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INTERNATIONAL JOURNAL OF AGRICULTURE & BIOLOGY
ISSN Print: 1560–8530; ISSN Online: 1814–9596
19–0824/2019/22–5–1102–1108
DOI: 10.17957/IJAB/15.1175
http://www.fspublishers.org
Full Length Article
To cite this paper: Ashraf, M.N., T. Aziz, M.A. Maqsood, H.M. Bilal, S. Raza, M. Zia, A. Mustafa, M. Xu and Y. Wang, 2019. Evaluating organic materials
coating on urea as potential nitrification inhibitors for enhanced nitrogen recovery and growth of maize (Zea mays). Intl. J. Agric. Biol., 22: 1102‒1108
Evaluating Organic Materials Coating on Urea as Potential Nitrification
Inhibitors for Enhanced Nitrogen Recovery and Growth of Maize (Zea
mays)
Muhammad Nadeem Ashraf1,2, Tariq Aziz1,3*, Muhammad Aamer Maqsood1, Hafiz Muhammad Bilal1,3, Sajjad
Raza4,8, Munir Zia5,6, Adnan Mustafa2, Minggang Xu2* and Yaosheng Wang7
1Institute of Soil and Environmental Sciences, University of Agriculture, Faisalabad-38040, Pakistan
2National Engineering Laboratory for Improving Quality of Arable Land, Institute of Agricultural Resources and Regional
Planning, Chinese Academy of Agricultural sciences (CAAS), Beijing-10081, China
3UWA School of Agriculture and Environment, The University of Western Australia, Australia
4College of Natural Resources and Environment, Northwest A&F University, Yangling-712100, Shaanxi, China
5R&D Department, Fauji Fertilizer Company (Pvt.) Ltd. Rawalpindi, Pakistan
6BGS-UoN Centre for Environmental Geochemistry, Keyworth, U.K.
7Institute of Environment and Sustainable Development in Agriculture, Chinese Academy of Agricultural Sciences (CAAS),
Beijing, China
8Department of Environmental Sciences, University of Okara, Pakistan
*For correspondence: draziz@uaf.edu.pk; xuminggang@caas.cn
Abstract
To slow down the urea hydrolysis and inhibition of nitrification process is a promising approach for enhancing crop
production and reducing environmental risk of nitrogen (N). Two separate (incubation and pot) experiments were conducted to
estimate the nitrification inhibition potential of coated urea and maize growth. The applied treatments included control,
ordinary urea, neem (Azadirachta indica) oil coated urea (NOCU1%) and NOCU2%, moringa (Moringa oleifera) oil coated
urea (MOCU1%) and MOCU2% and pomegranate (Punica granatum) leaves extract coated urea (PLECU0.5%) and
PLECU1%. In incubation experiment, changes in mineral-N were studied for 40 days by analyzing NH4+-N and NO3--N
contents in soil samples taken at 10-days intervals. The application of coated urea delayed the nitrification up to 30 days and
increased the plant available N pool compared to un-coated urea. Highest N losses (40–48%) were observed in case of un-
coated urea, while minimum NO3--N (7.40 mg kg-1) concentrations were recorded where, 2% NOCU was applied. Maize was
sown as a test crop in pot experiment with same treatments plan. Apparent N recovery ranged from 61–84% between coated
urea treatments than ordinary urea. Similarly, growth related parameters i.e. plant height, dry matter yield, chlorophyll a and b
significantly (P ≤ 0.05) increased with the application of natural nitrification inhibitors (NNIs) coated urea than ordinary urea,
respectively. Correspondingly, the relative growth rate (RGR) was increased by 11–89% and 30–70% in all NNIs coated urea
than control and ordinary urea, respectively. In conclusion, application of NNIs seemed highly effective to reduce N losses and
sustaining better crop productivity. © 2019 Friends Science Publishers
Keywords: Apparent N recovery; Mineral-N; Nitrification inhibitors; Nitrified N; Relative growth rate
Introduction
Global food security is a key challenge to feed the 9 billion
people by 2050, while world food demand would be
increased up to 60% (FAO, 2017). Almost, 50% of the world
population utilize nitrogen (N) fertilizers for crop production
(Erisman et al., 2008). It is an important phytonutrient and is
the main yield limiting factor in majority of crops (Galloway
et al., 2004) including maize (Zea mays L.). Most of the soils
lack plant available N, consequently, external sources of
fertilization have to be applied for sustaining crop growth
and yield (Jones et al., 2005).
