1.2.18
SV-TOXIN, PARTIALLY PURIFIED FROM CULTURE FILTRATES OF STEMPHYLIUM VESICARIUM, INDUCES AN INCREASE IN OXYGEN UPTAKE AND IN THE ACTIVITY OF PLASMA MEMBRANE NADH:FERRICYANIDE REDUCTASE IN PEAR

R. BUGIANI 1, N. NEGRINI2, M. COCUCCI2, I. PONTI1 and P. CAVANNI1

1Servizio Fitosanitario - Regione Emilia-Romagna, Bologna, Italy; 2 Dip. Fisiologia Piante Coltivate e Chimica Agraria, University of Milan, Milan, Italy

Background and objectives
The pathogenic activity of Stemphylium vesicarium (Wallr.) E. Simmons, the agent of brown spot of European pear (Pyrus communis L.), seems to be mediated by a toxin able to produce necrotic spots on leaves and fruits of susceptible cultivars. The toxin, which is present in the culture filtrates of the fungus, induces the release of electrolytes [1]. Apart from this effect, the mechanism of action of the toxin is still unknown. The aim of this work was to investigate early signalling events of the pathogen attack and therefore we analysed the effect of the toxin, partially purified from culture filtrates of Stemphylium vesicarium, on pear leaf tissue.

Materials and methods
Pear plantlets from in vitro cultures of susceptible (Abate Fètel) and resistant (William) cultivars were used. SV toxin was partially purified from culture filtrates by methanol extraction, chromatography on a DEAE column, FPLC and HPLC. Leaf protoplasts were obtained by enzymatic digestion and separation on a discontinous Percoll gradient. Plasma membrane vesicles were purified by the two-phase partition method [2].

Results and conclusions
The toxin did not directly affect membrane transport activities when tested at the same concentrations inducing the appearance of necrotic spots and the release of electrolytes into the medium. The respiration rates measured on leaf tissue sections indicated that in vivo the toxin was able to stimulate, in the susceptible cultivar, a rapid and early increase in oxygen uptake, but it reduced neither ATP nor ADP levels in the tissue. In vitro the toxin induced a slight decrease in respiration rate of isolated mitochondria. Under these conditions, however, measurement of safranine O quenching ruled out the possibility that the toxin could act as an uncoupler. In protoplasts isolated from leaf tissue of a susceptible cultivar, a slight inhibitory effect of the toxin on oxygen uptake was also evident.

The activity of NADH:ferricyanide reductase of plasma membrane vesicles purified from leaf tissue of the susceptible cultivar incubated for different times in the absence and in the presence of the toxin increased during the time of incubation. This increase did not occur when the toxin was administered in the assay medium. These preliminary data on the effect in vivo, but not in vitro, of the toxin on the increase in oxygen uptake and ferricyanide reduction suggest the possibility that the mechanism of action of the toxin concerns the activation of some membrane activities leading to the production of active oxygen species.

References
1. Cavanni P, Bugiani R, Zanotti M et al., 1993. Proceedings of the 6th International Congress of Plant Pathology, Montreal, 1993, p. 223 (abstr).
2. Larsson C, Widele S, Kjellbom P, 1987. Methods in Enzymology 148, 558-568.