Preliminary Study On Synergistic Activity And Mechanisms Of Mixtures Of Boscalid And Enestroburin Against Botrytis Cinerea

Tomato gray mold is an important disease caused by Botrytis cinerea.At present,the control of this disease still depends mainly on chemical fungicides.The chemical control occupies an indispensable position.Due to the rapid propagation and high fitness,resistance of B.cinerea is easy to occur.In addition,unreasonable using of fungicides,the B.cinerea has developed resistance to various fungicides with different mechanism,leading to a reduction in the efficacy of these fungicides.Therefore,it is urgent to develop fungicides with a novel mechanism or to screen synergistic combination to control the damage of gray mold.Boscalid is mainly used to control of various plant diseases such as gray mold,powdery mildew and so on.Enestrobin is mainly used for controlling powdery mildew and downy mildew.In this study the optimal virulence synergistic ratio of boscalid and enestrobin against B.cinerea was screened out.The effect of the optimal synergistic ratio(6:1)on that such as the different growth stages,mycelium morphological character,cell membrane permeability,respiratory rate,and the content of intracellular ATP,soluble protein and sugar of B.cinerea.This research can provide a theoretical basis for revealing the synergistic mechanism of compound of boscalid and enestrobin,and effective control of gray mold.The main results are as follows.1.This study was measured the joint toxicity of mixtures of boscalid and enestrobin to B.cinerea by the mycelium growth rate method.The results showed that when the proportion of boscalid and enestrobin was 6:1,the virulence of the mixture to the mycelium growth of B.cinerea was the highest and this ratio was defined as optimum synergistic ratio.The Inhibition of agent of optimal synergistic ratio against the other five strains of B.cinerea was also significantly higher than boscalid or enestrobin and they all showed synergistic inhibition.2.germination of conidia method was used to observed the inhibiton of mixture of boscalid and enestrobin on the germination of B.cinerea.The results showed that the agent of optimal synergistic ratio inhibited the spore germination of B.cinerea evidently higher than the two single agents,showing obvious synergistic action.The inhibition of the mycelium yield of agent of optimal synergistic ratio by weighing method.The results showed that the inhibition rate of the agent of optimal synergistic ratio to mycelium yield of B.cinerea was over 80% at different time,and higher than two single agents.The influence of the agent of optimal synergistic ratio on the spore capacity of B.cinerea was assayed by blood cell counting method.The results showed that the agent treatment of optimal synergistic ratio had no spore and the spore yield inhibition of agent of mixing against B.cinerea was obviously better than that of the two single agents.The influence of the agent of optimal synergistic ratio on sclerotia formation to B.cinerea was measured by weighing method.The results showed that when the concentration was higher than 2.5 ?g/mL,the agent treatment of optimal synergistic ratio had no sclerotia,and had stronger inhibition effect on sclerotia compared with the two single agents.3.The protective and curative activity of the agent of optimal synergistic ratio on the gray mold of tomato was determined by the in vitro leaf method.The results showed that the agent of best synergistic ratio was better than the single agent for the Protective and curative activity against B.cinerea and was synergy.The protective effect was better than curative effect.The field experiment showed that the control effect of agent of optimal synergistic ratio against tomato gray mold was 80.84%~88.07%,which was significantly superior to pyrimethamine and enestrobin.4.The results of mycelium morphology observation showed that the mycelium branches of B.cinerea by the agent treatment of optimum synergistic ratio was increased significantly.The top of some hyphae of B.cinerea treated with boscalid was inflated.The separation spacing of hyphae of B.cinerea treated with enestrobin was increased and shortened obviously,and the small substances suspected inclusion leakage on the surface of mycelium was observed.5.The integrity of cell membrane of B.cinerea conidia of optimal synergistic ratio was determined by fluorescence microscope.The results showed that the membrane of B.cinerea conidia was damaged by chemical treatment.The permeability of B.cinerea cell membrane was measured by conductivity method.The results showed that the agent of optimum synergistic ratio increased the cell membrane permeability of B.cinerea.6.The influence of the agent of optimum synergistic ratio and its single agent on the respiratory rate and ATP content of B.cinerea was measured by oxygen electrode method and high performance liquid chromatography method respectively.The results showed that the respiration of B.cinerea conidia treated with the agent of optimal synergistic ratio was strongly inhibited and the inhibition rate reached 90.68%.The ATP content of B.cinerea intracellular decreased greatly and the energy synthesis of B.cinerea conidia was inhibited.The influence of optimal synergistic ratio to the content of soluble protein and sugar in the mycelium of B.cinerea was observed by colorimetric method.The results showed that the synthesis of soluble protein and sugar was inhibited treated with the agent of optimal synergistic ratio at the early stage.7.The optimal synergistic ratio enhances the inhibition of the respiration of B.cinerea and hinders the synthesis of energy in vivo,which leads to the disorder of substance metabolism and demonstrating synergistic effect.It is preliminarily inferred that this may be one of the mechanisms on physiological and biochemical levels of synergistic action of the mixture of boscalid and enestrobin.