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Influence of arbuscular mycorrhizal mycelial exudates on soil bacterial growth and community structure

Jonas F. Toljander, Björn D. Lindahl, Leslie R. Paul, Malin Elfstrand, Roger D. Finlay
DOI: http://dx.doi.org/10.1111/j.1574-6941.2007.00337.x 295-304 First published online: 1 August 2007

Abstract

Plant root systems colonized by arbuscular mycorrhizal (AM) fungi have previously been shown to influence soil bacterial populations; however, the direct influence of the AM extraradical mycelium itself on bacterial growth and community composition is not well understood. In this study, we investigated the effects of exudates produced by AM extraradical mycelia on the growth and development of an extracted soil bacterial community in vitro. The chemical composition of the mycelial exudates was analysed using proton nuclear magnetic resonance spectrometry. Following the addition of exudates to a bacterial community extracted from soil, bacterial growth and vitality were determined using a bacterial vitality stain and fluorescence microscopy. Changes in community composition were also analysed at various times over the course of 3 days by terminal restriction fragment length polymorphism analysis, in combination with cloning and sequencing of 16S rRNA genes. Mycelial exudates increased bacterial growth and vitality and changed bacterial community composition. Several Gammaproteobacteria, including a taxon within the Enterobacteriaceae, increased in frequency of occurrence in response to AM mycelial exudates. This study is the first attempt to identify carbohydrates from the extraradical mycelium of an AM fungus, and demonstrates the direct effects of mycelial exudates on a soil bacterial community.

Keywords
  • arbuscular mycorrhiza (AM)
  • extraradical mycelium
  • exudates
  • TRFLP
  • soil bacteria
  • bacterial community

Introduction

In addition to increasing the nutrient absorptive surface area of host plant root systems, the extraradical mycelium of arbuscular mycorrhizal (AM) fungi also provides a direct pathway for translocation of photosynthetically derived carbon to microenvironments in the soil. The continuous provision of energy-rich compounds, coupled with the large surface area of the mycelium that interacts with the surrounding soil environment (hyphosphere) provide important niches for bacterial colonization and growth. A rapid turnover of mycorrhizal mycelium (Staddon et al., 2003) constitutes one possible pathway by which mycorrhizal fungi may transfer photo-assimilates into the soil. However, exudation of carbohydrates by living hyphae may enable a more direct and reciprocal interaction between mycorrhizal fungi and other microorganisms. In a previous study we tested the ability of a number of soil bacterial isolates to attach to and colonize living and dead AM fungal hyphae (Toljander et al., 2006). We found that some bacterial strains showed a greater tendency to attach to vital hyphae, whereas other bacterial strains tended to attach to nonvital hyphae. This suggests that some bacteria have a biotrophic strategy, utilizing exudates released by vital fungal hyphae, while other bacterial species utilize the hyphae themselves as substrate. Compounds released may also function as chemo-attractants or stimulatory compounds for some bacteria (Grayston et al., 1997; Sood, 2003). An increased knowledge of the functional responses of bacteria to mycelial exudation is necessary for understanding the effects of management practices on soil microbial communities.

While there is some information available on the metabolism and translocation of carbon within AM fungal tissues (Bago et al., 2002), the composition of exudates from AM fungal extraradical mycelia, and their availability to soil microbial communities have not been studied (Bending et al., 2006). Filion et al. (1999) studied the effect of mycelial exudates on the growth of individual isolates of fungi and bacteria, using a split Petri dish system to extract soluble substances released by the extraradical mycelium of Glomus intraradices. They found that these extracts could have antagonistic as well as stimulatory effects on the tested microorganisms, suggesting that mycelial products may play an important role in direct interactions between AM fungi and other soil microorganisms. However, basic knowledge on the differential effects of mycelial products on bacterial communities is still lacking. In this study we employed an axenic system to produce AM mycelial exudates in order to examine the potential of these exudates to stimulate an extracted community of soil bacteria as well as to modify its composition.

