Toxicity Mechanism Of CuO NPs To Phytophthora Nicotianae And Its Control Effect On Tobacco Black Shank

Tobacco Black Shank is a soil-borne vascular disease caused by Phytophthora nicotianae,which is a prominent problem in tobacco plant protection.Once the disease breaks out,it often causes the death of a large area of tobacco plants,resulting in huge economic losses.Theoretically,the selection of resistant varieties is the most environmentally friendly and effective means to control black shank disease in tobacco.However,due to the demand of industry and the differentiation of Phytophthora nicotianae,the wide spread of tobacco varieties resistant to black shank disease has been limited.Secondly,the use of conventional chemical agents to control Tobacco Black Shank,such as metalaxyl,metalaxgl mancozeb,etc.,due to its single action site against Phytophthora nicotianae,long-term use often causes the disease to produce strong resistance.In addition,biological antagonistic agents,such as Trichoderma harzianum and Pythium Oligandrum,were used to control Tobacco Black Shank.Although the inhibition effect of these antagonists on Phytophthora nicotianae was very obvious under indoor conditions,the field control effect of these antagonistic bacteria was not very satisfactory.It is difficult to achieve the expected control effect.Therefore,the scientific and efficient control strategy of Tobacco Black Shank disease has become a prominent problem in tobacco production.Metal Nanoparticles(MNPs) is a kind of important antibacterial material.Because of its unique physical and chemical properties,it is difficult to develop resistance to pathogens because of their diverse action sites.It has been reported that the main toxic mechanism of MNPs to pathogenic microorganisms is that it causes cells to produce excessive relative reactive oxygen species(ROS),which leads to intracellular metabolism disorder and DNA degradation.Due to its strong toxicity and low price,copper oxide nanoparticles(CuO NPs) has become an important MNPs to study the mechanism of interaction between MNPs and pathogenic microorganisms in recent years.At present,the antifungal mechanism of CuO NPs against Phytophthora nicotiana has not been reported.Therefore,the antifungal effects of CuO NPs and Mirco-copper oxide(MCuO) on Phytophthora nicotianae were compared and analyzed systematically in this paper,and the mechanism of toxicity of CuO NPs to Phytophthora nicotianae was further studied.To explore the key technology of CuO NPs in the prevention and control of Tobacco Black Shank disease.The results provide theoretical and practical support for the prevention and control of CuO NPs in Tobacco Black Shank.The main results of this paper are as follows:1.CuO NPs had a significant antifungal effect on Phytophthora nicotianae,and there was a dose-effect relationship.In this study,the inhibition effects of CuO NPs and MCuO on Phytophthora nicotianae mycelium growth at the same concentration were compared by classical hyphal growth rate method.It was found that,CuO NPs significantly inhibited the mycelial growth of Phytophthora nicotianae in the concentration range of 25~100 mg/L.And with the increase of treatment concentration,the inhibitory effect was enhanced,and the inhibitory effect was significantly higher than that of MCuO.At the same time,CuO NPs had significant inhibitory effect on sporangium production and zoospore germination of Phytophthora nicotianae in the range of concentration,and the inhibitory effect was significantly higher than that of MCuO.The results showed that CuO NPs were much more toxic to Phytophthora nicotianae than MCuO under the same concentration.2.The toxic mechanism of CuO NPs to Phytophthora nicotianae includes physical damage and chemical oxidation.According to the SME-EDS study,CuO NPs treated a large amount of CuO NPs on the mycelium surface after treatment with Phytophthora nicotianae.The surface of the hyphae showed irregularities and collapses.The TEM observation and PI fluorescence staining proved that CuO NPs can adsorb on the surface of mycelium and destroy the integrity of cell membrane.In addition,through DCFH-DA fluorescence staining,antioxidant addition test and SOD activity assay,it is proved that CuO NPs can cause a large amount of ROS in Phytophthora nicotianae,which leads to its died.3.Pot experiments showed that,CuO NPs had a good control effect on Tobacco Black Shank and had good safety on tobacco seed germination and tobacco seedling growth at the same time.The results of greenhouse experiment showed that CuO NPs had good control effect on Tobacco Black Shank,and 100 mg/L and 200 mg/L CuO NPs were applied respectively.After 6 days,the average control effect on Tobacco Black Shank was 43.66 % and 63.24%,respectively.The effect of CuO NPs on the germination of tobacco seeds was determined by laboratory.The results showed that CuO NPs with concentrations between 25 and 200 mg/L could promote the germination of tobacco seeds,increase the germination potential of tobacco seeds,and shorten the germination time.With the increase of CuO NPs concentration,the germination potential of tobacco seeds also increased,and the germination time was shortened.In addition,CuO NPs had no significant negative effects on the growth of tobacco seedlings.4.The synergistic antifungal effect of CuO NPs combined with commercial fungicides.Field experiment proved that CuO NPs have synergistic effect with metalaxgl mancozebThe inhibition effects of different concentrations of CuO NPs and two fungicides(metalaxyl and mancozeb) on hyphal growth of Phytophthora nicotianae were evaluated by Abbott.The results showed that CuO NPs could enhance the antifungal toxicity of metalaxyl to Phytophthora nicotianae.It shows additive effect.CuO NPs had both additive and antagonistic effects on the antifungal toxicity of mancozeb.Low concentration showed additive effect and higher concentration showed antagonistic effect.The results of field experiments showed that 100 mg/L of CuO NPs could enhance the control effect of metalaxgl mancozeb on Tobacco Black Shank.