Fire blight is a devastating disease of apple and pear caused by the bacterium Erwinia amylovora . One of its main symptoms is canker formation on perennial tissues which may lead to the death of limbs and/or the entire tree. E. amylovora overwinters in cankers which play an important role in initiating fire blight epidemics. However, knowledge of pathogen biology in cankers is scarce, in part due to limitations of classical microbiology methods and the inability of most molecular techniques to distinguish live from dead cells. In this work, a viability digital PCR (v-dPCR) protocol using propidium monoazide (PMA) was developed, allowing for the first time the selective detection and absolute quantification of E. amylovora live cells in apple and pear cankers collected in two time periods. Some key factors affecting the v-dPCR performance were the maceration buffer composition, the target DNA amplicon length, the thermal cycle number and the use of sodium dodecyl sulfate or PMA enhancer for Gram-negative bacteria to improve the effect of PMA. In the future, this methodology could shed light on E. amylovora population dynamics in cankers and provide clues on the effect of management practices, host cultivar, host water/nutritional status, etc., on bacterial survival.

Erwinia amylovora causes fire blight in economically important plants of the family Rosaceae. This bacterial pathogen spends part of its life cycle coping with starvation and other fluctuating environmental conditions. In many Gram-negative bacteria, starvation and other stress responses are regulated by the sigma factor RpoS. We obtained an E. amylovora rpoS mutant to explore the role of this gene in starvation responses and its potential implication in other processes not yet studied in this pathogen. Results showed that E. amylovora needs rpoS to develop normal starvation survival and viable but nonculturable (VBNC) responses. Furthermore, this gene contributed to stationary phase cross-protection against oxidative, osmotic, and acid stresses and was essential for cross-protection against heat shock, but nonessential against acid shock. RpoS also mediated regulation of motility, exopolysaccharide synthesis, and virulence in immature loquats, but not in pear plantlets, and contributed to E. amylovora survival in nonhost tissues during incompatible interactions. Our results reveal some unique roles for the rpoS gene in E. amylovora and provide new knowledge on the regulation of different processes related to its ecology, including survival in different environments and virulence in immature fruits.