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Plant and fungal responses to elevated atmospheric carbon dioxide in mycorrhizal seedlings of Pinus sylvestris
Environmental and Experimental Botany 40 (1998) 237 – 246
Plant and fungal responses to elevated atmospheric carbon dioxide in mycorrhizal seedlings of Pinus syl6estris H. Rouhier *, D.J. Read Department of Animal and Plant Sciences, The Uni6ersity of Sheffield, PO Box 601, Sheffield S10 2TN, UK Received 26 March 1998; received in revised form 20 June 1998; accepted 2 July 1998
Abstract The effects of elevated CO2 concentration upon the mycorrhizal relationships of Scots pine (Pinus syl6estris) seedlings were investigated. Plants were grown for 4 months with their shoots exposed to ambient (CAMB = 360 ml l − 1) or elevated (CELEV =700 ml l − 1) CO2 environments while their root systems, either colonised by the mycorrhizal fungi Paxillus in6olutus or Suillus bo6inus, or left in the non-mycorrhizal condition, were maintained in sealed dishes. In one series of these plants the effects of CELEV upon the extent of mycorrhizal development and upon their growth and nutrition were determined, while another series were transferred from the dishes after 1 month, to transparent observation chambers before being returned to the two CO2 environments. In these chambers, the effects of CELEV upon development of the external mycelial systems of the two mycorrhizal fungi was determined by measuring the advance of the hyphal fronts of the mycorrhizal fungi across non-sterile peat from the colonised plants. The dry mass and number of mycorrhizal tips were significantly higher in CELEV than in the CAMB condition in plants colonised by both fungi in the dishes. Yields of whole plants and of shoots were higher in the CELEV treatment whether or not they were grown in the mycorrhizal condition, but the greater yields were not associated in these sealed systems with enhanced nutrient gain. The dry mass of non-mycorrhizal plants was greater than that of those colonised by mycorrhizal fungi under elevated CO2. This is thought to be attributable to the energetic cost of production of the larger mycorrhizal systems in this treatment. The extent of development of the mycorrhizal mycelial systems of both fungi was greatly increased in CELEV relative to that in CAMB environments. It is hypothesised that increased allocation of carbon to mycorrhizal root systems and their associated mycelia would provide the potential for enhancement of nutrient acquisition in open systems of greater fertility. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Carbon allocation; CO2; Mycorrhiza; Nitrogen; Paxillus in6olutus; Phosphorus; Pinus syl6estris; Suillus bo6inus
1. Introduction * Corresponding author. Present address: Department of Microbial Ecology, Lund University, Ecology Building, S-223 62 Lund, Sweden. Tel.: +46 46 2223758; Fax: +46 46 2224158; E-mail [email protected]
It is no longer in doubt that increases of atmospheric CO2 concentration can exert profound influences upon the growth and carbon allocation
S0098-8472/98/$ - see front matter © 1998 Elsevier Science B.V. All rights reserved. PII S0098-8472(98)00039-2
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of trees. Average increases of biomass of 64 tree species exposed to various elevated CO2 regimes were reported by Ceulemans and Mousseau (1994) to be 63% for deciduous and 38% for coniferous species. However, the mechanisms involved in the facilitation of these responses are not understood. It is generally accepted that the additional carbon fixed by plants exposed to elevated CO2 is allocated to roots (Bazzaz, 1990), the expectation being that this would promote the ability of the plant to capture the nutrients necessary to sustain the enhanced growth. In this case the ultimate destination of much of the assimilate allocated below ground is likely to be the mycorrhizal roots and mycelium but these parts of the carbon pathway are relatively poorly understood. Among those who have considered the mycorrhizal component some have observed increases in mycorrhiza formation in response to elevated CO2 (Norby et al., 1987; Tingey et al., 1995, 1996, 1997) while others have found any such effect to be short-lived (O’Neill et al., 1987b; Lewis et al., 1994). Occasionally even reductions of mycorrhiza formation have been reported (Walker et al., 1997). Those investigating the biology of mycorrhiza are increasingly aware of the pivotal role played by the external mycelium of mycorrhizal roots in nutrient capture by plants (Smith and Read, 1997). It is this distal part of the carbon pathway which is likely to be critical in determining the success of nutrient scavenging processes. While there have been reports of changes in the population structure (Godbold and Bernston, 1997; Rey and Jarvis, 1997) and of increased fungal occurrence under elevated CO2 (Tingey et al., 1997) only one study (Ineichen et al., 1995) has specifically shown increased production of mycorrhizal hyphae under these conditions. This study revealed a doubling of the biomass of external mycelium of Pisolithus tinctorius growing from colonised roots of Pinus syl6estris grown at 600 ml l − 1. In addition, a triplication of the number of mycorrhizas themselves was observed in this treatment. The experiments of Ineichen et al. (1995) were carried out using an unnatural substrate, cardboard, to support mycorrhiza development.
In the present study we set out to determine the effects of elevated CO2 upon biomass gain and nutrient relations of P. syl6estris grown, using Sphagnum peat as substrate, in mycorrhizal (M) association with two fungi, Suillus bo6inus and Paxillus in6olutus, and in the non-mycorrhizal (NM) condition. We paid particular attention to the relationship between mycorrhizal colonisation, development of the external mycelium, and the response of the plants grown under ambient (CAMB) and elevated (CELEV) CO2 (700 ml l − 1). We hypothesised that growth under CELEV would lead to enhancement of below ground carbon allocation that would be detectable in the form of greater mycelial and mycorrhizal root development but that different fungal symbionts would respond differently.
