Diversity and Plant Growth-Promoting Potential of (un)culturable Bacteria in the Hedera Helix Phylloplane

doi:10.21203/rs.3.rs-52944/v1

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Diversity and Plant Growth-Promoting Potential of (un)culturable Bacteria in the Hedera Helix Phylloplane

https://doi.org/10.21203/rs.3.rs-52944/v1

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published 27 Feb, 2021

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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 five 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) profile 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 significant 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.

General Microbiology

Hedera helix

phylloplane diversity

plant growth promotion

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 (flowers) 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, flora, meso- and macrofauna, temperature, ultraviolet light exposure, pollution, fertilization and water limitations [4]. The community composition has also a constant component, influenced 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 identified 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 identified 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 five different growth media solidified 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.

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 to taxonomically identify the phylloplane 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 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 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 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 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. 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 classified within these four major phyla. This could be expected, given the taxonomic structure of the H. helix phylloplane and the fact that the vast majority of cultured bacteria are affiliated with these phyla [25]. All selected growth media more or less equally favored Actinobacteria and Gammaproteobacteria, with average relative abundances of 19.0% and 34.7%, respectively. Bacteroides 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 50 most abundant ASVs in the uncultured bacterial phylloplane samples and their phylogenetic relationship are shown in FIGURE 3. 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 media, expressed in colony-forming units (CFU) per gram of fresh leaf material, is shown in FIGURE 4B. 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 are most varied (rich) with regard to nutrients thereby making it easier for r-selected species 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 1-aminocyclopropane-1-carboxylic 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 4A. 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 [26]. The volatile phytohormone acetoin was already shown to promote growth and induce systemic resistance in Arabidopsis thaliana [27, 28], and ACC deaminase reduces ethylene levels, which is related to plant growth promotion [29]. 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 4C). 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. Most isolated Curtobacterium and Methylobacterium species 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 [2].

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 [30] and abundant occurrence in extreme environments, characterized by acidic/alkaline pH, low or high temperatures, salinity and radiation, and low levels of moisture and nutrients [31]. For example, Frigoribacterium and Glaciibacter are typical psychrophilic genera containing a rare group of B-type peptidoglycan [32, 33], and also Frondihabitans species are well-adapted to colder and ultraviolet light-exposed environments such as the phylloplane [34].

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)” [35]. A further pangenomic study including a comparison with all publicly available genomes to understand which properties are specific to the H. helix phylloplane is on its way and planned to be further elaborated on in the future.

In this study, we cultured bacteria from the phylloplane of H. helix using five different growth media (LB, LB01, YEx, YMA and YFlour), and compared the results with the uncultured bacterial phylloplane. The uncultured phylloplane samples contained the highest intra-sample diversity, and inter-sample bacterial diversity shifts from media most rich in nutrients to the more selective growth media. The majority of ASVs of uncultured phylloplane samples could be taxonomically classified within the four major phyla Proteobacteria, Actinobacteria, Bacteroidetes and Firmicutes, which furthermore were fully represented within growth medium samples. The PGP profile that we obtained by testing 200 isolates can help to select candidates with advantageous traits within microbe-assisted approaches, such as improved biomass production, plant protection or phytoremediation. Our isolation efforts also resulted in a significant collection of bacterial strains underrepresented in public databases, mostly from the phylum Actinobacteria, which are currently under further investigation in a pangenomic approach aiming to expand our understanding of bacteria living in the phylloplane.

Abbreviations

ACC 1-aminocyclopropane-1-carboxylic acid

ASV amplicon sequence variant

CFU colony-forming unit

IAA indole-3-acetic acid

PGP plant growth promotion

Collection and preparation of phylloplane samples

Leaves (n = 80) of H. helix growing in the wild were collected at four sites around Hasselt, Belgium (coordinates in WGS84 format: 50.936546, 5.317226; 50.928680, 5.332674; 50.940104, 5.438675; 50.921694, 5.433951). Permission for sampling was obtained by local legislation and performed in accordance with institutional and international guidelines. Plant leaves were identified as specimens belonging to H. helix by Vincent Stevens; voucher specimens are available from Hasselt University. Leaves were cut from the plants at shoulder height using sterile forceps, put in sterile tubes (five leaves per tube) filled with autoclaved phosphate buffer (50 mM Na₂HPO₄∙7H₂O, 50 mM NaH₂PO₄∙H₂O, 0.8 mM Tween 80, pH 7.0) and immediately transferred to the laboratory. Leaf weight was determined gravimetrically and microbial cells were detached from the leaf surface by sonication (100 W, 42 kHz, 3 min), followed by shaking on an orbital shaker (240 rpm, 30 min). Next, leaf wash suspensions were centrifuged (4000 rpm, 15 min) and the resuspended pellets were randomly pooled into four samples. For each sample, an aliquot was immediately stored at -80 °C until DNA isolation; another aliquot was stored overnight at 4 °C for culturing of phylloplane bacteria.