One of the key agricultural practices is the higher
application of nitrogenous fertilizers because of its
immediate response to crop uptake. Yet, the use efficiency
of the applied N in agriculture is considerably low and about
70% of the applied N fertilizer in the field is lost through
one or more of different process like leaching, runoff,
volatilization and de-nitrification (Subbarao et al., 2006).
These conspicuous processes not only contribute to
economic losses but also have serious concerns of
environmental deterioration. For instance, increased N2O
which is one of the dominant greenhouse gas can have a
considerable effect on global climate change (Zhang et al.,
Nitrification Inhibitors to Improve N Recovery and Maize Growth / Intl. J. Agric. Biol., Vol. 22, No. 5, 2019
1103
2019). Moreover, nitrate-N (NO3--N) accumulation in water
bodies is a leading cause of eutrophication (Dinnes et al.,
2002). Therefore, it is a dire need to introduce
environmental friendly approaches that can minimize soil N
losses and improve efficiency of applied N fertilizers.
A number of strategies are available for enhanced
efficiency of fertilizers in agriculture including agronomic,
molecular and genetic approaches. Use of urease and
nitrification inhibitors (NIs) is one of the sustainable
approaches to reduce N losses and to improve crop growth
and yield (Zhang et al., 2019; Li et al., 2018). The NIs are
the chemicals or organic compounds that slow down the
oxidation of urea fertilizers from ammonium (NH4+) to
nitrate (NO3-) thereby delaying the activities of
Nitrosomonas and Nitrobacter (Subbarao et al., 2006).
Whereas, various chemical compounds are used as
nitrification inhibitors, such as dicyandiamide (DCD)
(Wakelin et al., 2014), 3,4-dimethylpyrazole phosphate
(DMPP) and Nitrapyrin (Zerulla et al., 2001), but most of
them are relatively expensive and have adverse effects on
beneficial soil microorganisms (Dong et al., 2013).
Therefore, it is crucial to explore the natural substances that
possess the nitrification inhibitory activities for augmenting
N recovery efficiency of applied fertilizers and to reduce the
N losses. For example, Patra et al. (2002) reported that
neem oil coated urea demonstrated an increase in apparent
N recovery by 20–30% compared to the uncoated urea. In
this regard, utilizing natural nitrification inhibitor(s) would
be a viable technique to reduce soil N losses in
environmental friendly manner (Prasad, 2009). Moreover,
no information is available on the potential of locally
available and economically feasible natural substances to
inhibit nitrification and urea hydrolysis specifically, their
effects on N recovery and maize growth have not been
assessed. Therefore, this study was conducted (i) to evaluate
the nitrification inhibition potential of selected natural
substances and (ii) to determine the N recovery efficiency
and impact of controlled release urea on maize growth.
Materials and Methods
Incubation Experiment
An incubation study was conducted in the wire house of
Institute of Soil and Environmental Sciences (ISES),
University of Agriculture, Faisalabad (U.A.F.) (31º43´ N,
73º60´ E). The soil (200 g) was taken in plastic cups from
the research farm area of UAF. The soil texture was sandy
clay loam (sand, 47.98%; silt, 29.52%; clay, 22.50%) and
had soil pH of 7.7, soil organic matter of 0.78%, CEC of
14.5 cmolc kg-1, EC of 1.2 dS m-1, total nitrogen (Kjeldahl
method) of 0.06%, NO3--N (mg kg-1) of 4.0 mg kg-1, NH4+-N
of 2.7 mg kg-1.
Urea was developed as neem oil coated urea, moringa
oil coated urea. For this 100 g of granular urea was coated
with 1 mL and 2 mL of respective oil extracts representing 1
and 2% coating on v/w basis. The pomegranate leaf extract
(PLE) was used with urea at the time of application into two
percentages on urea granules as 0.5 and 1% (v/w). Then
coated urea was allowed to dry at room temperature in Soil
Fertility and Plant Nutrition Laboratory, ISES, UAF. The
treatments applied were T1: control, T2: ordinary urea, T3:
neem oil coated urea (NOCU) (1%), T4: NOCU (2%), T5:
moringa oil coated urea (MOCU) (1%), T6: (MOCU) (2%)
and T7: pomegranate leaves extract coated urea (PLECU)
(0.5%), T8: PLECU (1%).
