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Chemistry and Ecology

ISSN: 0275-7540 (Print) 1029-0370 (Online) Journal homepage: http://www.tandfonline.com/loi/gche20

Assessment of soil quality under different tillage


practices during wheat cultivation: soil enzymes
and microbial biomass
Divya Pandey, Madhoolika Agrawal & Jitendra Singh Bohra
To cite this article: Divya Pandey, Madhoolika Agrawal & Jitendra Singh Bohra (2015)
Assessment of soil quality under different tillage practices during wheat cultivation:
soil enzymes and microbial biomass, Chemistry and Ecology, 31:6, 510-523, DOI:
10.1080/02757540.2015.1029462
To link to this article: http://dx.doi.org/10.1080/02757540.2015.1029462

Published online: 23 Jun 2015.

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Date: 21 September 2016, At: 01:22

Chemistry and Ecology, 2015


Vol. 31, No. 6, 510523, http://dx.doi.org/10.1080/02757540.2015.1029462

Assessment of soil quality under different tillage practices


during wheat cultivation: soil enzymes and microbial biomass
Divya Pandeya , Madhoolika Agrawala and Jitendra Singh Bohrab
a Department of Botany, Laboratory of Air Pollution & Global Climate Change, Banaras Hindu
University, Varanasi 221005, India; b Department of Agronomy, Institute of Agricultural Sciences, Banaras
Hindu University, Varanasi, India

(Received 3 March 2014; final version received 9 March 2015)


Microbial processes, particularly enzyme activities, play crucial functional roles in soil ecology, hence
serving as sensitive indicators of soil quality. We assessed the temporal dynamics of microbial biomass
and selected soil enzymes (-d-glucosidase, cellobiohydrolase, polyphenol oxidase, urease, glycineaminopeptidase and alkaline phosphatase) during wheat cultivation, under four different tillage practices
in the ricewheat system. The four practices involved conventional tilling of soil before cultivating
each crop (CTR-CTW); no tilling before cultivating rice but conventional tillage before wheat (NTRCTW); conventional tilling before cultivating rice but no tilling before wheat (CTR-NTW) and no tilling
before cultivation of each crop (NTR-NTW). Microbial biomass and activities of hydrolytic enzymes
increased under NTR-NTW followed by CTR-NTW and NTR-CTW with respect to the conventional
practice CTR-CTW, thus reflecting improvement in microbial activities with reduced tillage frequency.
Enzyme activities generally depended on soil moisture and temperature, but nature of relationships varied
among different practices. Nutrient demand appeared to be the strongest driver of alkaline phosphatase
and urease, and soil temperature for glycine-aminopeptidase. Under CTR-CTW, activities of most of the
extracellular enzymes were related with -d-glucosidase or urease, but such relations altered under rest
of the practices. The study showed that extracellular soil enzymes respond sensitively to tillage practices
as well as environmental variables, particularly soil temperature and moisture and hence can serve as a
sensitive indicator of changes in soil processes. Considering improvement in microbial biomass and enzymatic activities as indicators of better soil quality, adoption of no tillage apparently improved soil quality.
Still, more number of field studies are required under tillage managements to explore the relationships
between different enzyme activities and environmental factors.
Keywords: extracellular soil enzymes; conventional tillage; no tillage; microbial biomass; wheat
cultivation

1.

Introduction

Enzymes play highly specialised and vital roles in controlling different biochemical processes in ecosystems. In soils, actions of enzymes become even more complex, yet interesting
because they are secreted into the external environment, catalysing reactions not only for a
single organism but the entire microbial community. Roles of extracellular enzymes in relation to biogeochemistry of nutrients are being investigated for nearly a century; environmental
controls over enzyme activities and coherence between different enzymes are still not well
*Corresponding author. Email: madhoo.agrawal@gmail.com
2015 Taylor & Francis

