Specific rhizosphere bacterial and fungal groups respond differently to elevated atmospheric CO₂

Abstract

Soil community responses to increased atmospheric CO₂ concentrations are expected to occur mostly through interactions with changing vegetation patterns and plant physiology. To gain insight into the effects of elevated atmospheric CO₂ on the composition and functioning of microbial communities in the rhizosphere, Carex arenaria (a non-mycorrhizal plant species) and Festuca rubra (a mycorrhizal plant species) were grown under defined atmospheric conditions with either ambient (350 p.p.m.) or elevated (700 p.p.m.) CO₂ concentrations. PCR-DGGE (PCR-denaturing gradient gel electrophoresis) and quantitative-PCR were carried out to analyze, respectively, the structure and abundance of the communities of actinomycetes, Fusarium spp., Trichoderma spp., Pseudomonas spp., Burkholderia spp. and Bacillus spp. Responses of specific functional groups, such as phloroglucinol, phenazine and pyrrolnitrin producers, were also examined by quantitative–PCR, and HPLC (high performance liquid chromatography) was employed to assess changes in exuded sugars in the rhizosphere. Multivariate analysis of group-specific community profiles showed disparate responses to elevated CO₂ for the different bacterial and fungal groups examined, and these responses were dependent on plant type and soil nutrient availability. Within the bacterial community, the genera Burkholderia and Pseudomonas, typically known as successful rhizosphere colonizers, were significantly influenced by elevated CO₂, whereas the genus Bacillus and actinomycetes, typically more dominant in bulk soil, were not. Total sugar concentrations in the rhizosphere also increased in both plants in response to elevated CO₂. The abundances of phloroglucinol-, phenazine- and pyrrolnitrin-producing bacterial communities were also influenced by elevated CO₂, as was the abundance of the fungal genera Fusarium and Trichoderma.

Introduction

Since the advent of the industrial revolution, the concentration of atmospheric CO₂ in the Earth's atmosphere has increased by 31%, and it is expected to rise at an annual rate of 0. 5% (Alley et al., 2007). This rapid rise in CO₂ concentration has led to considerable interest in the potential of biological systems to mitigate the effects of rising atmospheric CO₂ concentration by enhanced carbon (C) sequestration. It is generally believed that under increasing atmospheric CO₂ conditions the largest input of C to the soil from the atmosphere is through photosynthesis (Ainsworth and Long, 2005). Particularly for C₃ plants (Long et al., 2004), stimulation of plant production results in enhanced fluxes of organic compounds into the soil (King et al., 2004). Changes in soil-borne C pools, acting as a C sink or source of CO₂, can potentially affect the CO₂ concentration in the atmosphere and therefore influence global climate. It is estimated that, under current atmospheric CO₂, up to 40% of the C fixed by plants can be transferred through root turnover in the soil (Lynch and Whipps, 1990). Under elevated atmospheric CO₂ levels, rhizodeposition is generally expected to increase (Darrah, 1996) and its composition to be altered (Hodge and Millard, 1998) due to changes in plant carbohydrate status (Barron-Gafford et al., 2005).

Rhizodeposition is the key process of C input into the soil, occurring through secretions (including enzymes and mucilage), sloughing off of dead cells, root turnover and root exudation, releasing low molecular-weight, water-soluble substances, such as sugars, aminoacids and organic acids (Nguyen, 2003). In addition, C flux can be mediated through arbuscular mycorrhizal fungi (AMF). These symbiotic fungi, which infect the majority of land plants (Smith and Read, 1997), have been shown to influence C flow in response to elevated CO₂, thereby affecting soil microbial-community abundance and structure (Jones et al., 1998; Drigo et al., 2007; Phillips, 2007).

Previous studies on the effects of elevated atmospheric CO₂ concentrations have either examined total microbial-community patterns (Griffiths et al., 1998; Kandeler et al., 1998; Marilley et al., 1999; Montealegre et al., 2002; Jossi et al., 2006; Lipson et al., 2006; Drigo et al., 2007), metabolic patterns (Grayston et al., 1998; Hodge and Millard, 1998; Tarnawski et al., 2006) or have focused on the structure of specific microbial communities of interest, such as Rhizobium species (Schortemeyer et al., 1996; Montealegre et al., 2000), Pseudomonas species (Marilley et al., 1999; Tarnawski et al., 2006), ß/α-Proteobacteria or Acidobacteria (Lipson et al., 2006) and mycorrhizal fungi (Gamper et al., 2005). However, there is still a general lack of knowledge with respect to the relative responses of different specific microbial groups in response to elevated atmospheric CO₂ conditions.

