The Construction And Synergistic Mechanism Of Azoxystrobin Nanoformulations

Pesticides are imperative materials to guarantee the steady growth of agricultural yields.Due to the low efficiency of foliar deposition and dose transfer on target crops,conventional pesticide formulations are prone to droplets falling off,dust drifting,and poor dispersion during spraying.The low effective utilization rates of pesticides lead to food safety and ecological issue,which have seriously hampered the sustainable development of agriculture.As most strobilurins,azoxystrobin is a high-efficiency and broad-spectrum insoluble fungicides.It is widely used in prevention of the disease caused by fungus and the first product of sales in 2016.In the development of low-cost and water-soluble novel pesticide formulations,nanoscience and nanotechnology have provided dominant potential to improve the solubility and bioefficiency.In the research,the azoxystrobin nanosuspensions were fabricated by wet media milling.The azoxystrobin polylactic acid?PLA?microspheres?MS?with controllable particle sizes were prepared by solvent evaporation.The physicochemical characterization and bioassay tests of the nanoformulations were carried out.In addition,the antifungal mechanism and biosafety of the new azoxystrobin formulations were analyzed.The main results were as follows:1.The nanosuspension?6%azoxystrobin?was prepared by wet media milling.The mean particle size,polydispersity index?PDI?and zeta potential of the nanosuspension measured by dynamic light scattering?DLS?were 238.1±1.5 nm,0.17±0.02 and-31.8±0.3 mV,respectively.The morphology of the azoxystrobin nanoparticles were characterized by scanning electron microscopy?SEM?and transmission electron microscopy?TEM?.The nanoparticles exhibited irregular bulk and rhombus shape.After storage at 25?,4?and 54?for 14 days,the decomposition rates of azoxystrobin were 2.05%,1.37%and 2.91%,respectively,both below 5%.The contact angles of the azoxystrobin nanosuspension on cucumber and cabbage leaves were smaller than those of water dispersible granules-A?WDG-A?and WDG-B.Meanwhile,the retention volumes of the nanosuspension on cucumber and cabbage surfaces were higher than the WDG formulation.Furthermore,significantly different contact angles and retention volumes were observed.2.Simulated release rate experiment in vitro,the cumulative release rates of azoxystrobin nanosuspension,WDG-A and WDG-B were 52.88%,36.01%and 27.56%,respectively,in the first 0.5 h.The result indicated that water solubility and dissolution rate of azoxystrobin could be improved by nanocystalization.The azoxystrobin nanosuspension exhibited the highest toxicity with a LC₅₀ of 1.4243?g/mL,followed by WDG-A and WDG-B with the medial lethal concentration(LC₅₀)values of 2.4668?g/mL and 2.4221?g/mL,respectively.The Fusarium oxysporum were observed by light microscope.In the control group,Fusarium oxysporum maintained intact,chiseled septa and a strong budding reproductive capacity.In comparison,the hyphae tended to intertwine when exposed to the azoxystrobin nanosuspension.The morphologies of Fusarium oxysporum were examined by SEM.The mycelia treated with the azoxystrobin nanosuspension became severely shrunk and crumpled.In the control group,mycelia presented smooth walls and intact surfaces without shrinkage.In exposed group,the total soluble protein content,superoxide dismutase?SOD?and catalase?CAT?activities initially increased and then decreased with prolonging incubation time.This results manifested the azoxystrobin nanosuspension could reduce the defensive antioxidant capability of Fusarium oxysporum and generate excessive reactive oxygen species?ROS?.At the application concentration of 600?g/mL,the azoxystrobin nanosuspension was safe for cucumber seedlings,without phytotoxicity symptoms.3.Assisted by membrane emulsification,the size-controlled azoxystrobin PLA MS were prepared by solvent evaporation.The PLA MS characterized by TEM were presented a uniform regular spherical shape and monodisperse,without adhesive.The hydrodynamic mean particle sizes of the aqueous diluted MS1,MS2,and MS3 were 130.9±0.2 nm,353.4±6.3 nm and 3078.0±336.6 nm,respectively.The drug loading and encapsulation efficiency the azoxystrobin MS showed a positive correlation with the particle sizes.However,the release rate and percentage of cumulative release were inversely relationship with the MS particle sizes.In the stability tests at 25?,4?and 54?,the decomposition rates of azoxystrobin MS1 were all less than 5%.The contact angles of the azoxystrobin MS1,MS2and MS3 on cucumber and cabbage leaves were decreased with the reduction of particle sizes.The result revealled that reducing particle sizes of azoxystrobin MS was able to improve the wettability on cucumber and cabbage leaves,further enhancing their adhesion.4.Simulated release behavior test in vitro,the release rate and cumulative release of azoxystrobin from MS were negatively related to particle sizes.The lowest LC₅₀ value of the azoxystrobin MS1?130.9 nm?indicated that its toxicity index was 10.11-fold that of the azoxystrobin MS3?3078.0 nm?size against Colletotrichum higginsianum Saccare.The mycelial intracellular uptake efficiencies of azoxystrobin MS were determined by the fluorescent intensity of coumarin-6.The coumarin-6 fluorescent values of MS1 and MS2were approximately 7.25 and 3.13 times that of MS3 in Colletotrichum higginsianum Saccare,respectively.To estimate the effect of MS1,MS2,and MS3 on mycelia oxidative stress,intracellular ROS of Colletotrichum higginsianum Sacc were tested by 2',7'-dichlorofluorescin diacetate?DCFH-DA?assay.The DCF fluorescent values of MS1 and MS2 were approximately 6.27 and 2.57 times that of MS3.Compared to the control check?CK?group,a significant decrease of the soluble protein content was analyzed in the azoxystrobin treated groups.The similar experimental results emerged in the activities of SOD and CAT.The lower activities of SOD and CAT indicated that the azoxystrobin MS was capable of causing more seriously oxidative damage,even leading to cell apoptosis,against Colletotrichum higginsianum Sacc.Comapared with CK group,significant decreases of SOD and CAT activities were analyzed in the azoxystrobin-treated groups?25?g/mL or 100?g/mL?.These findings indicated that the lettuce roots were subjected to oxidative stress.