Enhancing naked oat (Avena nuda L.) productivity with minimal indirect nitrogen loss and maximum nitrogen use efficiency through integrated use of different nitrogen sources
Enhancing naked oat (Avena nuda L.) productivity with minimal indirect nitrogen loss and maximum nitrogen use efficiency through integrated use of different nitrogen sources
Tariful Alam Khan 0 1
Faisal Nadeem 1
Lili Chen 0 1
Xiaofen Wang 0 1
Zhaohai Zeng 0 1
Yuegao HuID 0 1
0 College of Agronomy, China Agricultural University , Beijing , China , 2 College of Resources and Environmental Sciences, China Agricultural University , Beijing , China
1 Editor: P. Pardha-Saradhi, University of Delhi , INDIA
Data Availability Statement: All relevant data are
within the manuscript and its Supporting
Funding: This study was supported by the China
Agriculture Research System (CARS-08-B-1). YH
received this funding award. The funders had no
role in study design, data collection and analysis,
decision to publish, or preparation of the
Oat (Avena nuda L.) is a nutritious grain crop, rich in dietary fibers and phytochemicals.
Application of efficient nitrogen (N) sources and dose is very important to obtain higher crop
productivity and to achieve environmental sustainability. The exploitation of natural
beneficial microbes and organic nitrogen in combination with chemical nitrogen would be effective
to boost soil N for plant uptake. Hence, a field experiment was conducted during 2016 and
2017 with the aim to ameliorate the use of chemical N (CN) with organic nitrogen (ON) and
microbial fertilizer (MBF) without compromising the productivity of oat. T1 = control, T2 =
100% CN, T3 = 100% CN+MBF, T4 = 75% CN+ 25% ON+MBF, T5 = 50% CN+ 50% ON
+MBF, T6 = 100% ON+MBF, T7 = 100% ON were the treatments. 50% CN + 50% ON +
MBF treatment proved to be an efficient combination regarding enhanced biomass and
grain yield, nitrogen uptake and NUE as compared to rest of the treatments in both years.
During the critical stages of the crop, when most of the applied CN was leached from the top
20 cm soil depth, a substantial N came from the PM mineralization through enhanced
microbial activity by the addition of MBF. Lastly, the application of ON supplemented with MBF
improved the rhizosphere soil properties, i.e. mineral N concentration, total N (TN), soil
organic carbon (SOC), microbial biomass carbon (MBC), microbial biomass nitrogen
(MBN), soil respiration rate and enzymatic activity. A balanced and source conscious
application of CN, ON and MBF reduced N losses and added a substantial amount of N into the
soil N pool. We concluded that organic N combined with chemical N and MBF proved to be
effective in improving soil properties ensuring less N loss and increasing oat production in
the semi-arid region.
Agriculture, which plays a pivotal role in meeting the food demands of the growing world
population, is becoming increasingly dependent on chemical fertilizers [
], as a good correlation
has been found by the application of industrially manufactured fertilizers with crop yields
]. Plant growth and development necessarily depend on nitrogen as it is a crucial
component of chlorophyll required for photosynthesis, nucleic acids, amino acids, proteins and some
organic acids [
]. However, the over application of synthetic nitrogenous fertilizers is not
effective for increasing crop yields, because of higher vulnerability to nitrogen losses in the form of
volatilization (emitting oxides of nitrogen), denitrification, leaching, (polluting underground
water reserves), and eutrophication (polluting surface water bodies), not only increase the cost
of production but also lead to serious environmental health hazards [
]. Hence, high
levels of soil N are susceptible to losses [
] which ultimately can reduce the N use efficiency
]. Nitrogen losses from NH3 volatilization, NO3 leaching and denitrification were
estimated to be 23, 18 and 2% respectively in wheat-maize rotation system in the North China
Plain (NCP). On the other hand, about 50% of the applied CN is lost by different processes in
paddy fields [
], and the N loss through leaching is the major contributor . Therefore,
improved management of the N application rate with appropriate N sources and their
combinations is the key to increase crop NUE [
]. Organic nitrogen (ON) and microbial
fertilizers (MBF) in a combination with chemical nitrogen may prove to reduce the cost of
production, environmental hazards without compromising crop yield.
Among organic manures, poultry manure (PM) is easily available, environment friendly, rich
in nutrients with quick release, capable of increasing soil organic carbon and supplying all
essential macronutrients (N, P, K, Ca and Mg) for plant growth and development [
organic manure has the limitations of slow release of nutrients with lesser nutrient utilization, it
also has the benefits of boosting soil physiochemical processes by increasing microbial activity.
Microbial fertilizers (MBF) are comprised of living cells of distinct micro-organisms which
are usually applied as seed inoculants or liquid spray to the root rhizosphere. These
microorganisms colonize in the root zone and nourish plant growth by converting unavailable
fundamental soil nutrients (N, P) into available forms through biological processes [
increase soil microbial biomass (N and C) in green house [
] as well as field conditions [
The microbes especially bacteria, present in microbial fertilizer, not only benefit leguminous
crops through nodulation and atmospheric nitrogen fixation, but their beneficial association
with non-legumes especially cereals has also been reported [
]. However, it is not possible to
rely on MBF only by completely replacing the use of chemical fertilizer, due to their unequal
distribution in soil [
]. The intermingling of microbial fertilizers and chemical fertilizers
in different agro climatic conditions of different countries revealed a higher performance of
microbial fertilizers in the presence of lower dose of chemical fertilizers .
There have been previous studies reporting nitrogen use efficiency in cereals like maize,
rice, wheat and foxtail millet [
]. Naked oat (Avena nuda L.) and hulled oat (A. sativa
L.) are two types of oats belonging to the Avena genus and Gramineae family cultivated in
China. The fast-growing nature of oats (Avena nuda L.) gives them the capacity to produce
ample quantities of fresh fodder within a short period with high nutritional values. The high
protein and ?-glucone content along with the presence of hypo-glycemic fiber content in oat
grain increase its value as a human food. On the other hand, rice, wheat, maize and other high
yielding cereal crops generally occupy most of the agricultural land but oat can be cultivated in
marginal land with less fertilizer and water requirements.
This study aims to reduce the use of chemical N fertilizers, obtain high N use efficiency and
limit environmental impact from polluting. The main objectives of this experiment were to
2 / 22
evaluate yield of naked oat under the provision of chemical N and organic N supplemented
with microbial fertilizer, to examine the NUE in oat production system, and to explore the
changes in soil N, C and enzymatic activity.
Materials and methods
Experimental site description
The experiment was conducted during 2016 and 2017 at Baicheng Academy of Agricultural
Sciences in Baicheng City (45? 37?N, 122? 48?E, 155 m above sea level), Jilin province, China.
No human, animal or plant species were harmed during the experiment. The region is a typical
semi-arid area located in Northeast China, with a mean annual precipitation of 407 mm
(mostly occurs from April to September). The area has a temperate, semiarid and continental
climate, with a mean temperature of 18.02?C during the cropping season and a frost-free
period of 125?135 days. The average annual effective accumulated temperature is 2915?C. The
soil is light Chernozem. The former crop was sunflowers. The basic soil physicochemical
properties and average daily air temperature and daily rainfall during the oat growing season at the
study site (April to October) are shown in S1 Fig.
Experimental treatments, design and field management
The experiment comprised of seven fertilizer treatments: T1 = control- without chemical
nitrogen (CN), organic nitrogen (ON) and microbial fertilizer (MBF), T2 = 100% CN, T3 =
100% CN + MBF, T4 = 75% CN + 25% ON + MBF, T5 = 50% CN + 50% ON+ MBF, T6 =
100% ON + MBF, T7 = 100% ON. The experimental arrangement was a randomized complete
block design (RCBD) with four replications. There were 28 plots in total. Every plot had a size
of 20 m2 (5 m ? 4 m) and 12 rows with row to row distance of 30 cm. Every plot received 300 g
of oat seeds (25g or around 750 seeds/row). The recommended dose of N (i.e. 90 kg N ha?1 as
practiced by the farmers in this area) was considered as 100% dose. High quality seeds of oat
cultivar ?Bai Yan 2? were obtained from Baicheng Academy of Agricultural Science, at Jilin
Province in China.
Chemical N and other nutrients application. Urea (46.3% N) was used as a source of
chemical N, and 90, 67.5 and 45 kg N ha-1 were applied as 100, 75 and 50% N doses. In
addition, P was applied at the rate of 55 Kg ha-1 P2O5 Whereas K was applied at the rate of 45 Kg
ha-1 K2O. The source of P was Ca (H2PO4)2 H2O and that of K was K2SO4, which were also
applied to all experimental treatments including control (S3 Table). N, P, and K were applied
as basal dose, broadcasted and incorporated into the top 20 cm soil layer before seed sowing.
