Impacts Of Long-term Different Fertilization Regimes On Community Structures Of Denitrifiers And Function

As one of the major pathways of nitrogen loss from fertilized agricultural soils and an importantsource of greenhouse gas N₂O emission, denitrification has been receiving more and more attention.However, limited information has been available on effects of different fertilziiation regimes ondenitrifying communities in upland soils. Thus, in order to explore the long-term effects of differentorganic and inorganic fertilizers on compositions of nirK, nirS and nosZ-type denitrifiers communitiesin northeast dryland soils of China, soils collected from the following four treatments in three long-termexperimental sites (i.e., Haerbin, HEB; Gonghzuling, GZL; Shenyang, SY.) were analyzed: nofertilization, CK; organic manure, OM, chemical fertilization (NPK), and combination of organicmanure and chemical fertilization (MNPK). The compostion was analysed using terminal restrictionfragment length polymorphism (T-RFLP). In addition, the activities of denitrifying community weremeasured in terms of potential denitrification enzyme activity (DEA). Results are as follows:1) OM treatments significantly increased DEAs in soils from GZL and SY, repectively, while itexcerted less influences on the DEA from HEB; NPK treatments decreased DEAs from HEB and SY,repectively. There was no difference between CK and NPK in GZL except that DEA from GZL wasincreased under MNPK treatment. No significant differences were observed in DEAs in soils collectedfrom HEB and SY between CK and MNPK treatment.2) Community structures of nirK-type denitrifiers were changed by treatments with NPK and MNPKin HEB and SY, respectively, in which the community structures showed similar T-RFLP patterns, whileOM treatments caused minor changes compared to CK treatments. On the contrary, OM and MNPKtreatments significantly changed the community structures of nirK-type denitrifiers in GZL, while NPKand CK treatments showed similar T-RFLP pattern. Phylogenetic analysises showed that most of nirKgenes cloned in the soils collected from these3sites had high similarity to the uncultured bacteriumfrom soils with the groups responseive to fertilization being clustered into one branch. However, thegroups responsive to fertilization from these sites were different based on the postioning of sequences inthe branch. Correlation analysis indicated that in HEB, except C_org/N, organic matter, total nitrogen(TN), moisture, C_org/P, total phosphorus (P), Olsen P and pH were significantly correlated to the changeof community structure (r=0.323-0.789, P<0.05), whereas DEAs were uncoupled to the change ofcommunity structure (P>0.05). In GZL, except moisture, C_org/N, C_org/P, DEA, P, Olsen P, organic matter,TN and pH were correlated to the change of community structure (r=0.461-0.657, P<0.05). In SY, onlyDEA and pH were correlated to the change of community structure (r=0.335-0.455, P<0.05).Comprehensive analysis of the data from HEB, GZL and SY showed that, except moisture, theremaining parameters were correlated to the changes of community structures of nirK-type dentirifiers(r=0.128-0.604, P<0.05).3) NPK and MNPK treatments in HEB and SY signifcantly changed community structures ofnirS-type denitrifiers, whereas both OM and CK treatments did not. On the contrary, OM and MNPKtreatments in GZL caused significant changes, whereas NPK treatment showed similar pattern to CK. Phylogenetic analysises indicated that most of sequences showed low similarity to the cultutred bacteria,but showed high similarity to the uncultured bacteria from soils. Part of groups responsive tofertilization among these3sites took the same position in phylogenetic trees, however, the major groupsresponsive to fertilization among these sites were not identical. Correlation analyses indicated that, inHEB, except C_org/N, organic matter, DEA, C_org/P, moisture, TN, P, Olsen P and pH were correlatedto the change of community structure (r=0.263-0.55, P<0.05); in GZL, except moisture and C_org/N,DEA, TN, Olsen P, organic matter, P and pH were correlated to the change of community structure(r=0.381-0.836, P<0.05); P, Olsen P and pH were correlated to the change of community structureof SY (r=0.255-0.511, P<0.05); Comprehensive analysis of the data from HEB, GZL and SY showedthat all parameters were correlated to changes of community structures of nirS-type denitrifiers(r=0.078-0.350, P<0.05).4) Under NPK and MNPK treatments community structures of nosZ-type denitrifiers were changed inHEB and SY, respectively. OM treatments caused minor changes in HEB, but not in SY. In GZL OMtreatments caused significant changes of community structure, while NPK treatments showed similatiryto CK treatments. Phylogenetic analysises indicated that most of sequences were affiliated toRhizobiales in α-proteobacteria. Furthermore, the groups responsive to fertilization among these siteswere identical. Correlation analyses indicated that, in HEB, except DEA and C_org/N, organic matter, TN,moisture, C_org/P, P, pH and Olsen P were correlated to the change of community structure(r=0.307-0.734, P<0.05); in GZL, except C_org/N and moisture, C_org/P, DEA, P, TN, pH, Olsen P andorganic matter were correlated to the change of community structure (r=0.490-0.703, P<0.05); in SY,except DEA, moisture and C_org/N TN, C_org/P, organic matter, P, Olsen P and pH were correlated to thechange of community structure (r=0.246-0.492, P<0.05); Analysis of the data from HEB, GZL and SYshowed that all parameters were correlated to differences of community structures of nosZ-typedenitrifiers among these three sites (r=0.093-0.470, P<0.05).In conclusion, the results indicate that different long-term fertilzaition regimes affect both communitystructure of nirK, nirS and nosZ-type denitrifers and acitivty with change of community structurepattern being site-specific and mainly related to pH and Olsen P. The relationship between DEA andcommunity structure is both site-and gene-dependent, and community structures of nirK, nirS andnosZ-type denitirifers respond differently to changes of soil physical-chemical properties.