φRSM1 and φRSM3 (φRSM phages) are filamentous phages (inoviruses) that infect Ralstonia solanacearum, the causative agent of bacterial wilt. Infection by φRSM phages causes several cultural and physiological changes to host cells, especially loss of virulence. In this study, we characterized changes related to the virulence in φRSM3-infected cells, including (i) reduced twitching motility and reduced amounts of type IV pili (Tfp), (ii) lower levels of β-1,4-endoglucanase (Egl) activity and extracellular polysaccharides (EPS) production, and (iii) reduced expression of certain genes (egl, pehC, phcA, phcB, pilT, and hrpB). The significantly lower levels of phcA and phcB expression in φRSM3-infected cells suggested that functional PhcA was insufficient to activate many virulence genes. Tomato plants injected with φRSM3-infected cells of different R. solanacearum strains did not show wilting symptoms. The virulence and virulence factors were restored when φRSM3-encoded orf15, the gene for a putative repressor-like protein, was disrupted. Expression levels of phcA as well as other virulence-related genes in φRSM3-ΔORF15-infected cells were comparable with those in wild-type cells, suggesting that orf15 of φRSM3 may repress phcA and, consequently, result in loss of virulence.

Ralstonia solanacearum is the causative agent of bacterial wilt in many important crops. ϕRSS1 is a filamentous phage that infects R. solanacearum strains. Upon infection, it alters the physiological state and the behavior of host cells. Here, we show that R. solanacearum infected by ϕRSS1 becomes more virulent on host plants. Some virulence and pathogenicity factors, such as extracellular polysaccharide (EPS) synthesis and twitching motility, increased in the bacterial host cells infected with ϕRSS1, resulting in early wilting. Tomato plants inoculated with ϕRSS1-infected bacteria wilted 2 to 3 days earlier than those inoculated with wild-type bacteria. Infection with ϕRSS1 induced early expression of phcA, the global virulence regulator. phcA expression was detected in ϕRSS1-infected cells at cell density as low as 10(4) CFU/ml. Filamentous phages are assembled on the host cell surface and many phage particles accumulate on the cell surface. These surface-associated phage particles (phage proteins) may change the cell surface nature (hydrophobicity) to give high local cell densities. ϕRSS1 infection also enhanced PilA and type IV pilin production, resulting in increased twitching motility.

The first Ralstonia -infecting bacteriophage from soil of the United States, designated RsoM1USA, was isolated from a tomato field in Florida. Electron microscopy revealed that phage RsoM1USA is member of the genus P2virus in the family Myoviridae with an icosahedral head of about 66 nm in diameter, a contractile tail of about 152 nm in length, and a long “neck.” Phage RsoM1USA infected 12 of the 30 tested R. solanacearum species complex strains collected worldwide in each of the three Ralstonia species: R. solanacearum , R. pseudosolanacearum , and R. syzygii . The phage completed its infection cycle 180 min post infection with a burst size of about 56 particles per cell. Phage RsoM1USA has a genome of 39,309 nucleotides containing 58 open reading frames (ORFs) and is closely related to Ralstonia phage RSA1 of the species Ralstonia virus RSA1 . The genomic organization of phage RsoM1USA is also similar to that of phage RSA1, but their integrases share no sequence homology. In addition, we determined that the integration of phage RsoM1USA into its susceptible R. solanacearum strain K60 is mediated by the 3′ 45-base portion of the threonine tRNA (TGT), not arginine tRNA (CCG) as reported for phage RSA1, confirming that the two phages use different mechanism for integration. Our proteomic analysis of the purified virions supported the annotation of the main structural proteins. Infection of a susceptible R. solanacearum strain RUN302 by phage RsoM1USA resulted in significantly reduced growth of the infected bacterium in vitro , but not virulence in tomato plants, as compared to its uninfected RUN302 strain. Due to its differences from phage RSA1, phage RsoM1USA should be considered the type member of a new species with a proposed species name of Ralstonia virus RsoM1USA.