Vertical transmission explains the specific Burkholderia pattern in Sphagnum mosses at multi-geographic scale

The betaproteobacterial genus Burkholderia is known for its versatile interactions with its hosts that can range from beneficial to pathogenic. A plant-beneficial-environmental (PBE) Burkholderia cluster was recently separated from the pathogen cluster, yet still little is known about burkholderial diversity, distribution, colonization, and transmission patterns on plants. In our study, we applied a combination of high-throughput molecular and microscopic methods to examine the aforementioned factors for Burkholderia communities associated with Sphagnum mosses – model plants for long-term associations – in Austrian and Russian bogs. Analysis of 16S rRNA gene amplicons libraries revealed that most of the Burkholderia are part of the PBE group, but a minor fraction was closely related to B. glathei and B. andropogonis from the pathogen cluster. Notably, Burkholderia showed highly similar composition patterns for each moss species independent of the geographic region, and Burkholderia-specific fluorescent in situ hybridization of Sphagnum gametophytes exhibited similar colonization patterns in different Sphagnum species at multi-geographic scales. To explain these patterns, we compared the compositions of the surrounding water, gametophyte-, and sporophyte-associated microbiome at genus level and discovered that Burkholderia were present in the Sphagnum sporophyte and gametophyte, but were absent in the flark water. Therefore, Burkholderia is a part of the core microbiome transmitted from the moss sporophyte to the gametophyte. This suggests a vertical transmission of Burkholderia strains, and thus underlines their importance for the plants themselves.


INTRODUCTION
The genus Burkholderia, which was described by Yabuuchi et al. (1992), encompasses a diverse group of Betaproteobacteria with currently more than 60 validly described species. Burkholderia species are known for their beneficial as well as pathogenic interaction with plants, animals, and humans (Coenye and Vandamme, 2003). In the past, most studies focused on the pathogenic species for their enormous clinical importance (Mahenthiralingam et al., 2005). Recently, a specific plantbeneficial-environmental (PBE) Burkholderia cluster that contains non-pathogenic species was divided from the cluster that comprises human, animal and plant pathogens (Caballero-Mellado et al., 2007;Suárez-Moreno et al., 2010. However, there is no clear border between both groups especially within the plant-associated species; for example B. glathei was suggested to be transfered from the pathogenic to the PBE group (Verstraete et al., 2013). Many PBE members belong to Burkholderia species symbiotic to tropical plants; each nodulating plant species is colonized by a single unique endophytic Burkholderia species (Van Oevelen et al., 2002;Lemaire et al., 2011). Several species from the PBE cluster share characteristics that are of use in association with plants, such as quorum sensing systems, the presence of nitrogen fixation and/or nodulation genes, and the ability to degrade aromatic compounds (Suárez-Moreno et al., 2012), and many of them are characterized by an endophytic lifestyle (Sessitsch et al., 2005;Gasser et al., 2009;Mitter et al., 2013). While single strains of the PBE cluster are already well-characterized, little is known about the ecology and colonization pattern of Burkholderia species on plants.
Plants have been recognized as meta-organisms due to their close symbiotic relationship with their microbiome that fulfills important host functions (Berg, 2009;Bulgarelli et al., 2012;Hirsch and Mauchline, 2012;Lundberg et al., 2012;Berg et al., 2013). These advances were driven by both "omic"-technologies guided by next-generation sequencing (NGS) and microscopic insights (Berendsen et al., 2012;Jansson et al., 2012). Mosses belong to the phylogenetically oldest group of land plants on Earth, and their long-term intense relationship with their associated microbes has contributed to the co-evolution of a highly specific microbiome (Opelt and Berg, 2004;Opelt et al., 2007c;Bragina et al., 2012). Therefore, mosses are important models in studying plant-microbe interactions and the ecology of plant-associated bacteria. The genus Sphagnum is among the most abundant and cosmopolitan of bog vegetation in the Northern hemisphere, and greatly contributes to both global carbon turnover and global climate (Raghoebarsing et al., 2005;Dise, 2009). The ecological significance of bogs is directly related to the physical, morphological, and chemical characteristics of Sphagnum peat mosses which set Sphagnum apart from other mosses in practically every stage of the life cycle (Daniels and Eddy, 1985). Burkholderia species play an important role for Sphagnum mosses and peatland ecosystem (Opelt et al., 2007a,b), and new Burkholderia species, which belong to the PBE cluster, have recently been isolated from these mosses (Vandamme et al., 2007). However, their composition and occurrence on Sphagnum at various geographical scales-ranging from the moss gametophyte and sporophyte up to continental level-is not yet understood. We hypothesize that Sphagnum species are colonized by specific Burkholderia from the PBE cluster independent from the geographic region.
To study this hypothesis and understand the ecological role, composition, colonization, as well as distribution pattern on plants, we studied Burkholderia communities on two Sphagnum species (S. magellanicum and S. fallax) associated with different a-biotic parameters from different bogs in Austria and Russia. We used an assortment of methods combining the analysis of Burkholderia-specific 16S rRNA gene pyrosequencing libraries with FISH-CLSM analysis. Furthermore, we compared the compositions of water, gametophyte-, and sporophyte-associated microbiomes to understand the transmission and distribution patterns of the Burkholderia communities.