The aim of this study was to assess the plant-driven impact of elevated atmospheric CO₂ concentration on shifts in the composition of specific bacteria and fungi inhabiting F. rubra and C. arenaria rhizosphere in dutch coastal dune ecosystems. To address this objective, we examined the effect of elevated atmospheric CO₂ on plant-root exudation (high performance liquid chromatography (HPLC)) and on the community structure and abundance of a number of specific soil-borne groups with presumabely contrasting life-history strategies. For specific groups, analyses targeted Pseudomonas spp. (Lugtenberg et al., 2001) and Burkholderia spp. (Berg et al., 2005), as bacteria well adapted to the rhizosphere environment and actinomycetes and the genus Bacillus, as group showing typical bulk-soil ecological strategies (Smalla et al., 2001). Two important fungal genera, Trichoderma and Fusarium, were similarly examined. As an example of potential functional impacts of elevated atmospheric CO₂, we also examined the densities of selected genes involved in antibiotic production (phloroglucinol, phenazine and pyrrolnitrin) related to potentially antagonistic activity of Pseudomonas spp. and Burkholderia spp. All results were subsequently analyzed by multivariate statistical methods to estimate the relative impact of elevated CO₂ treatment in comparison to plant species and soil-origin effects on the different microbial targets (Borcard et al., 1992; Ter Braak and Verdonschot, 1995; Filion et al., 2000).

Materials and methods

DNA isolation and PCR-DGGE analyses

Plant production, incubation conditions at ambient and elevated CO₂ and harvesting procedures are explained in detail in Drigo et al. (2007). Briefly, 4-week-old sterilized seedlings of F. rubra (a mycorrhizal plant) and C. arenaria (a non-mycorrhizal plant) were planted in a coastal-dune soil (Middelduinen), a former beach plain soil (Kwade Hoek) or a river-dune soil (Bergharen). A total of 240 pots (750 cm³) were filled with 1 kg of soil and adjusted to 10% volumetric water content (on the basis of dry weight). Together with 72 unplanted soils, 4-week-old seedlings of F. rubra and C. arenaria were selected for uniformity and transferred to containers (three seedlings per container), which were equally distributed and incubated in four CO₂ flow cabinets, which controlled the temperature and moisture conditions. Two cabinets were maintained at atmospheric condition of 350 p.p.m. CO₂, and two cabinets were maintained at 700 p.p.m. CO₂. A detailed description of the CO₂ flow cabinets is provided in Drigo et al. (2007). F. rubra and C. arenaria rhizosphere and bulk soils were harvested, respectively. at 73 and 62 days after germination, when plants were dominated by shoot growth. In C. arenaria, the root–soil systems were first separated from rhizomes. On harvest, roots from each pot were shaken gently to remove loosely adhering soil. Approximately 80% of the initial soil remained in the pots, and this portion was considered ‘bulk soil’. Pieces of roots with remaining adhering soil were cut and taken as a combined rhizosphere sample (Drigo et al., 2007).

