长期不同施肥处理对反硝化菌群落结构及功能的影响
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摘要
作为农田氮肥损失的重要途径和温室气体N_2O排放的重要来源，农田反硝化作用研究受到越来越多的关注，然而目前只有有限的报道是关于不同施肥处理对旱地反硝化菌种群影响机制的研究。为探索长期不同施肥处理对旱地反硝化作用影响的微生物分子生态机制，采集哈尔滨（HEB）、公主岭（GZL）和沈阳（SY）等三处长期定位试验点的土壤，采用末端限制性片段长度多态性（T-RFLP）技术，结合土壤反硝化潜势（DEA）和理化性质的测定，研究了长期不同施肥处理对东北旱地土壤nirK、nirS和nosZ型反硝化菌群落结构的影响。三处试验点皆设不施肥（CK）、单施有机肥（OM）、单施无机肥（NPK）和有机和无机肥混施（MNPK）等4个处理。结果如下：
1）OM处理显著增加了公主岭和沈阳的DEA，而对哈尔滨的DEA无显著影响；NPK处理显著降低了哈尔滨和沈阳的DEA，对公主岭的DEA无显著影响；除公主岭的MNPK处理显著增加了土壤的DEA外，在哈尔滨和沈阳，MNPK处理对土壤DEA无显著影响。
2）哈尔滨及沈阳的NPK和MNPK处理改变了nirK型反硝化菌的群落结构，且NPK和MNPK处理的群落结构相似，OM处理对nirK型反硝化菌群落结构影响较小；在公主岭OM和MNPK处理显著改变了nirK型反硝化菌的群落结构，而NPK和CK处理的群落结构相似。系统发育分析表明，三个地点的大多数nirK克隆序列与土壤中未培养的反硝化细菌相似度较高，在三个地点施肥处理中显著变化的片段长度集中分布在进化树的一个大簇内。相关性分析表明，在哈尔滨，除C_(org)/N外，有机质（OM）、全氮（TN）、含水量（Moisture）、C_(org)/P、全磷（P）、速效磷（OlsenP）和pH依次与nirK型反硝化菌群落结构的变化显著相关（r=0.323~0.789，P<0.05），DEA与群落结构的变化不相关；在公主岭，除含水量、C_(org)/N、C_(org)/P和DEA外，环境因子依P、OlsenP、有机质（OM）、TN和pH与群落结构的变化显著相关（r=0.461~0.657，P<0.05）；在沈阳只有DEA和pH与群落结构的变化显著相关（r=0.335~0.455，P<0.05）；综合分析显示除Moisture外，其余指标皆与地点间的群落结构差异显著相关（r=0.128~0.604，P<0.05）。
3）哈尔滨及沈阳的NPK和MNPK处理显著改变了nirS型反硝化菌的群落结构，OM与CK处理的群落结构相似；公主岭的OM和MNPK处理显著改变了nirS型反硝化菌的群落结构，NPK处理对群落结构无显著影响。系统发育分析显示多数的nirS克隆序列与已培养的微生物相似度较低，但与来自土壤中的nirS序列相似度较高，三个地点中施肥处理显著改变的部分片段长度位于进化树相近位置，但施肥影响的大多数片段长度所代表的反硝化菌并不一致。相关性分析显示，在哈尔滨，除C/N、有机质（OM）和DEA外，C/P、含水量、TN、P、Olsen P和pH依次与群落结构的变化显著相关（r=0.263~0.55，P<0.05）；在公主岭，除含水量和C_(org)/N外，DEA、TN、Olsen P、有机质（OM）、P和pH与群落结构的变化显著相关（r=0.381~0.836，P<0.05）；在沈阳只有Olsen P和pH与群落结构的变化显著相关（r=0.255~0.511，P<0.05）；综合分析显示，全部指标皆与地点间的群落结构差异显著相关（r=0.078~0.350，P<0.05）。
4）哈尔滨和沈阳的NPK和MNPK处理显著改变了nosZ型反硝化菌的群落结构，OM处理对哈尔滨和沈阳nosZ型反硝化菌的群落结构影响较小；公主岭的OM和MNPK处理显著改变了nosZ型反硝化菌的群落结构，而NPK处理无明显影响。系统发育显示，多数的nosZ克隆序列属于α-变形菌纲（α-proteobacteria）的根瘤菌目（Rhizobiales），三个地点中对施肥显著响应的片段长度代表的反硝化菌一致。相关性分析显示，在哈尔滨，除DEA和C_(org)/N外，有机质（OM）、TN、Moisture、C_(org)/P、P、pH和Olsen P与群落结构的变化显著相关（r=0.307~0.734，P<0.05）；在公主岭，除C_(org)/N和Moisture外，C_(org)/P、DEA、P、TN、pH、Olsen P和有机质依次与nosZ型反硝化菌群落结构的变化显著相关（r=0.490~0.703，P<0.05）；在沈阳，除DEA、Moisture和C_(org)/N外，TN、C_(org)/P、有机质（OM）、P、Olsen P和pH依次与群落结构的变化显著相关；综合分析显示，全部指标皆与地点间的nosZ型反硝化菌群落结构显著差异相关（r=0.093~0.470，P<0.05）。
综上所述，本研究表明，长期不同施肥处理影响了nirK、nirS和nosZ型反硝化菌的群落结构和活性，群落结构对施肥处理响应的模式依地点的不同而不同，土壤pH和Olsen P的变化是导致三种类型反硝化菌群落结构改变的主要原因，且三种类型反硝化菌对土壤理化性质变化的敏感程度不同。
As one of the major pathways of nitrogen loss from fertilized agricultural soils and an importantsource of greenhouse gas N_2O 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.
