Xanthomonas euvesicatoria is a Gram-negative phytopathogenic bacterium and is one of the four representative Xanthomonas species (e.g., X. euvesicatoria, X. gardneri, X. perforans, and X. vesicatoria) responsible for bacterial spot disease (BSD) in pepper and tomato that cause significant economic losses worldwide (Sharma et al., 2014; Osdaghi et al., 2021). Traditionally, BSD management has relied on chemical pesticides, antibiotics, and copper-based bactericides in agricultural practices (Hernández-Huerta et al., 2021). However, the emergence of antimicrobial-resistant phytopathogens and growing environmental concerns have led to the development of alternative control methods (Lugo et al., 2013; Bosmans et al., 2016). Bacteriophages (phages) have been proposed as a novel biocontrol strategy for phytopathogens (Garvey, 2022), and several studies have demonstrated their effectiveness in controlling BSD caused by Xanthomonas species in different crops, including tomato (Flaherty et al., 2000) and pepper (Gašić et al., 2018). Despite the global assessment and demonstrated capacity of phages to control BSD in economically important crops, limited information is available regarding phages that specifically infect X. euvesicatoria. In Korea, a recent study reported that X. euvesicatoria is the prevalent cause of BSD in peppers (Kyeon et al., 2016), therefore, finding a novel phage to control this pathogen is urgently needed. Here, we report the complete genome and detailed information of a new phage, vB_XeuM-YB23, that infects X. euvesicatoria.

Xanthomonas phage vB_XeuM-YB23 was isolated from a sewage sample collected in Cheongju, South Korea, using X. euvesicatoria KACC 18722, a bacterial strain originally isolated from the Korean chili pepper (Capsicum annuum), as the host. The morphological characteristics, biological properties, bacteriolytic activity, and environmental stability of the phage were examined as previously described (Park et al., 2023). Phage genomic DNA was extracted using the phenol-chloroform method and sequenced on an Illumina HiSeq X-10 platform by preparing a DNA library using a TruSeq Nano DNA library kit (Illumina). The resulting paired-end reads (5,739,563,152 bp; 38,010,352 reads) were assembled de novo using SPAdes (v.3.13.0), and the genome sequences were annotated using RAST (https://rast.nmpdr.org). Putative open reading frames (ORFs) were functionally analyzed using BLASTP (https://blast.ncbi.nlm.nih.gov), InterProScan (https://www.ebi.ac.uk/interpro), DeepTMHMM (https://dtu.biolib.com/DeepTMHMM), and SignalP (https://services.healthtech.dtu.dk/services/SignalP-5.0). The presence of tRNAs was determined using ARAGORN (https://www.trna.se), while virulence factors (VFs) and antimicrobial resistance genes (ARGs) were predicted using VFDB (http://www.mgc.ac.cn/cgi-bin/VFs/v5) and ARG-ANNOT (https://ifr48.timone.univ-mrs.fr/blast/arg-annot_nt.html), respectively. The phage genome map was visualized using Proksee (https://proksee.ca), and a genome-based phylogenetic tree was constructed using the VICTOR software (https://proksee.ca). Genomic comparisons were performed using VIRIDIC (http://viridic.icbm.de) and EasyFig (https://mjsull.github.io/Easyfig) to identify similarities and differences between the vB_XeuM-YB23 genome and those of other related phages.

The phage vB_XeuM-YB23 had an icosahedral head and a long contractile tail, indicating that it belongs to the family Myoviridae (Supplementary data Fig. S1A). Additionally, its broad host range (Supplementary data Table S1), bacteriolytic activity, and environmental stability (Supplementary data Fig. S1B–D) indicated its potential as a biocontrol agent against BSD.

The circular genome of phage vB_XeuM-YB23 was 63,787 bp in length, composed of 68.1% G + C content, 80 protein-coding genes, and one tRNA gene (Table 1). Functional annotation of the 80 predicted putative ORFs identified 16 genes related to DNA replication and nucleotide metabolism, eight genes encoding tail proteins, two genes associated with host cell lysis, 15 genes responsible for structure and packaging, one gene with additional function, and 38 genes encoding hypothetical proteins (Fig. 1A and Supplementary data Table S2). Genome-based phylogenetic analysis of phage vB_XeuM-YB23 and other phage genomes in the subfamily Bradleyvirinae demonstrated that the phage clustered with the genus Bosavirus (Fig. 1B). Moreover, the VIRIDIC heatmap analysis represented the intergenomic similarities among six phages of the genus Bosavirus, and phage vB_XeuM-YB23 exhibited the highest nucleotide identity (99.4%) with Xanthomonas phage Xp12 (Supplementary data Fig. S2A). A BLASTN analysis also revealed that the genome of phage vB_XeuM-YB23 had the highest similarity (99.4% with > 99.0% coverage) to Xanthomonas phage Xp12 (NC_073033). Further comparative analysis showed that 47 proteins (58.8%) encoded by phage vB_XeuM-YB23 were almost identical to those encoded by phage Xp12, with similarities ranging from 95.2 to 100%. Although phage vB_XeuM-YB23 encoded putative endolysin (ORF 50) with 99.4% similarity to that of phage Xp12, the gene encoding o-spanin (ORF 51) was absent from the Xp12 genome. Endolysins and spanins are key components of the lysis mechanisms of phages that infect several Gram-negative bacteria (Gontijo et al., 2021). In particular, spanin is essential for disrupting the bacterial outer membrane, which allows for the release of phage progeny from host cells (Cahill and Young, 2019). Therefore, phage vB_XeuM-YB23 could possess a distinct lysis system when compared to the previously reported Xanthomonas phages (Gašić et al., 2018).

