The plant bacterial wilt caused by Ralstonia solanacearum has occurred widely and caused huge economic losses. It 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, 22]. As R. solanacearum species complexes(RSSC) were constituted by three different Ralstonia subspecies, R. solanacearum strains are highly variable and rich in genetic diversity[14]. Consequently, it is difficult to be effectively controlled by traditional managements. So that people have been looking for new control methods all the time. Using bacteriophages to control bacterial diseases could be a good choice. Bacteriophage is a virus and it was first discovered by British bacteriologist Frederick Twort (1915) and Canadian bacteriologist Felix d'Herelle (1917) [13] . The both of them found that the bacteria could be killed when they were infected by a lytic bacteriophage. In subsequent years, phages had been considered as a potentially control candidate for the managements of bacterial diseases. It also is an alternative replacement of chemical control due to the advantages of selectivity and specificity. Moreover, phage-based control therapy could minimize using chemical agents and reduce environmental pollution and chemical residues in crops. Therefore, more efforts have been made to use phage-based therapy to limit bacterial diseases in global crop cultivation and production [3, 13], which has resulted in many R. solanacearum phages have been isolated up to the present, including ϕRS551 [2], ϕRSY1 [5], ϕRS138 [20], ϕRSF1 [7], RsoP1IDN [1] et al.
In this study, we isolated a novel phage RPZH3 which could infect R. solanacearum with the method of the double agar overlay plaque assay [15] from the soil of tobacco field in Nanping city, Fujian province, the South China, using R. solanacearum strain TBRS-5 as a host bacterium. To obtain phage solution, the strain TBRS-5 was cultured first in broth, when the optical density (OD) value of the bacterial suspension reached 0.8 at wave length of 600 nm, a 1mL phage solution was added into the bacterial suspension, then cultured for further 18 h, the cultured suspension was filtered with 0.22-µm microporous membranes, then the phage solution was obtained ready.
The host range of phage RPZH3 was examined by spot test. The results showed that the eighteen R. solanacearum strains could be lysed by the phage RPZH3 among 30 tested strains (Table S1). Phage particles were precipitated with 10% PEG8000, and their corresponding morphology was observed under a transmission electron microscope HT7700 (Hitachi Co., Ltd. Japan). The data showed that phage RPZH3 had an icosahedron head with 75 ± 5 nm in diameter and a short tail of 15 ± 5 nm in length (Fig. 1A), indicating that phage RPZH3 maybe belong to the family Podoviridae in the order Caudovirales.
To determine the optimal multiplicity of infection (MOI), the solution of the R. solanacearum strain TBRS-5 in exponential growth phase were mixed with equal volumes of in different titers, then put in a shaker, shaking for 12 h in 130 r/min, at 28 °C, and assayed the phage titer. The results showed that the optimal MOI of phage RPZH3 was 0.001 and the progeny titer was 2.37×1010 pfu/mL. The infection cycle of phage RPZH3 was characterized with one-step growth experiment, a 5-mL sample was taken at 10 min-intervals over a period of 2.5 h, and determine the phage titer, the latent and rise periods of phage RPZH3 were about 80 min and 60 min respectively, and the average burst size was 27 pfu/cfu (Fig. 1B). Besides, the thermal and acid-base stability of phage RPZH3 at different temperatures (45 °C - 90 °C) and pH value (pH1 - 13) was tested by the double-layer method with incubation for 1 h. The results showed that the phage could maintain high activity at the temperatures of 45°C -60℃ or pH 4-12 (Fig. 1C, 1D).
The genomic DNA of the phage RPZH3 was extracted from phage particles using protein K and sodium dodecyl sulfate (SDS)[19]. The whole genome sequencing of phage RPZH3 was performed by de novo sequencing based on the second-generation high-throughput sequencing strategy (Oebiotech, Shanghai China). For raw data, we used the software package Trimmatic to remove low-quality bases, adapters, and low-quality reads[8]. Genome assembly was performed with MITObim Software[11]. Gene annotation and open reading frame prediction were performed with GeneMarks (http://exon.biology.gatech.edu/ GeneMark/)[6]. tRNA prediction was carried out by using tRNAscan-SE (http://lowelab.ucsc. edu/tRNAscan-SE/)[16]. Homology search was performed by using BLAST on the NCBI website (https://blast.ncbi.nlm.nih.gov/ Blast.cgi) [4]. The genome circle map was drawn in software of DNAplotter[9].
