Effects of combined nitrification inhibitors on soil nitrification, maize yield and nitrogen use efficiency in three agricultural soils.

Application of nitrification inhibitors (NIs) with nitrogen (N) fertilizer is one of the most efficient ways to improve nitrogen use efficiency (NUE). To fully understand the efficiency of NIs with N fertilizer on soil nitrification, yield and NUE of maize (Zea mays L.), an outdoor pot experiment with different NIs in three soils with different pH was conducted. Five treatments were established: no fertilizer (Control); ammonium sulfate (AS); ammonium sulfate + 3, 4-dimethyl-pyrazolate phosphate (DMPP) (AD); ammonium sulfate + nitrogen protectant (N-GD) (AN); ammonium sulfate + 3, 4-dimethyl-pyrazolate phosphate + nitrogen protectant (ADN). The results showed that NIs treatments (AD, AN and ADN) significantly reduced soil nitrification in the brown and red soil, especially in AD and ADN, which decreased apparent nitrification rate by 28% - 44% (P < 0.05). All NIs treatments significantly increased yield and NUE of maize in three soils, especially ADN in the cinnamon soil and AD in the red soil were more efficiency, which significantly increased maize yield and apparent nitrogen recovery by 5.07 and 6.81 times, 4.39 and 8.16 times, respectively. No significant difference on maize yield was found in the brown soil, but AN significantly increased apparent nitrogen recovery by 70%. Given that the effect of NIs on both soil nitrification and NUE of maize, DMPP+N-GD was more efficient in the cinnamon soil, while N-GD and DMPP was the most efficiency in the brown and red soil, respectively. In addition, soil pH and soil organic matter play important role in the efficiency of NIs.

Introduction

Nitrogen (N) fertilizers can increase food production by almost 50%. Hence, tons of N fertilizers were applied to obtain high yield of grain, which made a significant contribution in alleviating the global food shortage [1]. However, plants were rarely able to absorb more than 50% of the N fertilizer applied to cropping systems, and most of them were lost through NO3- leaching, NH3 volatilization and N2O emission due to the excessive application of N fertilizers, which caused many problems, such as lower nitrogen use efficiency (NUE), economical loss, and environment pollution [2]. It is necessary, therefore, to find out an efficient way to improve NUE, increase crop yield and mitigate environmental pollution.

Adding nitrification inhibitors (NIs) into ammonium-based fertilizer is one of the considerably effective technologies that can inhibit nitrification, reduce N loss, thus improving crop yield and NUE in agricultural systems [3,4]. Nitrification is a major process impacting N cycling in the high-production agricultural systems [5]. NIs are compounds that delay the process of nitrification of NH4+ to NO3- and subsequent by depressing the activities of nitrifiers and denitrifiers in soil [6]. Nitrogen protectant (N-GD) is a mixture of DCD and DMPP in a certain proportion. Two commonly used NIs are 3,4-dimethylpyrazol phosphate (DMPP) and dicyandiamide (DCD). Many studies documented that the application of DMPP or DCD with organic fertilizer (cattle slurry, cow urine) and inorganic fertilizer (urea, ammonium chloride, ammonium sulfate) effectively reduced N loss and improved crop and grass yield and NUE in both agricultural and pasture system [7-11]. The application of DCD with urea significantly improved NUE by 12.9% and 14.6%, where reduced NO3- leaching by 58.5% and 35.2% and N2O emission factor by 83.8% and 72.7% in two soils (Huangzongrang and chaotu soils), respectively [3]. Fan et al found that the application of NIs with urea increased ammonium concentrations by 0.3% - 41.1% and decreased nitrate concentrations by 6.3% - 34.4% [12]. However, there has been little research on the effect of NIs combined with ammonium sulfate in soils. It has been reported that sulfate (SO4-) is possibly reduced to thiosulfate (S2O32-), which is a nitrification inhibitor [13]. Moreover, the synergistic effect of anions and NIs might improve the inhibition effect of NIs [14]. In addition, DCD was more widely used in some countries especially in New Zealand as it was less volatile, easily souble in water and relatively cheaper than other NIs [15]. While DMPP has lower application of one-tenth of DCD dose, and it also has lower phytotoxicity to the plant [16,17]. DMPP is indiscriminately binding with ammonium monooxygenase, while DCD is blocking the electron transport in the cytochromes, which deactivate the enzyme responsible for the first step of nitrification [18]. Therefore, the present study addresses a combined application of different kinds of NIs to refine the use of NIs in soils.

The main factors affecting the effect of NIs are the properties of NIs, soil physicochemical properties (soil organic carbon (SOC), pH), fertilizer types (organic or inorganic), and field management [19,20]. It is worth to mention that soil pH is one of the main factors to influence the soil nitrification and the efficiency of NIs. In general, nitrification is easy in soils of pH ≥ 6.0, but not in soils of pH ≤ 5.0 [19]. A laboratory incubation experiment indicated that DMPP with ammonium chloride was more effective in inhibiting nitrification in the neutral soil (pH: 7.0; 93.5%) than in the alkaline soil (pH: 8.0; 85.1%) and acid soil (pH: 4.6; 70.5%) [21]. Another laboratory incubation experiment had shown that DMPP with urea maintained higher NH4+-N concentrations and lower NO3–-N concentrations, thus reducing N loss and improving NUE in the brown soil (pH: 6.31) [22]. While a field experiment study reported that DCD and urease inhibitor with urea had no significant difference in NH4+-N or NO3–-N in an acid soil, but in grain yield significantly higher than those of control treatment, and no significant difference in grain yield was found between NIs and without NIs [23]. Another field experiment showed that DMPP significantly increased the annual crop yield by 6% relative to the urea treatment in an alkaline soil [24]. A meta—analysis showed that both DCD and DMPP were effective in increasing soil NH4+-N content combined with ammonium sulfate (AS), urea or organic fertilizer. DMPP was also effective in increasing NH4+-N content and decreasing NO3—N when combined with AS [25]. Many previous studies focused on the effect of NIs alone [26-28], and the responses of different NIs on yield and NUE are various due to the different soils. Therefore, it is urgent to fully understand the effect of different NIs on soil nitrification, yield and NUE of maize in different soils to provide theoretical basis for choosing better NIs. In addition, this is the first research to study the effect of N-GD in different soils.

In this study, an outdoor pot experiment with different types of NIs additions in the above three soils was conducted. The objectives of this study were: 1) to examine the effect of NIs and their combination with ammonium sulfate on soil nitrification in three soils with different pH; 2) to compare the effect of NIs and their combinations in three soils with different pH; 3) to identify the effect of NIs on maize yield and NUE. We hypothesized that the combinations of NIs would be more efficient in three soils with different pH soil and soil pH may be the main factor affecting the effect of nitrification inhibitors.

