Isolation and characterization of Pseudomonas syringae subsp. savastanoi mutants defective in hypersensitive response elicitation and pathogenicity

Olive strain ITM317 of Pseudomonas syringae subsp. savastanoi, the causal agent of 'Olive and Oleander knot disease' was mutagenized by random transposon (Tn5) insertion. Of the 1 400 transconjugants, four were altered in their ability to induce a hypersensitive reaction (HR) on tobacco; Southern blot analysis showed that a single copy of the Tn5 element was present in their chromosomes. In particular, mutants ITM317-69, ITM317-1010 and ITM317-1194 did not elicit HR whereas mutant ITM317-916 induced a variable response. When assayed for pathogenicity on olive, mutants ITM317-916 and ITM317-1010 induced knots comparable both in size and morphology to those caused by the parental strain. Prototrophic mutant ITM317-1194, still able to produce indole-3-acetic acid and cytokinins, did not cause any knot formation on olive; furthermore, it was unable to multiply in host tissue. Auxotrophic mutant ITM317-69 caused the formation of smaller-sized knots and its prototrophic revertant fully regained the parental phenotypes, suggesting that a single Tn5 insertion had a pleiotropic effect on the mutated phenotypes. Tn5-containing EcoRI fragments from mutants ITM317-69, ITM317-916, ITM317-1010 and ITM317-1194 were cloned into the plasmid vector pBR322. Hybridization of these clones with the hrp gene cluster of P. s. pv. syringae strain 61 was not detected. These results suggest that genes different from those of the above gene cluster might be involved in the interaction of P. s. subsp. savastanoi with olive and with the non-host plant tobacco.