SAMPLING DESIGN
To analyze the diversity and distribution pattern of the Sphagnum-associated Burkholderia community, S. magellanicum BRID. (section Sphagnum) and S. fallax H. KLINGGR. (section Cuspidata) were selected. Both bryophytes are members of the typical and cosmopolitan vegetation in peat bogs (Daniels and Eddy, 1985). Adult gametophytes of mosses were sampled in three acidic peat bogs in Austria and in three acidic peat bogs in Russia in September 2009 and July 2010, respectively (Table S1). Four single replicates per Sphagnum species (15-20 plantlets) were collected in each of the investigated bogs at a minimum distance of about 40 m from each other. The plant samples were placed into sterile plastic bags and transported to the laboratory. In addition, two sporophyte samples of S. fallax consisting of enclosed spore capsules, and one water sample from a small wet depression (flark) were collected into sterile screw cap tubes and processed separately.

TOTAL-COMMUNITY DNA ISOLATION
The microbial fractions associated with moss gametophytes and sporophytes were extracted as previously described (Bragina et al., 2012). In short, 5 g of plant material were physically disrupted and resuspended in 10 ml of 0.85% NaCl. 2 ml of suspension were centrifuged at 13,000 r.p.m. for 20 min at 4 • C and the supernatant was discarded. For extraction of the sporophyte-associated microbial community, 10 enclosed spore capsules per sample were surface-sterilized and ground with 1.5 ml of 0.85% NaCl. The ground suspension was centrifuged at 13,000 r.p.m. for 20 min at 4 • C and the supernatant was discarded. The pellet from the flark water sample was obtained through several rounds of centrifugation at 10,000 r.p.m. for 15 min at 4 • C until a constant pellet size was obtained. The resulting cell pellets were applied for isolation of the total-community DNA using the FastDNA® SPIN Kit for Soil (MP Biomedicals, Solon, OH, USA). Final aliquots of the total-community DNA were further used for a deep sequencing-approach.