Soil genomic DNA was isolated from 500 mg (wet weight) rhizosphere soil using the PowerSoil™ DNA Isolation kit according to the manufacturer's specifications (MoBio Laboratories, Solana Beach, CA, USA). DNA was eluted in 50 μl of deionized water and stored at −20 °C until use. We randomly selected 216 DNA extracts obtained from rhizosphere soil samples (54 samples per CO₂ treatment, resulting in a total of 108 samples per plant) and 36 from unplanted soil under two different CO₂ treatments for molecular analyses of microbial communities (see below). All PCRs were carried out in a PTC-200 thermal cycler (MJ-Research, Waltham, MA, USA) in 25 μl volumes containing 2.5 μl of 10 × PCR buffer, 2.5 μl of bovine serum albumin (BSA; 4 mg ml⁻¹), 0.75 μl of each primer (30 pM), 2.5 μl of dNTPs mix (8 mM) and 0.056 U of Expand High Fidelity polymerase (Roche, Mannheim, Germany). To reduce variation caused by pipetting errors, PCR mixtures were prepared using a Corbett Robotics CAS-1200 precision liquid handling system (Sydney, NSW, Australia). Table 1 summarizes the primers, thermocycling regimes and electrophoresis conditions used to analyze different target communities. All PCRs were carried out according to the touchdown protocols described in Table 1. In addition to the target soil DNA, a negative control sample (without DNA) was included with every PCR run. PCR products were examined by standard 1.5% (w/v) agarose 0.5 TBE (Tris/Borate/EDTA) gel electrophoresis with ethidium-bromide staining, to confirm product integrity and estimate yield. Approximately 0.5 μg of PCR product was used for DGGE analysis, using the method of Muyzer et al. (1993) as modified by Kowalchuk et al. (2002), using the linear gradients indicated in Table 2. DGGE was carried out using a D-Code Universal Mutation Detection System (Bio-Rad, Hercules, CA). All gradient gels were topped with 10 ml of acrylamide containing no denaturant, and electrophoresis was carried out at 60 °C and 200 V for 10 min followed by an additional 16 h at 70 V. Gels were stained in ethidium bromide and digital images captured using an Imago apparatus (Gentaur, Brussels, Belgium) on UV transillumination. Owing to the number of samples, multiple DGGE gels were run for the three different soil origins (Bergharen, Middelduinen and Kwade Hoek) and the different plants species (C. arenaria and F. rubra). Each sample was assessed in at least two different runs to confirm the reproducibility of the DGGE fingerprint across gels. To facilitate comparative statistical analysis, all gels of the same community were combined into a composite image using Corel PHOTO-PAINT 12 before further analysis (Corel Corporation, Fremont, CA, USA, 2003). Gel images were normalized with respect to migration pattern of the standard lanes of each gel, before pattern comparison using the Image Master 1D program (Amersham Biosciences, Roosendaal, The Netherlands) with rolling-circle (r=10) background subtraction and automated band detection. Matching of bands was carried out in reference to a hypothetical composite lane containing bands at all positions found across each dataset. Owing to potential PCR bias and the non-quantitative nature of DGGE band densities, bands were scored on a presence or absences basis. DGGE fingerprints were hereby transformed into binary codes and used in statistical analysis as ‘species’ presence–absence matrices. The influence of plant species (C. arenaria versus F. rubra), soil origin (Bergharen, Kwade Hoek and Middelduinen), CO₂ concentration (ambient versus elevated) and the interactions between these three factors on the community structure, as examined by PCR-DGGE, was tested by distance-based redundancy analysis (Legendre and Anderson, 1999). Jaccard's coefficients of similarity were first calculated between samples and used to compute principal coordinates in the R-package (Casgrain and Legendre, 2001). When necessary, eigenvectors were corrected for negative eigenvalues using the procedure of Lingoes (1971), and all principal coordinate axes were exported to Canoco version 4.5 (Ter Braak and Šmilauer, 2002) and treated as ‘species’ data. To test the effects of the three groups of factors (plants, soil origin and CO₂), these were entered as dummy binary variables. One group of factors was entered as the explaining variables in the model, whereas the other two groups of factors were entered as co-variables. The significance of such models was tested with a Monte Carlo test based on 999 permutations restricted for split-plot design, with whole plots being the CO₂ flow cabinets. Further distance-based redundancy analyses were also carried out as described above on subsets of the whole dataset, by either analyzing plant species or soil origins separately.

Table 1 Primers, PCR, DGGE and real-time PCR conditions used in this paper

Table 2 Bacterial isolates used for the production of standard curves in the quantification of actinomycetes, Pseudomonas spp., Burkholderia spp., Bacillus spp., Fusarium spp., Trichoderma and potential producers of PRN, DAPG and PCA

The percentage of variation in the dataset related to the different factors was determined by variation-partitioning analysis (Borcard et al., 1992), using the same strategy as for distance-based redundancy analyses. The percentage of variation explained by a factor was the trace of the analysis constrained using this factor (redundancy analysis) divided by the trace of the unconstrained analysis (phenazine 2-carboxylic acid (PCA)). Unexplained variation was calculated by subtracting the trace of the overall amount of explained variation by the different factors in the model from the total inertia.

