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Soil Biol. Biochem. Vol. 30, No. 3, pp. 315321, 1998 # 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0038-0717/98 $19.00 + 0.00 S0038-0717(97)00133-8


Cellular and Environmental Physiology Department, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA U.K. and 2Department Plant and Soil Science, University of Aberdeen, Meston Building, Aberdeen AB9 2UE U.K. (Accepted 8 April 1997)

SummaryThe structure of microbial communities in the rhizospheres of ryegrass and wheat, growing at an elevated atmospheric CO2 concentration, was investigated using broad-scale DNA techniques. Community DNA hybridisation and %G + C base proling by thermal denaturation assess changes at the whole microbial community level. DNA analysis of the rhizosphere of ryegrass grown in soil microcosms for 28 or 42 d, showed only minor dierences between plants grown at 450 or 720 ml CO2 l1. In a second experiment with ryegrass, 5 of 10 replicate microcosms were pulse labelled with 14CO2 and 5 simultaneously sampled for DNA analysis. Carbon partitioning below ground showed changes due to the elevated CO2, notably an increased proportion of xed carbon in non-microbial biomass residue in the rhizosphere. There was again no eect of elevated CO2 on rhizosphere microbial community structure. Community DNA hybridisation indicated that the rhizosphere communities under ambient and elevated CO2 were 86% similar (unlikely to be a biologically relevant change), with indistinguishable %G + C proles. Wheat was grown to maturity (129 d) in a dierent soil microcosm design, and rhizosphere microbial communities from plants grown at 350 and 700 ml CO2 l1 were identical according to the DNA analyses. In these experiments rhizosphere microbial community structure at the broad scale was unaected by the interactions occurring below ground as a result of elevated concentrations of CO2. # 1998 Elsevier Science Ltd. All rights reserved


One of the eects of increased concentrations of atmospheric CO2 upon growing plants is alteration of C ow from the roots into the rhizosphere. Both the total amount of C (Billes et al., 1993) and the proportion of C partitioned below ground (Rattray et al., 1995) are aected. This extra C entering the soil can increase rhizosphere microbial biomass (Diaz et al., 1993; Zak et al., 1993) and more generally increases microbial activity in the rhizosphere. This is evident from enhanced soil respiration, increased enzyme activity, more rapid cycling of C through the soil and increased N mineralisation under elevated CO2 concentrations (Ko rner and Arnone, 1992; Zak et al., 1993; Ross et al., 1995). Although C released from the roots is readily assimilated into microbial biomass, the proportion of root-derived C retained by the microbial biomass is reduced under elevated CO2 (Rattray et al., 1995). This indicates that where elevated CO2 aects plant growth, there are likely to be concur*Corresponding author: Tel: 01382 562731; Fax: 01382 562426; Email bgri@scri.sari.ac.uk.

rent eects on rhizosphere microbial communities (Paterson et al., 1996). Diaz et al. (1993) argued that shifts in species composition should be anticipated in the microbial components of a large number of plant communities, in response to increasing atmospheric CO2 concentrations. Zak et al. (1996) also suggested that greater plant litter production at elevated CO2 should shift microbial community composition as fungal populations increase in response to greater substrate availability. However, little attention has been paid to microbial community structure in the rhizosphere and how this may change under the inuence of increasing CO2 (O'Neill, 1994). One of the important hypotheses to test is that rhizosphere community composition and dynamics will be altered and stimulated under plants growing in high CO2 atmospheres (Rogers et al., 1994). Phospholipid fatty acid analysis, however, indicated that there was no change in the rhizosphere microbial communities of Populus grandidentata in response to elevated CO2 concentration (Zak et al., 1996). Techniques based on the analysis of microbial DNA (community hybridisation and thermal dena-



B. S. Griths et al.

turation proles) have been developed to categorise microbial community structure in soil (Ritz and Griths, 1994; Griths et al., 1996; Ritz et al., 1997). These techniques are broad scale (i.e. cover all micro-organisms rather than specic groups) and are more representative of the whole microbial community than cultural techniques. We used these techniques to test whether there was an eect of elevated CO2 upon the rhizosphere microbial community structure of ryegrass and wheat.

