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Applied and Environmental Microbiology, May 2009, p. 2820-2830, Vol. 75, No. 9
0099-2240/09/$08.00+0     doi:10.1128/AEM.02110-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Migratory Response of Soil Bacteria to Lyophyllum sp. Strain Karsten in Soil Microcosms{triangledown}

J. A. Warmink and J. D. van Elsas*

Department of Microbial Ecology, Centre for Ecological and Evolutionary Studies, University of Groningen, Kerklaan 30, 9750RA Haren, The Netherlands

Received 11 September 2008/ Accepted 5 March 2009


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ABSTRACT
 
In this study, the selection of bacteria on the basis of their migration via fungal hyphae in soil was investigated in microcosm experiments containing Lyophyllum sp. strain Karsten (DSM2979). One week following inoculation with a bacterial community obtained from soil, selection of a few specific bacterial types was noticed at 30 mm in the growth direction of Lyophyllum sp. strain Karsten in sterile soil. Cultivation-based analyses showed that the migration-proficient types encompassed 10 bacterial groups, as evidenced by (GTG)5 genomic fingerprinting as well as 16S rRNA gene sequencing. These were (>97% similarity) Burkholderia terrae BS001, Burkholderia sordidicola BS026, Burkholderia sediminicola BS010, and Burkholderia phenazinium BS028; Dyella japonica BS013, BS018, and BS021; "Sphingoterrabacterium pocheensis" BS024; Sphingobacterium daejeonense BS025; and Ralstonia basilensis BS017. Migration as single species was subsequently found for B. terrae BS001, D. japonica BS018 and BS021, and R. basilensis BS017. Typically, migration occurred only when these organisms were introduced at the fungal growth front and only in the direction of hyphal growth. Migration proficiency showed a one-sided correlation with the presence of the hrcR gene, used as a marker for the type III secretion system (TTSS), as all single-strain migrators were equipped with this system and most non-single-strain migrators were not. The presence of the TTSS stood in contrast to the low prevalence of TTSSs within the bacterial community used as an inoculum (<3%). Microscopic examination of B. terrae BS001 in contact with Lyophyllum sp. strain Karsten hyphae revealed the development of a biofilm surrounding the hyphae. Migration-proficient bacteria interacting with Lyophyllum sp. strain Karsten may show complex behavior (biofilm formation) at the fungal tip, leading to their translocation and growth in novel microhabitats in soil.


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INTRODUCTION
 
Bacterial-fungal interactions are common in a wide variety of habitats like decaying wood, human bodies, and marine and soil environments (7, 12, 13, 15, 18). Especially in soil, interactions are likely to occur frequently, as members of both kingdoms abound in this system and depend on strategies that allow them to utilize the sparse carbonaceous nutrients that are available (6, 22, 26, 27). Interactions may be deleterious, neutral, or even beneficial for either or both of the partners. In particular, the putative beneficial effects exerted by soil fungi on associated bacteria may enhance bacterial fitness and thus provide a selective force on these (4, 5, 11, 14, 29). A range of different mechanisms is thought to play a role in the putative bacterial selection, in which particular fungus-released compounds may exert key effects in this selection (1, 10, 14, 28). In addition, changes in the structure of the local (soil) habitat effected by either of the partners (2) and/or production of antibacterial substances by the fungal partner (7, 9) may play roles.

The capacity of soil bacteria to use fungal hyphae as a means to reach and colonize novel microhabitats in soil has been proposed as a mechanism for pollutant-degrading bacteria to become efficient in polluted soil (16). However, the study addressed only bacterial migration with fungi via so-called fungal highways in non-soil systems like agar plates and glass bead systems. Clearly, such fungal highways might be used by bacteria to cross air gaps (23) during growth and movement in soil, but evidence for this is lacking. Movement of the bacterial partner was probably driven by motility of the bacterial cells in the water film surrounding the fungal hyphae. The observation of bacteria moving along fungal highways was supported by an earlier study that addressed bacterial motility via dead hyphae of an oomycete in soil (32). Together, these studies suggest that bacteria can utilize the mycosphere (here defined as the fungal hyphal network) in soil to reach and colonize novel microhabitats. However, these studies do not allow an in-depth assessment of which bacteria get selected by growing fungi and how they mechanistically make use of fungal highways.

In the current study, we assessed the putative selection of organisms from a soil bacterial community that was able to migrate in the mycosphere of Lyophyllum sp. strain Karsten, a close saprotrophic relative of the ectomycorrhizal fungus Laccaria proxima. We initially assessed the selection of particular bacterial species by L. proxima (29), which was an abundant ectomycorrhizal species with hazel trees. Thus, we developed a microcosm system composed of three compartments, which allowed the outgrowth of fungal hyphae from a nutrient source into sterile soil. Different aspects of bacterial migration along with the fungal front were studied. Based on these findings, a mechanism for bacterial migration in which biofilm formation plays a role is proposed.


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MATERIALS AND METHODS
 
Soil.
We used fresh soil sampled in Gieterveen, The Netherlands, denoted G soil (28). The G soil, of sandy texture, had a pH of 4.0 and total carbon (C) and total nitrogen (N) contents of 2.8% and 0.8%, respectively. For some experiments, we added 0.5% CaCO3 to the soil, which raised the pH to 5.0. For several experiments, the soil was sterilized twice at intervals of 3 days by autoclaving at 115°C for 45 min. Following each autoclaving step, a 60-min exposure to sterile air was used to release any volatile toxic compounds produced. The soil was sterile, as evidenced by dilution plating soil suspensions prepared in 0.85% NaCl on R2A agar (Becton, Dickinson, and Company, Sparks, MD), incubating plates at 23°C, and assessing any putative microbial growth over a prolonged incubation time.

