SURFACE STERILIZATION OF GRAPE PHYLLOXERA EGGS IN PREPARATION FOR IN-VITRO CULTURE WITH VITIS SPECIES

The development of an in vitro dual culture system with grape phylloxe ra (Daktulosphaira vitifoliae Fitch) and Vitis species is dependent up on effective surface sterilization of the eggs to prevent microbial co ntamination of the system. A range of commercial bleach (5.25% sodium hypochlorite) and ethanol solutions were tested for their effect on ph ylloxera eggs. Treatments were judged by how they affected egg hatch a nd nymph survival and how well they prevented microbial contamination in sterile culture. About 80% of the eggs survived a seven-minute trea tment in either 50% ethanol or 50% bleach with no surface contaminatio n detected in any of the replicates after seven days. However, all of the bleach treatments required rinsing with sterile water to avoid gre atly reduced egg hatch. Fifty percent ethanol for seven minutes had th e advantage of not needing a sterile water rinse after treatment, thus simplifying the procedure and reducing further risk of contamination. This sterilization procedure allows in vitro dual culture studies of phylloxera biology, phylloxera resistance and susceptibility within Vi tis, and development of a rapid screening tool for grape rootstock bre eding.