Organic fertilizer application. A composted, crushed, sieved (<1mm) and properly
mixed poultry manure (PM) was collected from Shijiazhuang animal breeding station, Hebei
province, China and used as a source of ON. The N, P and K content of the poultry manure
were 18.68 g kg-1, 8.17g kg-1 and 5.41 g kg-1, respectively.
Microbial fertilizer. Microbial fertilizer with the effective viable count of 2 million g-1
used in this study was supplied by the Beijing Liuhe Shenzhou Biotechnology Co., Ltd. It was a
mixed microbial fertilizer with lignite as the base material. Microbial fertilizer was applied at
the rate of 20 kg ha-1 and was divided into two slots; First, the seed was inoculated with 15 kg
ha-1 MBF and secondly, it was sprayed (5 kg ha-1 MBF mixed with water) in the rhizosphere
just after tillering stage. The high-throughput sequencing of the MBF was done by Shanghai
Meiji Biomedical Technology Company. High throughput sequencing was performed using
3 / 22
the MiSeq sequencing platform. DNA was extracted from 0.3 g of fresh soil using the E.Z. N.A.
Soil DNA Kit Omega Bio-teck Inc. Norcross, GA, USA, according to manufacturer?s
instructions. DNA from each sample was extracted in three replicates and pooled to form one mixed
DNA sample. The extracted DNA was checked on a 1% agarose gel and the concentration was
determined using a NANO Quant (Tecan, Ma?nnedorf, Switzerland). Bacterial consortia were
further analyzed by sequencing the V3-V4 hypervariable region of the 16S rRNA gene.
Universal primers 338F (5?-ACTCCTACGGGAGGCAGCA-3?) and 806R (5?-GGACTACH
VGGGTWTCTAAT-3?) were used to amplify V3-V4 region. Whereas, 817F (5?-TTAGC
ATGGAATAATRRAATAGGA-3?) and 1196R (5?-TCTGGACCTGGTGAGTTTCC-3?)
were the primers used to amplify the hypervariable regions of the fungal 18S rRNA gene using
a thermocycler PCR system (GeneAmp 9700, ABI, U.S.A.). A reaction mixture, containing 5X
FastPfu Buffer, dNTPs (2.5 mM), forward and reverse primers (5 ?M), FastPfu Polymerase and
template DNA was prepared in triplicate for PCR reaction. The PCR reactions were conducted
using the following program: denaturation at 95?C for 3 min followed by 35 cycles of 30 s at
95?C, annealing at 55?C for 30 s, elongation at 72?C for 45 s, with a final extension of 10 min at
72?C. The results of the high-throughput sequencing of the beneficial functional flora contained
in the microbial fertilizers used in this experiment are displayed in S2 Fig. For detecting the
relative abundance of nifH gene the PCR amplification of nifH gene was performed on an ABI
GeneAmp 9700 PCR thermocycler (Applied Biosystems) using the primers nifH-F/nifH-R
(50-barcode-AAA GGY GGW ATC GGY AARTCC ACC AC-30/50-TTG TTS GCS
GCRTAC ATS GCC ATC AT-30[
]. Seeds were soaked in water overnight dried on blotting
paper and mixed with microbial fertilizers and finally sown in the field manually.
Sampling, measurements and chemical analysis
Plant sampling and analysis. Plant samples were collected at harvesting stage to define
the aboveground biomass (including grain weight). From each experimental plot, above
ground plant parts were harvested randomly from an area of 100 cm2 in duplicate (as two
technical replicates) with four replicates per treatment to estimate aboveground fresh biomass
yield. After that, each sample was weighed and then oven-dried at 105?C for 30 min and then
dried at 70?C for 3 days to get constant weight to calculate the moisture gravimetrically and to
estimate dry biomass. For grain yield, an area of 2 m2 (edge rows were avoided) was selected
randomly from each plot (in duplicate), and all plants were harvested, sun dried, and threshed
to get grains. A moisture meter was used to measure the grain moisture contents, and grain
yield was standardized at 12.5% moisture content. All the yield contributing parameter like
plant height, ear length, effective spike number per hectare, grain number per spike, and 1000
grain weight were also investigated at maturity. After that, grains were oven-dried at 105?C for
30 minutes and then at 75?C until constant weight. Oven dried Straw and grains were ground
into powder using a Wiley mill, passed through a 0.5-mm mesh and stored at 4?C for N
analysis. The total N content in straw and grains was estimated by a semi-micro Kjeldahl digestion
and distillation method [
]. The concentration of other mineral elements (P, K, Ca, Mg, Mn,
Zn, Cu and Fe) in root and shoot were determined using ICP-MS according to the method
described by Masson et al., (2010) [
Soil sampling and analysis. Concurrently, soil samples (0?20 and 20?40 cm soil depth)
were also collected at pre-seeding, tillering, jointing-booting and after final harvest stage. The
samples were taken from 10 different points randomly from each plot and homogenized to get
a composite sample. These composite soil samples were sieved through 2 mm mesh, and
moisture content was determined. Soil moisture content was determined gravimetrically after
drying for 24 hours at 105?C. Each soil sample was divided into two parts. One part was placed in
4 / 22
plastic bag and stored in a refrigerator at -4?C to analyze mineral N, dissolved organic carbon
(DOC), dissolved organic nitrogen (DON), microbial biomass Nitrogen (MBN), microbial
biomass carbon (MBC) and enzymatic activity. The other part of the soil sample was air dried,
preserved in plastic bags and placed in a refrigerator at 4?C for the analysis of total N, organic
carbon, and pH. Soil pH was measured by preparing 1:2.5 soils: water suspension. Soil
NH4+N and NO3--N were analyzed by using a continuous flow analyzer (AA3 type, company: Seal,
Norderstedt, Germany). Walkley-Black method was used to determined soil organic carbon
]. Soil microbial biomass C and N were detected by chloroform fumigation-
extraction method [
]. After destructive sampling, 20 g of carefully mixed soil was directly extracted
using 80 ml of 0.5 M K2SO4 by shaking for ? h at 180 revs/min. Another 20 g soil was
fumigated with chloroform for 24 h and extracted in the same manner. The extracts were analyzed
for the total C concentration using a 2100 TOC/TIC analyzer (Analytik Jena, Germany). The
extracts of the non-fumigated samples were used to detect DOC and DON [
]. The biomass
was calculated based on the difference of K2SO4 extract between fumigated and non-fumigated
soil samples by applying two conversion factor 0.54 for MBN (kEN) and 0.45for MBC (kEC), in
the following equation [
MBC ? EC
MBN ? EN
Where, EC and EN refer to the different organic carbon and total N amount between
fumigated and non-fumigated treatment, respectively.
The soil respiration (SR) was estimated by the sealed jar incubation method, where the soil
was pre-incubated at 20?C for 7 days in dark condition by adjusting 55% water holding
capacity and measured the rate of CO2 derived from the moist soil. NaOH trap was used to measure
the CO2 for next 7 days and then titrated with HCl [
Enzymatic activities of soil. The activity of dehydrogenase (DHA, Enzyme Commission
number 1.1.1) enzyme was measured by using 2, 3, 5-triphenyltetrazolium chloride as a
]. ?-benzoyl-N-argininamide was used as a substrate to detect hydrolyzing benzoyl
argininamide (BAA-protease EC 3.4.4) [
]. p-nitrophenyl phosphate was used to determine
acid phosphatase (EC126.96.36.199) [
], while p-nitrophenyl-?-d-glucopyranoside and
p-nitrophenyl sulphate were used as a substrate to measure ?-glucosidase [
] and arylsulphatase [
Calculation. Nitrogen uptake in straw and grain was calculated by multiplying the
nitrogen concentration with its dry weight. The components of NUE were calculated with the
PAagrrtoianlofmacitcorniptrroodgeunctuivsietyef(fmicaierngcinya(lm)(aPrFgPin;aklg) (kAgN-1UNE);=kNgYAkg-1 N) = YNAY0
Apparent nitrogen recovery efficiency (marginal) (ANRE; %) = UNAU0 100
Physiological efficiency (marginal) (PNUE; kg kg-1 N) = Y Y0
Y = yield of a harvested portion (grain+ biomass) of the crop with N applied; Y0 = yield
without N application; NA = amount of total N applied (CN + ON); U = total N uptake by
crop with N application; U0 = total N uptake by crop without N application
Statistical analysis was performed using a windows software package (IBM SPSS Statistics 22).
Means of different treatments were compared using the least significant difference at a 0.05 or
5 / 22
ns means non-significant effects
, and represent significant effects at the P <0.05, P <0.01 and P <0.001, respectively.
0.01 level of probability. Correlation analysis was performed using Pearson correlation
function of SPSS. Graphical rendering was carried out using Sigma plot 13.0 windows software. All
the data are presented as means ? standard errors (SE) of four replicates.
