Characterization and complete genome sequence analysis of the novel phage RPZH3 infecting Ralstonia solanacearum

A novel lytic Ralstonia phage, RPZH3, was isolated from the soil of a tobacco field via a double agar overlay plaque assay. The phage has an icosahedral head 75 ± 5 nm in diameter with a short tail about 15 ± 5 nm in length. It was able to infect 18 out of 30 tested strains of R. solanacearum isolated from tobacco, sweet potato, tomato, pepper, and eggplant. The latent period of the phage was 80 min, and the burst period was 60 min with a burst size of about 27 pfu/cell. The phage was stable at pH 4–12 at 28°C, and it was also stable at temperatures from 45°C to 60°C at pH 7.0. The complete genome of phage RPZH3 consists of 65,958 bp, with a GC content of 64.93%. The genome contains 93 open reading frames (ORFs) and encodes a tRNA for cysteine. Nucleotide sequence alignment and phylogenetic analysis indicated that RPZH3 is a new member of the genus Gervaisevirus belonging to the class Caudoviricetes.

new control methods are sought. Using bacteriophages to control this bacterial disease could be a good choice. Bacteriophages are viruses that were first discovered by British bacteriologist Frederick Twort (1915) and Canadian bacteriologist Felix d'Herelle (1917) [13]. Both found that bacteria were killed when they were infected by lytic bacteriophages. In subsequent years, phages were considered as potential control agents for bacterial pathogens and as an alternative to chemical control due to their advantages of selectivity and specificity. Their use has also reduced environmental pollution and chemical residues in crops. Therefore, increasing efforts have been made to use phagebased therapies to limit bacterial diseases in global crop cultivation and production [3,13]. Many R. solanacearum phages have been isolated, including ϕRS551 [2], ϕRSY1 [5], ϕRS138 [24], ϕRSF1 [7], and RsoP1IDN [1].
In this study, R. solanacearum strain TBRS-5 was selected as a phage host. Phage RPZH3 was successfully isolated from the soil of a tobacco field in the city of Nanping, Fujian province, southern China, using a double agar overlay plaque assay [15]. To obtain a phage lysate, strain TBRS-5 was cultured in CPG liquid medium (peptone, 10 g; casein, 1 g; glucose, 5 g; water, 1000 mL; pH 7.0) until its optical density (OD 600 ) reached 0.8. A 1-mL phage Plant bacterial wilt caused by Ralstonia solanacearum occurs widely and causes huge economic losses. The pathogen has a broad host range and can infect more than 450 species of plants in 54 families, including many economically important crops, such as tobacco, tomato, pepper, and potato [12,27]. As R. solanacearum is a species complex composed of three different Ralstonia subspecies, R. solanacearum strains are highly variable and rich in genetic diversity [14]. Consequently, it is difficult to effectively control the pathogen through traditional management, and  Table S1). Phage particles were precipitated with 10% PEG 8000 and purified by centrifugation in 30% sucrose. Examination using an HT7700 transmission electron microscope (Hitachi Co., Ltd. Japan) showed that phage RPZH3 has an icosahedral head with a diameter of 75 ± 5 nm and a short tail about 15 ± 5 nm in length (Fig. 1A).
To determine the optimal multiplicity of infection (MOI) [18], R. solanacearum strain TBRS-5 was cultured in NB (nutrient broth) medium at 28°C until its concentration was 1 × 10 8 cfu/mL. It was then mixed with an equal volume of phage suspensions of different titers (1×10 9 , 1×10 8 , 1×10 7 , 1×10 6 , 1×10 5 , and 1×10 4 pfu/mL), resulting in different MOIs (10, 1, 0.1, 0.01, 0.001, and 0.0001 pfu/cfu). After incubation for 12 h at 28°C with rotary shaking at 130 rpm, the phage titers were immediately determined using the double agar overlay plaque assay. The results showed that the optimal MOI for phage RPZH3 infection was 0.001 (pfu/cfu), and the highest progeny titer was 2.37 × 10 10 pfu/ mL. The infection cycle of phage RPZH3 was characterized using a one-step growth experiment. A 0.5-mL sample was taken every 10 min for 2.5 h, and the phage titer was determined. The results showed that the latent and burst periods of phage RPZH3 were about 80 min and 60 min, respectively, and the average burst size was 27 pfu/cell (Fig. 1B).
