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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.

Cationic lipodepsipeptides from Pseudomonas spp. have been characterized for their structural and antimicrobial properties. In the present study, the structure of a novel lipodepsipeptide, cormycin A, produced in culture by the tomato pathogen Pseudomonas corrugata was elucidated by combined protein chemistry, mass spectrometry and two-dimensional NMR procedures. Its peptide moiety corresponds to L-Ser-D-Orn-L-Asn-D-Hse-L-His- L-aThr-Z-Dhb-L-Asp(3-OH)-L-Thr(4-Cl) [where Orn represents ornithine, Hse is homoserine, aThr is allo-threonine, Z-Dhb is 2,3- dehydro-2-aminobutanoic acid, Asp(3-OH) is 3-hydroxyaspartic acid and Thr(4-Cl) is 4-chlorothreonine], with the terminal carboxy group closing a macrocyclic ring with the hydroxy group of the N-terminal serine residue. This is, in turn, N-acylated by 3,4-dihydroxy-esadecanoate. In aqueous solution, cormycin A showed a rather compact structure, being derived from an inward orientation of some amino acid side chains and from the ‘hairpinbent’ conformation of the lipid, due to inter-residue interactions involving its terminal part. Cormycin was significantly more active than the other lipodepsipeptides from Pseudomonas spp., as demonstrated by phytotoxicity and antibiosis assays, as well as by red-blood-cell lysis. Differences in biological activity were putatively ascribed to its weak positive net charge at neutral pH. Planar lipid membrane experiments showed step-like current transitions, suggesting that cormycin is able to form pores. This ability was strongly influenced by the phospholipid composition of the membrane and, in particular, by the presence of sterols. All of these findings suggest that cormycin derivatives could find promising applications, either as antifungal compounds for topical use or as post-harvest biocontrol agents.