Response Of Anaerobic Ammonia-Oxidizing Bacteria And Methanotrophs To Different Tillage Modes In Paddy Fields

The paddy fields plays an important role in the carbon and nitrogen cycle,and have great amount of organic material,which can be decomposed by miccroorganisms under anaerobic conditions to produce large amounts of CH4 and N2O.Anaerobic ammonia oxidation(Anammox)reaction and anaerobic methane oxidation(Nr-DAMO)reaction with nitrite as electron acceptor will significantly impact the greenhouse effect,which has aroused people's concern about the ecological environment of paddy fields.However,the responses of the two microorganisms to different tillage modes in paddy fields need to be further studied.In this study,samples from different soil layers(0-20 cm,20-40 cm,40-60 cm,60-80 cm,80-100 cm)in three different tillage modes which are conventional paddy-upland rotation tillage(CT),combing ridge with no-tillage(RNT)and flooded paddy field(FPF)were collected as the research object.The community structure and abundance of Anammox and Nr-DAMO were studied by laboratory methods,real-time quantitative PCR,cloning and sequencing techniques.SPSS,Origin 2016,Mega 4,Canoco 4.5,R software were used for data analysis.The main results are as follows:(1)The response of Anammox bacteria to different soil layers in different tillage modes:The NO3--N,NH4+-N,MBC/N,nitrification potential were measured by the indoor cultivation method,and the five indexes were gradually decreasing with the deepening of the soil layers,but the general tendency was RNT>CT>FPF.It was found by quantitative PCR that there were a lot of Anammox in different tillage modes,and the hzsB gene copy numbers were 1.57�105-7.33�105 gene copies/(g�dry soil)(CT),2.35�105-6.01�106 gene copies/(g�dry soil)(RNT),1.74�105-7.05�105 gene copies/(g-dry soil)(FPF),respectively.The abundance of Anammox bacteria in different soil layers of the same tillage modes was 80-100 cm<0-20 cm<20-40 cm<40-60 cm<60-80 cm,The abundance of Anammox bacteria was the highest in RNT,and there was no significant difference between the other two tillage modes.Based on the construction of clone library of Anammox bacteria,it was found that the soil of different tillage modes had different community composition.Ca.Brocadia fulgia,Ca.Brocadia anammoxidans and Ca.Kuenenia.sp.were detected in the soil of CT,and Ca.Brocadia fulgia and Ca.Brocadia were found in RNT,and Ca.Brocadia fulgia and Ca.Jettenia.sp.were found in FPF,it is worth noting that Ca.Brocadia was found in all three tillage modes.The results of Pearson correlation analysis and R software mapping showed that NO3--N,NH4+-N and SOM were the main environmental factors affecting the abundance of Anammox community,and it can be seen from RDA analysis that NO3--N,MBC,SOM and TN were the main environmental factors affecting the diversity of Anammox community.(2)The response of anaerobic methane oxidizing archaea to different soil layers in different tillage modes:Study on the activity of anaerobic methane oxidizing archaea by laboratory cultivation experiments,and the results showed that the rate of anaerobic methane oxidation mediated by ANME-2d archaea in different soil layers of the same tillage mode was 40-60 cm>60-80 cm>80-100 cm>20-40 cm>0-20 cm,but the rate of anaerobic methane oxidation was the highest in RNT(0.32�0.03-3.33�0.27 ?gCH4�g-1�d-1),and the difference between the other two modes was not significant,the values were 0.26�0.03-2.12�0.28 ?gCH4�g-1�d-1(CT)and 0.26�0.03-2.21 �0.27 ?gCH4�g-1�d-1(FPF).The highest value of each treatment rate appeared in the soil layer of 60-80 cm.It indicated that RNT is conducive to the reduction of CH4 emissions and the anaerobic methane oxidation is mainly done in the anaerobic layers.Cloning sequencing and qPCR analysis revealed that a large number of ANME-2d archaea were detected in soils of different tillage modes.The 16S rRNA gene copy number of ANME-2d archaea were 3.68�105-2.20�106gene copies/(g�dry soi)(CT),4.92�105-3.54�106 gene copies/(g�dry soi)(RNT)and 5.79�105-5.19�106gene copies/(g�dry soi)(FPF),and gene copy numbers increased with increasing of soil depth in all tillage modes.The results of Pearson correlation analysis and R software mapping showed water content,MBC/MBN,NO3--N,NH4+-N were the main environmental factors affecting the activity,community structure,and abundance of ANME-2d archaea.(3)This study showed that RNT can significantly improve soil fertility,microbial activity,community structure and abundance,and reduce greenhouse gas emissions.This study can provide theoretical basis for the promotion of RNT.