Effect Of Long-term Fertilization On Ammonia Oxidation Archaea And Anammox Bacteria Community Structure In Calcareous Purplish Paddy Soil

The relationship between soil microbes' functional gene diversity and nitrogen cycle of the farmland ecosystem is the highlight of re-evaluation of the soil quality and function.Calcareous purple paddy soil is an important agricultural soil resource in Sichuan province and China.For the purpose of maintaining its quality,a NPK Long Term Fertilization Experiment was established at Chuanshan,Suining city,Sichuan province in the 1980s.Based on this experiment station,combined with chemical analysis and modern molecular analytical methods such as denaturing gradient gel electrophoresis(DGGE),terminal restriction fragment length polymorphism of(terminal restriction fragment length polymorphism,T-RFLP),quantitative PCR,were used to evaluate the community structure,biodiversity and vertical distribution on the 16S rRNA gene of the anammox and ammonia oxidation archaea(AOA).The anticipated results will reveal mechanisms on how the AOA and anammox 16S rRNA gene responded to different fertilization strategies,and the characteristics of their distribution.The results will be useful for a better understanding of the nitrogen cycling in the paddy soil micro-ecosystem,to develop sound fertilization protocol,and will provide solid foundation for maintaining soil quality.The results of this study are as follows:(1)The physico-chemical properties of calcareous purple paddy soil under different fertilization systems were analyzed by chemical analysis.Compared to non fertilizer treatment(CK),fertilization decreased the pH and the content of nitrate,while increased the content of soil organic matter,total nitrogen and ammonia nitrogen in calcareous purple paddy soil.(2)The diversity and structure of AOA communities in calcareous purple paddy soil under different fertilization systems were analyzed by DGGE.The results showed that different fertilization regimes in different soil depths had different impact on AOA community in calcareous purple paddy soil.Under different fertilization systems,the AOA community structure was not significantly changed in the 0-20 cm depth.With the increasing of soil depth,the AOA community structure varied obviously under different fertilization systems.Among the eight fetilizer systems,the AOA community under CK and N fertilizer treatments was relatively simple.The diversity index and richness of AO A community decreased with the increase of soil depth.The AOA was belong to thaumarchaeota,and was closely related to those uncultured AOA from different soil and water environment,and was phylogentically similar to the identified AOA Candidatus Nitrososphaera gargensis(GI:EU281321).Redundant gradient analysis showed that pH(P=0.012)was the main reason for the difference of AOA community structure in calcareous purple paddy soil.(3)The abundance of anammox bacteria in calcareous purple paddy soil under different fertilization systems was analyzed by quantitative PCR.The results showed that the abundance of anammox bacteria in the 0-20 cm depth was the highest among the four soil depths,the abundance of anammox bacteria in the 20-40 cm depth was the lowest.In each soil depth,the abundance of anammox bacteria under nitrogen treatment was the highest.The abundance of anammox bacteria under no fertilizer treatment(CK)and inorganic fertilizer treatments(N,NP,NPK)were higher than farmyard manure(M)and farmyard manure plus inorganic(MN,MNP,MNPK)treatments.Pearson correlation analysis reported that the copies number of anammox bacterial 16S rRNA was significantly correlated with pH(p=0.01),TN and SOM(p=0.05).(4)The diversity and structure of ana?mox bacteria communities in calcareous purple paddy soil under different fertilization systems were analyzed by DGGE and T-RFLP.The results showed that anammox bacteria most existed in the 0-20 cm depth of the calcareous purple paddy.The diversity of anammox bacteria communities in the 0-20 cm depth was the highest among the four soil depths.The diversity of anammox bacteria communities in the 20-40 cm depth was the lowest.The structure of anammox bacteria communities was significantly different under fertilization systems.In the 0-20 cm depth,the diversity index of anammox bacteria under N treatment was the lowest,while the NP treatment was the highest.The number of anammox bacteria under no fertilizer treatment(CK)and inorganic fertilizer was higher than farmyard manure treatment and farmyard manure plus inorganic treatment in the other soil depth(20-40,40-60 and 60-90 cm).The dominant anammox bacteria species in calcareous purple paddy soil were similar to Brocadia genera,followed by Scalindula,Redundant gradient analysis(RDA)showed that the soil pH is the dominant factor in shaping the anammox bacterial communities in calcareous purple soil.The research revealed that the structure of the anammox bacterial communities obviously varied distribution in the calcareous purple paddy soil.In summary,this study explored the impact of different long-term fertilization systems on the shift of the structure,diversity and vertical distribution of AOA and anammox bacterial community in calcareous purple paddy soil.The results showed that fertilization could significantly affect the AOA and anammox bacteria in calcareous purple paddy soil.Farmyard manure plus mineral fertilizer is beneficial to the development of AOA community structure.Despite the mere nitrogen fertilizer increased the abundance of anammox bacteria,but decreased the diversity of anammox bacterial community structure.Both AOA and anammox bacteria were mainly distributed in 0-20 cm.Among the measured soil properties,pH is the dominant factor in shaping the AOA and anammox bacterial community structure in calcareous purple paddy soil.