2. Materials and methods
2.1. Production of mycorrhizal and non-mycorrhizal plants of P. syl6estris A Sphagnum-peat (P) of high C:N ratio (40:1) was mixed with vermiculite (V) in the ratio 0.5 parts peat to 3.5 parts vermiculite, after passing the two components through a sieve of 2 mm mesh size. The PV mixture was moistened in 2-l batches with 0.8 l of modified Melin Norkrans nutrient solution (Brun et al., 1995) in which N and P concentrations were applied at 1/10 strength. The MMN nutrient solution contained (mg l − 1): KH2PO4 (50), (NH4)2HPO4 (25), CaCl2 (50), MgSO4 · 7H2O (150), FeCl3 · 6H2O (1). The mixture was adjusted to pH 4.7, autoclaved at 120°C for 45 min and transferred aseptically into 9-cm Petri dishes (50 ml per dish). At this stage the dishes were inoculated with agar discs supporting mycelia of the mycorrhizal fungi P. in6olutus (Batsch) Fr. or S. bo6inus L. Kuntze. At the same time surface sterilised seeds of P. syl6estris were placed on water agar in Petri dishes to provide sterile germinated seedlings. After 15 days, by which time the fungal inoculum discs had commenced colonisation of the PV, these seedlings were transferred to the Petri dishes with (M) or without (NM) the presence of the
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fungi. The seedlings were planted so that their shoots emerges through a groove cut in vertical wall of the Petri dish to be exposed to the atmosphere of the growth cabinet. The groove was sealed with sterile lanolin, and gas permeable Parafilm was used to wrap the junction between upper and lower portions of the dishes which were then transferred to different CO2 treatments. The total number of seedlings used for the present experiment was 40 (eight NM; 16 M (P. in6olutus); and 16 M (S. bo6inus)), 20 seedlings in each CO2 treatment.
2.2. Experiment 1: effect of CO2 on plant growth, nutrition and mycorrhizal formation The Petri dish systems were placed in two controlled environment cabinet (Fitotron, Sanyo– Gallenkamp PLC, UK) either at ambient CO2 concentration (CAMB: 350 ml l − 1) or in an atmosphere in which CO2 concentration was enriched to 700 ml l − 1 (CELEV). The CO2 concentration in each cabinet was monitored by infra-red gas analysers (ADC 2000, ADC PLC, Hoddesdon, UK) which were used to control introduction of pure CO2 in the CELEV treatment from an external cylinder. Lighting in the cabinets was provided by a combination of fluorescent tubes and incandescent tungsten lamps. The photon flux density at plants height was 400 mmol m − 2 s − 1. All plants received a 16-h day length with a day temperature of 20°C and a night temperature of 15°C. To eliminate chamber effects the plants and their CO2 treatments were switched at weekly intervals from one growth cabinet to the other, the position of the plants being randomised within the chambers at the time of each transfer.
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chambers (Finlay and Read, 1986). Each observation chamber was then returned to the CO2 environment from which the plants growing in it had been taken. Mycelial extension growth was determined by sequential measurements of the distance from the colonised seedling to the mycelial front which, as described by Read (1992), advanced in a fan-like manner across the peat from the plant. Measurements were made at 28, 55 and 87 days for S. bo6inus and at 28 and 55 days for P. in6olutus. Measurements at 87 days for P. in6olutus were not possible because the extramatrical mycelium covered the entire surface of the peat before that date.
2.4. Har6est and analysis After 4 months of growth in the cabinets, the remaining seedlings (eight M (four P. in6olutus and four S. bo6inus) and four NM) were harvested from each of the two CO2 treatments. Their shoots were excised from the root-soil system, oven dried at 80°C for 24 h and weighed. Roots were washed and in the case of M plants, mycorrhizal tips were counted and excised from the root system. Roots and mycorrhizal tips were oven dried (72 h) and weighed separately. Dried plant material (shoots, roots and mycorrhizal tips) was wet digested in sulphuric acid, its N and P concentrations being determined in duplicate aliquots of the digest mixture. Nitrogen was determined using the indo-phenol method (Kedrowski, 1983) and phosphorus using the phospho-molybdate-blue method in presence of a lithium sulfate/copper sulfate catalyst mixture (Boltz and Lueck, 1958).
2.5. Statistical analysis 2.3. Experiment 2: effect of CO2 on fungal growth After 1 month of exposure to the CO2 conditions described above, by which time all seedlings growing in the inoculated dishes were fully mycorrhizal, four seedlings colonised by each fungal species were selected from Petri dishes in each of the two CO2 environments. They were transferred to transparent 20 × 20 cm plexiglas observation
Data on dry weights, N and P content were subjected to a two-way analysis of variance (ANOVA) of randomised design using P\ 0.05 as the critical level of significance. The main experimental parameters, i.e. elevated CO2, mycorrhiza colonisation and their interaction were tested by F-test. The effect of CO2 treatment on fungal colonisation and growth was assessed by comparisons of the means using a Student’s t-test.
