A role for oxidative stress in the Citrus limon/Phoma tracheiphila interaction

Phoma tracheiphila, the agent of citrus 'mal secco', releases a toxic complex called malseccin into the plant. Oxidative stress was studied, both regarding the reaction of lemon (Citrus limon) leaves to the infiltration of fungal extracellular proteins, and the analysis of the pathogen's ability to cause symptoms in leaves of lemon cultivars with different degrees of susceptibility. Studies were perfomed in planta on three lemon cultivars: cv. Interdonato, partially tolerant to 'mal secco'; cv. Monachello, tolerant; and cv. Femminello, susceptible. When inoculated with P. tracheiphila extracellular proteins, cv. Interdonato leaves showed chlorosis and necrosis symptoms and an increase in lipoxygenase and glutathione peroxidase of up to 100 and 76.5 fold, respectively. Furthermore, extracellular proteins of P. tracheiphila infiltrated into leaves induced lipoperoxide formation 10 fold in cvs Interdonato and Femminello and 3 fold in cv. Monachello compared to the control, with Monachello reacting earlier. In vitro analyses indicate that the presence of lyophilized twigs and leaves (LTV) of cv. Monachello in the fungal growth medium, stimulated concentrations of superoxide dismutases (similar to 20-60 fold), glutathione peroxidase (similar to 115 fold) and catalase (similar to 15 fold) in fungal mycelia. When LTV of cv. Femminello was added, the pathogen produced a higher quantity of hydrolytic enzymes in vitro, polygalacturonase (similar to 40 fold) and laccase (similar to 110 fold). The MALDI-TOF and TOF/TOF MS analyses performed on 60-70 KDa extracellular proteins of P. tracheiphila after inoculation in malseccin conducive media showed the presence of a monoamine oxidase enzyme able to release NH4+ and H2O2 in the cell. Overall the results suggest oxidative stress occurs in this interaction.