Endopathogenic lifestyle of Pseudomonas savastanoi pv. savastanoi in olive knots

The endophytic phase of Pseudomonas savastanoi pv. savastanoi in olive stems and the structural and ultrastructural histogenesis of olive knots have been studied. Construction of a stable plasmid vector expressing the green fluorescent protein, in combination with the use of in vitro olive plants, allowed real-time monitoring of P. savastanoi pv. savastanoi infection. The infection process was also examined by bright field and epifluorescence microscopy as well as by scanning and transmission electron microscopy. Hypertrophy of the stem tissue was concomitant with the formation of bacterial aggregates, microcolonies and multilayer biofilms, over the cell surfaces and the interior of plasmolysed cells facing the air-tissue interface of internal opened fissures, and was followed by invasion of the outer layers of the hypertrophied tissue. Pathogenic invasion of the internal lumen of newly formed xylem vessels, which were connected with the stem vascular system, was also observed in late stages of infection. Ultrastructural analysis of knot sections showed the release of outer membrane vesicles from the pathogen surface, a phenomenon not described before for bacterial phytopathogens during host infection. This is the first real-time monitoring of P. savastanoi disease development and the first illustrated description of the ultrastructure of P. savastanoi-induced knots.