Molecular and biological characterization of ϕRs551, a filamentous bacteriophage isolated from a race 3 biovar 2 strain of Ralstonia solanacearum.

A filamentous bacteriophage, designated ϕRs551, was isolated and purified from the quarantine and select agent phytopathogen Ralstonia solanacearum race 3 biovar 2 strain UW551 (phylotype IIB sequevar 1) grown under normal culture conditions. Electron microscopy suggested that ϕRs551 is a member of the family Inoviridae, and is about 1200 nm long and 7 nm wide. ϕRs551 has a genome of 7929 nucleotides containing 14 open reading frames, and is the first isolated virion that contains a resolvase (ORF13) and putative type-2 phage repressor (ORF14). Unlike other R. solanacearum phages isolated from soil, the genome sequence of ϕRs551 is not only 100% identical to its prophage sequence in the deposited genome of R. solanacearum strain UW551 from which the phage was isolated, but is also surprisingly found with 100% identity in the deposited genomes of 10 other phylotype II sequevar 1 strains of R. solanacearum. Furthermore, it is homologous to genome RS-09-161, resulting in the identification of a new prophage, designated RSM10, in a R. solanacearum strain from India. When ORF13 and a core attP site of ϕRs551 were either deleted individually or in combination, phage integration was not observed, suggesting that similar to other filamentous R. solanacearum ϕRSM phages, ϕRs551 relies on its resolvase and the core att sequence for site-directed integration into its susceptible R. solanacearum strain. The integration occurred four hours after phage infection. Infection of a susceptible R. solanacearum strain RUN302 by ϕRs551 resulted in less fluidal colonies and EPS production, and reduced motilities of the bacterium. Interestingly, infection of RUN302 by ϕRs551 also resulted in reduced virulence, rather than enhanced or loss of virulence caused by other ϕRSM phages. Study of bacteriophages of R. solanacearum would contribute to a better understanding of the phage-bacterium-environment interactions in order to develop integrated management strategies to combat R. solanacearum

Susceptibility of <i>R</i>. <i>solanacearum</i> strains to ϕRs551.

<p>Susceptibility of <i>R</i>. <i>solanacearum</i> strains to ϕRs551.</p

Molecular and biological characterization of ϕRs551, a filamentous bacteriophage isolated from a race 3 biovar 2 strain of <i>Ralstonia solanacearum</i>

<div><p>A filamentous bacteriophage, designated ϕRs551, was isolated and purified from the quarantine and select agent phytopathogen <i>Ralstonia solanacearum</i> race 3 biovar 2 strain UW551 (phylotype IIB sequevar 1) grown under normal culture conditions. Electron microscopy suggested that ϕRs551 is a member of the family <i>Inoviridae</i>, and is about 1200 nm long and 7 nm wide. ϕRs551 has a genome of 7929 nucleotides containing 14 open reading frames, and is the first isolated virion that contains a resolvase (ORF13) and putative type-2 phage repressor (ORF14). Unlike other <i>R</i>. <i>solanacearum</i> phages isolated from soil, the genome sequence of ϕRs551 is not only 100% identical to its prophage sequence in the deposited genome of <i>R</i>. <i>solanacearum</i> strain UW551 from which the phage was isolated, but is also surprisingly found with 100% identity in the deposited genomes of 10 other phylotype II sequevar 1 strains of <i>R</i>. <i>solanacearum</i>. Furthermore, it is homologous to genome RS-09-161, resulting in the identification of a new prophage, designated RSM10, in a <i>R</i>. <i>solanacearum</i> strain from India. When ORF13 and a core <i>attP</i> site of ϕRs551 were either deleted individually or in combination, phage integration was not observed, suggesting that similar to other filamentous <i>R</i>. <i>solanacearum</i> ϕRSM phages, ϕRs551 relies on its resolvase and the core <i>att</i> sequence for site-directed integration into its susceptible <i>R</i>. <i>solanacearum</i> strain. The integration occurred four hours after phage infection. Infection of a susceptible <i>R</i>. <i>solanacearum</i> strain RUN302 by ϕRs551 resulted in less fluidal colonies and EPS production, and reduced motilities of the bacterium. Interestingly, infection of RUN302 by ϕRs551 also resulted in reduced virulence, rather than enhanced or loss of virulence caused by other ϕRSM phages. Study of bacteriophages of <i>R</i>. <i>solanacearum</i> would contribute to a better understanding of the phage-bacterium-environment interactions in order to develop integrated management strategies to combat <i>R</i>. <i>solanacearum</i>.</p></div

Effect of ϕRs551 on virulence of <i>R</i>. <i>solancearum</i> strain RUN302 in tomato.

<p>Three-week-old tomato plants were inoculated by pouring 50 ml (5 x 10⁷ cells/ml) of wild-type or ϕRs551-infected <i>R</i>. <i>solanacearum</i> RUN302 strain into each pot. A, appearance of the inoculated plants 10 days after inoculation with the wide type (left) or ϕRs551-infected (right) RUN302 strain. B, virulence of the uninfected wild type (diamonds) and ϕRs551-infected (squares) RUN302 strains. Plants were rated on a disease index ranging from 0 (healthy) to 4 (76 to 100% leaves wilted) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185034#pone.0185034.ref031" target="_blank">31</a>]. Points shown are means of three separate experiments, each containing 10 plants per treatment. Bars indicate standard errors.</p

List of primer pairs designed and used in this study.

<p>List of primer pairs designed and used in this study.</p

Electron micrograph of the purified ϕRs551 virions.

<p>The black scale bar represents 200 nm.</p

List of annotated ORFs of ϕRs551and their BLASTp results.

<p>List of annotated ORFs of ϕRs551and their BLASTp results.</p

Genomic organization of the phage ϕRs551 and the prophage RSM10, with reference to M13 for general structure and comparison to related <i>Ralstonia</i> phages ϕRSM3 and ϕRS603.

<p>The numbers in parentheses indicate the length (aa) of the ORF. The arrows represent the direction of transcription of ORFs. ORFs similar to the ones in M13 are in gray. ORFs annotated as a putative integrase and phage repressor are in black and darker gray, respectively. Types 1 and 2 putative phage repressors are differentiated with horizontal lines for the former and vertical ones for the latter within the darker grey arrows. ORFL stands for ORF-like in ϕRSM10. The location of the core <i>att</i>P site is indicated only in ϕRs551.</p

Impact of ϕRs551 infection on <i>R</i>. <i>solanacearum</i> strain RUN302 host cells.

<p>Growth in CPG liquid medium (A) and on TZC plates (B), twitching (C), swimming (D), and swarming (E) activities of the wt (diamonds in A, left in B to E) and ϕRs551-infected (squares in A, right in B to E) RUN302 strains. Bacterial growth in CPG was measured at the absorbance of 600 nm and graphed every three hours for 48 hours. Bars indicate standard errors. Three microliters of bacterial suspension (10⁸ cells/ml) were inoculated in the center of the twitching (minimal agar medium), swimming (CPG with 0.3% (w/v) agar), and swarming (CPG with 0.7% (w/v)) assay plates, incubated in the dark at 28°C and photographed five days after inoculation (D and E) under a microscope (C).</p