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, Volume 26, Issue 1, pp 75–82 | Cite as

In planta recovery of Erwinia amylovora viable but nonculturable cells

  • R. D. Santander
  • J. F. Català-Senent
  • E. Marco-Noales
  • E. G. BioscaEmail author
Original Paper

Abstract

Little is known about the survival mechanisms of Erwinia amylovora outside its hosts. It has been demonstrated that it enters the viable but nonculturable state (VBNC) when exposed to different types of stress. In the VBNC state, bacterial cells remain viable but unable to grow on the solid general media where they usually do, and are thus undetectable by conventional culture-dependent methods. In this work, we have evaluated the recovery of E. amylovora VBNC cells by passage through pear plantlets, in comparison with other recovery methods commonly used for this pathogen: incubation in KB broth and inoculation of immature fruits. VBNC cells were obtained by exposure of bacterial cells to different types of stress (oligotrophy, nutrient deprivation and chlorine), and recovery assays were performed at 26°C. In all cases, the recovery of VBNC cells was more effective in plantlets than in liquid KB or immature fruits. In fact, when cells were exposed to chlorine for more than 30 min, only passage through host plant gave positive result, enabling recovery of E. amylovora cells few days after inoculation of plants. These results suggest a higher effectiveness of in planta recovery than those performed with liquid KB or detached fruits. Our results support the hypothesis of the VBNC state being part of the E. amylovora life cycle. The potential existence of this physiological state in nature should be taken in consideration in epidemiological studies of fire blight, with the aim to optimize the management and control of this disease.

Keywords

Fire blight Oligotrophy Nutrient deprivation Chlorine VBNC Recovery 

Notes

Acknowledgments

This work was supported through project AGL2008-05723-C02-02 from the Ministerio de Ciencia e Innovación of Spain, and was performed in the framework of COST 864. R.D. Santander thanks a FPU predoctoral fellowship from the Ministerio de Educación of Spain. E. Marco-Noales has a contract from the Spanish Ministry of Education and Science (Programa INIA-CCAA) co-funded by European Social Fund. The authors wish to specially thank J.L. Díez for statistical analysis. We also thank “Servicio Central de Soporte a la Investigación Experimental” (SCSIE), from the University of Valencia, specially to A. Flores and C. Navajo for their expert technical assistance; as well as A. Palacio-Bielsa (CITA, Gobierno de Aragón) and M. A. Cambra (CPV, Gobierno de Aragón) for providing the immature pear fruits.