Materials and methods

Study site and soils

An outdoor pot experiment was carried out at the National Field Observation and Research Station of Agroecosystems in Shenyang, Liaoning province (41˚31’N, 123˚24’E), in which Dongdan-6531 spring maize (Zea mays L., from May to October, 2018) was planted. The mean annual air temperature is 7–8°C, and the mean annual precipitation is approximately 700 mm. The frost-free period is 147–164 days. The soils used in this study were obtained from three sites: a brown soil (Hap-Udic Luvisol in the FAO (Food and Agriculture Organization of the United Nations) WRB (World Reference Base) classification system) at Changtu country (42°25’N, 123°28’E), Liaoning Province of Northeast China, a cinnamon soil (Hap-Ustic Luvisol in the FAO WRB system) at Chaoyang City (41°49’N, 122°48’E), Liaoning Province of Northeast China, and a red soil (Haplic Luvisol in the FAO WRB system) at Yingtan National Agro-ecosystem Field Experiment Station (28°15′N, 116°55′E) of the Chinese Academy of Sciences in Jiangxi Province, China. Brown soil, cinnamon soil and red soil are the typical agricultural soils with different pH in China. The sampling sites were planted with maize and regularly fertilized. At each site, surface soil (0–20 cm) was collected, sieved to pass through a 5-mm mesh, and homogenized thoroughly. Soil physicochemical properties are shown in Table 1.

Table 1

Physicochemical properties of the three agricultural soils (0–20 cm soil layers).

Soil property	Cinnamon soil	Brown soil	Red soil
pH	7.90 ± 0.08	5.30 ± 0.02	4.70 ± 0.83
Total C(g kg-1)	12.30 ± 0.20	9.24 ± 0.02	6.86 ± 0.35
Total N(g kg-1)	1.07 ± 0.03	0.98 ± 0.08	0.89 ± 0.14
NH4+-N(mg kg-1)	16.70 ± 0.13	13.83 ± 0.71	13.20 ± 0.83
NO3--N(mg kg-1)	21.28 ± 0.28	10.34 ± 0.25	11.53 ± 0.98
Available P(mg kg-1)	5.70 ± 0.63	11.73 ± 1.04	13.70 ± 1.41
Available K(mg kg-1)	91.32 ± 2.77	61.87 ± 2.89	131.49 ± 7.13
SOM (g kg-1)	21.21 ± 0.34	15.94 ± 0.03	11.83 ± 0.60

Values indicate mean ± standard deviations (n = 3). Total C, total soil carbon; Total N, total soil nitrogen; NH4+-N, ammonium nitrogen; NO3--N, nitrate nitrogen; P, phosphorus; K, potassium; SOM: Soil organic matter.

Experimental design

Five treatments were established: 1) no N fertilizer (control); 2) ammonium sulfate (AS); 3) AS+3, 4-dimethyl-pyrazolate phosphate (DMPP) (AD); 4) AS+nitrogen protectant (N-GD) (AN); 5) AS+DMPP+N-GD (ADN). Each treatment had six replications. The fertilizer ammonium sulfate, triple superphosphate and potassium chloride were applied with an application rate of 0.3g N, 0.12 g P2O5 and 0.15 g K2O per kg soil, respectively. The application rates of DMPP and N-GD were 0.5% and 1.5%, respectively on the w/w basis of N, and the application rate of every single NI was reduced by 50% in NIs combinations treatments. All the amendments were basal dressed, and air-dried soil (equivalent to 8 kg of oven-dry weight) was thoroughly mixed with the corresponding amendments before added a plastic pot of a 28-cm diameter with a 26-cm height. Soil moisture content was adjusted with deionized water to 60% of the maximum water-holding capacity (WHC), and watering was carried out every day during the maize growth period. Five seeds were sown in each pot, and the seedlings were thinned to one per pot after germination and seedling establishment. DMPP and N-GD were supplied by Zhejiang Chemical Institute, China and Spanish corporation, respectively.

Sampling of soil and plant

Soil samples were collected at the seedling stage, elongation stage, filling stage, and maturity stage of maize, i.e. 30, 59, 95 and 135 days after planting, respectively, with three replicates for each treatment. For each pot, five soil cores were collected by using a soil auger (2.5 cm in diameter), and bulked into a composite sample. The samples were packed with ice pack and transported to laboratory, and passed through a 2 mm sieve before determining inorganic N (NH4+-N and NO3--N) and moisture content.

The whole plant was harvested at the mature stage of maize from another three replications, and separated into straw and grain. The plant will be placed in oven, dried to constant weight at 65°C to calculate aboveground biomass of maize, then ground to pass through a 0.25 mm sieve for the analysis of total nitrogen (TN).

Analytical methods

Soil pH was tested in 1:2.5 soil-water ratio by using potentiometric method with a pH meter (METTLER TOLEDO S200, Shanghai, China), and TN of maize was determined by dry combustion using an elemental analyzer (Vario EL III, Germany) [29]. Soil available phosphorus (AP) was extracted with 0.5 mol L-1 NaHCO3 and analyzed by the molybdenum blue method [30], soil available potassium (AK) was extracted with 1 mol L-1 NH4OAc and determined by the flame photometry method [30]. The soil NH4+-N and NO3--N content were determined by extracting a 5-g soil subsamples with 50 ml of 2 mol L-1 potassium chloride (KCl) and the samples were shaken for 1 h on a reciprocal shaker, filtered and the extract analyzed on a Continuous Flow Analyzer (AA III, Germany) [31].

Calculations and statistical analysis

The soil apparent nitrification rate (ANR, %) is usually used to characterize the intensity of soil nitrification, which was calculated using Eq (1) [28]:

Agronomic nitrogen use efficiency (ANUE, g g-1) and apparent nitrogen recovery (AR, %) were calculated by Eq (2) and Eq (3): Where: Y, grain yield with N fertilizer; YC, grain yield with no fertilizer; NF, the amount of N fertilizer applied; U, plant nitrogen uptake in the aboveground parts with N fertilizer; UC, plant nitrogen uptake in the above-ground parts with no N fertilizer [32].

A three -way ANOVA was used to analyse the impact of soil types (S), nitrification inhibitor (NI) and days after planting (D) and their interactions ((S*NI), (S*D), (NI*D) and (S*NI*D)) on the content of NH4+-N, NO3--N and apparent nitrification rate (ANR). A two-way ANOVA was used to analyze the effects of soil types (S), nitrification inhibitor (NI) and their interactions (S*NI) on grain yield, ANUE and AR at the maturity stage of maize. Multiple comparisons among the treatments were further explained using Duncan test. Differences were considered significant at P < 0.05. All statistical analyses were performed using SPSS Version 22.0. Graphs were prepared with Origin 9.0.