Amplicon metagenomics of the bacterial phylloplane

Leaf wash suspensions were centrifuged (13200 rpm, 20 min, 4 °C) and genomic DNA was isolated using the NucleoSpin Soil kit (Macherey-Nagel, Düren, Germany). The V3–V4 hypervariable region of the bacterial 16S rRNA gene was PCR-amplified using 341F (5’-CCTACGGGNGGCWGCAG-3’) and 785R (5’-GACTACHVGGGTATCTAATCC-3’) primers with attached GS FLX Titanium adaptors, the sequencing key TCAG, and a sample-specific multiplex identifier. PCR products were purified by gel electrophoresis (1.5% agarose gel, 90 V, 45 min) and the 514 bp bacterial amplicon was excised and further purified using the UltraClean GelSpin DNA extraction kit (Mo Bio Laboratories, Carlsbad, CA, USA). Samples were brought to an equimolar concentration (10¹⁰ molecules µL^− 1) using the Quant-iT PicoGreen dsDNA assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Correct amplicon size and integrity were checked on an Agilent 2100 Bioanalyzer system (Agilent Technologies, Santa Clara, CA, USA), followed by sequencing on a Genome Sequencer FLX system (Roche Applied Science, Penzberg, Germany) with GS FLX Titanium series reagents by Macrogen Europe (Amsterdam, The Netherlands).

High-throughput characterization of cultured phylloplane bacteria

Leaf wash suspension aliquots were pooled and inoculated on 120 × 120 mm square Petri dishes containing LB [20], LB01 (1/10 dilution of LB), YMA [21], YFlour [22] or YEx (this study) and incubated at 30 °C (10 replicates per growth medium). Gellan gum was used as solidifying agent and phosphate-containing components were separately autoclaved to prevent the formation of growth-inhibiting molecules such as H₂O₂ [19]. The composition of the growth media is summarized in Table 1. Four weeks after inoculation, the number of CFU per gram of fresh leaf material on the growth media was determined on eight replicates per growth medium and subsequently all present colonies were suspended in autoclaved 10 mM MgSO₄ followed by centrifugation (4000 rpm, 15 min). Resuspended pellets were pooled into four samples per growth medium. Pellets were immediately stored at -80 °C until DNA isolation. Similar as described previously, genomic DNA was isolated and the V3–V4 hypervariable regions of the bacterial 16S rRNA genes were PCR-amplified, purified and prepared for sequencing.

Table 1

**Composition of the growth media.** Products are given in g L^− 1 distilled water. Products marked in grey were filter-sterilized before being added to the other autoclaved components. Phosphate-containing components (underlined) were separately autoclaved. All growth media have pH 7.0.
	LB [20]	LB01 (1/10 dilution of LB)	YEx (this study)	YMA [21]	YFlour [22]
CaCO₃					0.3
CoCl₂			1.9 × 10^− 4
CuCl₂			1.7 × 10^− 5
Fe(III)NH₄ citrate			4.8 × 10^− 3
H₃BO₃			6.2 × 10^− 5
K₂HPO₄				0.1
KH₂PO₄				0.4
MgSO₄ ∙ 7H₂O	1	0.1	1	1	1
MnCl₂			1 × 10^− 4
Na₂HPO₄ ∙ 2H₂O			0.04
NaCl	10	1	0.1	0.1
NaMoO₄			3.6 × 10^− 5
NiCl₂			2.4 × 10^− 5
Tris			6.1
ZnSO₄ ∙ 7H₂O			1.4 × 10^− 4
D-Fructose			0.5
D-Glucose			0.5
Gluconate			0.7
Lactate			0.4
Mannitol				10
Succinate			0.8
Sucrose					0.3
Plain flour					6
Tryptone	10	1
Yeast extract	5	0.5	0.4	0.4	0.3
Gellan gum	7.5	7.5	7.5	7.5	7.5
ADDITIONAL FIGURE A1 Rarefaction analysis of all samples. Rarefaction plots indicating the average number of amplicon sequence variants (ASVs) for uncultured bacterial phylloplane samples (A; n = 4) and growth medium samples (B; n = 20).

