Study On The Mechanism Of Methane Cycle And Nitrogen Cycle In Paddy Soil Caused By Thifluzamide Mediating Soil Microorganism

Pesticides have been widely utilized for decades to improve crop yields,but their application poses numerous potential risks to agricultural ecosystems worldwide.The pesticide thifluzamide helps protect rice plants in paddy fields against rice sheath blight,but its use constitutes a potential hazard to non-target soil microorganisms,methane（CH₄）and nitrogen transformations,affect CH₄ and N₂O emissions,and result in potential environmental contamination.In this study,we performed indoor culture and greenhouse plot experiments,and combined qPCR and amplicon sequencing to clarify the effects of thifluzamide stress on soil microbes with respect to their community structure,interaction networks,and the gene abundance ofnitrogen cycling and CH₄ metabolism.1.The greenhouse plot experiment was conducted to ascertain the effects of thifluzamide on CH₄ and N₂O emissions as well as methanogen genes and nitrogen cycling genes.Thifluzamide temporarily promoted CH₄ emissions but did not significantly affect N₂O emissions.Thifluzamide increased the copy number of mcrA in rice rhizosphere soil,and CH₄ emissions on day 7 after application were significantly positively correlated with mcrA copy number.However,thifluzamide did not significantly affect the copy number of nifH,AOA-amoA,or narG but reduced the copy number of nxrA,AOB-amoA,napA,nirK,nirS,and qnorB in rice rhizosphere soil and increased the copy number of nosZ.nxrA/amoA,was denoted as the completed nitrification process,was the determining factor during whole experimental session.2.A greenhouse plot experiment was conducted to ascertain the dynamics of thifluzamide degradation and the effects of thifluzamide on the structure of the bacterial and archaeal communities.The half-life of thifluzamide in the paddy soil was 31.49 days.Thifluzamide temporarily inhibited bacterial copy number but temporarily increased the copy number of fungi and archaea in rhizosphere paddy soil.Principal component analysis revealed that thifluzamide altered the structure of bacterial communities in rhizosphere paddy soil.At the phylum level,thifluzamide significantly increased the relative abundance of Actinobacteriota,Firmicutes,and Desulfobacterota but significantly reduced the relative abundance of Proteobacteria,Gemmatimonadota,and Nanorchaeota.At the genus level,thifluzamide significantly increased the relative abundance of Candidatus＿Solibacter,Anaerolinea,Methanocella,Methanosaeta,and Rice＿Cluster＿I but significantly reduced that of Rhodanobacte,Nitrospira,Novosphingobium,Candidatus＿Methanoperedens,and Candidatus＿Nitrososphaera,among other species.Thifluzamide increased the abundance of methanogenic archaea such as Methanoregula and Methanosaeta,thus increasing the abundance of methanogenic genes and leading to increased CH₄emissions from rice fields.3.A 3-month-long indoor cultivation experiment was conducted to ascertain the dynamics of thifluzamide degradation in soil,i.e.,at initial concentrations of 0.05mg/kg soil,0.5 mg/kg soil,and 5 mg/kg soil,as well as the effects of thifluzamide on functional groups involved in nitrogen cycling and CH₄ cycling in three typical paddy soils from Guiyang（GY）,Hangzhou（HZ）,and Jiansanjiang（JSJ）.The degradation dynamics in the three soils could be described by a bi-exponential kinetic model.The respective half-life of thifluzamide at the three different concentrations in the three soil types varied from 162.2 to more than 1000 days for GY soil,252.1 to 350.7 days for HZ soil,and 152.0 to 419.6 days for JSJ soil.The genes involved in nitrogen and CH₄ cycling were impacted transiently.Thifluzamide increased the abundance of genes governing nitrogen cycling in the GY paddy soil but decreased the abundance of nitrate-reducing and nitrite-reducing microorganisms in JSJ paddy soil.Thifluzamide increased the abundance of each of qnorB,nxrA,and nirK in HZ paddy soil but decreased the abundance of nifH,amoA,and nitrate-reducing microorganisms.Thifluzamide increased the abundance of mcrA and pmoA in samples of all three paddy soils.The ratio of mcrA to pmoA increased in JSJ paddy soil,which may have increased the CH₄ emissions from paddy fields,with potential implications for the environment.4.A 3-month-long indoor cultivation experiment was conducted to ascertain the effects of thifluzamide on the structure of bacterial and archaeal communities in three typical paddy soils.Thifluzamide significantly increased the abundance of bacteria and decreased he abundance of fungi in GY paddy soil,increased the abundance of bacteria,fungi,and archaea in HZ soil,and decreased the abundance of bacteria,fungi,and archaea in JSJ loam sandy soil transiently.Thifluzamide also transitorily significantly altered theαdiversity of both the bacterial and archaeal communities.Principal component analysis revealed that the structure of the overall microbial community in thifluzamide-treated soils was affected by both soil type and the duration of thifluzamide exposure.Network analysis revealed that thifluzamide-treated soils had more total nodes,total links,average degree,and keystones and resulted in more complex microbial communities in GY T10,HZ,and JSJ soils.However,thifluzamide decreased the close association and the complexity of the network structure in the GY T1 and T100 networks.Moreover,thifluzamide increased the keystones in the three types of paddy soil,and the keystones were associated with CH₄ production in the HZ and JSJ soils.Finally,we carried out microcosm and greenhouse pot experiments combined with 16S rRNA high-throughput sequencing and qPCR.We systematically researched the residue characteristics of thifluzamide and the influence of thifluzamide on the microbial structure in paddy soils,and revealed the mechanism material cycling caused by thifluzamide mediating paddy-soil microorganisms.This research provides a theoretical basis for the rational use of thifluzamide and ecological security assessment.