Spatial Differentiation Of Anaerobic Methane-oxidizing Archaea In Flooded Paddy Fields

Winter flooded paddy fields,a kind of artificial wetlands with rich organic matter and anaerobic conditions after carbon and nitrogen cycles,would produce large amounts of CH₄ and N₂O.Nitrite-dependent anaerobic methane oxidation(N-DAMO)carried OTU by microorganisms drives the coupling reaction of carbon and nitrogen cycles,has significant effects on the mitigation of greenhouse gas emissions.Soil physical and chemical properties and other environmental factors of the winter flooded paddy fields were also different due to the difference of water status between waterloggogenic and gley.The effects of environmental factors on the distribution,abundance and diversity of the ANME-2d involved in anaerobic methane oxidation microorganisms in different types of winter flooded paddy fields will be further investigated.In this study,the waterloggogenic paddy soil of two district winter flooded paddy fields was collected as the research object,and the paddy soil of gleyed winter flooded paddy fields was collected as the control,to explore the influence of different types ofwinter flooded paddy fields on the distribution,abundance and diversity of NR-DAMO arabea.The waterloggogenic paddy soil was collected from Hechuan District(HC)and Shapingba District(FH)of Chongqing,and the gley paddy soil was collected from the National Purple Soil Fertility and Fertilizer Efficiency Monitoring and Testing Base of Southwest University Beibei District(JD),Chongqing.The soil layers of different depths were collected according to the soil formation layer 1.The soil of waterloggogenic paddy soil,which was divided into ploughing layer(A):0-15 cm,ploughing layer(P):15-25 cm and waterloggogenic layer(W):25-40 cm.2.The gley paddy soil is divided into plough layer(A):0-22 cm,plough bottom layer(P):22-38cm and gley layer(G):38-55 cm.Through anaerobic culture,cloning sequencing and quantitative PCR,the spatial differentiation characteristics of anaerobic methane-oxidizing archaea(ANME-2d),as well as the influence of environmental factors and trace metal elements on the spatial differentiation characteristics of ANME-2d were explored.The measured data were further analyzed.The main results were as follows:1.The anaerobic methane oxidation rate in different soil layers at different sampling sites was measured by indoor culture.The results showed that the variation trend of anaerobic methane oxidation rate was waterloggogenic(gley)W(G)>plough bottom layer(P)>plough layer(A),the overall JD was the highest in different winter paddy fields(0.406±0.051-12.133±0.401?g CH₄·g^-1·d^-1).2.Nest PCR was used to amplify the 16S r RNA gene of Anaerobic methane-oxidizing archaea(ANME-2d)in winter paddy fields from different sampling sites.A total of 270 effective sequences were obtained from 9 sampling sites,and 9 OTUs were obtained according to 3%sequence dissimilarity.Phylogenetic analysis revealed that they were highly similar to ANME-2d,and all formed within the same cluster.3.Real-time fluorescent quantitative PCR showed that the abundance of anaerobic methane-oxidizing archaea(ANME-2d)16S r RNA gene was 4.82×10⁶-9.30×10⁷ gene copies/(g·dry soil).The 16S r RNA gene abundance in gley layer(G)of Bei Bei(JD)was the highest,and that in plough layer(A)of He Chuan(HC)was the lowest.Correlation analysis showed that the the 16S r RNA gene abundance of anaerobic methane-oxidizing archaea(ANME-2d)was positively correlated with soil p H and the trace metal element copper(P<0.05),and negatively correlated with organic matter,water content and cation exchange capacity(P<0.05).