The Cannabis plant has been cultivated for its nutritional value, durable fibers, medicinal use, and recreation for thousands of years (1). The ratio of cannabidiol (CBD) and tetrahydrocannabinol (THC) is important for both medicinal and recreational use, and the yields of these metabolites could be impacted by the microbial communities associated with the cannabis plant (2). Although bacterial isolates have been recovered and sequenced from Cannabis flowers (3), few have been reported from the Cannabis rhizosphere. Such bacterial isolates may have applications in biotechnology or agriculture.

Here we present the draft genome sequences of six bacterial isolates (Table 1) obtained from the rhizosphere of cultivated Cannabis plants (Anandia Labs; Vancouver, Canada). One gram of soil, recovered from Cannabis roots, was suspended in 10 mL of double-distilled water. The soil particulates were sedimented by centrifugation and the soil water extract spread-plated on Pseudomonas isolation agar (PIA, BD Difco). After incubation at 37ºC, single colonies were isolated by three rounds of single-colony isolation with streaking, again on PIA agar.

Brain-heart infusion (BHI) agar (BD Difco) was used to culture bacterial isolates at room temperature (~25⁰C) for 48 h. Single colonies were suspended in 2 mL BHI broth and grown to an OD₆₀₀ of 1.0. Cells were pelleted by centrifugation (10,000× g for 20 min at 4⁰C) and genomic DNA was extracted using a DNeasy Blood & Tissue kit (QIAGEN) according to the recommended protocol for Gram-negative bacteria. DNA purity was confirmed using a NanoDrop 2000 spectrophotometer (Thermo Fisher) and quantified using a Qubit dsDNA BR kit (Thermo Fisher). The G-tube fragmentation spin-column protocol (Covaris) was used to shear 1 µg genomic DNA into 400–700 bp fragments. These were then sequenced in paired ends (2 × 300 bp) on a MiSeq apparatus (Illumina) using the KAPA HyperPrep kit (Roche Sequencing Solutions) with Illumina TruSeq 3-PE adapters and the manufacturer’s recommended protocol for bacterial WGS sequencing. Read quality was visualized with FastQC (version 0.11.8 (4)) and quality filtering/adapter removal was performed with TRIMMOMATIC (version 0.39–2 (5)), keeping sequences below Q15 quality score over a minimum read length of 36 bp. Trimmed Illumina reads were assembled with the A5 pipeline (version 20150522 (6)). Annotation was done via the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) version 6.4 (7). Taxonomy was assigned to draft genomes with whole-genome in silico digital DNA-DNA hybridization (dDDH) via the TYGS (8). Genome completeness was evaluated with CheckM (9). All software was run with default parameters unless otherwise specified.

Our results include two potential new species with dDDH d4 below 70% (Table 1). Further genomic analysis of these strains will facilitate a greater understanding of the molecular mechanisms of plant-microbe interactions in the growth of the Cannabis plant and the potential impact of secondary metabolite production.