Values were recorded daily at midday (Table 1). 2.4. Patch material Lolium perenne L. shoots dual-labelled with 15N and 13C were generated as previously described (Hodge et al., 1998). In Expt 1, 1 g of this dried and milled labelled shoot material was mixed with 5 g of autoclaved (121
C; 30 min), oven-dried sand and 4 g fresh weight (FW) of sieved (710 mm) loam soil, placed inside a 10  10 cm, 20 mm mesh bag and added to the microcosm compartment as the patch. The loam soil (pH 6.8 in 0.01 M CaCl2) was collected from an experimental garden at the University of York, UK, and which has had no fertiliser addition for at least 10 years. The patch material contained 41 mg N (15.5 At% 15N) and 664 mg C (2.5 At% 13C). In Expt 2, the patch added to the compartment was 1 g of the labelled shoot material mixed with 11 g of an autoclaved, oven-dried sand:Terragreen® mix (1:1 v/v) and placed in a similar mesh bag. In Expt 2 the patch contained 25 mg N (19.1 At% 15N) and 200 mg of C (2.6 At% 13C). 2.5. Plant and fungal measurements In Expt 2 for the 36 d non-destructive harvest, a soil core was taken 2 cm from the edge of the patch. From this, two sub-samples of 4 g each were used to determine the hyphal length density surrounding the patch. A second core was taken from the plant compartment, from which roots were extracted, washed and stained to determine mycorrhizal root colonisation (as Hodge, 2003a). The other harvests in both Expts 1 and 2 were destructive and in each the dry weight (DW) of shoots and roots were recorded. Root sub-samples were stained and percentage root length colonization (%RLC) assessed at  250 magnification; vesicle and arbuscule frequency was also recorded for a minimum of 100 intersections (McGongle et al., 1990). The abundance of internal

mycorrhizal hyphae at each intersect was estimated to determine colonisation intensity. The proportion of the root cortex colonised with internal hyphae was assigned a class (1e25, 25e50, 50e75, 75e100%) and the result was expressed as a percentage by calculating (25x þ 50y þ 75z þ 100t)/(x þ y þ z þ t), where x, y, z, t, were the number of intersects in each class (adapted from Plenchette and Morel, 1996). The patch was divided into three and the FW recorded. Two of these sub-samples (of 4 g each) were used to determine external hyphal length densities (ERM growth), while the third was dried at 65
C until a constant DW. External mycorrhizal hyphae were extracted using a modified membrane filter technique (Hodge, 2003b) and a minimum of 50 fields of view counted at 125 magnification using the gridline intercept technique (Hodge, 2001). AMF hyphal lengths (m) were then converted to hyphal densities per g DW of the patch material contained within the mesh bags (see above). The dried root, shoot and patch material were milled to a fine powder for measurement of total C, N 13C and 15N using continuous flow-isotope ratio mass spectrometry (CF-IRMS). Enrichment of the root and shoot material with 15N and 13C was calculated accounting for the natural abundance of the stable isotopes. In Expt 2, dried plant material was also used to determine shoot phosphorus concentration after triple acid digestion using the molybdenum blue method (Allen, 1974). 2.6. Data analysis In both experiments plant and fungal data were analysed with ANOVA using SPSS v. 16.0 for Windows (SPSS Inc., Chicago, IL, USA) after testing for normality and transforming where appropriate. When interactions occurred between factors Duncan's multiple range post hoc tests were applied to establish differences between treatment means. In Expt 1 at 105 d, one plant in the treatment in which hyphae were permitted access to a cooled patch compartment (C þ AMF) died and was therefore excluded from the analysis. In Expt 2, the mean daily temperature of each replicate patch compartment over the duration of the experiment was calculated. The relationship between this and plant and fungal variables was then determined separately for the two AMF species using a linear or quadratic best fit line where appropriate. Where more than one order of polynomial could be used to describe a significant relationship (e.g. linear and quadratic) an F-ratio test (Sokal and Rohlf, 1981; Potvin et al., 1990) was performed to determine whether using the higher order polynomial was appropriate. Relationships between internal colonisation (% RLC, % arbuscules and % vesicles) and plant P and 15N contents and patch N and C were also determined in the same way. In both experiments, Spearman rank-order correlation was used to test the association between internal and external AM fungal growth vs. plant 15N content, N concentration, N content and biomass. In Expt 2, the percentage of patch 15N detected in the plant tissue were analysed using non-parametric tests (i.e. KruskaleWallis test (H) for the influence of the temperature gradient and the ManneWhitney U test for the effect of AMF species) as the data was not normally distributed even after transformation. Differences referred to in the text are statistically significant with P < 0.05 unless otherwise stated. 3. Results 3.1. The effect of compartment temperature on AMF hyphal growth In Expt 1, although fungal hyphae were observed in the organic patches where G. hoi hyphae was excluded, hyphal length densities were much lower compared to the G. hoi access permitted