References

  1. Alexander E, Pham D, Steck TR (1999) The viable-but-nonculturable condition is induced by copper in Agrobacterium tumefaciens and Rhizobium leguminosarum. Appl Environ Microbiol 65:3754–3756PubMedGoogle Scholar
  2. Álvarez B, López MM, Biosca EG (2008) Survival strategies and pathogenicity of Ralstonia solanacearum phylotype II subjected to prolonged starvation in environmental water microcosms. Microbiology 154:3590–3598. doi: 10.1099/mic.0.2008/019448-0 PubMedCrossRefGoogle Scholar
  3. Anonymous (1984) Resolución de 23 de Abril de 1984, de la subsecretaria, por la que se aprueba la Lista positiva de aditivos y coadyuvantes tecnológicos autorizados para tratamientos de las aguas potables de consumo público. Ministerio de Sanidad y Consumo de España. BOE. 111/1984 p12675Google Scholar
  4. Anonymous (2000) Council Directive 2000/29/EC of 8 May 2000 on protective measures against the introduction into the Community of organisms harmful to plants or plant products and against their spread within the Community. Off J Eur Communities, L169, 43:1–112Google Scholar
  5. Anonymous, EPPO, European and Mediterranean Plant Protection Organization (2004) Diagnostic protocols for regulated pests: Erwinia amylovora. In EPPO Standards PM 7/20 (1). Bull OEPP/EPPO Bull 34:159–171Google Scholar
  6. Biosca EG, Marco-Noales E, Ordax M, López MM (2006) Long-term starvation-survival of Erwinia amylovora in sterile irrigation water. Acta Hortic 704:107–112Google Scholar
  7. Bonn GW, van der Zwet T (2000) Distribution and economic importance of fire blight. In: Vanneste JL (ed) Fire blight the disease and its causative agent, Erwinia amylovora. CABI Publishing, Wallingford, pp 37–54CrossRefGoogle Scholar
  8. Boulos L, Prévost M, Barbeau B, Coallier J, Desjardins R (1999) LIVE/DEAD® BacLight™: application of a new rapid staining method for direct enumeration of viable and total bacteria in drinking water. J Microbiol Methods 37:77–86PubMedCrossRefGoogle Scholar
  9. Byrd JJ, Xu H-S, Colwell RR (1991) Viable but nonculturable bacteria in drinking water. Appl Environ Microbiol 57:875–878PubMedGoogle Scholar
  10. Cabrefiga J, Montesinos E (2005) Analysis of aggressiveness of Erwinia amylovora using disease-dose and time relationships. Phytopathology 95:1430–1437. doi: 10.1094/PHYTO-95-1430 PubMedCrossRefGoogle Scholar
  11. Cappelier JM, Magras C, Jouve JL, Federighi M (1999) Recovery of viable but nonculturable Campylobacter jejuni cells in two animal models. Food Microbiol 16:375–383CrossRefGoogle Scholar
  12. del Campo R, Russi P, Mara P, Mara H, Peyrou M, Ponce de Leon I, Gaggero C (2009) Xanthomonas axonopodis pv. citri enters the VBNC state after copper treatment and retains its virulence. FEMS Microbiol Lett 298:143–148. doi: 10.1111/j.1574-6968.2009.01709.x PubMedCrossRefGoogle Scholar
  13. Gauthier MJ (2000) Environmental parameters associated with the viable but nonculturable state. In: Colwell RR, Grimes DJ (eds) Nonculturable microorganisms in the environment. American Society for Microbiology, Washington, pp 87–112Google Scholar
  14. Ghezzi JI, Steck TR (1999) Induction of the viable but non-culturable condition in Xanthomonas campestris pv. Campestris in liquid microcosms and sterile soil. FEMS Microbiol Ecol 30:203–208PubMedCrossRefGoogle Scholar
  15. Grey BE, Steck TR (2001) The viable but nonculturable state of Ralstonia solanacearum may be involved in long-term survival and plant infection. Appl Environ Microbiol 67:3866–3872. doi: 10.1128/AEM.67.9.3866-3872.2001 PubMedCrossRefGoogle Scholar
  16. Hoben HJ, Somasegaran P (1982) Comparison of the pour, spread, and drop plate methods for enumeration of Rhizobium spp. in inoculants made from presterilized peat. Appl Environ Microbiol 44:1246–1247PubMedGoogle Scholar
  17. Ishimaru C, Klos EJ (1984) New medium for detecting Erwinia amylovora and its use in epidemiological studies. Phytopathology 74:1342–1345CrossRefGoogle Scholar
  18. King EO, Ward MK, Rainey DE (1954) Two simple media for the demonstration of pyocyanin and fluorescein. J Lab Clin Med 44:301–307PubMedGoogle Scholar
  19. Lelliot RA (1967) The diagnosis of fireblight (Erwinia amylovora) and some diseases caused by Pseudomonas syringae. EPPO Publ Ser A 45-E:27–34Google Scholar
  20. Llop P, Caruso P, Cubero J, Morente C, López MM (1999) A simple extraction procedure for efficient routine detection of pathogenic bacteria in plant material by polymerase chain reaction. J Microbiol Methods 37:23–31PubMedCrossRefGoogle Scholar
  21. López MM, Bertolini E, Caruso P, Penyalver R, Marco-Noales E, Gorris MT, Morente C, Salcedo C, Cambra M, Llop P (2005) Advantages of an integrated approach for diagnosis of quarantine pathogenic bacteria in plant material. Phytopathol Pol 35:49–56Google Scholar
  22. Manahan SH, Steck TR (1997) The viable but nonculturable state in Agrobacterium tumefaciens and Rhizobium meliloti. FEMS Microbiol Ecol 22:29–37CrossRefGoogle Scholar
  23. Marco-Noales E, Bertolini E, Morente C, Lopez MM (2008) Integrated approach to detection of nonculturable cells of Ralstonia solanacearum in asymptomatic Pelargonium spp. cuttings. Phytopathology 98:949–955. doi: 10.1094/PHYTO-98-8-0949 PubMedCrossRefGoogle Scholar
  24. Moreno Y, Piqueres P, Alonso JL, Jiménez A, González A, Ferrús MA (2007) Survival and viability of Helicobacter pylori after inoculation into chlorinated drinking water. Water Res 41:3490–3496. doi: 10.1016/j.watres.2007.05.020 PubMedCrossRefGoogle Scholar
