plant surface microbiology.pdf

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Daniel Knoll and Lukas Schreiber
Fig. 7. Penetration of
Pseudomonas fluorescens
through cuticular membranes
of Vinca major.The
amount of penetrated cuticles
increases with incubation
time. After 9.5 days
50 % of the inoculated cuticles
are penetrated by P.
fluorescens. At the end of
the kinetic there is a linear
increase of penetrated cuticles
with a rate of 6.1 %
penetrated CM per day in
relation to the total amount
of inoculated cuticles
barrier functions of the cuticle for microbial penetration. Once these values
are known, barrier properties of cuticles of different plant species that differ
in their morphology like cuticle thickness or chemistry like wax composition
can be compared. Another attractive application is to measure penetration of
different microbial strains or mutants that differ in their array of extracellular
enzymes like cutinase activity.
Several control experiments need to be conducted to ensure bacterial penetration
through isolated cuticles. (1) When glass slides are mounted into the
chambers in place of cuticles there was never any bacterial growth detectable
in the nutrition solution. This gives evidence that bacteria are not able to
bypass the glass surface via the silicone grease seal. (2) In addition, no bacterial
growth was detected in the nutrition solution when cuticles were inoculated
with sterile PBS indicating that the system itself is sterile and no other
origins for bacterial growth are possible except from the inoculus on the outer
cuticle surface. (3) Finally, a third control consists of applying 200 ml of dead
bacteria. Cells are cultivated as described above and subsequently killed with
paraformaldehyde and stained with the fluorescent dye DAPI. It was checked
that all bacterial cells were killed.After the inoculation period of 6 h the nutrition
solution is checked for the presence of DAPI-stained cells with fluorescence
microscopy. A fraction of about 10 % of the inoculated cuticles was
apparently leaky for dead cells. This might be due to mechanical injuries to
the cuticular membranes during the process of isolation or during the mounting
of cuticular membranes in the chambers. Those membranes were sorted
out and not considered any further. Furthermore, cuticular water permeability
measured prior to inoculation with bacterial cells was very low (Table 1),
indicating that the membranes form high effective barriers for the transport
of water on the molecular level. This also suggests that they build intact barriers
for microbial cells as well. Basically, all control experiments confirmed