1.8.23
PURIFICATION, BIOLOGICAL AND CHEMICAL CHARACTERIZATION OF A HOST-SPECIFIC SV-TOXIN FROM CULTURE FILTRATES OF STEMPHYLIUM VESICARIUM CAUSING BROWN SPOT OF EUROPEAN PEARS

P SINGH1, R BUGIANI2, P CAVANNI2, H NAKAJIMA3, M KODAMA3, H OTANI1 and K KOHMOTO3

1United Graduate School of Agricultural Sciences, Tottori University, Tottori 680-0945, Japan; 2Osservatorio per le Malattie delle Piante, Bologna, Italy; 3Laboratory of Plant Pathology, Faculty of Agriculture, Tottori University, Tottori 680-0945, Japan

Background and objectives
Stemphylium vesicarium causes brown spot of European pear (Pyrus communis), a disease of economic importance in Mediterranean pear-producing areas in Europe. Infection and necrosis occur on leaves, fruit and twigs. Most of the highly susceptible cultivars are of great commercial interest in Europe [1]. Despite the importance of the disease, little information on mode of pathogenesis is available. The current study was undertaken to determine the primary factors involved in the disease development. The pathogen from our studies most probably produces a host-specific toxin (HST), named SV-toxin in culture filtrates, which selectively induces necrosis only on susceptible cultivars.

Materials and methods
Four pathogenic strains (named IT35, IT36, IT37 and IT38) and two non-pathogenic strains (IT39 and IT42) of S. vesicarium and European pear cultivars (Yoshimoto, Passe Crassane, Conference, La France, Le Lectier, General Leclerc and Bartlett) , Japanese pear cultivars (Nijisseiki, Osa Nijisseiki and Chojuro) and other non-host plants were used in this study. SV-toxin was purified from culture filtrates by Amberlite XAD-2 resin adsorption, Silica Gel ODS-Q3 column and HPLC. The biological activity and host specificity of toxin were determined by leaf necrosis assays; 1H- , 13C-NMR and FAB-MS analyses were conducted for structural determination of purified SV-toxin.

Results and conclusions
Purified SV-toxin showed high host-specific toxicity only to European pear cultivars, which were found to be highly susceptible to the pathogen. The susceptible cultivars showed veinal necrosis at 0.1 p.p.m., whereas resistant cultivars were found insensitive to the pure toxin at about 104-fold increased concentration. Yoshimoto, a Japanese cultivar of European pear, was found to be highly susceptible to the pathogen and sensitive to SV-toxin at ca 0.01 p.p.m. concentration. A positive correlation was obtained between toxin productivity and pathogenicity of the different pathogenic and non-pathogenic strains under investigation. Since S. vesicarium invades host tissues intercellularly, SV-toxin was also detected in the intercellular fluids of infected leaves. The partial structure of SV-toxin appears to have benzaldehyde ring attached to some unknown group(s). FAB-MS reveals that SV-toxin is a small molecule. We are currently in the process of elucidating the structure of SV-toxin. Characterization of the mechanisms involved in disease development by SV-toxin is in progress.

References
1. Montesinos E, Moragrega C, Llorente I, Vilardell P, 1995. Plant Disease 79, 471-473.