The soil was incubated for 40 days in which normal
urea and urea coated with different compounds (neem
seeds-extract oil, moringa seeds-extract oil and pomegranate
leaves extract) were applied. These cups were incubated in
the wire house of ISES, U.A.F., having daily average
temperature of 22°C (20–25oC) and arranged in a
completely randomized design (CRD) and each treatments
was replicated three times. The soil moisture was adjusted
by weighing the soil pot after every 2 days and lost water
(>0.05 g) was added as distil water.
Nitrogen was applied in the form of urea (200 mg N
kg-1). Soil NH4+-N and NO3--N were analyzed in soil
samples taken at 10 days intervals by AB-DTPA extract and
indophenol blue methods, respectively, by using
Spectrophotometer, following the International Center for
Agricultural Research in Dry Areas (ICARDA) Manual
(Estefan et al., 2013).
The nitrified N was calculated by using the following
equation (Majumdar et al., 2001):
The percent inhibition of nitrification for the evaluation of
natural substances on coated urea was calculated as
proposed by Sahrawat (1980), using the following equation:
% inhibition of nitrification =
Where, C is the NO3--N content in urea treated soil while S
is NO3--N content in urea treated soil with natural
nitrification inhibitors.
Pot Experiment
The same soil was used for the pot experiment. Four kg
soil was taken in plastic pots and same treatments were
applied as discussed previously in the incubation study. In
pot study, N was applied in the form of urea (200 mg N
kg-1) while basal dose of phosphorus and potassium
fertilizers were added in the form of superphosphate (100
mg P2O5 kg-1) and potassium chloride (100 mg K2O kg-1).
The soil was mixed thoroughly immediately after
fertilizers application in each pot. Eight seeds of maize
cultivar (Sygenta-8441) were sown and 4 plants were
maintained at seedling stage. To maintain the constant
soil moisture, distilled water was used throughout the
experimental period. Two plants were harvested after 21
Ashraf et al. / Intl. J. Agric. Biol., Vol. 22, No. 5, 2019
1104
days of sowing and remaining two plants at second
harvest (after 42 days of sowing). Data regarding growth
traits like fresh and dry shoot weight, root weight, plant
height, chlorophyll a & b, and total N from plants were
recorded. All the laboratory analyses were carried out
according to ICARDA Manual (Estefan et al., 2013). The
plants were harvested at two different stages and relative
growth rate (RGR) was calculated following the equation
proposed by Hunt (1978) as:
RGR (mg g-1 d-1) =
Whereas, W1 is the short dry weight at first gravest (g), W2
is the shoot dry weight at second harvest (g) and ΔT is the
time interval between two harvest (days).
The N recovery was calculated by using the equation
proposed by Singh and Shivay (2003):
Where Nt is an amount of N taken up from the test pot (mg
kg-1), No is amount of N taken up from the control plot (mg
kg-1) and Na is the quantity of added N (mg kg-1).
Statistical Analysis
All the statistical analyses were performed using the SPSS
v20.0 (IBM SPSS Statistics; Armonk, N.Y., U.S.A.) and
graphs were plotted using Sigma-Plot v12.5. A one-way
ANOVA was used to analyze the data and Duncan’s
multiple range comparisons (DMR) were performed to
separate the difference between the means at P ≤ 0.05.
Results
Incubation Experiment
Temporal changes in ammonical N (NH4+-N): The
concentration of ammonium N (NH4+-N) in the soil
increased with time under all treatments; However, increase
varied significantly among the various treatments.
Maximum NH4+-N contents were recorded after 20 days of
incubation (Table 1). Maximum NH4+-N concentration
(57.1 mg kg-1 soil) was observed in treatment receiving
MOCU (2%) followed by NOCU (2%) and PLE (1%).