Bacterial cell suspension for migration experiments.
To prepare soil bacterial cell suspensions for inoculation, freshly sampled G soil was first homogenized and then added to sterile 0.85% NaCl (saline) in a 1:10 proportion (0.5 g of soil/5 ml of saline). The resulting suspensions were homogenized by vigorous mixing on a vortex shaker (at full speed three times for 1 min). Following this treatment, soil particles were allowed to settle for 1 min on the bench, after which 50 µl of the supernatant, containing roughly 108 cells as estimated by direct microscopy, was used for inoculation in migration experiments.

To prepare suspensions of cultured bacterial strains (Table 1) for inoculation, bacteria were grown overnight in 3 ml of R2A medium (Becton, Dickson and Company, Sparks, MD) at 23°C, with shaking. The cells were spun down for 5 min at 4,000 x g, washed, and resuspended in 1 ml of 0.85% NaCl; this was repeated two times. The final cell suspensions were diluted to an OD660 of 0.05 (containing an estimated 107 cells/ml, using dilution plating on R2A agar). In total, 50 µl of this bacterial suspension (thus containing roughly 5 x 105 cells) was used directly for inoculation of soil in the migration experiments.


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  		TABLE 1. Bacterial strains used in this study

Growth and maintenance of used fungi.
The (basidiomycetous) fungi used in this study, i.e., Lyophyllum sp. strain Karsten (DSM2979), Hebeloma cylindrosporum Romagnesi (provided by I. van Aarle), Paxillus involutus, and Laccaria bicolor (strain S238N) (the last two provided by P. Frey-Klett) were grown on oat flake agar plates, prepared with 30 g of oat flake (local pet shop) and 15 g of agar (Duchefa, Haarlem, The Netherlands), filled with water to 1 liter, and sterilized at 121°C for 21 min. Once every 4 weeks, all fungal strains were transferred to fresh oat flake plates for maintenance.

Microcosm and migration experiments.
A microcosm system was designed that consisted of three-compartment petri dishes (Greiner Bio-one, Frickenhausen, Germany). Two compartments (Fig. 1) were filled each with 9 g of moist, sterilized G soil (moisture content of 17%, corresponding to 60% of water holding capacity, bulk density [wet wt/vol] of about 1.3), yielding layers of 4 mm. The third compartment was filled with oat flake agar. The physical barriers between the oat flake and the two soil compartments prevented compounds from the oat flake from reaching the soil compartments. The barriers were overcome by the fungal hyphae, and hence outgrowth of a fungus from the nutrient-rich oat flake environment into the soil was achieved. The system was inoculated with the different basidiomycetous fungi on the oat flake medium and incubated at 28°C, thereby allowing the colonization of the oat flake plus about 1 to 4 mm of the sterile soil (prior to introduction of bacterial inocula).


Figure 1
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  		FIG. 1. Microcosm model based on a three-compartment petri dish.

By means of a pipette, washed bacterial suspensions (50 µl) were evenly distributed in one 3-mm-wide streak in the sterile soil in each soil compartment directly adjacent to the front of the growing hyphae. After incubation at 23°C, soil samples (100 mg) were taken at different time points and sites within the soil compartments by removing 4-mm-diameter cores from the soil. Samples were taken of the soil both in and against the hyphal growth direction in relation to the inoculation location. Samples (quadruplicates; two from each microcosm) were then used for further analyses by dilution plating and DNA extraction (see below).

Analysis of total bacterial communities by molecular means.
DNA was extracted from G soil and microcosm samples with a PowerSoil DNA extraction kit (MoBio Laboratories, Carlsbad, CA) according to the manufacturer's instructions. DNA of sufficient purity, size (~20 kb), and amount (20 µg/g of soil) was obtained for further analyses. These DNA extracts were used for further analyses via PCR-denaturing gradient gel electrophoresis (DGGE) to analyze the bacterial communities using primers 2 and 3 based on the 16S rRNA gene, as described previously (19).

Analysis of the culturable bacterial community.
Bacterial cell suspensions from microcosm samples and fresh G soil were prepared as described above and used for dilution plating on R2A agar (Becton, Dickinson and Company, Sparks, MD). The dilution plates were incubated at 23°C for 2 weeks and regularly monitored for colony development. Enumeration of the CFU was done after 2 weeks.

From the dilution plates prepared from the microcosm samples as well as fresh G soil, randomly picked colonies were streaked to purity, resulting in 84 isolates from the microcosm and 176 isolates from G soil. All isolates were grown overnight in (3 ml) R2A medium, after which they were resuspended in 17% (vol/vol) glycerol and stored at –80°C.

Genomic fingerprinting of the bacterial isolates.
To group the isolates and discern replicates, all were subjected to genomic fingerprinting by colony PCR targeting repetitive elements in the genome using primer (GTG)5 (20). Clustering of the patterns generated for all isolates was achieved using the unweighted pair group method with mathematical averages with the program Gelcompar II (Applied Maths, Sint-Martens-Latem, Belgium). Representative members (three per group, or just one if the group contained one to two isolates) of each group were used for further analyses.