Results

The contents of NH4+-N and NO3--N in soils

The contents of NH4+-N and NO3--N were significantly affected by soil types, nitrification inhibitor and days after planting (Table 2, P < 0.05). The application of N fertilizer and NIs significantly increased the soil inorganic N content in all three agricultural soils (Fig 1). However, there was no significant differences in soil NH4+-N content of all treatments at 135 days after maize planting, which decreased about 11 mg kg-1, 10 mg kg-1 and 24 mg kg-1 in the cinnamon soil, brown soil and red soil, respectively (Fig 1A, 1C and 1E, P > 0.05). There was the same pattern between of NO3--N content with the NH4+-N content, which increased at 30 days after maize planting and gradually declined followed the growth of maize (Fig 1B, 1D and 1F). In the cinnamon soil, the addition of NIs significantly increased soil inorganic N expect for AN. AD maintained higher NH4+-N content for a long time, while ADN had higher NO3--N content during the growth of maize (Fig 1A, P < 0.05). In addition, ADN had higher inorganic nitrogen than AN (Fig 1B).

Table 2

Three-way ANOVA (P < 0.05) of soil types (S), nitrification inhibitor (NI) and days after planting (D) and their interactions ((S*NI), (S*D), (NI*D) and (S*NI*D)) on the content of NH4+-N, NO3—N and apparent nitrification rate (ANR).

Factors	DF	NH4+-N			NO3--N			ANR
		SS	F	p	SS	F	P	SS	F	p
S	2	13347.4	4094.4	***	1037.9	1540.3	***	14333.3	1465.1	***
NI	3	1073.1	219.5	***	49.6	49.0	***	74.5	5.1	**
D	3	30164.2	6168.7	***	9501.7	9400.4	***	8221.6	560.3	***
S*NI	6	1088.5	111.3	***	283.2	140.1	***	874.9	29.8	***
S*D	6	9895.4	1011.8	***	665.2	329.0	***	3300.3	112.4	***
NI*D	9	1555.7	106.1	***	190.4	62.8	***	700.0	15.9	***
S*NI*D	18	1243.7	42.4	***	734.3	121.1	***	1341.6	15.2	***
Model	47	58368.1	761.9	***	12462.2	787.0	***	28846.2	125.5	***
Error	96	156.5			32.3			469.6

SS, the sum of squares.

F value, the ratio of mean squares of two independents samples.

*** Indicates significance at P < 0.001

** Indicates significance at P < 0.01.

Fig 1

Dynamic changes of NH4+-N and NO3−-N of different treatments in three soils.

Treatment: Control, no fertilizer and nitrification inhibitors; AS, ammonium sulphate; AD, AS+3, 4-dimethylpyrazole phosphate (DMPP); AN, AS+nitrogen protectant (N-GD); ADN, AS+3, 4-dimethylpyrazole phosphate (DMPP)+nitrogen protectant (N-GD). Error bars represented standard deviations (n = 3).

In the brown soil, the NH4+-N content in AD was the highest than that in other treatments at30 days after maize planting (Fig 1C, P < 0.05). No significant differences were detected between AN and ADN for NH4+-N content (Fig 1C, P > 0.05). At 59 days after maize planting, the NH4+-N content in AD and AN was higher compared with ADN (Fig 1C). AN also maintained higher NH4+-N content until the 135 days after maize planting. All treatments with NIs significantly decreased NO3--N content at 30 days after maize planting, especially AN was lower in NO3--N content at the later stages (Fig 1D). Moreover, AN and ADN maintained higher nitrification inhibition during the four sampling periods (S1 Table).

In the red soil, all treatments with NIs significantly increased NH4+-N content compared with AS at 30 days after maize planting (Fig 1E, P < 0.05). In addition, ADN maintained higher NH4+-N content for a long period. Lower NO3- -N values in AD and ADN were found compared with AS, while ADN had no significant difference in NO3- -N content with AS (Fig 1F, P >0.05).

Apparent nitrification rate

Apparent nitrification rate (ANR) indicates the intensity of soil nitrification process. The lower the value is, the weaker the inhibition intensity of nitrification inhibitors on soil nitrification process is; the higher the value is, the higher the nitrification process intensity is. Soil types, nitrification inhibitor and days after planting significantly affected ANR (Table 2, P < 0.05). ANR was higher in the cinnamon soil than that in the other two soils (Fig 2). In the cinnamon soil, higher ANR was observed in treatments of adding NIs into N fertilizer, especially in ADN, which showed that NIs significantly increased soil inorganic nitrogen (Fig 2A). In the brown soil, all treatments with NIs significantly reduced ANR over AS during the whole stage of maize growth. AD was the lowest at 30 days after maize planting, followed by AN (Fig 2B). In the red soil, the lowest values were observed in both AD and ADN (Fig 2C, P < 0.05). In addition, ADN significantly inhibited nitrification during the whole growth stage of maize (Fig 2C).

Fig 2

Changes of apparent nitrification rate of three soils during four sampling periods.

Treatment: AS, ammonium sulphate; AD, AS+3, 4-dimethylpyrazole phosphate (DMPP); AN, AS+nitrogen protectant (N-GD); ADN, AS+3, 4-dimethylpyrazole phosphate (DMPP)+nitrogen protectant (N-GD). Error bars represented standard deviations (n = 3).

Aboveground biomass and nitrogen uptake of maize

Aboveground biomass

In general, NIs with ammonium sulfate significantly increased aboveground biomass compared to AS in all the tested soils (Fig 3, P < 0.05). Grain yield of maize was significantly affected by both soil types and nitrification inhibitor (Table 3, P < 0.01). Both grain and straw biomass had the highest value in the brown soil than those in the cinnamon soil and red soil (Fig 3). In the cinnamon soil, the highest value of grain yield was found in ADN than that in other treatments (Fig 3A, P < 0.05). AN resulted in significant increase in grain yield when compared to AS (P < 0.05), while there was no significant difference between AD and AS for grain yield of maize (P > 0.05).

Fig 3

Aboveground biomass of maize under different treatments in three soils.

Treatment: AS, ammonium sulphate; AD, AS+3, 4-dimethylpyrazole phosphate (DMPP); AN, AS+nitrogen protectant (N-GD); ADN, AS+3, 4-dimethylpyrazole phosphate (DMPP)+nitrogen protectant (N-GD). Error bars represented standard deviations (n = 3). Different letters indicate significant differences between different treatments at P < 0.05 by Duncan test.

Table 3

Two-way ANOVA (P < 0.05) of soil types (S) and nitrification inhibitor (NI) on grain yield, agronomic nitrogen use efficiency (ANUE) and apparent nitrogen recovery (AR) of maize.

