Long-term Fertilization Affects Ammonia-oxidizing Prokaryotes In Acidic And In Neutral Paddy Soils

Paddy fields account for75%of worldwide rice yield, breeding as many as one hundred million people. Long-term fertilization acts as a model system to reveal the feedback and adaptation of ecosystem function to anthropogenic disturbance. It also clarifies the respond of ammonia-oxidizing prokaryotes to fertilization. Studies have shown that a long-term fertilization has an effect on ammonia-oxidizing prokaryotes. But owing to technical constraints, the studies do not in-depth. Wheat soil and rice soil were studied to explore the nucleic acid extraction method for air-dried soil, providing a foreshadowing for analysing air-dried soil in molecular methods. We also set Yingtan acidic paddy soil in Jiangxi province and Changshu neutral paddy soil in Jiangsu province as study objectives. In these areas, inorganic fertilizers had applied. The qPCR, the stable isotope probing-DGGE, the DNA-SIP and454pyrosequencing were used to study the effect of long-term fertilization on the community abundance, structure and active composition of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB).In the present study, the DNA of air-dried or fresh paddy soil and wheat soil were extracted using three extraction methods, including Griffiths method, freezing-thawing method and kit extraction method. The results show that the best extraction method of DNA extraction is kit extraction method. DNA yields in the three successive extractions accounted for more than80%of total DNA. The three consecutive extractions can at the utmost reflect soil microbial abundance. The DNA abundance of the air-dried soil is lower than that in the fresh soil. The variation in the DNA abundance of air-dried soil could to some extent reflect that of the fresh soil.The AOA copy numbers of NPK treatment and CK treatment in acidic and in neutral soils don’t have significantly difference, but the AOB copy numbers of NPK treatment is significantly higher than CK treatment. AOA copy numbers are higher than AOB copy numbers in both acidic soil and neutral soil. To some extent, long-term fertilization contributed to the growth of AOB in paddy soils.AOA of acidic and neutral paddy soils shows that NPK treatment and CK treatment had no significant difference. After8weeks incubation added urea, Group1.1b was added in AOA community structure of acidic and neutral paddy soils, which changes the community structure of AOA. It’s the same between NPK treatment and CK treatment in acidic paddy soil, but that is different in neutral paddy soil. The AOB community structure was totally different before incubation. After8weeks incubation, the community structure didn’t change, but the differences between NPK treatment and CK treatment remain.In acidic and neutral paddy soils, active crenarchaeota of NPK treatment is lower than that of CK treatment. It illustrates that long-term fertilization inhibited the growth of crenarchaeota, which including AOA, under certain circumstances. After8weeks incubation added urea, the active AOB of NPK treatment is higher than that of CK treatment. It indicates that the long-term fertilization favors the growth of active AOB. Active crenarchaeota abundance is higher than active AOB abundance in acidic and in neutral paddy soils. It hints that active archaea plays a leading role in two soils.In acidic and neutral paddy soils,89%of active AOA in NPK treatment and88%of active AOA in CK treatment before incubation clustered to Group1.1a-associated, there are several clustered to Group1.1b and Group1.1c. In neutral paddy soil,99%of AOA in NPK and CK treatments before incubation clustered to Group1.1b.100%of AOB in CK treatment before incubation clustered to Nitrosospira Cluster3of acidic paddy soil and neutral paddy soil. In acidic paddy soil,63%of AOB in NPK treatment before incubation clustered to Nitrosomonas communis lineage,37%clustered to Nitrosospira Cluster3. In neutral paddy soil,96%of AOB in NPK treatment before incubation clustered to Nitrosospira Cluster3,4%clustered to Nitrosomonas communis lineage. It indicates that long-term fertilization leads to changes of AOB community groups in acidic and in neutral paddy soils, but it didn’t change AOA community groups.After8weeks incubation in acidic paddy soil,93%of active AOA in NPK treatment clustered to Group1.1b,6%clustered to Group1.1a-associated;96%of active AOA in CK treatment clustered to Group1.1a-associated,4%clustered to Group1.1b. It indicates that the active AOA main groups in NPK and CK treatments are different respond to added urea. After8weeks incubation in neutral paddy soil,100%of active AOA in NPK and CK treatments clustered to Group1.1b.95%of active AOB in NPK treatment clustered to Nitrosomonas communis lineage,5%clustered to Nitrosospira Cluster3;99%of active AOB in CK treatment clustered to Nitrosomonas communis lineage,1%clustered to Nitrosospira Cluster3. It indicates that the added urea stimulates the growth of Nitrosomonas communis lineage, which belongs to AOB group.Our study clarifies the community structure and function of AOA and AOB, also indicates the relativity between them in paddy soil ecosystem. We aim to illustrate long-term fertilization has an effect on the community structure of AOA and AOB, and to enrich the understanding of ammonia-oxidizing prokaryotes in soil and the interaction in ecosystem. The stratergy, methods and results in our study have feature and innovation.