Ecological Characteristics Of Ammonia-Oxidizing Microorganisms In Black Soils Of Northeast China

Ammonia-oxidizing archaea（AOA）and ammonia-oxidizing bacteria（AOB）play an important role in the natural nitrogen cycle.Due to the complex species and niche differences of ammonia-oxidizing microorganisms,the abundance,relative nitrification contribution and community structure of the same kind of ammonia-oxidizing microorganisms may vary greatly in different habitats.In recent years,the discovery of complete ammonia-oxidizing bacteria（comammox）has subverted the traditional understanding of two-step nitrification,making it more difficult to distinguish the nitrification contributions of various ammonia-oxidizing microorganisms in the environment.However,there are few studies on amoA gene abundance,activity,community structure and environmental influencing factors of various ammonia-oxidizing microorganisms in northeast black soil under different environmental conditions.Therefore,in this study,the typical northeast black soil was used as the test material.Firstly,the Illumina Miseq high-throughput sequencing results of four groups of comammox specific primers in black soil samples were evaluated.Then,the abundance of amoA gene,nitrification contribution,community characteristics and environmental influencing factors of AOA,AOB and comammox in different habitats were investigated.Finally,the influence of key environmental factors on the activity of ammonia-oxidizing microorganisms in black soil was verified by the DNA stable-isotope probing（DNA-SIP）technique.The main research results are as follows:（1）PCR amplification and Illumina Miseq high-throughput sequencing of comammox amoA gene in farmland and natural soil samples from two sampling sites in the Northeast black soil region by primers primer sets Ntsp-amoA 162F/359R,comamoA F/R,CA377f/C576r and CB377f/C576r.It was found that all four specific primer sets selected in this study could be applied to Illumina Miseq high-throughput sequencing of Comammox in the northeast black soil.Compared with the primer set comamoA F/R,Ntsp-amoA 162F/359R obtained more OTU numbers,and showed more advantages in the analysis of comammox diversity and community structure.Primer sets CA/B377f/C576r can be used for high-throughput sequencing of comammox Clade A and Clade B in the northeast black soil,respectively,which is suitable for the analysis of comammox Clade A or Clade B community characteristics,separately.（2）Nitrification inhibitor experiment and Illumina Miseq high-throughput sequencing technology were used to analyze the differences of amoA gene abundance,relative nitrification contribution and community characteristics of various ammonia-oxidizing microorganisms in farmland and natural soil at regional scale in the northeast black soil region.The results showed that autotrophic nitrification dominated the nitrification process of farmland and natural black soil in northeast China,and AOB played an important role in the autotrophic nitrification of farmland soil.Soil reclamation decreased the abundance of AOA and comammox Clade B amoA genes,but increased the abundance of AOB amoA genes in northeast black soil.Soil AK,p H and NO₃^--N contents were the most important environmental factors affecting the abundance of AOA,AOB and comammox in this study,while TP and p H were the most important environmental factors affecting the nitrification potential of AOA+comammox（NP-AOA+Com）and AOB（NP-AOB）,respectively.The high-throughput sequencing results showed that soil reclamation increased the OTU number of AOA and decreased the OTU number of AOB and comammox.Nitrososphaera,Nitrosospira,and comammox Clade B are the dominant populations in the northeast black soil.Soil reclamation significantly changed the community structure of autotrophic ammonia-oxidizing microorganisms including AOA,AOB and comammox at each sampling site,but soil geographical differences mainly affected the community structure of AOA,and soil reclamation mainly affected the community structure of AOB and comammox.The distribution of amoA genes in black soil was determined by various soil environmental factors,among which TP,AK and TC were the main environmental factors affecting the community structure of AOA,AOB and comammox,respectively.（3）Nitrification inhibitor experiment and Illumina Miseq high-throughput sequencing technology were used to analyze the effects of different fertilization regimes on the abundance of amoA gene,relative nitrification contribution and community characteristics of various ammonia-oxidizing microorganisms in the soil of three long-term positioning stations in the northeast black soil region.It was found that although geographical separation was the main factor determining the abundance and community structure of various ammonia-oxidizing microorganisms in soil,long-term different fertilization management also affected the abundance,total nitrification potential and community structure of various ammonia-oxidizing microorganisms in soil.The nitrification process of soil with or without fertilization was mainly autotrophic.AOA and comammox dominated autotrophic nitrification in unfertilized acidic soil,while AOB dominated autotrophic nitrification in neutral soil.Long-term application of organic fertilizer and inorganic fertilizer increased the nitrification contribution of AOB.The abundance,activity and community structure of ammonia-oxidizing microorganisms were mainly affected by soil pH and inorganic nitrogen content.（4）DNA-SIP microcosmic culture was used to trace the species and community structure of active ammonia-oxidizing microorganisms in acidic and neutral black soils with and without nitrogen,and it was found that AOA dominated the nitrification process in acidic soil.AOA,AOB and comammox Clade A participated in the nitrification process of neutral soil.High ammonia condition reduced the soil pH and increased the nitrification intensity of AOB in neutral soil.Acid low nitrogen,acid high nitrogen and neutral treatments soil active AOA are mainly related to Nitrososphaera subcluster 1.1,Nitrososphaera subcluster sister 2 and Nitrososphaera subcluster sister 1.1,respectively;AOB and comammox Clade A belong mainly to Nitrosospira cluster 9 and Clade A.2 in neutral soils.