After 30 days of incubation, the NH4+ concentration in
coated treatments was still high (average 35 mg kg-1 soil)
than non-coated urea treatment. In the soil where uncoated
urea was applied, NH4+ concentration increased during the
first 10 days and later decreased gradually with time and
very little plant available NH4+ was left (6.5 mg kg-1 soil) by
the end of the incubation period (Table 1). At the end of the
incubation period, the treatments PLECU1% and
MOCU2% maintained higher NH4+-N contents (20.3 and
21.1 mg kg-1 soil, respectively) compared to other
treatments.
Temporal Changes in NO3--N
The NO3--N concentration remained statistically unchanged
till 20 days after incubation and then decreased continuously
in control (Table 1). The concentration of NO3--N increased
during incubation period and reached to the maximum value
of 12.61 mg kg-1 soil at 30 days and then decreased suddenly
to 4.99 mg kg-1 soil in the treatments where ordinary urea
was applied. Later on, the NO3--N concentration increased
continuously, however, values varied with treatments though
best performance in terms of relatively lower NO3--N
concentration was calculated with MOCU2% (Table 1).
Percent Inhibition of Nitrification
The nitrification inhibition (NI) potential of natural
substances coated on urea varied significantly among the
treatments (Table 2). At 10 days of interval NOCU1%
showed highest (67%) NI potential while 66% NI potential
was observed in PLECU0.5% at 20 days. Similarly, at 30
days and 40 days of incubation, PLECU1% and MOCU2%
showed highest NI by 53.9 and 43.9%, respectively.
Pot Experiment
Plant Growth (1st Harvest)
The plant height was significantly (P ≤ 0.05) increased
under all the urea coated treatments compared with
uncoated urea (Table 3). The plant height was recorded
maximum in PLECU0.5% (39.9 cm). Progressive increase
in height was observed with increasing the level of coating
of each treatment, except for PLECU 1% treatment in which
decreased from 39.9 to 37.1 cm. The dry matter yield was
not significantly different among treatments at 1st harvest.
Plant Growth (2nd Harvest)
The response of maize growth and related parameters
significantly varied (P ≤ 0.05) compared to the control
(Table 3–4). Urea coated with different natural nitrification
inhibitors produced higher dry matter, plant height and
chlorophyll contents (especially chlorophyll a) relative to the
ordinary urea. The maximum plant height (98 cm) was
recorded in NOCU2% followed by NOCU1% (94 cm) and
PLECU0.5% (91 cm) treatments. The moringa oil coated
urea produced almost similar plant height at both levels i.e.
MOCU1% (86.7 cm) and MOCU2% (86.3 cm). The dry
matter yield of 5.09 g and 4.99 g per plant was recorded at
PLECU0.5% and PLECU1%, respectively, followed by
MOCU1% (3.59 g), MOCU2% (3.43 g), NOCU1% (3.42 g)
and NOCU2% (3.97 g), respectively (Table 3). Similarly,
root dry matter production was recorded (0.29–0.52 g plant-1)
in between coated urea treatments than ordinary urea (0.37 g