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511

understood.[1,2] It is however known that extracellular enzyme activities in soil respond sensitively to environmental changes and human interventions, and hence they have been proposed
as key indicators for expressing soil quality.[3,4] The most commonly assessed soil enzymes
include those which participate in mineralisation of C, N and P. Most of these decompose
constituents of plant litter [5,6] and hydrolyse protein and other complex organics.[79] Secretion and activity of these enzymes are found to depend on demand of nutrients, availability
of substrates and activities of microbes.[10] In cultivated soils, enzyme activities can be used
to understand the impact of different practices on natural elemental transformations and their
influence on soil quality.
In India, no tillage practice is becoming popular among the farmers as a resource-conserving
technique.[11] This practice involves no ploughing before sowing and often is accompanied by
leaving the crop residues over the soil. This increases organic matter input in the soil accompanied by reduced mineralisation due to soil disturbance, thereby improving soil carbon stock,
nutrient contents, stabilised soil structure and enhanced biological activities.[1217] It has been
observed that higher organic matter content in the soil promotes better microbial biomass, diversity and activity,[1821] but climatic conditions and other physiochemical conditions of the soil
affect short-term responses.[18,2225]
The available studies indicate that activities of most of the enzymes including dehydrogenase, -glucosidase, -glucosidase, arylsulfatase, acid phosphatase, alkaline phosphatase,
invertase, amidase and urease generally tend to increase under no tillage compared to conventional tillage.[18,2629] But, there is limited information on how enzyme activities might
respond to tillage during cultivation of one crop and no tillage during the other crop. This is
important because practicing no tillage in every crop and over a long term is not preferred by
farmers in many systems, including the ricewheat systems in Indo-Gangetic plains.[29]
Based on the findings of different studies in the scientific literature as discussed earlier, we
hypothesise that soil under continued no tillage (NTR-NTW) will have the highest microbial
biomass and will also exhibit the highest activities of all the enzymes, followed by CTR-NTW,
NTR-CTW and CTR-CTW. Furthermore, enzyme activities also depend strongly on environmental conditions. Temperature, pH, moisture and oxido-reduction potential (ORP) are among
the most important environmental factors that control biochemical processes in soil, and hence
they have been found to be significantly related with enzyme activities.[10,2427] Therefore, we
further hypothesise that all the extracellular enzyme activities will show significant relationships
with temperature, moisture and ORP of soil and nature of relationships will remain similar under
all the practices.
We carried out temporal monitoring of selected enzyme activities participating in transformations of C, N and P in soil under a long-term experimental field maintained under different
practices of tillage and no tillage in the ricewheat system. An attempt has been made to identify synchronies between different enzyme activities, and their relationships with environmental
factors under different tillage practices.

2.
2.1.

Materials and methods


Experimental set-up

A long-term field experiment was established in 2003 under all India Coordinated Research
Project on Integrated Farming Systems at the agricultural farm of the Institute of Agricultural
Sciences, Banaras Hindu University, Varanasi, India (25.14N latitude, 82.03E longitude and
76.19 m above mean sea level). The field was maintained under four practices of conventional
and no tillage in the rice (cv PHB-71)wheat (cv HD-2824) system since 2003 in a randomised

512

D. Pandey et al.

Table 1. Average soil properties at the beginning of the tillage experiment in 2003 and before sowing of wheat in 2010
(mean 1 SE). Different letters in a row indicate that means of 2010 are different from each other in accordance with
DMRT (p < .5).
2003a

pH
Dry bulk density (g/cm3 )
Soil organic C (%)
Total N (mg/g)
Available P (g/g)
Texture
Sand (%)
Silt (%)
Clay (%)

2010

CTR-CTW

CTR-CTW

NTR-CTW

CTR-NTW

NTR-NTW

7.30 0.01
1.43 0.01
0.33 0.02
0.32 0.02
1.23 .07
Sandy loam
59.51
14.19
26.30

7.36
1.47 0.01c
0.38 0.02d
0.38 0.03d
1.03 0.04d
Sandy loam
62.57
13.65
23.78

7.42
1.49 0.01c
0.60 0.02c
0.61 0.02c
1.44 0.08b
Sandy loam
63.74
13.74
22.52

7.36
1.53 0.10b
0.77 0.02b
0.51 0.02b
1.44 0.03b
Sandy loam
63.23
11.11
25.66

7.33 0.03ns
1.75 0.01a
0.96 0.05a
0.59 0.03a
1.82 0.03a
Sandy loam
60.45
17.24
22.31

0.06ns

0.05ns

0.10ns

Notes: CTR-CTW = tillage before transplanting rice and sowing of wheat; NTR-CTW = no tillage before sowing of rice and tillage
before sowing of wheat; CTR-NTW = tillage before transplanting rice but no tillage before sowing of wheat; NTR-NTW = no tillage
before sowing of both rice and wheat.
a
Taken from Pandey et al. [29].