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treatment (i.e. 0.19 ± 0.02 m g1; G. hoi excluded vs. 1.31 ± 0.50 m g1; G. hoi access), and showed no increase through time or with temperature. In contrast, where G. hoi was permitted access there was an interaction between patch compartment temperature and time (F1,11 ¼ 4.54, P ¼ 0.05) (although temperature alone was not a significant factor) because, while growth at 30 d after patch addition was unaffected by temperature (0.21 ± 0.05 m g1, mean of the two temperature treatments), by 105 d c. 5 times more hyphae was present in the ambient (3.89 ± 1.10 m g1) compared with the cooled (0.80 ± 0.19 m g1) patch compartments. In Expt 2, hyphal length densities at the first harvest (36 d) were low for both AMF species (0.93 ± 0.22 m hyphae g1 for G. hoi and 0.22 ± 0.14 m hyphae g1 for G. intraradices) and unaffected by patch compartment temperature (data not shown). Between the 36 d and 86 d harvests hyphal densities within the patch increased 2.5  for G. hoi and more than 35  for G. intraradices. Thus, counter to our third hypothesis that G. intraradices hyphal growth would be more sensitive to lower temperatures than that of G. hoi, at the destructive harvest (86 d) G. intraradices consistently produced at least double the hyphal length density of G. hoi in the patch compartment (i.e. 2.93 ± 0.95 m g1 G. hoi v 7.94 ± 1.26 m g1 G. intraradices). Moreover, even at the coolest patch compartment temperature (position 1; c. 11
C), G. intraradices produced greater hyphal lengths than G. hoi (i.e. 2.41 ± 1.13 m hyphae g1 for G. hoi and 6.43 ± 0.82 m hyphae g1 for G. intraradices). Although mean hyphal length densities tended to be higher at position 3 (c. 18
C), than the coolest position (i.e. position 1; c. 11
C) for both AMF species, considerable variation in hyphal lengths within temperature treatments meant that this effect was not significant (data not shown). 3.2. The effect of compartment temperature on AMF colonisation Whilst patch compartment temperature had a substantial effect on the external hyphal growth of G. hoi by the end of Expt 1, this had little impact on the intraradical mycelium development. The % RLC by G. hoi actually decreased (F1,23 ¼ 13.25, P < 0.001) between 30 d (59 ± 4%) and 105 d (43 ± 3%) and neither %RLC, colonisation intensity or arbuscule frequency were significantly influenced by patch compartment temperature or patch access. Similarly, in Expt 2, root colonisation by both AMF species was not affected by the patch compartment temperature experienced by the ERM. There was, however, significant differences in the total %RLC (20 ± 2% G. hoi v 59 ± 3% G. intraradices), colonisation intensity (46 ± 2% G. hoi v 59 ± 1% G. intraradices) and arbuscule frequency (5 ± 1% G. hoi v 20 ± 2% G. intraradices) between the two AMF species.

