J.A. Warmink

Mechanisms that promote bacterial fitness in fungal‐affected soil microhabitats

Soil represents a very heterogeneous environment for its microbiota. Among the soil inhabitants, bacteria and fungi are important organisms as they are involved in key biogeochemical cycling processes. A main energy source driving the system is formed by plants through the provision of plant-fixed (reduced) carbon to the soil, whereas soil nitrogen and phosphorus may move from the soil back to the plant. The carbonaceous compounds released form the key energy and nutrient sources for the soil microbiota. In the grossly carbon-limited soil, the emergence of plant roots and the formation of their associated mycorrhizae thus create nutritional hot spots for soil-dwelling bacteria. As there is natural (fitness) selection on bacteria in the soil, those bacteria that are best able to benefit from the hot spots have probably been selected. The purpose of this review is to examine the interactions of bacteria with soil fungi in these hot spots and to highlight the key mechanisms involved in the selection of fungal-responsive bacteria. Salient bacterial mechanisms that are involved in these interactions have emerged from this examination. Thus, the efficient acquisition for specific released nutrients, the presence of type-III secretion systems and the capacity of flagellar movement and to form a biofilm are pinpointed as key aspects of bacterial life in the mycosphere. The possible involvement of functions present on plasmid-borne genes is also interrogated.

Selection of bacterial populations in the mycosphere of Laccaria proxima: is type III secretion involved?

The bacterial communities in the Laccaria proxima mycosphere (soil from beneath fruiting bodies) and the corresponding bulk soil were compared by cultivation-dependent and cultivation-independent methods. To assess the distribution of type III secretion systems (TTSS), a PCR-based system for the broad detection of a highly conserved gene involved in TTSS, that is hrcR, was developed and used to assay the cultured bacteria from the L. proxima mycosphere and surrounding bulk soil. PCR-DGGE based on the 16S ribosomal RNA gene showed the selection of presumably mycosphere-specific bacterial groups in the mycosphere of L. proxima compared to the bulk soil in 3 sampling years. Moreover, plate counts revealed that the numbers of culturable heterotrophic bacteria were increased in the mycosphere as compared to the bulk soil. Strikingly, the percentage of randomly picked isolates that carried the hrcR gene showed a significant increase, from 2.8 in the bulk to 13.4 in the mycosphere soil. The increase could be mainly attributed to the emergence of a hrcR positive Pseudomonas fluorescens, denoted BS053, which constituted the most dominant species in the culturable mycosphere communities. This organism was, together with a hrcR-positive Burkholderia terrae BS110, exclusively found in mycosphere soil. Direct detection of hrcR genes using a cultivation-independent approach showed the selection of several hrcR gene types uniquely in the mycosphere, indicating the selection of several TTSS-harboring bacterial species. Thus, different bacteria were found to be enriched in the L. proxima mycosphere and TTSS can be involved in some of the interactions with the fungal host.

Universal and species-specific bacterial 'fungiphiles' in the mycospheres of different basidiomycetous fungi.

In previous work, several bacterial groups that show a response to fruiting bodies (the mycosphere) of the ectomycorrhizal fungus Laccaria proxima were identified. We here extend this work to a broader range of fungal fruiting bodies sampled at two occasions. PCR-DGGE analyses showed clear effects of the mycosphere of diverse fungi on the total bacterial and Pseudomonas communities in comparison with those in the corresponding bulk soil. The diversities of the Pseudomonas communities increased dramatically in most of the mycospheres tested, which contrasted with a decrease of the diversity of the total bacterial communities in these habitats. The data also indicated the existence of universal (i.e. Pseudomonas poae, P. lini, P. umsongensis, P. corrugata, P. antarctica and Rahnella aquatilis) as well as specific (i.e. P. viridiflava and candidatus Xiphinematobacter americani) fungiphiles, defined as bacteria adapted to the mycospheres of, respectively, three or more or just one fungal species. The selection of such fungiphiles was shown to be strongly related to their capacities to use particular carbonaceous compounds, as evidenced using principal components analyses of BIOLOG-based substrate utilization tests. The differentiating compounds, i.e. L-arabinose, L-leucine, m-inositol, m-arabitol, D-mannitol and D-trehalose, were tentatively linked to compounds known to occur in mycosphere exudates.

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

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.

Selection of Sphingomonadaceae at the Base of Laccaria proxima and Russula exalbicans Fruiting Bodies

ABSTRACT The dense hyphal network directly underneath the fruiting bodies of ectomycorrhizal fungi might exert strong influences on the bacterial community of soil. Such fruiting bodies might serve as hot spots for bacterial activity, for instance by providing nutrients and colonization sites in soil. Here, we assessed the putative selection of specific members of the Sphingomonadaceae family at the bases of the fruiting bodies of the ectomycorrhizal fungi Laccaria proxima and Russula exalbicans in comparison to the adjacent bulk soil. To do so, we used a previously designed Sphingomonadaceae-specific PCR-denaturing gradient gel electrophoresis (DGGE) system and complemented this with analyses of sequences from a Sphingomonadaceae-specific clone library. The analyses showed clear selective effects of the fruiting bodies of both fungi on the Sphingomonadaceae community structures. The effect was especially prevalent with R. exalbicans. Strikingly, similar fungi sampled approximately 100 m apart showed similar DGGE patterns, while corresponding bulk soil-derived patterns differed from each other. However, the mycospheres of L. proxima and R. exalbicans still revealed divergent community structures, indicating that different fungi select for different members of the Sphingomonadaceae family. Excision of specific bands from the DGGE patterns, as well as analyses of the clone libraries generated from both habitats, revealed fruiting body-specific Sphingomonadaceae types. It further showed that major groups from the mycospheres of R. exalbicans and L. proxima did not cluster with known bacteria from the database, indicating new groups within the family of Sphingomonadaceae present in these environments.

The type three secretion system facilitates migration of Burkholderia terrae BS001 in the mycosphere of two soil-borne fungi

The type three secretion system (T3SS) is known to play a critical role in several bacterial-eukaryotic cell interactions. Recent indirect evidence has also pointed to a role of this system in bacterial-fungal interactions in soil. In the current study, we examine if the T3SS of the fungal-interactive Burkholderia terrae strain BS001 can aid in the interaction of this bacterium with two soil fungi, i.e., Lyophyllum sp. strain Karsten and Trichoderma asperellum 302. We first analyzed the T3SS of strain BS001 and then constructed a knockout mutant of the essential sctD gene. The selected sctD mutant strain did not show any differences to the wild-type strain with respect to its growth and nutrient utilization behavior, excluding polar effects of the mutation. Then, the migration ability of the sctD mutant strain along with the hyphae of Lyophyllum sp. strain Karsten growing through presterilized soil was tested, revealing hampered comigration as compared to the wild-type strain. The effect was also observed with T. asperellum 302. However, the migration impairment was only noticed in mixed-inoculation experiments, whereas it remained unnoticed when the two strains were inoculated in separate. These data demonstrate that the T3SS assists B. terrae BS001 in its interaction with two soil fungi, without being essential for these interactions. As far as we know, this is the first time that the role of a T3SS in the comigration of bacteria along with soil-exploring fungi is verified directly.