Two-Way ANOVA analysis
Analysis of variance showed the significance of treatments on all parameters except N
concentration in grain and physiological nitrogen use efficiency (PNUE). Years had mixed trend with
above ground biomass yield, grain weight straw weight, N concentration in straw, N
accumulation in above ground plant parts and N accumulation in straw showing significance while
the rest were non-significant. The interactive effect of year and treatment was significant for
partial factor productivity (PFP), agronomic nitrogen use efficiency (ANUE), apparent N
recovery efficiency (ANRE) and above ground biomass yield while non-significant for others
(Table 1). A strongly positive correlation between N accumulation and above ground biomass
yield was found in the year 2016, 2017 and both (Fig 1A, 1B and 1C).
Crop yield and yield components
We found significantly increased grain yield by the combined application of CN and ON
supplemented with MBF which was consistent with the increased number of effective spike per
hectare, number of grain per spike and 1000 grain weight under T4 and T5 treatment
(Table 2). Besides, plant height and ear length were also enhanced significantly under T3, T4
and T5 treatment combinations contributing to higher biomass yield. Grain yield in
treatments T2, T3, T4 and T5 was 39%, 48%, 57%, and 64% higher in 2016 whereas 53%, 63%,70%
and 77% higher in 2017, respectively as compared to control treatment (T1) (Fig 2A and 2B).
T5 treatment produced 4% more grain yield than T4 in 2016 and this amount was not less
than 3.5% in 2017. Moreover, 17% and 14% more grain yield was produced by T5 treatment in
2016 and 2017, respectively, compared to T2 treatment. The effective spike, grain number per
spike and 1000 grain weight were non-significant in T5 treatment with respect to T4 treatment
6 / 22
but were significantly higher than either of T2 treatment or T7 treatment across both years
(Table 2). In both years, straw yield ranged from 6008 to 7263kg ha-1 and 5883 to 7599 kg ha-1,
respectively with T5 being the highest. Furthermore, the replacement of 25% CN with 25%
Fig 1. Above ground biomass with respect to N accumulation. The relation between above ground biomass yield
and N accumulation in the year (A) 2016, (B) 2017 and (C) both year pooled data.
7 / 22
ON in T4 treatment revealed 16.8 and 25% increased straw yield relative to control in 2016
and 2017, respectively (Fig 2A and 2B). The replacement of 50% CN with 50% ON reinforced
with MBF (T5) showed 20.90% and 29.17% more straw yield than the control in 2016 and
2017, respectively and 4.63% more straw yield in 2017. Above ground biomass yield in T5 was
10658 kg ha-1 (in 2016) and 11151 kg ha-1 (in 2017) which was approximately 31.98% and
41.33% higher than control (T1) in 2016 and 2017, respectively (Table 2). 11.50% more
aboveground biomass yield was obtained in T5 treatment than T2 (100% CN) treatment across both
years. The harvest index increased from 0.256 (T1) to 0.319 (T5) in the year 2016 while 0.254
(T1) to 0.318 (T5) in the year 2017 with T5 being the highest across both years (Table 2).
Nitrogen concentration and accumulation in oat tissues
Our results showed that the reduction of CN and addition of ON supplemented with MBF
increased nitrogen concentration in above ground plant parts. Highest N concentrations in
grain and straw were recorded in T5 treatment, which were 17.29 g kg-1 and 7.12 g kg-1 in 2016
while 18.86 g kg-1 and 8.28 g kg-1 in 2017, respectively. Grain nitrogen concentration was
significantly higher than Straw nitrogen concentration across the treatments in both years with
control having the least of them (Fig 2C and 2D). Consistently, nitrogen accumulation in grain
remained significant to nitrogen accumulation to straw in 2016 as well as in 2017 (Fig 2E and
2F). The total nitrogen accumulation ranged from 55.62 to 110.59 kg ha-1 in 2016 and 50.44 to
130.27 kg ha-1 in 2017, respectively. Nitrogen uptake in the treatment T5 was higher than T2
(34.39% in 2016 and 26.08% in 2017) and T6 (54.92% in 2016 and 50.27% in 2017). Above
ground N accumulation in T5 was almost double than control in 2016 and even more in 2017.
In case of nitrogen partitioning, the accumulation of N in grain was higher than straw,
which ranged from 31.86 to 58.91 kg ha-1 in 2016 and 28.03 to 67.35 kg ha-1 in 2017.In 2016 N
uptake in grain at T5 treatment was 28.82%, 60.95% and 84.90% higher than T2, T7 and
control (T1), respectively. On the other hand, grain N accumulation in T5 treatment was 21.68%
and 68.41% higher than in T2 and T7 treatment, respectively, and was more than double than
control in 2017. N accumulation, in straw showed significant differences among the treatments
in both years, ranged from 23.76 to 51.68 kg ha-1 and 22.41 to 62.92 kg ha-1 in 2016 and 2017,
respectively. In 2017 straw N accumulation showed the highest value in T5 treatment which
was 21.74% higher than the same treatment in the previous year. Straw N accumulation in
control was 6.02% decreased in 2017 compared to 2016 (Fig 2E and 2F).
8 / 22
Fig 2. Above ground biomass yield with respect to nitrogen concentration and accumulation under different treatment across the year. (A, B) Changes in above
ground biomass yield partitioning in grain and straw during 2016 & 2017, (C, D) changes in partitioning nitrogen concentration in grain and straw during 2016 &
2017, (E, F) changes in partitioning nitrogen accumulation in grain, and straw during 2016 & 2017. Bars indicate standard errors (n = 4). Treatments with different
letters for the same variable analyzed (straw or grain) are significantly different according to the LSD test (P <0.05). T1 = Control, T2 = 100% CN, T3 = 100% CN
+ MBF, T4 = 75% CN + 25% ON + MBF, T5 = 50% CN + 50% ON+ MBF, T6 = 100% ON + MBF and T7 = 100% ON.
9 / 22
Nitrogen use efficiency
The components of nitrogen use efficiency (NUE) such as PFP, ANUE and ANRE of oat are
shown in Table 3. PFP in treatment combination T4 and T5 was higher than the rest of the
treatments in our experiment. Although both treatments were non-significant in 2016, but
PFP of T5 treatment was significantly higher than T4 in 2017. As far as ?ANUE?, ?ANRE? and
?PNUE? are concerned they remained non-significant between T4 and T5 treatment but
significantly higher compared to all other treatment combinations in respective years. Treatment
T5 showed highest PFP, ANUE and ANRE ratio, compared with T4, T3, and T2 treatments,
indicating gentle decline of NUE with the increasing application of CN. ANRE illustrated
highest values (0.55?0.80) in T5 whereas gradual drop in T2 and T3 which were comparable in
magnitude with the treatment T4 and T5. Treatment T6 and T7 showed lower PFP, ANUE
and ANRE but the higher PNUE than the other treatments. The decrement of CN, increased
PNUE in T6 treatment.
Nitrogen dynamics in soil
In our experiment, we determined ammonium nitrogen (NH4+-N) and nitrate nitrogen
(NO3--N) content of top 20 cm and 40 cm depth soil at four growth stages of the oat. At
tillering stage, NH4+?N content in soil reached the peak point with T3 treatment being the
maximum (4.33 mg kg-1), then T2 (4.16 mg kg-1) and T4 (3.04 mg kg-1) treatment in 2016 (Fig 3A
and 3B). In 2016, the soil NH4+-N contents in T4 and T5 at jointing- booting stage were 3.53
mg kg-1 and 3.99 mg kg-1, respectively. NH4+-N content was the highest in T5 treatment soil at
jointing-booting stage and harvesting stage. In both years the trend of NH4+-N content
remained the same except residual NH4+-N being higher in 2017 than 2016 (Fig 3A and 3B
and S2 Table). The NO3--N contents of top 20 cm soil increased rapidly and were 21.48 mg
kg-1 in 2016 and 23.86 mg kg-1 in 2017 for T2 treatment, respectively. Soils of T2 and T3
treatment observed decrement of NO3--N contents at jointing-booting stage which were increased
rapidly in T4 and T5 treatment at the same depth. Generally, NO3--N content of top 20 cm soil
increased in all the treatments except T1, T2 and T3 till jointing-booting stage but decreased at
the harvesting stage. At jointing-booting stage, NO3--N content was the highest (19.16 and
22.76 mg kg-1, in both years, respectively) in soil of T5 treatment at 20 cm soil depth (Fig 3C
and 3D). On the other hand, NO3--N content at 40 cm soil depth was significantly higher in
T2 and T3 treatment at every stage, maximizing at jointing-booting stage (Fig 3E and 3F).