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3. Results
3.1. Effects of ele6ated CO2 on biomass of M and NM seedlings of P. syl6estris The whole plant biomass of seedlings in both M and NM treatment were increased by exposure to elevated CO2 (Fig. 1(a)). Growth enhancement was significantly (P B 0.01) greater for plants inoculated with P. in6olutus than S. bo6inus. The shoot yields in all three categories of plants were all significantly (P B 0.01) higher in CELEV than CAMB. There were no differences between the two fungi in the terms of their effects on shoot yield (Fig. 1(b)). In the case of root biomass NM plants and those colonised by P. in6olutus showed significantly (PB 0.01) greater production but increased CO2 supply did not lead to significant impact upon root yield of plants colonised by S. bo6inus (Fig. 1(c)). Despite the absence of a response in the whole root yield under CELEV the S. bo6inus colonised plants produced significantly (P B0.01) greater biomass of mycorrhizal tips (Fig. 1(d)) than those grown in CAMB. The same was true for plants with P. in6olutus mycorrhiza. ANOVA analysis showed that effect of CO2 enrichment upon biomass was significant (P B0.01) in all compartments for P. in6olutus and for shoots and mycorrhizal tips for S. bo6inus. Comparison of the effects of CELEV on whole plant, shoot and root biomass production, showed that in each of these tissue categories the yields were greater in the NM than in the M plants. ANOVA analysis showed that these negative effects upon plants of the M relative to the NM type were significant at PB 0.01 in the case of total and root dry weights of plants colonised by S. bo6inus, and P B 0.05 in the case of root dry weight of plants with Paxillus mycorrhizas. As discussed later, smaller yields in M relative to NM seedlings may be attributable to the costs of initiating the mycelial systems associated with the mycorrhizal roots.
3.2. Effects of ele6ated CO2 on plant tissue N and P concentration Analysis of the nitrogen and phosphorus con-
centrations of whole plant (Fig. 2(a and e)), shoot (Fig. 2(b and f)), and root tissues (Fig. 2(c and g)) and of mycorrhizal tips (Fig. 2(d and h)) showed that exposure to elevated CO2 resulted either in lower N and P concentrations or to a lack of significant difference between them. In only three cases, increases of nutrient concentration were observed under elevated CO2. These were all in non-mycorrhizal plants where the total N and P contents of tissues were also significantly increased (data not presented). No such increases were observed in M plants associated with either fungus. The CO2 effect was significant (PB 0.01) for whole plant N and P concentrations in plants with both mycorrhizal fungi except in the case of N concentration in plants colonised by S. bo6inus. The ANOVA revealed significant (PB 0.01) decreases of P concentrations in all compartments of mycorrhizal plants. These decreases were also seen in the concentration of N in P. in6olutus colonised plants, but not in the case of those colonised by S. bo6inus. Interaction effects (CO2 × mycorrhiza) were significant (PB0.05 and PB 0.01) for all N and P concentrations in P. in6olutus colonised plants but were insignificant for those colonised by S. bo6inus.
3.3. Effects of ele6ated CO2 on the number of mycorrhizal tips In seedlings colonised by either fungi there was a significant (PB 0.01) increase in the number of mycorrhizal tips formed per plant under CELEV (Fig. 3(a)), where the total number of mycorrhizal tips was 62% higher in the plants colonised by either S. bo6inus or P. in6olutus than in their counterparts grown under CAMB. When expressed as number per unit dry weight of root, however, only in the case of S. bo6inus colonised plants was there a significant (PB0.01) effect of CELEV (Fig. 3(b)).
3.4. Effects of ele6ated CO2 upon de6elopment of extraradical mycelium Following transfer of the mycorrhizal plants to peat in transparent observation chambers, devel-
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Fig. 1. Effect of elevated CO2 and mycorrhizal colonisation by P. in6olutus (P. inv.) or S. bo6inus (S. bov.) on the dry weights of P. syl6estris seedlings. (a) Whole plant, (b) shoots, (c) roots and (d) mycorrhizal tips. Two-week-old transplanted mycorrhizal (M) and non-mycorrhizal (NM) plants were grown for 4 months at ambient ( =350 ml l − 1) and elevated ( = 700 ml l − 1) CO2 concentration. Error bars represent 95% confidence limits (n = 4) and statistical differences between means (*P B0.05; **P B 0.01; n.s., not significant P\ 0.05) are the result of a t-test.
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Fig. 2. Effect of elevated CO2 and mycorrhizal colonisation by P. in6olutus or S. bo6inus on nitrogen (a – d) and phosphorus (e–h) concentrations in whole plant, shoots, roots and mycorrhizal tips of P. syl6estris seedlings. Two-week-old transplanted mycorrhizal (M) and non-mycorrhizal (NM) plants were grown for 4 months at ambient ( =350 ml l − 1) and elevated ( =700 ml l − 1) CO2 concentration. Error bars represent 95% confidence limits (n =4) and statistical differences between means (*P B0.05; **P B 0.01; n.s., not significant P \0.05) are the result of a t-test.
