Complete Genome Sequences of 10 Xanthomonas oryzae pv. oryzae Bacteriophages

Xanthomonas oryzae pv. oryzae is the causative agent of bacterial leaf blight of rice. The application of bacteriophages may provide an effective tool against this bacterium. Here, we report the complete genome sequences of 10 newly isolated OP2-like X. oryzae pv. oryzae bacteriophages.

28°C. All isolates were purified by three successive single-plaque isolation methods using the classical drop-on-lawn technique (15).
Phage nucleic acid was isolated by using the High Pure viral nucleic acid kit (Roche Diagnostics GmbH, Germany), according to the manufacturer's instructions. Genomic sequences of the X. oryzae pv. oryzae phages of this study were determined with MiSeq (Illumina, Inc., USA) next-generation sequencing (NGS) equipment, using Nextera XT kit (Illumina Inc.) for paired-end library preparation and Illumina V2 sequencing kit (Illumina Inc.), according to the guidelines of the manufacturer, resulting in 2,649,822 (250-bp-long) reads. The mean coverages were between 409ϫ (XPP8) and 10,461ϫ (XPP2).
The next-generation reads were analyzed for quality using the FastQC program (Babraham Bioinformatics, version 0.11.5), with default parameters. Low-quality bases and reads were trimmed and/or removed using the Trim Galore! (Babraham Bioinformatics, version 0.4.4 with paired mode) and Trimmomatic (version 0.36 with paired mode and using the CROP:150 MINLEN:150 parameters) programs (16). The qualityfiltered reads were assembled using the MyPro software package (17). In the assembly processes, we used the Assembly.py and Integrate.py python scripts for all samples.
Genome annotation was performed using the RAST server (18), with manual curation. Each hypothetical or conserved protein-encoding gene was subjected to a search using NCBI blastx against the nonredundant protein (nr) database (19). Results were accepted when the E value was lower than eϪ10 and the coverage was higher than 75% as a cutoff for notable similarity.
The complete genomes of all 10 X. oryzae pv. oryzae phages were assembled. Table 1 contains the sequence lengths and GϩC mol% of the newly isolated 10 phages and of the reference OP2. The GϩC mol% contents of the newly isolated phages were in the range of 60.0 to 62.4 mol%, similar to that of OP2, with the exception of XPV2, for which it was higher (64.3 mol%; Table 1). The presence of 77-to 3,411-bp direct terminal repeats was detected (with a self dot plot, using the Geneious 8.0.5 software) in 6 newly isolated phages ( Table 1). The complete genome nucleotide sequences of the phages were compared by pairwise alignments using the Geneious 8.0.5 software. Phage genome circularity was analyzed in silico. SAMtools/bcftools (20) was used to map the (raw) reads against the complete genomic sequence of the phage from whose genome sequence the reads originated. The positions of the mate-paired reads were investigated with IGV 2.5.2 (21). The circularity of the genomes was determined by observing mate-paired reads where the distance between the two reads spanned the whole genome. All of the investigated genomes were determined to be circular.
Genome sequencing of the newly isolated phages proved that these are OP2-like, with whole-genome nucleotide sequence similarities of 90.7 to 91.6% compared to OP2. XPV phages isolated from the Mekong Delta (Vietnam) had sequence identities of 93.2 to 96.3% compared to each other and identities of 93.0 to 94.7% compared to XPP phages isolated from the Philippines. Differences between the complete genome nucleotide sequences of the XPP phages were limited. To the best of our knowledge, this work was the first in which the complete genomes of OP2-like X. oryzae pv. oryzae phages were determined.
Data availability. The complete genome sequences of newly sequenced X. oryzae pv. oryzae bacteriophages have been submitted to GenBank, and accession numbers (listed in Table 1) were assigned. The BioProject number is PRJNA529058.