Effects Of Different Management Patterns On Nitrification And Nitrifying Microorganisms In Paddy Soils

Nitrification is considered a central biological process in the global nitrogen cycle.Nitrification in soil is an important cause for nitrogen loss,which affects the effective use of nitrogen by plants.Nitrate and Nitrous Oxide?N₂O?produced in the process of nitrification have some negative effects on the natural environment.The traditional recognition of nitrification is accomplished by two different microbial communities,ammonia-oxidizing microorganisms and nitrite-oxidizing bacteria.But the discovery of Comammox?Complete ammonia oxidizers?is changing our traditional perception,it can directly complete the transformation from ammonia to nitrate.This requires more work to re-evaluation of the nitrification in soil.Paddy soil is one of the main cultivated soils in China.At present,the utilization rate of nitrogen fertilizer in rice is low in China.Fertilization has an important impact on nitrification and nitrifying microorganisms in paddy soil.In this experiment,we selected two paddy soils under different management patterns,i.e.tillage and fertilization?T?and fallow?F?.A 56-day incubation experiment was conducted with stable isotope probe,in order to explore the Comammox in paddy soil.Soil nitrification potential was determined by fresh samples just collected.Three treatments were set for each soil sample under the two conditions of without adding nitrogen source?-CK?and adding nitrogen source?-N?,including ¹²CO₂ control,¹³CO₂ labeling and ¹³CO₂+C₂H₂ control.The incubation of soil samples were carried out in a constant temperature incubator at 28?,added N 50mg·kg^-1 and corresponding volume of gas per week according to the requirements of each treatment.After the end of incubation,samples from the initial stage?Day-0?and 56 days of incubation?Day-56?were analyzed.The amoA gene abundances of ammonia-oxidizing bacteria?AOB?,ammonia-oxidizing archaea?AOA?,Comammox Clade A and Clade B were quantified by real-time quantitative PCR?Q-PCR?.The judgment of marker results was completed in Institute of Soil Science,Chinese Academy of Sciences,Nanjing.Analysis of total microbial community structure was carried out by Hiseq sequencing.Results showed that T was higher than F in nitrification potential,which reached 24.97mg·kg^-1·d^-1 and 2.11 mg·kg^-1·d^-1,respectively.After 56 days of incubation,in the T that added nitrogen source?T-N?,the nitrate content of ¹²CO₂ and ¹³CO₂ treatments were 359.46 and 361.51mg·kg^-1,respectively.In the T that no nitrogen source was added?T-CK?,the nitrate content of¹²CO₂ and ¹³CO₂ treatments were 81.81 and 68.45 mg·kg^-1,respectively.Compared with 13.63mg·kg^-1 at the initial stage of incubation,nitrate content in ¹²CO₂ control treatment and ¹³CO₂labeling treatment increased significantly.Nitrification in the T were obvious during the incubation period,and the addition of nitrogen source promoted the nitrification.In the F that added nitrogen source?F-N?,the nitrate content of ¹²CO₂ and ¹³CO₂ treatments from 9.5 mg·kg^-1 at the initial stage of incubation to 87.44 mg·kg^-1 and 88.71 mg·kg^-1,respectively.In the F that no nitrogen source was added?F-CK?,the nitrate content of ¹²CO₂ and ¹³CO₂ treatments were 11.04 and 12.89 mg·kg^-1,respectively.There was no significant change compared with the initial stage,indicating that nitrification did not occur.The main reason for the absence of nitrification in F was the lack of substrates.In the treatment of ¹³CO₂+C₂H₂,the nitrate content did not increase significantly,indicating that C₂H₂ had a significant inhibitory effect,the nitrification in the paddy soil was mainly of the autotrophic type.At the initial stage of incubation,the amoA gene abundances of AOB,AOA and Comammox Clade A in T were higher than that in F,while Comammox Clade B was lower than that in F.After56 days of incubation,whether nitrogen source was added or not,the copy number of amoA genes of AOB and AOA increased in ¹²CO₂ and ¹³CO₂ treatments in T,but decreased in ¹³CO₂+C₂H₂treatment.In F-N,the copy number of AOA amoA genes in ¹²CO₂ and ¹³CO₂ treatments increased significantly compared with the initial stage.After incubation,the copy number of Comammox Clade A amoA genes decreased in different treatments,but the copy number of Comammox Clade B amoA genes increased significantly under¹²CO₂ and ¹³CO₂ treatments in T-CK and T-N.The total DNA extract was then subjected to isopycnic centrifugation to separate ¹³C-DNA from native ¹²C-DNA.Real-time quantitative PCR was used to quantify amoA genes in purified DNA of each layer,and further identified the labeling degree of ammonia-oxidizing microorganisms.The results showed that AOA and AOB were labeled in T-N during the incubation period;AOB,AOA and Comammox Clade B were labeled in T-CK;AOA was labeled only in F-N.Sequencing of amoA functional genes of ¹³C-DNA according to the marker results.The sequencing results showed that Nitrosospira Cluster 3 was the main active AOB,and Group1.1b was the main active AOA in T,while Group1.1a and Group1.1b were the main active AOA in F.Hiseq sequencing of soil samples at the initial stage of incubation and after incubation showed that the diversity of microbial communities in T was better than that in F.Bacteria of Proteobacteria,Acidobacteria,Chloroflexi,Actinobacteria,Nitrospirae,Planctomycetes and Gemmatimonadetes were dominant microorganisms in paddy soils that were selected.This study focused on the Comammox in paddy soil and the major participants in nitrification in two paddy soils under different management patterns.The main conclusions of this study were as follows:?1?Compared with fallow,tillage and fertilization significantly increased the nitrification potential of paddy soil;?2?Long-term fallow weakens nitrification in paddy soil,and the addition of substrate nitrogen promotes nitrification;?3?Higher abundance of Comammox were detected in paddy soil;?4?Compared with fallow paddy soil,tillage and fertilization can promote the abundance of AOB,AOA and Comammox Clade A in paddy soil;?5?AOB and AOA participated in nitrification in T.Among them,AOB was mainly Nitrosospira Cluster 3 and AOA was mainly Group1.1b.Comammox Clade B also participated in nitrification without adding external nitrogen sources,and the ammonia nitrogen used may came from soil mineralization.In F,nitrification was dominated by AOA,that is Group1.1a and Group1.1b.