Diversity and Plant Growth-Promoting Potential of (un)culturable Bacteria in the Hedera Helix Phylloplane Vincent Stevens ( [email protected] ) Universiteit Hasselt https://orcid.org/0000-0002-3725-1597 Soe Thijs Universiteit Hasselt Jaco Vangronsveld Universiteit Hasselt Research article Keywords: Hedera helix, phylloplane diversity, plant growth promotion Posted Date: August 10th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-52944/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published on February 27th, 2021. See the published version at https://doi.org/10.1186/s12866-021-02119-z. Page 1/19 Abstract Background An abundant and diverse community of microorganisms naturally exists on the phylloplane, the surface of leaves. It is one of the most prevalent microbial habitats on earth and bacteria are by far the most abundant members, living in a community that is highly dynamic. To increase our knowledge about the diversity and function of microbial communities living in the phylloplane, culture-dependent and - independent approaches help us a great deal. Results Here we isolated bacteria from the phylloplane of Hedera helix (common ivy), a widespread evergreen, using ve different growth media. We further included a comparison with the uncultured phylloplane, which we show to contain the highest intra-sample diversity. Inter-sample bacterial diversity shifts from growth media most rich in nutrients to those which are more selective. The four major phyla Proteobacteria, Actinobacteria, Bacteroidetes and Firmicutes comprised the vast majority of phyla in the uncultured H. helix phylloplane, which furthermore were fully represented within growth medium samples. The plant growth promotion (PGP) prole we obtained by testing 200 isolates can help to select candidates with advantageous traits within various microbe-assisted approaches. Our isolation effort also resulted in a signicant collection of bacterial strains underrepresented in public databases, mostly from the phylum Actinobacteria. Conclusions This study contributes as a case study of bacterial culturability and its relation with functional characteristics such as PGP potential which also is an important step towards understanding the ecological and functional role of microbial members living in the H. helix phylloplane. Background Interactions between plant-associated microorganisms and their host are being extensively studied. Increased knowledge of plant–microbe interactions enables a better understanding of their role during plant growth and development [1] and the translation into improved biomass production and microbe- assisted phytotechnologies [2]. An abundant and diverse community of microorganisms naturally exists on the surface of above-ground parts of plants, also known as the phyllosphere. The phyllosphere can be further subdivided into the caulosphere (stems), phylloplane (leaves), anthosphere (owers) and carposphere (fruits). It is one of the most prevalent microbial habitats on earth and bacteria are by far the most abundant and constant members, with a typical cell density of 106-107 cells cm− 2 [3, 4]. The composition of this community is dynamic, responding to environmental factors, both biotic and abiotic, such as leaf age and the co-presence of other microorganisms, ora, meso- and macrofauna, temperature, ultraviolet light exposure, pollution, fertilization and water limitations [4]. The community Page 2/19 composition has also a constant component, inuenced by stable factors such as host plant genotype [5, 6] and geographical location [7–9]. Hedera helix (common ivy), an evergreen plant known for its hardiness and wall/tree climbing ability [10], has a widespread occurrence in natural and urban environments and constitutes an excellent model for studying plant–microbe interactions in the phylloplane. To enhance our understanding about the diversity and function of microbial communities living in the phylloplane, culture-independent approaches are very useful. However, most microbes identied in this way remain uncultured [11]. An extensive meta-analysis covering 347 experiments in 26 studies of samples from lakes, rivers, drinking water, seawater, marine and terrestrial subsurfaces, animal hosts and soils found that a median of 0.5% of bacterial cells identied in direct counts could be grown in culture using standard techniques [12]. This shows that the “great plate count anomaly”, formulated decades ago and which states that less than 1% of environmental microbial cells are culturable with standard methods [13], still holds true today. It is reasonable to hypothesize that some members of these not-yet- cultured taxa are key ecological players, and it remains indispensable