Site description
Experiments were carried out at the research fields of Embrapa Agrossilvipastoril (Sinop, MT, Brazil), which comprise an area of flat relief with a clay textured soil classified as Haplic Ferralsol (Dystric) (IUSS WRB 2022) as described by Viana et al. (2015). According to the Köppen classification system, the climate of the region is tropical Aw with precipitation concentrated during the spring/summer seasons (Alvares et al. 2013). The average daily air temperatures and accumulated rainfalls during the two study years (2017 and 2018) are shown in Table 1.The chemical characteristics of the 0 to 10 cm soil layer were: pH (H2O) 5.8, total organic C 2.12%, total organic N 0.15%, base sum (S) 2.6 cmolc dm− 3, and 42% base saturation in 2017; and pH (H2O) 5.48, total organic C 2.38%, total organic N 0.17%, S 3.2 cmolc dm− 3, and 43% base saturation in 2018. The average amounts of sand, silt and clay were 33, 10 and 57%, respectively.
Table 1
Climatic conditions during the off-season cultivation of maize in 2017 and 2018
Period of measurement
|
Mean daily air temperature (°C)
|
|
Accumulated rainfall (mm)
|
2017
|
2018
|
|
2017
|
2018
|
15 days after TDF
|
25.5
|
25.1
|
|
122
|
246
|
30 days after TDF
|
25.4
|
24.9
|
|
198
|
319
|
During entire period after TDF
|
25.1
|
24.5
|
|
320
|
355
|
During entire maize growing cycle
|
25.0
|
24.7
|
|
475
|
515
|
Abbreviation: TDF, top dressing fertilization
Crop configuration and experimental design
The experimental plots measured 25 m² (5 x 5 m). Seeds of Optimum® Leptra® hybrid maize with insect resistance traits (Corteva Agriscience, Indianapolis, IN, USA) were sown during the recommended period for the region (Pereira et al. 2018) with row spacing of 0.45 m and a seeding rate that provided a population of 66,667 plants ha− 1. Maize hybrid P3431VHY was cultivated in the first year (sowing date 3 March 2017) with base fertilization comprising 14 kg ha− 1 of N, 105 kg ha− 1 of P2O5 and 56 kg ha− 1 of K2O. Hybrid 2B810PW was planted in the second year (sowing date 16 February 2018) with base fertilization comprising 14 kg ha− 1 of N, 70 kg ha− 1 of P2O5 and 70 kg ha− 1 of K2O. In each of the two years, corn was cultivated immediately after the soybean harvest.
The randomized experimental block design consisted of seven treatments and four repetitions per treatment as follows: T1- soil cultivated with maize control; T2 - soil cultivated with NFB-inoculated maize; T3 - soil cultivated with maize and granulated urea for top dressing fertilization (TDF); T4 - soil cultivated with NFB-inoculated maize and granulated urea for TDF; T5 - soil cultivated with maize and granulated urea containing DCD (concentration of 5% on N basis) for TDF; T6 - soil cultivated with maize and granulated urea containing NBPT (530 ppm) for TDF; and T7 - soil cultivated with maize and granulated urea containing DCD and NBPT for TDF. A suspension of Azospirillum brasilense was used to inoculate seed at a dose of 100 mL ha− 1. TDF was applied at the rate of 120 kg ha− 1 urea (45% N) at the V6 growth stage of maize.
Measurement of N2O emissions
A rectangular static chamber, comprising a metal base and a polyethylene top (0.60 x 0.40 x 0.09 m; length, width, height) was deployed in the working area of each experimental plot. A three-way tap was attached at the center of the chamber top for gas sampling, while a plastic tube was installed on the side of the chamber to serve as a ventilation port (Parkin and Venterea 2010). Samples of headspace gases were extracted from the chamber using 20 mL syringes. Two samplings were performed before TDF, following which samples were collected at two day intervals for 16 days. At the end of this period, samplings were performed weekly for six consecutive weeks, then every two weeks until the end of the maize cycle. Gas collections were always carried out in the mornings, with samples being extracted at four consecutive 20 min intervals over a 60 min period between 08.00 and 11.00 h (Alves et al. 2012). The internal temperature of the chamber was monitored at the time of gas collection with the aid of a digital thermometer.
Air samples were transferred from the syringes to 20 mL evacuated glass vials and subsequently analyzed on a model GC-2014 chromatograph (Shimadzu, Tokyo, Japan) equipped with an electrical conductivity detector. The amounts of N2O present were established from calibration curves constructed with N2O standards of known concentrations (383, 808 and 2027 nmol mol− 1) analyzed using the same equipment and parameters as the samples. The results were used to adjust the linear model by relating the variations of gas concentrations in the chamber headspace as a function of time. Soil N2O fluxes were calculated according to Eq. (1) as proposed by Hutchinson and Livingston (1993):
$${\text{N}}_{2}\text{O} \text{f}\text{l}\text{u}\text{x} ({\mu }\text{g} \text{N}-{\text{N}}_{2}\text{O} {\text{m}}^{-2}{\text{h}}^{-1}\text{)}= \frac{dC}{dt} \times \frac{V}{A} \times \frac{m}{vm}$$
1
where dC/dt is the change in gas concentration in the chamber as a function of time, V is the volume of the chamber (L), A is the area of the chamber (m²), m is the molecular weight (g) and vm the molecular volume (L) of the gas. The results were used to calculate the cumulative N2O emissions over the evaluation period using the Newton-Cotes numerical integration method (Rochette et al. 2015).