Identification and properties of selected isolates.
Representatives of each (GTG)5 group were subjected to identification by (partial) 16S rRNA gene sequencing as described previously (29). Furthermore, an indication for the presence of a type III secretion system (TTSS) was obtained by PCR-based detection of the hrcR gene as described previously (29). Metabolic tests using Biolog (GN2) assays (Biolog Inc., Hayward, CA) were performed on all representatives of the (GTG)5 groups according to the manufacturer's protocol. Intrinsic flagellar motility was tested using microscopic analyses of overnight cultures. Antagonism against Lyophyllum sp. strain Karsten was tested in a dual culture assay according to Berg et al. (4a) on oat flake medium. The inhibition zones were observed after incubation at 23°C for 7 days.


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RESULTS
 
Design of a microcosm system for the assessment of bacterial migration with fungal hyphae.
The microcosm made out of a three-compartment petri dish was used to establish a base compartment (the oat flake compartment) that would support growth of fungal mycelium in addition to two adjacent compartments containing G soil as the experimental matrix. The system allowed above-ground contact between the base and soil compartments and careful control of temperature and soil moisture. The compartments could be sampled using microsamplers without disturbing the experiment. The experiment thus allowed time courses to be performed and was easily replicable.

The basidiomycetous fungi Lyophyllum sp. strain Karsten (DSM2979), H. cylindrosporum Romagnesi, P. involutus, and L. bicolor (S238N) were tested for their performance in the microcosm experiments. All five attempts to obtain migratory growth of Lyophyllum sp. strain Karsten hyphae through nonsterile G soil failed, whereas the fungus grew readily through sterilized G soil. Similar results were obtained with H. cylindrosporum Romagnesi, P. involutus, and L. bicolor in nonsterile soil. This lack of extension through nonsterile soil was probably caused by suppression by the indigenous microbial community. It led us to perform all subsequent work in sterilized microcosms. Lyophyllum sp. strain Karsten was selected as the preferred fungus as the other fungal strains grew poorly. Lyophyllum sp. strain Karsten showed high movement of the hyphal front in the sterile soil (5 mm per day), forming a dense hyphal network in the soil. Lyophyllum sp. strain Karsten was also preferred as it is a close saprotrophic relative of L. proxima, the ectomycorrhizal fungus studied in our previous work (29). It thus supported previous assessments of L. proxima-associated bacteria in field G soil, in which strong selection of particular bacteria was shown (29) (Table 1).

Migration of soil bacteria via growing fungal hyphae.
Bacterial cell suspensions prepared from fresh G soil in 2005 were used in the initial migration selection experiment through sterilized G soil, whereas the experiment was repeated in 2008 with another, fresh G soil sample to determine the reproducibility of bacterial selection under circumstances identical to those in 2005. The fungal hyphae did serve as catalysts of the movement and growth of particular bacteria through the soil as in the fungal-containing plates; the CFU counts at 30-mm distance were, on average, log 7.2 ± 0.5 CFU/g of dry soil in the first experiment and log 7.2 ± 0.5 CFU/g of dry soil in the second one. Control samples taken from the soil compartments that had been inoculated with a bacterial suspension but had not been colonized with Lyophyllum sp. strain Karsten at a 30-mm distance from the inoculation site never showed any CFU on R2A plates (detection limit, 100 CFU per g of soil). In addition, the soil bacterial suspension added to the sterile G soil did not detectably hamper hyphal extension through the soil, as found by a comparison between bacterium-inoculated and uninoculated systems, presumably as a result of the lack of strong fungistasis in such a colonizing community.

Analysis of community migrators.
A suite of 84 isolates was randomly picked from plates (105 dilution) prepared from the 30-mm site and streaked to purity. (GTG)5-based fingerprinting analysis of these strains, collectively denoted as community migrators, showed their distribution among 10 distinct groups, denoted groups I through X (Table 2). Groups I to IV encompassed most (86%) of the strains. Phylogenetic analysis of representatives of these groups (three isolates per group) showed that the group I, III, and IV organisms were affiliated with Dyella japonica strain RB28 (>98% similarity; 60% of all 84 migrating isolates), whereas the isolates belonging to group II were affiliated with Burkholderia terrae BS110 (100% similarity; 26.5% of all 84 migrating isolates). The other migrators (in total, 13.5% of the total) fell into six different species, as follows: group V, Burkholderia sordidicola SNU 0201230 (98% similarity); group VI, Burkholderia sediminicola HU2-65W (100% similarity); group VII, "Sphingoterrabacterium pocheensis" 0032 (96% similarity); group VIII, Ralstonia basilensis ER121 (100% similarity); group IX, Sphingobacterium daejeonense JP10 (96% similarity), and group X, Burkholderia phenazinium HG14 (100% similarity). The repeat experiment performed in 2008 with a fresh G soil bacterial suspension again showed selection of group II, B. terrae, as well as group III, D. japonica, strains based on (GTG)5 fingerprint patterns, indicating a highly consistent selection mechanism for the same bacteria. The most abundant group (I) from 2005 was, however, not found in 2008 (Table 2).