Materials and methods

Generation of root organ cultures from white clover

The binary vector p35S GUS INT (Lindroth et al., 1999) was introduced into Agrobacterium rhizogenes LBA 9402 (generously supplied by David Clapham, Swedish University of Agricultural Sciences, Uppsala, Sweden) by electroporation. Transformed colonies arose on yeast extract mannitol broth supplemented with kanamycin (200 mg L−1) after 2 days cultivation at 28°C (Lindroth et al., 1999). The presence of the appropriate constructs in the recovered transformed colonies was verified by PCR. Transformed roots of white clover (Trifolium repens L.) were generated according to the methods of Elfstrand et al. (2005). One fast-growing culture ‘LP1’ supporting amplification of the gus insert, without amplifying the 800-bp virD3 fragment, was selected. Clover root cultures were maintained on fresh modified M (MM) medium, and were subcultured every 8–10 weeks. The modification of the M medium consisted of an increased concentration of vitamins: 3.6 mg L−1 glycine, 120 μg L−1 thiamine hydrochloride, 120 μg L−1 pyridoxine hydrochloride, 600 μg L−1 nicotinic acid, 60 mg L−1 myoinositol. The medium was supplemented with 1% (w/v) sucrose. To produce mycorrhizal root cultures, clover root organ cultures were inoculated with spore plugs from axenic cultures of Glomus sp. MUCL 43205 (GINCO, Lovain-la-Neuve, Belgium). Mycorrhizal root cultures were grown on MM media, supplemented with 0.5% (w/v) sucrose. Root cultures were kept at 25°C in the dark.

Establishment of two-compartment root organ cultures

Mycorrhizal and nonmycorrhizal two-compartment cultures (Fig. 1) were established in Petri dishes (90 × 15 mm), as described by Filion et al. (1999). Twenty-five milliliters of MM medium was added to the root compartment, and 8 mL of no-carbon (minus sucrose, EDTA and vitamins) M medium was added to the nonroot compartment, creating a slope from the bottom of the dish to the top of the plastic divider. The plastic divider constituted a barrier against diffusion from the root compartment to the nonroot compartment. Cultures were inspected on a regular basis, and roots appearing close to the plastic divider were moved before they were able to grow into the nonroot compartment.

1

Schematic view of mycorrhizal root organ culture in the split Petri dish system that was used to produce mycelial exudates. In the nonroot compartment, mycelia were allowed to proliferate in liquid minimal media without any added carbon source. Liquid medium that had been exposed to growing mycelia for 4 weeks was collected and used in incubation studies together with bacterial communities extracted from soil samples.

Production and analysis of liquid extracts from two-compartment cultures

When extensive amounts of extraradical hyphae had colonized the medium slope of the nonroot compartment, 7 mL of liquid (without gellan gum) MM medium was added to the bottom of the nonroot compartment, allowing hyphae to grow out into the liquid medium. The same procedure was carried out for nonmycorrhizal cultures. After 4 weeks, when mycelium covered c. 50% of the nonroot compartment in the mycorrhizal cultures, liquid was extracted from each of the separate mycorrhizal (n=4) and nonmycorrhizal (n=4) systems, and was filtered through sterile Acrodisc® syringe filters (0.2-μm HT Tuffryn® Membrane; Pall Corporation, Ann Arbor, MI).

A 2-mL portion of the liquid from each culture was vacuum-centrifuged at 40°C until dry. The dry residues were washed with 5 mL of deuterium oxide (D2O) and then vacuum-centrifuged again. The washing procedure was repeated three times. Washed and dried samples were frozen at −80°C before proton nuclear magnetic resonance (HNMR) spectrometry. The HNMR analysis was performed using a Bruker DRX400 spectrometer (Bruker Spectrospin GmbH, Rheinstetten, Germany) operating at 400 MHz and at 70°C. D2O was used as solvent, and chemical shifts (δ) were determined relative to HDO (δ 4.28). A pulsed sequence for presaturation of the water peak was used to increase the sensitivity of the analysis.