The genomic DNA of phage RPZH3 was extracted from phage particles using protein K and sodium dodecyl sulfate (SDS) [22]. The complete genome sequence was determined by de novo sequencing based on a second-generation high-throughput sequencing strategy (Oebiotech, Shanghai China). The software package Trimmomatic was used to remove low-quality bases, adapters, and low-quality reads from the raw data [8]. Genome assembly was performed using MITObim software [11]. Gene annotation and open reading frame prediction were performed using GeneMarkS (http://exon.biology.gatech.edu/GeneMark/) [6]. tRNAencoding genes were identified using tRNAscan-SE (http:// lowelab.ucsc.edu/ tRNAscan-SE/) [16]. A homology search of the NR database was performed using BLASTn, and putative functions of the proteins encoded by the ORFs were annotated by searching the non-redundant protein database on the NCBI website (https://blast.ncbi.nlm.nih. gov) [4] and the Conserved Domain Database (https://www. ncbi.nlm.nih.gov/cdd/) [17]. The genome organization and annotation were visualized using Easyfig software [23].
The double-stranded DNA sequence of phage RPZH3 consists of 65,958 bp with a GC content of 64.93%.
Analysis of the raw Illumina reads using PhageTerm [9] did not allow the packaging mechanism of the phage to be predicted, and the genome did not have obvious termini. This situation has been reported previously for other phages such as Enterobacteria phage T4 [9]. A BLAST search of the NR database using the complete genome sequence showed that phage RPZH3 had a high degree of similarity to three phage strains: Ralstonia phage Gervaise (81% query coverage, 96.06% identity, accession ID: NC_054963), Ralstonia phage Darius (79% query coverage, 96.03% identity, accession ID: MT740732), and Ralstonia phage GP4 (78% query coverage, 94.84% identity, accession ID: MH638294) [10,19,26]. According to the latest ICTV taxonomy report, all of these bacteriophages belong to the genus Gervaisevirus of the class Caudoviricetes. Analysis using VIRIDIC software [20] showed that the phage RPZH3 has 78.5% intergenomic similarity and a 0.9 genome length fraction with phage Gervaise. These results suggest that phage RPZH3 may be considered a new member of the genus Gervaisevirus.
Ninety-three putative ORFs, ranging from 99 to 9345 bp in length, were identified in the phage RPZH3 genome, which resulted in a coding percentage of 89.1%. The start codon of most of the predicted ORFs (84 ORFs, 90.23%) was ATG, five ORFs (ORF 15, 30, 58, 76, and 86) started with GTG, and three ORFs (ORF 12, 14, and 75) started with TTG, representing 5.38% and 3.23% of the ORFs respectively. Only ORF 93 started with CTG. Eighteen ORFs were found in the sense strand and 75 ORFs were found in the antisense strand. Among the 93 ORFs, 46 were identified using NCBI BLASTp as functional genes, and 47 were identified as encoding hypothetical proteins with unknown functions (Fig. 2, Supplementary Table S2). Using CDD search, structural domains were identified in only 35 ORFs (Supplementary Table S2). Among the 46 functional genes (Fig. 2), five ORFs were identified as putative structural genes, encoding a phage capsid protein (ORF 5), portal proteins (ORFs 12 and 14), and tail fiber proteins (ORFs IQ-tree version 1.6.8 were used to construct a phylogenetic tree by the maximum-likelihood (ML) method with LG + F + G4 as the best-fit model with 1000 bootstrap replicates [21,28]. The phylogenetic analysis showed that different phages clustered in different branches. Phage RPZH3 was associated with phage GP4 and phage Gervaise (Fig. 3), which belong to the genus Gervaisevirus of the class Caudoviricetes. The combination of the results of the complete genome sequence alignment and the phylogenetic analysis indicated that phage RPZH3 is a novel member of the genus Gervaisevirus virus infecting R. solanacearum.
In conclusion, the novel lytic Ralstonia phage, RPZH3, was isolated and characterized. It has a double-stranded DNA genome 65,958 bp in size and is most closely related to the phages Gervaise and GP4. Its morphology and the results of genomic and phylogenetic analyses show that phage RPZH3 is a novel member of the genus Gervaisevirus in the class Caudoviricetes. In order to analyze relationships between phage RPZH3 and other bacteriophages based on the phage major capsid protein (ORF 5) sequences, PhyloSuite version 1.2.1 and