Factors	DF	Grain yield			ANUE			AR
		SS	F	p	SS	F	p	SS	F	p
S	2	22787.7	201.8	***	3955.7	201.8	***	1274.2	31.8	***
NI	3	12958.1	76.5	***	2250.0	76.5	***	7682.6	127.8	***
S*NI	6	32174.4	95.0	***	5585.7	95.0	***	13024.2	108.4	***
Model	11	67920.2	109.3	***	11791.4	109.3	***	21981.0	99.8	***
Error	24	1355.4			235.3			480.8

SS, the sum of squares.

F value, the ratio of mean squares of two independents samples.

*** Indicates significance at P < 0.001.

In the brown soil, grain yield was the highest in AN followed by AD and AN, which were significantly higher than AS (Fig 3B, P < 0.05). No significant differences were detected in the application of NIs with ammonium sulfate (Fig 3B, P > 0.05).

In the red soil, the grain yield in NIs treatments was higher compared with AS (Fig 3C, P < 0.05). The highest value was found in AD in comparison to AN and ADN (Fig 3C).

Nitrogen uptake of maize

In the three agricultural soils, all treatments with NIs significantly increased N uptake by maize compared with control (Fig 4, P < 0.05). The highest value of grain and total nitrogen uptake of maize was found in the cinnamon soil (Fig 4). In the cinnamon soil, the highest N uptake of maize value was detected in ADN, which was significantly higher than other treatments (Fig 4A, P < 0.05). AN had higher N uptake of maize than that of AS, while AD had no significant difference from AS (Fig 4A, P < 0.05).

Fig 4

Nitrogen uptake of maize under different treatments in three soils.

Treatment: AS, ammonium sulphate; AD, AS+3, 4-dimethylpyrazole phosphate (DMPP); AN, AS+nitrogen protectant (N-GD); ADN, AS+3, 4-dimethylpyrazole phosphate (DMPP)+nitrogen protectant (N-GD). Error bars represented standard deviations (n = 3). Different letters indicate significant differences between different treatments at P < 0.05 by Duncan test.

In the brown soil, there was no significant difference between AD and AN, which were significantly higher in N uptake of maize than other treatments (Fig 4B, P < 0.05). N uptake of maize from ADN was no significantly different from AS (Fig 4B, P > 0.05).

In the red soil, NIs with N fertilizer significantly increased N uptake of maize compared with AS. AD significantly increased N uptake of maize compared with other treatments (Fig 4C, P < 0.05), while ADN was better than AN (Fig 4C).

Agronomic nitrogen use efficiency and recovery of apparent nitrogen

Agronomic nitrogen use efficiency (ANUE) and apparent nitrogen recovery (AR) are different aspects to explain the utilization of nitrogen fertilizer in maize. Soil types and nitrification inhibitor significantly affected ANUE and AR (Table 3, P < 0.05). ANUE was the highest in the brown soil, while the highest value of AR was found in the red soil (Fig 5). In the cinnamon soil, the highest values in both ANUE and AR were from ADN, which was significantly different from other treatments. AN was higher in NUE than AD and AS. AD in AR was significantly higher than AS, while ANUE from AD had no significant differences from AS treatment (Fig 5A, P > 0.05).

Fig 5

Nitrogen use efficiency (ANUE and AR) under different treatments in three soils.

ANUE, agronomic nitrogen use efficiency; AR: Apparent nitrogen recovery. Treatment: AS, ammonium sulphate; AD, AS+3, 4-dimethylpyrazole phosphate (DMPP); AN, AS+nitrogen protectant (N-GD); ADN, AS+3, 4-dimethylpyrazole phosphate (DMPP)+nitrogen protectant (N-GD). Error bars represented standard deviations (n = 3). Different letters indicate significant differences between different treatments at P < 0.05 by Duncan test.

In the brown soil, all treatments with NIs significantly improved ANUE and AR than those of AS (Fig 5B, P < 0.05), while there were no significant differences among them in ANUE. AR in AD and AN was significantly higher compared with ADN and AS. No significant differences in AR were detected between ADN and AS (Fig 5B, P > 0.05).

In the red soil, AD had the highest value in both ANUE and AR among all treatments, which was significantly different with the other treatments (Fig 5C, P < 0.05). No significant differences in ANUE were found between AN and ADN (Fig 5C, P > 0.05), while higher value in AR was found in ADN than AN.

Discussion

Effects of NIs on soil nitrification

The application of NIs have the potential to slow soil nitrification, thus increasing NH4+-N and decreasing NO3--N content, and improving crop yield, aboveground biomass, N uptake and NUE. It is worth to mention that nitrification in the red soil has become stronger after tillage in recent decades, which is according with Lu et al [33]. In the present study, NIs treatments considerably reduced soil nitrification for all three soils, especially in both brown soil and red soil (S1 Table), a result which was mainly driven by lower pH and fertility. The efficacy of NIs in inhibiting nitrification was reduced because of the rapid hydrolysis of NIs at high soil pH [34].

The effect of NIs varied among different types of soil. NIs significantly increased soil NH4+ in both soils expect for cinnamon soil, which is consistent with Guo et al [35], who found that DCD with urine had the potential to increase NH4+ content and maintained it higher for a long time. However, NIs reduced ANR in all three soils, which is in line with Gong et al [36], who found that DCD and DMPP with urea significantly suppressed potential nitrification rate. DMPP significantly inhibited nitrification in all three soils. DMPP is indiscriminately binding and interaction with ammonium monooxygenase to inhibit the first rate-limiting step of soil nitrification [37]. In addition, DMPP is a heterocyclic N compound with the advantages of low mobility, slow biodegradation and persistence [38]. At the later stage of maize, NH4+-N content declined gradually, which is in line with Zaman et al [9]. The main reasons were the soil nitrification (Fig 1) (decomposition of NIs at the later stage of maize) [9], N uptake of maize (Fig 4) [36] and microbial immobilization [39,40].

Effects of NIs on maize yield and NUE

There are many studies to test the effect of NIs on crops production and N uptake, but it is difficult to draw general conclusions because the performance of the options varied across sites due to soil type (pH, SOM), NIs type, the forms of N fertilizer (organic N fertilizer (urine), inorganic N fertilizer (urea, ammonium sulfate et al) and so on [18]. In the current research, NIs significantly increased yield and N uptake of maize compared with the application of ammonium sulfate among the three soils, especially DMPP and N-GD in the brown soil, DMPP+N-GD in the cinnamon soil and DMPP in the red soil. Cui et al also found that DCD with urea significantly increased the yield of radish and N concentrations by 10.4%–36.2% in vegetables [3]. A field experiment also showed that the application of DMPP increased the grain yield of rice and rape by 4.2%–4.7% and 6.6%–7.5%, respectively [41], which is in line with the present study. NIs can maintain higher NH4+-N content for a longer period, which improve crop more available N absorption [42]. Ammonium ions are usually adsorbed by clay particles or soil organic matter, thus decreasing N loss [25]. Moreover, NIs with N fertilizer can increase the synchrony between N supplied and crop demand [43]. However, the results of Huérfano et al and Guardia et al showed that there was no any effect of NIs on maize yield [44,45], which is in contrast to the results. The possible explanation is the effect of NIs mainly due to environmental factors (pot, field) and the type of fertilizer (ammonia sulfate, ammonia sulphate nitrate, calcium ammonium nitrate) [46]. The higher values of plant N uptake imply higher NUE for NIs. Obviously, NIs also significantly improved NUE in this study. The results are agreement with He et al [47], which also found NIs with urea increased NUE.