454-PYROSEQUENCING AND BIOINFORMATIC PROCESSING
The diversity of the Sphagnum-associated microbiome with a special focus on the genus Burkholderia was investigated using a barcoded pyrosequencing technology. For this purpose, 16S rDNA amplicons were generated using Taq-&Go™ Ready-to-use PCR Mix (MP Biomedicals, Solon, OH, USA). The totalcommunity DNA of gametophyte samples was selectively amplified with Burkholderia-specific primers BKH143Fw/BKH1434Rw (Schönmann et al., 2009) followed by amplification with universal bacterial primers Unibac-II-515f/Unibac-II-927r (Lieber et al., 2003). In addition, the total-community DNA of S. fallax gametophyte samples from the bog Pürgschachen Moor (Table S1) and flark water sample was amplified with universal bacterial primers Unibac-II-515f/Unibac-II-927r. The totalcommunity DNA of sporophyte samples was amplified with universal bacterial primers 799f/1492r (Lane, 1991;Chelius and Triplett, 2001) because application of the Unibac-II-515f/Unibac-II-927r achieved mostly plant-derived sequences (data of preliminary experiments). Primer sequences are listed in Table 1. Duplicate PCR products from all templates were purified with Wizard® SV Gel and PCR Clean-Up System (Promega, Madison, WI, USA). Amplicons derived from the same Sphagnum sp. and sampling site were pooled in equimolar ratios and subjected to pyrosequencing using the Roche 454 GS FLX and FLX+ Titanium platforms performed by LGC Genomics (Berlin, Germany) and Eurofins MWG (Ebersberg, Germany), respectively. In total, we produced 12 pyrosequencing libraries specific for Burkholderia and four general bacterial pyrosequencing libraries.
The 16S rDNA pyrosequencing libraries were processed using the open source software package Quantitative Insights Into Microbial Ecology (QIIME) version 1.6.0 (Caporaso et al., 2010) with default parameters. The raw datasets were de-multiplexed, the primer sequences were truncated, and the datasets were filtered by removing sequences of low-quality (quality score, <25), short sequences (<200 bp), and sequences containing ambiguous characters and/or homopolymers (>6 bp). The qualityfiltered datasets were de-noised and chimeras were removed if present. Sequences were clustered into operational taxonomic units (OTUs) using the UCLUST algorithm with a 97% similarity cut-off (Schloss and Handelsman, 2006;Edgar, 2010). The most abundant sequence within each OTU was taxonomically assigned using the Ribosomal Database Project (RDP) classifier with 80% confidence threshold (Wang et al., 2007). To refine the analysis, generated OTU-tables were filtered based on taxonomic metadata: OTUs classified to genera other than Burkholderia and OTUs containing chloroplast-derived sequences were removed from the burkholderial and general bacterial OTU-tables, correspondingly. Rarefaction analysis was performed for the complete datasets, while richness and diversity estimations were performed by calculating Chao1 and Shannon (H ) indices for the datasets normalized to the same number of sequences. For the general bacterial datasets, the occurrence of bacterial taxa was analyzed using the normalized datasets. Beta-diversity of the burkholderial datasets was analyzed using weighted UniFrac distance metric (Lozupone et al., 2010) and jackknife re-sampling (1,781 sequences per sample × 100 times). Statistical analysis was performed for the normalized datasets using the adonis test with 999 permutations (http://qiime.org/tutorials/category_comparison.html). Representative sequences of the burkholderial OTUs were aligned with reference sequences from the non-redundant nucleotide database (nt) of the NCBI server using the BLASTN algorithm. A bootstrapped neighbor-joining phylogenetic tree of the representative sequences and the closest database matches was constructed using software packages ClustalX version 2.0.12 (Larkin et al., 2007), Phylip version 3.69 (Felsenstein, 1989), and MEGA version 4.0 (Tamura et al., 2007) as previously described (Bragina et al., 2012).

SEQUENCE ACCESSION NUMBERS
The raw pyrosequencing data was deposited in the European Nucleotide Archive (ENA) under the project number PRJEB4660 with the accession numbers ERR361316-ERR361331.

FLUORESCENT in situ HYBRIDIZATION AND CONFOCAL LASER SCANNING MICROSCOPY
Single plants of S. magellanicum and S. fallax were fixed with 4% paraformaldehyde/phosphate buffered salt (3:1, v/v) and stained by in-tube FISH (Grube et al., 2009). The samples were hybridized with rRNA-targeting probes (genXpress, Wiener Neudorf, Austria) specific for Burkholderia and with a set of universal bacterial probes. Hybridization was carried out at 41 • C. The probes and corresponding stringency conditions are listed in Table 1. Confocal laser scanning microscopy (CLSM) was performed with a Leica TCS SPE confocal microscope (Leica Microsystems, Mannheim, Germany) as previously described (Bragina et al., 2012) followed by volume rendering of confocal stacks using the software Imaris 7.3 (Bitplane, Zurich, Switzerland).

