Complete Genome Sequence of Erwinia amylovora Strain TS3128, a Korean Strain Isolated in an Asian Pear Orchard in 2015

ABSTRACT Erwinia amylovora causes fire blight, a devastating disease of apples and pears. Here, we report the complete genome sequence and annotation of E. amylovora strain TS3128, which was isolated from Anseong, South Korea, where fire blight first occurred in 2015, using the PacBio RS II system.

E rwinia amylovora causes fire blight, one of the most serious bacterial diseases of apple and pear trees. To understand the genomic characteristics of E. amylovora strains isolated in South Korea, complete genome sequencing of E. amylovora strain TS3128 was performed; this is a virulent strain that was isolated from an infected pear tree in Anseong, South Korea, in 2015 (1).
To isolate a single strain, infected pear leaf tissues were surface sterilized using 70% ethanol and were dissected into samples (5 by 5 mm). The dissected samples were macerated in 10 mM phosphate-buffered saline (PBS) (pH 7.2) (Biosesang, South Korea) in a sterile mortar. Single colonies observed after 48 h of incubation on King's medium B at 27°C were isolated and subsequently purified. The purified strain was presumptively identified as E. amylovora by 16S rRNA and recA gene sequencing. Moreover, the primer pair AMSbL and AMSbR, based on the chromosomal amsB gene (2), was used to identify the strain as E. amylovora.
Total DNA was extracted from an overnight culture grown at 26°C in LB medium using the Wizard genomic DNA (gDNA) purification kit (Promega Corp., Madison, WI, USA). The integrity of gDNA was checked by agarose gel electrophoresis and quantified using a Qubit 2.0 fluorometer (Invitrogen, Carlsbad, CA, USA). The sequencing libraries were then prepared according to the manufacturer's instructions for 20-kb template preparation using the BluePippin system (Sage Science, Beverly, MA, USA) with a DNA template preparation kit v1.0 (Pacific Biosciences [PacBio], Menlo Park, CA, USA). Briefly, 10 mg of gDNA was sheared to 20 kb using g-TUBEs (Covaris, Woburn, MA, USA), purified, and end repaired; blunt-end SMRTbell adapters were then ligated. The libraries were quantified using a Qubit 2.0 fluorometer (Invitrogen) and qualified using the DNA 12000 chip (Agilent Technologies, Waldbronn, Germany). The libraries were then sequenced using PacBio P6C4 chemistry in an 8-well SMRT Cell v3 in the PacBio RS II SMRT platform (3).
The complete genome of E. amylovora TS3128 comprises a circular chromosome of 3,804,041 bp and one plasmid of 28,288 bp, with average G1C contents of 53.6% and 50.2%, respectively. The chromosome and plasmid contain a total of 3,477 genes, including 3,237 protein-coding genes and 11 protein-coding genes, respectively, annotated with the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) v4.11 (7). A total of 22 rRNA and 77 tRNA sequences were identified in the chromosome ( Table 1). The plasmid in E. amylovora TS3128 shares 99% sequence identity (100% coverage) with pEA29 (8).
Data availability. The genome sequence of E. amylovora strain TS3128 has been deposited in GenBank under accession numbers CP056034.1 (chromosome) and CP056035.1 (plasmid). Raw PacBio data have been deposited in the NCBI Sequence Read Archive (SRA) under the BioProject accession number PRJNA639906.