The double DNA sequences of phage RPZH3 contained a length of 65,958 bp with a GC mean content of 64.93%. The complete genome sequence of phage RPZH3, as query input, was run by BLASTn in NCBI website and results based on sequence similarity hit seven strains of phages, including Ralstonia phage GP4 (78% query cover, 94.84% identity, accession ID: MH638294), Burkholderia phage Bcep22 (15% query cover, 80.54% identity, accession ID: AY349011), Burkholderia BcepIL02 (14% query cover, 80.82% identity, accession ID: FJ937737), Burkholderia BcepMigl (14% query cover, 80.22% identity, accession ID: JX104231), DC1 (13% query cover, 81.22%, accession ID: JN662425), Ralstonia phage phiRSP (2% query cover, 83.45% identity, accession ID: MH252365) and Ralstonia phage RSK1 (3% query cover, 92.98% identity, accession ID: AB863625) [10, 17, 21]. All of these seven bacteriophages belonged to the Bcep22-like viruses of family Podoviridae in the order Caudovirales. Although the Phage RPZH3 showed high homology to phage GP4, the whole genome length of phage RPZH3 was longer by 4,829 bp than that of phage GP4, and the GC content was higher by about 0.93%. All the data above suggested that phage RPZH3 may be a new member of family Podoviridae.
In complete genome sequence of phage RPZH3, a total of 93 putative ORFs were identified. The ORFs’ sequences ranged in length from 99 to 9,345 bp and the lengths of predicted encoded proteins significantly varied from 33 to 3,115 amino acids. The total length of all the ORFs covered 58,768 bp which represent 89.1% of complete genome sequence. In the initiation codons of all the ORFs, a total of 84 ORFs initiated with the sequence of ATG, 5 with GTG, 3 with TTG, and 1 with CTG. Besides, 18 ORFs were found in sense strand and 75 ORFs were found in the antisense strand. Among the 93 ORFs (Fig. 2, Table S2), 5 ORFs were identified as structural genes encoding proteins such as phage capsid protein (ORF 5), portal protein (ORF 12, 14), and tail fiber protein (ORF 83, 84); 27 ORFs were identified as functional genes, encoding proteins such as phage virion associated protein (ORF 4, 77, 79, 82, 85, 87, 88, 90, 91), DarB-like antirestriction protein (ORF 45, 75, 76), DNA-binding protein (ORF 53, 54, 61, 62), DnaC-like protein (ORF 45), RecT-like protein (ORF 60), DUF protein (ORF 50, 72 and pyocin activator protein (ORF 65); 10 ORFs were identified as enzyme genes which encoded proteins such as phage terminase subunit (ORF 16, 35), HNH homing endonuclease II (ORF 37), RecB-like endonuclease (ORF 59), DNA polymerase III subunit beta (ORF 63), tyrosine recombinase (ORF 67), and acyl-CoA N-acyltransferase (ORF 80); 5 ORFs were identified as phage transcriptional genes that encoded corresponding proteins as transcriptional regulator (ORF 26, 28, 55, 57, 58); and 46 ORFs were responsible for encoding hypothetical proteins with unknown functions. In addition, the rests of three ORFs were not found in the GenBank database.
In order to analyze the relationship on the phage major capsid protein (ORF 5) sequences between phage RPZH3 and other reported bacteriophages, PhyloSuite version 1.2.1 and IQ-tree version 1.6.8 were used to construct a phylogenetic tree with LG+F+G4 as the best model by maximum-likelihood method (ML), and with the 1000 bootstrap replicates[18, 23]. The phylogenetic tree showed that different phages congregated on different branches. The phage RPZH3 was congregated with phage GP4, phage Bcep22, phage BcepIL02, phage BcepMigl, and phage DC1 (Fig. 3). These five bacteriophages belonged to the Bcep22-like viruses of family Podoviridae in the order Caudovirales. The combination of the results of complete genome sequence alignment in the GenBank database, the identification and phylogenetic tree of phage RPZH3 further indicated that it was a novel Bcep22-like virus infecting R. solanacearum.
In conclusion, the novel lytic Ralstonia phage RPZH3 was isolated and characterized which had a double-stranded DNA genome of 65958 bp in size and was most closely related to the phage GP4.The morphology and the results of genomic and phylogenetic analysis showed that the phage RPZH3 was a novel Bcep22-like virus of family Podoviridae in the order Caudovirales
Nucleotide sequence accession number
The complete genome sequence of phage RPZH3 was deposited in GenBank under the accession number of MZ870514.