Draft Genome Sequences of Two Xanthomonas fragariae Strains

Xanthomonas fragariae is the causal agent of angular leaf spot of strawberry. Short-read sequences were generated for two X. fragariae strains with different virulence phenotypes on the Illumina HiSeq 2000 platform. The genome sequences will contribute to a better understanding of pathogen evolution and the genes contributing to virulence in X. fragariae.

ABSTRACT

Xanthomonas fragariae is the causal agent of angular leaf spot of strawberry. Short-read sequences were generated for two X. fragariae strains with different virulence phenotypes on the Illumina HiSeq 2000 platform. These genome sequences will contribute to a better understanding of pathogen evolution and the genes contributing to virulence in X. fragariae.

ANNOUNCEMENT

Xanthomonas fragariae causes angular leaf spot of strawberry, a problematic disease in strawberry nursery production (1–4). Genome sequences of X. fragariae strains with similar virulences have been published (1–3). Here, we present the draft genome sequence assemblies of two X. fragariae strains reported as having different virulence phenotypes. Xf100 and Xf1431 were isolated in the 1990s from symptomatic plants in Florida; Xf1431 was reported as nonpathogenic on cultivar Dover (5, 6). The strains were received from John Hartung (USDA-ARS) and stored at −80°C.

A single colony per strain was transferred from a 3- to 4-day-old culture on sucrose peptone agar into sucrose peptone broth for 1 to 2 days at 25°C; genomic DNA was extracted from the colonies using a DNeasy blood and tissue kit (Qiagen, Germantown, MD) and sent to BGI (Shenzhen, Guangdong, China) for sequencing on the Illumina HiSeq 2000 system. For each strain, two libraries (∼470-bp and ∼6,300-bp inserts) were prepared (7) and sequenced with 90- and 50-bp reads, respectively. The sequences were processed with bioinformatics tools using default parameters unless stated otherwise. The raw reads were quality controlled with Trimmomatic v0.39 (8). After eliminating the low-quality reads, the clean reads (>160× coverage per strain) were used for de novo genome assembly with SOAPdenovo r242 (9, 10). Gene prediction and annotation were performed with the prokaryotic genome annotation program PROKKA v1.14.5 (11). The similarity of amino acid sequences to the LMG-25863 reference genome (GenBank accession no. GCA_000376745.1) (3) was measured using AAI-Profiler (12). The trimmed and paired reads were aligned to LMG-25863 with HISAT2 v2.2.1 (13); genomic variants were identified with the GATK haplotype caller v4.1.9.0 (14). Variants in the coding regions were functionally annotated with SnpEff v5.0 (15). The LMG-25863 protein sequences from the types II, III, IV, and VI secretion systems and the toxin-antitoxin (TA) system were compared against those of Xf100 and Xf1431 using BLASTP.

Xf100 and Xf1431 are highly similar to LMG-25863 (Table 1); all strains lacked several critical genes for pathogen-host interactions found in other xanthomonads, including the xcs genes in the type II secretion system, phenolics degradation I and II, glyoxylate shunt, and xylan degradation clusters I to III. All three strains share the same type II-xps, III, IV, and VI secretion systems, except for the ClpB T6SS protein present in Xf100 and Xf1431 but highly fragmented in LMG-25863. For genes in the TA system, Xf1431 harbors two copies of RelB and RelE, while Xf100 and LMG-25863 each have one; DinJ and YafO in Xf100 shared 44% and 88% identity to those in Xf1431 and LMG-25863. Only 148 variant sites (142 single nucleotide polymorphisms [SNPs], 3 insertions, and 3 deletions) were found between Xf100 and Xf1431, which may explain their different virulence phenotypes, but they shared increased variation (685 SNPs, 30 insertions, and 34 deletions) from LMG-25863 (isolated in Belgium), which could be attributed to their geographical distance. The genome sequences of Xf100 and Xf1431 will be important additional resources for understanding virulence in X. fragariae.

TABLE 1.

Sequencing statistics of Xf100 and Xf1431 in comparison to the reference X. fragariae strain LMG-25863

Strain	No. of reads	No. of bases (Mbp)	Assembly size (bp)	No. of contigs	N50 (kbp)	GC content (%)	No. of protein-coding genesa
Xf100	5,365,874	755	4,155,724	136	77.3	62.30	3,841
Xf1431	4,678,916	688	4,203,715	121	91.5	62.27	3,896
LMG-25863	8,185,858	1,022	4,182,545	96	131.4	62.20	3,919

^aAs predicted with PROKKA.

Data availability.

The assembled sequences are available under GenBank assembly accession no. GCA_016792245.1 and GCA_016792185.1; the raw reads are available under SRA accession no. SRR13617564 and SRR13617565 for Xf100 and SRR13618019 and SRR13618020 for Xf1431.