| Exploring the relationship between soil denitrifiers diversity and nitrogen biogeochemical cycling of the farmland ecosystem is the highlight of re-evaluation of the soil quality and function.Calcareous purple paddy soil is an importmant agricultural soil resource in Sichuan province and even China.In order to protect its quality,a NPK Long-term Fertilization Experiment was established at Chuanshan,Suining city,Sichuan province in the 1980s.Based on this experimental station,combined with chemical analysis and molecular techniques including denaturing gradient gel electrophoresis(DGGE),terminal restriction fragment length polymorphism of(T-RFLP),DNA sequencing,and quantitative PCR were used to reveal the response of the abundance,composition,and phylogenetically diveristy of the nirS-and nosZ-denitrifiers communities to different fertilizer treatments in different soil depth.The main results of this study are as follows:(1)According to chemical analysis,the physico-chemical properties of calcareous purple paddy soil under different fertilization systems were analyzed.The results showed that compared to the unfertilizer control(CK),fertilizations decreased the pH and nitrate content,while increased soil organic matter,total nitrogen and ammonia nitrogen.With increasing of the soil depth,soil pH also increased,while the content of total nitrogen and nitrate nitrogen decreased.No obvious changes were observed for the soil ammonia.Based on the residual quantity method of nitrate,the results showed that denitrifying enzyme activities of topsoil(0-20 cm)was obviously higher than that of other soil depth.Besides,nitrogen plus phosphorus(NP)fertilizer obviously improved denitrifying enzyme activity(DEA).The Pearson correlation analysis showed that DEA was negatively and significantly correlated with the soil pH(r = 0.46,p<0.01),and positively and significantly correlated with nitrate(r = 0.785,p<0.01).The DEA was significantly correlated with nosZ-denitrifiers community structure(p<0.05 by Monte Carlo Algorithms)but not correlated with nirS and nosZ gene copy number.(2)Fluorescence qPCR was used to determine the number of 16S rRNA and nirS,nosZ genes,the results showed that in the topsoil(0-20 cm),nirS and nosZ gene copy number were significantly higher than other three depths.Fertilizations contributed to the three determined genes copy number.Combination use of mineral fertilizer and farmyard manure would affect the 16S rRNA and nosZ genes has more obviously,while no effect was observed about the nirS gene.Based on the ratio of the nirS and nosZ gene to the 16S rRNA gene proportion,the abundance of nosZ-denitrifiers is higher than nirS-denitrifiers in the calcareous purple paddy soil.Meanwhile,the Pearson correlation analysis showed that nirS and nosZ gene copy number were positively correlated with soil organic matter.(3)DGGE was applied to analyze the nirS-denitrifiers under different fertilizer treatments.The results showed the community structure of nirS-denitrifiers varied obviously under different fertilizations.In 0-20 cm soil depth,the diversity and richness of nirS-denitrifiers in soil amended with mineral fertilizer plus manure was higher than those in mineral fertilizers,but in the other depths there was no obvious difference.In the 60-90 cm soil depth,the diversity and richness of nirS-denitrifiers was the lowest.Nine sequences were successfully retrieved from the DGGE bands,and among which,three sequences were unidentified.The rest sequences were all phylogenetically affiliated to Proteobacteria and most of them were ?-Proteobacteria.(4)T-RFLP technique was used to analyze the composition of nirS-and nosZ-gene containing denitrifers communities.The richness of nirS gene in different soil depth was not obviously varied,while those of nosZ genes in the topsoil were significantly higher than those in other soil depths.Compared to the nirS-and the nosZ-denitrifers community structure in the subsoil,those in the surface soil which were directly affected by fertilizer treatments were more complex.The dominant nirS T-RFs were 75 bp,80 bp,and 115 bp.The dominant T-RFs 115 bp of nirS-denitrifiers community was similar to Magnetospirillum(AB937700),while 75 bp was similar to Pseudomonas stutzeri(CP011854).Among four soil depths,there was not mutual T-RFs.The 90-and 355 bp T-RFs of nosZ-gene denitrifiers in 0-20 cm and 20-40 cm soil depth were similar to Azospirillum(Rhodospirillales).And the 300 bp T-RFs in 40-60 cm and 60-90 cm soil depth were similar to Bradyrhizobium(Rhizobiales).All the identified nosZ gene sequences belonged to Proteobacteria,and with?-Proteobacteria bacteria being the most dominant denitrifying bacteria species.(5)Canonical correspondence analysis(CCA)and redundancy analysis(RDA)were used to analyze the correlations between denitrifying bacterial community structure and soil physico-chemical parameter,the results showed that soil pH(p<0.05)and NH4+-N(p<0.05)were the key factors in shaping the nirS gene community composition.And soil organic matter,total nitrogen and pH(p<0.05)were critical to nosZ gene community compostion.In total,long-term fertilizations had different effect on DEA and nirS-and nosZ-denitrifiers communities and their vertical distribution in calcareous purple paddy soil.The inorganic fertilizer more promoted DEA.Both nirS-and nosZ-denitrifiers were mainly distributed in topsoil(0-20 cm),in which the community structure of nosZ-and nirS-denitrifiers were distinctly different with those in the other soil depths.Chemical fertilizers plus farmyard manure would increase the copy number and diversity index of nosZ-denitrifiers,while the copy numbers of nirS genes were not significantly affected by different fertilizers.Both the nirS-and nosZ-denitrifying bacteria are similar to Proteobacteria.And the dominant nirS-denitrifying bacteria were ?-Proteobacteria while the dominant nosZ-denitrifying bacteria species belonged to a-Proteobacteria.Among the measured soil parameters,soil pH was a key factor in shaping the nirS and nosZ denitrifying bacterial community composition. |
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