Evaluation of plant growth promotion potential

Bacterial phylloplane colonies were randomly picked from two replicates per growth medium. Isolated colonies (n = 200, 40 per growth medium) were checked for purity by streaking and grown for 24 h in their respective liquid growth medium at 30 °C on a shaker (150 rpm), washed and resuspended in 2 mL autoclaved 10 mM MgSO4 solution to obtain suspensions containing bacteria in mid-exponential phase (OD_{600 nm} = 0.4). Next, 20 µL of this bacterial suspension was used for the detection of IAA production using the Salkowski’s reagent method [36], for the detection of acetoin production using the Voges–Proskauer test [37], and for assessing ACC deaminase activity by monitoring the amount of α-ketobutyrate that was generated by the enzymatic hydrolysis of ACC [38]. Genomic DNA of all isolates was extracted using the MagMAX DNA Multi-Sample Kit (Life Technologies, Carlsbad, CA, USA) and a MagMAX Express-96 Deep Well Magnetic Particle Processor (Life Technologies, Carlsbad, CA, USA). The bacterial 16S rRNA gene was partially PCR-amplified using 27F (5’-AGAGTTTGATCMTGGCTCAG-3’) and 1492R (5’-TACGGYTACCTTGTTACGACTT-3’) primers and 20 µL of the PCR product was used for unidirectional Sanger sequencing using the 27F primer by Macrogen Europe (Amsterdam, The Netherlands).

Processing of sequencing data

Sequencing data were received in FASTQ format with GS FLX Titanium adaptors and the sequencing key TCAG trimmed from all sequences, demultiplexed based on the sample-specific multiplex identifier and further processed with DADA2 1.12.1 [39] for single-end analysis. All reads were quality-filtered by truncation to 340 bp (discarding all reads with less than 340 bp) and subsequently trimming 40 bp from the 5’-side with maxEE = 2 (maximum number of expected errors), resulting in a data set consisting only of high-quality V3–V4 16S rRNA gene sequences of exactly 300 bp with all irrelevant sequence parts removed. For error model learning, dereplication, sample inference and chimera removal default parameter settings were used. Taxonomy was assigned to each resulting ASV with IDTAXA [40] using the RDP 16S rRNA training set v16 [41]. The ASV table with assigned taxonomy was imported into phyloseq 1.28.0 [42] for making phylogenetic bar charts and rarefaction and diversity analysis. Intra-sample diversity was assessed with ASV observations, Shannon diversity and Simpson diversity. Inter-sample diversity was measured with principal coordinates analysis (PCoA) on a Bray–Curtis dissimilarity matrix, and different outcomes were tested using permutational multivariate analysis of variance (PERMANOVA, 999 permutations). Phylogenetic tree construction was done with PhyML 3.1 [43] after alignment of the sequences with MUSCLE 3.8.31 [44]. Differences in CFU between growth media were tested with a Kruskal–Wallis test followed by pairwise comparisons using a Wilcoxon rank-sum test with Benjamini–Hochberg correction. Sanger sequencing data were processed using sangerseqR 1.20.0 and sangeranalyseR 0.1.0 [45], and resulting high-quality sequences were taxonomically assigned with BLAST + 2.9.0 [46] using the RDP 16S rRNA training set v16 [41]. All data handling was done within R 3.6.1 [47].

Ethics approval and consent to participate

Not applicable

Consent for publication

Not applicable

Availability of data and materials

The metagenomic libraries of the bacterial phylloplane communities and sequences of the cultured bacteria on all growth media are available from the Short Read Archive (SRA) of the National Center for Biotechnology Information (NCBI) under project accession number PRJNA626008 and individual FASTQ sample identifiers SAMN14614763–86. All partial 16S rRNA gene sequences obtained by Sanger sequencing are available from NCBI under accession numbers MT360054–253.

Competing interests

The authors declare that they have no competing interests.

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).

Authors’ contributions

VS and ST conceived the study. VS performed the experiments, analysed the data, prepared the figures and wrote the manuscript. ST and JV made suggestions to improve the manuscript. All authors approved the final draft of the manuscript.

Acknowledgements

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.

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Diversity and Plant Growth-Promoting Potential of (un)culturable Bacteria in the Hedera Helix Phylloplane

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Abstract

Figures

Background

Results And Discussion

Characterization of the uncultured bacterial phylloplane and its culturable fraction

Functional and taxonomical characteristics of isolates from the selected growth media

Conclusions

Methods

Collection and preparation of phylloplane samples

Amplicon metagenomics of the bacterial phylloplane

High-throughput characterization of cultured phylloplane bacteria

Evaluation of plant growth promotion potential

Processing of sequencing data

Declarations

Authors’ contributions

Acknowledgements

References

Supplementary Files

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