SPHAGNUM MOSSES ARE PREFERENTIALLY COLONIZED BY BURKHOLDERIAL STRAINS FROM THE PBE CLUSTER AND A MINOR COMMUNITY FRACTION BELONGS TO THE PLANT-PATHOGENIC CLUSTER
High-throughput analysis of the Burkholderia community was achieved via an amplicon pyrosequencing approach targeting the V4-V5 region of the 16S rRNA gene. The pyrosequencing of 12 amplicon libraries of S. fallax and S. magellanicum samples from Austria and Russia retrieved 149,024 raw sequences ( Table 2). After initial processing, 87,917 quality sequences (average length, 405 bp) specific for Burkholderia genus were subjected to a detailed investigation. Rarefaction analysis of the pyrosequencing libraries, which were clustered with 97% sequence similarity, resulted in similar saturation profiles for all Sphagnum samples ( Figure S1). Richness estimation of the normalized datasets revealed that the current pyrosequencing survey attained 81.1-100% of the estimated richness ( Table 2). Low values of the Shannon diversity index (0.21-0.90, Table 2) indicated that the retrieved burkholderial communities contained a low number of highly abundant phylotypes. Through the use of automatic classification of the representative sequences, these phylotypes were assigned to B. bryophila, B. andropogonis, and B. glathei with several of them remaining unclassified at species level (Figure 1) To achieve a deeper insight into burkholderial diversity, we performed a phylogenetic analysis of the partial 16S rRNA www.frontiersin.org December 2013 | Volume 4 | Article 394 | 3 gene sequences from pyrosequencing libraries and closely related environmental strains (Figure 2). The closest database matches showed ≥96% of sequence identity to representative burkholderial sequences from pyrosequencing libraries. Clustering of the representative and reference sequences on the phylogenetic tree reflected several ecological traits of the examined burkholderial community. Specifically, cluster 2 was formed from representative sequences (this study) and the B. bryophila strain LMG 23648, a plant growth-promoting and antagonistic bacterium that was originally isolated from mosses in a nature reserve bog in Germany (Vandamme et al., 2007). This cluster also contained burkholderial strains PB1, F4W, F4, and SB1 which were isolated from acidic peat bogs in Russia (Belova et al., 2006). The phylogenetic clusters 1, 3, 4, 5, and 6 were represented by various endophytic and rhizospheric bacteria. These bacteria included the endophytic mycorrhizal B. phenazinium clone WT1 5, burkholderial clones sen290 and mat480 associated with lupin cluster roots (Weisskopf et al., 2011), and burkholderial endophytes M1U5b and M1U23 that were isolated from the arctic plants (Nissinen et al., 2012). Within the clusters 5 and 6, we detected B. andropogonis strain W20, a causative agent of the leaf spot in betel palm, and SFecto-B3clone1 clone of B. glathei species, a freeliving or moss-associated bacterium that is not considered a member of the PBE cluster (Opelt et al., 2007a;Suárez-Moreno et al., 2012). Interestingly, the representative sequence in these clusters showed higher sequence similarity (98-100%) with the harmless burkholderial strains M1U5b and M1U23 than with B. andropogonis and B. glathei sequences. Furthermore, cluster 7 contained bacteria from acidic and alpine soils. Overall, the phylogenetic analysis revealed that Sphagnum-associated Burkholderia are phylogenetically closely related to plantbeneficial and non-pathogenic Burkholderia from various acidic habitats, especially peat bogs, but also potential plant pathogens.

BURKHOLDERIA COMMUNITIES OF SPHAGNA EXHIBIT SIMILAR DISTRIBUTION AND COLONIZATION PATTERNS INDEPENDENT OF THE GEOGRAPHIC REGION
Biogeographical distribution of the Burkholderia communities was examined for the peat mosses S. fallax and S. magellanicum collected from Austrian and Russian bogs www.frontiersin.org December 2013 | Volume 4 | Article 394 | 5 (Table S1). Burkholderia showed highly similar distribution patterns for the analyzed moss species independent of the geographic region (Figure 3). An average weighted UniFrac distance was 0.47% with a maximum value of 1.08% for S. magellanicumassociated communities in Russian bogs ( Table S2). The statistical analysis using an adonis test confirmed that neither Sphagnum species (P = 0.261) nor geographic position (P = 0.363) had a significant influence on the beta-diversity of burkholderial communities. The general distribution of Burkholderia was confirmed through fluorescent in situ hybridization (FISH) of Sphagnum gametophytes with genus-specific and universal bacterial probes (Figure 4). Sphagnum mosses are characterized by a unique morphology (Daniels and Eddy, 1985) which makes them easily accessible for microbial colonization (Bragina et al., 2012). CLSM observation of hybridized plants showed that the Burkholderia community inhabited the leaves, but not the stem tissues of mosses. Straight and slightly curved rods of Burkholderia were detected in hyalocyte cells of leaves being likely attached to their cell walls. Inside the hyalocytes, Burkholderia remained as individual cells or formed microcolonies composed of few cells. Burkholderial cells were also found in association with other bacteria of unidentified taxonomy as shown in Figure 4A. Analysis of FISH-CLSM data showed that Burkholderia communities established similar colonization patterns in different Sphagnum species across the examined geographic scales.