113

In Expt 2, the impact of patch compartment temperature on host plant 15N content varied between the two AMF species. Root 15N content in plants colonised with G. hoi (but not G. intraradices) was positively and linearly related to temperature (Fig. 2). As in Expt 1 this impact of temperature did not occur in the shoots. There were also clear intraspecific differences in host 15N content depending on the AMF species the plants were associated with. In contrast, the percentage of patch N subsequently detected in the host plant was not significantly (H3 ¼ 1.625; P ¼ 0.654) affected by the temperature of the patch compartment. At 86 d, plants colonised by G. intraradices had captured on average 3 times more 15N from the organic material than those colonised by G. hoi (Table 2). This was the case even when G. intraradices was growing at the coolest patch compartment temperature (position 1; c. 11
C) indicating G. intraradices retained the ability to capture and transfer N at lower temperatures, again counter to our third hypothesis. As for Expt 1 13 C levels in the host plant shoot or root material was never enriched above background levels. In both experiments, no correlation was found between hyphal length density in the organic patch and host plant 15N content. In contrast, both root (Fig. 3a) and shoot 15N capture (Fig. 3b) were positively related to %RLC in plants colonised by G. intraradices. Similarly, vesicle frequency was also positively and linearly related to 15N capture in plants colonised by G. intraradices (mg 15 N ¼ 0.0722 þ 0.0249 %Vesicles, R2 ¼ 46%, F1,14 ¼ 11. 46, P ¼ 0.004). 3.4. Impact of AM fungal compartment temperature on host plant growth and nutrient status In Expt 1, plants produced more total biomass (F1,23 ¼ 5.54, P ¼ 0.028) when the AMF (G. hoi) had access to the patch compartment (3.2 ± 0.4 g) compared to when it was excluded (2.5 ± 0.2 g), although patch compartment temperature had no effect on plant biomass or total N content. The patch compartment
temperature experienced by G. hoi (i.e. 24 v 11 C) did, however, impact upon plant N concentration, which was higher (F1,11 ¼ 6.57, P ¼ 0.026) when plants were associated with G. hoi ERM accessing the ambient (11.61 ± 1.0 mg g1), than the cooled (8.6 ± 0.6 mg g1) patch compartment. In contrast to Expt 1, in Expt 2 total plant biomass was influenced by the temperature of the patch compartment experienced

3.3. The impact of temperature on fungal nitrogen transfer from the organic patch In Expt 1, host plant 15N content was c. 20 times greater when AMF hyphae were permitted access to the organic patch (0.21 ± 0.05 mg 15N) compared with when they were excluded (0.01 ± 0.001 mg 15N), thus supporting our first hypothesis, and also suggesting that N transfer via diffusion or mass flow was low in this system. In contrast, no 13C enrichment was detected in the plant tissue. Patch compartment temperature did have some impact on the amount of AMF 15N transferred from the patch to the host roots, but not to the shoots, as whilst there was little impact of temperature on root 15N content at 30 d, by 105 d it was higher when G. hoi had access to an ambient (0.15 ± 0.03 mg 15N) compared with a cooled (0.06 ± 0.02 mg 15N) patch compartment. Thus, there was only weak support from Expt 1 for our second hypothesis that AMF effectively capture nutrients even when temperature is reduced.

Fig. 2. The relationship between mean patch compartment temperature (
C) and root 15 N content (mg) in plants colonised by Glomus hoi at 86 d in Expt 2 (Log10 mg 15N ¼ ()2.48 þ 0.07
C, R2 ¼ 41%, F1,14 ¼ 9.89, P ¼ 0.007; n ¼ 16). Note the log scale on the Yaxis.