to have microorganisms cultured under laboratory conditions to further investigate, validate and exploit their functional potential. Therefore, one of the challenges for microbiologists today remains to develop strategies to “culture the uncultured”. In recent years, some effective strategies have been developed to isolate hard-to-culture microbes utilizing optical tweezers and laser microdissection, microbioreactors and diffusion chambers [14]. However, simpler and cheaper alternatives for retrieving a wide diversity of bacteria remain effective, such as the choice of a different solidifying agent [15], longer incubation periods, growth in low nutrient media [16, 17], addition of host (plant) extracts [18], and separate preparation of media components [19]. Once a collection of bacterial isolates is obtained and maintained in the laboratory, their functional characteristics such as plant growth promotion (PGP) potential can be evaluated including the biosynthesis of PGP hormones and enzymatic activities that can interfere with the plant stress status. Here we report the isolation of bacteria from the phylloplane of H. helix using ve different growth media solidied with gellan gum after an incubation period of four weeks, including a comparison with the uncultured bacterial phylloplane. LB [20], LB01 (1/10 dilution of LB), YMA [21], YFlour [22] and YEx were selected to cover a wide variety of bacterial taxa. For each growth medium, representative bacterial colonies were picked and their PGP potential was evaluated. This study contributes as a case study of bacterial culturability and its relation with functional characteristics such as PGP potential which also is an important step towards a better understanding of the ecological and functional role of microbial members living in the H. helix phylloplane. Results And Discussion Characterization of the uncultured bacterial phylloplane and its culturable fraction Page 3/19 Phylloplane samples from H. helix plants were analysed in a culture-dependent and -independent way. Amplicon metagenomics to taxonomically identify the phylloplane of H. helix and high-throughput characterization of the culturable fraction using ve different growth media resulted in a total of 177,872 high-quality 300 bp V3–V4 16S rRNA gene sequences, representing 1,482 amplicon sequence variants (ASVs). Bacterial intra-sample diversity was estimated by rarefaction analysis (FIGURE A1) and by calculating three alpha diversity indices: (i) the observed number of ASVs, (ii) Shannon’s diversity index and (iii) Simpson’s diversity index (FIGURE 1B). The uncultured phylloplane samples contained the highest intra-sample diversity, and while diversity in LB01 and YMA was higher compared to LB, YEx and YFlour, diversity in all growth medium samples was low relative to uncultured phylloplane samples. This observation relates to a key concept: only a fraction of environmental microbial cells are culturable with standard methods, also known as the “great plate count anomaly” [13] To infer bacterial inter-sample diversity, we employed PCoA on a Bray–Curtis dissimilarity matrix (FIGURE 1A). Statistical analysis revealed that the choice of growth medium is a signicantly contributing diversity-determining factor (R2 = 0.2925, p < 0.001). Moreover, visual examination of the PCoA plot shows that inter-sample bacterial diversity shifts when considering LB and to a lesser extent LB01, which are most varied (rich) with regard to nutrients, to the other growth media which are more selective. Bacterial diversity of the uncultured phylloplane appears to be different from the cultured bacterial diversity; these differences are further illustrated in FIGURE 2. At the phylum level (FIGURE 2A), for uncultured phylloplane samples on average 90.7% of ASVs could be taxonomically classied within the four major phyla with following relative abundances: Proteobacteria (51.1%; subdivided as 30.9% Alphaproteobacteria, 14.9% Gammaproteobacteria and 5.9% Betaproteobacteria), Actinobacteria (15.5%), Bacteroidetes (19.2%) and Firmicutes (4.9%). 5.6% of ASVs was classied within 12 other phyla (Acidobacteria, Armatimonadetes, Chlamydiae, Cyanobacteria, Deinococcus–Thermus, Fusobacteria, Gemmatimonadetes, Nitrospirae, Planctomycetes, Saccharibacteria, Verrucomicrobia and candidate phylum WPS-1) and the remaining 3.7% could not be classied at phylum level. This predominance at phylum level of Proteobacteria (with classes Alphaproteobacteria and Gammaproteobacteria in particular), Actinobacteria, Bacteroidetes and Firmicutes is typically observed in the phyllosphere of different plant species [4, 9, 23, 24]. For growth medium samples, 100% of ASVs could be taxonomically classied