Summary The abundance and composition of soil ammonia‐oxidizing bacteria (AOB) and ammonia‐oxidizing archaea (AOA) were investigated by using quantitative real‐time polymerase chain reaction, cloning and sequencing approaches based on amoA genes. The soil, classified as agri‐udic ferrosols with pH (H2O) ranging from 3.7 to 6.0, was sampled in summer and winter from long‐term field experimental plots which had received 16 years continuous fertilization treatments, including fallow (CK0), control without fertilizers (CK) and those with combinations of fertilizer nitrogen (N), phosphorus (P) and potassium (K): N, NP, NK, PK, NPK and NPK plus organic manure (OM). Population sizes of AOB and AOA changed greatly in response to the different fertilization treatments. The NPK + OM treatment had the highest copy numbers of AOB and AOA amoA genes among the treatments that received mineral fertilizers, whereas the lowest copy numbers were recorded in the N treatment. Ammonia‐oxidizing archaea were more abundant than AOB in all the corresponding treatments, with AOA to AOB ratios ranging from 1.02 to 12.36. Significant positive correlations were observed among the population sizes of AOB and AOA, soil pH and potential nitrification rates, indicating that both AOB and AOA played an important role in ammonia oxidation in the soil. Phylogenetic analyses of the amoA gene fragments showed that all AOB sequences from different treatments were affiliated with Nitrosospira or Nitrosospira‐like species and grouped into cluster 3, and little difference in AOB community composition was recorded among different treatments. All AOA sequences fell within cluster S (soil origin) and cluster M (marine and sediment origin). Cluster M dominated exclusively in the N, NP, NK and PK treatments, indicating a pronounced difference in the community composition of AOA in response to the long‐term fertilization treatments. These findings could be fundamental to improve our understanding of the importance of both AOB and AOA in the cycling of nitrogen and other nutrients in terrestrial ecosystems.

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Nitrous oxide (N 2 O) is a potent greenhouse gas and, in New Zealand, about one-third of the total greenhouse gas emissions from the agricultural sector are of N 2 O, mostly derived from animal excreta in grazed pasture soils. The aim of this study was to determine the effectiveness of a nitrification inhibitor, dicyandiamide (DCD), in reducing N 2 O emissions from animal urine patches in four different soils located in different regions of New Zealand with different soil, climatic and management conditions. The four soils are Templeton fine sandy loam and Lismore stony silt loam in Canterbury in the South Island, Horotiu silt loam in the Waikato region and Taupo pumice sand near Lake Taupo, both in the North Island. Results showed that the application of a fine-particle suspension nitrification inhibitor, DCD, to grazed pasture soils was very effective in reducing N 2 O emissions in all four different soils. Total N 2 O emissions (over 69-137 days) from animal urine patches ranged from 1 to 20.9 kg N 2 O-N ha )1 without DCD. These were reduced to 0.31-5.7 kg N 2 O-N ha )1 by the use of DCD, representing 61-73% reductions (with an average of 70% reduction). The N 2 O-N emission factor from animal urine N, EF 3 , was reduced from an average of 0.9 to 0.3% by the use of DCD. These results demonstrate the potential of using nitrification inhibitors to mitigate N 2 O emissions in a wide range of grazed pasture soils under different climatic and management conditions.

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The nitrification inhibitor dicyandiamide (DCD) has recently been shown to be effective in reducing nitrate leaching from grazed pasture soils. The objective of this study was to determine the influence of temperature and application rate on the effectiveness of DCD in nitrification inhibition. Possible effects on soil microbial biomass were also determined. The soil, Lismore silt loam (Pallic orthic brown soil; Udic Haplustept loamy skeletal), was incubated at a moisture content near field capacity under 2 temperatures (8 or 20 • C). Urea was applied at 25 kg N/ha and dairy cow urine at 1000 kg N/ha. DCD was applied at 2 rates equivalent to 7.5 or 15 kg/ha. The results show that at a soil temperature of 8 • C, the half-life of DCD was 111-116 days. The half-life of NH 4 + changed from 44 days without DCD to 243-491 days when DCD was applied. In contrast, at a soil temperature of 20 • C the half-life of DCD was 18-25 days. The half-life of NH 4 + changed from 22 days without DCD to 64-55 days with DCD. The 2 different rates of DCD had a small effect on the NH 4 + concentration in the soil. The application of DCD did not have a significant effect on soil microbial biomass. DCD would therefore be most effective in inhibiting nitrification and thus reducing nitrate leaching in late autumn-winter-early spring in most parts of New Zealand when daily average soil temperatures are generally below 10 • C and when drainage is high.