EF values, which refer to the amount of N2O released from the soil in relation to the amount of N applied, were calculated according to Eq. (2) over a period of up to 30 days after TDF, during which time the emissions are influenced by the fertilizer (IPCC 2019).
$$EF \left(\%\right)= \frac{\text{k}\text{g} {\text{N}}_{2}\text{O}\text{-}\text{N} \text{f}\text{r}\text{o}\text{m} \text{f}\text{e}\text{r}\text{t}\text{i}\text{l}\text{i}\text{z}\text{e}\text{d} \text{s}\text{o}\text{i}\text{l} - \text{k}\text{g} {\text{N}}_{2}\text{O}\text{-}\text{N} \text{f}\text{r}\text{o}\text{m} \text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l} \text{s}\text{o}\text{i}\text{l}}{\text{k}\text{g} \text{a}\text{p}\text{p}\text{l}\text{i}\text{e}\text{d} \text{N} \text{x} 100}$$
2
Soil sampling and analysis
Soil samples were collected, with the aid of a clean Dutch auger, from the 0 to 10 cm layer at 4, 8, 12, 16, 25, 39, 53, 67 and 81 days after TDF in both study years (2017 and 2018). Each sample was divided into two portions, the first of which was used to obtain the mass of fine air-dried soil (FADS) and pH, while the second was stored at 5ºC until required for the determination of NO3− and NH4+ concentrations. Samples of FADS were obtained by drying soil at room temperature, crushing and passing through a 2 mm mesh sieve. FADS samples were mixed with deionized water at a ratio of 1:2.5 (m/v) and pH values determined by the potentiometric method (Teixeira et al. 2017).
Extraction of NO3− and NH4+ from soil samples was carried out by mixing 1 g of soil with 5 mL of KCl solution (1 mol L− 1) in a 15 mL Falcon tube, shaking the suspension for 60 min and centrifuging at 4,500 rpm for 5 min. An aliquot (1.5 mL) of the supernatant was transferred to a 2 mL microcentrifuge tube and centrifuged at 14,000 rpm for 5 min (Cantarella and Trivelin 2001; Li et al. 2012). The concentration of NO3− was determined by measuring the absorbences of the extract at 220 and 275 nm in a UV/VIS spectrophotometer (Miyazawa et al. 1985; Olsen 2008), and the net absorbance of the solution corrected for the presence of dissolved organic matter according to Eq. (3).
Anet = A220 nn – (2 x A275 nn) (3)
For the quantification of NH4+, soil extracts were diluted 1:1 (v/v) with deionized water and 100 µL aliquots transferred to a 96-well microplate together with 100 µL each of reagents A and B. solutions. Reagent A was prepared by mixing 0.125 g sodium hydroxide and 0.75 mL sodium hypochlorite in a 25 mL volumetric flask and completing to volume with deionized water. Reagent B was prepared by mixing 0.125 g sodium hydroxide and 2.5 g sodium salicylate with 0.01 g sodium nitroprusside in a 25 mL volumetric flask and completing to volume with deionized water. Reaction mixtures were left to stand for 25 min, after which the color intensity of the blue reaction product was measured at 650 nm using a microplate reader (Sattolo et al. 2016). The concentrations of NO3− and NH4+ were determined from the net or actual absorbance values, respectively, using calibration curves (absorbance vs. concentration) constructed using standard compounds.
Samples of undisturbed soil collected in 98 cm³ cylinders throughout both years of maize cultivation cycles were used to determine soil bulk density. For each sampling date, the gravimetric moisture content of each soil sample was determined by subjecting the samples to a forced-air oven until a constant weight was achieved. These variables, together with the presumed soil particle density of 2.54 g mL for the 0 to 10 cm layer (Nascimento et al. 2021), were used to calculate the water-filled pore space (WFPS) as described by Linn and Doran (1984).
Statistical analysis
According to the Shapiro-Wilk test, data relating to N2O fluxes did not follow a normal distribution even after log transformation, therefore the non-parametric test of mean standard error was applied (Alfaro et al. 2015). Cumulative N2O emission and EF values obtained during the two maize growing seasons were submitted to one-way analysis of variance (ANOVA) and the significance of the differences determined using the Tukey test at 5% probability. In order to show the relationship between N2O fluxes and the evaluated soil attributes, an XY plot was constructed. Principal component analysis (PCA) was performed the variability between the treatments as a function of soil attributes and N2O fluxes during the first 30 days after TDF. For all statistical analysis were used R-studio software (R Development Core Team, 2010).