BURKHOLDERIA ARE VERTICALLY TRANSMITTED WITHIN THE CORE MICROBIOME OVER ENTIRE LIFE CYCLE OF THE HOST PLANTS
The 16S rDNA pyrosequencing libraries from the moss sporophyte, gametophyte, and flark water samples were compared to reveal potential transmission mechanisms of Sphagnumassociated bacteria with a special focus on the genus Burkholderia. The libraries were rarefied as shown in Figure S1. The pyrosequencing survey achieved 42.6-63.4% of total richness as estimated by the Chao1 index ( Table 2). Classification of the normalized datasets revealed the occurrence of certain bacterial taxa in S. fallax and water microhabitats (Figure 5). Thus, Proteobacteria, Bacteroidetes, and Acidobacteria were among the most abundant phyla in all examined microhabitats. At class level, Alphaproteobacteria (within the phylum Proteobacteria) comprised the dominant portion of the plant-associated microbiome, while the water microbiome was dominated by Sphingobacteria (Bacteroidetes). Furthermore, a comparison of microbiome structure at family level revealed several different occurrence patterns. For instance, Acidobactriaceae (within the class Acidobacteria) were ubiquitously distributed unlike the family Xanthomonadaceae (Gammaproteobacteria) that specifically colonized the moss-associated microhabitats, gametophyte, and sporophyte. Moreover, several bacterial taxa, namely Methylocystaceae (Alphaproteobacteria), inhabited moss gametophytes and flark water. To study the occurrence of Burkholderia in various microhabitats, we compared the compositions of water, gametophyte-, and sporophyte-associated microbiomes at genus level. Consequently, Burkholderia were detected in the Sphagnum sporophyte and gametophyte, but were absent in the flark water. To ensure that normalization did not influence the Burkholderia occurrence pattern, the non-normalized pyrosequencing libraries were checked for the presence of this genus. The occurrence pattern of Burkholderia coincided between the normalized and nonnormalized datasets (data not shown). However, pyrosequencing of the flark water microbiome achieved partial coverage of the estimated diversity ( Table 2) and therefore additional experiments would be required to confirm this finding. Altogether, the obtained results indicated that the moss microbiome exhibits potential for both water-mediated and host-mediated transmission and that Sphagnum-associated Burkholderia are potentially transmitted over the entire life cycle of the host plants.

