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Elevated CO2 concentrations in the atmosphere are leading to an increase in global mean temperature, and substantial climatic changes are expected over the next century (Houghton et al., 2001). Elevated CO2 generally increases plant growth (Ceulemans et al., 1999; Norby & Luo, 2004). Temperature effects, and especially the effects of soil temperature, have been less studied in the context of global environmental change.
Most experiments on the impacts of rising temperature on ecosystems have been conducted under controlled conditions and have used nonmycorrhizal plants, although most plants live in association with mycorrhizal fungi (Trappe, 1987) and two-thirds of plants are in symbiosis with arbuscular mycorrhizal (AM) fungi (Fitter & Moyersoen, 1996). AM fungi (Glomeromycota) grow both inside roots and in soil as an extra-radical mycelium (ERM), which directly experiences wide variations in soil environment such as pH, soil moisture and temperature. The AM symbiosis appeared contemporaneously with land plants (Remy et al., 1994) and must therefore have experienced major climate changes in the past. However, we still do not understand the basic ecology of this ubiquitous symbiosis (Fitter et al., 2000; Bever et al., 2001; Fitter et al., 2004). The fungi are obligate symbionts but form a mutual relationship with the host plant in which the fungus receives its entire carbon requirement from the host plant but provides the host with nutrients that are poorly mobile in soil, such as phosphate, or with other benefits (Newsham et al., 1995; Clark & Zeto, 2000). Hence, this symbiosis plays a key role in linking above- and below-ground carbon cycling (Finlay & Söderström, 1992).
Most of the carbon supply to the fungus seems to be recently fixed carbon (Jakobsen & Rosendahl, 1990), and the biochemical pathways 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 that affect plant photosynthesis, for example reduced light availability (Tester et al., 1986), affect fungal growth indirectly. Yet the fungus itself might respond to changes in the soil environment directly, independently of host plant responses; soil temperature is likely to be an important factor affecting growth and respiration. As fungal carbon supply can consume up to 20% of net photosynthesis (Smith & Read, 1997), any temperature responses of fungal growth and respiration might therefore have a significant influence on carbon input and cycling in the soil.
Elevated CO2 often increases internal colonization as a result of an increase in plant growth. Consequently, the amount of 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), and directly, through the fungal response (Heinemeyer & Fitter, 2004). Yet, to date, hyphal respiration rates have never been measured directly for AM fungi (Rillig & Allen, 1999) and it is not known how much ERM respiration contributes to the carbon budget of the plant and whether it shows similar acclimation to temperature changes as do roots (Gifford, 1995; Atkin et al., 2000) and soil respiration (Luo et al., 2001).
Ecological studies increasingly use the stable isotope 13C to follow a carbon signal through different trophic layers (Ostle et al., 2000; Radajewski et al., 2000), including AM fungi (Staddon et al., 1999a; Miller & Kling, 2000). We combined a compartment study with the application of two 13CO2 pulse labels during a period of warming the ERM. We aimed to measure (i) the sensitivity of the respiration of the ERM of an AM fungus to temperature and whether it displayed acclimation; (ii) how much of the carbon allocated to the fungus from the root carbon pool is recently fixed carbon; and (iii) the carbon costs for the host plant of the ERM and the dynamics of carbon flux from the roots to the ERM.
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- Materials and Methods
We report here the first quantitative estimate of the respiration of the ERM of an AM fungus and its response to soil warming, excluding background respiration. However, there is also surprisingly little information on ectomycorrhizal respiration. Ettema et al. (1999) and Hedlund & Augustsson (1995) reported basidiomycete (which are generally thicker as they tend to form rhizomorphs) hyphal respiration of 40 µg C g−1 d−1 and 0.055 µg C m−1 h−1, respectively, in comparison with which our findings of 0.2 µg C g−1 d−1 and 0.003 µg C m−1 h−1 seem to be very low. However, their results were obtained from field soil with the addition of glucose and antibiotics or in axenic culture, respectively.
We used gas sample tubes, which enabled us to calculate fungal compartment ERM respiration, and demonstrated an 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 temperature shown by roots (Atkin et al., 2000). However, in a warmer environment, longevity of hyphae might decline as reported for roots (Fitter, 1996; Norby & Jackson, 2000), leading to accumulation of dead hyphae, which were not measured in this study; the significantly lower respiration rate per unit hyphal length (LERM) after 2 wk of soil warming is consistent with the accumulation of dead hyphae, whereas the increased ratio of 13C to total carbon respired (Fig. 5b) is not, as it indicates more rapid carbon allocation to the ERM. Both observations could be explained by an increase in younger, and therefore thinner but also more active, mycelium under soil warming; such an increase is likely to have occurred. Nonetheless, there was a residue of 13C label in the ERM by the time the second label was applied. Although values in 13C-labelled treatments had returned to near starting values (c. +13 and +30‰ for nonmycorrhizal and mycorrhizal treatments, respectively), the enrichment in the second pulse was very high (c. +1900‰), and consequently any residue effect was negligible.