Table 1

Features of Xanthomonas phage vB_XeuM-YB23 genome

Features	Value
Genome size (bp)	63,787
G + C content (%)	68.1
Contigs	1
Total genes	81
Protein-coding genes (CDS)	80
tRNA	1

Fig. 1. Genomic characteristics of Xanthomonas phage vB_XeuM-YB23. (A) A map of the whole genome of phage vB_XeuM-YB23 constructed with Proksee. The outer color ring represents the ORFs with predicted functions: hypothetical proteins (light gray), tail proteins (red), structure and packaging proteins (pink), DNA replication and metabolism-related proteins (purple), lysis-related proteins (light blue), and additional functional proteins (blue). The middle ring indicates G + C content and the inner rings colored in light pink and yellow indicate the positive and negative G + C skew, respectively. (B) Genome-based phylogenetic tree of phage vB_XeuM-YB23 was generated with 17 Bradleyvirinae phages using VICTOR and the Pelczarvirinae phage PaoPu (NC_052932) as an outgroup.

The presence of both endolysin and o-spanin genes suggests a more efficient lysis mechanism that is a unique lysis system of phage vB_XeuM-YB23. This finding has significant implications for its potential application as a biocontrol agent for X. euvesicatoria, which causes BSD in economically important crops such as pepper.

Nucleotide sequence accession number

The complete genome sequence of Xanthomonas phage vB_XeuM-YB23 was deposited in GenBank under the accession number PP756300 and the phage has been deposited in the Korean Collection for Type Cultures (KCTC) under the accession number KCTC 4851.

고추 세균성점무늬병(bacterial spot disease, BSD)의 주요 원인균인 Xanthomonas euvesicatoria는 전 세계적으로 고추와 토마토 작물에 심각한 경제적 손실을 초래하는 식물병원성 세균이다. 우리는 국내 고추에서 분리한 X. euvesicatoria KACC 18722를 숙주로 하는 신규 박테리오파지 vB_XeuM-YB23의 전체 유전체 서열을 보고한다. vB_XeuM-YB23의 유전체는 63,787 bp 크기에 G + C 함량이 68.1 %이며, 80개의 단백질 코딩 유전자와 1개의 tRNA 유전자를 포함하고 있다. 유전체 기반 계통 분석 결과, 분리된 파지는 Bradleyvirinae 아과의 Bosavirus 속과 함께 군집을 이루었으며, endolysin과 o-spanin 유전자를 모두 보유하여 효율적인 용균성 기작을 나타내는 독특한 용균 시스템을 지니고 있었다. 이는 근연종인 Xanthomonas 파지 Xp12와 비교했을 때 보다 효과적인 용균 기작을 암시한다. 이 파지는 넓은 숙주 범위를 보였으며, 용균 활성과 환경 안정성을 입증하여 고추 및 관련 작물의 BSD에 대한 생물학적 방제제로서의 가능성을 시사했다. 이러한 결과를 바탕으로, 새로 분리된 Xanthomonas 파지 vB_XeuM-YB23은 Bosavirus 속의 새로운 구성원임을 확인하였으며, 이 파지의 유전체 정보는 Xanthomonas 파지의 생물다양성과 분류에 대한 통찰력을 제공할 뿐만 아니라, 고추 작물에서 X. euvesicatoria에 의해 발생하는 BSD 제어를 위한 화학 농약의 대안으로서 잠재적 응용 가능성을 시사한다.

This research was supported by the Development of Technology for the Biomaterialization of Marine Fishery Byproducts of the Korea Institute of Marine Science & Technology Promotion (KIMST) funded by the Ministry of Oceans and Fisheries (KIMST-20220128) and also supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (RS-2024-00336046).

All authors disclose no conflicts of interest relevant to this research.