Skip to main content

Genomic and Biological Characterization of Ralstonia solanacearum Inovirus Brazil 1, an Inovirus that Alters the Pathogenicity of the Phytopathogen Ralstonia pseudosolanacearum

Abstract

Filamentous bacteriophages contain a single-stranded DNA genome and have a peculiar lifestyle, since they do not cause host cell lysis, but establish a persistent association with the host, often causing behavioral changes, with effects on bacterial ecology. Over the years, a gradual reduction in the incidence of bacterial wilt has been observed in some fields from Brazil. This event, which has been associated with the loss of pathogenicity of Rasltonia spp. isolates due to infection by filamentous viruses of the inovirus group, is widely reported for Ralstonia spp. Asian isolates infected by inoviruses. In an attempt to elucidate which factors are associated with the phenomenon reported in Brazil, we investigated one isolate of R. solanacearum (UB-2014), with unusual characteristics for R. solanacearum, obtained from eggplant with mild wilt symptoms. To verify if the presence of filamentous bacteriophage was related to this phenotype, we performed viral purification and nucleic acid extraction. The phage genome was sequenced, and phylogenetic analyses demonstrated that the virus belongs to the family Inoviridae and was named as Ralstonia solanacerarum inovirus Brazil 1 (RSIBR1). RSIBR1 was transmitted to R. pseudosolanacearum GMI1000, and the virus-infected GMI1000 (GMI1000 VI) isolate showed alterations in phenotypic characteristics, as well as loss of pathogenicity, similarly to that observed in R. solanacearum isolate UB-2014. The presence of virus-infected UB-2014 and GMI1000 VI plants without symptoms, after 3 months, confirms that the infected isolates can colonize the plant without causing disease, which demonstrates that the phage infection changed the behavior of these pathogens.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Data Available

All data underlying the findings are fully available without restriction.

References

  1. 1.

    Roux S, Krupovic M, Daly RA et al (2019) Cryptic inoviruses revealed as pervasive in bacteria and archaea across Earth’s biomes. Nat Microbiol. https://doi.org/10.1038/s41564-019-0510-x

    Article  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Addy HS, Askora A, Kawasaki T et al (2012) The filamentous phage ϕRSS1 enhances virulence of phytopathogenic Ralstonia solanacearum on tomato. Phytopathology 102:244–251. https://doi.org/10.1094/PHYTO-10-11-0277

    Article  PubMed  Google Scholar 

  3. 3.

    Yamada T (2013) Filamentous phages of Ralstonia solanacearum: double-edged swords for pathogenic bacteria. Front Microbiol 4:1–7. https://doi.org/10.3389/fmicb.2013.00325

    Article  Google Scholar 

  4. 4.

    Mai-Prochnow A, Hui JGK, Kjelleberg S et al (2015) Big things in small packages: the genetics of filamentous phage and effects on fitness of their host. FEMS Microbiol Rev 39:465–487. https://doi.org/10.1093/femsre/fuu007

    Article  PubMed  Google Scholar 

  5. 5.

    Koonin EV, Dolja VV, Krupovic M et al (2020) Global organization and proposed megataxonomy of the virus world. Microbiol Mol Biol Rev 84:1–33. https://doi.org/10.1128/MMBR.00061-19

    Article  Google Scholar 

  6. 6.

    Ilyina TS (2015) Filamentous bacteriophages and their role in the virulence and evolution of pathogenic bacteria. Mol Genet Microbiol Virol 30:1–9. https://doi.org/10.3103/S0891416815010036

    Article  Google Scholar 

  7. 7.

    Hay ID, Lithgow T (2019) Filamentous phages: masters of a microbial sharing economy. EMBO Rep 20:1–24. https://doi.org/10.15252/embr.201847427

  8. 8.

    Waldor MK, Mekalanos JJ (1996) Lysogenic conversion by a filamentous phage encoding cholera toxin. Science 272:1910–1914. https://doi.org/10.1126/science.272.5270.1910

    CAS  Article  PubMed  Google Scholar 

  9. 9.

    Addy HS, Askora A, Kawasaki T et al (2012) Loss of virulence of the phytopathogen Ralstonia solanacearum through infection by φRSM filamentous phages. Phytopathology 102:469–477. https://doi.org/10.1094/PHYTO-11-11-0319-R

    CAS  Article  PubMed  Google Scholar 

  10. 10.