Background, aim, and scope As the second most important greenhouse gas, methane (CH 4 ) is produced from many sources such as paddy fields. Methane-oxidizing bacteria (methanotrophs) consume CH 4 in paddy soil and, therefore, reduce CH 4 emission to the atmosphere. In order to estimate the contribution of paddy fields as a source of CH 4 , it is important to monitor the effects of fertilizer applications on the shifts of soil methanotrophs, which are targets in strategies to combat global climate change. In this study, real-time polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) based on 16S rRNA and pmoA genes, respectively, were used to analyze the soil methanotrophic abundance and community diversity under four fertilization treatments: urea (N), urea and potassium chloride (NK), urea, superphosphate, and potassium chloride (NPK), and urea, superphosphate, potassium chloride, and crop residues (NPK+C), compared to an untreated control (CON). The objective of this study was to examine whether soil methanotrophs responded to the long-term, different fertilizer regimes by using a combination of quantitative and qualitative molecular approaches. Materials and methods Soil samples were collected from the Taoyuan Experimental Station of Agro-ecosystem Observation at Changde (28°55′ N, 111°26′ E), central Hunan Province of China, in July 2006. Soil DNAs were extracted from the samples, then the 16S rRNA genes were quantified by real-time PCR and the pmoA genes were amplified via general PCR followed by DGGE, cloning, sequencing, and phylogenetic analysis. The community diversity indices were assessed through the DGGE profile. Results Except for NPK, other treatments of N, NK, and NPK+C showed significantly higher copy numbers of type I methanotrophs (7.0-9.6×10 7 ) than CON (5.1×10 7 ). The copy numbers of type II methanotrophs were significantly higher in NPK+C (2.8×10 8 ) and NK (2.5×10 8 ) treatments than in CON (1.4×10 8 ). Moreover, the ratio of type II to type I methanotrophic copy numbers ranged from 1.88 to 3.32, indicating that the type II methanotrophs dominated in all treatments. Cluster analyses based on the DGGE profile showed that the methanotrophic community in NPK+C might respond more sensitively to the environmental variation. Phylogenetic analysis showed that 81% of the obtained pmoA sequences were classified as type I methanotrophs. Furthermore, the type I-affiliated sequences were related to Methylobacter, Methylomicrobium, Methylomonas, and some uncultured methanotrophic clones, and those type IIlike sequences were affiliated with Methylocystis and Methylosinus genera. Discussion There was an inhibitory effect on the methanotrophic abundance in the N and a stimulating effect in the NK and NPK+C treatments, respectively. During the ricegrowing season, the type II methanotrophs might be more profited from such a coexistence of low O 2 and high CH 4 concentration environment than the type I methanotrophs.

Nitrous oxide (N2O) is a potent greenhouse gas, and nitrate () is a water contaminant. In grazed grassland, the major source of both leaching and N2O emissions is nitrogen (N) deposited in animal excreta, particularly in the urine. The objective of this study was to determine the effectiveness of two nitrification inhibitors: (i) a solution of dicyandiamide (DCD) and (ii) a liquid formulation of 3,4‐dimethylpyrazole phosphate (DMPP) for reducing N2O emissions and leaching from urine patch areas in two grazed pasture soils under different environmental conditions. In the Canterbury Templeton soil, the nitrification rate of ammonium from the animal urine applied at 1000 kg N/ha was significantly decreased by the application of DCD (10 kg/ha) and DMPP (5 kg/ha). N2O emissions, measured over a 3‐month period, from dairy cow urine applied to the Canterbury Templeton soil were 1.14 kg N2O‐N/ha, and this was reduced to 0.43 and 0.39 kg N2O‐N/ha by DCD and the liquid DMPP, respectively. These are equivalent to 62–66% reductions in the total N2O emissions. Nitrate leaching losses from dairy cow urine applied to the Waikato Horotiu soil lysimeters were reduced from 628.6 kg ‐N/ha to 400.6 and 451.5 kg ‐N/ha by the application of DCD (10 kg/ha) or DMPP (1 kg/ha), respectively. There was no significant difference between the DCD solution and the liquid DMPP in terms of their effectiveness in reducing N2O emissions or leaching under the experimental conditions of this study. These results suggest that both the liquid formulations of DCD and DMPP have the potential to be used as nitrification inhibitors to reduce N2O emissions and leaching in grazed pasture soils.