Soil warming increased ERM growth in the hyphal compartment after 2 d and by nearly twofold after 2 wk (Table 1), as previously found (Heinemeyer & Fitter, 2004); yet there was no difference in LRC or LR and hence total LRC, suggesting that internal mycelium length does not determine growth of ERM as suggested by Tinker (1975) and Staddon et al. (2004). However, it is still debatable whether an increased ERM mass enters a slow turnover carbon pool in soils (Treseder & Allen, 2000; Rillig et al., 2001; Staddon et al., 2003) and it is known that the ERM length varies considerably among species (Smith et al., 2004; Munkvold et al., 2004). We suggest that as temperature rises ERM mass will become a greater carbon sink than soil respiration, which seems to acclimate, as shown in prairie and forest studies (Grace & Rayment, 2000; Luo et al., 2001); further, even if productivity is expected to increase in a warmer climate (Parton et al., 1995), rates of decomposition of soil organic matter seem to be 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% given by Jakobsen & Rosendahl (1990). However, we were only able to estimate the ERM in the plant compartment and did not account for root internal colonization with dense colonization of thicker hyphae and storage vesicles; total fungal carbon costs might then increase to over 5%, as reported by Snellgrove et al. (1982), Koch & Johnson (1984) and Cooper (1984). Further, we had to assume a similar hyphal mass for our G. mossea strain to that used by Harley & Smith (1983), which is somewhat uncertain. In our study, the carbon cost for ERM respiration was slightly higher than for growth, as estimated by Harris & Paul (1987). Further, although the ratio of 13C:12C respired (Fig. 5) was the same in ambient and heated treatments in the first pulse, it increased slightly under soil warming in the second pulse. This shows (i) that there was rapid carbon transport to the ERM in < 10 h, (ii) that only c. 10% of the carbon respired came from recently fixed carbon, and (iii) that there was faster carbon allocation inside the ERM under higher temperature, possibly as a result of increased production of young hyphae.
The finding that control plants did not show any 13C enrichment demonstrates that we successfully prevented contamination of unlabelled plants via 13C leakage from labelling chambers; roots of labelled plants increased 13C content by c. 18 mg (Fig. 6b), sufficient to follow a 13C signal into the hyphal compartment. Mycorrhizal plants had less labelled carbon than nonmycorrhizal plants in the roots, but not in the shoots. Although the difference in root 13C was only marginally significant (P = 0.089), it may demonstrate allocation of fixed carbon from mycorrhizal roots to the AM fungus (Jakobsen & Rosendahl, 1990) without affecting the shoot carbon pool. However, warming of the ERM did not result in a further decrease.
Diurnal differences in measured CO2 concentrations (Fig. 2a) reflected different ERM activities, corresponding to the carbon fixation of the plants: the concentration of root sucrose, which is believed to be one of the main forms of carbon transported to the fungus (Bago et al., 2003), increases during the day, consistent with peak activity of the AM fungus during and shortly after the photoperiod. Further, as ERM respiration clearly depended on short-term PAR effects, our findings could be explained both by a transfer mechanism based on leakage of carbon compounds into the apoplast followed by fungal capture, and by a transfer mechanism with greater regulation by the plant. There are two technical problems in measuring hyphal respiration as influenced by the plant compartment. First, mycorrhizal roots may have higher respiration rates than nonmycorrhizal roots (Solaiman & Saito, 1997); because we assumed that the 24% greater root length in the nonmycorrhizal plants (Fig. 7b) should have counteracted such an effect, we might have underestimated ERM respiration. Secondly, δ13C signals in the first pulse were 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 in the growth of the hyphal front or differences in ERM activity among replicates.
Plants showed typical mycorrhizal responses – increased leaf area, increased SLA and reduced root length (Harris & Paul, 1987; Gavito et al., 2001) – and mean net photosynthesis ranging between 4.5 and 8.8 µmol m−2 s−1 was very similar to data for this species obtained by Staddon et al. (1999a) under saturated light. Hyphal length densities were between 3 and 7 m g−1, within the range reported for glomalean fungi in other compartment experiments (Jakobsen et al., 1992; Hodge et al., 2001) but lower than values obtained in pot experiments or field data. The latter sometimes exceed 100 m g−1 (Sylvia, 1990), although much of this may be either dead (Sylvia, 1988) or nonmycorrhizal (Sylvia, 1986) material.
Photosynthesis is expected to increase under higher temperature and elevated CO2 (Ceulemans et al., 1999) if water supply is not limited. Plants will then need an increased nutrient supply to maintain higher growth rates (Olesniewicz & Thomas, 1999). The greater amount of ERM in the warmer environment found here will increase the effectiveness with which the host plant explores soil and if, as suggested by Staddon & Fitter (1998) for elevated CO2, more carbon were available to the fungus, the positive growth response of the ERM of AM fungi might well supply this increased nutrient demand in a warmer climate.
In this study, we have obtained evidence for faster carbon allocation to and increased respiration of the ERM under higher soil temperature, but also rapid acclimation and an immediate response to changes in available carbon from a previously mixed root carbon pool. The positive growth response of the ERM to higher temperature together with acclimation of its respiration might lead to a significant increase in carbon accumulation in soils when the climate becomes warmer. However, light seems to be the overall controlling factor in carbon allocation to the fungus. This study underlines the important role of the AM fungal symbionts in global carbon cycling and suggests that a more mycocentric view in ecological studies should be considered in future climate modelling.