In terms of cost, the price of DMPP is so expensive that its wide application is limited [25]. In the present study, DMPP had the similar efficiency with N-GD in the brown soil, while in the cinnamon soil DMPP+N-GD was more effective than DMPP alone, thus the best choice in the brown soil and cinnamon soil is the application of N-GD and DMPP+N-GD, respectively.

Effects of soil properties on NIs use

The efficiency of NIs differed in different agricultural soils may be attributed to the differences in physicochemical properties of three tested soils, especially soil pH and SOM. The results of Pearson correlation revealed that soil pH and SOM were negatively correlated with soil NH4+-N and NO3--N contents (Table 4, P < 0.05). The results confirmed our hypothesis that soil pH was the main factor influenccing the effect of nitrification inhibitors. Soil pH has been considered as one of the most important factors affecting the availability of NIs, because pH has potential to impact the mobility and degradation rate of the NIs in soils [48]. A meta—analysis showed that acidic soils (pH ≤ 6) showed a higher positive response to inhibitor application than neutral (pH 6–8) and alkaline soils (pH ≥ 8) [49]. The efficiency of DMPP was more stable in lower pH soil than that in alkaline soil. DMPP also performed better in neutral than in alkaline soil [21], which is similar to our results. It may be due to that the microbial activity in soils with higher pH is generally higher, which will accelerate the degradation rate of nitrification inhibitors. Additionally, the ANR in lower pH soils was lower than in alkaline soils, which is in accordance with the result of Lu et al [32]. In addition to soil pH, SOM also affected the effectiveness of NIs. SOM can absorb NIs and provide energy source for the microorganisms, which leads to the degradation of NIs, decreases the ability of NIs to inhibit nitrification [50]. The results of this paper are similarly to that of the literature, SOM was higher in the cinnamon soil than that in both brown and red soil, so the efficiency of DMPP in the cinnamon soil was lower than in the other two soils. While the effect of DMPP+N-GD had little influenced by SOM.

Table 4

Pearson correlation between soil pH, soil organic matter (SOM), NH4+-N and NO3—N.

Item	NH4+-N	NO3--N
pH	-0.175	-0.563**
SOM	-0.415*	-0.362*
NH4+-N	1	-0.518**

* Correlation is significant at the 0.05 level (two-tailed)

** Correlation is significant at the 0.01 level (two-tailed).

In addition, there is less study of N-GD, so more research should be required to understand its effect and mechanism. Moreover, the effect of NIs on the N cycle in a range of soil types, cultivated vegetation types, and climatic condition should be fully elucidated.

Conclusions

Nitrification inhibitors (NIs) with ammonium sulfate can significantly influence soil inorganic nitrogen, improve yield and nitrogen use efficiency (NUE) of maize. NIs combination 3, 4-dimethyl-pyrazolate phosphate (DMPP) + nitrogen protectant (N-GD) was the best way to increase soil available nitrogen and improve NUE in the cinnamon soil, while N-GD was more efficient in decreasing soil nitrification, improving yield and NUE of maize in the brown soil, DMPP was the most efficient in lowering soil nitrification and increasing maize yield and NUE in the red soil. Additionally, soil pH and soil organic matter are the main factors affecting the efficiency of NIs.

Nitrification inhibition rate of different treatments in three agricultural soils during four sampling periods.

(DOCX)

Click here for additional data file.

16 Jul 2021

PONE-D-21-09541

Effects of combined nitrification inhibitors on soil nitrification, maize yield and nitrogen use efficiency in three agricultural soils

PLOS ONE

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

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**********

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Reviewer #4: No

**********

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Reviewer #1: This manuscript described the effects of different nitrification inhibitors on soil nitrification and maize yield in various agricultural soils with different physicochemical properties. The subject is interesting and the paper is well organized. The introduction gave a satisfactory literature survey. The research methods used in this paper were described in detail and the data was well statistically analyzed. Appropriate figures were given to make the paper understood easily.

From my point of view, the work is well-done and provides theoretical basis to utilize fertilizer nitrogen effectively on maize in three soils with different nitrification inhibitors. The paper conclusions provided critical support for the theory that different nitrification inhibitors have different effects in different physical and chemical properties of soils. In addition, it seeks to find an economical way to improve NUE with new nitrification inhibitor N-GD due to higher price of DMPP. The results also showed that soil pH plays an important role in the effectiveness of DMPP, N-GD and their combination in influencing soil nitrification, yield and NUE of maize. Therefore, this article deserves to be published.

Some minor modifications should be made before publication:

1. In this manuscript, "decrease nitrification" should be revised as “inhibit/suppress nitrification".

2. Page 2, line 24, please revise “The application” to “ Application”.

3. Page 2, line 25, please revise “the most efficiency” to “the most efficient”.

4. Page 2, line 38, please add “that” after “than”.

5. Page 3, line 59, please delete “which”.

6. Page 4, line 78, please add “and” in front of “field”.

7. Page 4, line 82, please add parentheses.

8. Page 5, line 90, please delete “are”.

9. Page 7, line 139, please revise “sampling” to “samples”.

10. Page 11, line 216, Page 16, line 332, please revise “was” to “were”.

11. Page 17, line 341, please add “of maize” after “NUE”.

12. Page 17, line 343, “improving” should be revised to “improve”.

Reviewer #2: To understand the efficiency of nitrification inhibitors with N fertilizer on soil nitrification, yield and nitrogen use efficiency of maize (Zea mays L.), an outdoor pot experiment with different nitrification inhibitors in three soils with different pH were conducted in the paper. The reviewer think that the problems of this paper are as follows:

1.There are some sentences with irregular grammar or incomplete meaning: Such as:

(1)L39:”DMPP and N-GD were more effective in the red soil than in the cinnamon soil”What is more effective?

(2)L40 and 41:“… than other two soils, … than other two soils” were changed to“…than that in other two soils,… than that in other two soils.”

(3)L169:“NH4+-N content and NO3--N content” should be changed to“Contents of NH4+-N and NO3—N in soils”.

(4)L197:“Apparent nitrification rate (ANR) indicates that intensify of soil nitrification”, the sentence is not a clause, it should be changed to “Apparent nitrification rate (ANR) indicates the intensify of soil nitrification” .