Plant growth conditions for rye grass The experimental design was largely as reported by Rattray et al., 1995 and Paterson et al., 1996. Six seedlings of perennial ryegrass (Lolium perenne L., cv. Merlinda) were transferred when 7 d old to soil microcosms. These comprised a section of PVC pipe (length 20 cm, internal dia 7.5 cm), with a shoot chamber constructed from clear Perspex piping, to allow separation of shoot and root compartments and pulse labelling with 14CO2. A batch of loamy sand (Paterson et al., 1996) was sieved (3 mm) and stored moist at 48C before packing into microcosms. Plants were grown in a growth cabinet (Fitotron, Sanyo Gallenkamp PLC, Leicester, U.K.) with a 16 h photoperiod of 700 mmol m2 s1 at a constant temperature of 158C and relative humidity of 85%. CO2 concentrations were maintained at 720 ml l1 (718 mmol mol1 2 28 (day) or 216 (night) with a mean 24 h range 224) or 450 ml l1(451 mmol mol1 221 (day) or 14 (night) with a mean 24 h range 219). A single growth cabinet was used, therefore while CO2 treatments were applied in consecutive rather than concurrent experiments, all other growth conditions were identical. Two experiments were performed with ryegrass, using dierent control treatments with which to assess the eect of elevated CO2. Experimental design for ryegrass Experiment 1 The microcosms were packed to a bulk density of 1 Mg m-3, maintained at a matric potential of 50 kPa by the addition of sterile distilled water, and allowed to equilibrate for 3 wk before planting. Ten replicate test microcosms with ryegrass were grown under 720 ml CO2 l1. Rhizosphere samples were taken, as detailed below, when the plants were 28 and 42 d old. Soil, from the original batch stored moist at 48C, was then used to prepare another set of microcosms. Plant growth and rhizosphere samplings were repeated under 450 ml CO2 l1. Control microcosms were set up simultaneously with the test microcosms, and were incubated under ambient concentrations of CO2 in a completely separate growth cabinet. In this case, ambient CO2 concentrations were 342 2 22 ml l1 in the room housing the growth cabinet, while in the

cabinet CO2 concentrations declined linearly with plant growth, from ambient after 21 d to 230 ml l1 after 42 d. The light level in this cabinet was 550 mmol m2 s1. In this experiment the eect of elevated CO2 in the ``test'' microcosms was assessed in relation to the ``control'' microcosms. Experiment 2 Ten replicate microcosms were prepared with a fresh batch of loamy sand soil, as above, and ryegrass grown under 720 ml CO2 l1 until the plants were 28 d old. At this time 5 microcosms were pulse labelled with 14CO2, (Rattray et al., 1995; Paterson et al., 1996) and 5 microcosms used for rhizosphere sampling. Soil, from the same batch stored moist at 48C, was then used to prepare another set of microcosms. Plant growth and pulse labelling/rhizosphere samplings were repeated under 450 ml CO2 l1. Unplanted microcosms were also incubated under the same conditions. In this experiment the eect of elevated CO2 in the ``test'' microcosms was assessed in relation to the behaviour of the unplanted microcosms, the batch of stored soil and the pulse labelling reactions. Rhizosphere sampling The plants and soil were gently removed from the microcosms, the shoots removed and excess soil (i.e. non-rhizosphere) shaken from them. The rhizosphere soil was washed from the roots with 1/4 strength Ringer's solution, and concentrated by centrifugation at 900 g for 30 min. The soil was transferred in 2 g aliquots into sterile micro-centrifuge tubes and stored at 208C. The washed roots and shoots were dried at 708C overnight for dry weight determinations. Plant growth conditions and experimental design for wheat Soil samples were also taken from wheat roots which had been growing for 18 wk under controlled CO2 concentrations in a dierent experimental design. Briey, plants were grown in microcosms consisting of 1.2 m lengths of electrical cable trunking 25 50 mm in cross-section (Robinson et al., 1994). These microcosms t into two specially constructed, glasshouse-located chambers in which the concentration of CO2 was monitored by an infra-red gas analyser and maintained by injection of CO2 from a cylinder (Gordon et al., 1995). Air and soil temperatures in these chambers were controlled separately. Eight microcosms were lled with 1.35 kg oven-dry equivalent of a clay loam soil (Carpow Association, Carpow Series; Laing, 1976), and fertilised with N, P and K equivalent to 220, 18 and 35 mg kg1 dry soil respectively. A spring wheat seedling (Triticum aestivum L. cv. Tonic) was planted in each microcosm, and four microcosms were assigned randomly to each chamber, one at 350 and one at 700 ml CO2 l1 (these formed part of a larger experiment (M. van Vuuren et al., unpubl.