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Table 2 The effect of arbuscular mycorrhizal (AM) fungus (Glomus hoi or G. intraradices) on host plant biomass and nutrient content from Expt 2. Plant variables were measured at the destructive (86 d) harvest. Plant biomass and N and P concentrations were analysed by a two-way ANOVA with the temperature gradient and AM fungal species as the main factors. The temperature gradient was only a significant factor in the case of plant biomass (see Fig. 4), while the interaction between AM fungi and temperature gradient was never significant. Thus, only the differences between the two AM fungi are shown in the Table. The percentage of total patch15N captured by the plant did not show a normal distribution, even after transformation and therefore was analysed by the non-parametric ManneWhitney U test. Data are means ± standard errors (n ¼ 16 for each AM fungal species). G. hoi Percentage (%) of total patch15N captured Plant N concentration (mg g1)a Plant P concentration (mg g1)a Plant biomass (g)a a

G. intraradices

Test statistic

P

4±1

15 ± 3

Z ¼ () 3.241

0.001

12.9 ± 0.8

14.6 ± 0.7

F1,24 ¼ 2.994

0.096

2.1 ± 0.1

3.0 ± 0.2

F1,24 ¼ 18.435

<0.001

3.4 ± 0.3

3.1 ± 0.2

F1,24 ¼ 0.555

0.464

Data were log10 transformed before statistical analysis.

by their associated AMF symbiont although, strikingly, this difference only occurred when their associated AMF experienced the coolest patch compartment (at position 1, c. 11
C) compared to the patch compartment temperature at position 3 (c. 18
C) (Fig. 4). In contrast, there was no significant difference in plant biomass when the associated AMF partner was present in the coolest (position 1, c.

11
C) and warmest (position 4, c. 21
C) patch compartment (Fig. 4). This effect occurred in both root and shoot biomass separately (data not shown), and was independent of the species of AMF partner involved. Again, there was no evidence that biomass of plants associated with G. intraradices was reduced compared to plants associated with G. hoi when experiencing the lowest patch compartment temperature. When the relationship between total plant, root or shoot biomass and temperature was tested for plants colonised by each AMF species, a significant relationship was found only for plants colonised by G. hoi and only in plant shoots. This relationship was quadratic rather than linear (Fig. 5a). The AMF species with which the plants were associated had no overall mean effect on plant biomass. Total plant N content and concentration were not affected by the temperature the AMF partner experienced in the patch compartment. However, plant P content was lowest in plants whose AMF partner experienced the coolest patch temperature (position 1 c. 11
C; 6.25 ± 0.48 mg P) compared to with those growing at position 3 (c. 18
C; 9.23 ± 0.84 mg P). There was no evidence that plants colonised by G. intraradices were receiving less N and P at cooler patch compartment temperatures than those associated with G. hoi. Furthermore, plants colonised by G. intraradices consistently had higher N and P concentrations than those colonised by G. hoi irrespective of patch compartment temperature (Table 2), although in the case of N concentration this effect was only very weakly significant (P < 0.1). There was a quadratic relationship between total P content for plants colonised by G. hoi and patch compartment temperature (Fig. 5b). There was no such relationship for plants colonised by G. intraradices and patch compartment temperature. 4. Discussion Given that most soils experience temperatures of less than 15
C for the majority of the year (Tibbett and Cairney, 2007), surprisingly high (c. 25e30
C) temperature optima have been reported for soil €inen et al., 2005; Ba rcenasbacterial and fungal growth (Pietika Moreno et al., 2009). Perhaps importantly for AMF ERM development, fungal growth may be preferentially favoured over bacterial €inen et al., 2005). Moreover, growth at lower temperatures (Pietika global temperature changes are thought to be due to an increase in

Fig. 3. Relationship between percentage root length colonisation (% RLC) and a) shoot 15 N content (mg 15N ¼ () 0.637 þ 0.0141% RLC, R2 ¼ 42%, F1,14 ¼ 9.99, P ¼ 0.007) and b) root 15N content (mg 15N ¼ () 0.388 þ 0.0101% RLC, R2 ¼ 60%, F1,14 ¼ 20.66, P < 0.001) for plants colonized with Glomus intraradices in Expt 2, at 86 d. Data for the temperature experienced by the extra-radical mycelium are distinguished by the different symbols with position 1, c. 11
C, circles; position 2, c. 14
C, squares; position 3, c. 18
C, diamonds; position 4, c. 21
C, triangles. Data are fitted with a best fit linear regression line.

