Immuno-Assessment of «Pseudomonas syringae» Lipodepsipeptides (Syringomycins and Syringopeptins)

Abstract

Following a previous work on the immunological detection of syringopeptins (SPs), polyclonal antibodies
with a high specificity for syringomycins (SRs) were raised in rabbits and purified. Assayed in a competitive ELISA,
the most common forms of SR, i.e. SR-E and SR-G, were recognised with a detection limit of 0.1 mg per well, whereas
other structurally related bacterial lipodepsipeptides (LDP), such as SPs, pseudomycins (PSs) and syringotoxins
(STs) were not recognised. The immuno-assay (competitive ELISA) method developed in this work is about 100 times
more sensitive than the current chromatographic (HPLC) method and requires no previous extraction of the toxin.
The production of LDP in culture by strains of three pathovars of Pseudomonas syringae (pv. aptata, pv. lachrymans
and pv. syringae) was found to range from 0.026 to 0.055 mg ml-1 for SRs and from 0.02 to 0.06 mg ml-1 for SPs. Both
the concentration of LDP in aqueous extracts from zucchini cotyledons infected by P. syringae pv. lachrymans and the
severity of symptoms were shown to increase progressively after infection. The immunologically estimated concentration
of SRs in the infected cotyledons averaged 0.22 mg g-1 f wt after 12 hours, and 0.39 mg g-1 after 4 days. The
corresponding values for SPs were 0.11 and 0.37 mg g-1. In a recovery experiment, solutions of pure toxins (0.22 mg
SR-E and 0.14 mg SP25A g-1 f wt) were injected in healthy cotyledons. After 2 days, overestimation due to toxin
complexing in planta was of 10% for (SR-E) and 40% for (SP25A). Applying these percentages to the values estimated
for infected cotyledons, the net concentrations were as follows: 12 h after inoculation: 0.20 (SRs) and 0.07 (SPs)
mg g-1 f wt; four days after inoculation: 0.35 (SRs) and 0.22 (SPs) mg g-1 f wt. The values obtained with aqueous
extracts from infected plants are relatively high if compared to the figures of the in vitro experiments. It is assumed
that the high reactivity of ELISA to the immune-LDP-related compounds present in the water extracts from infected
plants is due to the presence of high molecular weight LDP complexes having a cross-reactivity with antibodies
substantially higher than that of free toxins.