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Fig. 3. Effect of elevated CO2 on production of mycorrhizal tips by P. syl6estris seedlings inoculated with P. in6olutus or S. bo6inus. (a) Number of mycorrhizal tips per plant; and (b) number of tips colonised per unit (mg) of root. Two-week-old transplanted mycorrhizal (M) plants were grown for 4 months at ambient ( =350 ml l − 1) and elevated ( =700 ml l − 1) CO2 concentration. Error bars represent 95% confidence limits (n = 4) and statistical differences between means (*P B0.05; **P B0.01; n.s., not significant P \0.05) are the result of a t-test.
opment of their extraradical mycelium systems could be seen to respond rapidly to CO2 elevation (Fig. 4). The hyphal fronts of both fungi grew, from the colonised roots, more rapidly under CELEV than CAMB condition from time zero. By the time of the first measurement of replicate plates after 28 days, the total areas of peat surface occupied by the mycelial fans were significantly (P B 0.01) greater in CELEV than CAMB in both fungi. Differences between the two treatments continued to diverge at the second harvest (55 days) in the case of P. in6olutus by which time this fungus occupied 444% greater total area of peat, and through to the third harvest (87 days) in S. bo6inus where the final difference between areas occupied by mycelium in CAMB and CELEV was 230%.
4. Discussion Under elevated concentrations of atmospheric CO2 enhancements were observed in total biomass production of both M and NM seedlings. This confirms the pattern of response reported in several previous studies (Walker et al., 1995b; Hodge, 1996; Lewis and Strain, 1996; Godbold et al., 1997; Tingey et al., 1997). Of particular interest was the observation that under these conditions the plants grown with their mycorrhizal symbionts produced greater number of mycorrhizal lateral roots and much more extraradical mycelium systems than did those grown at CAMB. These results thus provide further support for the view that mycorrhizal fungi act as a sink for assimilates and that under conditions where pho-
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Fig. 4. Effect of elevated CO2 on the development in mycelial systems of (a) P. in6olutus; or (b) S. bo6inus growing with P. syl6estris seedlings in mini-rhizotrons. Plants were grown at ambient (350 ml l − 1 — open symbols) and elevated (700 ml l − 1 — closed symbols) CO2 concentration. Error bars represent 95% confidence limits (n =4).
tosynthate availability to roots increases, a significant amount of these resources may be allocated to the fungus. It is evident, however, that despite the greater number of mycorrhizal tips in the Petri dish experiment, seedlings grown with their symbionts under elevated CO2 produced less biomass than did their NM counterparts. It has frequently been observed even under ambient CO2 conditions, that in the earliest stages of growth, during which the vegetative mycelium of the fungal partner is developing, the biomass of mycorrhizal plants can be lower than that of uncolonised seedlings (Dosskey et al., 1990; Stenstro¨m et al., 1990; Newton, 1991; Colpaert et al., 1992). This effect is thought to arise from the carbon demands placed upon the mycorrhizal seedling by its fungal symbiont (Smith and Read, 1997).
If, as appears likely from this study, fungal growth, and hence carbon demand, is enhanced when more CO2 is available to the plant, these effects on its biomass are likely to be exaggerated. The period of mycelial development is also one of maximal demand by the fungus for N and P to enable hyphal growth. There might in effect, be competition between fungal and plant tissues for these nutrients during establishment of the vegetative mycelium and this would explain the observed reductions in shoot and root N and P concentrations of M relative to NM plants under CELEV. Under our experimental conditions these competitive effects would be exacerbated by the confinement of the seedlings within the dishes which will have reduced the effectiveness of nutrient foraging by the mycelium.
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It has also been observed that plants grown under elevated CO2 show reduced tissue nutrient concentrations, those of N being particularly susceptible to these effects (Norby et al., 1987; O’Neill et al., 1987a; Johnson et al., 1995). Indeed, the results of Walker et al. (1995a) suggest that the responses of Pinus ponderosa seedlings to elevated CO2 are entirely determined by nitrogen availability since only when supplementary N was added to a containerised potting mix did the mycorrhizal plants respond positively to atmospheres with enriched CO2. Our observations of mycorrhizal mycelial development are in general accord with those of Ineichen et al. (1995) in so far as they confirm enhancement of mycorrhizal development under conditions of elevated CO2. However, in a number of respects they differ and these differences may be explicable in term of the contrasting experimental design employed in the two studies. Ineichen et al. (1995) observed no differences in extraradical mycelial development in M seedlings exposed to CELEV in early harvests, increases being recorded only after c 100 days of growth. However, their seedlings were not mycorrhizal when introduced into the growth chambers, so the early stage of development of the mycorrhizas themselves may, as indicated above, have consumed any excess of carbon being allocated to fine roots. In their case, also, a preformed mycelium would act as a sink for carbon before new biomass could be produced. This situation contrasts strongly with that in our observation chambers to which seedlings with preformed mycorrhizas were added. In this case the ‘cost’ of mycorrhiza formation, and of sustaining a large external mycelium, was eliminated so enabling the increased carbon flow to M roots under elevated CO2 to be allocated immediately for the support of new mycelial extension growth. The observed delay in response of the extraradical mycelium in the Ineichen et al. (1995) study is also consistent with their observation that the number of mycorrhizas formed in CELEV did not differ from those of CAMB until the second harvest c 60 days after inoculation with P. tinctorius. It must be recognised that there will be some interspecific differences between the fungal symbionts in their
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responses to elevated carbon availability but in these cases it seems that contrasting histories of mycorrhiza formation are more likely to explain the differences in the observed patterns of response to CELEV. It can be hypothesised that in nature, where mycorrhizal mycelial systems are free to forage without spatial constraints through larger volume of soil, increased allocation of carbon under CELEV, by enabling production of more extensive hyphal networks, would provide increased access to N. This hypothesis is currently being tested using observation chambers with patchily distributed remote sources of available N.
