Interactions Of Four HPPD Inhibitor Herbicides With Humic Acid And Maize Root Exudates

4-Hydroxyphenylpyruvate dioxygenase（HPPD）inhibitors are one of the most important herbicides with unique mechanisms of action,high biological activity,and a wide spectrum of herbicides.However,due to the widespread promotion of new HPPD resistant transgenic crops and the abuse of HPPD inhibitors,problems such as increased weed resistance,crop damage,and environmental pollution have been caused.Humic acid and root exudates are important media of environmental soil,which can interact with pollutants in adsorption,desorption and degradation,and affect the migration of pollutants on the soil solid phase,thus changing the effectiveness of soil organisms.In this thesis,the interaction mechanism,binding capacity of humic acid,maize root exudates and four HPPD inhibitor herbicides,namely,sulcotrione,tembotrione,topramezone,mesotrione,as well as the impact of HPPD inhibitors on maize growth were investigated by means of UV visible absorption spectrum,fluorescence spectrum,time-resolved fluorescence spectrum,infrared spectrum,dynamic light scattering technology,DFT theoretical calculation,and 2D-COS analysis technology.The results provide a reference for the behavior and fate of sulcotrione,tembotrione,topramezone,mesotrione in the environment.The binding capacity can provide a theoretical basis for revealing the persistence of these four herbicides in the soil.The research results of maize root exudates are of great significance for plant safety,and also provide a reference for the scientific and rational use of HPPD inhibitor herbicides.The main research results are as follows:1.Explore the fluorescence quenching mechanism of the interaction between HPPD inhibitors and humic acid.According to the fluorescence emission spectrum data at different temperatures,the K_svwas calculated by the Stern-Vlomer equation,and the fluorescence lifetime values was measured by time-resolved fluorescence spectroscopy.It can be concluded that the quenching mechanism of the complex formed by the combination of sulcotrione,tembotrione,topramezone and mesotrione with humic acid is static quenching.2.Determine the binding ability and mode of HPPD inhibitors with humic acid and maize root exudates.According to the magnitude of the binding constant K_aobtained from the modified Stern-Volmer equation,the binding ability of these four herbicides to humic acid is as follows:mesotrione>tembotrione>sulcotrione>topramezone.DFT calculated the O^2-electrostatic potential of sulcotrione,tembotrione,topramezone and mesotrione as topramezone（-50.24 kcal/mol）>sulcotrione（-52.76 kcal/mol）>tembotrione（-56.50kcal/mol）>mesotrione（-63.59 kcal/mol）.Based on the principle that the greater the electronegativity,the stronger the binding ability of humic acid,it can also be concluded that mesotrione has the strongest binding ability with humic acid and the weakest binding ability of topramezone.According to the degree of decrease in the intensity of protein like fluorescence peaks in the three-dimensional fluorescence spectrum of maize root exudates,it can be obtained that the binding ability of these four pesticides to maize root exudates is as follows:mesotrione>sulcotrione>tembotrione>topramezone.In addition,the van’t Hoff equation obtains negativeΔH,positiveΔS and negativeΔG.This indicates that the four pesticides combine with humic acids to form complexes through electrostatic interaction,and the reaction is spontaneous.3.Explore the conformational changes and functional group changes of HPPD inhibitors on humic acid and maize root exudates.Three-dimensional fluorescence spectroscopy showed a blue or red shift in the excitation and emission wavelengths of sulcotrione,tembotrione,topramezone and mesotrione with humic acid fluorescence peak A,and the fulvic acid like peak（peak A）and protein like peak（peak B）in maize root exudates,revealing that the conformations of humic acid and maize root exudates changed after the addition of these four pesticides.Fourier transform infrared spectroscopy combined with 2D-COS analysis revealed that humic acid binds to HPPD inhibitors through aromatic C-H,C=O,and C-Cl,and the first binding functional group is C=O;Among maize root exudates,C-O,C-Cl,and C-C have the strongest binding ability,while C=C and C=O of aromatic rings,quinones,or ketones occurred earliest between HPPD inhibitor herbicides and maize root exudates.4.Reveal the effect of p H on the interaction mechanism between HPPD inhibitors and humic acids.The binding constant K_aand the number of binding sites n were calculated using the Stern-Volmer equation,and the results showed that both K_aand n reached their maximum at p H 7.The Zeta potential and hydrodynamic radius were measured by DLS,and the results showed that the Zeta potential was the lowest and the hydrodynamic radius was the largest at p H 7.Therefore,under neutral conditions,humic acid is most likely to form stable complexes with sulcotrione,tembotrione,topramezone and mesotrione.5.Explore the effects of HPPD inhibitors on maize growth.After 10 days of stress on maize plants with sulcotrione,tembotrione,topramezone and mesotrione,the chlorophyll content of maize leaves was measured using an ultraviolet visible spectrophotometer,and the fresh weight of their roots,stems,and leaves was analyzed.The results showed that when the concentrations of the four pesticides were higher than 5 mg/L,the chlorophyll content of maize leaves decreased compared to the blank group,while the fresh weight of roots,stems,and leaves decreased.Therefore,when HPPD inhibitors reach a certain concentration（higher than 5 mg/L）,they have a negative impact on maize growth.