DISCUSSION
The genus Burkholderia is very important for plant and human health (Coenye and Vandamme, 2003;Suárez-Moreno et al., 2012;Mitter et al., 2013). We found that the microbiome of our model Sphagnum plant is preferentially enriched by the plantbeneficial and non-pathogenic Burkholderia from the PBE cluster, but also contains minor fraction of potential plant pathogens. We have provided new ecological insights into these important plant inhabitants including their composition, distribution, colonization, and transmission pattern.
Our hypothesis that Sphagnum species are colonized by specific Burkholderia from the PBE cluster has to be slightly revised. Although the most abundant B. bryophila species belongs to the plant-beneficial cluster, a minor fraction of B. andropogonis and B. glathei species are within the pathogen cluster (Suárez-Moreno et al., 2012). However, the phylogenetic analysis was crucial for elucidating their intra-specific diversity and ecological background (Figure 2). Amazingly, the resolved phylogenetic clusters contained plant-beneficial and non-pathogenic burkholderial strain as well as environmental clone sequences. This fact led us to the conclusion that Burkholderia members from the PBE cluster are of a great importance for the health and growth of Sphagnum plants. Our conclusion was supported by the isolation of B. bryophila and B. phenazinium beneficial strains from Sphagnum mosses at the same sampling sites by Shcherbakov et al. (2013). The minor fraction of the burkholderial community was formed by Burkholderia spp. from the plant-pathogenic cluster sensu Suárez-Moreno et al. (2012). However, the collected Sphagnum plants did not exhibit any disease symptoms. Therefore, we support the transfer of B. glathei the PBE cluster as recently suggested by Verstraete et al. (2013), who identified the species as common endosymbiont in plants of the Rubiaceae family. In contrast, Burkholderia andropogonis is the causal agent of numerous plant diseases affecting a wide range of monocot and dicot plants, e.g., sweet and field corn, blueberry, sorghum, carnation, coffee, statice, rye, and clover. Bacterial leaf stripe is one of the three major bacterial diseases of sorghum, and strict quarantine regulations against importation of B. andropogonisinfested sorghum feed grains and seeds are imposed by numerous countries (Ramundo and Claflin, 2005). Sphagnum mosses seem to be a natural reservoir for this plant pathogen. This is important because dry Sphagnum is often used for orchid and ornamental cultivation and transferred world-wide. On the other side, there are also hints that saprophytic B. andropogonis exists (Estrada-de los Santos et al., 2013), and many disease outbreaks depend on the abundance of pathogens and the diversity of the indigenous microbiome. At last, it is impossible to predict any pathogenic or beneficial effect from 16S rDNA analysis, and additional studies would be required to prove or contradict the pathogenicity of Sphagnum-associated B. andropogonis.
In this study, we discovered similar distribution patterns of Sphagnum-associated burkholderial communities independent of the geographic region, which well-confirmed our hypothesis. To elucidate this distribution pattern, we aimed to answer the question-what factors shape this community? In terrestrial habitats, pH serves as both a primary driver of microbiome structure as well as a specific determinant of the genus Burkholderia as it is known to exhibit pH tolerance as a general phenotypic trait (Lauber et al., 2009;Stopnisek et al., 2013). In our previous study, the same sampling sites in Austria were characterized as extremely to moderately acidic by means of Ellenberg's indicator values for pH (expressed as soil reaction) (Bragina et al., 2012). For sampling sites in Russia, the Ellenberg's values for pH varied at the same range (data not shown) and therefore all examined sites possessed favorable a-biotic conditions for the burkholderial colonization. Apart from the a-biotic factors, we previously demonstrated that various Sphagnum species determine the microbiome composition to different extents (Bragina et al., 2011(Bragina et al., , 2012. Through statistical analysis, we showed that neither geographic location nor Sphagnum species had a significant influence on the distribution of Burkholderia. Moreover, the similar colonization patterns of the moss-associated Burkholderia were verified using FISH-CLSM in a semi-quantitative way. For a better understanding of the distribution and colonization pattern revealed for Sphagnum-associated Burkholderia, we addressed the issue of bacterial transmission in the peat bog ecosystems. Recently, Putkinen et al. (2012) described a water dispersal of methane-oxidizing bacteria in the peat bogs. Moreover, our previous study revealed that nitrogenfixing bacteria were transferred within the moss sporophyte  As a result, we hypothesized that either host-mediated or water-mediated transmission is possible for Sphagnum-associated Burkholderia. Through the comparison of microbiome composition in various bog microhabitats, we found that burkholderial communities are potentially transmitted by the host plants. The violent spore discharge and wind dispersal of the Sphagnum spores would enable associated bacteria to migrate over the long distances and support spore germination at a new site (Szövényi et al., 2008;Sundberg, 2010). Altogether, the detected host-mediated transmission underlines the importance of Burkholderia for Sphagnum mosses themselves and defines their distribution pattern.
In recent decades, burkholderial community was considered a typical and well-adapted component of acidic peat bogs (Belova et al., 2006). In this study, we demonstrated that Burkholderia associated with the main vegetation of peat bogs, Sphagnum mosses, contain both plant-beneficial but also potentially pathogenic Burkholderia that are transmitted by the host plants over their life cycle. However, global warming and human disturbance may significantly shift the environmental conditions in the peat bog ecosystems and lead to the elimination or substitution of the beneficial microbes (Dise, 2009). The obtained data supports our knowledge on native plant microbiomes and can help for the maintenance of climate-relevant bog ecosystems.

ACKNOWLEDGMENTS
We would like to thank Vladimir Chebotar and Andrey Shcherbakov (St. Petersburg) for their cooperation in the joint project and interesting discussions. We would also like to thank Ekaterina Kuzmina (St. Petersburg) for her bryological expertise during the sampling in Russia. We are grateful to Meg Starcher (Graz/Washington) for English revision of the manuscript. This study was supported by FWF Austrian Science Fund (grant no. E-1653100183) to Gabriele Berg.