Eect of CO2 on rhizosphere community structure


data)). The microcosms were watered to constant weight, equivalent to a matric potential of 10 kPa for this soil, twice a week. After 18 wk growth the roots were carefully removed from each microcosm, the soil was homogenised and representative subsamples collected and stored frozen at 208C. DNA extraction and analysis DNA was extracted from the frozen soil aliquots as described by Griths et al. (1997), with the alteration that caesium chloride was removed using Microcon concentrators (Amicon Inc, Beverly, U.S.A.) instead of a Sephadex column. DNA was then checked for purity by spectrophotometry, quantied by the diphenylamine reaction and thermal denaturation proles determined, as described by Griths et al. (1997). Signicant dierences between thermal denaturation proles were determined by principal component analysis (PCA) of the parameters of the curves, and analysis of variance of the PC scores (Griths et al., 1997). The rst derivative of the absorbancy vs temperature (increase in absorbancy/temperature interval) was calculated to provide a %G + C distribution prole (Ritz et al., 1997). For the community DNA hybridisation studies, equal amounts of DNA from each replicate within a treatment were pooled to give a composite sample representative of that treatment. These pooled samples were used in cross-hybridisations (Griths et al., 1996). The observed similarity index, S, was calculated as in the paper by Lee and Fuhrman (1990), i.e. S = (Rs Rb)/ (Rc Rb) 100% where Rs = signal from sample (target) DNA, Rc = signal from control (probe) DNA and Rb = background signal. The lowest of the hybridisation couplets (i.e. sample X as probe versus sample Y as target = S1; sample Y as probe versus sample X as target = S2) was taken to be the true value of similarity (Lee and Fuhrman, 1990; Ritz and Griths, 1994). The mean similarity and 95% condence interval were determined by a bootstrap procedure. DNA was also extracted from ryegrass seedlings, prior to planting in soil, and cross-hybridised with DNA extracted from soil. This was a calibration to determine the concentration of plant DNA in rhizosphere soil samples. The sensitivity of the technique was also calibrated by comparing DNA from rhizosphere and non-rhizosphere soil.

There was a signicant eect of the rhizosphere on microbial community structure. Rhizosphere DNA was 29 (c.i. 2363)% similar to DNA from unplanted soil, and had a signicantly dierent thermal denaturation prole (ANOVA of PC scores, P < 0.05), which indicated a reduced median %G + C content of the rhizosphere microbial community (Fig. 1). Plant growth and

C partitioning

Ryegrass Experiment 1 Plants grown under 720 ml CO2 l1 had a signicantly greater mass than plants grown under 450 ml CO2 l1 after 28 d. Total plant dry wt's at this time were 600 mg (root-to-shoot ratio = 0.69) and 360 mg (r-to-s = 0.69) (P < 0.001) respectively. After 42 d there was no signicant dierence in plant weights under 720 ml l1 (1.62 g, r-to-s = 0.63) and 450 ml l1 (1.74 g, r-to-s = 0.81). Plant weights were log-transformed for analysis, and the standard error of the dierence of the mean of the transformed data was 0.11 (n = 40). Ryegrass Experiment 2 There was a signicant (P < 0.001) increase in plant growth at the higher concentration of CO2 after 28 d (Table 1). There was, however, signicantly (P < 0.01) less rhizosphere soil associated with the roots (rhizosphereto-root ratio) of plants grown at 720 than 450 ml l1 (Table 1). The carbon budget revealed that a greater proportion of xed carbon was retained in or respired from the shoots at the higher concentration of CO2 (Table 1). The converse was true for the roots. The proportion of 14C detected in the rhizosphere microbial biomass was not aected by CO2 enrichment, but a signicantly (P < 0.01) greater proportion of xed C was detected in the rhizosphere residue (i.e. C in the rhizosphere which


Calibration measurements of microbial community structure Ryegrass DNA showed small amounts at hybridisation to rhizosphere soil (similarity, S, =4%, with a 95% condence interval (c.i.) 35%) and unplanted soil (S =6%, c.i. 58%).
Fig. 1. %G + C distribution of microbial community DNA from unplanted soil () and ryegrass rhizosphere (w).