However, the total mineral N (NH4+-N + NO3--N) at 20 cm soil depth was highest at T2 and
10 / 22
Fig 3. Variations in N content at different depths in different growth stages under different N treatments during 2016 and
2017. (A, B) Ammonium nitrogen (NH4+-N) content at 20 cm depth soil, (C, D) Nitrate nitrogen (NO3--N) content at 20 cm depth
soil, (E, F) Nitrate nitrogen (NO3?N) content at 40 cm depth soil and (G, H) Mineral nitrogen (NH4+-N + NO3--N) content at 20
cm depth soil. The bars denote standard deviation of four replicates. T1 = Control, T2 = 100% CN, T3 = 100% CN + MBF,
T4 = 75% CN + 25% ON + MBF, T5 = 50% CN + 50% ON+ MBF, T6 = 100% ON + MBF and T7 = 100% ON.
11 / 22
In each column lower case lettering is used to show the significant differences between different types of treatments at P <0.05 level. Values show Standard errors (SE) ?
mean of four replicates. T1 = Control, T2 = 100% CN, T3 = 100% CN + MBF, T4 = 75% CN + 25% ON + MBF, T5 = 50% CN + 50% ON+ MBF, T6 = 100% ON + MBF,
T7 = 100% ON
T3 treatment (25.64 and 25.25 mg kg-1 in 2016, and 27.92 and 26.99 mg kg-1 in 2017,
respectively) at tillering stage, but at jointing-booting stage it was the highest in T5 treatment (Fig
3G and 3H).
Soil pH, Total N, Organic C, Dissolved Organic Nitrogen (DON) and
Dissolved Organic Carbon (DOC)
The diverse nitrogen sources illustrated a significant effect on fertility status of the
experimental soil without affecting soil pH too much. Sole application of CN did not improve pH and
fertility status of the differentially treated soils rather, it slightly decreased the soil pH. Combined
use of CN and ON with MBF neutralized the acidifying effect of CN and revealed an increasing
tendency of soil pH (Table 4). Total Nitrogen (TN) and organic carbon (OC) content of the
soil during 2017 harvesting time were analyzed (Table 4 and S2 Table). The levels of TN and
OC content varied markedly among the different treatment soil after harvesting of oat in 2017.
The highest level of soil TN was observed in T2 and T3, followed by T4 treatment, without
significance. Contrarily, compared to control, 77.92% and 66.23% more TN was observed in T2
and T5 treatments, respectively. On the other hand, organic C contents in soil were the highest
in the treatments T6 and T7followed by T5 treatment. The SOC lowered with the increasing
CN and vice versa whereas dissolved organic nitrogen (DON) and dissolved organic carbon
(DOC) differed significantly between treatments in both years. DON and DOC in T6
treatment were higher by 63.41% and 35.1%, respectively, compared to T3 treatment (Table 4).
Soil microbial biomass and soil respiration
Soil microbial biomass nitrogen (MBN) and microbial biomass carbon (MBC) under different
treatment at jointing-booting stage were represented as mean values of two years in Fig 4A
and 4B. MBC increased significantly in the treatment fertilized with ON+MBF and only ON
against their control during the jointing-booting stage, with the highest increment of MBC
consistently occurring in the T6 treatment (Fig 4B). Difference between T6 and other
treatments for MBC was highly significant except T7. The different N sources also affected MBN in
soil, which varied between different treatments (Fig 4A). Similarly, treatment T6 had the
highest MBN content compared to other treatments. Nevertheless, no significant differences were
displayed by T5, T6 and T7 treatments. Soil with different fertilization treatments showed
diverse respiration rates and higher cumulative CO2 production compared to those found in
12 / 22
Fig 4. Soil Microbial biomass and respiration rate. Mean values (2016?17) of (A) Soil microbial biomass N (MBN),
(B) soil microbial biomass C (MBC) and (C) soil respiration rate, in different treatment during the jointing booting
stage of oat. Bars indicate standard errors (n = 4). Treatments with different letters are significantly different according
to the LSD test (P <0.05). T1 = Control, T2 = 100% CN, T3 = 100% CN + MBF, T4 = 75% CN + 25% ON + MBF,
T5 = 50% CN + 50% ON+ MBF, T6 = 100% ON + MBF and T7 = 100% ON.
13 / 22
In each column lower case lettering is used to show the significant differences between different types of treatments at P <0.05 level. Values show Standard errors (SE) ?
mean of four replicates. T1 = Control, T2 = 100% CN, T3 = 100% CN + MBF, T4 = 75% CN + 25% ON + MBF, T5 = 50% CN + 50% ON+ MBF, T6 = 100% ON + MBF,
T7 = 100% ON
control treatment (Fig 4C). A significantly higher respiration rate was observed in T6
treatment, whereas T5 and T7 were at sequentially lower order compared to other treatments. The
respiration rate in T6 was approximately 82% higher than in control. Similarly, compared to
treatment T2 28% more respiration rate was observed in T7 treatment.
In current study, we analyzed dehydrogenase, acid phosphatase, arylsulphatase, ?-glucosidase
and BAA-protease in soil under applied treatments. Soil under T5, T6 and T7 treatments
showed high levels of aforementioned enzyme activities with dehydrogenase showing
significantly higher activity at T6 treatment than any other treatment except T7 (Table 5). Acid
phosphatase activity was significant at T5 treatment with T6 being non-significant to T5.
Arylsulphatase, ?-glucosidase and BAA-protease activities were higher in T5, T6 and T7 treatments
compared to all other treatments but the effect remained non-significant within themselves
Yield attributes influenced by different amendments
The average grain and straw yield with nitrogen use efficiency over two years indicated that
different treatments of nitrogen applied either alone or combined in different proportions,
showed significant differences in grain and biomass yield, and in the nitrogen recovery rate. In
this study higher oat grain and straw yield were obtained by the combined use of CN and ON
with MBF as evident from increased number of effective spikes per hectare, number of grains
per spike and 1000 grain weight (Table 2) and this finding is consistent with the previous
work result [
]. On the whole, yield results indicated that application of CN and ON with
MBF could significantly increase oat yield compared to 100% CN alone. Higher grain yield
and 1000 grain weight in the 100% CN + MBF (T3) treatment as compared to 100% CN (T2)
whereas lower grain yield in 100% ON compared to 100% ON+ MBF in 2016 and 2017
respectively (Fig 2A and 2B) were consistent with previous experiment which reported that maize
grain yield and 1000-grain weight were increased 13% and 10% respectively, with the
application of MBF + 88kg N ha-1 in comparison with the 100% dose (175 kg N ha-1) of CN [
Moreover, the yield results of current study illustrated the effectiveness of MBF when applied
with CN and ON owing to the fact that the application of microbial fertilizer could
significantly increase the number of tillers per plant (10?21%), straw yield (22?39%) and finally
grain yield (15?43%) [
] as compared to individual application of MBF or ON [
It has also been reported that the application of MBF along with organic and chemical
14 / 22
fertilizers increased the growth of cotton and maize [
]. Consistent with several previous
reports, our findings further elaborated that higher yield cannot be achieved solely depending
on organic manure [
]. In this experiment higher grain and straw yield under combined
application of CN and ON with MBF would have resulted from enough nutrient removal and
improved retention capacity of soil [
]. Furthermore, organic manure releases N slowly
into the soil which could really match with the demand of N in different growth stages of crop,
and this is very important off late to ameliorate environmental problems by reducing the
application of CN and establishing organic agriculture [
]. Previously, researchers used 50%
less mineral NPK with OM and effective MBF in cotton field and saved 50% mineral N
compared to the use of full dose of NPK . The higher grain and biomass yield due to the
application of MBF supplemented with organic and chemical fertilizer could also be explained by
the possible involvement of N2-fixation mechanism [
], excretion of ammonia ,
solubilization of phosphorous [
] and production of growth hormones [
Plant mineral contents and Nitrogen Use Efficiency (NUE)
Plants uptake N either in the form of nitrate or ammonium depending upon their availability
in the soil solution. Many reasons can influence their availability, one of them is soil organic
matter and the second one is microbial activity [
]. It is essential to coordinate crop N
demand and supply to improve the NUE. In our study, NUE was higher as is obvious from
increased PFP, ANUE and ANRE in T5 treatment compared to all other treatments across the
years (Table 3). 50% CN + 50% ON + MBF (T5) treatment improved the nitrogen recovery
and uptake compared to full dose of CN (T2) (Table 3) consistent with rice and wheat
], elaborating a positive interaction between organic and mineral N reinforced by
MBF to reduce nitrogen loss in the environment and increase its efficiency [
]. On the other
hand, the use of MBF could have increased the nutrient accumulation in oat similar to various
crops such as maize [
], cotton, pea [
], and wheat [
]. Nitrogen accumulation in plant is
associated with higher biomass production including greater N concentration in plant. The
increased N accumulation in plants due to the combined application of chemical and organic
fertilizer with MBF might be interpreted by N fixation mechanisms, solubilization of
phosphorous, production of IAA of bacterial strains [
]. Approximately 8?10% and 9?18% increase in
grain and straw nitrogen in both years, respectively (Fig 2E and 2F) showed the effectiveness
of microbes to make nitrogen available to crop plants for uptake [
]. Higher N efficiency was
found in T5 treatment indicating that the level, sources and timing of applied nitrogen firmly
matched the N demand of the oat crop. This N level reduced the loss of nitrogen in the
environment through leaching, runoff or other possibly associated mechanism during critical
period of oat growth (jointing-booting stage) and increased the uptake of N and hence, oat
yield. Additionally, as CN was applied with ON and MBF in the combination treatment, initial
N requirement of the oat was ensured by CN whereas, slow mineralization of OM and likely
increased N fixation as indicated by higher abundance of nifH gene (S3 Fig) with higher
enzymatic activities (Table 5), supplied N during the later growth stages of oat. This regulation of
nitrogen supplies under T5 treatment favored the mineral composition of oat for effective
biomass and yield production (S4 Table).
