The rhizosphere of plants is enriched in nutrients facilitating growth of microorganisms, some of which are recruited as endophytes. Endophytes, especially Actinobacteria, are known to produce a plethora of bioactive compounds. We hypothesized that Leontopodium nivale subsp. alpinum (Edelweiss), a rare alpine medicinal plant, may serve as yet untapped source for uncommon Actinobacteria associated with this plant. Rhizosphere soil of native Alpine plants was used, after physical and chemical pretreatments, for isolating Actinobacteria. Isolates were selected based on morphology and identified by 16S rRNA gene-based barcoding. Resulting 77 Actinobacteria isolates represented the genera Actinokineospora, Kitasatospora, Asanoa, Microbacterium, Micromonospora, Micrococcus, Mycobacterium, Nocardia, and Streptomyces. In parallel, Edelweiss plants from the same location were surface-sterilized, separated into leaves, roots, rhizomes, and inflorescence and pooled within tissues before genomic DNA extraction. Metagenomic 16S rRNA gene amplicons confirmed large numbers of actinobacterial operational taxonomic units (OTUs) descending in diversity from roots to rhizomes, leaves and inflorescences. These metagenomic data, when queried with isolate sequences, revealed an overlap between the two datasets, suggesting recruitment of soil bacteria by the plant. Moreover, this study uncovered a profound diversity of uncultured Actinobacteria from Rubrobacteridae, Thermoleophilales, Acidimicrobiales and unclassified Actinobacteria specifically in belowground tissues, which may be exploited by a targeted isolation approach in the future.

Fig.1.fas

Sanger sequence alignment of all experimental sequences (prefix -RLA) of Actinobacteria other than the genus Streptomyces with their Genbank accession numbers, closest matches in Genbank Blast search and type strains (T) of closest matches from rhizosphere isolates of Leontopodium nivalesubsp. alpinum. Alignments were performed with MEGA version 7.0.18 using ClustalW algorithms (Kumar et al., 2016).

Fig.1.newick

Maximum composite likelihood analysis (MCL) of Actinobacteria isolates from the rhizosphere of Leontopodium nivalesubsp. alpinumbased on Tamura and Nei (1993) model with Gamma distribution and invariate sites using Mega software version 7.0.18 (Kumar et al., 2016). The phylogenetic tree contains experimental sequences (RLA) with their Genbank accession numbers, their closest matches in Genbank Blast search and respectively closest type strains (T). The tree is drawn to scale with branch length representing the substitutions per site. Bootstrap support values were calculated with 1000 replicates. Isolates identify by the prefix -RLA followed by an individual isolate number and Genbank accession numbers in brackets. 

Fig.2.fas

Sanger sequences alignment of all experimental sequences (prefix -RLA) of the genus Streptomyces with their Genbank accession numbers, closest matches in Genbank Blast search and type strains (T) of closest matches from rhizosphere isolates of Leontopodium nivalesubsp. alpinum. Alignments were performed with MEGA version 7.0.18 using ClustalW algorithms (Kumar et al., 2016).

Fig.2.newick

MCL clustering of Streptomycesisolates from Edelweiss rhizosphere. Phylogenetic tree reconstruction was performed with Tamura (1992) as best fitting evolutionary model with invariant sites and Gamma distribution using Mega software version 7.0.18 (Kumar et al., 2016).Further specifications of the phylogeny are congruent to Figure 1.

Supplem.Fig.S1.fas

Alignment of curated metagenomic sequence data of Actinobacteria endophytes from Leontopodium nivalesubsp. alpinumresulting from Illumina HiSeq2500. Amplicons were acquired with the primers 357F and 806R and entailed the V3 and V4 region of 16S rDNA as paired end reads of 250 bases. Sequences were processed according to the Mothur MiSeq SOP by using mothur 1.39.5 (Schloss et al.,2009; Kozich et al.,2013). The full analysis script is available on GitHub (https://github.com/JackyHess/Leontopodium_metagen).

Supplem.Fig.S1.RAxML

ML analysis sequences of Actinobacterial endophyte metagenome of Edelweiss. Phylogenetic relationships include endophytes from all host plant tissues.Data are clustered into operational taxonomic units (OTUs) with an identity cutoff of 97% and taxonomic assignments are based on RDB classifier and Silva database (Wang et al., 2007).

The submitted data contain the alignments of sequences used for Fig. 1, 2 and Supplem. Fig. S1 in the format of .fas. Moreover, the submission entails the tree files in in the format of .newick (Fig. 1, 2) and .RAxML (Suppl. Fig S1).