PEROXIDASE-GENERATED HYDROGEN-PEROXIDE AS A SOURCE OF ANTIFUNGAL ACTIVITY INVITRO AND ON TOBACCO LEAF-DISKS

Hydrogen peroxide was generated in vitro by horseradish peroxidase in the presence of NADH or NADPH. When peroxidase was inactivated by heating (30 min at 100 C) or catalase was added to the reaction mixture, little or no hydrogen peroxide was generated as measured by a guaiacol oxidation assay. In the absence of either H2O2 or peroxidase, guaiacol was also not oxidized. Guaiacol oxidation was also observed when either NADH was replaced by NAD and malate in the presence of malate dehydrogenase or NADPH was replaced by NADP and glucose-6-phosphate in the presence of glucose-6-phosphate dehydrogenase. Oxidation was not observed when the enzymes were heat-inactivated. Hydrogen peroxide at 2.61 X 10(-5) M totally inhibited the spore germination of Peronospora tabacina, Cladosporium cucumerinum, and Colletotrichum lagenarium in vitro and markedly inhibited germination at 6.5 X 10(-6) M. When a sporangiospore suspension in pH 7.0 phosphate buffer was incubated with NADH or NADPH and peroxidase at 18-20 C for 18 h, the germination of sporangiospores of P. tabacina was inhibited by 72-78%. When the same mixture of sporangiospores with NADH or NADPH and peroxidase was applied to the upper surface of tobacco leaf disks, blue mold development was markedly inhibited. If peroxidase was inactivated or catalase was added, the inhibitory effects on spore germination and disease development were abolished. When NADH was replaced by NAD and malate in the presence of malate dehydrogenase, spore germination was inhibited in vitro and disease development on leaf disks was reduced.