ISHS Acta Horticulturae 273: V International Workshop on Fireblight
POPULATION DYNAMICS OF NONPATHOGENIC MUTANTS OF ERWINIA AMYLOVORA IN APPLE HOST TISSUE |
| Authors: | J.L. Norelli, M.T. Gilbert, H.S. Aldwinckle, C.H. Zumoff, S.V. Beer |
| Abstract:
Mutants of Erwinia amylovora that are altered in specific pathogenicity determinants were produced by transposon mutagenesis and are being used to study the molecular biology of pathogenicity. The purpose of this research was to characterize the ability of these mutants to grow in host tissue. The E. amylovora strain CFPB1367 was mutagenized with a mini-tet transposon derived from Tn10 (Way et al. 1984). Mutants were characterized for their ability to cause fire blight symptoms on inoculated immature pear fruits (Path), cause a hypersensitive response in tobacco leaves (HR) and produce slimy colonies on minimal media (taken as an indication of exopolysaccharide production [EPS]). Two mutants unaffected in pathogenicity (Path+/HR+/EPS+), three nonpathogenic mutants that do not cause a HR in tobacco (Path-/HR-/EPS+), two nonpathogenic mutants that cause a HR in tobacco but do not produce EPS (Path-/HR+/EPS-), and one nonpathogenic mutant that causes an HR in tobacco and produces EPS (Path-/HR+/EPS+) were characterized for their ability to grow in apple shoots. Overnight broth cultures were grown in Kado 523 medium amended with 20 ug of tetracycline per ml and the concentration of bacteria adjusted to 5 X 108 colony forming units (cfu) per ml. The first fully unfolded leaf (30% to 80% expanded) of actively growing McIntosh apple shoots were bissected with scissors. A 2 ul drop of inoculum (containing approximately 106 cfu) was placed on the cut surface of the midrib. The droplet was absorbed or dried in 1 to 10 min. At 0, 6, 24, 48, 72, and 144 hr after inoculation leaves were harvested at the petiole stem junction. Leaf and petiole were homogenized in 1 ml of sterile 0.05 M phosphate buffer, pH 6.5, with a sterile mortar and pestle. An additional 9 ml of buffer was added and 0.1 ml amounts of serial buffer dilutions of the suspension were spread in petri dishes containing Luria-Bertani medium amended with 20 ug tetracycline per ml. 5 replicate leaves were assayed at each time for each mutant strain. Dishes were incubated at 28 C for 2 days before colonies were counted. The populations of all mutant strains decreased in apple shoots 6 hr after inoculation. From 1 to 6 days following inoculation, the populations of Path+/HR+/EPS+ mutants and the wild type strain, CFPB1367, increased 2 to 3 orders of magnitude. The populations of Path-/HR-/EPS+ mutants and the saprophyte Erwinia herbicola remained | |
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