Genome-Annotated Bacterial Collection of the Barley Rhizosphere Microbiota

ABSTRACT A culture collection of 41 bacteria isolated from the rhizosphere of cultivated barley (Hordeum vulgare subsp. vulgare) is available at the Division of Plant Sciences, University of Dundee (UK). The data include information on genes putatively implicated in nitrogen fixation, HCN channels, phosphate solubilization, and linked whole-genome sequences.

T he microbial communities thriving at the root-soil interface, that is, the rhizosphere microbiota, represent an untapped resource of plant probiotic functions (1,2). Bacterial members of the microbiota capable of enhancing a plant's mineral uptake from soil and pathogen protection, namely, plant growth-promoting rhizobacteria (PGPRs), have gained prominence in both basic scientific and translational applications (3)(4)(5). As a resource for comparative investigations of the plant microbiota across host species, we present a collection of 41 bacterial strains encompassing 15 genera with the presence of at least 5 putative plant growth-promoting (PGP)-associated gene orthologs, including, dinG, hcn, nif, pho, and pqq (Table 1) (6).
Strains were isolated from the rhizosphere of cultivated barley (Hordeum vulgare L. subsp. vulgare), the fourth most cultivated cereal worldwide (7), which was grown in an agricultural soil used for previous barley-microbiota investigations (8,9). Bacterial rhizosphere fractions were obtained by detaching the soil adhering the uppermost 6 cm of barley roots by vortexing in phosphate-buffered saline (PBS) buffer. Serial dilutions were plated onto R2A and nutrient agar media and incubated at 20°C for 48 to 72 h (10,11). Individual CFUs were selected for isolation based on morphological variation; clean isolate liquid cultures were stored at 280°C in 70% glycerol following 24 to 48 h of shaking incubation at 27°C.
DNA was extracted as per the manufacturer's instructions using the FastDNA spin kit for soil (MP Biomedicals, USA). Individual bacterial isolates were subjected to wholegenome sequencing using the "standard service" of MicrobesNG (Birmingham, UK). Briefly, bacterial genomic DNA libraries were prepared using the Nextera XT library prep kit (Illumina, USA) following the manufacturer's protocol with the following modifications: 2 ng of DNA were input, and PCR elongation time was increased to 1 min. DNA quantification and library preparation were conducted on a Hamilton Microlab STAR automated liquid handling system. Pooled libraries were quantified using the Kapa Biosystems library quantification kit for Illumina on a Roche light cycler 96 quantitative PCR (qPCR) machine. Libraries were sequenced by using an Illumina HiSeq instrument with a 250-bp paired-end protocol. Reads were adapter trimmed using Trimmomatic (v0.30) with a sliding window quality cutoff of Q15 (12). De novo assembly was performed using SPAdes (v3.7), and contigs were annotated using Prokka Strain taxonomy reflects the lowest and unique rank as defined by GTDBTK (v1.6.0) with data version r202. c The identification in each bacterial genome is depicted by the plus sign.
(v1.12) (13,14). On the basis of GC content, unambiguous taxonomic annotations generated using amphora classification (15) and whole-genome average nucleotide identity (ANI) to identify individual "founder" members (ANI cutoff, 96%) yielded 41 genomes retained for downstream analyses. To compare only components of characterized metabolic pathways, predicted genes were concatenated and annotated with eggNOG-Mapper (v1.0.3) (16,17 (Fig. 1) was constructed using bcgTree (v1.1.0) and RAxML (v8.2.12), using RAxML's GTRGAMMA model and 100 bootstrap iterations (22,23); default parameters were used for all analyses unless otherwise noted. The collection is available as frozen isolates preserved in 300 to 500 mL of nutrient or R2A medium containing 50% glycerol and maintained at 270°C. To revive the frozen cultures, we recommend using a sterile inoculating loop to transfer a small amount (e.g., 50 mL equivalent) of the frozen culture onto a nutrient or R2A agar medium base following standard microbiological procedures. The plates should be incubated at 27°C for 24 to 48 h.
Data availability. The genomic sequences reported in this study are deposited in the European Nucleotide Archive (ENA). Accession numbers for the individual genomes are provided in Table 1. To acquire isolates, or for questions or suggestions, please contact Davide Bulgarelli at d.bulgarelli@dundee.ac.uk.