Complete Genome Sequences of Brenneria rubrifaciens Strain 6D370 and Brenneria nigrifluens Strain ATCC 13028, Causative Agents of Bark Cankers in Walnut

Brenneria rubrifaciens and Brenneria nigrifluens, respectively, cause deep and shallow bark canker disease in walnut. B. rubrifaciens exhibits quorum sensing-controlled virulence and rubrifacine pigment production. The complete genome sequences of these species will be useful for studying the role of genes regulated by quorum sensing, including pathways mediating pathogenesis.

ABSTRACT

Brenneria rubrifaciens and Brenneria nigrifluens, respectively, cause deep and shallow bark canker disease in walnut. B. rubrifaciens exhibits quorum sensing-controlled virulence and rubrifacine pigment production. The complete genome sequences of these species will be useful for studying the role of genes regulated by quorum sensing, including pathways mediating pathogenesis.

ANNOUNCEMENT

Brenneria rubrifaciens and Brenneria nigrifluens, respectively, cause deep and shallow bark cankers in English walnut (1, 2). While these cankers do not kill the trees, they weaken trunks and branches over time and, combined with other stresses, reduce walnut production. These bacterial species can coinfect walnut and often live as quiescent endophytes that become pathogenic when trees experience environmental stress (3, 4). Here, we report the genome sequences of B. rubrifaciens strain 6D370, isolated by Azad and Kado (5), and B. nigrifluens strain ATCC 13028, isolated by Wilson et al. (2). Both species were isolated from symptomatic walnut trees in the Central Valley of California (2, 5).

Brenneria rubrifaciens strain 6D370 is available upon request. B. nigrifluens strain ATCC 13028 was obtained from the American Type Culture Collection. For both strains, a single colony was inoculated into tryptic soy broth medium and incubated for 24 hours at 28°C and 210 rpm. Genomic DNA was isolated using the MasterPure complete DNA purification kit (Epicentre, Madison, WI) and submitted to MR DNA (Shallowater, TX) for library preparation and 2 × 250-bp paired-end sequencing on an Illumina MiSeq instrument. This generated ∼4.7 million (B. rubrifaciens) and 5.7 million (B. nigrifluens) paired-end reads. We also sequenced both genomes at the Michigan State University Research Technology Support Facility (RTSF) Genomics Core on a GridION instrument using one flow cell and the RBK004 rapid barcoding kit (Oxford Nanopore Technologies, San Francisco, CA) and obtained 2 Gbp (B. rubrifaciens) and 0.42 Gbp (B. nigrifluens) with average lengths of 10,925 and 10,783 bases, respectively. Raw sequences from both platforms were quality trimmed, filtered, and used for hybrid assembly via Unicycler v0.4.4 under default parameters (6). This yielded complete circular chromosomes of 4.03 Mbp (G+C content, 52.6%) for B. rubrifaciens strain 6D370 and 4.89 Mbp (G+C content, 55.9%) for B. nigrifluens strain ATCC 13028.

Both genomes were annotated via the NCBI Prokaryotic Genome Annotation Pipeline (7). The B. rubrifaciens genome contained 3,613 predicted genes, including 3,354 protein-coding sequences, 7 complete rRNA gene operons, 73 tRNAs, 5 noncoding RNAs (ncRNAs), and 159 pseudogenes. The B. nigrifluens genome consisted of 4,600 predicted genes, including 4,366 protein-coding sequences, 7 complete rRNA gene operons, 72 tRNAs, 7 ncRNAs, and 163 pseudogenes. We previously identified quorum-sensing (QS) genes encoding an acyl homoserine lactone (AHL) synthase (bruI) and an AHL-responsive transcription factor (bruR) in B. rubrifaciens that control production of the red pigment rubrifacine and virulence (4, 8, 9). B. rubrifaciens with a disrupted bruI is rubrifacine negative, but growth of the mutant in the presence of B. nigrifluens, which produces similar QS signal molecules, restores pigment production (4). Homologs of bruI and bruR were present in B. nigrifluens and are candidates for future studies to assess the role of QS in pathogenicity on walnut. Both genomes also encoded type III secretion systems (10). The B. rubrifaciens genome harbored two nearly identical gene clusters encoding a type IV secretion system (11). These secretion systems are important for virulence in plant-pathogenic bacteria and may be involved in enabling the development of the bark cankers caused by these Brenneria species. The complete genome sequences of both species also will facilitate our understanding of how environmental and physiological factors affect development of bark canker diseases in walnut.

Data availability.

The complete genome sequences are available in the NCBI database under BioProject number PRJNA478423 and GenBank accession numbers CP034035 (B. rubrifaciens strain 6D370) and CP034036 (B. nigrifluens strain ATCC 13028). The raw sequence reads are available under the Sequence Read Archive accession number SRP199005. The versions described in this paper are the first versions.