Efficacy of AAC00-1ΔhrcC as a blossom protectant to prevent
seed infestation by A. citrulli. The ability of AAC00-1ΔhrcC
to protect female blossoms from invasion by A. citrulli was evaluated.
Watermelon plants (Crimson Sweet) were maintained under
standard greenhouse conditions in 15-liter pots containing 90%
composted pine bark and 10% vermiculite until anthesis. At anthesis,
female blossoms were manually pollinated as described above,
then immediately inoculated by depositing 10 μl of a suspension
containing AAC00-1ΔhrcC, AAA99-2 at approximately 1 × 108
CFU/ml, or PBS as a negative control onto the stigmas. Five hours
after inoculation, blossoms were challenged with 10 μl of a cell
suspension containing AAC00-1 at 1 ×109 CFU/ml. Each
treatment was applied to 10 blossoms, each on a separate plant,
and fruit were allowed to develop for 35 days after pollination.
At harvest maturity, fruit were surface sterilized and seed were
manually extracted (seed from each fruit were maintained as a
separate lot), air dried at 25°C for 24 h, and stored at 4°C. To
determine the level of seed infestation by A. citrulli, samples (n =
100 seed) from each lot were subjected to the modified seedling
grow-out assay as described above. Seedlings were monitored
daily for 14 days and germination and BFB seedling transmission
percentages were recorded daily. This experiment was conducted
twice and mean BFB seedling transmission percentage data were
plotted over time and used to generate AUDPC data. Germination
data were also collected and used to determine the effect of
biocontrol blossom treatments on seed physiology. AUDPC data
were analyzed using the Tukey-Kramer’s HSD to determine the
significance of blossom protection on seed infestation by A.
citrulli.