Soil enzymatic activity as affected by l (2)
european journal of soil biology 44 (2008) 309–315
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Original article
Soil enzymatic activity as affected by long term application
of farm yard manure and mineral fertilizer under
a rainfed soybean–wheat system in N-W Himalaya
Supradip Saha*, Ved Prakash, Samaresh Kundu, Narendra Kumar, Banshi Lal Mina
Vivekananda Institute of Hill Agriculture, Indian Council of Agricultural Research, Almora – 263 601, Uttarakhand, India
article info
abstract
Article history:
Long-term experimental sites are expected to provide important information regarding soil
Received 8 June 2007
properties as affected by management practices. This study was designed to examine the
Accepted 29 February 2008
effects of continuous fertilization, and manuring on the activities of enzymes involved in
Published online 26 March 2008
mineralization of C, N, and P on a long term (33 years) field trial under sub-temperate
conditions in India. Treatments at the site included application of recommended doses
Keywords:
of nitrogen and phosphorus (NP), nitrogen and potassium (NK), nitrogen, phosphorus
Long-term experiment
and potassium (NPK), farmyard manure (FYM) with N (N þ FYM), FYM with NPK
Soil carbohydrate
(NPK þ FYM) and un-amended control (C). The study was done under rainfed soybean–
Soil enzymes
wheat rotation. Manure application increased soil carbohydrate, dehydrogenase, acid
Nutrient dynamics
and alkaline phosphatases, cellulase, and protease activity significantly. Urease activity
was not influenced by the manure treatment and the activity was highest in controls.
Both acid and alkaline phosphatase activities were negatively influenced by chemical
fertilizer treatment. Almost all the enzymes studied were significantly correlated with
soil C content. The results suggest that application of FYM directly or indirectly influences
the enzyme activity and it in turn regulates nutrient transformation.
ª 2008 Elsevier Masson SAS. All rights reserved.
1.
Introduction
In the effort to achieve sustainable agricultural production
while maintaining and preserving the environment, it is
crucial that soil biological health be improved or maintained.
Agricultural practices that improve soil quality and agricultural sustainability have been receiving more attention from
researchers and farmers [14]. Inorganic fertilizer, especially
N, P and K, not only serve to maintain, but their application
directly or indirectly causes changes in chemical, physical
and biological properties of the soil. These changes, in the
long term, are believed to have significant influences on the
quality and productive capacity of soils.
Soil microorganisms, particularly microbiota, play an
essential role in the cycling of elements and stabilization of
soil structure [5,32]. They also act as both a source and sink
of labile nutrients and C [20,47]. The mineralization of organic
matter is carried out by a large community of microorganisms
and involves a wide range of metabolic processes. Soil
enzymes are believed to be able to discriminate between soil
* Corresponding author. Tel.: þ91 5962 230060.
E-mail address: s_supradip@yahoo.com (S. Saha).
1164-5563/$ – see front matter ª 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejsobi.2008.02.004
310
european journal of soil biology 44 (2008) 309–315
management practices probably because they are related to
microbial biomass, which is sensitive to such treatments
[32]. Soil enzymes regulate the transformation process of
elements required for plant growth in soil [6]. The activity of
soil enzyme, whether extracellular or intracellular depends
on crop rotation, amendments, tillage and agricultural
management [14,24]. Transformation of N in soils includes
different processes, which are regulated by a number of extracellular degradative enzymes [26,50]. Transformation of
organic P through enzymatic reactions and the immobilization of P in the biomass itself play a fundamental role in P
cycling [35,36,46] and are likely to be affected by P amendments [10,27].
Although some researchers commented that soil organic
matter was not a plant growth requirement in-and-of itself
[38], almost all have realized its ecological significance in
overcoming constraints to crop growth, particularly, in the
areas of nutrient supply, nutrient and moisture relation, soil
structural stability and detoxification [42]. Information is
also needed to assess the contribution of the organic matter
in maintaining the soil microbial community and which in
turn, is related to specific enzymatic reactions, i.e. to biochemical processes involved in different nutrient cycling for assessing sustainability.
Long-term field treatments provide a means of investigating the influences of organic amendments on soil characteristics [8]. However, relatively limited consistent information is
available concerning changes in soil biological properties, under long-term field conditions of a N-W Himalayan ecosystem.
The objective of this study was to ascertain changes in soil
enzymatic activity in response to repeated applications of
organic amendment vis-a`-vis inorganic nutrients under field
conditions over several years. For this reason, soil enzymatic
properties, especially those related to the cycles of C, N, and
P in the soil, was compared in soils from a long-term field
experiment.
2.
Materials and methods
2.1.
Experimental site
A long-term field experiment was initiated in June 1973 on
a Typic Haplaquept at the experimental farm of Vivekananda
Institute of Hill Agriculture, located in the Indian Himalayan
region at Hawalbagh (29 360 N and 79 400 E at 1250 m above
mean sea level), in the state of Uttarakhand, India. Soil characteristics based on analysis of preserved soil samples taken in
1973 are given in Table 1. The climate is sub-temperate,
characterized by a moderate summer (May–Jun), extreme
winter (Dec–Jan) and general dryness, except during the
southwest monsoon season (Jun–Sep). Based on the records
for the period 1973–2006, average kharif (monsoon) season
rainfall was 684 mm (e.g. 68% of the average annual rainfall).
During soybean growth, average monthly rainfall (mm) was:
Jun (146), Jul (257), Aug (187), Sep (119), whereas, during wheat
growth the average monthly rainfall (mm) was: Oct (27), Nov
(6), Dec (28), Jan (53), Feb (56), Mar (48), Apr (42). The mean
monthly temperature ranged from a minimum of 0.4 C in
January to a maximum of 31.5 C in May.
Table 1 – Initial soil physico-chemical properties
(0–15 cm)
Properties
pH (soil:water, 1:2.5)
EC (dS m1)
CEC (cmol (pþ) kg1 soil)
Bulk density (Mg m3)
Total organic C (g kg1 soil)
C:N
Available N (mg kg1 soil)
Available P (mg kg1)
Available K (mg kg1)
Texture
Taxonomic classification
2.2.
