RESUMO
Gray mold disease caused by Botrytis cinerea infection is one of the major crop diseases. The application of environmental-friendly fungicides to control gray mold disease has been drawing great attention. Thymol, a natural compound, showed strong antifungal activity against Botrytis cinerea. We investigated the role of polyamine oxidase (PAO)-dependent hydrogen peroxide (H2O2) production in thymol-inhibited B. cinerea growth by using physiological and biochemical approaches. The results showed that: 1) Thymol significantly inhibited the growth of B. cinerea, with remarkable increases in H2O2 content, malondialdehyde (MDA) content, and PAO activity in mycelium. 2) Inhibition of PAO activity (addition of specific inhibitor MDL, N,N'-butanedienyl butanediamine) resulted in significant decreases in the contents of H2O2 and MDA as well as the partial recovery of mycelial growth under thymol treatment, suggesting that thymol might trigger PAO-dependent H2O2 accumulation resulting in oxidative injury and thus inhibit the growth of mycelium. 3) A PAO homologue gene BcPAO was cloned from B. cinerea. Multi-alignment combined with phylogenetic analysis showed that BcPAO protein had typical conserved domain of PAO family members. 4) Thymol at low concentrations did not affect the transcriptional level of BcPAO. However, the transcription of BcPAO was up-regulated remarkably by thymol at high concentration. This suggested that thymol-stimulated PAO activity may be resulted from the regulation of BcPAO. We conclude that oxidative injury caused by PAO-dependent H2O2 production is one of the possible antifungal modes of thymol against B. cinerea. The antifungal mode of thymol found in this study may provide basis for the application of environmental-friendly fungicides.
