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Applied and Environmental Microbiology, May 2006, p. 3482-3488, Vol. 72, No. 5
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.5.3482-3488.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Survival Strategy of Erwinia amylovora against Copper: Induction of the Viable-but-Nonculturable State

Mónica Ordax,1 Ester Marco-Noales,1 María M. López,1 and Elena G. Biosca2*

Instituto Valenciano de Investigaciones Agrarias (IVIA), Apartado Oficial, 46113 Moncada, Valencia, Spain,1 Departamento de Microbiología y Ecología, Universidad de Valencia, Av. Dr. Moliner, 50, 46100 Burjassot, Valencia, Spain2

Received 14 December 2005/ Accepted 28 February 2006

Copper compounds, widely used to control plant-pathogenic bacteria, have traditionally been employed against fire blight, caused by Erwinia amylovora. However, recent studies have shown that some phytopathogenic bacteria enter into the viable-but-nonculturable (VBNC) state in the presence of copper. To determine whether copper kills E. amylovora or induces the VBNC state, a mineral medium without copper or supplemented with 0.005, 0.01, or 0.05 mM Cu2+ was inoculated with 107 CFU/ml of this bacterium and monitored over 9 months. Total and viable cell counts were determined by epifluorescence microscopy using the LIVE/DEAD kit and by flow cytometry with 5-cyano-2,3-ditolyl tetrazolium chloride and SYTO 13. Culturable cells were counted on King's B nonselective solid medium. Changes in the bacterial morphology in the presence of copper were observed by scanning electron microscopy. E. amylovora entered into the VBNC state at all three copper concentrations assayed, much faster when the copper concentration increased. The addition of different agents which complex copper allowed the resuscitation (restoration of culturability) of copper-induced VBNC cells. Finally, copper-induced VBNC cells were virulent only for the first 5 days, while resuscitated cells always regained their pathogenicity on immature fruits over 9 months. These results have shown, for the first time, the induction of the VBNC state in E. amylovora as a survival strategy against copper.

* Corresponding author. Mailing address: Departamento de Microbiología y Ecología, Universidad de Valencia, Av. Dr. Moliner, 50, Burjassot 46100, Valencia, Spain. Phone: 34 96 354 3194. Fax: 34 96 354 4570. E-mail: elena.biosca{at}uv.es.

Applied and Environmental Microbiology, May 2006, p. 3482-3488, Vol. 72, No. 5
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.5.3482-3488.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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