长期施用有机和无机肥对黑土nirS型反硝化菌种群结构和丰度的影响

Influences of Long-term Application of Organic and Inorganic Fertilizers on the Composition and Abundance of nirS-type Denitrifiers in Black Soil

利用末端限制性片段长度多态性(T-RFLP)和实时荧光定量PCR(real-time quantitative PCR,Q-PCR)技术,结合反硝化潜势(DEA)和土壤理化性质的测定,探索了长期施用有机和无机肥对公主岭黑土nirS型反硝化细菌的群落结构和丰度的影响.试验设不施肥(CK)、单施有机肥(OM)、单施无机肥(NPK)以及有机肥和无机肥混施(MNPK)等4个处理.结果表明,长期施用有机肥显著增加了土壤的DEA,其中OM、NPK和MNPK处理分别为CK处理的5.92、1.81和6.03倍,而NPK和CK间无差异.有机肥处理增加了黑土nirS型反硝化细菌的丰度,OM、NPK和MNPK处理中nirS基因的拷贝数分别为CK的2.73、1.30和3.98倍;NPK处理对nirS基因的拷贝数影响不显著.T-RFLP图谱显示施用有机肥改变了nirS反硝化细菌的群落结构;相比于非有机肥处理,有机肥处理中增加了一类79 bp的片段类型,显著降低了84 bp的片段类型,并完全抑制了一类99 bp的片段类型,而有机肥处理间和非有机肥处理间的nirS群落结构分别相似.系统发育分析表明:黑土中nirS型反硝化菌主要由α、β和γ-变形菌纲及一些尚未培养的微生物组成,79 bp的片段类型与γ-变形菌纲的假单胞菌科(Pseudomonadaceae)和β-变形菌纲的伯克氏菌目(Burkholderiales)相似,84 bp片段类型与Burkholderiales和红环菌目(Rhodocyclales)相似.相关性分析表明,pH、全磷(TP)、全氮(TN)、总有机碳(TOC)、硝态氮(NO3--N)和铵态氮(NH4+-N)依次与nirS型反硝化细菌的种群丰度(r为0.724～0.922,P<0.05)和DEA(r为0.453～0.938,P<0.01)显著相关,DEA与nirS型反硝化细菌的种群丰度显著线性正相关(r=0.85,P<0.01);冗余度分析表明,除含水量外,TN、TP、pH、TOC、NH4+-N和NO3--N(r为0.440～0.862,P<0.01)依次与nirS型反硝化细菌群落结构的变化显著相关,DEA的变化和nirS型反硝化细菌群落结构的变化亦显著相关(r=0.863,P<0.01).本研究表明相比于无机肥处理,公主岭黑土中nirS型反硝化菌的群落结构与丰度对有机肥处理有更显著的响应,且其群落结构的改变与种群丰度的增加与DEA的提高显著相关.

The objectives of this study were to explore the effects of long-term organic and inorganic fertilizations on the composition and abundance of nirS-type denitrifiers in black soil.Soil samples were collected from 4 treatments（i.e.no fertilizer treatment,CK;organic manure treatment,OM;chemical fertilizer treatment（NPK） and combination of organic and chemical fertilizers treatment（MNPK）） in Gongzhuling Long-term Fertilization Experiment Station.Composition and abundance of nirS-type denitrifiers were analyzed with terminal restriction fragment length polymorphism（T-RFLP） and real-time quantitative PCR（Q-PCR）,respectively.Denitrification enzyme activity（DEA） and soil properties were also measured.Application of organic fertilizers（OM and MNPK） significantly increased the DEAs of black soil,with the DEAs in OM and MNPK being 5.92 and 6.03 times higher than that in CK treatment,respectively,whereas there was no significant difference between NPK and CK.OM and MNPK treatments increased the abundances of nirS-type denitrifiers by 2.73 and 3.83 times relative to that of CK treatment,respectively.The abundance of nirS-type denitrifiers in NPK treatment was not significantly different from that of CK.The T-RFLP analysis of nirS genes showed significant differences in community composition between organic and inorganic treatments,with the emergence of a 79 bp T-RF,a significant decrease in relative abundance of the 84 bp T-RF and a loss of the 99 bp T-RF in all organic treatments.Phylogenetic analysis indicated that the nirS-type denitrifiers in the black soil were mainly composed of α,β and γ-Proteobacteria.The 79 bp-type denitrifiers inhabiting exclusively in organic treatments（OM and MNPK） were affiliated to Pseudomonadaceae in γ-Proteobacteria and Burkholderiales in β-Proteobacteria.The 84bp-types were related to Burkholderiales and Rhodocyclales.Correlation analysis indicated that pH,concentrations of total nitrogen（TN）,total phosphorus（TP）,total organic carbon（TOC）,nitrate（NO-3-N） and ammonia（NH＋4-N） were significantly related to abundances of nirS-denitrifers（r=0.724-0.922,P0.05） and the DEA（r=0.453-0.938,P〈0.01）.In addition,the DEAs were linearly and positively correlated with the abundances of nirS-type denitrifers（r=0.85,P〈0.01）.Redundancy analysis showed that except moisture,pH and concentrations of TP,TP,TOC,NH＋4-N and NO-3-N were significantly correlated with the community structure of nirS-type denirifiers（r=0.440-0.862,P〈0.01）.Furthermore,the DEAs were significantly correlated with the compositions of nirS-denirifiers（r=0.863,P〈0.01）.In conclusion,the nirS-type denitrifiers in the black soil are more responsive to the organic treatments than to the inorganic treatments in terms of community composition and abundance,both of which are correlated with the changes of DEAs.