Fig. 4. Total biomass (g) for plants associated with arbuscular mycorrhizal fungi (AMF) extra-radical mycelium that had been grown at a four temperature treatments (position 1, c. 11
C; position 2, c. 14
C; position 3, c. 18
C and position 4, c. 21
C). Data were analysed by a two-way ANOVA with temperature gradient position and AMF species as the main factors. Neither AMF species nor the interaction term were significant but the temperature gradient position was (F3,24 ¼ 4.460; P ¼ 0.013). Different letters denote significant differences among treatments due to the position on the temperature gradient as determined by a Duncan's multiple range post hoc test. Data shown are means ± SE bars (n ¼ 8).

G. Barrett et al. / Soil Biology & Biochemistry 78 (2014) 109e117

Fig. 5. The relationship between mean patch compartment temperature (
C) and a) shoot biomass (g) and b) plant P content (mg) in plants colonised by G. hoi at the 86 d harvest. Both the shoot DW (g shoot DW ¼ () 2.681 þ 0.5937
C e 0.01771
C2, R2 ¼ 40%, F2,13 ¼ 4.28, P ¼ 0.037) and plant P content data (P ¼ ()10.13 þ 2.065
C 0.05909
C2, R2 ¼ 40%, F2,13 ¼ 4.39, P ¼ 0.035) were fitted with a best fit quadratic line.

daily minima as opposed to maximum temperature (Easterling et al., 1997). Consequently, responses at lower temperatures are more relevant to predicting future changes in how AMF might adapt to climate change. In the case of AMF external hyphal growth has been reported to be greatly reduced at temperatures below 15
C (Liu et al., 2004; Hawkes et al., 2008), and Gavito et al. (2005) concluded from their work on root organ cultures that temperatures greater than 18
C and less than 30
C were required for good AMF growth and development. Gavito et al. (2005) also concluded that root growth was much more tolerant than AMF growth to temperatures below 18
C. However, the temperature treatments employed by Gavito et al. (2005) would also influence root growth directly and thus, any indirect effects of the roots response to temperature impacting on AMF growth cannot be ruled out. In this study we avoid such issues of indirect effects of temperature on AMF growth by only exposing the external AMF hyphae to varied temperature treatments. Our approach also has relevance to AMF growth in soil given variation in temperature down the soil profile. Thus AMF may experience different temperature regimes than its associated plant partners. In both experiments reported here, temperature was not a significant factor on ERM growth although, in Expt 1, the interaction between temperature and time was significant as by the end of the experiment (105 d) there was 5 more AMF hyphal length density