Acknowledgements We thank The Human Capital and Mobility program of the European Commission for providing the grant (contract No. ERBCHBICT941011) which supported H. Rouhier during the course of this work.
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Plant and fungal responses to elevated atmospheric carbon dioxide in mycorrhizal seedlings of Pinus syl6estris H. Rouhier *, D.J. Read Department of Animal and Plant Sciences, The Uni6ersity of Sheffield, PO Box 601, Sheffield S10 2TN, UK Received 26 March 1998; received in revised form 20 June 1998; accepted 2 July 1998
Abstract The effects of elevated CO2 concentration upon the mycorrhizal relationships of Scots pine (Pinus syl6estris) seedlings were investigated. Plants were grown for 4 months with their shoots exposed to ambient (CAMB = 360 ml l − 1) or elevated (CELEV =700 ml l − 1) CO2 environments while their root systems, either colonised by the mycorrhizal fungi Paxillus in6olutus or Suillus bo6inus, or left in the non-mycorrhizal condition, were maintained in sealed dishes. In one series of these plants the effects of CELEV upon the extent of mycorrhizal development and upon their growth and nutrition were determined, while another series were transferred from the dishes after 1 month, to transparent observation chambers before being returned to the two CO2 environments. In these chambers, the effects of CELEV upon development of the external mycelial systems of the two mycorrhizal fungi was determined by measuring the advance of the hyphal fronts of the mycorrhizal fungi across non-sterile peat from the colonised plants. The dry mass and number of mycorrhizal tips were significantly higher in CELEV than in the CAMB condition in plants colonised by both fungi in the dishes. Yields of whole plants and of shoots were higher in the CELEV treatment whether or not they were grown in the mycorrhizal condition, but the greater yields were not associated in these sealed systems with enhanced nutrient gain. The dry mass of non-mycorrhizal plants was greater than that of those colonised by mycorrhizal fungi under elevated CO2. This is thought to be attributable to the energetic cost of production of the larger mycorrhizal systems in this treatment. The extent of development of the mycorrhizal mycelial systems of both fungi was greatly increased in CELEV relative to that in CAMB environments. It is hypothesised that increased allocation of carbon to mycorrhizal root systems and their associated mycelia would provide the potential for enhancement of nutrient acquisition in open systems of greater fertility. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Carbon allocation; CO2; Mycorrhiza; Nitrogen; Paxillus in6olutus; Phosphorus; Pinus syl6estris; Suillus bo6inus
1. Introduction * Corresponding author. Present address: Department of Microbial Ecology, Lund University, Ecology Building, S-223 62 Lund, Sweden. Tel.: +46 46 2223758; Fax: +46 46 2224158; E-mail [email protected]
It is no longer in doubt that increases of atmospheric CO2 concentration can exert profound influences upon the growth and carbon allocation
S0098-8472/98/$ - see front matter © 1998 Elsevier Science B.V. All rights reserved. PII S0098-8472(98)00039-2
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of trees. Average increases of biomass of 64 tree species exposed to various elevated CO2 regimes were reported by Ceulemans and Mousseau (1994) to be 63% for deciduous and 38% for coniferous species. However, the mechanisms involved in the facilitation of these responses are not understood. It is generally accepted that the additional carbon fixed by plants exposed to elevated CO2 is allocated to roots (Bazzaz, 1990), the expectation being that this would promote the ability of the plant to capture the nutrients necessary to sustain the enhanced growth. In this case the ultimate destination of much of the assimilate allocated below ground is likely to be the mycorrhizal roots and mycelium but these parts of the carbon pathway are relatively poorly understood. Among those who have considered the mycorrhizal component some have observed increases in mycorrhiza formation in response to elevated CO2 (Norby et al., 1987; Tingey et al., 1995, 1996, 1997) while others have found any such effect to be short-lived (O’Neill et al., 1987b; Lewis et al., 1994). Occasionally even reductions of mycorrhiza formation have been reported (Walker et al., 1997). Those investigating the biology of mycorrhiza are increasingly aware of the pivotal role played by the external mycelium of mycorrhizal roots in nutrient capture by plants (Smith and Read, 1997). It is this distal part of the carbon pathway which is likely to be critical in determining the success of nutrient scavenging processes. While there have been reports of changes in the population structure (Godbold and Bernston, 1997; Rey and Jarvis, 1997) and of increased fungal occurrence under elevated CO2 (Tingey et al., 1997) only one study (Ineichen et al., 1995) has specifically shown increased production of mycorrhizal hyphae under these conditions. This study revealed a doubling of the biomass of external mycelium of Pisolithus tinctorius growing from colonised roots of Pinus syl6estris grown at 600 ml l − 1. In addition, a triplication of the number of mycorrhizas themselves was observed in this treatment. The experiments of Ineichen et al. (1995) were carried out using an unnatural substrate, cardboard, to support mycorrhiza development.
In the present study we set out to determine the effects of elevated CO2 upon biomass gain and nutrient relations of P. syl6estris grown, using Sphagnum peat as substrate, in mycorrhizal (M) association with two fungi, Suillus bo6inus and Paxillus in6olutus, and in the non-mycorrhizal (NM) condition. We paid particular attention to the relationship between mycorrhizal colonisation, development of the external mycelium, and the response of the plants grown under ambient (CAMB) and elevated (CELEV) CO2 (700 ml l − 1). We hypothesised that growth under CELEV would lead to enhancement of below ground carbon allocation that would be detectable in the form of greater mycelial and mycorrhizal root development but that different fungal symbionts would respond differently.