    Chopin MC, Rouault A, Dusko Ehrlich S, Gautier M (2002) Filamentous phage active on the gram-positive bacterium Propionibacterium freudenreichii. J Bacteriol 184:2030–2033. https://doi.org/10.1128/JB.184.7.2030-2033.2002

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Mingzhi L, Ling X, Ziling S, Yongquan L (2007) Isolation and characterization of a phytotoxin from Xanthomonas campestris pv. retroflexus. Chin J Chem Eng 15:639–642. https://doi.org/10.1016/S1004-9541(07)60138-4

    Article  Google Scholar 

  12. 12.

    Rice SA, Tan CH, Mikkelsen PJ et al (2009) The biofilm life cycle and virulence of Pseudomonas aeruginosa are dependent on a filamentous prophage. ISME J 3:271–282. https://doi.org/10.1038/ismej.2008.109

    CAS  Article  PubMed  Google Scholar 

  13. 13.

    Webb JSJ, Lau M, Kjelleberg S (2004) Bacteriophage and phenotypic variation in Pseudomonas aeruginosa biofilm development. J Bacteriol 186:8066–8073. https://doi.org/10.1128/JB.186.23.8066-8073.2004

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Álvarez B, Biosca EG, López MM (2010) On the life of Ralstonia solanacearum, a destructive bacterial plant pathogen. In: Méndez-Vilas A (ed) Technology and education topics in applied microbiology and microbial biotechnology, 2nd edn. Formatex Research Center, Spain, pp 267–279

    Google Scholar 

  15. 15.

    Ahmad AA, Stulberg MJ, Huang Q (2017) Prophage Rs551 and its repressor gene orf14 reduce virulence and increase competitive fitness of its Ralstonia solanacearum carrier strain UW551. Front Microbiol 8:1–10. https://doi.org/10.3389/fmicb.2017.02480

    Article  Google Scholar 

  16. 16.

    Askora A, Kawasaki T, Fujie M, Yamada T (2014) Insights into the diversity of phirSM phages infecting strains of the phytopathogen Ralstonia solanacearum complex: regulation and evolution. Mol Genet Genomics 289:589–598. https://doi.org/10.1007/s00438-014-0835-3

    CAS  Article  PubMed  Google Scholar 

  17. 17.

    Addy HS, Askora A, Kawasaki T et al (2012) Through infection by φRSM filamentous phages. Phytopathology 102:469–477. https://doi.org/10.1094/PHYTO-11-11-0319-R

    CAS  Article  PubMed  Google Scholar 

  18. 18.

    Fegan M, Prior P, Allen C, Hayward AC (2005) How complex is the “Ralstonia solanacearum species complex”? Bact wilt Dis Ralstonia solanacearum species complex

  19. 19.

    Safni I, Cleenwerck I, De Vos P et al (2014) Polyphasic taxonomic revision of the Ralstonia solanacearum species complex: proposal to emend the descriptions of Ralstonia solanacearum and Ralstonia syzygii and reclassify current R. syzygii strains as Ralstonia syzygii subsp. syzygii subsp. nov., R. s. Int J Syst Evol Microbiol 64:3087–3103. https://doi.org/10.1099/ijs.0.066712-0

    CAS  Article  PubMed  Google Scholar 

  20. 20.

    Prior P, Ailloud F, Dalsing BL et al (2016) Genomic and proteomic evidence supporting the division of the plant pathogen Ralstonia solanacearum into three species. BMC Genomics 17:90. https://doi.org/10.1186/s12864-016-2413-z

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Gonçalves OS, de Souza F, O, Bruckner FP, et al (2021) Widespread distribution of prophages signaling the potential for adaptability and pathogenicity evolution of Ralstonia solanacearum species complex. Genomics 113:992–1000. https://doi.org/10.1016/j.ygeno.2021.02.011

    CAS  Article  PubMed  Google Scholar 

  22. 22.

    Yamada T, Kawasaki T, Nagata S et al (2007) New bacteriophages that infect the phytopathogen Ralstonia solanacearum. Microbiology 153:2630–2639. https://doi.org/10.1099/mic.0.2006/001453-0

    CAS  Article  PubMed  Google Scholar 

  23. 23.

    Murugaiyan S, Bae JY, Wu J et al (2011) Characterization of filamentous bacteriophage PE226 infecting Ralstonia solanacearum strains. J Appl Microbiol 110:296–303. https://doi.org/10.1111/j.1365-2672.2010.04882.x

    CAS  Article  PubMed  Google Scholar 

  24. 24.