Total exuded-sugar analysis

The HPLC procedure outlined by Stipanovic et al. (1998) was used to analyze sorbitol, mannitol, trehalose, glucose, fructose, melibiose and sucrose concentrations in the rhizosphere of C. arenaria and F. rubra grown at ambient and elevated CO₂ conditions. A total of 80 pot leachate samples (40 of F. rubra and 40 of C. arenaria), each comprised of 100 mg of freeze-dried water-extracted exudates, were harvested 73 and 62 days after germination, respectively. All the samples were shaken for 30 min in a capped 125 ml Erlenmeyer flask with 15 ml of glass beads, 10 ml of 3:1 hexane:ethyl acetate and 100 μl of 10% HCl. The solution was filtered over a glass-filter funnel into a 50 ml pear-shaped flask, and the beads and residue were rinsed three times with 3 ml 3:1 hexane:ethyl acetate. The solvent was left to evaporate in a hot water bath and the residue in the flask was re-dissolved with four 150-μl washes. Each wash was transferred to a maxi-clean silica cartridge (Alltech, Breda, The Netherlands). The silica cartridge was dried using compressed air, and the soil solutions were eluted with 5 ml of isopropyl alchol:acetonitrile:water:ethyl acetate (35:21:39:5). The eluent was filtered through a 45-μm nylon filter and transferred to a crimp top vial for HLPC analysis. Twenty microliters of each sample were injected into a DIONEX HPLC system (DIONEX Corp., Sunnyvale, CA, USA), equipped with a single wavelength absorbance detector and a 250 mm × 4.6 mm i.d. ALLtima C-18 column (Alltech, Breda, The Netherlands). The column was eluted with ethanol mix at a flow rate of 1.25 ml min⁻¹ and kept at 55 °C during analysis according to the protocol described by Stipanovic et al. (1998). Detection was carried out at 272 nm. Standards of sorbitol, mannitol, trehalose, glucose, fructose, melibiose and sucrose were used to assess the retention times of individual sugar. As control treatments, the effect of mechanical root damage on sugar concentration in the rhizosphere and the natural abundance of the sugars in the bulk soil at ambient and elevated CO₂ were considered. To detect the effect of root damage on exudation patterns, roots were washed and separated into main and lateral roots and a metal blade was used to damage the root tissue. The material was immediately freeze-dried after injury, ground and extracted for sugar analysis.

The concentration (S) of each single sugar in the rhizosphere soil of C. arenaria and F. rubra plants grown in Bergharen, Kwade Hoek and Middelduinen at ambient and elevated CO₂ was calculated by subtracting the natural abundance of each singular sugar (S_n) as recovered in the bulk soil and the sugar concentration due to root damage (S_r). S_tot represents the total sugar concentration derived from HPLC analysis:

To determine the differences in sugar concentrations between ambient and elevated CO₂ treatment in the different rhizosphere soils, the data were examined by using analysis of variance. The analyses were carried out according to a split-plot design as described by Filion et al. (2000). The F statistic used for testing the significance of main effects of the CO₂ treatment applied to whole plots (CO₂ flow cabinets) was obtained by dividing the treatment mean square by the mean square for CO₂ flow cabinets nested within CO₂ treatments. The error term, to test for the interactions between CO₂ and soil origin or plant species, was based on the mean square of the interaction between these treatments and cabinets nested in CO₂ (Filion et al., 2000). The analyses were carried out in Statistica 7.0 (StatSoft Inc., Tulsa, OK, USA). Normality was tested using the Shapiro–Wilks test and by inspection of residuals, and variance homogeneity by Levene's test. When data failed to satisfy one of these tests, an appropriate transformation was applied (log or square-root transformation). Tukey's honestly significant difference method and the modified version for unequal sample size (Unequal N honestly significant difference in Statistica) were used for post-hoc comparisons with a 0.05 grouping baseline. Analyses of variance were carried out in Statistica 8.0 to test for significant differences between the soil characteristics and the effect of elevated CO₂. A block structure was used for the ambient and elevated CO₂ data sets. After analysis of variance, least significant differences were calculated at a significance level of P=0.05.

Real-time PCR

Quantitative real-time PCR assays were carried out in 25-μl reaction volumes with the ABsolute QPCR SYBR green mix (AbGene, Epsom, UK) on a Rotor-Gene 3000 (Corbett Research) to quantify actinomycetes, Burkholderia spp., Pseudomonas spp., Bacillus spp., Fusarium spp. and Trichoderma spp. Small subunit (SSU) rRNA gene densities were quantified using the primers and conditions described in Table 1. Quantitative real-time PCR assays were also carried out for the detection of the pyrrolnitrin (PRN), 2, 4 diacetylphloroglucinol (DAPG), phenazine 2-carboxylic acid (PCA) production loci (Table 1). All mixes were made using a CAS-1200 pipetting robot (Corbett Research). Standards were made from full-length PCR-amplified 16S-, 18S-rRNA and antibiotic production genes from the pure isolates described in Table 2. By using 10-fold increments, the standard concentrations were adjusted from 10⁹ to 10¹ gene copies per μl for each species. In all the runs, the amplification efficiency (Eff) was calculated using the software manufactured by Corbett Research by the formula:

The efficiency of the different real-time PCR ranged from 90–100% (−3.6>slope>−3.1). The length of the amplicons ranged between 500 to 800 bp and secondary structures were not encountered in any of the runs. The threshold of each single run was placed above any baseline activity and within the exponential increase phase. The cycle thresholds (C_T) were determined by a mathematical analysis of the resulting curve using the software manufactured by Corbett Research. The C_T values of the non-template controls were always around 40, indicating no amplification. Dissociation curves were determined for quantitative real-time PCR products to confirm product integrity, and random samples were run on 2% agarose gels to confirm the absence of spurious PCR products.

