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RESEARCH PRODUCT

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

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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. Fire blight, caused by the bacterium Erwinia amylovora (Burrill) Winslow et al. (39) and reported in more than 40 countries around the world, is a very serious and destructive disease of pome fruits and many ornamental plants from the Rosaceae family (34). Copper compounds, widely utilized against fire blight from the beginning of the last century (35), are still employed in many countries, especially in the European Union, where antibiotic utilization is restricted (2). Their use is one of the most common methods for controlling bacterial plant diseases, but it has led many bacteria to develop different strategies against copper ions (31). Until now, very little information on the interaction between E. amylovora and Cu2+ ions has been available (4, 15, 41). Copper treatments have traditionally been considered as bactericides in agriculture (29, 34, 35), their effectiveness often being measured by the absence of bacterial growth on a solid medium (12, 13). However, recent studies have shown the induction of the viable-but-nonculturable (VBNC) state by copper in several plant-pathogenic bacteria, such as Agrobacterium tumefaciens (1), Xanthomonas campestris pv. campestris (16), and Ralstonia solanacearum (17). This state, in which cells progressively lose their culturability on nonselective solid medium but still remain viable, is considered to be a bacterial survival strategy under adverse environmental conditions (30). Therefore, the failure to produce a visible colony may not necessarily mean that the bacterial cell is dead. Furthermore, it has been reported that VBNC cells can maintain pathogenicity (17, 19). In this respect, it has been suggested that copper-induced VBNC cells of some phytopathogenic bacteria could be related to the persistent nature of infections in copper-treated fields (17). A similar situation could occur with E. amylovora, since fire blight remains difficult to control as a disease (26), which has been related to the ability of this bacterium to survive and spread in different ways (32). Moreover, some bacteria, under favorable environmental conditions, can revert from the VBNC state to a culturable one, in a process usually called resuscitation. This reversion is considered a confirmation that the VBNC state is a bacterial survival strategy (19, 28). The nature of the VBNC state, however, is still the topic of an intense debate in the literature, and some authors argue that this condition may be a physiological state prior to cell death (7, 23). In spite of previous work concerning the toxic effect of copper ions for E. amylovora (15), many questions on the survival of this bacterium in the presence of this metal remain unanswered. Thus, the aim of this work has been to determine whether copper kills or induces the VBNC state of E. amylovora cells and if such VBNC cells retain their pathogenicity. Furthermore, the possible reversion of this bacterium from the nonculturable state has been studied as well as whether resuscitated cells could regain their pathogenic potential