plant-1) and control (0.27 g plant-1), respectively (Table 3).
Nitrification Inhibitors to Improve N Recovery and Maize Growth / Intl. J. Agric. Biol., Vol. 22, No. 5, 2019
1105
Chlorophyll a and b also differed in each treatment
(Table 4). The nitrification inhibitors (NIs) significantly (P
≤ 0.05) increased chlorophyll contents (chlorophyll a and b)
as compared to application of ordinary urea. The lowest
chlorophyll a and b (21.57 and 31.14 mg g-1) respectively,
were recorded by application of ordinary urea. The
chlorophyll a produced by MOCU (1 and 2%) and PLECU
(0.5 and 1%) was not statistically significant. The highest
Table 1: Temporal release of NH4+-N and NO3- -N from urea fertilizer in a soil medium as affected by different natural nitrification
inhibitor(s) (Incubation study)
NH4+-N (mg kg-1)
NO3--N (mg kg-1)
Treatments
10 D
20 D
30 D
40 D
10 D
20 D
30 D
40 D
Control
2.81 ± 0.10 f
1.70 ± 0.18 f
1.34 ± 0.09 g
1.31 ± 0.08 e
2.99 ± 0.06 f
1.81 ± 0.12 e
1.63 ± 0.24 e
1.58 ± 0.17 f
Ordinary Urea
20.21 ± 1.22 a
16.54 ± 0.68 e
12.25 ± 0.61 f
9.22 ± 0.63 d
4.62 ± 0.33 a
9.07 ± 0.32 a
12.61 ± 1.07 a
4.99 ± 0.61 d
NOCU 1%
7.70 ± 0.28 d
44. 89 ± 2.16 d
30.85 ± 0.56 c
15.06 ± 0.42 b
2.04 ± 0.70 de
6.36 ± 0.74 b
9.44 ± 0.70 b
10.40 ± 0.61 a
NOCU 2%
6.95 ± 0.40 d
51.44 ± 0.53 b
35.79 ± 0.55 b
12.25 ± 0.23 c
1.52 ± 0.32 e
6.22 ± 0.49 b
8.03 ± 0.84 bcd
7.40 ± 0.35 c
MOCU 1%
11.69 ± 0.55 b
50.01 ± 0.46 c
26.29 ± 0.92 f
14.94 ± 0.55 b
3.18 ± 0.17 b
3.43 ± 0.56 c
6.76 ± 1.29 cd
10.28 ± 0.24 a
MOCU 2%
10.36 ± 0.32 c
57.12 ± 0.29 a
39.54 ± 0.50 a
21.16 ± 0.45 a
2.80 ± 0.13 bc
3.06 ± 0.13 c
6.54 ± 0.78 d
7.55 ± 0.51 c
PLECU 0.5%
5.08 ± 0.53 e
49.41 ± 0.60 c
28.59 ± 0.64 e
15.16 ± 0.87 b
2.29 ± 0.43 cd
3.13 ± 0.76 c
8.27 ± 0.62 bc
9.04 ± 0.94 b
PLECU 1%
3.47 ± 1.20 f
50.83 ± 0.70 bc
30.23 ± 0.65 c
20.36 ± 0.99 a
2.50 ± 0.23 cd
2.15 ± 0.42 d
7.47 ± 0.50 cd
10.28 ± 0.24 a
The mean values ±SE (n=3), different lower case letters show the significant difference among the treatments at (P < 0.05), NH4+-N and NO3--N at 10 days, 20 days, 30 days and
40 days
Here Control; ordinary urea; NOCU 1% and NOCU 2%, 1% and 2% urea coated with neem oil; MOCU 1% and MOCU 2%, 1% and 2% urea coated with moringa oil; PLECU
0.5% and PLECU 1%, 0.5% and 1% urea coated with pomegranate leaves extract
Table 2: Nitrified N and percent inhibition of nitrification after the application of natural nitrification inhibitors
Nitrified N (%)
Nitrification inhibition (%)
Treatments
10 D
20 D
30 D
40 D
10 D
20 D
30 D
40 D
Ordinary urea
18.60 ± 0.8 c
35.44 ± 1.7 a
50.69 ± 1.0 a
29.56 ± 2.4 c
NOCU 1%
20.74 ± 6.2 c
12.42 ± 1.6 b
23.40 ±1.1 b
40.81 ± 2.0 a
55.88 ± 8.2 ab
38.72 ± 1.3 d
32.24 ± 2.9 c
20.69 ± 3.0 c
NOCU 2%
17.90 ± 3.6 c
10.72 ± 0.8 b
18.30 ± 1.6 d
37.64 ± 1.3 a
67.15 ± 6.9 a
43.52 ± 2.5 d
40.09 ± 3.0 b
34.94 ± 2.4 b
MOCU 1%
21.40 ± 1.0 c
6.41 ± 1.0 c
20.35 ± 2.5 cd
40.77 ± 0.9 a
31.11 ± 3.8 d
51.74 ± 3.7 c
49.17 ± 3.0 a
33.54 ± 2.4 b
MOCU 2%
21.27 ± 1.1 c
5.08 ± 0.1 cd
14.17 ± 1.3 e
26.28 ± 1.3 c
39.41 ± 2.7 cd
57.23 ± 1.6 bc
52.87 ± 3.1 a