1）OM处理显著增加了公主岭和沈阳的DEA，而对哈尔滨的DEA无显著影响；NPK处理显著降低了哈尔滨和沈阳的DEA，对公主岭的DEA无显著影响；除公主岭的MNPK处理显著增加了土壤的DEA外，在哈尔滨和沈阳，MNPK处理对土壤DEA无显著影响。
2）哈尔滨及沈阳的NPK和MNPK处理改变了nirK型反硝化菌的群落结构，且NPK和MNPK处理的群落结构相似，OM处理对nirK型反硝化菌群落结构影响较小；在公主岭OM和MNPK处理显著改变了nirK型反硝化菌的群落结构，而NPK和CK处理的群落结构相似。系统发育分析表明，三个地点的大多数nirK克隆序列与土壤中未培养的反硝化细菌相似度较高，在三个地点施肥处理中显著变化的片段长度集中分布在进化树的一个大簇内。相关性分析表明，在哈尔滨，除C_(org)/N外，有机质（OM）、全氮（TN）、含水量（Moisture）、C_(org)/P、全磷（P）、速效磷（OlsenP）和pH依次与nirK型反硝化菌群落结构的变化显著相关（r=0.323~0.789，P<0.05），DEA与群落结构的变化不相关；在公主岭，除含水量、C_(org)/N、C_(org)/P和DEA外，环境因子依P、OlsenP、有机质（OM）、TN和pH与群落结构的变化显著相关（r=0.461~0.657，P<0.05）；在沈阳只有DEA和pH与群落结构的变化显著相关（r=0.335~0.455，P<0.05）；综合分析显示除Moisture外，其余指标皆与地点间的群落结构差异显著相关（r=0.128~0.604，P<0.05）。
3）哈尔滨及沈阳的NPK和MNPK处理显著改变了nirS型反硝化菌的群落结构，OM与CK处理的群落结构相似；公主岭的OM和MNPK处理显著改变了nirS型反硝化菌的群落结构，NPK处理对群落结构无显著影响。系统发育分析显示多数的nirS克隆序列与已培养的微生物相似度较低，但与来自土壤中的nirS序列相似度较高，三个地点中施肥处理显著改变的部分片段长度位于进化树相近位置，但施肥影响的大多数片段长度所代表的反硝化菌并不一致。相关性分析显示，在哈尔滨，除C/N、有机质（OM）和DEA外，C/P、含水量、TN、P、Olsen P和pH依次与群落结构的变化显著相关（r=0.263~0.55，P<0.05）；在公主岭，除含水量和C_(org)/N外，DEA、TN、Olsen P、有机质（OM）、P和pH与群落结构的变化显著相关（r=0.381~0.836，P<0.05）；在沈阳只有Olsen P和pH与群落结构的变化显著相关（r=0.255~0.511，P<0.05）；综合分析显示，全部指标皆与地点间的群落结构差异显著相关（r=0.078~0.350，P<0.05）。
4）哈尔滨和沈阳的NPK和MNPK处理显著改变了nosZ型反硝化菌的群落结构，OM处理对哈尔滨和沈阳nosZ型反硝化菌的群落结构影响较小；公主岭的OM和MNPK处理显著改变了nosZ型反硝化菌的群落结构，而NPK处理无明显影响。系统发育显示，多数的nosZ克隆序列属于α-变形菌纲（α-proteobacteria）的根瘤菌目（Rhizobiales），三个地点中对施肥显著响应的片段长度代表的反硝化菌一致。相关性分析显示，在哈尔滨，除DEA和C_(org)/N外，有机质（OM）、TN、Moisture、C_(org)/P、P、pH和Olsen P与群落结构的变化显著相关（r=0.307~0.734，P<0.05）；在公主岭，除C_(org)/N和Moisture外，C_(org)/P、DEA、P、TN、pH、Olsen P和有机质依次与nosZ型反硝化菌群落结构的变化显著相关（r=0.490~0.703，P<0.05）；在沈阳，除DEA、Moisture和C_(org)/N外，TN、C_(org)/P、有机质（OM）、P、Olsen P和pH依次与群落结构的变化显著相关；综合分析显示，全部指标皆与地点间的nosZ型反硝化菌群落结构显著差异相关（r=0.093~0.470，P<0.05）。
综上所述，本研究表明，长期不同施肥处理影响了nirK、nirS和nosZ型反硝化菌的群落结构和活性，群落结构对施肥处理响应的模式依地点的不同而不同，土壤pH和Olsen P的变化是导致三种类型反硝化菌群落结构改变的主要原因，且三种类型反硝化菌对土壤理化性质变化的敏感程度不同。
As one of the major pathways of nitrogen loss from fertilized agricultural soils and an importantsource of greenhouse gas N_2O 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.
引文
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