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  		TABLE 2. Community migrating strains isolated in this study

To assess the prevalence of community migrators in the initial G soil bacterial suspension, this suspension was plated on R2A agar (105 dilution), and 176 colonies were randomly picked from the plates and subjected to (GTG)5 fingerprint analyses. Clustering of the resulting profiles (unweighted pair group method with mathematical averages) resulted in 128 different profile groups. A cross-comparison of these with the profiles of the community migrators revealed that only the group X (B. phenazinium) profile could be found in the initial soil community, making up 2.2% of the initial culturable community (4/176 isolates).

Cultivation-independent assessment of bacterial community development and migration.
PCR-DGGE analysis of the bacterial community developing with Lyophyllum sp. strain Karsten at the soil inoculation site over 14 days showed the emergence, over time, of a number of specific bands (Fig. 2A). The soil bacterial cell suspension used as the inoculum showed a highly complex PCR-DGGE profile, which contained at least 70 bands. However, the profiles obtained shortly (3 h) after inoculation (day zero) differed from the inoculum, presumably as a result of the initial processes of colonization of, and adaptation to, the soil. After 14 days, the profiles consisted of eight dominant bands (Fig. 2A, bands B1 to B8). Samples of the soil that had not received bacterial inoculum produced no discernible profiles. None of the bands emerging after 14 days under the influence of Lyophyllum sp. strain Karsten was detectable in the day zero profile or the inoculum profiles. This indicated a density of the underlying, putatively selected bacteria in these samples below the limit of detection of PCR-DGGE analysis. Furthermore, increases in profile complexity over time and the appearance and disappearance of particular bands were also observed, as evident in the profiles generated from the day 5 and 7 samples (Fig. 2A).


Figure 2
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  		FIG. 2. PCR-DGGE analyses of development of the bacterial community at the inoculation (A) as well as hyphal front (B) in time. Markers for 16S rRNA gene fragments of (from top to bottom): Listeria innocua, Enterobacter cloaceae, Mesorhizobium sp., Burkholderia cepacia, and Arthrobacter sp. B1 through B8 represent key bands in the profiles that were (partially) presumptively identified (see the text).

The PCR-DGGE profiles obtained from the hyphal growth contained five major bands. These profiles also showed particular bands emerging over time (Fig. 2B). Some bands that emerged in the PCR-DGGE profiles obtained from the inoculation location were also found as emerging ones in these profiles, i.e., bands B1, B2, B3, B4, and B7 (Fig. 2). However, some bands found at the inoculation spot were missing from the fungal front profiles, i.e., bands B5, B6, and B8.

Technical difficulties did not allow the direct sequencing of bands. Hence, we presumptively identified specific selected bands by comparing the comigration of amplicons obtained from the organisms described above, i.e., B. terrae BS001 and D. japonica BS021, with the bands in the direct PCR-DGGE profiles. By this analysis, bands B1, B3, and B4 in combination were all ascribed to B. terrae BS001 as this organism yielded these multiple bands in PCR-DGGE. Band B5 was further presumptively identified as belonging to D. japonica BS021 based on the comigration with the derived band from this organism.

Single-strain migration.
Given the fact that the bacterial migration with the fungal hyphal front might have been due to either an intrinsic property of the organism itself or to a potential helper effect exerted by other migrating bacteria, representatives of all 10 community migrator groups were subjected to single-strain migration experiments performed in quadruplicate with Lyophyllum sp. strain Karsten in the same setup used above. Of the 10 groups, 6 could not migrate as single strains with Lyophyllum sp. strain Karsten, although control experiments showed good survival of all bacterial strains at the inoculation spot. These presumable obligatory community-dependent migrators were B. phenazinium BS028 (group X), B. sediminicola BS010 (group VI), B. sordidicola BS026 (group V), D. japonica BS013 (group IV), S. daejeonensis BS025 (group IX), and S. pocheensis BS024 (group VII). On the other hand, representatives of the remaining four groups showed a clear capacity to comigrate as a single population with growing Lyophyllum sp. strain Karsten hyphae. These were D. japonica BS021 (group I), D. japonica BS018 (group III), B. terrae BS001 (group II), and R. basilensis BS017 (group VIII) (Table 1).

The D. japonica group IV was closely related to the D. japonica groups I and III on the basis of its similar 16S rRNA gene sequence and also revealed some similarities in the (GTG)5 patterns (data not shown). Remarkably, strains of this group did not show any migration via the fungal hyphae, whereas group I and III strains revealed avid bacterial migration.

Properties of selected bacteria. (i) TTSSs.
For all strains, a clear correlation was found between the capacity to comigrate with Lyophyllum sp. strain Karsten as a single strain through soil and the presence of the hrcR gene (an indicative marker for the presence of a TTSS) as all four single-strain migrators possessed the hrcR gene, whereas four of the other six community migrators (except B. sordidicola BS026 and B. phenazinium BS028) did not possess the hrcR gene (Table 1). We also assessed the comigration capacity of 12 organisms (Table 1) obtained from the mycosphere of L. proxima (related to Lyophyllum) in natural G soil (29). Of these, only the hrcR-positive B. terrae BS110 (Table 1) revealed single-strain migration with Lyophyllum sp. strain Karsten. The (GTG)5 patterns and 16S rRNA gene sequence of this strain were similar to those of B. terrae BS001, representative of one of the most abundant single-strain migrator groups (group II) in this study.

(ii) Intrinsic flagellar motility.
All community migratory strains as well as strains obtained from the L. proxima mycosphere were microscopically analyzed in wet mounts for intrinsic (flagellar) motility. With the exception of Chryseobacterium piscium BS055 (obtained from the L. proxima mycosphere), all strains were positive as they showed active motility that was not caused by Brownian motion (Table 1).