(5)L253:“Agronomy nitrogen use efficiency and apparent nitrogen recovery” should be changed to“Utilization efficiency of agronomy ang recovery of apparent nitrogen”.

(6) L327:“was more stable in lower pH soil than in alkaline soil” should be changed to“was more stable in lower pH soil than that in alkaline soil”.

2.Improper expression or incomplete meaning of some section titles:

(1)L146:“Analytical procedures” should be changed to “Analytical methods” (Only the determination methods are given, and there are no determination procedures or operation processes)

(2)Note: To distinguish the relationship between the instrument and the method, the instrument only is the means of the method, but the instrument itself is not the method. Such as: “with a pH meter” in L147, “with an elemental analyzer” in L148, “determined by the flame photometer method” in L150-151, and “using a Continuous Flow Analyzer” in L151-152. Above all, these sentences should be expressed in the form of “methods” rather than “instruments”, especially, “determined by the flame photometer method” in L150-151 was a wrong expression, should be changed to “determined by the flame photometry method”.

(3) The meaning of “Apparent nitrification rate”in L196 was too general.

3.Some tense usageswere not correct,the past tense should be used in many sentences but used the present tense.

4.In the description of result and content discussion, the first person is used too much. Such as: "we conduct an outdoor.." in L99, “We clearly observed that…..” in L170(Figure 1 should be described first), “We also found that…” in L179, “we clearly observed……” in L340(It is more appropriate to have such a statement in the conclusion). It is suggested that the result or content described in the first person should be described in the passive voice.

5.“In our study” is used too often, which is to discuss the results of this paper, so it is unnecessary to emphasize it many times.

6. The expression of this comparison "The results was similarly to our study,....."in L332 is improper, it should be expressed "The results of this paper was similarly to that of the literature,.....".

7.The part of “conclusion” is an objective statement of the law presented by the research results, The expression of “In our study, we clearly observed” should be deleted.

8.The logical relationship between several experimental results in the result part is not very clear, moreover, the logical relationship between the results and the discussion is also fuzzy.

Reviewer #3: Opinion on manuscript: PONE-D-21-09541 major revision

Global food security is a key challenge of the modern world. The demand for food is growing exponentially and in this regard agricultural production has to be increased to meet global food demands. One of the key agricultural practices is the higher application of nitrogenous fertilizers. However, the use efficiency of applied fertilizers is currently very low. This low efficiency is attributed to the great losses of nitrogenous fertilizers after their application into the soil due to leaching, run off, volatilization and de-nitrification. Overall, the findings of this study have essential implications for the enhancing NUE and apparent nitrogen recovery. The comments are as follows, and most of them are suggestions to the authors that should be taken on board for a revised version.

Specific comments:

1. Page 2 - line 25 –“efficiency ways” should be changed into “efficient ways”

2. Page 2 - line 32 –“which decreased apparent nitrification rate 28%” should be changed into “which decreased apparent nitrification rate by 28%”

3. Page 2 - line 35 –“5.07 times” delete “times”

4. Page 4 - line 67,69, 88 – “But” “And” should not be the first word of the sentence and rewrite this.

5. Page 4 - line 81 – “A laboratory incubation experiment…” should be marked with a reference.

6. Page 8 - line 147 – “pH meter” should be marked with the mode.

7. Page 8 – line 149 – “molybdenum blue method” reference?

8. Page 10 - line 197 – “Apparent nitrification rate” should be abbreviated.

9. Page 13 - line 255 – “Agronomy nitrogen use efficiency” should be changed into “agronomic nitrogen use efficiency”.

10. Page 14 - line 289 – “which was line in with” Grammatical errors

10. Page 15 - line 295-297 the discussion is so simple, rewrite this part.

10. Page 14 - line 314 – “environmental factors, soil properties and the type of fertilizer” should be more detailed. Rewrite this part according to the results of this article.

10. Page 16- line 323 – “In our study, the efficiency of NIs differed in different agricultural soils, mainly due to the different pH”. The conclusion is far fetched and rewrite this sentence.

The effects of pH on nitrification should be more detailed.

11. Page 16- line 322 – Please add a “Table for correlation analysis between soil properties and NIs use” if possible.

12. please calculate the nitrification inhibition rate of NIs

13. The whole manuscript some of the expressions are too colloquial and the meaning is not clearly expressed, please check the grammar in the article carefully.

Reviewer #4: This manuscript had compared the effects of combined nitrification inhibitors on soil nitrification, maize yield and nitrogen use efficiency in three agricultural soils with pot experiment. Compared to the other studies, this research is not novel. Other issues that need revision or clarification include:

(1) In introduction: It mentioned in line 67-68 that "there has been little research on the effect of NIs combinedwith ammonium sulfate in soils". However, in this study, they did not compare ammonium sulfate with other N fertilizers, such as urea, or NH4Cl. It's better to summarize other studies in lines 82-90.

(2)M&M section：I suggest to arrange the orders of the three soils as Cinnamon soil, Brown soil, Red soil, i.e based on soil pH, from high to low, because soil pH is an very important parameter to affect nitrification.

The available K of Brown soil in Table 1 was as low as 5.7 mg/kg. It is too low. Please check it.

(3) Result: the pixels of the Figures are too low!

(4) Discussion: The mechanism behind the results need analysis.

Line 281-282,"It is worth to mention that nitrification in red soil becomes stronger, which was according with Lu et al [31], mainly due to the tillage during the decades." It seems that the nitrification of red soil was lower than the two other soils.

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Junhong Bai

Reviewer #4: No

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Submitted filename: Review comments for.docx

Click here for additional data file.

18 Aug 2021

Response to academic editor’s comments

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming.

We ensured that our manuscript meets PLOS ONE's style requirements, including those for file naming.

2. Thank you for stating the following in the Competing Interests section:

"The authors have declared that no competing interests exist."

We note that one or more of the authors are employed by a commercial company: North Huajin Chemical Industries Group Corporation, Jinxi Natural Gas Chemical Co. Ltd.

We ensured that we have specifically and accurately indicated the role(s) that these authors had in our study. We confirmed that this commercial affiliation does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement.

3. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager.

We ensured that we have an ORCID iD and that it is validated in Editorial Manager.

Response to reviewers’ comments

Reviewer #1: This manuscript described the effects of different nitrification inhibitors on soil nitrification and maize yield in various agricultural soils with different physicochemical properties. The subject is interesting and the paper is well organized. The introduction gave a satisfactory literature survey. The research methods used in this paper were described in detail and the data was well statistically analyzed. Appropriate figures were given to make the paper understood easily.