Value
6.2
0.08
8.7
1.32
5.8
12.8
127
12
65
Sandy loam
Typic Haplaquept
Experimental design
The experiment included two crops per year, soybean (Jun–
Sep) and wheat (Oct–Apr), with six treatment combinations
(in kg ha1): no fertilizer and no manure (un-amended
control); 20 N þ 35 P (NP); 20 N þ 33 K (NK); 20 N þ 35 P þ 33
K (NPK), 20 N þ FYM at 10 Mg ha1 (N þ FYM, commonly
used by the local farmers) and NPK þ FYM at 10 Mg ha1
(NPK þ FYM). The required doses of FYM and fertilizers were
given only to the soybean crop every year and the wheat
crop was allowed to grow on residual fertility. Treatments
were distributed in a randomized block design with six replications over three uniformly level terraces. The net plot size
was 5.4 2.0 m. Fertilizers used were urea for N, single
super-phosphate for P and murate of potash for K. Based on
the chemical analysis of every fifth year, FYM (C:N z 23.6:1)
had 370 g of moisture kg1 and contained 7.1–7.5 g N kg1,
2.1–2.4 g P kg1 and 5.3–5.8 g K kg1 on an oven-dry weight
basis. FYM was prepared using cattle dung mixed with urine
and bedding material. Oak (Quercus leucotrichophora) leaves
(1.1% N, 0.55% P, 0.74% K) were mostly used as bedding material in cattle sheds. These materials were thoroughly mixed
and stored in a heap under shade. The heap was plastered
with a 2-inch thick layer of soil and dung and was left undisturbed for 45 days. Full doses of N, P, K and FYM were incorporated into the upper 5 cm of soil during final land preparation.
2.3.
Soil sampling for chemical and
microbial analysis and preparation
Initial soil samples were collected in 1973 prior to the start of
the experiment. After harvest of wheat, soil samples were
taken from the surface layer (0–15 cm) of six treatments
with six replications in May, 2006, before the start of land
management for soybean. Random cores were taken from
each plot with a 5-cm diameter tube auger and bulked. The
moist soil samples were sieved (2 mm) after removing plant
material and roots. Half of the soil samples were air-dried
and stored at room temperature until chemical analysis. All
chemical results are means of triplicate analyses and are
expressed on an oven-dry basis. Soil moisture was determined
after drying at 105 C for 24 h.
The rest of the sieved soil (2 mm) was immediately transferred to the laboratory for microbiological analysis. Soil
311
european journal of soil biology 44 (2008) 309–315
samples were kept at 4 C in plastic bags for a few days to
stabilize the microbiological activity disturbed during soil
sampling and handling, and then analysed within 2 weeks.
All microbial results reported are means of six replicates and
are expressed on a moisture-free basis. Moisture content
was determined after drying at 105 C for 24 h.
the enzyme reaction was stopped by adding 4 ml of 0.5 M
NaOH and 1 ml of 0.5 M CaCl2 to prevent dispersion of humic
substances. After centrifugation at 4000 rpm for 10 min, the absorbance was measured in the supernatant at 400 nm; enzyme
activity was expressed as mg p-nitrophenol released g1 soil h1.
2.6.
2.4.
Soil was analyzed for pH in a 1:2.5 soil:water suspension [19],
soil texture was determined by Bouyoucos hydrometer [4] and
available K by 1 N NH4OAc using a flame photometer [19]. Soil
was analysed for oxidizable SOC by the method of Walkley
and Black [49], Kjeldahl N by FOSS Tecator (Model 2200), and
available P following the Olsen method [33]. A core sampler
was used for soil bulk density determination. Soil was analysed for total C and N using a CHN analyser (FOSS Heraeus
CHNORapid) following a dry combustion method.
Soil carbohydrate was estimated following the modified
method of Martens and Loeffelmann [29]. The soil sample
(100 mg) was first solubilized and then hydrolysed with 1 M
H2SO4 to get monosaccharides, which were estimated colorimetrically using the phenol–sulfuric acid method [11].
2.5.
Grain yield
Chemical analyses
Microbiological analyses
Soil dehydrogenase activity was estimated by reducing
2,3,5-triphenyltetrazolium chloride [7]. Five grams of soil
sample were mixed with 50 mg of CaCO3 and 1 ml of 3% (w/
v) 2,3,5-triphenyltetrazolium chloride (TTC) and incubated
for 24 h at 37 1 C. Dehydrogenase enzyme converts TTC to
2,3,5-triphenylformazan (TPF). The TPF formed was extracted
with acetone (3 15 ml), the extracts were filtered through
Whatman No. 5 and absorption was measured at 485 nm
with a spectrophotometer (Analytik Jena, Germany).
Cellulase activity was determined by estimating the
glucose equivalent after mixing the soil sample (10 g) with
15 ml of acetate buffer (2 M, pH 5.5) and 15 ml of 0.7% (w/v)
carboxy methyl cellulose and incubated for 24 h at 50 1 C
[39]. The glucose equivalent was estimated following the
DNS method [30].
Protease activity was assayed by determining the tyrosine
released when 1 g of the oven-dry equivalent of field-moist
soil sample was incubated with 5 ml of 50 mM Tris buffer
(pH 8.1) and 5 ml of 2% Na-caseinate at 50 1 C for 2 h [25].
The aromatic amino acids released were extracted and the
remaining substrate was precipitated with 0.92 M trichloroacetic acid and measured colorimetrically using Folin–Ciocalteu reagent at 700 nm. The activity of protease was expressed
as mg tyrosine produced g1 soil 2 h1.
Urease activity was measured following the method of
Tabatabai and Bremner [44]. Five grams of soil were incubated
with 5 ml of 0.05 M THAM buffer (pH 9.0) and 1 ml of 0.2% of
urea solution at 37 C for 2 h. Excess urea was extracted with
KCl–PMA solution and estimated colorimetrically at 527 nm.
Phosphomonoesterase (acid and alkaline phosphatase) activity was assayed using 1 g of soil (wet equivalent), 4 ml of
0.1 M modified universal buffer (pH 11 for alkaline phosphatase
and pH 6.5 for acid phosphatase), and 1 ml of 25 mM p-nitrophenyl phosphate [43]. After incubation for 1 h at 37 1 C
Both soybean and wheat were harvested at 5 cm above the soil
surface in the first week of October and the fourth week of
April, respectively. Grain yield was expressed on a 12 and 9%
moisture basis for wheat and soybean, respectively.
2.7.
Statistical analyses
Each sample was analyzed in triplicate and the values were
then averaged. Data were assessed by Duncan’s multiple
range tests (1955) with a probability P 0.05 [12]. Differences
between mean values were evaluated by a one-way analysis
of variance (ANOVA) (SPSS version 10.0). Pearson correlation
analyses were performed using the SPSS programme.
3.
Results
3.1.
Soil pH and oxidizable carbon
Table 1 gives the initial physico-chemical properties of the soil
used for the long term experiment. Taxonomically the soil
was classified as Typic Haplaquept. The pH values in
0–15 cm of surface soil ranged from 5.3 to 6.5, with that of
the untreated control soil around pH 5.7. Manure application
increased soil pH significantly, while chemical fertilizer application resulted in lowering of soil pH (Table 2). NK treatment
lowered the soil pH value up to 5.3.
In the surface 0–15 cm, soil oxidizable carbon was less in
the different fertilized treatments than in FYM treated plots
(Table 2). In different fertilized treatments, oxidizable-C followed the order NK < NP < NPK. Application of FYM along
with NPK contributed significantly to incorporation of C in
soil. Addition of FYM at 10 Mg ha1 year1 along with NPK
for 33 years doubled the organic C content than NPK alone.
3.2.
