Draft Genome Sequences for Bacteria Associated with Root Nodules of Alnus incana in New England

ABSTRACT Nine bacterial strains isolated from the root nodules of Alnus incana were sequenced to determine their potential roles in plant health. The selected bacterial isolates belonged to the genera Bacillus, Herbaspirillum, Pantoea, Paenibacillus, and Rothia. Here, we report the draft genome sequences.

B esides hosting endosymbionts, root nodules have other occupants or plant endophytes that appear to assist plant growth and health (1)(2)(3)(4)(5). Using a culture-independent approach, we elucidated the nodule microbiome of Casuarina glauca (1) and have extended this study to Alnus incana found in New England. We previously reported the genome sequences of 10 bacterial isolates obtained in 2018 (6). Here, we continue to isolate more endophytes of alder nodules.
In September and November 2019, root nodule samples were collected from A. incana found by Adams Point at Jackson's laboratory in Durham, New Hampshire. The root nodules were surface sterilized with hydrogen peroxide and rinsed several times with sterile distilled water. The nodule was cut into a fine powder with a sterilized razor, and dilutions were plated onto Czapek (7) and R2A (8) media. About 77 isolates were initially obtained, purified, and propagated on either Czapek or R2A media. These isolates were incubated overnight in their respective isolation medium (Table 1), and genomic DNA (gDNA) was extracted by cetyltrimethylammonium bromide (CTAB) DNA extraction protocol (9). RNA was removed by RNase treatment. The quality and quantity of the gDNA were verified by a Thermo Scientific NanoDrop. Nine isolates were chosen for whole-genome sequencing analysis to provide insight into their plant-microbe interactions, including potential plant growth-promoting activity.
Whole-genome sequencing was performed at the Hubbard Center for Genome Studies (University of New Hampshire, Durham, NH) using Illumina technology techniques (10). A paired-end library was constructed using a Nextera DNA library preparation kit (Illumina, San Diego, CA) and sequenced on an Illumina NovoSeq instrument to produce 250-bp paired-end reads. The total numbers of reads for all 9 strains are listed in Table 1. The Illumina sequence data were trimmed by Trimmomatic version 0.36 (11). TruSeq adapters were trimmed with an allowance of two mismatches. Leading and trailing bases below the quality of 3 were trimmed. The read was then scanned with a sliding window of 4 bp and trimmed if the average quality dropped below 30. Finally, reads were dropped if the length was less than 36 bp. Trimmed sequencing reads were assembled using SPAdes version 3.15.2 (12) with the default settings. Default parameters were used for all software unless otherwise specified. The assembled genomes were annotated via the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) (13). The assembly metrics and annotation features are given in Table 1.
The identities of the strains were determined by a whole genome-based taxonomic analysis via the Type (Strain) Genome Server (TYGS) platform (14) (https://tygs.dsmz.de), including digital DNA-DNA hybridization (dDDH) values (15). The type-based species clustering using a 70% dDDH radius around each of the type strains was used as previously described (16), while subspecies clustering was done using a 79% dDDH threshold as previously introduced (17). Among the nine isolates, the three Bacillus velezensis strains are identical to each other, and the type strain and Paenibacillus sp. alder61 isolates were characterized as belonging to Paenibacillus faecis. Although three Pantoea isolates are identical to each other, they represent a potential new species, as does the Herbaspirillum isolate. Data availability. The draft genome sequences of these bacterial strains have been deposited in GenBank under the accession numbers listed in Table 1. Both the assembly and raw reads are available at DDBJ/ENA/GenBank under BioProject accession number PRJNA748777.

ACKNOWLEDGMENTS
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