Xanthomonas campestris pv. campestris is an important bacterial plant pathogen of economic crops worldwide. It can infect cruciferous crops and cause black rot, leading to severe yield reduction [1]. Chemical treatment of bacterial infections can lead to the development of bacterial resistance and the accumulation of drugs in the soil, which can reduce control effectiveness or cause environmental problems [2]. Phages are a potential biocontrol agent that is environmentally safe and able to co-evolve with its host [3].
In this study, a novel bacteriophage AhaSv infecting X. campestris pv. campestris 8004 was isolated and characterized from lake water of Aha Lake National Wetland Park, Guiyang, Guizhou province, China. Enrichment, purification and lysate were prepared as previously described by James and Joseph [4]. Phage formed round clear plaques of about 0.8-1.5 mm in diameter on double-layer AGAR plates (Fig. 1A). The phage lysate was stained with 2% phosphotungstic acid, and the morphology of phage particles was observed using a JEM1400 transmission electron microscope (JEOL, Japan) at an accelerating voltage of 80 kV. Phage AhaSv was shown a siphoviral morphology with a icosahedral head (diameter of about 65.74 ± 1.46 nm) and a long noncontractile tail (length of about 219.91 ± 2.50 nm) (n=3) (Fig. 1B).
Genomic DNA was extracted from phage lysate using the TaKaRa Mini BEST Viral RNA/DNA Extraction Kit (Takara Clontech). Whole genome sequencing was performed using an Illumina NovaSeq sequencing platform (Shanghai Personalbio Technology Co., Ltd., China). Sequence data was evaluated for quality control using FastQC v0.11.7, the reads were filtered by AdapterRemoval v2.2.2 [5] and SOAPec v2.03 [6]. Contigs were assembled using A5-MiSeq v20160825 [7] and SPAdes v3.12.0 [8]. Open reading frames (ORFs) of the assembled genome were predicted by the RAST server (https://rast.nmpdr.org) [9]. The functions of ORFs were predicted by aligning the protein sequences using the NCBI server (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The tRNA genes in the genome sequences were searched online by tRNAscan-SE (http://lowelab.ucsc.edu/tRNAscan-SE/) [10].
The genome sequencing revealed that phage AhaSv had a linear dsDNA with a total length of 55,576 bp and a GC content of 63.23%. Phage AhaSv encodes a total of 71 ORFs (Supplementary Table S1), of which three ORFs (ORF2, 3 and 13) show no similarity to any of the annotated proteins. No tRNAs were found. These ORFs were classified into six groups according to their functions, including structure, DNA replication and repair, packaging, lysis, other functions, and hypothetical proteins. The conserved domains in the ORFs were identified using CD-Search server (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) (Supplementary Table S2). The genome mapping was perfomed using CGview server (https://proksee.ca/) [11] (Supplementary Fig. S1). Phage AhaSv was predicted to have a lytic lifestyle by the absence of integrase genes in its genome and the formation of clear plaques.
Multiple sequence alignments were performed using ClustalW algorithm, and the phylogenetic trees were generated by the neighbor-joining method using MEGA X software with 1,000 bootstrap replicates [12]. The proteome tree was constructed based on the whole genome sequence using VipTree (https://www.genome.jp/ viptree) [13]. The intergenomic similarities between phage AhaSv and the most closely related phages were calculated by Virus Intergenomic Distance Calculator (VIRIDIC) (http://rhea.icbm.uni-oldenburg.de/VIRIDIC/) [14]. Comparative genomic analysis was performed using Easyfig 2.2.3 [15].
BLASTn analysis showed that phage AhaSv had high sequence similarity with three phage strains: Xylella phage Salvo (NC_042345, 91% query coverage, 96.58% identity), Xanthomonas phage Seregon (ON189048, 95% query coverage, 94.39% identity), and Xylella phage Bacata (NC_052973, 91% query coverage, 96.24% identity). All three phages belong to the genus Salvovirus of the family Casjensviridae. Phylogenetic analysis based on the amino acid sequences of the terminase large subunit and DNA polymerase, as well as the whole genome sequences, showed that phage AhaSv was clustered and highly related to phages Seregon, Salvo and Bacata (Fig. 2), which was consistent with the results of BLASTn analysis.
According to VIRIDIC analysis, phage AhaSv shared the highest genomic identity of 90.6% with phage Seregon (Fig. 3A) and could thus be identified as a new species of an the genus Salvovirus (nucleotide similarity was less than 95% and greater than 70%) [16]. Comparative analysis of the whole genome sequences of phage AhaSv with those of phages Seregon and Salvo using Easyfig software showed that they were arranged withinin similar modules (Fig. 3B). Among the structure proteins, the tail fiber protein encoded by ORF39 of phage AhaSv showed low genetic homology with the other two phages, as indicated by the absence of gray shading in this region (Fig. 3B). Proteomic comparison using BLASTp server showed that of the 71 proteins encoded by AhaSv, 62 proteins shared identity with Seregon (minimum 61.08%, maximum 100%), and 62 proteins shared identity with Salvo (minimum 58.69%, maximum 100%).
In conclusion, a novel Xanthomonas phage AhaSv was isolated and identified as a member of the genus Salvovirus. The results provide a new genomic resource for future research of phage evolution and application.