Most of the samples, and all standards, were assessed in at least two different runs to confirm the reproducibility of the quantification. The SSU rRNA gene numbers were analyzed using analysis of variance as described previously (see sugar analysis).

Results

Effects of elevated CO₂ on Pseudomonas spp., Burkholderia spp., actinomycetes and Bacillus spp. community profiles

Plant species, soil origin, CO₂ concentration and the interactions between these factors all explained a significant (P<0.001) part of the variation in Pseudomonas spp. and Burkholderia spp. communities (Table 3 and Figure 1; Supplementary Figure S1A, B). Variation partitioning of PCR-DGGE datasets for these communities showed that CO₂ treatment, soil origin and plant species together explained about 55% of the variation in community profiles for Pseudomonas spp. and 62% for Burkholderia spp. (Table 3 and Figures 1a, b). These variables all explained a significant part of the variation, but their relative contribution differed between these two groups of organisms. For Pseudomonas spp., the greatest degree of variation was explained by elevated CO₂ (30%), whereas soil origin and plant species explained about 5 and 20%, respectively, of the total variation. For Burkholderia spp., the greatest degree of variation was explained by elevated CO₂ (35%) and plant species (22%), whereas soil origin explained only 2.8% of the total variation. For actinomycetes and Bacillus spp., soil origin was the only significant explanatory factor (98 and 97%, respectively; Table 3; Supplementary Figure S1C, D). Plant species, soil origin and the interactions between these factors did not explain a significant (Table 3) part of the variation for actinomycetes and Bacillus spp.

Table 3 Main PCR-DGGE distance-based redundancy analyses results for plant species (Carex arenaria and Festuca rubra), soil origin (Middelduinen, Kwade Hoek and Bergharen) and ambient and elevated CO₂ concentrations on Pseudomonas, Burkholderia, actinomycetes and Bacillus spp. communities based on 999 Monte Carlo permutations test and split-plot analysis of variance

Figure 1

Variation-partitioning representation of PCR-DGGE (PCR-denaturing gradient gel electrophoresis) analyses of (a) Pseudomonas spp. and (b) Burkholderia spp. rhizosphere communities under (c) Festuca rubra (mycorrhizal) plants only and (d) Carex arenaria (non-mycorrhizal) plants only, grown in Bergharen (river dune) Kwade Hoek (former beach) and Middelduinen (coastal dune). The numbers list the percentage of variance accounted for CO₂ levels, plant species, soil origin, unexplained variance and their interactions. The significance of all of the different factors on the microbial community structure was tested by db-RDA (distance-based redundancy analysis) based on 999 Monte Carlo permutations and split-plot analysis of variance (see material and methods). The area of the white square that is not covered by any of the other rectangles is proportional to the unexplained variance.

Effect of elevated CO₂ on the rhizosphere community densities

The SSU rRNA gene abundances for Pseudomonas spp. and Burkholderia spp., differed with respect to plant species, soil origin and elevated CO₂ treatments (Figures 2a, b). In Kwade Hoek and Middelduinen soil, elevated CO₂ had no effect on the Pseudomonas spp. and Burkholderia spp. rhizosphere community abundances associated with C. arenaria (F_1,2=6.79, P=0.12), but Burkholderia spp. target numbers in the F. rubra rhizosphere were affected by elevated CO₂ (F_1,2=533.40, P=0.002). In Bergharen soil, Pseudomonas and Burkholderia communities (F_2,4=10.30, P=0.026) had a significant CO₂ and soil interaction, indicating that the effects of CO₂ elevation were soil dependent and affected the two plants rhizospheres to a similar extent.

Figure 2

(a) Pseudomonas spp., (b) Burkholderia spp., (c) actinomycetes, (d) Bacillus spp., (e) Fusarium spp. and (f) Trichoderma spp. SSU (small subunit) ribosomal RNA genes abundance in the rhizosphere of Carex arenaria and Festuca rubra grown at elevated (ELEV) and ambient (AMB) CO₂ in Bergharen, river dune (b), Kwade Hoek, former beach (K) or Middelduinen, coastal dune (M) soils. Different letters within the graph refer to significantly different averages based upon a Tukey HSD (honestly significant difference) test.

More complex interactions between CO₂ concentration, plant species and soil origins were also observed. For C. arenaria, Burkholderia 16S-rRNA gene abundance decreased or remained stable with elevated CO₂ in all soils, whereas the opposite pattern was observed for F. rubra. Pseudomonas populations in C. arenaria and F. rubra increased or remained stable in Bergharen and Kwade Hoek soils, but decreased in Middelduinen soil.