Extraction of bacteria from field soil

Soil was collected in mid-June 2004 from a 10-year-old fallow field in Uppsala (59°N 49′, 17° 38′E), Sweden. The parent material of the soil is postglacial clay, and the dominant flora consists of Trifolium spp., Achillea millefolium L., Dactylis glomerata L. and Phleum pratense L. Five subsamples (c. 10 × 10 × 10 cm), dug out from the topsoil, were subsequently pooled and mixed while kept cold. Soil bacteria were extracted from a total of 60 g of soil using Nycodenz® (Medinor AB, Stockholm, Sweden), allowing density-gradient separation of prokaryotic cells from soil material and eukaryotic cells (Rickwood et al., 1982; Lindahl & Bakken, 1995). Extracted bacteria were suspended in sterile 0.1 M pH 5.8 potassium phosphate buffer. The pH of the buffer was similar to that of the mycorrhizal cultures and the soil from which the bacteria were originally extracted. The bacterial cell concentration was measured in a Bürker chamber, and the absence of fungal propagules in the suspension was confirmed using an Axioplan fluorescence microscope (Zeiss, Oberkochem, Germany). Bacteria were immediately used for assessment of the effects of mycorrhizal exudates.

Experimental treatments

The filtered liquid withdrawn from the four mycorrhizal and four nonmycorrhizal split Petri dish systems was added to the wells of 24-well plates (well diameter 15 mm) with lids. For each of the two treatments, four replicate samples containing bacteria and liquid extracts were set up for every time point of harvest, which was performed at 3, 6, 9, 20, 48, and 72 h postinoculation with bacteria. Samples for analysis of the bacterial community before exposure to liquid extracts (at 0 h) were frozen immediately. To each well, 180 μL of filtered liquid extract and 20 μL of bacterial suspension were added, resulting in a final bacterial concentration of 3.6 × 107 cells mL−1 in each well. The plates were incubated at room temperature and high humidity on a rotary table set on slow rotation, to provide aeration.

Harvest and quantification of bacterial growth and vitality

An aliquot (10 μL) from each well was withdrawn for immediate vitality staining, and the residual (190 μL) was immediately frozen and stored at −80°C until DNA extraction. The 10-μL aliquots were stained with a LIVE/DEAD®BacLight Bacterial Vitality Kit (Molecular Probes, Leiden, the Netherlands). Vital and nonvital cells in each replicate sample were quantified in a Bürker chamber using an Axioplan fluorescence microscope. Bacterial vitality for each sample was determined as the number of vital cells divided by the total number of cells.

DNA extraction, PCR and terminal restriction fragment length polymorphism analysis of the bacterial community

Bacterial cells were lysed by heating the frozen samples to 90°C, and DNA was subsequently extracted using a DNEasy® Plant Kit (Qiagen, Crawley, UK). The bacterial 16S rRNA gene was amplified using the following final concentrations of PCR reaction constituents: 0.2 mM of all four nucleotides, 0.1 μM of each primer, 1.5 mM MgCl2 and 0.025 U μL−1 of ThermoRed DNA polymerase (Saveen & Werner, Malmö, Sweden). DNA template was added as 50% of the final reaction volume. For terminal restriction fragment length polymorphism (TRFLP) analysis the primers 27f (5′-AGA GTT TGA TCC TGG CTC AG-3′) (Lane, 1991) 534r (5′-ATT ACC GCG GCT GCT GG-3′) (Muyzer et al., 1993), each labelled at the 5 end with WellRED dyes D3-PA and D4-PA, respectively, were used. The thermocycler program started with denaturation at 94°C for 5 min, followed by 25 cycles of 94°C for 30 s, 56°C for 45 s and 72°C for 60 s, and ended by a final extension for 7 min at 72°C. DNA concentrations were standardized by visually comparing the amounts of PCR products on 1% w/v agarose gel, and diluting samples to obtain similar concentrations of products. PCR products were digested overnight at 37°C using the restriction endonucleases CfoI, HaeIII and MspI (Promega Corporation, Madison, WI). The TRFLP profiles were analysed with a Beckman Coulter CEQ 8000 Genetic Analysis System using a CEQ DNA Size Standard Kit-600 (Beckman Coulter, Fullerton, CA).