43.96 ± 1.1 a
PLECU 0.5%
31.10 ± 5.1 b
5.93 ± 1.3 cd
22.43 ± 1.7 bc
37.34 ± 3.7 a
50.27 ± 9.2 bc
60.42 ± 8.6 ab
47.97 ± 6.8 a
36.14 ± 4.6 b
PLECU 1%
43.12 ± 8.2 a
4.05 ± 0.7 d
19.81 ± 1.3 cd
33.57 ± 1.5 b
45.87 ± 4.9 bcd
66.06 ± 4.1 a
53.93 ± 0.7 a
37.07 ± 1.1 b
Different lower case letters show the significant difference among the treatments (P < 0.05). Values are means ± S.E (n=3)
Here Control; ordinary urea; NOCU 1% and NOCU 2%, 1% and 2% urea coated with neem oil; MOCU 1% and MOCU 2%, 1% and 2% urea coated with moringa oil; PLECU
0.5% and PLECU 1%, 0.5% and 1% urea coated with pomegranate leaves extract
Table 3: Effects of various natural nitrification inhibitors coated urea on growth related parameters of maize
Treatments
Plant height (cm)
Shoot dry weight (g/plant)
Root dry weight (g/plant)
1st Harvest
2nd Harvest
1st Harvest
2nd Harvest
2nd Harvest
Control
25.4 ± 0.92 f
76.3 ± 1.25 g
0.28 ± 0.01 d
2.88 ± 0.08 e
0.27 ± 0.02 b
Ordinary Urea
27.6 ± 0.89 g
78.7 ± 0.47 f
0.32 ± 0.02 ab
3.01 ± 0.05 bc
0.34 ± 0.03 a
NOCU 1%
32.3 ± 0.67 a
94.0 ± 0.81 b
0.33 ± 0.03 ab
3.42 ± 0.05 de
0.36 ± 0.05 ab
NOCU 2%
38.2 ± 1.04 c
98.0 ± 0.82 a
0.35 ± 0.05 a
3.97 ± 0.04 b
0.41 ± 0.04 ab
MOCU 1%
34.7 ± 0.88 de
86.7 ± 1.25 cd
0.29 ± 0.02 ab
3.59 ± 0.03 bc
0.29 ± 0.04 ab
MOCU 2%
35.2 ± 0.8 c
86.3 ± 1.24 e
0.29 ± 0.03 ab
3.43 ± 0.04 cd
0.38 ± 0.03 ab
PLECU 0.5%
39.9 ± 1.12 e
91.0 ± 0.82 c
0.29 ± 0.02 ab
5.09 ± 0.09 a
0.47 ± 0.05 ab
PLECU 1%
37.1 ± 0.98 ab
88.0 ± 0.81 de
0.28 ± 0.02 b
4.99 ± 0.08 a
0.52 ± 0.04 a
Different lower case letters show the significance difference among the treatments (P < 0.05). Values are means ± S.E (n=3)
Here Control, ordinary urea; NOCU 1% and NOCU 2%, 1% and 2% urea coated with neem oil; MOCU 1% and MOCU 2%, 1% and 2% urea coated with moringa oil; PLECU
0.5% and PLECU 1%, 0.5% and 1% urea coated with pomegranate leaves extract
Table 4: Effects of various natural nitrification inhibitors coated urea (N fertilizer) on chlorophyll contents of maize
Treatments
Chlorophyll (mg g-1) fresh weight
Chlorophyll a
Chlorophyll b
Control
17.61 ± 0.95 e
26.93 ± 0.89 e
Ordinary Urea
21.57 ± 0.86 d
31.14 ± 0.50 d
NOCU 1%
26.11 ± 0.73 c
33.71 ± 0.61 d
NOCU 2%
30.26 ± 1.31 b
34.32 ± 0.81 d
MOCU 1%
32.56 ± 1.06 a
37.05 ± 0.48 c
MOCU 2%
32.73 ± 0.87 a
40.79 ± 0.43 b
PLECU 0.5%
33.58 ± 0.90 a
42.91 ± 0.20 a
PLECU 1%
32.50 ± 0.95 a
42.56 ± 0.48 a
Different lower case letters show the significant difference among the treatments (P < 0.05). Values are means ± S.E (n=3)
Here Control; ordinary urea; NOCU 1% and NOCU 2%, 1% and 2% urea coated with neem oil; MOCU 1% and MOCU 2%, 1% and 2% urea coated with moringa oil; PLECU
0.5% and PLECU 1%, 0.5% and 1% urea coated with pomegranate leaves extract
Ashraf et al. / Intl. J. Agric. Biol., Vol. 22, No. 5, 2019
1106
chlorophyll b (42.91 and 42.56 mg g-1) observed by
PLECU0.5% and PLECU1%, respectively. However,
chlorophyll a and chlorophyll b contents were significantly
different under PLECU, MOCU and NOCU application
compared to ordinary urea (Table 4).
