Effects of animal manure and nitrification inhibitor on N2O emissions and soil carbon stocks of a maize cropping system in Northeast China

Study area and soil properties

A field experiment was established in May 2012 at Shenyang Agro-Ecological Station (41°31′N, 123°22′E) of the Institute of Applied Ecology, Chinese Academy of Sciences, Northeast China. This region has a warm-temperate continental monsoon climate. The mean annual air temperature and annual precipitation are 7.5 °C and 680 mm, respectively. The soil is classified as Luvisol (FAO classification). The soil properties of the topsoil layer (0–20 cm) at the start of the experiment are as follows: SOC = 9.0 g kg⁻¹, available NH₄⁺–N = 1.18 mg kg⁻¹; available NO₃⁻–N = 9.04 mg kg⁻¹; Olsen-P = 38.50 mg kg⁻¹, available K = 97.90 mg kg⁻¹, bulk density = 1.25 g cm⁻³, and pH = 5.8. The determination method of soil was shown in “Soil analysis” section.

Field experiment

Three treatments were established in this experiment: (1) mineral fertilizers (NPK); (2) pig manure incorporation at a local conventional AM application rate of 15 Mg ha⁻¹ yr⁻¹ (NPKM, 126 kg N ha⁻¹ on dry weight); and (3) NPKM plus DMPP (3,4-Dimethylpyrazole phosphate) incorporation at a rate of 0.5% of applied urea (2.39 kg ha⁻¹, 220 kg N/the N content of urea (0.46) × 0.5%) (NPKI + M). The treatments were applied following a randomized design across three replicate field plots (4 m × 5 m). Plots of different treatments remained unchanged in the same locations for 4 years. Each year, the composted pig manure (213 g C kg⁻¹ and 22 g N kg⁻¹ based on dry weight on average, characteristics of pig manure was listed in Table S1) was broadcasted evenly onto the plots a few days before maize planting, and ploughed to a depth of 20 cm by machine (TG4, Huaxing, China). For the respective treatments, urea (220 kg N ha⁻¹ yr⁻¹), calcium superphosphate (110 kg P₂O₅ ha⁻¹ yr⁻¹), and potassium chloride (110 kg K₂O ha⁻¹ yr⁻¹) were applied on the same day as maize (Zea mays L.) was planted. The urea and inhibitor were fully mixed before application.

Maize (cultivar was Fuyou #9) was planted on 3rd May 2012, 3rd May 2013, 6th May 2014, and 10th May 2015, at a spacing of 37 cm and 60 cm between rows. No irrigation was applied throughout the experimental period. Maize was harvested on 13th September 2012, 29th September 2013, 29th September 2014, and 29th September 2015, respectively. At harvest, maize yield and aboveground biomass yield were measured by harvesting all plants (20 m²) in each plot. The straw and grain were removed after each harvest and the soil with about 5 cm maize stem was ploughed to a depth of approximately 20 cm in April each year.

Each cropping cycle, therefore, consisted of periods of maize (from May to September) and fallow (from October to April) of the following year.

The precipitation and air temperature data were acquired from the meteorological station of the Shenyang Agro-Ecological Station. The precipitation during the 2012/2013, 2013/2014, 2014/2015, and 2015/2016 periods were 911.9 mm, 621.7 mm, 485.7 mm, and 585.3 mm, respectively (Fig. 1). 72.3%, 75.5%, 66.5%, and 73.0% of these annual precipitations occurred during maize-growing period, respectively. The mean annual air temperatures in these years were 7.7 °C (− 21.2 to 27.5 °C), 8.1 °C (− 22.7 to 28.3 °C), 9.5 °C (− 21.7 to 28.2 °C) and 9.3 °C (− 17.1 to 27.0 °C), respectively. The soil temperature at a depth of 5 cm varied between − 14 and 35 °C during the four-year period (Fig. 2b). The change trend of soil surface temperature was the same as that of soil temperature at 5 cm depth (Fig. 2a). The mean soil WFPS (0–15 cm) varied between 15 and 73% (Fig. 2c).

Gas sampling and analysis

The gas was sampled between 3rd May 2012 and 14th April 2016 using a static closed chamber system as described by Dong et al.¹⁶. Briefly, a stainless-steel chamber base (56 cm length × 28 cm width) was inserted into the soil of each plot to a depth of approximately 10 cm, with its long edge perpendicular to the rows of maize. The top chamber (56 cm length × 28 cm width × 20 cm height) was also made of stainless steel. Gas samples were obtained using a syringe 0, 20, and 40 min after the chambers had been closed between 9:00 am and 11:00 am on each sampling day. Gas samples were collected every 2‒6 days and every 7‒15 days during the growing seasons and non-growing seasons, respectively. The first gas sampling time was on day 1, day 3, day 1, and day 3 after maize planting each year. The N₂O concentrations in gas samples were quantified using a gas chromatograph (Agilent 7890A, Shanghai, China) with an electron capture detector.