Construction of TRF reference database and TRFLP identification of bacteria

Two clone libraries were constructed by cloning PCR products from samples at the beginning and at a later part of the experiment (0 and 48 h, respectively) using a TOPO-TA cloning kit (Invitrogen, Carlsbad, CA). PCR of selected clones was performed as above, with the only modification that the primers were exchanged for 27f and 926r (5′-CCG TCA ATT CCT TTR AGT TT-3′), neither labelled with 5′ WellRED dye. Ninety-six (0 h) and 48 (48 h) clones from the two cloning reactions were selected for sequencing. Clone inserts were sequenced from both directions using the m13 primers provided with the cloning kit. Sequences were blasted and aligned with selected reference sequences from GenBank (http://www.ncbi.nlm.nih.gov/) using the clustalw algorithm in megalign version 5.07 (DNASTAR, Madison, WI). Reference sequences were selected to present the highest sequence match and the lowest Expect value (usually e=0.0). Sequence resemblances were analysed by neighbour-joining and bootstrap analysis in paup (Swofford, 2002). Clone sequences were registered in GenBank under accession numbers EF494540-EF494632. Submitted sequences were named using the following format: X_Yh_JTSP, where X is the clone number(s) associated with the sequence, and Y is the clone library (0 or 48 h), to which the sequence is affiliated.

The clones were subjected to TRFLP analysis (the same procedure as TRFLP described above) and compared with the community TRFLP patterns. To assign putative identities to TRFs obtained from community samples, comparisons were made with TRFs obtained from sequenced clones using the software tramp (Dickie et al., 2002), with the maximum acceptable error for fragment size set to ±2 bp.

Statistical analysis

Differences in bacterial growth (total number of cells), vitality (vital:total cell ratios), and numbers of recorded bacterial taxa were tested for statistical significance by two-way anova, with sampling time and presence of mycelial exudates as independent variables, and Fisher's protected least significant difference (PLSD) posthoc tests. Cell-number values were log-transformed, and bacterial vitality ratios were arcsine-transformed before analysis. Differences in bacterial community composition were represented graphically using correspondence analysis (CA) in past 1.42 (Hammer et al., 2006). Canonical correspondence analysis (CCA) followed by a Monte Carlo permutation test (n=999) in pc-ord 4.25 (McCune & Mefford, 1999) was used to test the statistical significance of the effects of sampling time and mycelial exudates on the bacterial community composition. In all statistical analyses the frequency of occurrence of each bacterial taxon identified by the TRF database was recorded in terms of presence/absence in samples. Only bacterial taxa occurring in two or more samples were included in the ordination analyses. χ2 tests were performed to test the hypothesis of equal distribution of individual taxa among samples with and without mycelial exudates. An overview of the results from the statistical analyses is presented in Table 1.

View this table:
1

Summary of results from statistical analyses: effects of time of sampling and presence of mycelial exudates on bacterial growth, vitality and community composition

Time of samplingPresence of exudatesInteraction (time × exudates)
Bacterial growth*P< 0.0001P=0.12P< 0.0001
Bacterial vitality*P< 0.0001P=0.20P< 0.0001
No. of bacterial taxa*P< 0.0001P=0.28P=0.14
Community composition*P< 0.01P< 0.001
  • * Results from two-way analysis of variance. While there were no overall significant effects of exudates on bacterial growth and vitality, there was a strongly significant (P<0.01) effect at 48 h.

  • Results from canonical correspondence analysis with a Monte Carlo permutation test (n=999).

  • P-values denote the level of statistically significant effect by the factors or by the interaction between factors.