Fate of nitrogen applied in soil
To achieve sustainability in agriculture, it is now mandatory to reduce N loss and manage high
yielding crops properly [
]. In previous studies, it was observed that the losses of N were
higher with the increased doses of applied CN up to 200 kg ha-1 [
]. Ammonia volatilization
is the most crucial way of N loss during the growing season [
], while leaching and
15 / 22
denitrification are the other ways of N loss. The application of ON is an effective way to
mitigate NH3 volatilization losses [
]. As illustrated by our experiment where NH4+-N
content of the top 20 cm soil increased in CN+ ON+ MBF (T5) and ON+MBF (T6) treatment
combinations which might be due to the mineralization of OM resulted from increased
microbial activity by MBF. Nitrogen mineralization from poultry manure was the highest at
jointing-booting stage of the crop (50?65 Days after sowing) (Fig 3A and 3B) which is consistent
with the previous studies where highest mineralization of N was observed after 63 days of
incubation involving poultry manure . Nitrate (NO3-) is often leached down from the
rhizosphere and deposited below at 40 cm depth as is obvious from the low and high NO3-- N
contents at 20cm and 40cm soil depth in treatments T2 and T3 during the jointing booting
stage in our experiment (Fig 3C?3F). The highest NO3--N content and total mineral N at
20cm depth during critical jointing-booting stage of oat across the years (Fig 3C?3D and
3G3H) further emphasized the point that combined use of ON and CN reduce the N loss by
transforming inorganic N to organic N forms (immobilization), thereby increase the fertilizer
efficiency compared with the sole application of CN [
Microbial biomass C and N
Microbial biomass is a crucial component of organic matter ensuring plant nutrient
]. Distinguishable differences in soil microbial biomass level were found between the
treatments in jointing-booting stage, with highest level in the treatment T6; and then T5 and
T7 were in the sequential lower order (Fig 4A and 4B). The C pool in the organic manure,
which can be easily metabolized, contributed to the soil microbial biomass [
affecting crop growth & development directly or indirectly . Soil microbial activity
enhancement was closely linked with high availably of soil N for crop plants [
increments in MBN and MBC in treatments T6 whereas decrement in treatment T2 elicited
the impact of combined application of OM and MBF (Fig 4A and 4B). We speculated that it
might be due to the balance between organic and inorganic pools of nitrogen in the soil, which
acted as a substrate for MBF to mineralization and immobilization available N reserves to
ensure a favorable C/N ratio upon which oat could dwell. Microbial activity is directly related
to soil respiration [
] which in our experiment was highest in T6 treatment (Fig 4C) at
jointing-booting stage. Respiration is considered to reflect the availability of C for microbial
maintenance and is a measure of the basic turnover rates in soil . We reasoned that amended
soil with PM and MBF derived substantial microbial biomass effective for mineralization and
thus increased soil available N for the oat.
Dehydrogenase and other soil hydrolytic enzyme activity
Microbial enzymes in the soil are actually induced by metabolic procedure, generally reflecting
the soil microbial activity level and the biochemical reaction intensities [
]. Current study
illustrated that different fertilizer treatments undoubtedly affected the enzyme activities in soil
(Table 5). Dehydrogenase (DHA) is an important indicator of microbial activity among the
oxyreductase enzyme, and has been treated as a biomarker to assess quality of soil under
]. In this experiment, compared to T2 treatment more than 60% and 70% higher
DHA activity was observed in T5 and T6 treatment respectively, which is highly significant.
Similar trend was observed for other enzymes with increased activities in T5 and T6
treatments (Table 5), mainly due to enhanced microbial activity when combined fertilization was
]. Greater DHA activity is the indicator of an ample availably of organic
degradable substrates in the soil, resulting in higher microbial activities. Dehydrogenase enzyme had
a positive correlation with SOC (P<0. 01, n = 21 and r = 0.95) and soil respiration (P<0. 01,
16 / 22
n = 21 and r = 0.977) (Data not shown) which means increased microbial growth and activity
due to organic substrate in the soil [
]. Enhanced activities of hydrolytic enzymes such as
acid phosphatase, arylsulphatase, ?-glucosidase also indicated that soil function was enhanced
with the incorporation of OM and MBF. On the whole, our results indicated that metabolic
processes were higher in the OM and MBF treated soil as compared to control or soil treated
with CN only, which affected biological transformation of soil N. Apart from reducing
nitrogen losses and increasing nitrogen use efficiency in oat production system, the findings of
current study can be combined with various environmental factors to calibrate or even generate
resource use efficient models for oat as well as other cereal crops in future [
It is necessary to understand the economics and environmental hazards associated with N
losses in agricultural fields. Experimental results of current study revealed the benefits of
combined application of CN, ON and MBF in terms of enhanced biomass and grain yield of oat,
owing to the betterment of soil properties in semi-arid regions of Northeast China. Integrated
application of CN, ON and MBF increased nitrogen availability in the rhizosphere and
reduced N loss, perhaps due to slow release of N from ON and on-time availability during the
most critical period of oat growth. Moreover, the increased microbial activity due to organic N
and MBF improved soil health in terms of soil respiration and contributed to microbial N pool
available to oat for uptake and translocation. Overall, we found 50% CN+50% ON+MBF as an
effective fertilizer dose to minimize nitrogen loss, and maximize NUE and oat production in
semi-arid region of China.
S1 Fig. The daily average temperature and precipitation during the oat growing period in
2016 and 2017.
S2 Fig. Microbial community structure of microbial fertilizers (A) phylum and (B)genus
S3 Fig. Abundance of nifH gene in rhizosphere soil samples under different treatments
(A), taxonomic distribution of the diazotrophic Phyla in different rhizosphere soil samples
(B) and percentage of ?, ? and ? Proteobacteria in different rhizosphere soil samples (C).
Values are mean ? SD (n = 3). Different letters indicate significant differences (P<0.05).
T1 = Control, T2 = 100% CN, T3 = 100% CN + MBF, T4 = 75% CN + 25% ON + MBF,
T5 = 50% CN + 50% ON+ MBF, T6 = 100% ON + MBF and T7 = 100% ON at the 2nd year
jointing booting stage of oat, respectively.
S1 Table. The physical and chemical properties of the soil in experimental field.
S2 Table. Soil chemical properties after 2nd harvesting of Oat. Soil pH, Soil total nitrogen,
NH4+-N and NO3- -N at different soil depths under different fertilizer treatments.
S3 Table. Fertilizer balance under different treatments.
17 / 22
S4 Table. Minerals content in Shoot and root after 2nd year harvesting of oat under
We thank Chen Wang, Qisheng Yang, Junwei Hu, Yadong Yang, Jiang Ying and the members
of Baicheng Academy of Agricultural Sciences for their kind help during field experiments.
Conceptualization: Tariful Alam Khan, Zhaohai Zeng.
Data curation: Tariful Alam Khan.
Formal analysis: Faisal Nadeem.
Funding acquisition: Yuegao Hu.
Investigation: Tariful Alam Khan.
Project administration: Yuegao Hu.
Resources: Lili Chen, Xiaofen Wang, Yuegao Hu.
Supervision: Xiaofen Wang, Yuegao Hu.
Validation: Xiaofen Wang.
Visualization: Zhaohai Zeng, Yuegao Hu.
Writing ? original draft: Tariful Alam Khan.
Writing ? review & editing: Faisal Nadeem, Yuegao Hu.
18 / 22
19 / 22
20 / 22
21 / 22
Wang Q, Feng-Rui L, Zhao L, Zhang E, Shi S, Zhao W, et al. Effect of irrigation and nitrogen application
rates on nitrate nitrogen distribution and fertilizer nitrogen loss, wheat yield and nitrogen uptake on a
recently reclaimed sandy farmland. Plant Soil. 2010; 337:325?339.