(iii) Antagonism.
The putative antagonistic properties of all community migrator (and L. proxima isolated) strains against Lyophyllum sp. strain Karsten were then tested in a dual-culture assay. Growth of Lyophyllum sp. strain Karsten was inhibited by only six strains, i.e., B. sediminicola BS010, Pseudomonas poae BS053, Chryseobacterium piscium BS055, Aquamonas fontana BS086, Chryseobacterium aurantiacum BS126, and Chryseobacterium joosteii BS181. All other bacterial strains did not have an influence on the development and growth of Lyophyllum. Interestingly, none of the strains except B. sediminicola BS010 showed antagonistic properties in microcosm experiments (data not shown).

On the other hand, Lyophyllum sp. strain Karsten present in the microcosm limited the survival of bacterial strains Arthrobacter ramosus BS066, Mycobacterium hodleri BS043, Paenibacillus polymyxa BS109 and C. joosteii BS126 (all L. proxima isolates). These bacterial species were not found at the inoculation spot (detection limit, <200 CFU/g of soil) after a 1-week incubation following the introduction at an inoculum density of ± 105 CFU/g of dry soil (Table 1) although these bacterial strains showed survival in the corresponding soil without the fungus.

Metabolic tests using Biolog.
Metabolic profiles were obtained via Biolog substrate utilization tests and cross-compared using principal components analyses (PCA). A strong clustering of (group II) B. terrae (strain BS001) and (group VI) B. sediminicola (BS010) with strains denoted as universal "fungiphiles" in previous work (27) was found (Fig. 3). This clustering was primarily based on the utilization of 15 of 18 compounds, which have previously been postulated to be often present in fungal exudates (28), in the Biolog assay (14, 21, 24, 25, 31). The compounds utilized by B. terrae (BS001) were L-arabinose, D-arabitol, {alpha}-D-glucose, M-inositol, D-mannitol, D-trehalose, citric acid, D-alanine, L-aspartic acid, L-glutamic acid, L-phenylalanine, L-proline, D-serine, L-threonine, and glycerol. In the PCA analyses, the three D. japonica strains, i.e., BS003, BS013 and BS021, clustered together, but they formed a cluster separate from the typical universal fungiphile cluster established earlier (Fig. 3). For these D. japonica strains, the utilization of such compounds was limited to D-trehalose, L-aspartic acid, L-glutamic acid, L-leucine, L-phenylalanine, L-proline and L-threonine. The other community migrators, i.e., R. basilensis (BS017), B. sordidicola (BS026), and B. phenazinium (BS028), did not show clear clustering and were found between the fungiphile groups and the bulk soil isolates based on metabolic profiles.


Figure 3
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  		FIG. 3. PCA of the metabolic profiles obtained with the Biolog assay. BSXXX codes (where X is a number 0 to 9) represent strains that were obtained in this study; P01 to P18 and B1 to B8 were obtained in a previous study (28) and represent Pseudomonas isolates from mycospheres and bulk soil isolates, respectively.

The migratory direction of B. terrae and D. japonica in soil microcosms.
We selected two strains, i.e., D. japonica (BS021; group I) and B. terrae (BS001; group II), representing the abundant single-strain migrators, for further studies, as these were likely most strongly selected by the Lyophyllum sp. strain Karsten hyphae. Both B. terrae BS001 and D. japonica BS021 were introduced as single strains (inoculum density of about 105 CFU/g of dry soil) at the hyphal growth front, and their migration either with or against the growth direction of the Lyophyllum sp. strain Karsten hyphae was determined.

Migration of both inoculant strains was shown to occur at the hyphal front of Lyophyllum sp. strain Karsten but only in the direction of hyphal growth (Table 3). Migration in the opposite direction was never observed, whereas the presence of fungal hyphae did not hamper bacterial outgrowth on plates (Table 3). The capacity of the two strains to migrate in any direction on the old hyphae (1 week after active fungal growth) was also tested by following the fate of an inoculum (±105 CFU/g of dry soil) placed in the middle of the hyphal mat. For both inoculant strains, bacterial cells persisted at the site of introduction at roughly 105 CFU per g of dry soil, whereas no migration (detection limit, about 100 CFU per g of dry soil) in either the direction of growth or the opposite direction was noticed (Table 3). These results strongly indicate the requirement of actively growing Lyophyllum sp. strain Karsten hyphae for single-strain migration of B. terrae BS001 and D. japonica BS021 in soil.


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  		TABLE 3. Summary of migrational behavior of B. terrae BS001 and D. japonica BS021 with Lyophyllum sp. strain Karsten hyphae through sterile G soil

Distribution and growth rate of B. terrae and D. japonica cells on fungal hyphae.
To assess the distribution of inoculant cells established on growing Lyophyllum sp. strain Karsten hyphae in the microcosm, 2 x 106 (B. terrae BS001) and 7 x 106 (D. japonica BS021) cells were introduced at the hyphal front, yielding after 3 h (locally) about log 5.3 CFU of B. terrae BS001 per g of dry soil and log 5.8 CFU of D. japonica BS021 per g of dry soil. After 1 week, the bacterial densities were determined at three different spots in the soil compartments, i.e., the introduction site as well as 15 and 30 mm (hyphal growing front) in the fungal growth direction. Bacterial CFU were never found from the 15- and 30-mm sites on plates prepared from control soil compartments (without the presence of the fungus).