B. S. Griths et al.

Table 1. Plant growth measurements and 14C distribution between plant and soil pools for 28-d-old ryegrass (Lolium perenne) grown at 450 and 720 ml CO2 l-1 concentrations. Mean of ve replicate microcosms with standard error in parenthesis 450 ml l-1 shoot root rhizosphere soil root-to-shoot ratio rhizosphere-to-root ratio shoot root shoot respiration root/soil respiration microbial biomass rhizosphere residue % microbial biomass* dry weights (g) 0.29 (0.022) 0.25 (0.020) 10.2 (0.83) 1.0 (0.17) 44.0 (4.7) % C distribution 72.1 (2.75) 25.8 (2.74) 0.42 (0.037) 1.57 (0.267) 0.091 (0.012) 0.059 (0.042) 70.0 (15.37)

720 ml l-1 0.629 (0.101) 0.59 (0.108) 12.0 (1.08) 1.1 (0.39) 22.0 (4.0) 76.3 (1.70) 21.3 (1.84) 1.33 (0.144) 0.78 (0.140) 0.087 (0.032) 0.154 (0.021) 34.1 (8.54)

* % of the 14C distribution in the rhizosphere that is in the microbial biomass (i.e. microbial biomass/microbial biomass + rhizosphere residue 100).

Fig. 3. %G + C distribution of ryegrass rhizosphere microbial community DNA from plants grown for 28 d at 450 ml CO2 l-1 (w) or 720 ml CO2 l-1 (.), in ryegrass Experiment 2.

was not part of the microbial biomass) under elevated CO2. Thus, under 720 ml l1 the proportion of microbial biomass 14C in the rhizosphere was about half that under 450 ml CO2 l1 (Table 1). Rhizosphere microbial community structure Ryegrass Experiment 1 The community DNA hybridisation assay showed that after 28 d the similarity between rhizospheres of plants grown under 720 or 450 ml l1 (S = 55%, c.i. 4074%) was not distinguishable from the similarity between the rhizospheres of control plants (S = 70%, c.i. 65 78%). There were no dierences in microbial community structure between rhizosphere DNA samples from the CO2 treatments after 42 d, with an S of

106% (c.i. 99113%) for 720 and 450 ml l1, and an S of 102% (c.i. 98107%) for the control plants. The %G + C distribution proles of the rhizosphere DNA from the CO2 treatments were also not signicantly (ANOVA of PC scores, P > 0.05) dierent from the control plants (Fig. 2). Ryegrass Experiment 2 The rhizosphere DNA samples from under 720 and 450 ml l1 had a similarity of 86 (c.i. 8488)%), and had statistically similar %G + C distribution proles (ANOVA of PC scores, P > 0.05; Fig. 3). The controls in this experiment were samples from the batch of sieved soil taken when the microcosms were prepared, and soil from unplanted microcosms incubated for 28 d. In both cases the microbial communities remained constant between the sets of microcosms, with S

Fig. 2. %G + C distribution of ryegrass rhizosphere microbial community DNA, from plants grown for 28 d at 450 ml CO2 l-1 (w) or 720 ml CO2 l-1 (.) and from plants grown simultaneously at ambient CO2 concentrations to give a 450 ml l-1 control (r) or a 720 ml l-1 control (W).

Fig. 4. %G + C distribution of wheat rhizosphere microbial community DNA from plants grown for 18 wk at 350 ml CO2 l-1 (q) or 700 ml CO2 l-1 (Q).

Eect of CO2 on rhizosphere community structure


values of 98 (c.i. 93104)% and 89 (c.i. 76104)% respectively and with statistically similar %G + C distribution proles (ANOVA of PC scores, P > 0.05). Wheat In this experiment microbial community structure from soil samples under 700 and 350 ml CO2 l1 was not signicantly dierent. The DNA samples were 102 (c.i. 86114)% similar and had statistically (ANOVA of PC scores, P > 0.05) similar %G + C distribution proles (Fig. 4).

The interpretation of microbial community structure from community hybridisation and thermal denaturation proles has been discussed by Griths et al. (1997). Both methods give complementary information and need to be interpreted together. The calibration measurements, which essentially revealed no signicant contamination of rhizosphere soil by plant DNA, and that there was a signicant eect of the plant on microbial community structure, showed that the broad-scale approach taken was suitable for the study of changes in rhizosphere microbial community structure. In the rst experiment although the rhizosphere microbial community structure under 720 ml l1 was signicantly dierent to that under 450 ml l1, the rhizosphere microbial community structures in the control plants also diered signicantly. This indicated that dierences could be attributed to the replication over time within the experiment and that any CO2-specic responses were relatively minor compared to inherent replicability over time. A dierence between replicate experiments is not unexpected, Gilbert et al. (1993) similarly found that applying the same treatment to rhizosphere soils at dierent times did not give consistent results. The second experiment was limited to 28 d, based on the results from Experiment 1, and was coupled with the simultaneous determination of carbon budgets. These conrmed that the characteristic changes associated with elevated CO2 (Rattray et al., 1995; Paterson et al., 1996) had taken place, namely that the rhizosphere microbial biomass assimilated less of the exuded carbon under elevated CO2. These changes to the C budget were not sucient, however, to aect rhizosphere microbial community structure to any substantial degree. The microbial communities at 450 and 720 ml CO2 l1 were 86% similar, which is equivalent to a change in similarity of 14% and is consistent with the value of 15% (i.e. 70%55%) from the rst experiment. A similarity of over 80% (or a dierence of less than 20%) is not considered to be a signicant change (Lee and Fuhrman, 1990). This was conrmed by the similarity of the thermal denaturation proles. The third measurement, in which replicated wheat plants had been growing under ambient and