Soil carbohydrate and dehydrogenase activity
Soil total polysaccharide content for six different treatments
are tabulated in Fig. 1. Carbohydrate content in control and
Table 2 – Soil pH, total SOC and nitrogen, C-to-N ratio of
a Typic Haplaquept (0–15 cm) affected by mineral
fertilizer and manure treatment
Treatment
pH
TOC (g kg1)
N (g kg 1)
C:N
C
NP
NK
NPK
N þ FYM
NPK þ FYM
5.7
5.6
5.3
5.4
6.4
6.5
6.34
7.51
6.88
8.06
11.09
11.89
0.560
0.692
0.581
0.651
0.782
0.856
11.3:1
10.9:1
11.8:1
12.4:1
14.2:1
13.9:1
312
1.4
Dehydrogenase
CHO ( )
40
dc
bd
cd 1.0
cc
30
cd
bc
ac
1.2
ac
0.8
0.6
bc
20
Glucose equivalent
g TPF produced g-1 h-1
50
0.4
10
ad
ad
ad
ad
NK
NPK
g urea hydrolysed g-1 h-1/
g tyrosine produced g-1 2h-1
european journal of soil biology 44 (2008) 309–315
100
bc
80
60
a
a
40
b
20
b
a
a
0
Control
N+FYM NPK+FYM
NP
NK
NPK
N+FYM NPK+FYM
NK treatment was at par. NPK þ FYM treatment (1.14%) was
best for contributing soil carbohydrate content. Values for
other treatments increased in the order NPK (0.67%) < N þ
FYM (0.74%) < NP (0.879).
Changes in dehydrogenase activity in differently treated
soils are shown to be distributed across a wide range (Fig. 1).
Only fertilized treatments along with controls showed significantly lower intracellular metabolism of soil microbes, which
depicts dehydrogenase activity. NP, NK and NPK showed
similar levels of activity, at par with the controls. FYM application increased enzymatic activity a few times when it was
applied along with N or NPK.
Soil enzymatic activities
Enzyme activities varied widely among the treatments studied. The treatment with the highest activity was found to
vary depending on the enzyme. The highest value for cellulase
activity was found in NPK þ FYM, followed by N þ FYM treatments that received organic amendments (Fig. 2). Invertase
activity did not vary much across treatments and the highest
value was found in treatments that received organic manure
along with the recommended fertilizer. Protease activity in
the soil followed a similar trend and was highest in manure
500
Cellulase
Invertase
400
c
b
bc
300
200
100
a
a
a
a
ab
ab
ab
b
b
0
Control
NP
NK
NPK
N+FYM NPK+FYM
Fig. 2 – Cellulase and invertase activities in soil with
different treatments. Bars sharing the same letter are not
significantly different (P < 0.05). Error bars represent
standard deviation.
Fig. 3 – Urease and protease activities in soil with different
treatments. Bars sharing the same letter are not
significantly different (P < 0.05). Error bars represent
standard deviation.
amended treatments. Unlike other enzymes, urease activity
was inhibited in the organic amended soils (Fig. 3). The highest value was found in non-amended controls followed by
NP and NPK þ FYM. Fig. 4 shows acid and alkaline phosphatase activities for all the treatments. As the pH of the soil
lies within the acidic range, it might be expected that acid
phosphatase is the dominant activity in this soil [13]. The
highest value was found in NPK þ FYM followed by N þ FYM.
For other treatments, the activity was far below these two
treatments. Activity of the manure treated soil was 2–3-fold
greater than that in soils from the other treatments. A similar
trend was observed in the case of alkaline phosphatase
activity. NPK treated soil was least and NPK þ FYM was
highest for this enzyme activity.
A correlation matrix (Table 3) shows some significant relationships among the enzymes studied. There was positive
correlation between cellulase and invertase, which are
involved in C transformation. Total organic carbon was correlated with all enzymes assayed except urease and dehydrogenase. Cellulase also showed a strong positive correlation with
protease and phosphatase. Acid phosphatase shared a strong
correlation with protease and alkaline phosphatase. Protease
activity was strongly correlated (r ¼ 0.953, P < 0.001) with the
N content of the soil. Cellulase activity was also correlated
with invertase activity (r ¼ 0.802, P < 0.01).
g p-nitrophenol produced g-1 h-1
NP
Fig. 1 – Dehydrogenase activity and carbohydrate content
in soil with different treatments. Bars sharing the same
letter are not significantly different (P < 0.05). Error bars
represent standard deviation. ac and ad represent
carbohydrate and dehydrogenase respectively.
mg glucose eq. g-1 24 h-1/mg
glu eq. g-1 h-1
c
c
0.0
Control
3.3.
bc
b
0.2
0
Urease
Protease
c
200
c
Acid Phosphatase
Alkaline Phosphatase
b
150
d
100
a
a
50
b
ab
c
a
a
a
a
0
Control
NP
NK
NPK
N+FYM
NPK+FYM
Fig. 4 – Acid and alkaline phosphatase activities in soil
with different treatments. Bars sharing the same letter are
not significantly different (P < 0.05). Error bars represent
standard deviation.
313
european journal of soil biology 44 (2008) 309–315
Table 3 – Linear correlation coefficient between nutrient and enzyme activities of soil
TOC
N
DA
Cell
Inv
Ure
Pro
AcP
N
DA
Cell
Inv
Ure
Pro
AcP
AkP
0.830**
0.881**
0.893**
0.910**
0.670
0.725
0.821**
0.901**
0.427
0.802*
0.240
0.121
0.092
0.040
0.245
0.922**
0.853**
0.411
0.887**
0.783*
0.294
0.872**
0.844**
0.301
0.801*
0.740*
0.418
0.909**
0.928***
0.804**
0.502
0.767*
0.708
0.439
0.810*
0.920***
TOC, total organic carbon; N, total N; DA, dehydrogenase; Cell., cellulase; Inv, invertase; Ure, urease; Pro, protease; AcP, acid phosphatase;
AkP, alkaline phosphatase. *P < 0.1, **P < 0.05, ***P < 0.001 respectively.
3.4.
Grain yield
Mean yield of soybean and wheat after 33 years of cropping is
presented in Table 4. Soybean yield varied from 0.56 Mg ha1
for C to 2.93 Mg ha1 for NPK þ FYM. In the case of wheat, it
ranged from 0.72 to 1.91 Mg ha1, highest for NPK þ FYM and
lowest for C.
4.
Discussion
Soil pH ranged from 5.3 in the NK plots, which is significantly
lower than for all other treatments, to 6.5 in the NPK þ FYM
plots. The pH of the N treated plots was significantly lower
than that of the C and manured plots. Long term applications,
especially N, therefore had acidifying effects resulting in
lowering of pH. This confirms earlier findings that most
N-containing fertilizers tend to acidify soil [1,2]. This is mainly
due to the fact that most fertilizers supply N as NHþ
4 first,
which upon oxidation releases Hþ ions [28].
