School of Engineering and Science - Jacobs University

Material and Methods
determination of bacterial population 20 such wounds were analysed per timepoint.
Disks of 7 mm diameter surrounding the wound-site were punched from
the leaflet using a cork-borer. The disks were grinded down in 20 ml of sterile
NaCl (0.9%). A dilution serious of the resuspension was plated on KB-agar and
cell-forming units were counted after 48 hours of incubation.
4.4.2 Spray inoculation
For spray inoculation experiments, bacterial inoculum was prepared by
resuspending cells from a fresh KB-agar plate in sterile water. OD 600nm of the
suspension was adjusted to 0.01. 100 ml of this suspension were sprayed on
three 4 weak old soybean plants. For determination of bacterial population
fresh soybean leaflets were cut and grinded down in sterile 0.9% NaCl (10ml of
NaCl per gram fresh weight). A dilution serious of the resuspension was plated
on KB-agar and cell-forming units were counted after 48 hours of incubation.
4.5 Analysis of siderophore-production
4.5.1 Uptake of 59 Fe
59 Fe can be utilized to monitor the bacterial uptake of iron/siderophore
complexes (Meyer et al., 2002). In this experiment, bacterial cells from 40 h of
culture in CAA medium were harvested by centrifugation, washed once with
distilled water, and resuspended at an OD 600nm of 0.33 in an incubation medium
made of succinate medium with the nitrogen source omitted. Label mix
containing 59 Fe/siderophore complex consisted of 5 µl of the commercial 59 Fe 3+
solution (iron chloride in 0.1 M HCl, specific activity 110 to 925 MBq/mg of iron;
Amersham) was diluted first with 100 µl of water and then mixed with 10 µl of a
6.5-mg/ml siderophore solution. Alternatively, 5 µl of the 59 Fe 3+ solution were
mixed with 100 µl of siderophore-containing culture supernatants. The final
volume of the label mix was adjusted after 30 min of incubation at room
temperature to 1 ml with incubation medium. Bacterial suspension (1.8 ml) was
mixed at time zero with 0.2 ml of label mix. After 20 min of incubation under
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