Relative Growth Rate (RGR)
The relative growth rate (RGR) was increased by 11–89%
and 30–70% in all coated treatments than control and
ordinary urea, respectively (Table 5). The maximum RGR
was determined in PLECU0.5% by 134.6 mg g-1 and it was
70% higher than ordinary urea followed by PLECU 1%
(65%), MOCU 2% (49%), MOCU 1% (47%), NOCU 2%
(36%) and NOCU 1% (30%), respectively. The RGR was
statistically significant (P ≤ 0.05) in case of PLECU 0.5%
than ordinary urea and control (Table 5).
Plant N uptake and Apparent N Recovery Efficiency
Shoot N concentration was increased from 34.5 to 56.8% in
treatments with natural nitrification inhibitors (NNIs) coated
urea compared to ordinary urea. The PLECU 0.5% showed
significantly higher shoot N concentration compared to
other coated material (Table 5). On the other hand,
maximum (83.98%) apparent N recovery was observed in
PLECU 0.5% followed by PLECU 1% (79.68%), NOCU
2% (75.99%), MOCU 2% (73.96%), MOCU 1% (65.45%),
NOCU 1% (61.01%), respectively, while lowest ANR
efficiency was recorded by 37.25% in ordinary urea (Table
5).
Discussion
Plant growth and development is mostly limited by lack of
available N. Therefore, management of N fertilization in
agriculture is a challenging task due to a range of its
influencing factors. About 70% of applied N is lost into the
environment and volatilization, de-nitrification and leaching
are main causes for this loss (Zakir et al., 2008).
Nitrification of ammonical N is the initial stage resulting in
loss of N to the environment. Results of this study showed
that decline in NH4+-N concentration in soil (especially from
20-days incubation period onward) might be due to limited
activities of Nitrosomonas and Nitrobacter by inhibition of
nitrification through applied natural substances which are
consistent with the other studies where, application of
nitrification inhibitors retarded the activities of nitrobacteria
(Dong et al., 2013; Zhang et al., 2019). As expected, 50%
of applied N disappeared within the 30 days of incubation
(Table 3). Moreover, during this period, the concentration of
NO3--N was increased and of NH4+-N was decreased (Table
4), confirming that much of the NH4+-N was transformed
into NO3--N in soil depicting that nitrification is the major
cause of N losses (Abbasi and Adams, 2000; Abbasi et al.,
2011). On the other hand, reduction in NH4+-N oxidation by
using natural nitrification inhibitors (NIs) as indicated by its
consistent concentration throughout a period of 30 days
(incubation) in this study maybe a promising approach to
inhibit the nitrification and improve the plant N uptake
while decreasing its losses. It is well established that using
natural substances such as neem oil having strong
triterpenoid compounds controls the loss of urea-N by
delaying the bacterial oxidation over a certain period of time
(4 to 10 weeks) (Subbarao et al., 2006; Prasad, 2009). This
delay in NH4+-N oxidation significantly varied with the
applied NNIs coated urea in present study (Table 3). At the
end of the incubation period (i.e., 40 days), the bacterial
oxidation of 47 to 49.5% was delayed in all coated urea than
ordinary urea, respectively. These findings, are in line with
another study showing that nitrification inhibitors delayed
the NH4+ oxidation for longer periods than urea application
without nitrification inhibitors (Herrmann et al., 2007; Dong
et al., 2013).
Further, the extent of nitrified N (NO3--N) as percent
of total N in soil was much higher in treatments receiving
uncoated urea (Table 2). Minimum nitrified N with NNIs
was observed after 20 days of incubation period and ranged
from 4.1 to 12.4% and further depends on the rate and type
of the coating material (Table 2). The PLECU 0.5% and
PLECU 1% proved to be stronger inhibitor in this study
followed by MOCU (1% and 2%) and NOCU (1% and 2%)
which is probably due to having phenolic functional groups
which play an important role in delaying the nitrobacteria
and urease activity (Viuda-Martos et al., 2010). Thus
PLECU is more effective having nitrification and properties
as indicated by the higher concentration of NH4+ for longer
period of time. Percent nitrification inhibition by “NOCU
and MOCU” ranged 49% to 40.9%, respectively, which is
in agreement to (Patra and Chand, 2009), they described that
coating urea with nitrification inhibitors significantly inhibit
the nitrification and decreased the N loss. These results
support the hypothesis that NNIs extends the time of N
availability to plants by reducing nitrification and NH3+
volatilization (Patra and Chand, 2009).