Results

Low-molecular-weight compounds in mycelial exudates

The HNMR spectrograms of liquid media that had been exposed to the growing mycorrhizal fungal mycelium (Fig. 2b) for 4 weeks differed in chemical composition from those from the nonmycorrhizal controls (Fig. 2a). Two signals, representing formiate (Peak 1, δ 8.43) and acetate (Peak 5, δ 1.87), were detected in media exposed to mycelia (Fig. 2b). In addition, signals representing α- and β-glucose (Peak 3, δ 5.20; Peak 4, δ 4.61), a starch-like compound (possibly glycogen) (Peak 2, δ 5.33), putative di- and oligosaccharides (δ 5.15, δ 4.90, δ 4.70 and δ 4.53), as well as an unassigned methyl group (δ 1.30) were detected in mycelium-exposed samples. Extracts from both treatments also produced a cluster of peaks with chemical shifts in the range of δ 3–4, representing polymeric compounds of the gellan gum. Samples with exudates also contained a number of unidentified peaks within this range, indicating the presence of polymeric compounds of mycorrhizal origin.

2

HNMR spectrograms of liquid extracts from the hyphal (nonroot) compartment of two-compartment root organ cultures (a) without and (b) with an arbuscular mycorrhizal fungus (Glomus sp. MUCL 43205). Numbers denote signals detected in mycorrhizal cultures: (1) formiate (δ 8.43), (2) starch-like compound, possibly glycogen (δ 5.33), (3) α-glucose (δ 5.20), (4) β-glucose (δ 4.61), (5) acetate (δ 1.87). In addition, weak signals representing putative di-/oligosaccharides (δ 5.15, δ 4.90, δ 4.70 and δ 4.53; not highlighted in figure), and an unassigned methyl group (δ 1.30; not highlighted in figure) were detected. The diagrams were standardized with respect to signal strength using the amplitude of the single peak with a chemical shift of 4.28 that represents the solvent. The cluster of tall peaks (δ∼3−4) in the middle of both diagrams represents polymeric signals produced by the gellan gum, a polymer used to solidify the ramp of medium present in the compartment from which the liquid was extracted. Additional peaks in the mycelial exudates in this range represent unidentified high-molecular-weight compounds of mycorrhizal origin.

Bacterial growth and vitality

There was a significant effect of time of sampling (time of incubation) on bacterial growth (P<0.0001), and a significant (P<0.0001) interaction between time of sampling and presence of exudates (Table 1). During the course of the experiment there was an overall increase in the concentration of bacterial cells (Fig. 3a), regardless of the presence of exudates, with significantly higher bacterial counts at 48 and 72 h incubation than at 3, 6 or 9 h (P<0.01). In the treatment with mycelial exudates, bacterial growth was either similar to or higher than that of the nonmycorrhizal treatment. At 48 h of incubation, the average cell concentration was more than one order of magnitude higher in the presence of exudates than in the control systems (P<0.01).

3

Bacterial community growth and vitality after various times of incubation in the presence and absence of mycelial exudates from an arbuscular mycorrhizal fungus (Glomus sp. MUCL 43205). (a) Log total number of cells counted at each sampling occasion. (b) Proportion of vital cells, calculated as the number of vital cells divided by the number of vital and nonvital cells. Closed and open circles symbolize mean values of samples with or without mycelial exudates, respectively. Error bars represent SEs of the means. Y-axes are broken and do not begin at zero.

A significant effect of time of sampling on bacterial vitality (the ratio of living to total bacteria) (P<0.0001) was observed, and there was also a significant interaction between time of sampling and mycorrhizal treatment (P<0.0001) (Table 1). An overall increase in bacterial vitality (Fig. 3b) was observed during the course of the experiment, regardless of the presence of exudates, with significantly higher vitality at 20, 48 and 72 h incubation than at 3, 6 or 9 h (P<0.0001). In the presence of exudates, bacterial vitality peaked at 48 h and was significantly higher then than at both 20 and 72 h (P<0.01 and P<0.0001, respectively). Bacterial vitality at 48 h was also significantly higher in the presence of exudates than in the control systems (P<0.01), but was significantly lower at 72 h (P<0.01).

