Measurement of active oxygen species generated in planta in response to elicitor AVR9 of Cladosporium fulvum

Injection of elicitor preparations containing the Avr9 gene product of Cladosporium fulvum (race 4) into tomato carrying the Cf9 gene induced leaf necrosis and production of active oxygen species (AOS). The nature and temporal development of AOS and its relationship to leaf necrosis was explored using in planta probes for AOS. Generation of O-2(-) was detected in elicitor-injected tomato leaves using a manganese/diaminobenzidine technique. To detect the generation of H2O2, the fluorescent probe, 2',7'-dichlorofluorescin diacetate (DCFH-DA), was injected into leaf areas previously injected with elicitor. The fluorescent compound, 2',7'-dichlorofluorescein (DCF), formed from oxidation of DCFH by H2O2 in the presence of peroxidases, was produced much faster in interveinal panels injected with elicitor than in those injected with control preparations (lacking the Avr9 product), or with the probe alone. Fluorescence intensity was compared visually under u.v. light and/or measured on a fluorimeter following extraction from the tissue. DCF formation in leaves on injection of known concentrations of H2O2 was verified. Elicitor-induced oxidation of DCFH was inhibited by co-injection of elicitor with catalase or an inhibitor of superoxide dismutase. Co-injection of DCFH-DA with the antioxidants, L-ascorbic acid and glutathione, or with KCN, an inhibitor of peroxidases, into leaves previously injected with elicitor also blocked fluorescence development. Oxidation of DCFH was detected in leaves injected with elicitor at concentrations lower than those that cause leaf necrosis. Necrosis development was partially inhibited and/or significantly delayed by injection of elicitor mixed with catalase and several other antioxidants or scavengers of AOS. These results provide in planta evidence for the involvement of AOS in the defence response of tomato to a race specific elicitor of C. fulvum. (C) 1998 Academic Press Limited.