    Askora A, Kawasaki T, Usami S et al (2009) Host recognition and integration of filamentous phage ϕRSM in the phytopathogen, Ralstonia solanacearum. Virology 384:69–76. https://doi.org/10.1016/j.virol.2008.11.007

    CAS  Article  PubMed  Google Scholar 

  25. 25.

    Askora A, Yamada T (2015) Two different evolutionary lines of filamentous phages in Ralstonia solanacearum: their effects on bacterial virulence. Front Genet 6:1–6. https://doi.org/10.3389/fgene.2015.00217

    CAS  Article  Google Scholar 

  26. 26.

    Horita M, Tsuchiya K (2001) Genetic diversity of Japanese strains of Ralstonia solanacearum. Phytopathology 91:399–407. https://doi.org/10.1094/PHYTO.2001.91.4.399

    CAS  Article  PubMed  Google Scholar 

  27. 27.

    Opina N, Tavner F, Hollway G et al (1997) A novel method for development of species and strain-specific DNA probes and PCR primers for identifying Burkholderia solanacearum (formerly Pseudomonas solanacearum). Asia Pac J Mol Biol Biotechnol 5:19–30

    Google Scholar 

  28. 28.

    Fegan M, Prior P (2005) How complex is the Ralstonia solanacearum species complex. In: Allen C, Prior P, Hayward AC (eds) Bacterial wilt disease and the Ralstonia solanacearum species complex. American Phytopathological Society, pp 449–461

  29. 29.

    Sambrook J, Russell DW, W Russell D (2001) Molecular cloning a laboratory manual, 3rd ed. Cold Spring Harbor Laboratory Press

  30. 30.

    Inoue-Nagata AK, Albuquerque LC, Rocha WB, Nagata T (2004) A simple method for cloning the complete begomovirus genome using the bacteriophage phi 29 DNA polymerase. J Virol Methods 116:209–211. https://doi.org/10.1016/j.jviromet.2003.11.015

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Besemer J, Borodovsky M (1999) Heuristic approach to deriving models for gene finding. Nucleic Acids Res 27:3911–3920. https://doi.org/10.1093/nar/27.19.3911

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Altschul SF, Madden TLTTL, Schäffer AA et al (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402. https://doi.org/10.1093/nar/25.17.3389

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Altschul SF, Gish W, Miller W et al (1990) Basic local alignment search tool. J Mol Biol 215:403–410. https://doi.org/10.1016/S0022-2836(05)80360-2

    CAS  Article  PubMed  Google Scholar 

  34. 34.

    Kumar S, Stecher G, Li M et al (2018) MEGA X: molecular evolutionary genetics analysis across computing platforms

  35. 35.

    Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425. https://doi.org/10.1093/oxfordjournals.molbev.a040454

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Kimura M (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16:111–120. https://doi.org/10.1007/BF01731581

    CAS  Article  PubMed  Google Scholar 

  37. 37.

    Muhire BM, Varsani A, Martin DP (2014) SDT: a virus classification tool based on pairwise sequence alignment and identity calculation. PLoS ONE 9:e108277. https://doi.org/10.1371/journal.pone.0108277

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  38. 38.

    Granada GA, Sequeira L (1983) Survival of Pseudomonas solanacearum in soil, rhizosphere, and plant roots. Can J Microbiol 29:433–440. https://doi.org/10.1139/m83-070

    Article  Google Scholar 

  39. 39.

    da Silva XA, da Silva FP, Vidigal PMP et al (2018) Genomic and biological characterization of a new member of the genus Phikmvvirus infecting phytopathogenic Ralstonia bacteria. Arch Virol 163:3275–3290. https://doi.org/10.1007/s00705-018-4006-4

    CAS  Article  PubMed  Google Scholar 

  40. 40.

    Adams MH (1959) Bacteriophages. Interscience Publishers, New York

    Google Scholar 

  41. 41.

    Huerta AI, Milling A, Allen C (2015) Tropical strains of Ralstonia solanacearum outcompete race 3 biovar 2 strains at lowland tropical temperatures. Appl Environ Microbiol 81:3542–3551. https://doi.org/10.1128/AEM.04123-14

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Van TTB, Yoshida S, Miki K et al (2014) Genomic characterization of ϕRS603, a filamentous bacteriophage that is infectious to the phytopathogen Ralstonia solanacearum. Microbiol Immunol 58:697–700. https://doi.org/10.1111/1348-0421.12203

    CAS  Article  PubMed  Google Scholar 

  43. 43.