For the Lyophyllum sp. strain Karsten systems that had received B. terrae BS001 cells, very similar strain BS001 CFU counts of approximately log 8.6 ± 0.15 per g of dry soil were found at all three sampling sites, and no significant differences (t test, P > 0.05) were observed between the CFU counts found at these locations. Also, D. japonica BS021 was detected at log 8.2 ± 0.19 CFU/g of soil, again showing no significant differences between the three locations in the soil compartments (Fig. 4). The B. terrae BS001 CFU counts were higher (t test, P < 0.05) than those of D. japonica BS021 under the same conditions (Fig. 4). Thus, for both strains, growth resulting in an estimated >1,000-fold population increase was observed in the total soil compartment in comparison to the cell density that had originally been introduced into this compartment. Specifically, this indicated that at least nine cell divisions of the introduced bacteria had taken place within a week.


Figure 4
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  		FIG. 4. The distribution of B. terrae BS001 and D. japonica BS021 at different locations in the microcosm. Both bacteria were separately introduced in the system. A, Introduction spot of bacteria; B, halfway between introduction spot and hyphal front (15 mm from introduction spot); and C, hyphal front (30 mm from introduction spot).

Bacterial growth and rescue from low pH by Lyophyllum sp. strain Karsten.
The influence of Lyophyllum sp. strain Karsten hyphae on the putative growth of B. terrae BS001 in G soil was monitored over a period of 9 days using CaCO3-amended (pH 5.0) as well as unamended (pH 4.0) soil. Both the inoculation site (initially at the hyphal front) and the moving hyphal front were sampled during this time period in addition to controls without the fungus. In the control samples, a strong decrease of B. terrae BS001 CFU numbers from about 105 to below the detection limit (200 CFU/g of dry soil) was noticed after day 2. This strong decrease in CFU numbers in the absence of Lyophyllum sp. strain Karsten hyphae was overcome, and growth of B. terrae BS001 was even observed when the soil pH had been raised from 4.0 to 5.0 (data not shown). Thus, in soil that contained growing Lyophyllum sp. strain Karsten hyphae, strong increases in the B. terrae BS001 CFU numbers were observed from the initial ± log 5 CFU/g of dry soil to a maximum of about log 7.3 CFU/g of dry soil. The latter value was supposed to roughly represent the maximum carrying capacity for B. terrae BS001 in the specific Lyophyllum sp. strain Karsten mycosphere system (Fig. 5). At the growing hyphal front, the level was reached after 3 days and did not change significantly afterwards, indicating a maximal level of bacterial density at the fungal hyphae. Starting with an inoculum of 105 cells per gram of soil, the inoculation site reached this maximum level at day 5, thus indicating that the bacterial growth rate was limited on these (older) hyphae.


Figure 5
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  		FIG. 5. Influence of the fungus Lyophyllum sp. strain Karsten on growth of strain BS001 in time at two sites (inoculation spot and fungal growth front) in the microcosm. The detection limit for this experiment was 200 CFU/g of dry soil. Values below this limit are shown as zero. Control, no fungal host; Fungus (I), system with fungus, inoculation spot; Fungus (F), system with fungus, fungal growth front.

Microscopic analysis of bacterial Lyophyllum sp. strain Karsten interactions.
Microscopic analyses of Lyophyllum sp. strain Karsten hyphae growing on water agar showed two morphological hyphal types, i.e., (i) a "normal" type, mainly found directly on the agar layer, and (ii) an aberrant type, identified as aerial hyphae growing into the air and not contacting the agar. The diameter of the aerial hyphal type (1.5 µm) was about half that of the normal hyphal type (3 µm) and showed hydrophobic properties, as evidenced by observing the behavior of small droplets of water added onto the fungal mat. These aerial hyphae were also observed on top of the G soil that was colonized by the fungus. The normal hyphae were probably present between the soil particles; however, microscopic analyses of these were impossible, given the fact that the hyphal structure in soil is destroyed during sample preparation.

Microscopic analyses of the migration of B. terrae (BS001) via Lyophyllum sp. strain Karsten hyphae on water agar showed the existence of a biofilm formed by bacterial cells on the growing hyphae (Fig. 6). The migration followed a similar pattern as in soil microcosms in that, as determined by plating and microscopic analyses, bacterial cells were always found on the extending hyphal tips, even at distances about 50 mm from the initial introduction spot. Controls without Lyophyllum sp. strain Karsten did not show any migration of B. terrae BS001 point inoculated on water agar. Biofilm formation was shown to occur only on the thicker (normal) Lyophyllum sp. strain Karsten hyphae as the aerial hyphae remained free of any visual bacterial colonization (Fig. 6).


Figure 6
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  		FIG. 6. Microscopic analyses of fungal hyphae with biofilm formation. (A) Inoculated with BS001. (B) Not inoculated. The arrow indicates biofilm aerial hyphae.


