USE OF A MU BACTERIOPHAGE DERIVATIVE TO CONSTRUCT MUTANTS OF ERWINIA AMYLOVORA ALTERED IN PATHOGENICITY

The purpose of this work was to isolate mutants of E. amylovora altered in pathogenicity.
Our earlier work (Vanneste et al., 1987) showed that Mu derivatives can be used as genetic tools in E. amylovora strain 1430. Therefore, we chose to use a Mu derivative for the mutagenesis, and we selected the Mud₁B^x::Tn9 (lac, Ap^r, Cm^r) constructed by Baker et al. (1983) and called Mu dX.

Mu lysates were prepared by induction of a lysogenic strain of Escherichia coli containing a Mu dX prophage and a Mu cts 62 prophage used as a helper phage.
Lysogenic clones were selected based on chloramphenicol resistance conferred by Tn9 carried by the Mu dX prophage.

Mutants affected in pathogenicity were identified by inoculation of apple root callus.
When the wild-type strain 1430 was applied, exudate appeared after 3 days of incubation at 28°C. Among 1400 lysogenic clones examined, 12 did not produce exudate or produced it only following prolonged incubation (Table 1). In addition, two mutants failed to induce a hypersensitive response when they were injected into tobacco leaves.

The possibility that the genome of avirulent mutants contained several transposable genetic elements, due to the presence of a Mu cts 62 prophage or due to the transposition of either the Mu dX prophage or Tn9 carried by the Mu dX prophage, was investigated by Southern blot hybridization.
Preparations of total DNA isolated from six mutants altered in pathogenicity were digested with the restriction endonuclease EcoRI, and the blots were probed with ³²P-labelled Mu cts 62 DNA to detect DNA sequences homologous to the Mu DNA (Mu dX or Mu cts 62 prophage) and with the ³²P-labelled pBR325 DNA to detect DNA sequences homologous to Tn9 (Figure 1). Analysis of the autoradiograms obtained with these two probes indicate the number and the nature of the transposable genetic element(s) present in the genome (Table 2). The presence of a single Mu dX prophage, revealed by a unique EcoRI border fragment detected with either probe, was shown for 5 of the 6 strains examined (Figure 2), while for the mutant

IV International Workshop on Fire Blight

J.L. Vanneste, D. Expert, J.P. Paulin

https://doi.org/10.17660/ActaHortic.1987.217.30