The 16S-rRNA gene abundances for Bacillus spp. differed with respect to plant species (F₁=20.080; P<0.001) and soil origin (F₂=9.69; P<0.001), whereas actinomycetes community abundances were not significantly affected by any variable (Figures 2c, d). For both plant species, elevated CO₂ had no effect on the community abundance for actinomycetes (F_1,2=1.019, P=0.42) or Bacillus spp. (F_1,2=0.366, P=0.61).

SSU rRNA gene abundances for Fusarium spp. and Trichoderma spp.

The SSU rRNA gene abundances for Trichoderma were generally much higher for F. rubra than for C. arenaria and differed with respect to soil origin. Fusarium densities showed an opposite trend with respect to plant species (Figures 2e, f). For Trichoderma (F_1,2=215.06, P<0.01) and Fusarium (F_1,2=1011.34, P<0.001), there was a significant CO₂–plant species interaction, indicating that CO₂ elevation had different effects on the two plants rhizospheres. More complex interactions between CO₂ concentration, plant species and soil origins were also observed. For C. arenaria, Fusarium 18S-rRNA gene abundance increased with elevated CO₂ across all three soil origins, but for F. rubra, these patterns were exactly the opposite.

Effect of elevated CO₂ on gene densities associated with PRN, DAPG and PCA production

The density of genes associated with DAPG and PRN production was generally much higher in F. rubra than in C. arenaria and differed with respect to soil origin (Figures 3a, b). Genes responsible for the production of PCA were present at low densities in the rhizospheres of F. rubra plants grown in Bergharen and Kwade Hoek soil, but relatively high in all C. arenaria rhizosphere samples (Figure 3c). For the DAPG-, PRN- and PCA-associated genes, there was a significant CO₂ elevation effect (F_1,2=15.766, P<0.028; F_1,2=11.35, P<0.03 and F_1,2=11.659, P<0.028, respectively), with the effects of CO₂ being dependent on the plant species and soil origin. Elevated CO₂ apparently increased the abundance of DAPG- and PRN-producing communities in both plant rhizospheres grown in Middelduinen, but only in F. rubra for Bergharen soil.

Figure 3

(a) Pyrrolnitrin production locus (PRN), (b) 2,4 diacetylphloroglucinol production locus (2,4-DAPG) and (c) phenazine 2-carboxylic acid production locus (PCA) Pseudomonas and Burkholderia spp. antibiotics functional genes in the rhizosphere of Carex arenaria and Festuca rubra grown at elevated (ELEV) and ambient (AMB) CO₂ in Bergharen, river dune (b), Kwade Hoek, former beach (K) or Middelduinen, coastal dune (M) soils. Different letters within the graph refer to significantly different averages based on a Tukey HSD (honestly significant difference) test.

Effect of elevated CO₂ on total soluble exuded sugars

On being averaged across all treatments and community types, the mean of total soluble sugars increased significantly under elevated CO₂ in the rhizosphere of both plant species (plant–CO₂: F_1,2=86.40; P<0.001) (Figure 4). Interestingly, although both plant species increased their total exudation of sugars (glucose, fructose, sucrose, mannitol, melibiose and sorbitol) at elevated CO₂, this effect was generally greater in the rhizosphere of F. rubra. For F. rubra, the exudation of trehalose, a major product of AMF, was also increased by four-fold at elevated CO₂, compared with ambient CO₂ conditions (Figure 4). As expected, no trehalose exudation was detected for the non-mycorrhizal plant, C. arenaria, at either level of atmospheric CO₂.

Figure 4

Means of (a) sorbitol, (b) mannitol, (c) glucose, (d) fructose, (e) melibiose and (f) sucrose at elevated CO₂, in Festuca rubra and Carex arenaria rhizosphere as determined by HPLC (high performance liquid chromatography) analysis. (g) Means of trehalose at ambient and elevated CO₂ in F. rubra rhizosphere, in Carex arenaria trehalose was hardly detected. Different letters within the graph refer to significantly different averages based on a Tukey HSD (honestly significant difference) test.

Discussion

Response of specific soil-borne microbial groups to plant growth at elevated concentrations of atmospheric CO₂

Specific bacterial community responses

Distance-based redundancy analysis of community profiles showed that increased atmospheric CO₂ exerted differential influences on the specific bacterial groups in the rhizosphere samples associated with C. arenaria and F. rubra. Large effects of elevated CO₂ were observed within the Pseudomonas and Burkholderia communities, but no significant effects were apparent for actinomycetes and Bacillus spp. These results are in agreement with previous observations that slow-growing soil microorganisms, such as actinomycetes, were unaffected by elevated CO₂ (Zak et al., 1996; Bardgett et al., 1999; Jossi et al., 2006). The observed impacts of elevated CO₂ were within a background of other significant sources of variation, such as effects of different plant species and soil origins, as examined by variation-partitioning analysis (Figure 1). CO₂ enrichment also influenced the abundance of the genera Pseudomonas and Burkholderia, and these effects were dependent on the soil origin and plant species (Figure 2). No such effects were observed for the abundances of the actinomycetes or the genus Bacillus.

