Impacts Of Chlorothalonil On Microbial Denitrification And Nitrous Oxide Release In Biochar-amended Soil

As an important part of the biochemical cycle of the earth,soil nitrogen cycle has been receiving much attention in the past few decades.The central issue regarding to soil nitrogen cycle involves how nitrous oxide?N₂O?production and depletion occurs to cause N₂O emission at the soil surface.The microbial denitrification process,as one of the important processes for the release of nitrous oxide from soil,has gradually become a research hotspot in recent years.As an important source of greenhouse gas N₂O,agricultural soils are expected to account for 59%of global N₂O emissions by 2030.The extensive use of pesticides in agricultural production leads to residues and accumulation in the soil.Studies have confirmed that pesticide residues have a negative impact on the environment and nitrogen cycle of soil.Biochar-amended soils could reduce the damage of pesticides to the soil environment and increase microbial activity in the soil.However,there are few studies on the effects of pesticide application on soil denitrification and N₂O release at present,and there is no systematic study on the effects of pesticide application on pesticide metabolism and soil environment in biochar-amended soilBased on the above,the fungicide chlorothalonil was used in this study.Using routine indicators at the macro level to investigate the effects of chlorothalonil on soil denitrification and N₂O emissions.Combining microscopic molecular and molecular indicators to analyze the mechanism of chlorothalonil affecting denitrification and N₂O release.At the same time,to explore the effects of chlorothalonil on the denitrification process and N₂O release in biochar-amended soil and the corresponding mechanism of influence through long-term biochar development experiments.?1?This study evaluated the variation of denitrification,N₂O release,denitrification gene abundance and denitrifying enzyme activity in soils after 72 hours exposure to chlorothalonil.The results showed that the nitrate removal rate was reduced from 74.6%to 54.1%and the N₂O release was increased from 48 mg N kg^-1 to 76 mg N kg^-1 with the increasing chlorothalonil concentration from 5 to 25 mg kg^-1.The decrease of transport system activity?ETSA?value and adenosine triphosphate?ATP?content indicates that chlorothalonil has an inhibitory effect on microbial metabolism.Enzyme activity studies showed that the reduction of nitrate reductase?NAR?,nitrite reductase?NIR?and nitric oxide reductase?NOR?activity is the main reason for inhibition of denitrification by chlorothalonil.In addition,the linera relationship between denitrifying reductase activity and intracellular metabolism indicates that the reduction of microbial metabolism could be another reason for the inhibition of denitrification by chlorothalonil.Overall,short-term exposure of soil to chlorothalonil inhibits soil denitrification and significantly increases N₂O release,which could disrupt soil nitrogen cycling and exacerbate global warming.?2?The nitrate removal rate decreased from 100%to 44.3%to 43.7%and the amount of N₂O released increased from 8.6,90.8 to 97.9 mg N kg^-1 with the increasing concentration of chlorothalonil from 0,5 mg kg^-1 to 25 mg kg^-1 in the long-term biochar experiment.The nitrates in the biochar treatments were all removed,and the N₂O emissions were 4.38,4.76 and 29.6 mg N kg^-1,respectively,which were 49.3%,94.8%and 69.7%lower than those in the non-biochar treatment treatments.By quantifying the activities of denitrification reductase,it was found that the biochar-amended soils could maintain and promote the denitrification process by maintaining denitrifying enzyme activity.The significant increase in NOS activity in biochar treatment may be the main reason for the decrease in N₂O release.?3?The short-term application of chlorothalonil had no significant effect on soil microbial community structure and abundance.Compared to the control treatment,the denitrifying microbial abundance of the 25 mg kg^-1 chlorothalonil treatment was lower than that of the blank treatment treatment and the abundance of denitrification functional gene nirK,nirS and nosZ in 25 mg kg^-1 chlorothalonil treatment was reduced by 31.6%,22.1%and 50.7%,respectively.Therefore,the significant decrease in abundance of denitrification functional genes may be another reason for the inhibition of soil denitrification by chlorothalonil.During the biochar long-term experiment,the soil microbial community structure and abundance in 25 mg kg^-1 chlorothalonil?C25?changed significantly compared with the blank treatment?C0?.However,the biochar-amended+25 mg kg^-1 chlorothalonil treatment?B25?have no significant differences with the control treatment in soil microbial community structure and abundance.Compared with the C25 and B25 treatments,the abundance of denitrification functional gene narG,nirK,nirS,norB and nosZ increased by 21%,4%,10%,8%and 47%in B25 treament,respectively.The above results indicate that repeated application of chlorothalonil to soil had a negative impact on denitrification and N₂O release.However,there was no significant change in gene abundance of biochar-amended treatments,indicating that biochar maintains the abundance of denitrifying functional genes and the structure and function of soil microbes.