Effect Of Long-term Fertilization On The Diversity Of Nitrite Reductase Genes (nirk And Nirs) In Paddy Soil

Denitrification is one of the most important mechanisms for nitrogen losses of paddy soil.It not only reduces the utilization of nitrogen fertilizer,but also increases the emissions of N₂O,which is involved in destruction of the stratospheric ozone layer and in global warming,and subsequently accelerates environmental degradation.Long-term fertilization has significant influence on denitrification,which is stimulated by the application of nitrogen fertilizer,resulting in an increase of N₂O emissions.In order to study the effect of long-term fertilization on soil denitrifier communities,the diversity of denitrifying communities was studied.The structurally different but functionally equivalent genes coding for nitrate reductases,a key enzyme of denitrification process, were used as a molecular marker for denitrifying bacteria.The research would contribute to increase the utilization of nitrogen,decrease the emission of greenhouse gases and provide scientific basis for sustainable development of paddy field productivity.The soils were collected from a long-term paddy field experiment(started in 1990) located in Taoyuan,China.PCR primers were designed for the amplification of both nirK and nirS fragments,which were successfully amplified from the soil samples of CK(no fertilizer),N(nitrogen fertilizer),NPK(N,P and K fertilizers) and NPKOM(NPK plus organic matter) plots.To assess the underlying nir genes diversity and community structure,PCR products were cloned and sequenced.The main findings are as follows:1) Fertilization treatments had various influence on soil denitrification rate which was highly stimulated by applying organic matter,and followed by nitrogen fertilizer. Compared with CK,application of NPK fertilizers slightly increased the soil denltrification rate without significant difference.2) The effect of long-term fertilization on the diversity of nirK and nirS gene was different.The Chaol analysis revealed that application of NPK achieved the highest diversity of nirK gene fragments,while nitrogen fertilizer increased the diversity of nirS gene significantly.3) The comparison of sequence composition among the clone libraries based on LIBSHUFF was performed,which showed that significant differences existed in different treatments for nirK gene libraries,and revealed an opposite situation for nirS gene. 4) Of the 249 sequenced clones 232 were related to known nirK gene i.e.a proportion of 93%,whereas the proportion for nirS gene was 79%.Compared with the NCBI database from the BLAST search,the identities of nirK fragments ranged from 80%to 95%(average identity of 90.7%) at nucleotide level,while the identities of nitS fragments ranged from 72%to 82%(average identity of 74.7%).Phylogenetic analysis of nucleotide fragments grouped the nirK sequences into eight major clusters with some OTUs from certain treatment clustered together.Whereas,the highly uniform distributed nirS sequences were grouped into five clusters with most of clones branching in one major cluster.In summary,fertilization treatments had various influence on the nir-containing bacterial communities.Balanced application of NPK fertilizers significantly increased nirK gene community diversity,whereas single nitrogen fertilizer had highest diversity of nirS-containing community.Although long-term fertilization obviously changed the community structure of nirK-containing denitrifiers,it had no significant effect on nirS gene community structure.The results showed nirK gene community structure was more sensitive to fertilization practices than that of nirS gene.