Previous studies have identified Pseudomonas and Burkholderia as highly rhizo-competent genera (Vancanneyt et al., 1996; Lugtenberg et al., 2001; Treonis et al., 2004; Berg et al., 2005). In contrast, actinomycetes and Bacillus spp. have been implicated to be typical bulk-soil inhabitants (Smalla et al., 2001), although recent studies suggest that these groups may represent only modest proportions of total soil microbial communities (Roesch et al., 2007). Thus, our results provide new evidence for the previously presumed levels of interaction of these groups with plants, with rhizosphere bacteria reacting more strongly to changes in plant physiology and exudation induced by elevated atmospheric CO₂ concentrations.

Antibiotic-production genes

To gain some insight as to whether observed changes in the rhizosphere communities might have functional significance, we also targeted a number of genes known to be involved in the antibiotic production of potential plant-growth-promoting bacteria. For example, PRN is an antibiotic produced by Pseudomonas and Burkholderia species (Raaijmakers et al., 2002), known to act upon plant pathogens, such Rhizoctonia solani and Fusarium spp. DAPG and PCA have also been shown to be involved in the biocontrol activity and ecological competence of several Pseudomonas strains. These two antibiotics are effective against the wheat root disease fungal pathogen Gaeumannomyces graminis var. tritici (take-all), as well as Pythium ultimum and Fusarium oxysporum (Raaijmakers et al., 2002).

Our results showed that CO₂ enrichment influenced the densities of PRN- and DAPG-producing genes in Pseudomonas and Burkholderia, with a smaller effect on PCA-producing gene densities (Figure 3). Similar to the changes in community composition described above, the density of antibiotic producers also strongly depended on the plant species and soil origin. For instance, significant increases related to PRN, DAPG and PCA producers were observed for C. arenaria in Middelduinen soil. Interestingly, for F. rubra under elevated CO₂, large changes were observed for PRN and DAPG producers in Bergharen and Middelduinen soils, whereas the PCA-producing Pseudomonas and Burkholderia spp. community was hardly detected, except in Middelduinen soil. Antibiotic-producing rhizosphere communities in Bergharen and Middelduinen soils showed marked CO₂ treatment responses, but no significant effects were observed in Kwade Hoek soil for either of the plants tested. These results suggest that the observed changes in bacterial communities, most notably in Pseudomonas and Burkholderia, which are known for their production of these antibiotics, may have functional consequences with respect to interactions with plant pathogens (Raaijmakers et al., 2002), and that these responses are plant and soil specific. It is interesting to note that Tarnawski et al. (2006) observed lower frequencies of Pseudomonas strains capable of producing hydrogen cyanide, a potential inhibitor of root parasitic fungi, in two perennial grassland systems (Lolium perenne and Medicago coerula) under elevated (600 p.p.m. vs 360 p.p.m.) CO₂ concentrations.

Changes in plant exudation patterns in response to elevated CO₂

The elevated CO₂ conditions generally resulted in increased sugar exudation in the rhizosphere (Figure 4). The level of this increase and composition of exudates depended on plant species and soil origin. For instance, although both plants showed increased total exudation of sugars (glucose, fructose, sucrose, mannitol, melibiose and sorbitol) at elevated CO₂, this effect was on average twice as large in F. rubra spp. arenaria (caespitose) rhizospheres. In addition, we found that F. rubra produced four times more trehalose under elevated CO₂ conditions as compared with that at ambient CO₂ conditions. C. arenaria did not seem to release appreciable amounts of this compound regardless of atmospheric CO₂ level (Figure 4). Although plant litter represents the dominant pathway by which plant C is transferred to soil, living roots also contribute significantly to this process through turnover of fine roots, sloughing of living cells and exudation (Matamala et al., 2003; Philips et al., 2006). Furthermore, root exudation probably exerts a disproportionate impact on rhizosphere communities, as it represents the most easily accessible C available to the soil microbes (Cardon, 1996). It should be realized that analysis of root secretions in natural soils is hampered by the fact that rhizosphere microorganisms continuously consume and produce easily metabolized compounds. Thus, our exudate measurements represent more the net flux of secretion (efflux) and uptake (influx), which itself depends on the biomass, affinity and consumption rates of consumers (Tarnawski and Aragno, 2006).

