Antifungal activity of non-induced, chitin-induced, and prey-induced pitcher liquid against plant fungal pathogens. (A) Chitin injection into closed pitchers induced a change in the liquid colour. Comparison of samples of non-induced (white dot), chitin-induced (red dot) and prey-induced (blue dot) pitcher liquid. (B) B. cinerea growth inhibition measured on excised tobacco leaves. Samples of 20 μl of 16.7-fold concentrated non-induced, chitin-induced, and prey-induced pitcher liquid were separately applied on the leaves. An agar plug containing B. cinerea young mycelium was placed on each spot and the leaves were incubated in a humid growth chamber at 22 °C for 6 d. (C) Inhibition of R. solani and (D) F. oxysporum mycelial growth. Paper discs were soaked either with 12 mg DCIL/100 μl H₂O or 100 μl H₂O and applied on the surface of malt agar/potato dextrose agar plates. An agar plug (9 mm²) of R. solani or F. oxysporum young mycelium was placed in the centre. The plates were incubated for 4 d at 22 °C. (E) Inhibition of M. graminicola conidia germination. M. graminicola conidia (2.5×10³ conidia in 100 μl) were incubated in microtitre plates in the presence of increasing amounts of DCIL dissolved in 100 μl H₂O at 18 °C for 6 d (the final concentration in mg ml⁻¹ is indicated in E). Thereafter 50 μl of each culture was plated onto a malt agar plate and incubated for an additional 6 d under the same conditions.

Inhibition of A. solani and B. cinerea spore germination by a droserone/Me-droserone mixture (3:1 molar ratio). A. solani (A, B) and B. cinerea (C, D) spores (2×10³) were incubated in PDB in microtitre wells in the presence of increasing concentrations of droserone/Me-droserone mixture (3:1 molar ratio). The droserone concentration was 49, 243, and 486 μg ml⁻¹ in the 66, 330, and 660 μg ml⁻¹ mixtures, respectively. Inhibition of spore germination was determined after 24 h by microscopic photographing (A, C) and after 44 h by measuring the density of germinating spores at 595 nm (B, D). (This figure is available in colour at JXB online.)

The effect of droserone and Me-droserone on the viability of human 293T cells. Human-embryo-kidney cells (line 293T) were grown at 37 °C under 5% CO₂ in DMEM containing 10% foetal calf serum, 4500 mg l⁻¹ glucose, and 2 mM glutamine. Cells were seeded at a density of 8×10³ per well in a 96-well plate and incubated for 24 h. The medium was then exchanged for media containing 60 μl of increasing concentrations of Me-droserone (light grey) or droserone (dark grey), purified by reversed phase column and identified by NMR. After 48 h incubation, cell viability was determined by staining with 0.4% (w/v) trypan blue. Each treatment contained two replicates.

Identification of droserone and 5-O-methyldroserone by NMR analysis. The structure of compounds (upper panel) and ¹H NMR spectra of (A) non-induced, (B) chitin-induced, and (C) prey-induced (open pitcher) liquid. The proton signals, indicative of droserone and 5-O-methyldroserone, consist of the aromatic proton between 7 ppm and 8 ppm and the methyl protons that appear as singlets between 1.8 ppm and 1.9 ppm. The 5-methoxy group resonates as singlet at 4.0 ppm. A ratio of 4:1 of droserone to 5-O-methyldroserone was observed in the sample of chitin-induced liquid while these metabolites were absent from the non-induced and prey-induced pitcher liquid. The ratio between droserone and 5-O-methyldroserone was determined by measuring the integration of the vinyl methyl at position 2 of the naphthoquinones, which resonate at 1.92 ppm for droserone, and 1.99 ppm for 5-O-methyldroserone.

Inhibition of A. solani spore germination by purified droserone and Me-droserone. A. solani spores (2×10³) were incubated in PDB in microtiter wells in the presence of increasing concentrations of droserone (A) or Me-droserone (B). Inhibition of spore germination was determined after 48 h by microscopic photographing.