    Gutarra L, Herrera J, Fernandez E et al (2017) Diversity, pathogenicity, and current occurrence of bacterial wilt bacterium Ralstonia solanacearum in Peru. Front Plant Sci 8:1–12. https://doi.org/10.3389/fpls.2017.01221

    Article  Google Scholar 

  44. 44.

    Ahmad AA, Stulberg MJ, Mershon JP et al (2017) Molecular and biological characterization of ϕRs551, a filamentous bacteriophage isolated from a race 3 biovar 2 strain of Ralstonia solanacearum. PLoS ONE 12:e0185034. https://doi.org/10.1371/journal.pone.0185034

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  45. 45.

    Ahmad S, Lee SY, Kong HG et al (2016) Genetic determinants for pyomelanin production and its protective effect against oxidative stress in Ralstonia solanacearum. PLoS ONE 11:e0160845. https://doi.org/10.1371/journal.pone.0160845

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  46. 46.

    Li P, Wang D, Yan J et al (2016) Genomic analysis of phylotype I strain EP1 reveals substantial divergence from other strains in the Ralstonia solanacearum species complex. Front Microbiol 7:1–14. https://doi.org/10.3389/fmicb.2016.01719

    Article  Google Scholar 

  47. 47.

    Shieh GJ, Charng YC, Yang BC et al (1991) Identification and nucleotide sequence analysis of an open reading frame involved in high-frequency conversion of turbid to clear plaque mutants of filamentous phage Cf1t. Virology 185:316–322. https://doi.org/10.1016/0042-6822(91)90779-B

    CAS  Article  PubMed  Google Scholar 

  48. 48.

    Cheng J, Zhou X, Chou TF et al (2009) Identification of the amino acid-AZT-phosphoramidase by affinity T7 phage display selection. Bioorganic Med Chem Lett 19:6379–6381. https://doi.org/10.1016/j.bmcl.2009.09.067

    CAS  Article  Google Scholar 

  49. 49.

    McLeod SM, Waldor MK (2004) Characterization of XerC- and XerD-dependent CTX phage integration in Vibrio cholerae. Mol Microbiol 54:935–947. https://doi.org/10.1111/j.1365-2958.2004.04309.x

    CAS  Article  PubMed  Google Scholar 

  50. 50.

    Askora A, Abdel-Haliem MEF, Yamada T (2012) Site-specific recombination systems in filamentous phages. Mol Genet Genomics 287:525–530. https://doi.org/10.1007/s00438-012-0700-1

    CAS  Article  PubMed  Google Scholar 

  51. 51.

    Bille E, Zahar JR, Perrin A et al (2005) A chromosomally integrated bacteriophage in invasive meningococci. J Exp Med 201:1905–1913. https://doi.org/10.1084/jem.20050112

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  52. 52.

    Kawai M, Uchiyama I, Kobayashi I (2005) Genome comparison in silico in Neisseria suggests integration of filamentous bacteriophages by their own transposase. DNA Res 12:389–401. https://doi.org/10.1093/dnares/dsi021

    CAS  Article  PubMed  Google Scholar 

Download references

Acknowledgements

We are thankful to the members of our laboratory for the helpful discussions and Dr. Caitilyn Allen (University of Wisconsin-Madison), for providing the Ralstonia pseudosolanacearum GMI1000 isolate used in this work.

Funding

This research was supported by grant APQ-01926–14 (FAPEMIG) to PAZ. ASX was the recipient of a FAPEMIG graduate fellowship, JCFA was the recipient of a CAPES graduate fellowship, and RSC was the recipient of a PNPD/CAPES post-doc fellowship.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Poliane Alfenas-Zerbini.

Ethics declarations

Ethics Approval

Not applicable.

Consent to Participate

Not applicable.

Conflict of Interest

The authors declare no competing interests.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 28535 KB)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

de Almeida, J.C.F., da Silva Xavier, A., Cascardo, R.d. et al. Genomic and Biological Characterization of Ralstonia solanacearum Inovirus Brazil 1, an Inovirus that Alters the Pathogenicity of the Phytopathogen Ralstonia pseudosolanacearum. Microb Ecol (2021). https://doi.org/10.1007/s00248-021-01874-w

Download citation

Keywords

  • Ralstonia spp.
  • Multitrophic interactions
  • Bacteriophages
  • Plant pathogenic bacteria