Previous studies have shown that, under elevated CO₂, especially C₃ plants (as used in our study) increase their total root exudation mainly through the expansion of their root systems (Rogers et al., 1994; Allard et al., 2005), yielding qualitative and quantitative changes in root exudation and other forms of rhizodeposition (Paterson et al., 1996; Hodge and Millard, 1998). Differences in plant exudation patterns between plants species is thought to exert differential selection in the rhizosphere, thereby shaping the size and structure of soil-borne communities (Bardgett et al., 1999; Smalla et al., 2001; Kowalchuk et al., 2002). The AMF infections are also known to affect exudation patterns (Frey-Klett et al., 2007), and the differential mycorrhizal status of the two plants in our study (F. rubra is mycorrhizal whereas C. arenaria is not; Greipsson and El-Mayas, 1999; Orlowska et al., 2005) may have contributed to differences in observed exudation patterns. The results with respect to trehalose exudation are of particular relevance in this respect, as this disaccharide is an important product of AMF. Trehalose has also been implicated in the selection of potential mycorrhizal helper bacteria, including several Pseudomonas and Burkholderia species (Frey-Klett et al., 2007), although it is premature to conclude that the observed changes in these genera are related to this function. The AMFs have been implicated as major conduits for C translocation from plants into the soil under different atmospheric CO₂ conditions, and our previous observation that the mycorrhizal plant (F. rubra) exerted greater influence on bacterial and fungal communities is consistent with this assertion (Drigo et al., 2007).

It should be noted that our study only considered the sugars, and the study of other exudates, such as amino acids and organic acids, would provide a more complete picture of exudation patterns. Moreover, to fully understand the flow of C to soil, root turnover and root biomass would need to be followed over time. In our experiment, we could observe roughly an increase of root biomass of the 30% for both plants in response to the elevated CO₂ treatment.

Effects of elevated CO₂ on fungal communities

Given the important role of fungal pathogens and antagonists in the functioning of plant–soil systems (Hagn et al., 2007), we also examined changes in two key fungal genera related to these activities. Fusarium spp. are common soil fungi, which can have important roles not only as plant pathogens, but also as saprotrophic competitors of other pathogenic fungi (Duffy et al., 2004; Yergeau et al., 2005). In this study, the density of Fusarium spp., as judged by quantitative real-time PCR targeting the 18S-rRNA gene, was significantly reduced in the rhizosphere of the mycorrhizal plant (F. rubra) exposed to elevated atmospheric CO₂ concentration. Interestingly, it has been suggested that AMF colonization, can reduce Fusarium community size (Filion et al., 2000). Direct interactions, such as competition between the symbiont and the pathogen for infections sites (Muchovej et al., 1991), or indirect interactions, such as alteration of root exudation and/or of the mycorhizosphere microbial community, were proposed as potential mechanisms of this observed reduction. The exact nature of these relationships requires further investigation in order to gain insight into the potential consequences of elevated CO₂ on plant disease conditions. We also observed an increase in genes associated with the production of antibiotics known to affect Fusarium species in response to elevated CO₂ (see above), but we are not yet able to show a causal link between this increase and the decline in Fusarium densities.

Fungi of the genus Trichoderma account for a major portion of fungal biomass in soils, and often act as important control agents for a wide range of phytopathogens (Harman and Björkmann, 1998; Hagn et al., 2007). In contrast to Fusarium spp., Trichoderma density increased under elevated CO₂ in the F. rubra rhizosphere. This pattern mirrored that previously observed for the total fungal community size (Drigo et al., 2007).

Concluding remarks

In a previous general examination of bacterial and fungal communities in the rhizosphere of plants subjected to elevated CO₂ (Drigo et al., 2007), it was observed that the effects of elevated CO₂ were dependent on plant species and soil type. Here, we examined more closely the specific microbial groups affected by elevated CO₂ and showed that presumably rhizo-competent bacteria and fungi are strongly affected by increased atmospheric CO₂ in contrast to typical bulk-soil representatives. These patterns were consistent with observed changes in the density of antibiotic production genes as well as changes in exudation patterns. It should be noted, however, that our controlled experimental design did not allow for long-term adaptation to changes in atmospheric CO₂ conditions, and effects of such adaption may be significant (Klironomos et al., 2005).

Although this study provides new insight into the specific plant–microbe interactions in the rhizosphere under elevated CO₂, knowledge is still rather scarce with respect to the relative flow of C to different biological groups of the plant–soil ecosystem (Olsson and Johnson, 2005; Carney et al., 2007; Kreuzer-Martin, 2007). Such knowledge is critical to not only our understanding of soil food web, but also for predicting the future impacts of increasing CO₂ levels.