Manure treated plots showed significantly greater net C
build up than fertilized treatments. Recommended fertilizer
application has been shown to result in modest increases in
labile organic matter content. Total SOC in NPK treatment
was higher than NP and NK treatments. This is attributable
to higher yields under fertilized conditions, which result in
increased inputs of organic matter to the soil in the form of
both root turnover and crop residues (Table 4). Furthermore,
Table 4 – Mean grain yield as influenced by fertilization
over 33 years of rainfed soybean–wheat cropping
Treatment
Mean grain yield (Mg ha1)
Soybean
Control
NP
NK
NPK
N þ FYM
NPK þ FYM
a
0.56
0.88c
0.64b
1.42d
2.42e
2.93f
Wheat
0.72a
0.92c
0.81b
1.10d
1.60e
1.91f
Columns sharing the same letter are not significantly different
(P < 0.05).
in a soybean–wheat cropping system, a significant amount
of leaf fall from soybean contributes to a greater amount of
added organic matter [23].
Soil invertase catalyses the hydrolysis of sucrose to glucose
and fructose, and is linked to the soil microbial biomass
[15,21,37]. In our experiment, organic amendment did not
contribute much to the hydrolysis of sucrose. The activity
was correlated with cellulase (P < 0.05). Cellulases play an
important role as a group of enzymes in global recycling of
the most abundant polymer, cellulose in nature. Though the
rate of cellulase activity was highest in NPK þ FYM followed
by N þ FYM and NPK, no clear effect of manures was visible.
Cellulase activity was positively correlated with protease
(P < 0.01), invertase (P < 0.05) and phosphatases (P < 0.05).
The activity of assayed enzymes was generally well
correlated with the organic C content because all of these
parameters were increased substantially by increasing
returns of organic residues. Indeed, in general, the activity of
soil enzymes is significantly correlated with organic C content
[17,40]. Being the substrate of enzymes, soil organic matter
plays a vital role in protecting soil enzymes since they form
complexes with clay and humus [45].
An increase in soil polysaccharide content in manure treatment has been interpreted because of more activity of soil
microflora, which can be correlated with dehydrogenase activity. There was an increasing trend of soil carbohydrate in NP
treatment, which is not explainable with the present state of
knowledge. Our results show that the activities of dehydrogenase, acid and alkaline phosphatases, cellulase, and protease
were highest in FYM treated soil. This maximum activity might
be linked to more substrate availability in these soils. This
reflects the greater biological activity in these plots and the stabilization of extracellular enzymes through complexation with
humic substances [6,9,16,22,31]. In contrast, urease and invertase activity followed a different pattern, where organic
manure had very little effect on the activity. Dehydrogenase
activity basically depends on the metabolic state of the soil
biota. A significant increase in dehydrogenase activity occurred
in the plots with organic treatments, especially with NPK.
Activity of urease was significantly higher in plots under
control followed by NPK þ FYM and NP treated plots. Dick
et al. [10] showed that urease activity decreased with increasing
application of NH3 based-N fertilizers. It was hypothesized that
the addition of the end product of the enzymatic reaction (NHþ
4)
314
european journal of soil biology 44 (2008) 309–315
suppressed urease synthesis. The N mineralization in soil was
characterized by measuring extracellular protease activity. The
rates of protease activity were higher with the organic amendments than in the other treatments. The high activity of
manure treated soil might be due to an increased growth of
the microbial community and it was supported by the study
of Kandeler et al. [22], which showed that biomass-specific
protease activities of different soils were not related to the
organic content or texture. More opinion was available for the
greater enzyme activity and it was suggested that strong binding of the enzyme to soil colloids protects from denaturation
[6]. Activity of protease was significantly and positively
correlated with cellulase (P < 0.01) and invertase (P < 0.05).
Acid phosphatase activity in NPK treatment was more than
4-fold lower than NPK þ FYM treatment and it was supported
by Haynes and Swift [18], who found that acid phosphatase
activity of soils generally decreases in response to fertilizer
application. Phosphatases play an important role in P cycling
where organic P is more due to limited biological mineralization of SOM as a result of formation of complexes of organic
P with active Al and Fe [48] and the amount of available P is
low. P transformation and cycle also depend on soil reaction.
Acid phosphatase activities in different treatments were
greatly regulated by soil pH values. In general, FYM applied
to soil has long been employed to enhance favourable soil
conditions in terms of pH and availability of P. In this study,
higher acid phosphatase activity was shown in soils with pH
of 6.5. The observed effect could be due to quantity, specific
activity, or stability of the enzyme at that particular soil pH.
Acid phosphatase activity was correlated with cellulase
(P < 0.05), invertase (P < 0.05) and protease (P < 0.01). The
significantly greater activities of alkaline phosphatase in the
manure treated soil might be attributed to enhanced microbial
activity and diversity due to manure input over the years.
Manure addition to a soil may have resulted in changes in
origin, states, and/or persistence of enzymes in the soil [34].
The inhibition of activities with mineral fertilizer could be
seen as well. Control soils exhibited higher activities than
the NPK and NK. This can be explained by the inhibition of
phosphatase synthesis by mineral fertilization. In slight
contrast to our results, the literature reported that this inhibition was due to mineral-P [3,41]. Alkaline phosphatase was
affected and was negatively correlated to soil P concentrations
and microbial biomass C and P [51].
Grain yield of soybean and wheat were significantly influenced by amendments. All the fertilized treatments significantly improved soybean yields relative to un-amended
control (Table 4). Wheat yields were also significantly influenced by the residual effect of the treatments. Application of
organic amendments along with inorganic nutrients (NPK þ
FYM) yielded 2.07 and 1.73 times more than inorganic nutrient
(NPK) only in the case of soybean and wheat respectively. The
yield data clearly demonstrate the superiority of the integrated use of FYM and chemical fertilizers, which provided
greater stability in crop production in comparison to NPK
treatment. The beneficial effect of integrated use of NPK and
FYM was more pronounced and effective in enhancing the
productivity with the advancement of year of cultivation.
This is attributed to the maintenance and improvement of
soil nutrient status as well as soil biological activity especially
soil enzymatic activity. Application of FYM along with NPK
improved soil organic carbon by 47.5 and 87.5% over NPK
alone and un-amended control. It also increased dehydrogenase, cellulase, protease and phosphatases by several fold
over inorganic nutrient alone. Improvement in soil dehydrogenase activity was more than five times in NPK þ FYM over
C treatment. Substantial improvement in soil chemical and
biological activity due to application of FYM must contribute
in sustaining the productivity and soil health.
5.
Conclusions
It is concluded that the accumulation of organic matter and
recycling of C have substantial effects on the activity of
enzymes involved in mineralization of C, N and P. Activities
of these studied enzymes are correlated with each other and
further correlated with soil organic matter content. Farm
yard manure along with recommended fertilizer improved
soil biological activity, depicted from several fold increase
in soil enzymatic activity. Improvement in soil organic matter and biological activity upon application of organic
amendments along with inorganic nutrients resulted in substantial increase in crop yield, which can be sustainable for
years.
