Ralstonia solanacearum, R. pseudosolanacearum, and R. syzygii are well known as causal agents of bacterial wilt, which results in severe agricultural losses, and are therefore considered important phytopathogenic bacteria [1, 2]. Because bacteriophages (phages) can infect and lyse Ralstonia spp., lytic phages have attracted considerable attention as a potential biocontrol agent for managing bacterial wilt disease [3]. Various phages capable of infecting Ralstonia spp. have been isolated [3]. Among them, six jumbo Ralstonia phages: RSL1, RSF1, RSL2, RP12, RP31, and RsoM2USA, having genomic DNA >200 kbp are considered to have a broad host range [4-8]. Furthermore, it has been reported that three jumbo Burkholderia phages: FLC6, FLC8, and FLC9, isolated from leaf litter compost have the ability to lyse multiple isolates of R. pseudosolanacearum [9-11]. To establish practical jumbo phage–based biocontrol agents with lytic potential against a broad range of pathogenic Ralstonia spp. in order to control bacterial wilt disease, further isolation of jumbo Ralstonia phages is necessary. In this study, two novel Ralstonia jumbo phages: FLC1-1B and FLC4-3B, were isolated from leaf litter compost, and their complete genomes were sequenced for proteomic tree analysis. Their host range, virus particle structure, and bacterial wilt disease–suppressive activity were also characterized.
Phage isolation, phage DNA extraction, electron microscopy observation of the phage particle, and host range analysis are described in the online Supplementary Information. Two phage strains, which were named FLC1-1B and FLC4-3B, were isolated from leaf litter compost. Electron microscope analysis revealed that both strains have icosahedral heads and contractile tails (Fig. S1), suggesting that they belong to the order Caudoviricetes.
The procedures for phage DNA library construction, DNA sequencing, and in silico data analysis are also described in the online Supplementary Information. The length of the complete genome DNA sequences of FLC1-1B and FLC4-3B were 290,008 bp (G+C content = 56.00%) and 291,257 bp (G+C content = 55.95%), respectively, and they were therefore classified as jumbo phages. A blastn search using the NCBI nt database was conducted as a nucleotide homology search of the complete genome sequence for FLC1-1B and FLC4-3B. The results indicated that the closest hit was Ralstonia phage RP12 (accession number NC_041911.1) in the genus Ripduovirus of the order Caudoviricetes. The query coverage and identity between RP12 and FLC1-1B were 35% and 77.40%, respectively. The query coverage and identity between FLC4-3B with RP12 were 35% and 77.38%, respectively. Meanwhile, 99.0% of the genomic sequence between FLC1-1B and FLC4-3B was similar (Fig. 1). According to a demarcation criterion proposed by a roadmap for genome-based phage taxonomy [12], if the product of the query coverage value times the identity value between RP12 and FLC1-1B or FLC4-3B is less than 0.70, then FLC1-1B or FLC4-3B should be classified into a different genus but not Ripduovirus. The product of the query coverage and identity of RP12 and FLC1-1B was 0.2709 (0.35 × 0.774 = 0.2709) and that of RP12 and FLC4-3B was 0.2708 (0.35 × 0.774 = 0.2708). Because both values were <0.07, FLC1-1B and FLC4-3B should be classified into a different genus. The number of predicted open reading frames (ORFs) in the complete genome of FLC1-1B and FLC4-3B were 309 and 310, respectively (Fig. 1). A total of 54 ORFs in each genome were annotated according to the annotation of RP12 (Fig. 1). Meanwhile 4 tRNA genes were detected in both genomes, whereas no rRNA genes were predicted (Fig. 1). A proteomic tree analysis also revealed that FLC1-1B and FLC4-3B belonged to the same clade (Fig. 2). Furthermore, a phylogenetic tree based on the amino acid sequences of the terminase large subunit and SbcC indicated a clade comprising FLC1-1B and FLC4-3B (Figs. S1 and S2), suggesting that their phylogenic relations are consistent with the relations of a proteomic tree. These results suggested that FLC1-1B and FLC4-3B should be classified into a different genus in the order Caudoviricetes. Host range analysis revealed that FLC1-1B and FLC4-3B lysed 5 and 3 out of 9 R. pseudosolanacearum isolates, respectively, while both phages were unable to lyse 3 isolates of R. syzygii subsp. indonesiensis (Table S2). Furthermore, treatment of tomato nursery plants with the Ralstonia phage FLC4-3B followed by inoculation with R. pseudosolanacearum significantly suppressed the occurrence of wilt disease (Fig. S3). These findings suggest that FLC1-1B and FLC4-3B can be used as biocontrol agents for managing bacterial wilt disease in tomato cultivation.