2.9.9
ATTEMPTS TO DETERMINE THE PRESENCE OF ENDOPHYTIC ERWINIA AMYLOVORA BACTERIA IN SYMPTOMLESS APPLE TISSUES

T VAN DER ZWET and EW BROWN

USDA, ARS, Appalachian Fruit Research Station, Keameysville, WV, USA

Background and objectives
A major limiting factor in the effective control of fire blight on apples and pears is the lack of knowledge in the detailed ecology and aetiology of Erwinia amylovora in the disease cycle. Migration of the bacterium in stem tissue has been shown to occur via the phloem [1]. First demonstration of the presence of E. ;amylovora in symptomless side-shoots of apple and pear trees following artificial inoculation was published in 1972 [2}. Since that time, numerous observations have been reported of unexplained fire blight symptoms during the growing season following recommended control strategies. The following research projects have been conducted to attempt elucidation of the systemic presence of the bacterium as an endophyte in symptomless apple tissues.

Materials and methods

In 1994 (experiment A), symptomless shoots on 30 'Jonathan'/M7 apple trees in the greenhouse were examined for the presence of endophytic E. ;amylovora, 10 ;months after the trees were artificially inoculated with the bacterium. Shoots were surface sterilized, and 3-mm pieces were plated on nutrient-yeast-dextrose agar. Crosse-Goodman selective medium [3] was used to confirm characteristic colony cratering.

In 1995 (experiment B), two 12-year-old 'Rome'/Sd1 apple trees were severely pruned and the remaining branches surface sterilized. In the spring of 1996, these trees were thoroughly sprayed with copper in the delayed dormant bud stage and were then placed indoors near sterile clear plastic arborspheres, complete with air supply systems and bacterial filters.

In 1997 (experiment C), attempts were made to isolate endophytic E. ;amylovora from symptomless shoots on four 22-year-old 'Rome Beauty'/M9 trees in the orchard. The trees showed moderate blight symptoms and samples were collected in June at five different locations on each tree.

Results and conclusions
In experiment A, of the 30 original 'Jonathan' trees, 20% contained E. ;amylovora in one or more of the symptomless side-shoots. Endophytic bacteria were also recovered from unopened blossom buds of three out of nine trees that had formed terminal flower buds. The bacterium remained latent for 10 ;months in neglected greenhouse trees.

In experiment B, on the uncovered, pruned 'Rome' trees, the first shoot blight symptoms appeared in June and continued until mid-July. No symptoms ever appeared on the trees in the arborspheres and no E. ;amylovora bacteria were recovered from symptomless shoots or on open Petri dishes lowered into the spheres. The bacterium did not appear to be present as a systemic pathogen.

In experiment C, more than 300 shoot discs were plated from 24 symptomless shoots (four replicates) most distal to any blight symptoms and 23.9% contained E. ;amylovora bacteria. Subsequent sampling in July and October were negative. The bacterium appeared to be temporarily present as an endophytic pathogen.

References
1. Lewis SM, Goodman RN, 1965. Phytopathology 55, 719-723.
2. Keil HL, van der Zwet, T, 1972. Phytopathology 62, 39-42.
3. Crosse JE, Goodman RN, 1973. Phytopathology 63, 1425-1426.