Detection of tomatinase from Fusarium oxysporum f. sp. lycopersici in infected tomato plants.

The antifungal glycoalkaloid alpha-tomatine of the tomato plant (Lycopersicon esculentum) is proposed to protect the plant against phytopathogenic fungi. Fusarium oxysporum f. sp. lycopersici, a vascular pathogen of tomato, produces a tomatinase enzyme which hydrolyses the glycoalkaloid into non-fungitoxic compounds. Detoxification of alpha-tomatine may be how this fungus avoids the plant glycoalkaloid barrier. As an initial step to evaluate this possibility we have studied the induction of tomatinase; (i) in fungal cultures containing extracts from leaf, stem or root of tomato plants; and (ii) in stem and root of tomato plants infected with the pathogen at different infection stages. The kinetics of tomatinase induction with leaf extract (0.6% dry weight) was similar to that observed with 20 micrograms ml-1 of alpha-tomatine. In the presence of stem extract, tomatinase activity was less than 50% of that induced with leaf extract, whereas in the presence of root extract tomatinase activity was very low. In the stem of infected tomato plants tomatinase activity was higher at the wilt stage than in previous infections stages and in root, tomatinase activity appeared with the first symptoms and was maintained until wilting. TLC analysis showed that the tomatinase induced in culture medium with plant extracts and in infected tomato plants had the same mode of action as the enzyme induced with pure alpha-tomatine, hydrolysing the glycoalkaloid into its non-fungitoxic forms, tomatidine and beta-lycotetraose. The antisera raised against purified tomatinase recognized in extracts of root and stem of infected tomato plants a protein of 50000 (45000 when proteins were deglycosylated), corresponding to the tomatinase enzyme. Therefore, it is concluded that F. oxysporum f. sp. lycopersici express tomatinase in vivo as a result of the infection of tomato plant.