1. Santos VB , Araujo SF , Leite LF , Nunes LA , Melo JW . Soil microbial biomass and organic matter fractions during transition from conventional to organic farming systems . Geoderma . 2012 ; 170 : 227 - 231 . https://doi:org/10.1016/j.geoderma. 2011 . 11 .007.
Wang Q , Feng-Rui L , Zhao L , Zhang E , Shi S , Zhao W , et al. Effect of irrigation and nitrogen application rates on nitrate nitrogen distribution and fertilizer nitrogen loss, wheat yield and nitrogen uptake on a recently reclaimed sandy farmland . Plant Soil . 2010 ; 337 : 325 - 339 . https://doi:10.1007/s11104-010- 0530-z.
3. Zaman M , Nguyen ML , Blennerhasssett D. The effect of different rates of urea with or without urease inhibitor (NBPT) on wheat yield and quality . Agric. J . 2010 ; 5 ( 5 ): 309 - 312 . https://doi:10.3923/aj. 2010 . 309 .312.
4. Zhao D , Reddy KR , Kakani VG , Reddy VR . Nitrogen deficiency effects on plant growth, leaf photosynthesis and hyperspectral reflectance properties of sorghum . Eur J Agron . 2005 ; 22 ( 4 ): 391 - 403 . https:// doi:org/10.1016/j.eja. 2004 . 06 .005
5. Emilsson T , Berndtsson JC , Mattsson JE , Rolf K. Effect of using conventional and controlled release fertilizer on nutrient runoff from various vegetated roof systems . Ecol. Eng . 2007 ; 2 ( 9 ): 260 - 271 . https://doi:org/10.1016/j.ecoleng. 2006 . 01 .001.
6. Chandna P , Khurana ML , Ladha JK , Punia M , Mehla RS , Gupta R . Spatial and seasonal distribution of nitrate-N in groundwater beneath the rice-wheat cropping system of India: a geospatial analysis Environ . Monit. Assess . 2011 ; 178 ( 1-4 ): 545 - 62 . https://doi:10.1007/s10661-010-1712-0.
7. Tilman D , Balzer C , Hill J , Befort BL . Global food demand and the sustainable intensification of agriculture . Proc. Natl Acad. Sci . U.S.A. 2011 ; 108 : 20260 - 20264 . https://doi.org/10.1073/pnas.1116437108 PMID: 22106295
8. Rawat SK , Singh RK , Singh RP . Remediation of nitrite in ground and surface waters using aquatic macrophytes . J. Environ. Biol . 2012 : 33 ( 1 ), 51 - 56 . PMID: 23033643
9. Ju X , Liu X , Zhang F , Roelcke M. Nitrogen fertilization, soil nitrate accumulation, and policy recommendations in several agricultural regions of China . Ambio; J. Human Env . 2004 ; 33 ( 6 ): 300 - 305 . PMID: 15387063 . https://doi:org/10.1579/ 0044 -7447- 33 .6. 30
10. Zhu ZL , Chen DL . Nitrogen fertilizer use in China contributions to food production, impacts on the environment and best management strategies . Nutr . Cycl. Agroecosys. 2002 ; 63 : 117 - 127 .
11. Ata-Ul-Karim ST , Zhu Y , Yao X , Cao W. Determination of critical nitrogen dilution curve based on leaf area index in rice . Field Crops Res . 2014 ; 167 : 76 - 85 . https://doi:10.1016/j.fcr. 2014 . 07 .010.
12. Pindi PK , Satyanarayana SDV . Liquid microbial consortium- a potential tool for sustainable soil health . J. Biofertil. Biopestic . 2012 ; 3 ( 4 ): 1 - 9 . https://doi:10.4172/ 2155 - 6202 . 1000124 .
13. Ju XT , Liu XJ , Zhang LJ . 2010 . Nitrogen cycling and eco environment effect of winter wheat-summer maize rotation in North China Plain . In: Fundamental Research on Fate and Efficient Use of Nitrogen Fertilizer in Main Agroecosystems . Science Press, China. pp. 55 - 106 . (in Chinese).
14. Gheysari M , Mirlatifi SM , Homaee M , Asadi ME , Hoogenboom G . Nitrate leaching in a silage maize field under different irrigation and nitrogen fertilizer rates . Agric. Water Manage . 2009 ; 96 : 946 - 954 . https:// doi:10.1016/j.agwat. 2009 . 01 .00.
15. Chandna P , Khurana ML , Ladha JK , Punia M , Mehla RS , Gupta R . Spatial and seasonal distribution of nitrate-N in groundwater beneath the rice-wheat cropping system of India: a geospatial analysis Environ . Monit. Assess . 2011 ; 178 ( 1-4 ): 545 - 62 . https://doi:10.1007/s10661-010-1712-0.
16. Tilman D , Cassman KG , Matson PA , Naylor R , Polasky S. Agricultural sustainability and intensive production practices . Nature . 2002 ; 418 : 671 - 677 . https://doi.org/10.1038/nature01014 PMID: 12167873
17. Yousaf M , Fahad S , Shah AN , Shaaban M , Khan MJ , Sabiel SAI . The effect of nitrogen application rates and timings of first irrigation on wheat growth and yield . Int. J. Agric. Innovat. Res . 2014 ; 2 ( 4 ): 645 - 65 .
18. Tewolde H , Sistani KR , Rowe DW . Broiler litter as a sole nutrient source for cotton: nitrogen, phosphorus, potassium, calcium, and magnesium concentrations in plant parts . J. Plant Nutr . 2005 ; 28 : 605 - 619 . https://doi:10.1081/PLN-200052633.
19. Bolan NS , Szogi AA , Chuasavathi T , Seshadri B , Rothrock MJ Jr., Panneerselvam P. Uses and management of poultry litter . World's Poult.Sci. J . 2010 ; 66 : 673 - 698 . https://doi:10.1017/ S0043933910000656.
20. Rokhzadi A , Toashih V . Nutrient uptake and yield of chickpea (Cicer arietinum L.) inoculated with plant growth promoting rhizobacteria . Aust. J. Crop Sci . 2011 ; 5 ( 1 ): 44 - 48 .
21. Chen GC , He ZL , Huang CY . Microbial biomass phosphorus and its significance in predicting phosphorus availability in red soils . Commun. Soil Sci. Plant Anal . 2000 ; 31 ( 5 &6): 655 - 667 . https://doi:org/10. 1080/0010362000937046.
22. Mandal A , Patra AK , Singh D , Swarup A , Masto RE . Effect of long term application of manure and fertilizer on biological and biochemical activities in soil during crop development stages . Bioresour. Technol . 2007 ; 98 : 3585 - 3592 . https://doi.org/10.1016/j.biortech. 2006 . 11 .027 PMID: 17207997
23. Bhardwaj D , Ansari MW , Sahoo RK , Tuteja N. Biofertilizers function as key player in sustainable agriculture by improving soil fertility, plant tolerance and crop productivity . J. of Microb. Cell Fact . 2014 ; 13 : 66 . https://doi:10.1186/ 1475 -2859-13-66.
24. Ardakani MR , Mazaheri D , Mafakheri S , Moghaddam A . Absorption efficiency of NPK through triple inoculation of wheat (Triticum aestivum L.) by Azospirillum brasilense, Streptomyces sp ., Glomus intaradices and manure application . Physiol. Mol. Biol. Plant . 2011 ; 17 ( 2 ): 181 - 192 . https://doi:10.1007/ s12298-011-0065-7.
25. Kumar M , Bauddh K , Sainger M , Sainger PA , Singh JS , Singh RP . Increase in growth, productivity and nutritional status of rice (Oryza sativa L. cv Bastmati) and enrichment in soil fertility applied with an organic matrix entrapped urea . J. Crop Sci. Biotechnol . 2012 ; 15 ( 2 ): 137 - 144 . https://doi:10.1007/ s12892-012-0024-z.
26. Kumar S , Bauddh K , Barman SC , Singh RP . Amendments of microbial bio fertilizer and organic substances reduces requirement of urea and DAP with enhanced nutrient availability and productivity of wheat (Triticum aestivum L.) . J.Eco. Engin . 2014 ; 71 : 432 - 437 . https://doi.org/10.1016/j.ecoleng. 2014 . 07 .007
27. Mondal SM , Mallikarjun M , Ghosh M , Ghosh DC , Timsina J . Influence of integrated nutrient management (INM) on nutrient use efficiency, soil fertility and productivity of hybrid rice . Arch. Agron. Soil Sci . 2016 ; 62 ( 11 ): 1521 - 1529 , https://doi:10.1080/03650340. 2016 .1148808
28. Afreha D , Zhanga J , Guan D , Liu K , Song Z , Zheng C , et al. Long-term fertilization on nitrogen use efficiency and greenhouse gas emissions in a double maize cropping system in subtropical China . Soil Till. Res . 2018 ; 180 : 259 - 267 . https://doi:org/10.1016/j.still. 2018 . 03 .016.