2. Materials and methods
2.1. Production of mycorrhizal and non-mycorrhizal plants of P. syl6estris A Sphagnum-peat (P) of high C:N ratio (40:1) was mixed with vermiculite (V) in the ratio 0.5 parts peat to 3.5 parts vermiculite, after passing the two components through a sieve of 2 mm mesh size. The PV mixture was moistened in 2-l batches with 0.8 l of modified Melin Norkrans nutrient solution (Brun et al., 1995) in which N and P concentrations were applied at 1/10 strength. The MMN nutrient solution contained (mg l − 1): KH2PO4 (50), (NH4)2HPO4 (25), CaCl2 (50), MgSO4 · 7H2O (150), FeCl3 · 6H2O (1). The mixture was adjusted to pH 4.7, autoclaved at 120°C for 45 min and transferred aseptically into 9-cm Petri dishes (50 ml per dish). At this stage the dishes were inoculated with agar discs supporting mycelia of the mycorrhizal fungi P. in6olutus (Batsch) Fr. or S. bo6inus L. Kuntze. At the same time surface sterilised seeds of P. syl6estris were placed on water agar in Petri dishes to provide sterile germinated seedlings. After 15 days, by which time the fungal inoculum discs had commenced colonisation of the PV, these seedlings were transferred to the Petri dishes with (M) or without (NM) the presence of the
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fungi. The seedlings were planted so that their shoots emerges through a groove cut in vertical wall of the Petri dish to be exposed to the atmosphere of the growth cabinet. The groove was sealed with sterile lanolin, and gas permeable Parafilm was used to wrap the junction between upper and lower portions of the dishes which were then transferred to different CO2 treatments. The total number of seedlings used for the present experiment was 40 (eight NM; 16 M (P. in6olutus); and 16 M (S. bo6inus)), 20 seedlings in each CO2 treatment.
2.2. Experiment 1: effect of CO2 on plant growth, nutrition and mycorrhizal formation The Petri dish systems were placed in two controlled environment cabinet (Fitotron, Sanyo– Gallenkamp PLC, UK) either at ambient CO2 concentration (CAMB: 350 ml l − 1) or in an atmosphere in which CO2 concentration was enriched to 700 ml l − 1 (CELEV). The CO2 concentration in each cabinet was monitored by infra-red gas analysers (ADC 2000, ADC PLC, Hoddesdon, UK) which were used to control introduction of pure CO2 in the CELEV treatment from an external cylinder. Lighting in the cabinets was provided by a combination of fluorescent tubes and incandescent tungsten lamps. The photon flux density at plants height was 400 mmol m − 2 s − 1. All plants received a 16-h day length with a day temperature of 20°C and a night temperature of 15°C. To eliminate chamber effects the plants and their CO2 treatments were switched at weekly intervals from one growth cabinet to the other, the position of the plants being randomised within the chambers at the time of each transfer.
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chambers (Finlay and Read, 1986). Each observation chamber was then returned to the CO2 environment from which the plants growing in it had been taken. Mycelial extension growth was determined by sequential measurements of the distance from the colonised seedling to the mycelial front which, as described by Read (1992), advanced in a fan-like manner across the peat from the plant. Measurements were made at 28, 55 and 87 days for S. bo6inus and at 28 and 55 days for P. in6olutus. Measurements at 87 days for P. in6olutus were not possible because the extramatrical mycelium covered the entire surface of the peat before that date.
2.4. Har6est and analysis After 4 months of growth in the cabinets, the remaining seedlings (eight M (four P. in6olutus and four S. bo6inus) and four NM) were harvested from each of the two CO2 treatments. Their shoots were excised from the root-soil system, oven dried at 80°C for 24 h and weighed. Roots were washed and in the case of M plants, mycorrhizal tips were counted and excised from the root system. Roots and mycorrhizal tips were oven dried (72 h) and weighed separately. Dried plant material (shoots, roots and mycorrhizal tips) was wet digested in sulphuric acid, its N and P concentrations being determined in duplicate aliquots of the digest mixture. Nitrogen was determined using the indo-phenol method (Kedrowski, 1983) and phosphorus using the phospho-molybdate-blue method in presence of a lithium sulfate/copper sulfate catalyst mixture (Boltz and Lueck, 1958).
2.5. Statistical analysis 2.3. Experiment 2: effect of CO2 on fungal growth After 1 month of exposure to the CO2 conditions described above, by which time all seedlings growing in the inoculated dishes were fully mycorrhizal, four seedlings colonised by each fungal species were selected from Petri dishes in each of the two CO2 environments. They were transferred to transparent 20 × 20 cm plexiglas observation
Data on dry weights, N and P content were subjected to a two-way analysis of variance (ANOVA) of randomised design using P\ 0.05 as the critical level of significance. The main experimental parameters, i.e. elevated CO2, mycorrhiza colonisation and their interaction were tested by F-test. The effect of CO2 treatment on fungal colonisation and growth was assessed by comparisons of the means using a Student’s t-test.
