长期施肥对酸性土壤氨氧化微生物群落的影响

Effect of long-term fertilization on community of ammonia oxidizers in acidic soil

【目的】长期施肥显著影响着酸性土壤的p H,研究由此引起的土壤中氨氧化古菌(ammonia-oxidizing archaea,AOA)和氨氧化细菌(ammonia-oxidizing bacteria,AOB)的变化,为土壤培肥提供理论依据。【方法】供试土壤为27年长期定位施肥试验的红壤,供试作物为玉米。选择不施肥对照(CK)、氮肥120 kg/(hm^2·a)(N)、氮磷钾肥(NPK)和猪粪2000 kg/(hm^2·a)(OM)4个处理采集土壤样品,测定了土壤基本理化性状;利用q PCR、PCR-DGGE方法,分析土壤AOA和AOB群落丰度与组成。【结果】1)长期定位施肥导致土壤p H值发生显著变化,N处理的土壤p H值最低,仅为4.03,其次是NPK和CK处理的土壤,OM处理土壤p H值最高,接近中性达6.40。2)与CK相比,长期施肥提高了土壤有机质、全氮、铵态氮和硝态氮含量。3)OM处理显著提高了土壤NH_3浓度,而其它处理对NH_3浓度无显著影响。4)施肥显著增加了土壤AOA的丰度,OM处理提升幅度最大;AOA丰度与土壤有机质碳、全氮呈极显著正相关(P<0.01),与铵态氮、土壤NH_3浓度呈显著正相关(P<0.05),与土壤p H、硝态氮关系不显著(P>0.05);施肥改变了AOA的群落结构,CK、N、NPK处理的群落结构差异不显著,OM处理与另外三个处理差别较大。主要AOA类群是Group 1.1b,少数属于Group 1.1aassociated。RDA分析表明,土壤p H值、有机质、总氮、铵态氮、土壤中NH_3浓度是导致AOA群落变化的主要环境因子。5)仅OM处理对AOB丰度和群落产生了显著影响,主要类群是Nitrosospira Cluster 3,少数属于Nitrosospira Cluster 9。AOB丰度与土壤NH_3浓度呈极显著正相关(P<0.01),与有机质碳、全氮呈显著正相关关系(P<0.05),与土壤p H、铵态氮、硝态氮关系均不显著(P>0.05)。【结论】长期施用不同肥料对酸性土壤的理化性质影响差异大,AOA和AOB的丰度和群落结构也发生了明显变化,尤其是施加有机肥之后。来自不同处理的大部分AOA属于Group 1.1b类群,少数属于Group 1.1a-associated类群。仅在OM处理中检测到AOB类群,大部分属于Nitrosospira Cluster 3,少数属于Nitrosospira Cluster 9。

[ Objectives ] Long-term fertilization significantly altered the soil nitrogen cycle and the community structure of ammonia oxidizers. However, the different fertilization treatments, especially organic manure, had the significant effect on the soil pH of acid soils. Systematical studies are needed to clarify the change in abundance and composition of ammonia oxidizing archaea （AOA） and ammonia oxidizing bacteria （AOB）. [ Methods ] An acidic upland soil, sited under long-term （27-year） fertilization, was chose for the investigation with maize as tested materials. Soil samples were collected from the four treatments of mineral N fertilizer （N）, mineral NPK fertilizer （NPK）, organic manure （OM） and an unfertilized control （CK）. The abundance and composition of AOA and AOB were determined using real-time polymerase chain reaction （PCR） and denaturing gradient gel electrophoresis （DGGE）. [ Results ] 1） Long term fertilization resulted in significant changes in soil pH values. The lowest soil pH was in treatment N, only 4.03, that in treatment NPK and CK were slightly higher, and that in treatment OM was the highest, which was 6.40, close to neutral. 2） Long-term fertilization improved the content of soil organic matter, total nitrogen, ammonium nitrogen and nitrate nitrogen. 3） The OM treatment significantly increased the soil NH3 concentration, the others did not. 4） Fertilization significantly increased the abundance of AOA, with the maximum amplitude in OM treatment. The AOA abundance was extremely significantly correlated with the contents of soil organic matter, carbon and total nitrogen （P 〈 0.01）, and significantly correlated with NH,^-N and soil NH3 concentrations （P 〈 0.05）, not significantly correlated with soil pH and NO~ -N （P 〉 0.05）. The community structure of AOA was changed, but that only in OM treatment was obvious. Phylogenetic analyses showed that most of the AOA sequences from the fertilization treatments were affiliated with group 1. l b thaumarchaea and a few with the group 1.1 a-associated thaumarchaea. The RDA analysis showed that soil pH, organic matter, total nitrogen, ammonium nitrogen and NH3 concentrations in soil were the main environmental factors leading to the change of AOA community. 5） Only OM treatment showed significant effect on the abundance and composition of AOB. The AOB sequences from OM treatment soil were affiliated with Nitrosospira Cluster 3 and some with and Nitrosospira Cluster 9. The AOB abundance was positively correlated with soil NH~ concentration （P 〈 0.01）, and positively correlated with organic matter, carbon and total nitrogen （P 〈 0.05）. [ Conclusions ] Long-term different fertilizations have caused different variation of soil physical and chemical properties, as well as the abundance and community structure of AOA and AOB in acid soil, especially the application of organic manure. Most of the AOA in all the treatments are affiliated with group 1. lb thaumarchaea and a few with the group 1.1 a-associated thaumarchaea. Only the AOB group was detected in the OM treatment, most of which were affiliated with Nitrosospira Cluster 3, and a few were affiliated with Nitrosospira Cluster 9.