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Abstract

To cure the fireblight pathogen Erwinia amylovora of a 29 kb plasmid (pEA29) that is common in strains of these bacteria, restriction fragments of this plasmid were inserted into plasmids based on replication functions of bacteriophage fd, which cannot replicate in bacteria without expression of viral gene 2-protein. A 4·4 kb PstI fragment including the unique BamHI site of pEA29 allowed pfd plasmids to be propagated in E. amylovora. Furthermore, selection for these plasmids removed the natural 29 kb plasmid, apparently because of plasmid incompatibility. The pfd plasmids with the 4·4 kb insertion had a high tendency to segregate in E. amylovora when grown without selective pressure. Plasmid-free E. amylovora strains were virulent in standard tests on slices of immature pears, but symptom development was delayed compared to the wild-type. When assayed on pear seedlings a deficiency in pathogenicity was observed. Furthermore, strains without pEA29 spread more slowly on a lawn of pear cells than did wild-type strains. Plasmid-free strains of E. amylovora were not auxotrophic, but synthesis of extracellular polysaccharide was altered under certain growth conditions.