29. Devkota KP , Devkotab M , Khadka L , Khadka A , Paudel G , Acharya S , et al. Nutrient responses of wheat and rapeseed under different crop establishment and fertilization methods in contrasting agroecological conditions in Nepal . Soil Till. Res . 2018 ; 181 : 46 - 62 . https://doi:10.1016/j.still. 2018 . 04 .001.
30. Nadeem F , Ahmad Z , Wang R , Han J , Shen Q , Chang F , et al. Foxtail Millet [Setaria italica (L.) Beauv.] Grown under Low Nitrogen Shows a Smaller Root System, Enhanced Biomass Accumulation, and Nitrate Transporter Expression . Front. Plant Sci . 2018 ; 9 : 205 . https://doi.org/10.3389/fpls. 2018 .00205 PMID: 29520286
31. Rosch C , Mergel A , Bothe H . Biodiversity of denitrifying and dinitrogen fixing bacteria in an acid forest soil . Appl. And Environ. Microbiol . 2002 ; 68 ( 8 ): 3818 - 3829 . PMCID: PMC124007 .
32. Nelson DW , Sommers LE . Total nitrogen analysis for soil and plant tissues . J Assoc Off Anal Chem . 1980 ; 63 : 770 - 778 .
33. Masson P , Dalix T , Bussi?re S. Determination of Major and Trace Elements in Plant Samples by Inductively Coupled Plasma-Mass Spectrometry , Communications in Soil Science and Plant Analysis . 2010 ; 41 : 3 , 231 - 243 . https://doi:10.1080/00103620903460757.
34. Nelson DW , Sommers LE . Total carbon, organic carbon and organic matter . In: Page A.L. , Miller R.H. , Keeney D.R . (Eds.), Methods of Soil Analysis. Part 2. Chemical and Microbiological Properties. Agronomy Monograph 9 , 2nd ed. Agronomy Society of America and Soil Science Society of America , Madison, WI. 1982 ; pp: 539 - 577 .
35. Vance ED , Brookes PC , Jenkinson DS . An extraction method for measuring soil microbial biomass C. Soil Biol . Biochem . 1987 ; 19 : 703 - 707 .
36. Smolander A , Kitunen V . Soil microbial activities and characteristics of dissolved organic C and N in relation to tree species . Soil Biol . Biochem . 2002 ; 34 : 651 - 660 . https://doi:org/10.1016/S0038- 0717 ( 01 ) 00227 - 9
37. Jenkinson DS , Brookes PC , Powlson DS . Measuring soil microbial biomass . Soil Biol . Biochem . 2004 ; 36 : 5 - 7 . https://doi:10.1016/j.soilbio. 2003 . 10 .002.
38. Salamanca EF , Raubuch N , Joergensen RG . Relationships between soil microbial indices in secondary tropical forest soils . Appl. Soil Ecol . 2002 ; 21 : 211 - 219 .doi:org/10.1016/S0929- 1393 ( 02 ) 00091 - 4 .
39. Casida LE Jr, Klein DA , Santoro R . Soil dehydrogenase activity . Soil Sci . 1964 ; 98 : 371 - 378 .
40. Gil-Sotres F , Trasar-Cepeda MC , Ciardi C , Ceccanti B , Leiro?s MC. Biochemical characterization of biological activity in very young mine soils . Biol. Fert. Soils . 1992 ; 13 : 25 - 30 .
41. Tabatabai MA , Bremner JM . Use of p-nitrophenyl phosphate for assay of soil phosphatase activity . Soil Biol . Biochem . 1969 ; 1 : 301 - 307 .
42. Eivazi F , Tabatabai MA . Glucosidases and galactosidases in soils . Soil. Biol.Biochem . 1988 ; 20 : 601 - 606 .
43. Tabatabai MA , Bremner JM . Arylsulfatase activity of soils . Soil Sci. Soc. Am.Proc . 1970 ; 34 : 225 - 229 .
44. Li P , Lu J , Hou W , Pan Y , Wang Y , Khan MR . Reducing nitrogen losses through ammonia volatilization and surface runoff to improve apparent nitrogen recovery of double cropping of late rice using controlled release urea . Environ. Sci. Pollut. Res . 2017 ; 24 : 11722 - 11733 . https://doi:10.1007/s11356017-8825- 8.
45. Naveed M , Khalid M , Jones DL , Ahmad R , Zahir ZA . Relative efficacy of Pseudomonas spp., containing ACC-deaminase for improving growth and yield of maize (Zea mays L.) in the presence of organic fertilizer . Pak. J. Bot . 2008 ; 40 : 1243 - 1251 .
46. Shaharoona B , Naveed M , Arshad M , Zahir ZA . Fertilizer dependent efficiency of Pseudomonads for improving growth, yield, and nutrient use efficiency of wheat (Triticum aestivum L.) . Appl Microbiol Biotechnol . 2008 ; 79 : 147 - 155 . https://doi.org/10.1007/s00253-008 -1419-0 PMID: 18340443
47. Bayu W , Rethman NFG , Hammes PS , Alemu G. Effects of farmyard manure and inorganic fertilizers on sorghum growth, yield, and nitrogen use in a semi-arid area of Ethiopia . J. Plant Nutr . 2006 ; 29 : 391 - 407 .https://doi:10.1080/01904160500320962.
48. Yang J , Gao W , Ren S. Long-term effects of combined application of chemical nitrogen with organic materials on crop yields, soil organic carbon and total nitrogen in fluvo-aquic soil . Soil Tillage Res . 2015 ; 151 : 67 - 74 . https://doi:10.1016/j.still. 2015 . 03 .008.
49. Wei W , Yan Y , Cao J , Christie P , Zhang F , Fan M. Effects of combined application of organic amendments and fertilizers on crop yield and soil organic matter: an integrated analysis of long-term experiments . Agric. Ecosyst. Environ . 2016 ; 225 : 86 - 92 . https://doi:10.1016/j.agee. 2016 . 04 .004.
50. Zhang J , Wang M , Cao Y , Liang P , Wu S , Leung AOW . Replacement of mineral fertilizers with anaerobically digested pig slurry in paddy fields: assessment of plant growth and grain quality . Environ. Sci. Pollut.Res . 2017 ; 24 : 8916 - 8923 . https://doi:10.1007/s11356-015-5125-z.
51. Naveed M , Khalid M , Jones DL , Ahmad R , Zahir ZA . Relative efficacy of Pseudomonas spp., containing ACC-deaminase for improving growth and yield of maize (Zea mays L.) in the presence of organic fertilizer . Pak. J. Bot . 2008 ; 40 : 1243 - 1251 .
52. Khaliq A , Abbasi MK , Hussain T . Effect of integrated use of organic and inorganic nutrient sources with effective micro-organisms (EM) on seed cotton yield in Pakistan . Bioresour. Technol . 2006 ; 97 : 967 - 972 . https://doi.org/10.1016/j.biortech. 2005 . 05 .002 PMID: 16023343
53. Seufert V , Ramankutty N , Foley JA . Comparing the yields of organic and conventional agriculture . Nature . 2012 ; 485 : 229 - 232 . https://doi.org/10.1038/nature11069 PMID: 22535250
54. Han KH , Choi WJ , Han GH , Yun SI , Yoo SH , Ro HM . Urea-nitrogen transformation and compost-nitrogen mineralization in three different soils as affected by the interaction between both nitrogen inputs . Biol. Fertil. Soils . 2004 ; 39 : 193 - 199 . https://doi:10.1007/s00374-003-0704-4.
55. Ke J , Xing X , Li G , Ding Y , Dou F , Wang S. Effects of different controlled-release nitrogen fertilizers on ammonia volatilization, nitrogen use efficiency and yield of blanket-seedling machine-transplanted rice . Field Crops Res . 2017 ; 205 : 147 - 156 . https://doi:10.1016/j.fcr. 2016 . 12 .027.
56. Dent D , Cocking E. Establishing symbiotic nitrogen fixation in cereals and other non-legume crops: The Greener Nitrogen Revolution . Agric. & Food Secur . 2017 ; 6:7 . https://doi:10.1186/s40066-016-0084-2.
