Genome-sequenced bacterial collection from sorghum epicuticular wax

ABSTRACT A collection of 44 isolates isolated from the epicuticular wax of stems of energy sorghum is available at the Great Lakes Bioenergy Researcher Center, Michigan State University, MI, USA. We enriched bacteria with putative plant-beneficial phenotypes and include information on their phenotypic diversity, taxonomy, and whole-genome sequences.

T he heat-and drought-tolerant and high-biomass crop bioenergy sorghum (Sorghum bicolor L. Moench) accumulates high levels of epicuticular wax on the outer surface of the cuticle to protect plants against various stresses (1)(2)(3).It has been shown that the sorghum epicuticular wax harbors specialized microbiome members with putative plant-beneficial capabilities (4).As a resource for genomic and functional diversity of bacteria associated with phyllosphere exudates, we present a collection of 44 bacterial strains isolated from sorghum epicuticular wax.These strains represent 13 families and include 17 genera with putative plant-beneficial capabilities, including nitrogen fixation, phosphate solubilization, resistance to terpenoids, use of methanol as carbon source, and tolerance to desiccation, based on the culture media in which the strains were originally isolated.
Bacterial strains were isolated from the epicuticular wax of stems of bioenergy sorghum hybrid TX08001 grown at the Texas A&M University Research Farm in College Station, Texas (30 o 55′5.55″N, 96 o 43′64.6″W).On 2 September 2020, the fifth and sixth fully elongated stem internodes were destructively collected using razor blades into sterile whirl-pack bags.Within 2 hours of collection, the samples were transported to the laboratory on ice and stored at −80°C.On 8 October 2020, samples were delivered to Michigan State University on dry ice and stored at -80°C until processing.Epicuticu lar wax was carefully scraped from stems with sterile razor blades and collected into sterile 1.5 mL Eppendorf tubes.Wax from different plants was pooled, and 100 mg was resuspended in 1 mL of sterile water.Resuspended wax was serially diluted from 10 −1 to 10 −4 .To capture a diversity of bacteria from the wax, we used a variety of cultivation media (Table 1).To select for anaerobic bacteria, plates were placed in anaerobic jars containing three bags of anaerobic gas generator (Mitsubishi AnaeroPack System).All plates were incubated at 25°C and 37°C for up to 14 days.Isolated colonies were transferred on new plates with the same medium as used for isolation to confirm purity.Bacteria were grown overnight on R2A broth at 28°C with shaking at 200 rpm, and glycerol stock (25% vol/vol) of pure bacteria isolates was stored at −80°C.Prior to performing whole-genome sequencing, we performed full-length 16S rRNA gene sequencing, with universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACGGTTACCTTGTTACGACTT-3′) (5), of each isolate using the Sanger protocol to link  Individual isolates or the full cryopreserved collections are available.Cultures can be regrown using standard microbiological procedures to transfer a sterile inoculation loop of freezer stock onto the original isolation medium and incubation time (Table 1).
The high molecular weight genomic DNA of each wax bacterial isolate was extrac ted by using a phenol/chloroform extraction protocol (6).The genomic DNA of each strain was submitted to the Joint Genome Institute, a U.S. Department of Energy User Facility, for library preparation and sequencing with the Pacific Biosciences (PacBio) platform (7).DNA was sheared to 10 kb using g-TUBE columns (Covaris) and subjected to library preparation using the SMRTbell Express Template Prep Kit 2.0 and sequenced on the PacBio Sequel platform.Samples that failed DNA quality control required by the PacBio pipeline were instead sequenced using the NovaSeq S4 Illumina platform (8).High-fidelity PacBio Circular Consensus Sequencing (CCS) reads >5 kb were assembled with Flye 2.8.3 (9) using default settings.Raw Illumina sequences were quality filtered using BBTools (10).Artifact-filtered and normalized Illumina reads were assembled with SPAdes v3.15.3 (11) (-phred-offset 33 -cov-cutoff auto -t 16 m 64 -careful -k 25,55,95), and contigs were discarded if the length was <1 kb (BBTools reformat.sh:minlength = 1,000 ow = t).Genome completeness and contamination were estimated with CheckM v1.2.2 (12).The assembly was annotated using Prokka 1.14.6 (13), and the phylogenetic tree was inferred from using Orthofinder 2.5.5 (14) and edited with iTOLs v.6.5.8 (15) (Fig. 1).

TABLE 1 -
Taxonomy, colony phenotype, and genome characteristics of 44 bacterial isolates from sorghum epicuticular wax, as described in this study a

TABLE 1 -
Taxonomy, colony phenotype, and genome characteristics of 44 bacterial isolates from sorghum epicuticular wax, as described in this study a