Studies to elucidate the cause of alteration in colony morphotype of Xylella fastidiosa subsp. pauca, ST53

Abstract: Isolation in pure culture of Xylella fastidiosa (Xf) strains from infected olives showing severe
decline in southern Italy, represented a major breakthrough for advancing the research on this first
and most severe bacterial outbreak which emerged a few years ago in Europe. However, during an
extensive isolation campaign, some of the cultured isolates (Xf subspecies pauca, ST53) started to
show an odd shape and growth pattern. Briefly, after an initial growth, the colonies started to become
translucent, of reduced size, roughness and adherent to the media. Passage of these isolates on new
agar-plates, regardless of the media, evolved in cell death. Microscope examination using the
BacLight LIVE/DEAD bacterial viability staining kit and observations at the Transmission Electron
Microscopy (TEM) revealed that the odd-shaped bacterial colonies consisted of dead cells, and the
presence of different bacteriophage-(like) particles were observed in the TEM preparations. Ultrathin
sections of these bacterial colonies showed a higher number of intracellular vesicles/endospores than
those observed in the cells with regular morphotype, and their content discharged into the extracellular
space. Filtrated suspensions prepared from altered colonies scraped from the plates, were able to
reproduce similar alterations when mixed with ST53-isolates (subsp. pauca) of the regular
morphotype. Conversely, no effects were noticed when strains belonging to other subspecies were put
in contact with the same filtrates.
Studies are in progress to understand the nature of these intracellular vesicles/endospores which
resemble a sort of ‘pseudolysogenic phages’ (Ripp S and Miller RV, 1997) induced in other bacterial
species as a strategy to enhance survival in unfavourable environmental conditions. Indeed, attempts
to purify phage particles from cultures displaying odd colonies are ongoing to verify whether they
originate by spontaneous induction from temperate phages, known to occur in the genomes of the
ST53-isolates causing the infections in the epidemic area of Apulia (southern Italy)