In planta recovery of Erwinia amylovora viable but nonculturable cells

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.