Complete genome sequences of Pseudomonas allii, pathogens causing bacterial rot in onion, tomato, Chinese yam, and lettuce in Japan

TL;DR

This paper presents the full genome sequences of four Pseudomonas allii strains that cause bacterial rot in crops like onion, lettuce, Chinese yam, and tomato in Japan.

Contribution

The study provides the first complete genome sequences of Pseudomonas allii strains associated with bacterial rot in these specific crops.

Findings

Four complete genome sequences of Pseudomonas allii strains were determined.

Each strain is linked to bacterial rot in a specific crop in Japan.

Abstract

Here, we report the complete genome sequences of four strains of Pseudomonas allii (MAFF 301514, MAFF 302050, MAFF 730042, and MAFF 730146) that represent pathogens causing bacterial rot in onion, lettuce, Chinese yam, and tomato, respectively, in Japan.

Tables

TABLE 1. Sequencing summary and statistics of the genome sequences of four strains of Pseudomonas allii

	Culture collection accession no.a
Feature	MAFF 301514	MAFF 302050	MAFF 730042	MAFF 730146
Isolation sources	Onion (Allium cepa)	Lettuce (Lactuca sativa var.capitata)	Chinese yam (Dioscoreapolystachya “Nagaimo”)	Tomato (Solanumlycopersicum)
Collection location	Hyogo	Tochigi	Aomori	Yamaguchi
Collection year	1980	1978	1977	1993
Original strain names	N-8042	NL 7843	YM 7702	To-1
Isolator	Akira Ohuchi	Yukio Tsuchiya	Manabu Umekawa	Tatsuhiko Karatsu
Sequencing summary
Nanopore	DRR636248	DRR636249	DRR636250	DRR636251
No. of raw readsb/bases (Mbp)	23,920/306	35,212/379	39,707/576	22,714/341
N50 (bp)	24,261	18,716	25,540	26,573
Illumina	DRR636252	DRR636253	DRR636254	DRR636255
No. of raw reads/bases (Gbp)	3,744,092/0.56	12,388,708/1.85	14,074,772/2.10	10,552,470/1.57
Mean coverage of depth in polypolish	28×	94×	103×	80×
Genome statistics
Total length (bp)	6,883,462	6,661,385	6,896,138	6,584,391
No. of circular replicons	1	1	1	1
Coverage of depth in Flye	32×	42×	62×	38×
G + C content (mol %)	60.9	60.9	60.8	61.0
No. of CDSs	6,238	6,015	6,274	5,939
Coding ratio (%)	89.7	89.8	89.6	89.8
No. of rRNA operons	5	5	5	5
No. of tRNA	71	73	73	72
CheckMc(Pseudomonas marker set)
Completeness (%)	100	100	100	100
Contamination (%)	0.72	0.72	0.72	0.72
OrthoANIud value (%)	–	99.1	98.9	99.1
dDDHe value (%)	–	92.6	91.5	92.8
GenBank accession no.	AP039447	AP039448	AP039449	AP039450

Full text

ANNOUNCEMENT

Bacteria-induced rot symptoms have been a challenge to onion cultivation in Japan, and Pseudomonas marginalis is considered as a causal agent (1). Previously, we re-identified P. marginalis strains obtained from onions as a novel species, Pseudomonas allii (2). During quality control in the Genebank Project of National Agriculture and Food Research Organization, three strains obtained from various crops (3–5, Table 1) showed nearly identical 16S rRNA and rpoD sequences to the type strain MAFF 301514. It is important to consider whether P. allii is a multi-host pathogen or an aggregate of heterogeneous strains, each with a limited host range (5). Here, we present the genome sequences of strains related to P. allii, including that of the type strain.

The strains were isolated from Honshu Island in Japan, including prefectures located at the northern (Aomori) and western (Yamaguchi) ends (Table 1), and were preserved under the Genebank Project as a lyophilized state. The strains were cultured on standard methods agar (Nissui) for 3 or 4 days at 28°C. Genomic DNA was extracted using the NucleoBond HMW DNA kit (Macherey-Nagel). Intact DNA was used for the library preparations.

A long-read sequencing library was prepared using the Rapid Barcoding Kit (SQK-RBK004, Oxford Nanopore Technologies [ONT]) and sequenced using PromethION with an R9.4.1 flow cell (FLO-PRO002, ONT). Raw ONT reads (Table 1) were filtered with >10 Phred quality score cutoff by Chopper version 0.5.0 (6). The filtered reads were assembled using Flye version 2.9.2 (7) with default options except for “-g 7 m --asm-coverage 50,” and the assembled circular genomes were polished using Medaka version 1.8.0 (https://github.com/nanoporetech/medaka) with the “r941_prom_hac_g507” model using the filtered reads.

Short-read sequencing libraries were prepared using Illumina DNA Prep (M) Tagmentation (Illumina), including the purification and size selection (ca. 500 bp), and 151 bp paired-end reads were sequenced using NovaSeq 6000 (Illumina) (Table 1). Adapter trimming and quality filtering were performed using fastp version 0.23.4 (8) with default settings except for the minimum length set to 151 and adapters specified by a sequencing company. Error corrections consisted of mapping with filtered Illumina reads using bwa-mem2 version 2.2.1 (9) with default except for “-a” option and polishing using Polypolish version 0.5.0 with default settings (10).

Genomes were annotated using the DDBJ Fast Annotation and Submission Tool (https://dfast.ddbj.nig.ac.jp/) with default settings except for the tRNAscan-SE and dnaA rotation function. Table 1 presents the statistics for the genomes determined in this study. OrthoANIu and digital DNA-DNA hybridization (dDDH) values (Table 1), calculated using the ANI Calculator (https://www.ezbiocloud.net/tools/ani) (11) and Genome-To-Genome Distance Calculator version 3.0 (https://ggdc.dsmz.de/ggdc.php#) (12), respectively, were higher than the Average Nucleotide Identity (ANI) (95%–96%) and dDDH (70%) thresholds for species delineation (13). These results show that the strains are the same species, and the genomes obtained here can be used as a starting point for analyzing intraspecific variations.