From my point of view, the work is well-done and provides theoretical basis to utilize fertilizer nitrogen effectively on maize in three soils with different nitrification inhibitors. The paper conclusions provided critical support for the theory that different nitrification inhibitors have different effects in different physical and chemical properties of soils. In addition, it seeks to find an economical way to improve NUE with new nitrification inhibitor N-GD due to higher price of DMPP. The results also showed that soil pH plays an important role in the effectiveness of DMPP, N-GD and their combination in influencing soil nitrification, yield and NUE of maize. Therefore, this article deserves to be published.

Some minor modifications should be made before publication:

RESPONSE: We appreciate the suggestions from the reviewer, which help us greatly improve our manuscript.

1. In this manuscript, "decrease nitrification" should be revised as “inhibit/suppress nitrification".

It was revised.

2. Page 2, line 24, please revise “The application” to “ Application”.

It was revised.

3. Page 2, line 25, please revise “the most efficiency” to “the most efficient”.

It was revised.

4. Page 2, line 38, please add “that” after “than”.

It was added.

5. Page 3, line 59, please delete “which”.

It was deleted.

6. Page 4, line 78, please add “and” in front of “field”.

It was added.

7. Page 4, line 82, please add parentheses.

It was added.

8. Page 5, line 90, please delete “are”.

It was deleted.

9. Page 7, line 139, please revise “sampling” to “samples”.

It was revised.

10. Page 11, line 216, Page 16, line 332, please revise “was” to “were”.

It was revised.

11. Page 17, line 341, please add “of maize” after “NUE”.

It was added.

12. Page 17, line 343, “improving” should be revised to “improve”.

It was revised.

Reviewer #2: To understand the efficiency of nitrification inhibitors with N fertilizer on soil nitrification, yield and nitrogen use efficiency of maize (Zea mays L.), an outdoor pot experiment with different nitrification inhibitors in three soils with different pH were conducted in the paper. The reviewer think that the problems of this paper are as follows:

RESPONSE: We appreciate the suggestions from the reviewer, which help us greatly improve our manuscript.

1. There are some sentences with irregular grammar or incomplete meaning: Such as:

(1) L39:”DMPP and N-GD were more effective in the red soil than in the cinnamon soil” What is more effective?

After careful checking, it was changed “DMPP was more effective in the red soil than in the cinnamon soil”.

(2)L40 and 41:“… than other two soils, … than other two soils” were changed to“…than that in other two soils,… than that in other two soils.”

It was changed.

(3)L169:“NH4+-N content and NO3--N content” should be changed to“Contents of NH4+-N and NO3--N in soils”.

It was changed.

(4)L197:“Apparent nitrification rate (ANR) indicates that intensify of soil nitrification”, the sentence is not a clause, it should be changed to “Apparent nitrification rate (ANR) indicates the intensify of soil nitrification”.

It was changed.

(5)L253:“Agronomy nitrogen use efficiency and apparent nitrogen recovery” should be changed to“Utilization efficiency of agronomy ang recovery of apparent nitrogen”.

It was changed.

(6)L327:“was more stable in lower pH soil than in alkaline soil” should be changed to“was more stable in lower pH soil than that in alkaline soil”.

It was changed.

2. Improper expression or incomplete meaning of some section titles:

(1)L146:“Analytical procedures” should be changed to “Analytical methods” (Only the determination methods are given, and there are no determination procedures or operation processes)

It was changed.

(2)Note: To distinguish the relationship between the instrument and the method, the instrument only is the means of the method, but the instrument itself is not the method. Such as: “with a pH meter” in L147, “with an elemental analyzer” in L148, “determined by the flame photometer method” in L150-151, and “using a Continuous Flow Analyzer” in L151-152. Above all, these sentences should be expressed in the form of “methods” rather than “instruments”, especially, “determined by the flame photometer method” in L150-151 was a wrong expression, should be changed to “determined by the flame photometry method”.

We have rewrite these sentences to express in the form of “methods”. It was changed.

(3) The meaning of “Apparent nitrification rate” in L196 was too general.

We revised the meaning of “Apparent nitrification rate” to make it more specific.

3. Some tense usages were not correct, the past tense should be used in many sentences but used the present tense.

It was corrected.

4. In the description of result and content discussion, the first person is used too much. Such as: "we conduct an outdoor.." in L99, “We clearly observed that…..” in L170(Figure 1 should be described first), “We also found that…” in L179, “we clearly observed……” in L340(It is more appropriate to have such a statement in the conclusion). It is suggested that the result or content described in the first person should be described in the passive voice.

We fully agreed with the suggestion of the reviewer. According the suggestions, we described in the passive voice to describe the result or content described in the first person. Such as “an outdoor… was conducted” . The first person have been deleted. In the conclusion, we deleted the sentence “we clearly observed……”.

5. “In our study” is used too often, which is to discuss the results of this paper, so it is unnecessary to emphasize it many times.

We fully agree with the suggestion of the reviewer. According the suggestions, we deleted “In our study”.

6. The expression of this comparison "The results was similarly to our study,....."in L332 is improper, it should be expressed "The results of this paper was similarly to that of the literature,.....".

It was changed.

7. The part of “conclusion” is an objective statement of the law presented by the research results, The expression of “In our study, we clearly observed” should be deleted.

We deleted the expression of “In our study, we clearly observed”.

8. The logical relationship between several experimental results in the result part is not very clear, moreover, the logical relationship between the results and the discussion is also fuzzy.

We reorganized the results and the discussion to make the content clear.

Reviewer #3: Opinion on manuscript: PONE-D-21-09541 major revision

Global food security is a key challenge of the modern world. The demand for food is growing exponentially and in this regard agricultural production has to be increased to meet global food demands. One of the key agricultural practices is the higher application of nitrogenous fertilizers. However, the use efficiency of applied fertilizers is currently very low. This low efficiency is attributed to the great losses of nitrogenous fertilizers after their application into the soil due to leaching, run off, volatilization and de-nitrification. Overall, the findings of this study have essential implications for the enhancing NUE and apparent nitrogen recovery. The comments are as follows, and most of them are suggestions to the authors that should be taken on board for a revised version.

RESPONSE: We appreciate the suggestions from the reviewer, which help us greatly improve our manuscript.

Specific comments:

1. Page 2 - line 25 –“efficiency ways” should be changed into “efficient ways”

It was changed.

2. Page 2 - line 32 –“which decreased apparent nitrification rate 28%” should be changed into “which decreased apparent nitrification rate by 28%”

It was changed.

3. Page 2 - line 35 –“5.07 times” delete “times”

It was deleted.

4. Page 4 - line 67,69, 88 – “But” “And” should not be the first word of the sentence and rewrite this.

We have already rewritten this.

5. Page 4 - line 81 – “A laboratory incubation experiment…” should be marked with a reference.

We have already marked the reference.

6. Page 8 - line 147 – “pH meter” should be marked with the mode.

We have already marked the mode.

7. Page 8 – line 149 – “molybdenum blue method” reference?

The reference has been marked.