115

in the ambient compared to the cooled organic patches. Heinemeyer et al. (2006) also only exposed the external AMF hyphae to differing temperatures (i.e. 12/20
C c.f. 18/26
C day/night respectively) and reported a doubling of Funneliformis mosseae (syn. Glomus mosseae) hyphal length densities at the higher temperature. The increase in hyphal length densities in the present study compared to that found by Heinemeyer et al. (2006) is probably due to increased hyphal proliferation as a result of an organic patch being present (see St John et al., 1983; Albertsen et al., 2006; Nuccio et al., 2013) as shown by the significant patch and temperature interaction. In contrast, in Expt 2 hyphal densities were highly variable within temperature treatments for both AMF species and no clear impact of patch compartment temperature was observed, even though two of the temperatures tested were similar to those in Expt 1. Although the patches used in Expt 1 had a higher C:N ratio to those used in Expt 2 (i.e. C:N 16:1 v 8:1), and the C:N ratio greatly influences patch decomposition (Hodge et al., 2000a, 2000b, 2000c) in Expt 1 the patches were mixed with fresh soil while in Expt 2 a microbially inert material (autoclaved sand:Terragreen) was used. AMF, unlike the fungi involved in the ecto- and ericoid mycorrhizal associations (Hodge et al., 1995; Read and Perez-Moreno, 2003), have very limited saprotrophic capability (Smith and Read, 2008; Leigh et al., 2011), thus, it is likely the soil microbial biomass present in the patches in Expt 1 enhanced patch decomposition particularly at the higher (ambient) temperature €inen et al., 2005) and the AMF hyphae responded by pro(Pietika liferation to this nutrient flush. In this study, internal colonisation (including total and intensity of colonisation) was unaffected by the temperature experienced by the external AMF hyphae (ERM) in the second (hyphal only) compartment in both experiments. Similarly, Heinemeyer and coworkers (Heinemeyer and Fitter, 2004; Heinemeyer et al., 2006) also found no impact on internal colonisation when only the ERM was exposed to differing temperatures. In contrast, when both the plant and the AM fungi experience an increase in temperature internal colonization is generally reported to increase (Smith and Roncadori, 1986; Wang et al., 2002), although this likely depends on the extent and duration of the temperature change (see Karasawa et al., 2012). This suggests that the change in internal colonisation is likely driven by an indirect effect of the temperature on the plant, possibly through an increase in carbon allocation belowground given AMF are large conduits for plant C (Hodge, 1996; Johnson et al., 2002). Strikingly, in Expt 2, the temperature experienced by the external mycelium phase of the AMF influenced plant (including both root and shoot) biomass and plant P content (Figs. 4 and 5) being greater when the AMF compartment was at 18
C than at 11
C but no different at 21
C. Previous studies have shown AMF colonisation leads to enhanced plant P capture compared to nonAM plants, but only at temperatures of 15
C and above (Wang et al., 2002; Kytoviita and Ruotsalainen, 2007). In this study however, all plants were colonised and there was no non-mycorrhizal controls. In Expt 1 only two temperatures (11
C or 24
C) were tested and no effect upon plant biomass or total N content was observed. This suggests that growing the AMF at 18
C had a more beneficial impact on host plant growth and P nutrition than higher (>21
C) temperatures, and is further supported by the quadratic relationships observed in G. hoi colonized plants (Fig. 5). Thus, while Gavito et al. (2005) suggested temperatures of 18e30
C are required for good AMF development, our results suggest the actual benefit to the host plant occurs only at their lower range and at far lower temperatures than those reported for soil bacterial and rcenas-Moreno et al., fungal optima (Pietik€ ainen et al., 2005; Ba 2009). Moreover, temperature did not significantly affect AMF ERM growth in Expt 2 suggesting that the ability of the fungus to

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G. Barrett et al. / Soil Biology & Biochemistry 78 (2014) 109e117