57. Hoffman BM , Lukoyanov D , Yang ZY , Dean DR , Seefeldt L . Mechanism of Nitrogen Fixation by Nitrogenase: The Next Stage . Chem. Rev . 2014 ; 114 ( 8 ): 4041 - 4062 . https://doi.org/10.1021/cr400641x PMID: 24467365
58. Narula N , Kumar V , Singh B , Bhatia R , Lakshminarayana K. Impact of Biofertilizers on grain yield in spring wheat under varying fertility conditions and wheat-cotton rotation . Arch. of Agron. And Soil Sci . 2011 ; 51 ( 1 ): 79 - 89 . https://doi:10.1080/03650340400029382.
59. Willmann M , Gerlach N , Buer B , Polatajko A , Nagy R , Koebke E , et al. Mycorrhizal phosphate uptake pathway in maize: vital for growth and cob development on nutrient poor agricultural and greenhouse soils . Front. Plant Sci . 2013 ; 4 : 533 . https://doi.org/10.3389/fpls. 2013 .00533 PMID: 24409191
60. Olanrewaju OS , Glick BR , Babalola OO . Mechanisms of action of plant growth promoting bacteria . World J. Microbiol. Biotechnol . 2017 ; 33 : 197 . https://doi.org/10.1007/s11274-017 -2364-9 PMID: 28986676
61. Yaduvanshi NPS . Substitution of inorganic fertilizers by organic manures and the effect on soil fertility in rice-wheat rotation on reclaimed sodic soil in India . J. Agric. Sci . 2003 ; 140 : 161 - 168 . https://doi:org/ 10.1017/S0021859603002934.
62. Egamberdiyeva D. The effect of plant growth promoting bacteria on growth and nutrient uptake of maize in two different soils . Appl. Soil Ecol . 2007 ; 36 ( 2-3 ): 184 - 189 . https://doi:org/10.1016/j.apsoil. 2007 . 02 .005.
63. Egamberdiyeva D , Ho?flich G. Effect of plant growth promoting bacteria on growth and nutrient uptake of cotton and pea in a semi-arid region of Uzbekistan . J. Arid Environ . 2004 ; 56 ( 2 ): 293 - 301 . https://doi: org/10.1016/S0140-1963( 03 ) 00050 - 8 .
64. Khan AM , Zaidi A . Synergistic effects of the inoculation with plant growth-promoting rhizobacteria and an arbuscular mycorrhizal fungus on the performance of wheat . Tur. J. Agric. For . 2007 ; 31 : 355 - 362 .
65. Salantur A , Ozturk A , Akten S , Sahin F , Donmez F. Effect of inoculation with non-indigenous and indigenous rhizobacteria of Erzurum (Turkey) origin on growth and yield of spring barley . Plant Soil . 2005 ; 275 : 147 - 156 . https://doi:10.1007/s11104-005-8094-z.
66. Zaidi A , Khan MS . Interactive effect of rhizotrophic microorganisms on growth, yield, and nutrient uptake of wheat . J. Plant Nutr . 2005 ; 28 : 2079 - 2092 . https://doi:10.1080/01904160500320897
67. Duan YH , Shi XJ , Li SL , Sun XF , He XH . Nitrogen use efficiency as affected by phosphorus and potassium in long-term rice and wheat experiments . J. Integr. Agric . 2014 ; 13 : 588 - 596 . https://doi:10.1016/ S2095- 3119 ( 13 ) 60716 - 9 .
68. Sepaskhah AR , Tafteh A . Yield and nitrogen leaching in rapeseed field under different nitrogen rates and water saving irrigation . Agric. Water Manage . 2012 ; 112 : 55 - 62 . https://doi:10.1016/j.agwat. 2012 . 06 .005.
69. Xu M , Li D , Li J , Qin D , Hosen Y , Shen H . Polyolefin-coated urea decreases ammonia volatilization in a double rice system of Southern China . Agron. J. 2013 ; 105 : 277 - 284 . https://doi:10.2134/agronj2012. 0222 .
70. Huang S , Lv W , Bloszies S , Shi Q , Pan X , Zeng Y. Effects of fertilizer management practices on yieldscaled ammonia emissions from croplands in China: a meta-analysis . Field Crops Res . 2016 ; 192 : 118 - 125 . https://doi:10.1016/j.fcr. 2016 . 04 .023.
71. Abbasi MK , Khaliq A . Nitrogen Mineralization of a Loam Soil Supplemented with Organic-Inorganic Amendments under Laboratory Incubation . Front. Plant Sci . 2016 ; 7 : 1038 . https://doi.org/10.3389/fpls. 2016 .01038 PMID: 27493649
72. Abbasi MK , Khaliq A , Shafiq M , Kazmi M , Ali I . Comparative effectiveness of urea N, poultry manure and their combination in changing soil properties and maize productivity under rain fed conditions in northeast Pakistan . Expl. Agric . 2010 ; 46 ( 2 ): 211 -230 C. https://doi:10.1017/S0014479709991050.
73. Rashid MI , Mujawar LH , Shahzade T , Almeelbi T , Ismail IM , Ovesa M. Bacteria and fungi can contribute to nutrients bioavailability and aggregate formation in degraded soils . Microbiol. Res . 2016 ; 183 : 26 - 41 . https://doi.org/10.1016/j.micres. 2015 . 11 .007 PMID: 26805616
74. Tejada M , Garcia C , Gonzalez JL , Hernandez MT . Use of organic amendment as a strategy for saline soil remediation: Influence on the physical, chemical and biological properties of soil . Soil Biol . Biochem . 2006 ; 38 : 1413 - 1421 . https://doi:10.1016/j.soilbio. 2005 . 10 .017.
75. Dijkstra FA , Bader NE , Johnson DW , Cheng W. Does accelerated soil organic matter decomposition in the presence of plants increase plant N availability? Soil Biol . Biochem . 2009 ; 41 : 1080 - 1087 .https://doi: org/10.1371/journal.pone. 0108555 .
76. Jannoura R , Joergensen RG , Bruns C . Organic fertilizer effects on growth, crop yield, and soil microbial biomass indices in sole and intercropped peas and oats under organic farming conditions . Europ. J. Agronomy . 2013 ; 52 : 259 - 270 . https://doi:org/10.1016/j.eja. 2013 . 09 .001.
77. Tu C , Ristaino JB , Hu S. Soil microbial biomass and activity in organic tomato farming systems: Effects of organic inputs and straw mulching . Soil Biol Biochem . 2006 ; 38 : 247 - 255 . https://doi:10.1016/j. soilbio. 2005 . 05 .002.
78. Melero S , Porras JCR , Herencia JF , Madejo?n E. Chemical and biochemical properties in a silty loam soil under conventional and organic management . Soil Till. Res . 2006 ; 90 : 162 - 170 . https://doi:10. 1016/j.still. 2005 . 08 .016.
80. Garc? ?a-Ruiz R , Ochoa V , Bele?n-Hinojosa M , Carreira JA . Suitability of enzyme activities for the monitoring of soil quality improvement in organic agricultural systems . Soil Biol . Biochem . 2008 ; 40 : 2137 - 2145 . https://doi:10.1016/j.soilbio. 2008 . 03 .023.
81. Liang Y , Nikolic M , Peng Y , Chen W , Jiang Y. Organic manure stimulates biological activity and barley growth in soil subject to secondary salinization . Soil Biol . Biochem . 2005 ; 37 : 1185 - 1195 . https:// doi:10.1016/j.soilbio. 2004 . 11 .017.
82. Madejo?n E , Murillo JM , Moreno F , Lo?pez MV, Arrue JL , Alvaro-Fuentes J , et al. Effect of long-term conservation tillage on soil biochemical properties in Mediterranean Spanish areas . Soil Till. Res . 2009 ; 105 : 55 - 62 . https://doi:10.1016/j.still. 2009 . 05 .007.
83. Caravaca F , Ceccanti B . Land use in relation to soil chemical and biochemical properties in a semiarid Mediterranean environment . Soil Till. Res . 2002 ; 68 : 23 - 30 . https://doi:org/10.1016/S0167-1987( 02 ) 00080 - 6 .
84. Peake A , Whitbread A , Davoren B , Braun J , Limpus S. The development of a model in APSIM for the simulation of grazing oats and oaten hay . In Global Issues. Paddock Action.? Proceedings of 14th Agronomy Conference: Adelaide , South Australia. 2008 .
85. Ojeda JJ , Caviglia OP , Irisarri JG , Agnusdei MG . Modelling inter-annual variation in dry matter yield and precipitation use efficiency of perennial pastures and annual forage crops sequences . Agricultural and forest meteorology. 2018a; 259 : 1 - 10 . https://doi.org/10.1016/j.agrformet. 2018 . 04 .014
86. Ojeda JJ , Pembleton KG , Caviglia OP , Islam MR , Agnusdei MG , Garcia SC . Modelling forage yield and water productivity of continuous crop sequences in the Argentinian Pampas . European Journal of Agronomy. 2018b; 92 : 84 - 96 . https://doi.org/10.1016/j.eja. 2017 . 10 .004