Bacterial community composition

In total, 72 TRFLP types (taxa) were distinguished by matching the TRFLP profiles against the TRF reference database. The nine most frequently detected taxa were all identified as Pseudomonas spp., each encountered in a wide range of samples (48–92%) without any significant responses to mycelial exudates. The number of taxa detected in the samples was significantly influenced by the time of sampling (P<0.0001), with decreasing numbers throughout the experiment, but not by presence of mycelial exudates (Table 1).

Correspondence analysis (Fig. 4) shows that the bacterial community composition changed with time, but differently so in the presence of mycelial exudates. At 6 h, samples containing mycelial exudates form a cluster in the ordination diagram that is separated from the control samples. Samples collected at 9 h or later are separated from earlier samples irrespective of the presence of exudates. At 9 and 20 h, samples from different treatments are not clearly separated, but at 48 and 72 h, samples containing mycelial exudates again form a cluster separate from the control samples. Canonical correspondence analysis with a Monte Carlo permutation test showed that community composition was correlated with both time of sampling (P<0.01) and the presence of exudates (P<0.001). The canonical axes together accounted for 12% of the total variance in species composition.

4

Ordination plot derived from sample scores from the correspondence analysis of bacterial community composition after various times (0, 3, 6, 9, 20, 48 and 72 h) of incubation with or without mycelial exudates from an arbuscular mycorrhizal fungus (Glomus sp. MUCL 43205). Closed and open symbols symbolize samples with or without mycelial exudates, respectively. Symbol shapes represent times of sampling: cross: 0 h; circle: 3 h; diamond: 6 h; triangle: 9 h; oval: 20 h; square: 48 h; tilted triangle: 72 h. Groupings highlight differences in bacterial community composition between samples with or without mycelial exudates at 6, 48 and 72 h. The dashed line transecting the diagram separates samples at the beginning (0–6 h) from those at the end (9–72 h) of the experiment.

Species scores from the correspondence analysis and the χ2 test of original data of taxon distribution in samples were used to single out bacterial taxa specifically associated with either treatment (Table 2 and Fig. 5).

View this table:
2

Bacterial taxa identified in samples by their TRFLP profiles by comparison with clone TRFLP profiles

Clone designationTaxonomic descriptionNo. of samples in which taxon was detectedχ2 test
NMMP-value
Taxa found only in NM
Clone 23*Flavobacterium sp.40.04
Clone 21*Unknown bacterium40.04
Clone 61*Alphaproteobacteria30.08
Clone 71*Putative Chloroflexi20.16
Clone 49*Betaproteobacteria20.16
Taxa found predominantly in NM
Clone 57*Betaproteobacteria730.20
Clone 60*Flavobacterium sp.620.16
Clone 89*Phenylobacterium sp.510.10
Clone 29*Myxococcales510.10
Clone 51*Acidobacteria310.32
Taxa found only in M
Clone 37*Legionella sp.40.04
Clone 35Pedobacter sp.40.04
Clone 52*Acidobacteria20.16
Taxa found predominantly in M
Clone 03Enterobacteriaceae5150.03
Clone 83*Betaproteobacteria370.21
Clone 25*Gammaproteobacteria150.10
Clone 64*Flexibacteraceae140.18
Clone 96*Betaproteobacteria140.18
  • * Clone library 0 h.

  • Taxa predominantly found in a treatment (NM or M) were detected more than twice as frequently in that treatment as in the other.

  • Clone library 48 h.

  • The Table shows sequenced clones with increased frequency of occurrence in response to the absence (NM) or presence (M) of mycelial exudates. For additional phylogenetic information, see Fig. 5.

