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.
由灰霉菌侵染导致的农作物灰霉病是农业重大病害之一,采用环境友好型杀菌剂防治灰霉病受到越来越多的关注。天然化合物麝香草酚对灰霉菌具有较强的抑菌活性,本文采用生理生化方法探讨了麝香草酚通过调控灰霉菌多胺氧化酶(PAO)产生过氧化氢(H2O2)的抑菌机理。结果表明: 1)麝香草酚抑制灰霉菌生长,并伴随着菌丝体中H2O2和丙二醛(MDA)的积累及PAO活性的升高。2)在麝香草酚处理的菌丝中加入特异性抑制剂MDL(N,N'-丁烷二烯基丁二胺)抑制PAO活性,导致H2O2和MDA显著下降,菌丝生长得到部分恢复。表明麝香草酚可能触发灰霉菌中依赖于PAO的H2O2累积,进而产生氧化损伤效应,导致部分菌丝生长受阻。3)从灰霉菌中克隆了一个编码PAO的基因BcPAO,序列比对和进化分析显示,BcPAO蛋白具有典型的PAO家族保守结构域特征。4)低浓度麝香草酚处理对BcPAO转录水平无显著影响,而高浓度麝香草酚处理则显著上调BcPAO的转录水平,说明麝香草酚对灰霉菌PAO活性的刺激作用可能源于对BcPAO的调控。研究表明,麝香草酚对灰霉菌的抑菌效应之一可能表现为:菌丝PAO活性上升导致H2O2大量产生,引发菌丝氧化损伤,生长受阻。本研究发现的麝香草酚抑菌模式可为环境友好型杀菌剂的应用提供理论依据。.
Keywords: Botrytis cinerea; antifungal mechanism; hydrogen peroxide; polyamine oxidase; reactive oxygen species; thymol.