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available at www.sciencedirect.com
journal homepage: http://www.elsevier.com/locate/ejsobi
Original article
Soil enzymatic activity as affected by long term application
of farm yard manure and mineral fertilizer under
a rainfed soybean–wheat system in N-W Himalaya
Supradip Saha*, Ved Prakash, Samaresh Kundu, Narendra Kumar, Banshi Lal Mina
Vivekananda Institute of Hill Agriculture, Indian Council of Agricultural Research, Almora – 263 601, Uttarakhand, India
article info
abstract
Article history:
Long-term experimental sites are expected to provide important information regarding soil
Received 8 June 2007
properties as affected by management practices. This study was designed to examine the
Accepted 29 February 2008
effects of continuous fertilization, and manuring on the activities of enzymes involved in
Published online 26 March 2008
mineralization of C, N, and P on a long term (33 years) field trial under sub-temperate
conditions in India. Treatments at the site included application of recommended doses
Keywords:
of nitrogen and phosphorus (NP), nitrogen and potassium (NK), nitrogen, phosphorus
Long-term experiment
and potassium (NPK), farmyard manure (FYM) with N (N þ FYM), FYM with NPK
Soil carbohydrate
(NPK þ FYM) and un-amended control (C). The study was done under rainfed soybean–
Soil enzymes
wheat rotation. Manure application increased soil carbohydrate, dehydrogenase, acid
Nutrient dynamics
and alkaline phosphatases, cellulase, and protease activity significantly. Urease activity
was not influenced by the manure treatment and the activity was highest in controls.
Both acid and alkaline phosphatase activities were negatively influenced by chemical
fertilizer treatment. Almost all the enzymes studied were significantly correlated with
soil C content. The results suggest that application of FYM directly or indirectly influences
the enzyme activity and it in turn regulates nutrient transformation.
ª 2008 Elsevier Masson SAS. All rights reserved.
1.
Introduction
In the effort to achieve sustainable agricultural production
while maintaining and preserving the environment, it is
crucial that soil biological health be improved or maintained.
Agricultural practices that improve soil quality and agricultural sustainability have been receiving more attention from
researchers and farmers [14]. Inorganic fertilizer, especially
N, P and K, not only serve to maintain, but their application
directly or indirectly causes changes in chemical, physical
and biological properties of the soil. These changes, in the
long term, are believed to have significant influences on the
quality and productive capacity of soils.
Soil microorganisms, particularly microbiota, play an
essential role in the cycling of elements and stabilization of
soil structure [5,32]. They also act as both a source and sink
of labile nutrients and C [20,47]. The mineralization of organic
matter is carried out by a large community of microorganisms
and involves a wide range of metabolic processes. Soil
enzymes are believed to be able to discriminate between soil
* Corresponding author. Tel.: þ91 5962 230060.
E-mail address: s_supradip@yahoo.com (S. Saha).
1164-5563/$ – see front matter ª 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejsobi.2008.02.004
310
european journal of soil biology 44 (2008) 309–315
management practices probably because they are related to
microbial biomass, which is sensitive to such treatments
[32]. Soil enzymes regulate the transformation process of
elements required for plant growth in soil [6]. The activity of
soil enzyme, whether extracellular or intracellular depends
on crop rotation, amendments, tillage and agricultural
management [14,24]. Transformation of N in soils includes
different processes, which are regulated by a number of extracellular degradative enzymes [26,50]. Transformation of
organic P through enzymatic reactions and the immobilization of P in the biomass itself play a fundamental role in P
cycling [35,36,46] and are likely to be affected by P amendments [10,27].
Although some researchers commented that soil organic
matter was not a plant growth requirement in-and-of itself
[38], almost all have realized its ecological significance in
overcoming constraints to crop growth, particularly, in the
areas of nutrient supply, nutrient and moisture relation, soil
structural stability and detoxification [42]. Information is
also needed to assess the contribution of the organic matter
in maintaining the soil microbial community and which in
turn, is related to specific enzymatic reactions, i.e. to biochemical processes involved in different nutrient cycling for assessing sustainability.
Long-term field treatments provide a means of investigating the influences of organic amendments on soil characteristics [8]. However, relatively limited consistent information is
available concerning changes in soil biological properties, under long-term field conditions of a N-W Himalayan ecosystem.
The objective of this study was to ascertain changes in soil
enzymatic activity in response to repeated applications of
organic amendment vis-a`-vis inorganic nutrients under field
conditions over several years. For this reason, soil enzymatic
properties, especially those related to the cycles of C, N, and
P in the soil, was compared in soils from a long-term field
experiment.
2.
Materials and methods
2.1.
Experimental site
A long-term field experiment was initiated in June 1973 on
a Typic Haplaquept at the experimental farm of Vivekananda
Institute of Hill Agriculture, located in the Indian Himalayan
region at Hawalbagh (29 360 N and 79 400 E at 1250 m above
mean sea level), in the state of Uttarakhand, India. Soil characteristics based on analysis of preserved soil samples taken in
1973 are given in Table 1. The climate is sub-temperate,
characterized by a moderate summer (May–Jun), extreme
winter (Dec–Jan) and general dryness, except during the
southwest monsoon season (Jun–Sep). Based on the records
for the period 1973–2006, average kharif (monsoon) season
rainfall was 684 mm (e.g. 68% of the average annual rainfall).
During soybean growth, average monthly rainfall (mm) was:
Jun (146), Jul (257), Aug (187), Sep (119), whereas, during wheat
growth the average monthly rainfall (mm) was: Oct (27), Nov
(6), Dec (28), Jan (53), Feb (56), Mar (48), Apr (42). The mean
monthly temperature ranged from a minimum of 0.4 C in
January to a maximum of 31.5 C in May.
Table 1 – Initial soil physico-chemical properties
(0–15 cm)
Properties
pH (soil:water, 1:2.5)
EC (dS m1)
CEC (cmol (pþ) kg1 soil)
Bulk density (Mg m3)
Total organic C (g kg1 soil)
C:N
Available N (mg kg1 soil)
Available P (mg kg1)
Available K (mg kg1)
Texture
Taxonomic classification
2.2.