Soil analysis

The soil temperature and volumetric water content (SVWC) were measured at depth of 0–15 cm using a bent stem thermometer and a time-domain reflectometry (Zhongtian Devices Co. Ltd, China), respectively. SVWC was converted to soil water-filled pore space (WFPS) using the following equation:

where BD is soil bulk density (g cm⁻³). Particle density was assumed to be 2.65 g cm⁻³.

Soil samples from the 0–20 cm layer were collected in each plot in April 2012 (before sowing) and October 2015 (maize harvest) using a 5 cm diameter stainless steel soil sampler. The five soil samples collected from different locations in each plot were mixed thoroughly. Visible roots were removed by hand and the samples were air-dried and sieved using a 0.15 mm sieve. SOC was then quantified using an elemental analyzer (Vario EL III, Elementar, Germany). Soil available NH₄⁺–N and NO₃⁻–N were extracted with 2 M KCl and measured colorimetrically using a continuous flow injection analyzer (Futura, Alliance, France)¹⁷. Soil Olsen-P was extracted with NaHCO₃ and colorimetrically measured using a spectrophotometer (Lambda 2, PerkinElmer, USA). Soil available K was extracted by 1 M CH₃COONH₄ and analyzed with a flame photometer (FP640, Jingmi, China). Soil pH was determined with deionized water (1:2.5) and analyzed using a pH meter (PHS-3C, LeiCi, China) with a glass electrode.

DNA extraction and real-time quantitative PCR

The soil samples for measuring the abundance of nitrification and denitrification functional genes were collected on May 20, 2015. Soil DNA was extracted with the soil DNA extracted kits (EZNA soil DNA Kit; Omega Bio-Tek Inc., U.S.A.). The copy numbers of nitrification and denitrification functional genes were determined by q-PCR with the Roche LightCyler® 96 (Roche, Switzerland). Additional details about the primers and amplification procedure can be found in Dong et al.¹⁶.

Data analysis

The N₂O flux (μg N₂O–N m⁻² h⁻¹) is calculated based on the increase of N₂O concentration per unit chamber area for a specific time interval¹⁸ as follows:

where F (μg N₂O–N m⁻² h⁻¹) is the N₂O flux, T (◦C) is the air temperature in the chamber, M (g N₂O–N mol⁻¹) is the molecular weight of N₂O–N, 22.4 (L mol⁻¹) is the molecular volume of the gas at 101.325 kPa and 273 K, H (m) is the chamber height, dc/dt (ppb h⁻¹) is the rate of change in the N₂O concentration in the chamber.

Cumulative N₂O emissions were calculated as follows:

where F is the N₂O emission flux (μg N₂O–N m⁻² h⁻¹), i is the ith measurement, (t_i+1 − t_i) is the number of days between two adjacent measurements, and n is the total number of the measurements. Annual N₂O emissions were calculated between the fertilization dates of each successive year.

The SOC stock (Mg ha⁻¹) in the topsoil was calculated as:

where BD is soil bulk density (g cm⁻³), D is the depth of the topsoil (0.2 m).

The topsoil SOC sequestration rate (SOCSR) (Mg ha⁻¹ yr⁻¹) was estimated using the following equation:

where C_stock2015 and C_stock2012 are the SOC stocks in 2015 and 2012, respectively, and t is the duration of the experiment (years).

Statistical analyses were performed using SPSS 13.0 (SPSS, Chicago, USA). The differences in cumulative N₂O emissions and maize yields within a year, and other factors among treatments were assessed using one-way Analysis of Variance (ANOVA) with least significant difference post-hoc tests and a 95% confidence limit. The effects of different treatments, years, and their interactions on N₂O emission, maize yield and aboveground biomass were examined using one-way repeated measures ANOVA. Pearson correlation analysis was used to analyze the relationships between cumulative N₂O emissions and precipitation (N = 12 (three data each year, four years)), as well as N₂O flux and soil available nitrogen content.

Statements of research involving plants

It is stated that the current research on the plants comply with the relevant institutional, national, and international guidelines and legislation. It is also stated that the appropriate permissions have been taken wherever necessary, for collection of plant or seed specimens. It is also stated that the authors comply with the ‘IUCN Policy Statement on Research Involving Species at Risk of Extinction’ and the ‘Convention on the Trade in Endangered Species of Wild Fauna and Flora’.

Source: Ecology - nature.com