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3. Results
3.1. Effects of ele6ated CO2 on biomass of M and NM seedlings of P. syl6estris The whole plant biomass of seedlings in both M and NM treatment were increased by exposure to elevated CO2 (Fig. 1(a)). Growth enhancement was significantly (P B 0.01) greater for plants inoculated with P. in6olutus than S. bo6inus. The shoot yields in all three categories of plants were all significantly (P B 0.01) higher in CELEV than CAMB. There were no differences between the two fungi in the terms of their effects on shoot yield (Fig. 1(b)). In the case of root biomass NM plants and those colonised by P. in6olutus showed significantly (PB 0.01) greater production but increased CO2 supply did not lead to significant impact upon root yield of plants colonised by S. bo6inus (Fig. 1(c)). Despite the absence of a response in the whole root yield under CELEV the S. bo6inus colonised plants produced significantly (P B0.01) greater biomass of mycorrhizal tips (Fig. 1(d)) than those grown in CAMB. The same was true for plants with P. in6olutus mycorrhiza. ANOVA analysis showed that effect of CO2 enrichment upon biomass was significant (P B0.01) in all compartments for P. in6olutus and for shoots and mycorrhizal tips for S. bo6inus. Comparison of the effects of CELEV on whole plant, shoot and root biomass production, showed that in each of these tissue categories the yields were greater in the NM than in the M plants. ANOVA analysis showed that these negative effects upon plants of the M relative to the NM type were significant at PB 0.01 in the case of total and root dry weights of plants colonised by S. bo6inus, and P B 0.05 in the case of root dry weight of plants with Paxillus mycorrhizas. As discussed later, smaller yields in M relative to NM seedlings may be attributable to the costs of initiating the mycelial systems associated with the mycorrhizal roots.
3.2. Effects of ele6ated CO2 on plant tissue N and P concentration Analysis of the nitrogen and phosphorus con-
centrations of whole plant (Fig. 2(a and e)), shoot (Fig. 2(b and f)), and root tissues (Fig. 2(c and g)) and of mycorrhizal tips (Fig. 2(d and h)) showed that exposure to elevated CO2 resulted either in lower N and P concentrations or to a lack of significant difference between them. In only three cases, increases of nutrient concentration were observed under elevated CO2. These were all in non-mycorrhizal plants where the total N and P contents of tissues were also significantly increased (data not presented). No such increases were observed in M plants associated with either fungus. The CO2 effect was significant (PB 0.01) for whole plant N and P concentrations in plants with both mycorrhizal fungi except in the case of N concentration in plants colonised by S. bo6inus. The ANOVA revealed significant (PB 0.01) decreases of P concentrations in all compartments of mycorrhizal plants. These decreases were also seen in the concentration of N in P. in6olutus colonised plants, but not in the case of those colonised by S. bo6inus. Interaction effects (CO2 × mycorrhiza) were significant (PB0.05 and PB 0.01) for all N and P concentrations in P. in6olutus colonised plants but were insignificant for those colonised by S. bo6inus.
3.3. Effects of ele6ated CO2 on the number of mycorrhizal tips In seedlings colonised by either fungi there was a significant (PB 0.01) increase in the number of mycorrhizal tips formed per plant under CELEV (Fig. 3(a)), where the total number of mycorrhizal tips was 62% higher in the plants colonised by either S. bo6inus or P. in6olutus than in their counterparts grown under CAMB. When expressed as number per unit dry weight of root, however, only in the case of S. bo6inus colonised plants was there a significant (PB0.01) effect of CELEV (Fig. 3(b)).
3.4. Effects of ele6ated CO2 upon de6elopment of extraradical mycelium Following transfer of the mycorrhizal plants to peat in transparent observation chambers, devel-
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Fig. 1. Effect of elevated CO2 and mycorrhizal colonisation by P. in6olutus (P. inv.) or S. bo6inus (S. bov.) on the dry weights of P. syl6estris seedlings. (a) Whole plant, (b) shoots, (c) roots and (d) mycorrhizal tips. Two-week-old transplanted mycorrhizal (M) and non-mycorrhizal (NM) plants were grown for 4 months at ambient ( =350 ml l − 1) and elevated ( = 700 ml l − 1) CO2 concentration. Error bars represent 95% confidence limits (n = 4) and statistical differences between means (*P B0.05; **P B 0.01; n.s., not significant P\ 0.05) are the result of a t-test.
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Fig. 2. Effect of elevated CO2 and mycorrhizal colonisation by P. in6olutus or S. bo6inus on nitrogen (a – d) and phosphorus (e–h) concentrations in whole plant, shoots, roots and mycorrhizal tips of P. syl6estris seedlings. Two-week-old transplanted mycorrhizal (M) and non-mycorrhizal (NM) plants were grown for 4 months at ambient ( =350 ml l − 1) and elevated ( =700 ml l − 1) CO2 concentration. Error bars represent 95% confidence limits (n =4) and statistical differences between means (*P B0.05; **P B 0.01; n.s., not significant P \0.05) are the result of a t-test.
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Fig. 3. Effect of elevated CO2 on production of mycorrhizal tips by P. syl6estris seedlings inoculated with P. in6olutus or S. bo6inus. (a) Number of mycorrhizal tips per plant; and (b) number of tips colonised per unit (mg) of root. Two-week-old transplanted mycorrhizal (M) plants were grown for 4 months at ambient ( =350 ml l − 1) and elevated ( =700 ml l − 1) CO2 concentration. Error bars represent 95% confidence limits (n = 4) and statistical differences between means (*P B0.05; **P B0.01; n.s., not significant P \0.05) are the result of a t-test.
opment of their extraradical mycelium systems could be seen to respond rapidly to CO2 elevation (Fig. 4). The hyphal fronts of both fungi grew, from the colonised roots, more rapidly under CELEV than CAMB condition from time zero. By the time of the first measurement of replicate plates after 28 days, the total areas of peat surface occupied by the mycelial fans were significantly (P B 0.01) greater in CELEV than CAMB in both fungi. Differences between the two treatments continued to diverge at the second harvest (55 days) in the case of P. in6olutus by which time this fungus occupied 444% greater total area of peat, and through to the third harvest (87 days) in S. bo6inus where the final difference between areas occupied by mycelium in CAMB and CELEV was 230%.