Value
6.2
0.08
8.7
1.32
5.8
12.8
127
12
65
Sandy loam
Typic Haplaquept
Experimental design
The experiment included two crops per year, soybean (Jun–
Sep) and wheat (Oct–Apr), with six treatment combinations
(in kg ha1): no fertilizer and no manure (un-amended
control); 20 N þ 35 P (NP); 20 N þ 33 K (NK); 20 N þ 35 P þ 33
K (NPK), 20 N þ FYM at 10 Mg ha1 (N þ FYM, commonly
used by the local farmers) and NPK þ FYM at 10 Mg ha1
(NPK þ FYM). The required doses of FYM and fertilizers were
given only to the soybean crop every year and the wheat
crop was allowed to grow on residual fertility. Treatments
were distributed in a randomized block design with six replications over three uniformly level terraces. The net plot size
was 5.4 2.0 m. Fertilizers used were urea for N, single
super-phosphate for P and murate of potash for K. Based on
the chemical analysis of every fifth year, FYM (C:N z 23.6:1)
had 370 g of moisture kg1 and contained 7.1–7.5 g N kg1,
2.1–2.4 g P kg1 and 5.3–5.8 g K kg1 on an oven-dry weight
basis. FYM was prepared using cattle dung mixed with urine
and bedding material. Oak (Quercus leucotrichophora) leaves
(1.1% N, 0.55% P, 0.74% K) were mostly used as bedding material in cattle sheds. These materials were thoroughly mixed
and stored in a heap under shade. The heap was plastered
with a 2-inch thick layer of soil and dung and was left undisturbed for 45 days. Full doses of N, P, K and FYM were incorporated into the upper 5 cm of soil during final land preparation.
2.3.
Soil sampling for chemical and
microbial analysis and preparation
Initial soil samples were collected in 1973 prior to the start of
the experiment. After harvest of wheat, soil samples were
taken from the surface layer (0–15 cm) of six treatments
with six replications in May, 2006, before the start of land
management for soybean. Random cores were taken from
each plot with a 5-cm diameter tube auger and bulked. The
moist soil samples were sieved (2 mm) after removing plant
material and roots. Half of the soil samples were air-dried
and stored at room temperature until chemical analysis. All
chemical results are means of triplicate analyses and are
expressed on an oven-dry basis. Soil moisture was determined
after drying at 105 C for 24 h.
The rest of the sieved soil (2 mm) was immediately transferred to the laboratory for microbiological analysis. Soil
311
european journal of soil biology 44 (2008) 309–315
samples were kept at 4 C in plastic bags for a few days to
stabilize the microbiological activity disturbed during soil
sampling and handling, and then analysed within 2 weeks.
All microbial results reported are means of six replicates and
are expressed on a moisture-free basis. Moisture content
was determined after drying at 105 C for 24 h.
the enzyme reaction was stopped by adding 4 ml of 0.5 M
NaOH and 1 ml of 0.5 M CaCl2 to prevent dispersion of humic
substances. After centrifugation at 4000 rpm for 10 min, the absorbance was measured in the supernatant at 400 nm; enzyme
activity was expressed as mg p-nitrophenol released g1 soil h1.
2.6.
2.4.
Soil was analyzed for pH in a 1:2.5 soil:water suspension [19],
soil texture was determined by Bouyoucos hydrometer [4] and
available K by 1 N NH4OAc using a flame photometer [19]. Soil
was analysed for oxidizable SOC by the method of Walkley
and Black [49], Kjeldahl N by FOSS Tecator (Model 2200), and
available P following the Olsen method [33]. A core sampler
was used for soil bulk density determination. Soil was analysed for total C and N using a CHN analyser (FOSS Heraeus
CHNORapid) following a dry combustion method.
Soil carbohydrate was estimated following the modified
method of Martens and Loeffelmann [29]. The soil sample
(100 mg) was first solubilized and then hydrolysed with 1 M
H2SO4 to get monosaccharides, which were estimated colorimetrically using the phenol–sulfuric acid method [11].
2.5.
Grain yield
Chemical analyses
Microbiological analyses
Soil dehydrogenase activity was estimated by reducing
2,3,5-triphenyltetrazolium chloride [7]. Five grams of soil
sample were mixed with 50 mg of CaCO3 and 1 ml of 3% (w/
v) 2,3,5-triphenyltetrazolium chloride (TTC) and incubated
for 24 h at 37 1 C. Dehydrogenase enzyme converts TTC to
2,3,5-triphenylformazan (TPF). The TPF formed was extracted
with acetone (3 15 ml), the extracts were filtered through
Whatman No. 5 and absorption was measured at 485 nm
with a spectrophotometer (Analytik Jena, Germany).
Cellulase activity was determined by estimating the
glucose equivalent after mixing the soil sample (10 g) with
15 ml of acetate buffer (2 M, pH 5.5) and 15 ml of 0.7% (w/v)
carboxy methyl cellulose and incubated for 24 h at 50 1 C
[39]. The glucose equivalent was estimated following the
DNS method [30].
Protease activity was assayed by determining the tyrosine
released when 1 g of the oven-dry equivalent of field-moist
soil sample was incubated with 5 ml of 50 mM Tris buffer
(pH 8.1) and 5 ml of 2% Na-caseinate at 50 1 C for 2 h [25].
The aromatic amino acids released were extracted and the
remaining substrate was precipitated with 0.92 M trichloroacetic acid and measured colorimetrically using Folin–Ciocalteu reagent at 700 nm. The activity of protease was expressed
as mg tyrosine produced g1 soil 2 h1.
Urease activity was measured following the method of
Tabatabai and Bremner [44]. Five grams of soil were incubated
with 5 ml of 0.05 M THAM buffer (pH 9.0) and 1 ml of 0.2% of
urea solution at 37 C for 2 h. Excess urea was extracted with
KCl–PMA solution and estimated colorimetrically at 527 nm.
Phosphomonoesterase (acid and alkaline phosphatase) activity was assayed using 1 g of soil (wet equivalent), 4 ml of
0.1 M modified universal buffer (pH 11 for alkaline phosphatase
and pH 6.5 for acid phosphatase), and 1 ml of 25 mM p-nitrophenyl phosphate [43]. After incubation for 1 h at 37 1 C
Both soybean and wheat were harvested at 5 cm above the soil
surface in the first week of October and the fourth week of
April, respectively. Grain yield was expressed on a 12 and 9%
moisture basis for wheat and soybean, respectively.
2.7.
Statistical analyses
Each sample was analyzed in triplicate and the values were
then averaged. Data were assessed by Duncan’s multiple
range tests (1955) with a probability P 0.05 [12]. Differences
between mean values were evaluated by a one-way analysis
of variance (ANOVA) (SPSS version 10.0). Pearson correlation
analyses were performed using the SPSS programme.
3.
Results
3.1.
Soil pH and oxidizable carbon
Table 1 gives the initial physico-chemical properties of the soil
used for the long term experiment. Taxonomically the soil
was classified as Typic Haplaquept. The pH values in
0–15 cm of surface soil ranged from 5.3 to 6.5, with that of
the untreated control soil around pH 5.7. Manure application
increased soil pH significantly, while chemical fertilizer application resulted in lowering of soil pH (Table 2). NK treatment
lowered the soil pH value up to 5.3.
In the surface 0–15 cm, soil oxidizable carbon was less in
the different fertilized treatments than in FYM treated plots
(Table 2). In different fertilized treatments, oxidizable-C followed the order NK < NP < NPK. Application of FYM along
with NPK contributed significantly to incorporation of C in
soil. Addition of FYM at 10 Mg ha1 year1 along with NPK
for 33 years doubled the organic C content than NPK alone.
3.2.
