Characterization of the uncultured bacterial phylloplane and its culturable fraction
Phylloplane samples from H. helix plants were analysed in a culture-dependent and -independent way. Amplicon metagenomics applied to taxonomically identify the phylloplane bacterial diversity of H. helix and high-throughput characterization of the culturable fraction using five 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 (alpha) 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, as expected.
To infer bacterial inter-sample (beta) diversity, we employed PCoA on a Bray–Curtis dissimilarity matrix (FIGURE 1A). Statistical analysis revealed that the choice of growth medium is a significantly 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 contain the highest amount of resources (especially nitrogen sources), to YMA and YFlour which are more selective and to YEx which is more limited but also most varied in resources. Differences in carbon/nitrogen ratio and carbon sources between the growth media likely contribute to the respective biodiversity and prevailing taxonomic groups observed, as it is the case in other bacterial (culture) systems [20-22].
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 classified 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 classified 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 classified at phylum level. It was previously reported for different plant species, including A. thaliana, L. sativa, G. max, T. repens and O. sativa, that the phyllosphere community mainly comprises bacteria belonging to phylum Proteobacteria (with classes Alphaproteobacteria and Gammaproteobacteria in particular), Actinobacteria, Bacteroidetes and Firmicutes, where members of Proteobacteria constitute ~50% of the community composition [2-8]. Here we show that this holds also true for H. helix, and it further strengthens the finding that the composition of the phyllosphere microbiome on higher taxonomic level is similar across various host plant species.
For growth medium samples, 100% of ASVs could be taxonomically classified within these the phyla Proteobacteria, Actinobacteria, Bacteroidetes and Firmicutes. This could be expected, given the taxonomic structure of the H. helix phylloplane and the general finding that the vast majority of cultured bacteria are affiliated with these phyla . All selected growth media more or less equally favored Actinobacteria and Gammaproteobacteria, with average relative abundances of 19.0% and 34.7%, respectively. Bacteroidetes however were only found on LB01, YEx and YMA. LB and LB01 greatly favored Firmicutes compared to the other growth media with an average relative abundance of 36.1% and 27.1%, respectively, and YFlour was most selective for Betaproteobacteria (20.6%). FIGURE 2B illustrates the relative abundances of the 10 most abundant genera across all growth medium samples and their relation with the uncultured bacterial phylloplane. LB favored the growth of Bacillus and Stenotrophomonas, with average relative abundances of 19.2% and 16.3%, respectively, while YFlour was most selective for Burkholderia (13.9%) and Curtobacterium (14.7%). On the other hand, Rhizobium could be found on all growth media except on LB. Also, in the uncultured phylloplane on average 18.1% of ASVs could not be classified at genus level. For growth medium samples this was 4.7% for LB, 5.4% for LB01, 9.0% for YEx, 11.0% for YFlour and 6.8% for YMA. In other words, most potentially novel bacterial species were cultured on YFlour, while LB and its 1/10 diluted version LB01 had the highest abundance of known bacteria.
The highest proportion of ASVs (76.3%) is unique for the growth media, 21.9% is shared between at least two of the growth media and only 1.8% is shared between all growth media (FIGURE 3). This highlights the importance of the use of varied growth media in the context of capturing most of the bacterial diversity. However, it is important to note that ASV abundance is not considered in this picture. The 50 most abundant ASVs in the uncultured bacterial phylloplane samples and their phylogenetic relationship are shown in FIGURE 4. Culturing was successful with at least one of the selected growth media for 18 of these top 50 ASVs. All top ASVs were classified within the phyla Proteobacteria, Actinobacteria, Bacteroidetes or Firmicutes except one (ASV 49) that was classified as Fusobacterium within the phylum Fusobacteria.
A comparison of the viable count of phylloplane bacteria growing on the growth media, expressed in colony-forming units (CFU) per gram of fresh leaf material, is shown in FIGURE 5B. LB and LB01 comprised a significantly higher number of bacterial colonies compared to YEx, YFlour and YMA (p < 0.05). From the selected growth media, LB and LB01 contain the highest amount of (nitrogen) resources thereby making it easier for r-selected species that are notably emphasized by high growth rates to grow, likely explaining the higher viable count on these growth media.