block design. The present study was conducted during wheat cultivation period of 20102011.
The four tillage practices were: (i) conventional tilling before transplantation/sowing of both
the crops (CTR-CTW), (ii) no tilling before sowing of rice and conventional tilling before sowing of wheat (NTR-CTW), (iii) conventional tilling before transplantation of rice and no tilling
before sowing of wheat (CTR-NTW) and (iv) no tilling before sowing of both rice and wheat
(NTR-NTW). Net size of each plot was 5.25 m 5.25 m with a border of 0.5 m around each of
the experimental plot. The entire experiment was maintained in triplicate. Under conventional
tillage, soil was ploughed up to 15 cm depth during land preparation for sowing/transplantation
of crop. In case of no tillage, the preceding crop was harvested by cutting only the ripened ears,
while the rest of the plant part ( > 20 cm) was left standing behind. The soil was left undisturbed
and no till drill sowing of the next crop was done. This involved drilling of 2.5 cm deep holes
between the standing residues of previous crop followed by placing the seeds into the holes and
covering them. A detailed description of crop raising and management is provided by Pandey
et al. [29]. General soil properties in the four practices at the beginning of the tillage experiment
in July 2003 and before sowing of wheat in 2010 are given in (Table 1).

2.2.

Soil sampling and analyses

Soil samples were collected for measurement of enzyme activities at five-day intervals, starting
from the date of sowing and continued till the date of harvesting. Soil samples were collected at three randomly selected sites per plot. Therefore, there were nine samples per tillage
practice. Soil samples were drawn from top 15 cm depth with the help of a core sampler
(diameter 6.5 cm and depth 15 cm) after removing loose litter. The sampled soils were stored
immediately in polyethylene bags in an insulated box maintained at 4C. The analyses were
completed the same day and all procedures involved in soil enzyme assays were carried out
in cold conditions ( 4C). The activities measured were cellobiohydrolase (EC 3.2.1.91), glucosidase (EC 3.2.1.21), polyphenol oxidase (EC 1.10.3.2), alkaline phosphatase (EC 3.1.3.1),
glycine-aminopeptidase (EC 3.4.11.-) and urease.
Activities of cellobiohydrolase, -glucosidase, polyphenol oxidase, alkaline phosphatase and
glycine-aminopeptidase were assayed following Allison and Jastrow.[30] After removing visible litter, each soil sample was gently homogenised with hand and sieved through 2 mm sieve.

Chemistry and Ecology

513

In all, 10 g of sieved soil was disrupted in cold ( 4C) Tris buffer maintained at the respective pH of the soil for one minute. The enzyme extract was separated through cold centrifugation
and incubated with substrates pNP--d-glucopyranoside for -glucosidase, pNP-cellobioside for
cellobiohydrolase, glycine-p-nitroanilide for glycine-aminopeptidase, pNP-phosphate for alkaline phosphatase and pyrogallol + EDTA for polyphenol oxidase in an incubator maintained
at temperature equal to the respective soil temperature (Ts). The enzymesubstrate reactions
led to coloured products, pNP in case of -glucosidase, cellobiohydrolase and alkaline phosphatase, p-nitroaniline (pNA) in case of glycine-aminopeptidase and purpurogallin (PPG) in case
of polyphenol oxidase. Absorbance of the coloured products was measured at respective wavelengths (460 nm for pNP and pNA and 420 nm for PPG) using a spectrophotometer (Visiscan
167, Systronics, India). Urease activity was determined taking urea as a substrate. For this, 10 g
soil extract in cold Tris buffer was incubated with 10% urea solution for 3 h at temperature equal
to Ts. NH+
4 thus released was estimated following sodium phenate method. All enzyme activities
1
dry soil h1 .
were expressed as the quantity of product formed (pNP, pNA, PPG or NH+
1)g
For determination of microbial biomass carbon (C) and nitrogen (N), soil samples were collected 1 day before land preparation and 30, 45, 75, 105 days after sowing (DAS) in each of the
tillage practices. Samples were collected in similar way as was done for soil enzymes. Microbial
biomass C and N were extracted following the chloroform fumigation and extraction method.[31]
For this, 10 g of fresh soil samples was fumigated under chloroform vapours in vacuum desiccators for 24 h. The difference between the C and N contents in the fumigated and unfumigated
samples measured following the wet oxidation [32] and ninhydrin reactive N,[33] was, respectively, proportional to microbial biomass C and N. Soil temperature (Ts) was measured in situ at
10 different positions per tillage practice with a mercury thermometer (least count 0.05C) at 15
cm depth.

2.3.