Soil carbohydrate and dehydrogenase activity
Soil total polysaccharide content for six different treatments
are tabulated in Fig. 1. Carbohydrate content in control and
Table 2 – Soil pH, total SOC and nitrogen, C-to-N ratio of
a Typic Haplaquept (0–15 cm) affected by mineral
fertilizer and manure treatment
Treatment
pH
TOC (g kg1)
N (g kg 1)
C:N
C
NP
NK
NPK
N þ FYM
NPK þ FYM
5.7
5.6
5.3
5.4
6.4
6.5
6.34
7.51
6.88
8.06
11.09
11.89
0.560
0.692
0.581
0.651
0.782
0.856
11.3:1
10.9:1
11.8:1
12.4:1
14.2:1
13.9:1
312
1.4
Dehydrogenase
CHO ( )
40
dc
bd
cd 1.0
cc
30
cd
bc
ac
1.2
ac
0.8
0.6
bc
20
Glucose equivalent
g TPF produced g-1 h-1
50
0.4
10
ad
ad
ad
ad
NK
NPK
g urea hydrolysed g-1 h-1/
g tyrosine produced g-1 2h-1
european journal of soil biology 44 (2008) 309–315
100
bc
80
60
a
a
40
b
20
b
a
a
0
Control
N+FYM NPK+FYM
NP
NK
NPK
N+FYM NPK+FYM
NK treatment was at par. NPK þ FYM treatment (1.14%) was
best for contributing soil carbohydrate content. Values for
other treatments increased in the order NPK (0.67%) < N þ
FYM (0.74%) < NP (0.879).
Changes in dehydrogenase activity in differently treated
soils are shown to be distributed across a wide range (Fig. 1).
Only fertilized treatments along with controls showed significantly lower intracellular metabolism of soil microbes, which
depicts dehydrogenase activity. NP, NK and NPK showed
similar levels of activity, at par with the controls. FYM application increased enzymatic activity a few times when it was
applied along with N or NPK.
Soil enzymatic activities
Enzyme activities varied widely among the treatments studied. The treatment with the highest activity was found to
vary depending on the enzyme. The highest value for cellulase
activity was found in NPK þ FYM, followed by N þ FYM treatments that received organic amendments (Fig. 2). Invertase
activity did not vary much across treatments and the highest
value was found in treatments that received organic manure
along with the recommended fertilizer. Protease activity in
the soil followed a similar trend and was highest in manure
500
Cellulase
Invertase
400
c
b
bc
300
200
100
a
a
a
a
ab
ab
ab
b
b
0
Control
NP
NK
NPK
N+FYM NPK+FYM
Fig. 2 – Cellulase and invertase activities in soil with
different treatments. Bars sharing the same letter are not
significantly different (P < 0.05). Error bars represent
standard deviation.
Fig. 3 – Urease and protease activities in soil with different
treatments. Bars sharing the same letter are not
significantly different (P < 0.05). Error bars represent
standard deviation.
amended treatments. Unlike other enzymes, urease activity
was inhibited in the organic amended soils (Fig. 3). The highest value was found in non-amended controls followed by
NP and NPK þ FYM. Fig. 4 shows acid and alkaline phosphatase activities for all the treatments. As the pH of the soil
lies within the acidic range, it might be expected that acid
phosphatase is the dominant activity in this soil [13]. The
highest value was found in NPK þ FYM followed by N þ FYM.
For other treatments, the activity was far below these two
treatments. Activity of the manure treated soil was 2–3-fold
greater than that in soils from the other treatments. A similar
trend was observed in the case of alkaline phosphatase
activity. NPK treated soil was least and NPK þ FYM was
highest for this enzyme activity.
A correlation matrix (Table 3) shows some significant relationships among the enzymes studied. There was positive
correlation between cellulase and invertase, which are
involved in C transformation. Total organic carbon was correlated with all enzymes assayed except urease and dehydrogenase. Cellulase also showed a strong positive correlation with
protease and phosphatase. Acid phosphatase shared a strong
correlation with protease and alkaline phosphatase. Protease
activity was strongly correlated (r ¼ 0.953, P < 0.001) with the
N content of the soil. Cellulase activity was also correlated
with invertase activity (r ¼ 0.802, P < 0.01).
g p-nitrophenol produced g-1 h-1
NP
Fig. 1 – Dehydrogenase activity and carbohydrate content
in soil with different treatments. Bars sharing the same
letter are not significantly different (P < 0.05). Error bars
represent standard deviation. ac and ad represent
carbohydrate and dehydrogenase respectively.
mg glucose eq. g-1 24 h-1/mg
glu eq. g-1 h-1
c
c
0.0
Control
3.3.
bc
b
0.2
0
Urease
Protease
c
200
c
Acid Phosphatase
Alkaline Phosphatase
b
150
d
100
a
a
50
b
ab
c
a
a
a
a
0
Control
NP
NK
NPK
N+FYM
NPK+FYM
Fig. 4 – Acid and alkaline phosphatase activities in soil
with different treatments. Bars sharing the same letter are
not significantly different (P < 0.05). Error bars represent
standard deviation.
313
european journal of soil biology 44 (2008) 309–315
Table 3 – Linear correlation coefficient between nutrient and enzyme activities of soil
TOC
N
DA
Cell
Inv
Ure
Pro
AcP
N
DA
Cell
Inv
Ure
Pro
AcP
AkP
0.830**
0.881**
0.893**
0.910**
0.670
0.725
0.821**
0.901**
0.427
0.802*
0.240
0.121
0.092
0.040
0.245
0.922**
0.853**
0.411
0.887**
0.783*
0.294
0.872**
0.844**
0.301
0.801*
0.740*
0.418
0.909**
0.928***
0.804**
0.502
0.767*
0.708
0.439
0.810*
0.920***
TOC, total organic carbon; N, total N; DA, dehydrogenase; Cell., cellulase; Inv, invertase; Ure, urease; Pro, protease; AcP, acid phosphatase;
AkP, alkaline phosphatase. *P < 0.1, **P < 0.05, ***P < 0.001 respectively.
3.4.
Grain yield
Mean yield of soybean and wheat after 33 years of cropping is
presented in Table 4. Soybean yield varied from 0.56 Mg ha1
for C to 2.93 Mg ha1 for NPK þ FYM. In the case of wheat, it
ranged from 0.72 to 1.91 Mg ha1, highest for NPK þ FYM and
lowest for C.
4.
Discussion
Soil pH ranged from 5.3 in the NK plots, which is significantly
lower than for all other treatments, to 6.5 in the NPK þ FYM
plots. The pH of the N treated plots was significantly lower
than that of the C and manured plots. Long term applications,
especially N, therefore had acidifying effects resulting in
lowering of pH. This confirms earlier findings that most
N-containing fertilizers tend to acidify soil [1,2]. This is mainly
due to the fact that most fertilizers supply N as NHþ
4 first,
which upon oxidation releases Hþ ions [28].