Functional and taxonomical characteristics of isolates from the selected growth media
Evaluation of PGP potential, determined as indole-3-acetic acid (IAA), 3-hydroxy-2-butanone (acetoin) and 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase production by bacterial isolates cultured with the selected growth media, is shown in FIGURE 5A. IAA is the most common phytohormone of the auxin class, and induces cell elongation and division with all subsequent results for plant growth and development . The volatile phytohormone acetoin was already shown to promote growth and induce systemic resistance in Arabidopsis thaliana [25, 26], and ACC deaminase reduces ethylene levels, which is related to plant growth promotion . In our study regarding phylloplane bacteria, those capable of IAA production were high-abundant on LB and LB01 and nearly absent on YFlour. YEx is characterized by bacterial isolates showing relatively high production of acetoin. Isolates producing ACC deaminase were low-abundant on all growth media. The bacterial 16S rRNA gene of all 200 isolates was partially sequenced and these sequences could be taxonomically assigned to genus level (FIGURE 5C). Most isolates were assigned to the genera Curtobacterium (41) and Methylobacterium (37). Frigoribacterium (16), Bacillus (13), Rathayibacter (11) and Sphingomonas (10) and Pantoea (9) were also common. That one-fifth of cultured bacteria are classified within the genus Curtobacterium may not be surprising, as this genus is ubiquitously reported to be associated within phyllosphere habitats [28-31]. In one comprehensive isolation study comprising 200 leaf samples of soybean and corn plants, Curtobacterium species could be isolated from every sample . Also, previous culture-independent phyllosphere studies paired with isolation have allowed the identification of representative bacterial strains from various genera, including Methylobacterium , Frigoribacterium , Sphingomonas [34, 35] and Pantoea . Most isolated Curtobacterium and Methylobacterium species in this study were able to produce IAA and acetoin, while regarding ACC deaminase such correlations are not that clear. This PGP profile can help to select bacterial isolates with specific PGP traits that can be exploited in microbe-assisted approaches, such as improved biomass production, plant protection or phytoremediation . However, it is important to note that evaluating PGP traits based on in vitro experiments solely has its caveats . For example, it is possible that the production of phytohormones does not occur in the natural plant–microbe partnership or that this production occurs in a pathogenic context . Follow-up in vivo inoculation experiments are necessary to conclusively evaluate PGP potential, but nevertheless in vitro PGP screening remains an important first step.
Our isolation of phylloplane bacteria on a variety of growth media resulted in a significant collection of bacterial strains underrepresented in public databases. Most isolates (104/200) were taxonomically classified within the phylum Actinobacteria, which represented 20 out of a total of 37 genera, including Curtobacterium (41), Frigoribacterium (16), Rathayibacter (11), Glaciibacter (5), Cellulomonas (4), Frondihabitans (4), Microbacterium (4), Nocardioides (3), Cellulosimicrobium (2), Leifsonia (2), Nocardia (2), Sediminihabitans (2), Arthrobacter (1), Brevibacterium (1), Flexivirga (1), Gordonia (1), Herbiconiux (1), Micrococcus (1), Patulibacter (1) and Rhodococcus (1). This is interesting given the fact that Actinobacteria members are well-known for their secondary metabolite production  and abundant occurrence in extreme environments, characterized by acidic/alkaline pH, low or high temperatures, salinity and radiation, and low levels of moisture and resources . For example, Frigoribacterium and Glaciibacter are typical psychrophilic genera containing a rare group of B-type peptidoglycan [41, 42], and also Frondihabitans species are well-adapted to colder and ultraviolet light-exposed environments such as the phylloplane .
In light of the coordinated efforts to expand our understanding about plant-associated bacteria and life in general, several strains from this study were selected for whole-genome sequencing in the framework of the U.S. Department of Energy (DOE) Joint Genome Institute (JGI) project the “Genomic Encyclopedia of Bacteria and Archaea (GEBA)” . A further pangenomic study including a comparison with all publicly available genomes to understand which properties are specific to the phylloplane is ongoing and planned to be further elaborated on in the nearby future.