elevated concentrations of CO2 simultaneously, also revealed no signicant eect on rhizosphere microbial community structure. There was also no eect of CO2 upon soil microbial biomass associated with these plants (M. van Vuuren et al., unpub. data). These measurements were taken from wheat rather than ryegrass, although elevated CO2 did have a similar eect on the below-ground C budget of these two species after 21 d (Paterson et al., 1996), and after a much longer period of plant growth. The similarity of these responses by two plant species suggests that the results obtained over the short term from ryegrass are typical. A concern is that these results, like all others from greenhouse studies may not be transferable to the eld, as a result of soil disturbance eects and soil moisture relations. C and nutrient release arising from soil disturbance may have masked any eects due to altered CO2 regimes, but this embraces an important point: eects on rhizosphere community structure due to the CO2 regimes may be so subtle that in disturbed systems (e.g. tilled soils) they are of little consequence. The initial calibration measurements showed that there was a large eect of the rhizosphere on microbial community structure. However, the more subtle alterations to exudate patterns caused by an altered C ow resulting from elevated CO2 concentrations did not aect the rhizosphere microbial community structure, as determined by the broad-scale measurements made in this study. There is little doubt that elevated CO2 aects C ow from roots. In wheat continuously-labelled with 14CO2 for 28 d C release below ground was enhanced, although this was attributed to extra root growth rather than modied root activity (Billes et al., 1993). The C budget in this study also revealed a shift in the balances of C entering the rhizosphere. There have been no consistent trends in the responses of microbial numbers or biomass to elevated CO2. O'Neill (1994) reviewed the few studies to date, and showed a positive eect of CO2 on microbial populations in some and no eect in other studies. These conicting results were ascribed to dierent plant species, plant community type, soils and methodology. In an earlier study, O'Neill et al. (1987) found a trend towards reduced populations in the rhizosphere of yellow poplar under elevated CO2. Runion et al. (1994) found no eect of elevated CO2 on microbial populations in cotton rhizosphere, while Rice et al. (1994) and Diaz et al. (1993) found increased microbial biomass. Changes in microbial biomass, however, give no information about microbial community structure. Thus, while the rhizosphere microbial biomass of Populus grandidentata increased under elevated CO2 there was no detectable change in microbial community structure (Zak et al., 1996).


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The interpretation of any eects of rhizosphere C ow are complicated by interactions with nutrients and water. Paterson et al. (1996) attributed the dierential utilisation of 14C-labelled rhizodeposits to a shift in the nutritional status of the rhizosphere soil, driven by altered quantity or quality of root-derived organic compounds. Elevated CO2 reduces plant water use due to increased stomatal closure (Lawlor and Mitchell, 1991), leaving the soil wetter between waterings than under ambient CO2. Dierences in water use may explain the greater amount of rhizosphere soil per unit of root under elevated CO2, in this and in other studies (Paterson et al., 1996). Microbial growth in the rhizosphere is directly aected by the depletion of mineral nutrients (Merckx et al., 1987), exudation is aected by soil nutrient status (Liljeroth et al., 1990) and there is an interaction between rhizosphere microbial biomass and soil moisture content (Rice et al., 1994). These interactions make it very dicult to attribute changes in the rhizospheres of plants grown under dierent concentrations of CO2 to any single factor, such as altered patterns of exudation. Our results, and those of Zak et al. (1996), indicate that rhizosphere microbial community structure at the broad scale was unaected by the interactions occurring below ground (i.e. amount of exudate, exudate composition, nutrient depletion, water movement) as a result of elevated concentrations of CO2.
Acknowledgements We thank Joyce Davidson for typing the manuscript and Margret van Vuuren for access to the wheat rhizosphere samples. This work was funded by the Scottish Oce Agriculture, Environment and Fisheries Department, under their Flexible Funding Scheme.


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