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DISCUSSION
 
Selection of particular soil bacteria by hyphae (forming the mycosphere) of soil fungi can encompass a range of mechanisms. For instance, the ability of bacteria to utilize particular substances secreted by the fungal host (10, 14, 28) can play a role as can bacterial activity to capture a carbon source directly from fungal hyphae via local cell wall degradation (8). Although not extensively studied in this paper, selective pressure by the fungus on the soil bacterial community can also be mediated by the provision of antibiotic substances in the mycosphere (7). Kohlmeier et al. (16) showed the migration of bacteria on fungal hyphae via intrinsic motility, and they propose the use of the so-called fungal highways to introduce pollutant-degrading bacteria into contaminated soil systems. We hypothesized that migration via hyphal structures in the soil can be used by bacteria to reach and colonize new sites in soil and thus be successful in the colonization of such newly emerging niches in soil. We thus set up experiments in sterile soil microcosms in order to study the selection, migration, and interaction of bacteria in the mycosphere of the soil fungus Lyophyllum sp. strain Karsten (DSM2979).

The microcosm systems were designed to contain sterilized soil as this opens opportunities to investigate bacterium-fungus interactions in bipartite or otherwise simple communities without interference from the complexity of the native soil inhabitants. Indeed, colonization of unsterilized G soil by the fungus used, Lyophyllum sp. strain Karsten, was not observed, and this may be attributed to the grossly fungistatic character of the native G soil, possibly linked to suppression of incoming fungal hyphae by the native microbiota that may have broad antagonistic properties against invading organisms (17).

The experiments in which soil bacterial communities were interrogated for their capacity to comigrate with Lyophyllum sp. strain Karsten through G soil revealed a strong reduction of migratory bacterial diversity, both at the culturable as well as at the total bacterial community levels. This indicates that a strong selective force was exerted on the bacterial community for migratory fitness (Table 1 and Fig. 2). The selection was clearly exerted by conditions provided by the growing fungus Lyophyllum sp. strain Karsten, as in the relevant nonfungal controls no bacterial migration or growth was ever observed. The selection was quite reproducible, since in an experiment executed with a G soil microbial community sampled 3 years later, two identical bacterial species were found in abundance after migration, i.e., B. terrae group II and D. japonica group IV types. The rise and fall of particular bacterial phylotypes over time, as seen in the PCR-DGGE profiles in this experiment, indicated the occurrence of competition between the different types, resulting in shifting balances between these over time. Hence, a glimpse of shifting fitness maxima across time, encompassing several bacterial species in the mycosphere, was given. In these fluctuating communities, B. terrae BS001 and D. japonica BS013 likely play important roles, given the consistency with which they were detected.

Migration of bacterial cells with Lyophyllum sp. strain Karsten hyphae through soil was not a commonality for all bacteria found to migrate in the initial selection experiments. In fact, an important part (25%) of the organisms that could migrate as part of the community could not do so when tested as single strains. The property to migrate as a single strain was thus found in only 4 of the 10 bacterial groups, i.e., D. japonica BS021 (group I), B. terrae BS001 (group II), D. japonica BS013 (group III), and R. basilensis BS017 (group VIII) (Table 1). Strikingly, these were the groups that contained most individuals (75%) and, hence, were most strongly (migration and growth) selected. By implication, the other, less abundant migrators (denoted community migrators) may have profited from the single-strain migrators by means of a postulated migration helper effect. This migration helper effect is defined by the possibility that community migrators benefit from single-strain migrators and are able to migrate, as outlined elsewhere (Warmink et al., unpublished data).

The capacity to actively migrate—and grow—with growing fungal hyphae through soil may confer a specific fitness asset that has emerged in particular soil bacteria, allowing them to avidly respond to a growing mycosphere and find their preferred ecological niche in it. This asset may actually be complex and involve capacities to adhere to and grow and migrate in the mycosphere. Novel ecological niches are presumably constantly created at growing hyphal tips, and bacteria that possess the migrational fitness trait can be highly competitive versus the other bacteria that are locally present. The colonization efficiency of bacteria with this presumed migrational fitness trait was, however, not similar between strains. This is shown (Fig. 4) for B. terrae BS001, which under similar circumstances was more successful and reached higher cell numbers than D. japonica BS021 in G soil microcosms.

The correlation between the presumptive presence of an active TTSS and the capacity to migrate as a single strain with hyphae of Lyophyllum sp. strain Karsten was striking. TTSSs may be involved in the active attachment of bacteria, coupled or not to an effect on the fungal cell wall to the extent that fungal cells are weakened, biochemical pathways are changed, and enhanced nutrients may become available in a way beneficial for the bacteria. Such mechanisms may have played a role in the observed migrational fitness. The hypothesis of active involvement of a TTSS is corroborated by the finding of the TTSS-negative D. japonica BS013 (group IV), which was [based on (GTG)5 fingerprinting and 16S rRNA gene sequence] similar to the single-strain migrator D. japonica BS021 (group I) but did not show the ability to migrate as a single strain (Table 1). In a previous study (28), we observed the selection of TTSS-containing bacteria under the influence of L. proxima, the ectomycorrhizal fungus related to Lyophyllum sp. strain Karsten, although a specific function of the TTSS was not indicated. Interestingly, one TTSS-positive species obtained from that study, B. terrae BS110, was similar to one of the most frequently selected organisms, the group II B. terrae BS001 in the current microcosm studies. Selection by migration may thus have played a role in the selective process that took place at the fruiting bodies of L. proxima in natural G soil (28). In this previous study (28), the prevalence of TTSSs in bulk soil communities (used for inoculation in this study) was found to be low (<3% of the culturable bacterial community). This low prevalence of TTSSs, together with the finding that all single-migrating strains are TTSS positive, provided evidence supporting a role of the TTSS in bacterial migration. Assessment of the behavior of a TTSS mutant versus the wild-type would provide much more compelling evidence for this contention. However, our attempts to produce such a mutant via integration of a selectable marker into the TTSS gene cluster were, unfortunately, not successful (data not shown).

