Diversity and plant growth-promoting potential of (un)culturable bacteria in the Hedera helix phylloplane
Background: A diverse community of microbes naturally exists on the phylloplane, the surface of leaves. It is one of the most prevalent microbial habitats on earth and bacteria are the most abundant members, living in communities that are highly dynamic. Today, one of the key challenges for microbiologists is to develop strategies to culture the vast diversity of microorganisms that have been detected in metagenomic surveys. Results: We isolated bacteria from the phylloplane of Hedera helix (common ivy), a widespread evergreen, using five growth media: Luria–Bertani (LB), LB01, yeast extract–mannitol (YMA), yeast extract–flour (YFlour), and YEx. We also included a comparison with the uncultured phylloplane, which we showed to be dominated by Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes. Inter-sample (beta) diversity shifted from LB and LB01 containing the highest amount of resources to YEx, YMA, and YFlour which are more selective. All growth media equally favoured Actinobacteria and Gammaproteobacteria, whereas Bacteroidetes could only be found on LB01, YEx, and YMA. LB and LB01 favoured Firmicutes and YFlour was most selective for Betaproteobacteria. At the genus level, LB favoured the growth of Bacillus and Stenotrophomonas, while YFlour was most selective for Burkholderia and Curtobacterium. The in vitro plant growth promotion (PGP) profile of 200 isolates obtained in this study indicates that previously uncultured bacteria from the phylloplane may have potential applications in phytoremediation and other plant-based biotechnologies. Conclusions: This study gives first insights into the total bacterial community of the H. helix phylloplane, including an evaluation of its culturability using five different growth media. We further provide a collection of 200 bacterial isolates underrepresented in current databases, including the characterization of PGP profiles. Here we highlight the potential of simple strategies to obtain higher microbial diversity from environmental samples and the use of high-throughput sequencing to guide isolate selection from a variety of growth media. ; Funding This work was supported by the Hasselt University Methusalem project 08M03VGRJ, by the project G0D0916N ("Plant–microbe associations to reduce particulate matter concentration and toxicity in urban areas: a multidisciplinary approach") financed by the Research Foundation – Flanders (FWO) and by a personal grant of VS provided by the Research Foundation – Flanders (FWO). The funding bodies had no role in the design of the study and collection, analysis, interpretation of data, or manuscript preparation. Acknowledgements We thank Prof. Jonathan Van Hamme for proofreading the language in this manuscript. We thank the VSC (Flemish Supercomputer Center), funded by the Research Foundation – Flanders (FWO) and the Flemish Government – department EWI for providing the computational resources and services used in this work.