4. Discussion Under elevated concentrations of atmospheric CO2 enhancements were observed in total biomass production of both M and NM seedlings. This confirms the pattern of response reported in several previous studies (Walker et al., 1995b; Hodge, 1996; Lewis and Strain, 1996; Godbold et al., 1997; Tingey et al., 1997). Of particular interest was the observation that under these conditions the plants grown with their mycorrhizal symbionts produced greater number of mycorrhizal lateral roots and much more extraradical mycelium systems than did those grown at CAMB. These results thus provide further support for the view that mycorrhizal fungi act as a sink for assimilates and that under conditions where pho-
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Fig. 4. Effect of elevated CO2 on the development in mycelial systems of (a) P. in6olutus; or (b) S. bo6inus growing with P. syl6estris seedlings in mini-rhizotrons. Plants were grown at ambient (350 ml l − 1 — open symbols) and elevated (700 ml l − 1 — closed symbols) CO2 concentration. Error bars represent 95% confidence limits (n =4).
tosynthate availability to roots increases, a significant amount of these resources may be allocated to the fungus. It is evident, however, that despite the greater number of mycorrhizal tips in the Petri dish experiment, seedlings grown with their symbionts under elevated CO2 produced less biomass than did their NM counterparts. It has frequently been observed even under ambient CO2 conditions, that in the earliest stages of growth, during which the vegetative mycelium of the fungal partner is developing, the biomass of mycorrhizal plants can be lower than that of uncolonised seedlings (Dosskey et al., 1990; Stenstro¨m et al., 1990; Newton, 1991; Colpaert et al., 1992). This effect is thought to arise from the carbon demands placed upon the mycorrhizal seedling by its fungal symbiont (Smith and Read, 1997).
If, as appears likely from this study, fungal growth, and hence carbon demand, is enhanced when more CO2 is available to the plant, these effects on its biomass are likely to be exaggerated. The period of mycelial development is also one of maximal demand by the fungus for N and P to enable hyphal growth. There might in effect, be competition between fungal and plant tissues for these nutrients during establishment of the vegetative mycelium and this would explain the observed reductions in shoot and root N and P concentrations of M relative to NM plants under CELEV. Under our experimental conditions these competitive effects would be exacerbated by the confinement of the seedlings within the dishes which will have reduced the effectiveness of nutrient foraging by the mycelium.
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It has also been observed that plants grown under elevated CO2 show reduced tissue nutrient concentrations, those of N being particularly susceptible to these effects (Norby et al., 1987; O’Neill et al., 1987a; Johnson et al., 1995). Indeed, the results of Walker et al. (1995a) suggest that the responses of Pinus ponderosa seedlings to elevated CO2 are entirely determined by nitrogen availability since only when supplementary N was added to a containerised potting mix did the mycorrhizal plants respond positively to atmospheres with enriched CO2. Our observations of mycorrhizal mycelial development are in general accord with those of Ineichen et al. (1995) in so far as they confirm enhancement of mycorrhizal development under conditions of elevated CO2. However, in a number of respects they differ and these differences may be explicable in term of the contrasting experimental design employed in the two studies. Ineichen et al. (1995) observed no differences in extraradical mycelial development in M seedlings exposed to CELEV in early harvests, increases being recorded only after c 100 days of growth. However, their seedlings were not mycorrhizal when introduced into the growth chambers, so the early stage of development of the mycorrhizas themselves may, as indicated above, have consumed any excess of carbon being allocated to fine roots. In their case, also, a preformed mycelium would act as a sink for carbon before new biomass could be produced. This situation contrasts strongly with that in our observation chambers to which seedlings with preformed mycorrhizas were added. In this case the ‘cost’ of mycorrhiza formation, and of sustaining a large external mycelium, was eliminated so enabling the increased carbon flow to M roots under elevated CO2 to be allocated immediately for the support of new mycelial extension growth. The observed delay in response of the extraradical mycelium in the Ineichen et al. (1995) study is also consistent with their observation that the number of mycorrhizas formed in CELEV did not differ from those of CAMB until the second harvest c 60 days after inoculation with P. tinctorius. It must be recognised that there will be some interspecific differences between the fungal symbionts in their
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responses to elevated carbon availability but in these cases it seems that contrasting histories of mycorrhiza formation are more likely to explain the differences in the observed patterns of response to CELEV. It can be hypothesised that in nature, where mycorrhizal mycelial systems are free to forage without spatial constraints through larger volume of soil, increased allocation of carbon under CELEV, by enabling production of more extensive hyphal networks, would provide increased access to N. This hypothesis is currently being tested using observation chambers with patchily distributed remote sources of available N.
Acknowledgements We thank The Human Capital and Mobility program of the European Commission for providing the grant (contract No. ERBCHBICT941011) which supported H. Rouhier during the course of this work.
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