The oxidative burst in tomato plants induced by race-specific elicitors of Cladosporium fulvum

One of the intriguing features of plant disease resistance is an initial oxidative burst. The present study, using the Cladosporium fulvum -tomato pathosystem, explored the oxidative burst in both leaf tissues and in cell cultures following treatment with race specific elicitors obtained from the fungus. The oxidative burst was detected by 2',7'dichlorofluorescin diacetate (DCFH-DA), a probe for detection of H₂O₂. A variety of tests, including the use of catalase, showed that the probe measured both intracellular and extracellular H2O2. Ile oxidative burst occurred within 1–2 hr in leaf tissues and within a few minutes in cell cultures, respectively, after elicitor treatment. In cell cultures, the oxidative burst was abolished by inhibitors of NADPH oxidase, suggesting that this enzyme is responsible for the generation of O₂−, a precursor of H₂O₂. As well, O₂− was detected in planta by the Mn2+/diaminobenzidine technique. Prevention of increased cytosolic Ca2+ appeared to block the increased H₂O₂ generation. Studies with other inhibitors suggested that protein kinase(s) and phospholipase C were involved in the oxidative burst, supporting the possibility that the signaling mechanism leading to the oxidative burst in tomato cells is similar to that observed in neutrophils. Activities of antioxidant enzymes, e.g., superoxide dismutase, catalase, and glutathione S-transferase, were not changed in leaves undergoing an oxidative burst. Total peroxidase activity, including ascorbate peroxidase, increased in elicitor-treated leaves but the activity of ascorbate peroxidase decreased by 4 hr. Elicitor-induced necrosis in leaves was significantly delayed, but not completely inhibited, by catalase or by scavengers of active oxygen species (AOS). An oxidative burst was still induced at elicitor dilutions which caused no visible necrosis and only high concentrations (ca 1M) of H₂O₂ could mimic the visible leaf necrosis induced by elicitor. Growth of C. fulvum germ tubes was inhibited in vitro by moderate levels of H₂O₂, suggesting a possible role for H₂O₂ in restricting colonization. A model is proposed which describes the relationship between AOS, generated from an oxidative burst, and the fate of both host cells and invading fungal cells.