8. Page 10 - line 197 – “Apparent nitrification rate” should be abbreviated.

We abbreviated “Apparent nitrification rate” as “ANR”.

9. Page 13 - line 255 – “Agronomy nitrogen use efficiency” should be changed into “agronomic nitrogen use efficiency”.

It was changed “Agronomic nitrogen use efficiency”.

10. Page 14 - line 289 – “which was line in with” Grammatical errors

It was corrected.

10. Page 15 - line 295-297 the discussion is so simple, rewrite this part.

We have rewritten this part.

10. Page 14 - line 314 – “environmental factors, soil properties and the type of fertilizer” should be more detailed. Rewrite this part according to the results of this article.

We have rewritten this part according to the results of this article.

10. Page 16- line 323 – “In our study, the efficiency of NIs differed in different agricultural soils, mainly due to the different pH”. The conclusion is far fetched and rewrite this sentence.

The effects of pH on nitrification should be more detailed.

We have rewritten this sentence. The effect of pH on nitrification has been described in detail.

11. Page 16- line 322 – Please add a “Table for correlation analysis between soil properties and NIs use” if possible.

The application of NIs have the potential to slow soil nitrification, thus affecting soil inorganic (NH4+-N and NO3--N) concentrations. Therefore, we added a “Table for correlation analysis among soil properties, NH4+-N and NO3--N”.

12. please calculate the nitrification inhibition rate of NIs

We already calculated the nitrification inhibition rate of NIs, and added a table in the supplementary material.

13. The whole manuscript some of the expressions are too colloquial and the meaning is not clearly expressed, please check the grammar in the article carefully.

We revised some of the expression of the manuscript to make the content clear. We checked the grammar in the article carefully, and corrected it.

Reviewer #4: This manuscript had compared the effects of combined nitrification inhibitors on soil nitrification, maize yield and nitrogen use efficiency in three agricultural soils with pot experiment. Compared to the other studies, this research is not novel. Other issues that need revision or clarification include:

RESPONSE: We appreciate the suggestions from the reviewer, which help us greatly improve our manuscript. The paper aims to compare the different effects of nitrification inhibitors in different physical and chemical properties of soils, and this is the first research to study the effect of new nitrification inhibitor (nitrogen protectant (N-GD)) in different soils.

(1) In introduction: It mentioned in line 67-68 that "there has been little research on the effect of NIs combined with ammonium sulfate in soils". However, in this study, they did not compare ammonium sulfate with other N fertilizers, such as urea, or NH4Cl. It's better to summarize other studies in lines 82-90.

We summarized other studies in the revised manuscript.

(2)M&M section：I suggest to arrange the orders of the three soils as Cinnamon soil, Brown soil, Red soil, i.e based on soil pH, from high to low, because soil pH is an very important parameter to affect nitrification.

The available K of Brown soil in Table 1 was as low as 5.7 mg/kg. It is too low. Please check it.

We fully agreed with the suggestion of the reviewer. We arranged the orders of the three soils as Cinnamon soil, Brown soil, Red soil. We checked the available K of brown soil, and corrected it.

(3) Result: the pixels of the Figures are too low!

We adjusted the pixels of the Figures.

(4) Discussion: The mechanism behind the results need analysis.

Line 281-282,"It is worth to mention that nitrification in red soil becomes stronger, which was according with Lu et al [31], mainly due to the tillage during the decades." It seems that the nitrification of red soil was lower than the two other soils.

It is well known that nitrification in natural red soil is extremely weak due to low pH and soil texture, but nitrification has been accelerated in many such soils following tillage in recent decades. However, the nitrification of red soil was lower than the two other soils. The reason of this phenomenon is its lower pH and soil organic matter.

Submitted filename: Response to reviewers.docx

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5 Oct 2021

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Manuel Joaquín Reigosa, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: I am happy to see that the authors have answered all the queries raised by the reviewers. After revision the MS is scientifically more sound than the first submitted form. In my opinion the MS is fit for publication.

Reviewer #2: The manuscript has been carefully revised by the author, but there are still several small problems as follows：

1. L26 and L102,103: The active voice such as “we conducted an outdoor pot ……” used in the abstract L26 is inconsistent with the passive voice such as “an outdoor pot experiment with different types of NIs additions in the above three soils was conducted” in the preface L102,103. It suggested that the passive voice should be used as much as possible in the full text.

2.L65，67: There are no percentage signs for some quantities such as “58.5 in L65” and “83.8” that need to be added with a percentage sign “%”, i.e. change 58.5 to 58.5% in L65 and change 83.8 to 83.8% in L67.

3. The meaning of some percentages such as “93.5% (pH: 7.0, 93.5%), 85.1% (pH: 8.0, 85.1%) and 70.5% (pH: 4.6, 70.5%) in L83 and 84 is not clear.

4. To make the meaning of the sentence of “Samples were shaken for 1 h on a reciprocal shaker, filtered and the extract analyzed on a Continuous Flow Analyzer “ clearer (i.e. the contents of NH4+-N and NO3—N in the extract of soil were analyzed on a Continuous Flow Analyzer), the two sentences “The soil NH4+-N and NO3—N content were determined by extracting a 5-g soil subsamples with 50 ml of 2 mol L-1 potassium chloride (KCl)” in L157 and “Samples were shaken for 1 h on a reciprocal shaker, filtered and the extract analyzed on a Continuous Flow Analyzer” in L158 should be combined into one sentence, for example, change the two sentences to “The soil NH4+-N and NO3—N content were determined by extracting a 5-g soil subsamples with 50 ml of 2 mol L-1 potassium chloride (KCl) and the samples were shaken for 1 h on a reciprocal shaker, filtered and the extract analyzed on a Continuous Flow Analyzer”.

5.L161：Is the concept “Apparent nitrification rate” in line 161 mentioned for the first time? If this concept is mentioned for the first time, the significance of calculating “Apparent nitrification rate” should be introduced briefly. Otherwise, the reader does not know why “Apparent nitrification rate” should be calculated in this paper.

6. The abbreviation “ANR” of “Apparent nitrification rate” should be clearly marked when it appears for the first time in L204. Moreover, what the reviewers don't understand is that the “Apparent nitrification rate” in the first sentence in L204 is abbreviated, but why does the “Apparent nitrification rate” in the second sentence in L204 and 205 use the full name?

Reviewer #3: This manuscript describes the effects of nitrification inhibitors on nitrification and maize yield in different agricultural soils, which is a very meaningful study. The authors have adequately addressed the comments raised in a previous round of review and this manuscript is now acceptable for publication. The manuscript has described a technically sound piece of scientific research with data that supports the conclusions, and the statistical analysis has been performed appropriately and rigorously. In my opinion, this work is well written and provides a theoretical basis for utilization. Therefore, I suggest that the article could be accepted and published in this journal.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.