Migration of bacteria via fungal hyphae has been shown previously (16). The authors showed the occurrence of migration via intrinsic bacterial motility through continuous liquid films surrounding the fungal hyphae. Our results indicate a second mechanism for migration via the fungal hyphae. We could not find any proof for bacterial migration via intrinsic motility in the water film surrounding fungal hyphae as the only factor for migration in our soil microcosm. In 11 non-single migrators, motility and also survival in mycosphere soil were observed (Table 1). Based on "the intrinsic motility migration hypothesis," these strains should be able to migrate. The lack of migration of all strains in the direction opposite to the Lyophyllum sp. strain Karsten hyphal growth direction also indicates a more complex mechanism in the migratory process than just the use of intrinsic motility for migration in soil via hyphal-bound water films.

We propose that bacterial biofilm formation, as observed in the cocultures of B. terrae BS001 and Lyophyllum sp. strain Karsten on water agar (Fig. 6), plays a key role in the migratory behavior of the fungus-responsive bacteria. The single-strain migrator bacteria can possibly move with the fungus by growing, as well as by using (twitching) motility, in association with the biologically active and extending hyphal tip (3) and in this way produce a biofilm, which stays behind on old hyphae but also moves/extends with newly emerging hyphae. This hypothesis was supported by the finding of a rather even abundance of bacterial cells on young and older Lyophyllum sp. strain Karsten hyphae in the migration study (Fig. 4), a potentially higher growth rate on the hyphal tip than on older hyphae (Fig. 5), and the lack of migration in the direction opposed to that of the growing hyphae (Table 1).

Moreover, intrinsic flagellar motility may be involved in the bacterial colonization of hyphal tips before attachment, given the fact that all bacteria with migration capacity showed flagellar motility (Table 1). Concerning hyphal differentiation, aerial hyphae of Lyophyllum sp. strain Karsten did not show biofilm formation with B. terrae BS001, as evidenced by microscopy. Aerial hyphae are known to produce hydrophobins in their walls and in this way gain hydrophobicity in order to escape from wet surfaces (30). It is known that most bacteria do not attach well to hydrophobic surfaces (16).

The formation of bacterial biofilms in contact with fungal hyphae has been observed previously (11, 13). However, our study indicates a relationship between the formation of bacterial agglomerates (here denoted as biofilms) at hyphal tips and the concomitant introduction of bacterial cells to novel niches by growing hyphal tips in soil. This is a novel finding that opens doors for direct studies on bacterium-fungus interactions and biofilm formation in soil, possibly extending into the natural soil environment.

The substrate utilization assays, in particular of the single-strain migrator B. terrae BS001 (Fig. 3), indicated an important role for particular compounds (some of them implied as components of fungal exudates) in the functioning of this strain in the mycosphere. Fifteen of 18 compounds that have been found in fungal exudates were utilized by strain BS001. However, our assumptions as to the release of such carbon sources by Lyophyllum sp. strain Karsten are based on knowledge of such releases from other (related) fungi (28). The utilization of this wide array of compounds supports our hypothesis of a passive or even active (using TTSS) capture of resources from Lyophyllum sp. strain Karsten by B. terrae (BS001). The clustering of this organism within the PCA-determined universal fungiphile cluster (Fig. 3) may indicate that it is probably successful in other mycospheres as well. On the other hand, the D. japonica isolates BS003, BS013, and BS021 did not cluster within the fungiphile cluster, in spite of the fact that these showed utilization of seven potentially fungus-released compounds. The issue of allocation of particular strains to fungus-interactive groups clearly requires further study; however, utilization of these seven compounds may be pinpointed as important in the selection of these strains.

In conclusion, a very restricted subset of a soil bacterial community was found to be able to comigrate as single strains with hyphae of Lyophyllum sp. strain Karsten through sterile G soil and presumably utilized a complex array of characteristics allowing this migration, which may have included a TTSS. This complex array of mechanisms is possibly not widespread in the bacterial community of bulk G soil, as members of only three bacterial species from among the total complexity, i.e., B. terrae, D. japonica, and R. basilensis, were found to possess this property, resulting in positive selection. These were detected after inoculation with the whole bacterial community and showed single-strain migration. For one strain, B. terrae BS001, indications were found for biofilm formation in the mycosphere and for the utilization of particular fungus-released compounds as potential driving forces behind successful comigration with Lyophyllum sp. strain Karsten hyphae.


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ACKNOWLEDGMENTS
 
We thank Wieger Rupert, Durk Strooisma, and Albert Ellens for their assistance in some practical parts of this study.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbial Ecology, Centre for Ecological and Evolutionary Studies, University of Groningen, Kerklaan 30, 9750RA Haren, The Netherlands. Phone: 31503632151. Fax: 31503632154. E-mail: j.d.van.elsas{at}rug.nl Back

{triangledown} Published ahead of print on 13 March 2009. Back


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Applied and Environmental Microbiology, May 2009, p. 2820-2830, Vol. 75, No. 9
0099-2240/09/$08.00+0     doi:10.1128/AEM.02110-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.




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