Further evidence for the role of the PE layer as a barrier to
embryo contamination in pericarp-inoculated seeds was provided
by immunofluorescence microscopy. Immunolabeled A. citrulli
cells were observed in both the inner layers of the PE envelope
and the intercellular spaces of parenchyma tissue of the cotyledons/
embryos in pistil-inoculated seeds (Fig. 3C). In contrast, A.
citrulli cells were not observed in the embryos of pericarpinoculated
seeds but on the surfaces of PE layers (Fig. 3D).
We also found that pistil-inoculation circumvented the PE layer
and deposited A. citrulli cells in the embryos of watermelon
seeds. This conclusion was supported by the observation that
significantly higher percentages of embryos were infested with A.
citrulli in pistil-inoculated seeds compared to pericarp-inoculated
seeds. Additionally, removal of PE layers and testae did not affect
BFB transmission for pistil-inoculated seeds, while BFB transmission
was significantly reduced for pericarp-inoculated seeds.
At present, details on the specific pathways of A. citrulli ingress
through watermelon blossom tissues are not available; however,
bacterial deposition might be influenced by the timing of development
of the different seed layers. To date, PE layer development
has not been described in detail for watermelon seed, but in
cucumber seeds the PE layer begins to form by 15 days post-anthesis (DPA) and matures by 35 to 45 DPA (25,41,42). At
15 DPA, a lipid layer differentiates in the epidermis of the four- to
five-layered nucellus. An immature callose layer appears on the
surface of the PE layer by 25 DPA, and by 35 DPA the lipid layer
matures and a callose layer is deposited on the endosperm surface
(41). At 45 DPA, the nucellar cells degrade to form a mature PE
layer. While similar details on watermelon PE layer development
are not available, preliminary experiments showed that inoculation
of female watermelon blossoms with A. citrulli resulted in
ovule contamination by A. citrulli within 24 h (data not shown).
Since the PE layer is not full developed by this time, it is possible
that bacteria deposited in the ovary pericarp can gain access to the
seed embryos. In contrast, Frankle et al. (7) showed that the
window for A. citrulli to migrate through the pericarp of immature
watermelon fruits is 14 to 21 DPA. If the bacterium invades
the ovule at this stage, it is likely that the PE layer will serve as a
barrier to embryo contamination