acquire nutrients and transfer them to their host was not simply due to greater hyphal length densities nor due to faster organic matter decomposition as a result of elevated temperature. Thus, while most other studies have grown the plant at differing temperatures and examined the impact on the AMF, here we demonstrate the temperature experienced by the AMF can impact upon plant growth and nutrition over relatively discrete temperature changes. In both Expt 1 and 2 while 15N from the patch was detected in the host plant tissue, no 13C enrichment of the plant tissue occurred. Therefore, it is likely that the N captured by the AMF was acquired after mineralization of the organic patch occurred, in agreement with the results of other studies using a similar experimental approach (e.g. Hodge et al., 2001; Herman et al., 2012). Had the AMF acquired organic N forms directly, (e.g. see Whiteside et al., 2012), 13C levels would be expected to be enriched in the colonized roots, even if the 13C was still retained in the fungal tissue following N transport from the AMF external to the internal mycelial phase, but no 13C enrichment of the root (or shoot) material occurred in either experiment conducted in the present study. Our results agree with the results of Hodge and Fitter (2010) who also did not detect any 13C enrichment in AMF hyphae extracted from near a similar organic patch as used in the present study. In Expt 1 N transfer from the patch to the roots was influenced by temperature, being lower when the patch compartment was cooled but importantly patch N transfer to the shoots was not affected. 15N detected in the shoots represents true transfer to the plant while that detected in the roots may still be held in the fungal tissue (see Hodge and Fitter, 2010). In Expt 2 the 15N content of roots colonized by G. hoi increased with temperature in the fungal compartment (Fig. 2), suggesting that G. hoi was moving N from the patch within its mycelium for its own requirements rather than passing this N to the plant as shoot 15N was not affected. In contrast, both shoot and root 15N content of plants colonized by G. intraradices was related to %RLC and was not related to temperature of the fungal compartment. Thus, this suggests, counter to our third hypothesis, that G. hoi was more sensitive to temperature changes experienced by its ERM than G. intraradices. Our data support the results of Leigh et al. (2009) who also found that G. intraradices transferred more N than G. hoi using a similar experimental system but under constant temperature conditions. Furthermore, Lekberg et al. (2011) found that in contrast to many other phylotypes, a G. intraradices RFLP was found to be a persistent component of AMF communities in vegetation under contrasting temperatures (due to geothermal influences) suggesting it is a generalist able to cope with wide variation in temperature. Consequently while our findings appear counter to Gavito et al. (2005) they are consistent with the field study of Lekberg et al. (2011). In conclusion, ERM growth of the two AMF used in this study was remarkably insensitive to changes in temperature. Similarly, root colonisation (i.e. intensity and total root length colonisation) were not influenced by the temperature experienced by the ERM. However, in Expt 2, there was a direct impact of ERM growth temperature on plant biomass and P content although, surprisingly, not at the highest growth temperature. The external phase of the ERM experiences more variation in temperature than the internal hyphal phase, thus may be more insensitive to altered temperature. Despite ERM growth not being significantly affected, nutrient capture from the organic material patch still occurred even at the lowest temperature studied (11
C), albeit at a lower amount. There was also some evidence for the AMF moving nutrients between the external and internal phase in the roots with varying temperature without a subsequent change in the amount transferred to the shoot. There was distinct differences between the two AMF in the