Manure treated plots showed significantly greater net C
build up than fertilized treatments. Recommended fertilizer
application has been shown to result in modest increases in
labile organic matter content. Total SOC in NPK treatment
was higher than NP and NK treatments. This is attributable
to higher yields under fertilized conditions, which result in
increased inputs of organic matter to the soil in the form of
both root turnover and crop residues (Table 4). Furthermore,
Table 4 – Mean grain yield as influenced by fertilization
over 33 years of rainfed soybean–wheat cropping
Treatment
Mean grain yield (Mg ha1)
Soybean
Control
NP
NK
NPK
N þ FYM
NPK þ FYM
a
0.56
0.88c
0.64b
1.42d
2.42e
2.93f
Wheat
0.72a
0.92c
0.81b
1.10d
1.60e
1.91f
Columns sharing the same letter are not significantly different
(P < 0.05).
in a soybean–wheat cropping system, a significant amount
of leaf fall from soybean contributes to a greater amount of
added organic matter [23].
Soil invertase catalyses the hydrolysis of sucrose to glucose
and fructose, and is linked to the soil microbial biomass
[15,21,37]. In our experiment, organic amendment did not
contribute much to the hydrolysis of sucrose. The activity
was correlated with cellulase (P < 0.05). Cellulases play an
important role as a group of enzymes in global recycling of
the most abundant polymer, cellulose in nature. Though the
rate of cellulase activity was highest in NPK þ FYM followed
by N þ FYM and NPK, no clear effect of manures was visible.
Cellulase activity was positively correlated with protease
(P < 0.01), invertase (P < 0.05) and phosphatases (P < 0.05).
The activity of assayed enzymes was generally well
correlated with the organic C content because all of these
parameters were increased substantially by increasing
returns of organic residues. Indeed, in general, the activity of
soil enzymes is significantly correlated with organic C content
[17,40]. Being the substrate of enzymes, soil organic matter
plays a vital role in protecting soil enzymes since they form
complexes with clay and humus [45].
An increase in soil polysaccharide content in manure treatment has been interpreted because of more activity of soil
microflora, which can be correlated with dehydrogenase activity. There was an increasing trend of soil carbohydrate in NP
treatment, which is not explainable with the present state of
knowledge. Our results show that the activities of dehydrogenase, acid and alkaline phosphatases, cellulase, and protease
were highest in FYM treated soil. This maximum activity might
be linked to more substrate availability in these soils. This
reflects the greater biological activity in these plots and the stabilization of extracellular enzymes through complexation with
humic substances [6,9,16,22,31]. In contrast, urease and invertase activity followed a different pattern, where organic
manure had very little effect on the activity. Dehydrogenase
activity basically depends on the metabolic state of the soil
biota. A significant increase in dehydrogenase activity occurred
in the plots with organic treatments, especially with NPK.
Activity of urease was significantly higher in plots under
control followed by NPK þ FYM and NP treated plots. Dick
et al. [10] showed that urease activity decreased with increasing
application of NH3 based-N fertilizers. It was hypothesized that
the addition of the end product of the enzymatic reaction (NHþ
4)
314
european journal of soil biology 44 (2008) 309–315
suppressed urease synthesis. The N mineralization in soil was
characterized by measuring extracellular protease activity. The
rates of protease activity were higher with the organic amendments than in the other treatments. The high activity of
manure treated soil might be due to an increased growth of
the microbial community and it was supported by the study
of Kandeler et al. [22], which showed that biomass-specific
protease activities of different soils were not related to the
organic content or texture. More opinion was available for the
greater enzyme activity and it was suggested that strong binding of the enzyme to soil colloids protects from denaturation
[6]. Activity of protease was significantly and positively
correlated with cellulase (P < 0.01) and invertase (P < 0.05).
Acid phosphatase activity in NPK treatment was more than
4-fold lower than NPK þ FYM treatment and it was supported
by Haynes and Swift [18], who found that acid phosphatase
activity of soils generally decreases in response to fertilizer
application. Phosphatases play an important role in P cycling
where organic P is more due to limited biological mineralization of SOM as a result of formation of complexes of organic
P with active Al and Fe [48] and the amount of available P is
low. P transformation and cycle also depend on soil reaction.
Acid phosphatase activities in different treatments were
greatly regulated by soil pH values. In general, FYM applied
to soil has long been employed to enhance favourable soil
conditions in terms of pH and availability of P. In this study,
higher acid phosphatase activity was shown in soils with pH
of 6.5. The observed effect could be due to quantity, specific
activity, or stability of the enzyme at that particular soil pH.
Acid phosphatase activity was correlated with cellulase
(P < 0.05), invertase (P < 0.05) and protease (P < 0.01). The
significantly greater activities of alkaline phosphatase in the
manure treated soil might be attributed to enhanced microbial
activity and diversity due to manure input over the years.
Manure addition to a soil may have resulted in changes in
origin, states, and/or persistence of enzymes in the soil [34].
The inhibition of activities with mineral fertilizer could be
seen as well. Control soils exhibited higher activities than
the NPK and NK. This can be explained by the inhibition of
phosphatase synthesis by mineral fertilization. In slight
contrast to our results, the literature reported that this inhibition was due to mineral-P [3,41]. Alkaline phosphatase was
affected and was negatively correlated to soil P concentrations
and microbial biomass C and P [51].
Grain yield of soybean and wheat were significantly influenced by amendments. All the fertilized treatments significantly improved soybean yields relative to un-amended
control (Table 4). Wheat yields were also significantly influenced by the residual effect of the treatments. Application of
organic amendments along with inorganic nutrients (NPK þ
FYM) yielded 2.07 and 1.73 times more than inorganic nutrient
(NPK) only in the case of soybean and wheat respectively. The
yield data clearly demonstrate the superiority of the integrated use of FYM and chemical fertilizers, which provided
greater stability in crop production in comparison to NPK
treatment. The beneficial effect of integrated use of NPK and
FYM was more pronounced and effective in enhancing the
productivity with the advancement of year of cultivation.
This is attributed to the maintenance and improvement of
soil nutrient status as well as soil biological activity especially
soil enzymatic activity. Application of FYM along with NPK
improved soil organic carbon by 47.5 and 87.5% over NPK
alone and un-amended control. It also increased dehydrogenase, cellulase, protease and phosphatases by several fold
over inorganic nutrient alone. Improvement in soil dehydrogenase activity was more than five times in NPK þ FYM over
C treatment. Substantial improvement in soil chemical and
biological activity due to application of FYM must contribute
in sustaining the productivity and soil health.
5.
Conclusions
It is concluded that the accumulation of organic matter and
recycling of C have substantial effects on the activity of
enzymes involved in mineralization of C, N and P. Activities
of these studied enzymes are correlated with each other and
further correlated with soil organic matter content. Farm
yard manure along with recommended fertilizer improved
soil biological activity, depicted from several fold increase
in soil enzymatic activity. Improvement in soil organic matter and biological activity upon application of organic
amendments along with inorganic nutrients resulted in substantial increase in crop yield, which can be sustainable for
years.
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