5

Neighbour-joining tree showing the sequence resemblance between cloned inserts of the 16S rRNA gene of soil bacteria and selected reference sequences denoted with GenBank accession numbers. The tree only comprises clones representing bacteria with an uneven distribution among samples with or without mycelial exudates from Glomus sp. MUCL 43205. M: taxon occurring exclusively or more frequently in samples with mycelial exudates. NM: taxon occurring exclusively or more frequently in samples without mycelial exudates. Solid lines represent bootstrap values ≥90.

A taxon within Enterobacteriaceae (Clone 03) was significantly (P<0.05) more frequent in samples with mycelial exudates, and was, in addition, more frequent in samples harvested at 20–72 h. Seven other taxa were either exclusively or predominantly detected in samples containing mycelial exudates: a Legionella sp. (Clone 37), a Pedobacter sp. (Clone 35), and taxa within Flexibacteraceae (Clone 64), Acidobacteria (Clone 52), Betaproteobacteria (Clones 83 and 96), and Gammaproteobacteria (Clone 25). In addition, three of these (Clones 35, 52, and 64) were detected in samples only at 3 and 6 h (Table 2, Fig. 5).

Ten bacterial taxa occurred exclusively, or showed a higher frequency of occurrence, in samples without mycelial exudates: two Flavobacterium spp. (Clones 23 and 60), an unknown taxon (Clone 21), a Phenylobacterium sp. (Clone 89), and taxa within Chloroflexi (Clone 71), Betaproteobacteria (Clones 49 and 57), Alphaproteobacteria (Clone 61), Myxococcales (Clone 29), and Acidobacteria (Clone 51). The two Flavobacterium spp. were detected in samples throughout the entire incubation period (0–72 h), whereas the other taxa with a higher frequency of occurrence in samples without mycelial exudates were detected only in samples harvested in the beginning of the experiment (3–9 h; Table 2, Fig. 5).

Discussion

This study was conducted in artificially assembled in vitro systems in which the effects of mycelial exudates, harvested from root organ cultures, on an extracted bacterial community could be studied in isolation from the complexity of the soil environment. By doing this we could demonstrate that AM fungal exudates may have a substantial quantitative and qualitative impact on bacterial communities. The relative importance of mycelial exudates for the development of soil microbial communities in the field remains to be investigated. In our systems, the bacteria and the fungus were never in physical contact with each other. In soils, the quantity and/or composition of mycelial exudates may be stimulated by the presence of bacteria in close proximity to the hyphae, involving a positive feedback in the interactions. Furthermore, because it has been demonstrated that bacteria can have positive feedbacks on mycorrhizal root colonization (e.g. Budi et al., 1999), it is interesting to speculate that bacteria may also stimulate the growth of extraradical mycelia, leading to increased exudation. Exudation of water and carbohydrates may create a favourable environment around the mycorrhizal fungal hyphae, and cell surface structures or mucilage of bacterial or fungal origin may provide an interface for physical and metabolic interactions (Johansson et al., 2004; Toljander et al., 2006). The existence of such effects has been postulated for bacteria–ectomycorrhiza interactions (Sun et al., 1999). The effects of mycelial exudates may, however, be diluted in the complex soil environment, where microbial communities are under the combined influence of mycelial exudates, root exudates, litter inputs, predators and abiotic factors.

The exudates from Glomus sp. MUCL 43205 contained low-molecular-weight sugars and organic acids, which were probably metabolised by bacteria, and also unidentified high-molecular-weight compounds that may have supported bacterial growth. In our experimental system, bacterial numbers increased over time, regardless of treatment, and the proportion of vital bacteria was high (Fig. 3a and b), suggesting that recycling of dead bacterial cells might have occurred as a result of the low availability of carbon and nutrients in the samples. When mycelial exudates were present, the proportion of vital cells was lower at the beginning of the experiment (at 3 and 9 h). The presence of mycelial exudates may have relaxed nutritional stress, reducing the recycling of bacterial cells. The observed maximum of cell counts and vitality at 48 h, in conjunction with the decline of both these parameters at 72 h, may indicate the depletion of mycelium-derived resources. The bacterial growth curves reflect the natural growth cycle of bacteria; however, it is evident that utilization of exudates by the bacteria resulted in quicker bacterial growth and a subsequent decline in bacterial numbers, whereas the stationary growth phase was delayed in samples without exudates. The slower increase in bacterial numbers in the absence of exudates may indicate a relatively slow utilization of less bio-available soil-derived carbon added together with the bacterial soil extract.