The percent inhibition of nitrification is the criterion to
evaluate the effectiveness of compounds to inhibit the
nitrification process for curtailing N losses (Hussain et al.,
2018). Lower values of nitrification inhibition have been
related to less availability of NO3- with the application of
NNIs (Sahrawat, 1980; Alonso-Ayuso et al., 2016) which is
useful to limit the N losses through leaching and associated
with the nitrification (Subbarao et al., 2006). On the other
hand, one of the reason for potential nitrification inhibition
in current study might be due to the fact that plant produces
certain compounds which inhibit the nitrification process by
blocking the activity of the ammonia monooxygenase
(AMO) enzyme (Subbarao et al., 2006), which had
profound role for augmenting nitrification rate (Jia et al.,
2013).
Use of NNIs coated urea not only reduces the N losses
in terms of nitrification but also increased the crop growth
Nitrification Inhibitors to Improve N Recovery and Maize Growth / Intl. J. Agric. Biol., Vol. 22, No. 5, 2019
1107
and yield (Patra et al., 2002). Relative to the control and
ordinary urea, growth related attribute like plant height, dry
matter yield (DMY) and chlorophyll content (a & b)
significantly increased in coated urea, respectively. We
proposed that urea coated with the NNIs significantly (P ≤
0.05) enhanced the growth related traits which was due to
sufficient availability of N and/or slow release of urea at
later stages of crop which reduce the N losses and thus
provide better N utilization by maize. Likewise, a recently
published meta-analysis showed that application of
chemical nitrification inhibitors and slow release urea
enhanced the crop growth and yield (Zhang et al., 2019).
Besides, relative growth rate (RGR) is actually the
“efficiency index” that depicts the total dry weight and per
unit increase in size of plant. Plants grown with coated urea
showed relatively higher RGR as compared to uncoated
urea (Table 5) and this may be attributed due to slow release
of N and maintaining uniform supply of N even at the later
stages of crop growth (Kaleem and Manzoor, 2013). These
NNIs slowed the release of N from urea granule (urea
hydrolysis) for better and gradual plant availability and thus
reduced the NO3- availability to denitrifying bacteria
(Alonso-Ayuso et al., 2016).
The relative increase in shoot N concentration in
plants grown with NNIs coated urea was between 52% and
139% over uncoated urea. Application of PLECU0.5%
exhibited the highest accumulation of N in shoot of maize
crop (Table 5). Generally, the plant N uptake can fluctuate
vary widely and depends on the availability of N in the soil.
The present study showed the higher N concentration which
was due to the higher NH4+ concentration provided by the
NNIs that retarded the bacterial nitrification and plant easily
assimilated the reduced nitrate to ammonium as inorganic
sources and then amino acids (Dechorgnat et al., 2010).
Conclusion
Relative to the ordinary urea, application of NNIs coated
urea inhibited the nitrification by 43–67% and increased
RGR by 30–70% and ANR recovered by 61–83%,
respectively. Higher shoot N concentration and increased N
recovery was mainly because of the inhibition of
nitrification process and delayed the urea hydrolysis under
application of urea coated with NNIs. This strategy not only
enhanced the crop growth but significantly reduced the N
losses and upholds the efficient use of applied N fertilizers.
Consequently, results suggested that use of organic
materials, which are inexpensive and easily available,
should be more widely used to reduce N losses for
sustaining better crop productivity.
Acknowledgements
We thank the Higher Education commission of Pakistan, the
International Research and Development Program
(2016YFE0112700) is gratefully acknowledged for their
financial support. We are also grateful to the anonymous
reviewers for their comments.
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[Received 16 May 2019; Accepted 29 May 2019; Published (online) 10 Nov 2019]
