Strain selection and improvement of gene transfer for genetic manipulation of Pseudomonas savastanoi isolated from olive knots

Research on diseases of herbaceous plants caused by Pseudomonads has been rapidly progressing; however, for most pathovars which infect woody plants, strains accessible to genetic manipulation have not yet been reported. At present, few studies have reported the transfer of genes to Pseudomonas savastanoi pv. savastanoi, the causal agent of olive knot disease. A collection of P. savastanoi pv. savastanoi isolates was tested for its ability to receive, by conjugation, the broad-host range plasmid pBBR1MCS-2; four of them, showing conjugation frequencies higher than 10−3 transconjugants/recipient, were selected. Differences in motility, colony size and morphology, and knot formation in olive explants were observed among the selected strains; nonetheless, amplification and sequencing of the 16S rRNA gene confirmed that they belonged to P. savastanoi species. Transformation frequency by electroporation of pBBR1MCS-2 into these strains was improved up to four orders of magnitude using plasmids isolated from a P. savastanoi strain and from an Escherichia coli modification/restriction-deficient strain. Three of the selected strains maintained pBBR1MCS-2 stably and compatibly with their native plasmids during at least 90 generations, allowing the use of this vector for gene expression studies. Transposition via conjugation of different mini-Tn5, with or without the reporter genes gfp or luxAB, yielded frequencies varying from 1 × 10−5 to 2.4 × 10−9 transconjugants/recipient. Southern analysis of mutants obtained in strain NCPPB 3335 using a collection of DNA sequence tag transposons indicated that transposition occurs randomly, and in most cases at single sites in the genome of this strain, allowing the utilization of transposon tools for cell tagging and for the construction of insertional mutations. Knots developed on one-year-old plants inoculated with a Gfp-tagged strain clearly showed green fluorescence.