In our experiment we also observed that bacterial community composition was significantly influenced by the presence of exudates. The Gammaproteobacteria were over-represented among the taxa that responded positively to mycelial exudates. This is in agreement with many studies reporting synergistic interactions between AM and Gammaproteobacteria, leading to increased mycorrhizal colonization and nutrient uptake in plants (see Artursson et al., 2006 and references therein). In the present study, a taxon within the Enterobacteriaceae (Clone 03) increased in response to the AM fungal mycelial exudates. Enterobacteriaceae bacteria have previously been shown to promote mycorrhizal establishment (Toro et al., 1997) and plant nitrogen and phosphorus uptake (Toro et al., 1997; Kim et al., 1998). Most other bacterial taxa that appeared to respond to the exudates were, however, only detected in a small number of samples.

The dominance of Gram-negative bacteria in our samples might indicate a possible bias against Gram-positive bacteria by the DNA extraction methods employed in this study. In most soils, however, Gram-negative bacteria typically represent the largest bacterial group (Kim et al., 2006). One of the most frequently detected bacterial groups in the present study was Pseudomonas spp., which increased over time regardless of the presence of mycelial exudates. Sequence data from the clone reference database showed that only 7% of clones from the original bacterial community were Pseudomonas spp., whereas the corresponding figure at 48 h of incubation was 58%. Many studies report an enrichment of pseudomonads in mycorrhizosphere soils (Andrade et al., 1997; Mansfeld-Giese et al., 2002). This phenomenon could be a result of the opportunistic nature of these bacteria, their high culturability, or the fact that they are favoured by in vitro conditions. It should be emphasized that the bacteria presented in this study are likely to represent a cultivable subset of a natural soil bacterial community, in which certain taxa have been enriched as a result of both in vitro conditions and exposure to exudates. A number of bacterial taxa were primarily associated with samples without mycelial exudates, suggesting that they were either inhibited by the exudates directly, or repressed by those bacteria that increased in response to the exudates. Two Flavobacterium spp. (Clones 23 and 60) and two Alphaproteobacteria (Clones 61 and 89) were notably more frequent among samples without exudates. Flavobacteria are common soil commensalists, and occasionally behave as opportunistic pathogens (Kim et al., 2006).

Our understanding of interactions between AM fungal extraradical mycelia and their biotic and abiotic environment is still in its infancy. The characterization of the composition of AM fungal exudates and the effects of these compounds on soil microbial communities may have wide implications for our understanding of the mechanisms behind nutrient cycling, biological control and bioremediation or phytochelation in soils. This study presents the first data on low-molecular-weight compounds in mycelial exudates from a Glomus species. The integrated effects of the mycorrhizosphere (roots and fungi) on bacterial communities have previously been demonstrated, but few attempts have so far been made to investigate the particular influence of AM fungal mycelia. Although there may be differences in the exudation patterns of different fungal species, the results of the current study highlight the possible importance of the AM fungal extraradical mycelium as a pathway for plant-derived carbohydrates to microorganisms in micro-sites distant from, and inaccessible to, the plant roots themselves.

Acknowledgements

We thank David Clapham (Department of Plant Biology & Forest Genetics, Swedish University of Agricultural Sciences [SLU], Uppsala, Sweden) for supplying the Agrobacterium rhizogenes LBA 9402 strain, and Rolf Andersson (Department of Chemistry, SLU, Uppsala, Sweden) for performing the NMR analysis. Financial support from the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (FORMAS) is gratefully acknowledged.

Footnotes

  • Editor: Karl Ritz

References

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