nutrient transfer to their host; the AMF (G. intraradices) which produced the most external hyphae also acquired the most nutrients, although there was no direct relationship between these two variables suggesting other factors such as the nutritional demand of the fungal symbiont or possible altered physiological uptake capacity by the ERM were also important. Further research needs to concentrate on a more mycocentric approach to fully understand the nutrient requirements of the fungus and the controls on nutrient transfer to the associated host plant. Acknowledgements This work was funded by a Macaulay Development Trust Studentship award to GB. CDC is funded by the Scottish Government, Rural Environment, Research and Analysis Directorate. References Albertsen, A., Ravnskov, S., Green, H., Jensen, D.F., Larsen, J., 2006. Interactions between the external mycelium of the mycorrhizal fungus Glomus intraradices and other soil microorganisms as affected by organic matter. Soil Biology & Biochemistry 38, 1008e1014. Allen, S.E., 1974. Chemical Analysis of Ecological Materials. Blackwell Scientific Publications, Oxford, UK. Atkin, O.K., Sherlock, D., Fitter, A.H., Jarvis, S., Hughes, J.K., Campbell, C., Hurry, V., Hodge, A., 2009. Temperature dependence of respiration in roots colonized by arbuscular mycorrhizal fungi. New Phytologist 182, 188e199. Atul-Nayyar, A., Hamel, C., Hanson, K., Germida, J., 2009. The arbuscular mycorrhizal symbiosis links N mineralization to plant demand. Mycorrhiza 19, 239e246. Baon, J.B., Smith, S.E., Alston, A.M., 1994. Phosphorus uptake and growth of barley as affected by soil temperature and mycorrhizal infection. Journal of Plant Nutrition 17, 479e492. Barrett, G., Campbell, C.D., Fitter, A.H., Hodge, A., 2011. The arbuscular mycorrhizal fungus Glomus hoi can capture and transfer nitrogen from organic patches to its associated host plant at low temperature. Applied Soil Ecology 48, 102e105. rcenas-Moreno, G., Go mez-Brando n, M., Rousk, J., Bååth, E., 2009. Adaptation of Ba soil microbial communities to temperature: comparison of fungi and bacteria in a laboratory experiment. Global Change Biology 15, 2950e2957. Easterling, D.R., Horton, B., Jones, P.D., Peterson, T.C., Karl, T.R., Parker, D.E., Salinger, M.J., Razuvayev, V., Plummer, N., Jamason, P., Folland, C.K., 1997. Maximum and minimum temperature trends for the Globe. Science 277, 364e367. Fitter, A.H., Heinemeyer, A., Staddon, P.L., 2000. The impact of elevated CO2 and global climate change on arbuscular mycorrhizas: a mycocentric approach. New Phytologist 147, 179e187. Fitter, A.H., Heinemeyer, A., Husband, R., Olsen, E., Ridgway, K.P., Staddon, P.L., 2004. Global environmental change and the biology of arbuscular mycorrhizas: gaps and challenges. Canadian Journal of Botany 82, 1133e1139. Fitter, A.H., Helgason, T., Hodge, A., 2011. Nutritional exchanges in the arbuscular mycorrhizal symbiosis: implications for sustainable agriculture. Fungal Biology Reviews 25, 68e72. Gavito, M.E., Schweiger, P., Jakobsen, I., 2003. P uptake by arbuscular mycorrhizal hyphae: effect of soil temperature and atmospheric CO2 enrichment. Global Change Biology 9, 106e116. Gavito, M.E., Olsson, P.A., Rouhier, H., Medina-Penafiel, A., Jakobsen, I., Bago, A., Azcon-Aguilar, C., 2005. Temperature constraints on the growth and functioning of root organ cultures with arbuscular mycorrhizal fungi. New Phytologist 168, 179e188. Gosling, P., Hodge, A., Goodlass, G., Bending, G.D., 2006. Arbuscular mycorrhizal fungi and organic farming. Agriculture, Ecosystems and Environment 113, 17e35. Hawkes, C.V., Hartley, I.P., Ineson, P., Fitter, A.H., 2008. Soil temperature affects carbon allocation within arbuscular mycorrhizal networks and carbon transport from plant to fungus. Global Change Biology 14, 1181e1190. Heinemeyer, A., Fitter, A.H., 2004. Impact of temperature on the arbuscular mycorrhizal (AM) symbiosis: growth responses of the host plant and its AM fungal partner. Journal of Experimental Botany 55, 525e534. Heinemeyer, A., Ineson, P., Ostle, N., Fitter, A.H., 2006. Respiration of the external mycelium in the arbuscular mycorrhizal symbiosis shows strong dependence on recent photosynthates and acclimation to temperature. New Phytologist 171, 159e170. Herman, D.J., Firestone, M.K., Nuccio, E., Hodge, A., 2012. Interactions between an arbuscular mycorrhizal fungus and a soil microbial community mediating litter decomposition. FEMS Microbiology Ecology 80, 236e247. Hetrick, B.A.D., Bloom, J., 1984. The influence of temperature on colonization of winter-wheat by vesicular arbuscular mycorrhizal fungi. Mycologia 76, 953e956. Hodge, A., 1996. Impact of elevated CO2 on mycorrhizal associations and implications for plant growth. Biology and Fertility of Soils 23, 388e398.

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