  25. Morita RY (1997) Bacteria in oligotrophic environments, starvation–survival lifestyle. Chapman & Hall, New York, pp 368–385Google Scholar
  26. Oliver JD (2005) The viable but nonculturable state in bacteria. J Microbiol 43 Spec No:93–110Google Scholar
  27. Oliver JD (2010) Recent findings on the viable but nonculturable state in pathogenic bacteria. FEMS Microbiol Rev 34:415–425. doi: 10.1111/j.1574-6976.2009.00200.x PubMedGoogle Scholar
  28. Oliver JD, Dagher M, Linden K (2005) Induction of Escherichia coli and Salmonella typhimurium into the viable but nonculturable state following chlorination of wastewater. J Water Health 3:249–257PubMedGoogle Scholar
  29. Ordax M, Biosca EG, Wimalajeewa S, López MM, Marco-Noales E (2009) Survival of Erwinia amylovora in mature apple fruit calyces through the viable but nonculturable (VBNC) state. J Appl Microbiol 107:106–116. doi: 10.1111/j.1365-2672.2009.04187 PubMedCrossRefGoogle Scholar
  30. Ordax M, Marco-Noales E, López MM, Biosca EG (2006) Survival strategy of Erwinia amylovora against copper: induction of the viable-but-nonculturable state. Appl Environ Microbiol 72:3482–3488. doi: 10.1128/AEM.72.5.3482-3488.2006 PubMedCrossRefGoogle Scholar
  31. Ordax M, Marco-Noales E, López MM, Biosca EG (2010) Exopolysaccharides favor the survival of Erwinia amylovora under copper stress through different strategies. Res Microbiol 161:549–555. doi: 10.1016/j.resmic.2010.05.003 PubMedCrossRefGoogle Scholar
  32. Roszak BD, Colwell RR (1987) Survival strategies of bacteria in the natural environment. Microbiol Rev 51:365–379PubMedGoogle Scholar
  33. Ruz L, Moragrega C, Montesinos E (2008) Evaluation of four whole-plant inoculation methods to analyze the pathogenicity of Erwinia amylovora under quarantine conditions. Int Microbiol 11:111–119PubMedGoogle Scholar
  34. Santander RD, Català-Senent JF, Ordax M, Marco-Noales E, Biosca EG (2009) Effect of the chlorine on survival of Erwinia amylovora in water. In: Abstracts of Cost Action 864 “Combining traditional and advanced strategies for plant protection in pome fruit growing” Valencia, Spain, p 21Google Scholar
  35. Santander RD, Català-Senent JF, Ordax M, Flores A, Marco-Noales E, Biosca EG (2011a) Evaluation of flow cytometry to assess Erwinia amylovora viability under different stress conditions. In Mendez-Vilas A (ed) Microorganisms in industry and environment. From scientific and industrial research to consumer products. World Scientific Publishing Company, Singapore, pp 51–54Google Scholar
  36. Santander RD, Marco-Noales E, Ordax M, Biosca EG (2011b) Erwinia amylovora colonization of host plants inoculated by irrigation. In Mendez-Vilas A (ed) Microorganisms in industry and environment. From scientific and industrial research to consumer products. World Scientific Publishing Company, Singapore, pp 48–50Google Scholar
  37. Sardessai YN (2005) Viable but non-culturable bacteria: their impact on public health. Curr Sci 89:1650Google Scholar
  38. Taylor RK, Guilford PJ, Clack RG, Foster RLS (2001) Detection of Erwinia amylovora in plant material using novel polymerase chain reaction (PCR) primers. N Z J Crop Hortic Sci 29:35–43. doi: 10.1080/01140671.2001.9514158 CrossRefGoogle Scholar
  39. Thomson SV (2000) Epidemiology of fire blight. In: Vanneste JL (ed) The disease and its causative agent, Erwinia amylovora. CABI Publishing, Wallingford, pp 9–36CrossRefGoogle Scholar
  40. van der Zwet T (2000) Distribution and economic importance of fire blight. In: Vanneste JL (ed) Fire blight: the disease and its causative agent, Erwinia amylovora. CABI Publishing, Wallingford, pp 37–54Google Scholar
  41. van der Zwet T, Beer SV (1995) Fireblight. Its nature, prevention and control: a practical guide to integrate disease management. US Department of Agriculture, Bulletin 631, 2nd edn, p 91Google Scholar
  42. Virto R, Mañas P, Álvarez I, Condon S, Raso J (2005) Membrane damage and microbial inactivation by chlorine in the absence and presence of a chlorine-demanding substrate. Appl Environ Microbiol 71:5022–5028. doi: 10.1128/AEM.71.9.5022-5028.2005 PubMedCrossRefGoogle Scholar
  43. Wang Y, Claeys L, van der Ha D, Verstraete W, Boon N (2010) Effects of chemically and electrochemically dosed chlorine on Escherichia coli and Legionella beliardensis assessed by flow cytometry. Appl Microbiol Biotechnol 87:331–341. doi: 10.1007/s00253-010-2526-2 PubMedCrossRefGoogle Scholar
  44. Weichart DH (1999) Stability and survival of VBNC cells-conceptual and practical implications. In: Bell CR, Brylinsky M, Johnson-Green P (eds) Microbial biosystems: new frontiers. Proceedings of the 8th international symposium on microbial ecology. Atlantic Canada Society for Microbial Ecology, Halifax, CanadaGoogle Scholar
  45. Winslow CEA, Broadhurst J, Buchanan RE, Krumwiede C, Rogers LA, Smith GH (1920) The families and genera of the bacteria; Erwineae. J Bacteriol 5:191–229PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • R. D. Santander
    • 1
  • J. F. Català-Senent
    • 1
  • E. Marco-Noales
    • 2
  • E. G. Biosca
    • 1
    Email author
  1. 1.Dpto. Microbiología y EcologíaUniversidad de ValenciaValenciaSpain
  2. 2.Instituto Valenciano de Investigaciones Agrarias (IVIA)ValenciaSpain

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