Statistical analysis

To test the differences between mean microbial biomass C and microbial biomass N between different tillage practices, one-way ANOVA with post hoc Duncans test was applied. In the same
experimental field, regular monitoring of gravimetric water content (GWC) and ORP was carried
out at five-day intervals starting from the day of sowing and are given by Pandey et al. [29]. Relationships between different enzyme activities, GWC, Ts and ORP were analysed through curve
fitting. In order to extract the principal components and classify the extracellular soil enzymes
and soil parameters, principal component analysis (PCA) was applied on the data of enzyme
activities, GWC, ORP and Ts with Varimax orthogonal rotation. All statistical analyses were
performed through SPSS version 10, IBM, USA.

3.

Results

At the time of sowing, Ts under all the practices ranged from 21C to 22C, which decreased
gradually to the minimum values of 14C at around 4550 DAS (Figure 1). Afterwards, temperatures again increased. Among different practices, NTR-NTW had the lowest Ts. The trends of
GWC and ORP are described elsewhere.[29] ORP values generally remained positive over the
study period, indicating that oxidising conditions prevailed.
At all the stages of crop development, microbial biomass C and N were the highest under NTRNTW followed by NTR-CTW, CTR-NTW and CTR-CTW (Figure 2). There were significant
temporal variations in microbial biomass C and N under all the tillage practices with decreasing

514

Figure 1.

D. Pandey et al.

Soil temperature under different tillage practices.

trend from sowing, becoming the minimum during the late vegetative stage, but increased later
along the crop development.
-Glucosidase activities were high during the early vegetative stages, and peak activities were
observed at 4550 DAS (from 49.47 g pNP g1 h1 under NTR-NTW to 58.19 g pNP g1
h1 under NTR-CTW). This stage corresponded to the initiation of the reproductive phase.
Among the four tillage practices, the highest activities of -glucosidase were recorded under
NTR-CTW on most of the monitoring dates except up to 2025 DAS, during which it was
the maximum under NTR-NTW. -Glucosidase activities were the minimum under CTR-CTW
among the four tillage practices. Cellobiohydrolase activities, on the other hand, increased gradually after the mid-vegetative stage of wheat plants (40 DAS) (Figure 3). Cellobiohydrolase
activities under different tillage practices were significantly different during the early vegetative
(030 DAS) and maturity (after 90 DAS) stages. During early vegetative stages, cellobiohydrolase activities were the lowest under CTR-CTW and highest under NTR-NTW practice whereas
during the maturity stage, it was generally low under NTR-NTW. Highest activities of cellobiohydrolase were recorded under NTR-CTW (88.75 g pNP g1 h1 ) at 86 DAS and the lowest
under CTR-CTW (7.01 g pNP g1 h1 ) at 21 DAS. Polyphenol oxidase activities fluctuated
between 14.2 g PPG g1 h1 under CTR-NTW and 31.5 g PPG g1 h1 under NTR-NTW up
to 40 DAS. Afterwards polyphenol oxidase activities increased to attain peaks between 50 and
70 DAS under all the practices.
Activities of alkaline phosphatase remained nearly constant under all the tillage practices up
to 30 DAS (Figure 3). Afterwards, alkaline phosphatase activities increased slightly under CTRCTW and CTR-NTW, but decreased under NTR-CTW and NTR-NTW. After 80 DAS however,
alkaline phosphatase activities increased sharply under all the tillage practices, with the highest
activity recorded under NTR-NTW (1.94 g pNP g1 h1 at 126 DAS) and the lowest under
NTR-CTW (0.08 g pNP g1 h1 at 52 DAS).
Enzymes involved in N mineralisation, namely, urease and glycine-aminopeptidase, followed
different temporal trends as presented in Figure 4. From the date of sowing, urease activities
increased gradually up to 15 DAS, but then declined sharply showing a dip at 20 DAS under

Chemistry and Ecology

515

Figure 2. Microbial biomass C and N under different tillage practices at different stages of wheat development. Bars
represent mean 1 SE (n = 9). Bars with different letters at a plant stage are significantly different from each other
according to DMRT (p < .05). DAS = Days after sowing.

all the tillage practices. After 40 DAS, urease activities again declined and then remained nearly
constant. Differences in urease activity under different practices could be observed only up to
40 DAS during which lowest activities were observed under NTR-CTW or NTR-NTW. Highest
1 1
h was recorded under CTR-NTW at 33 DAS. After 110
urease activity of 0.08 mg NH+
4 g
DAS, urease activities again increased. Glycine-aminopeptidase activities remained nearly constant and similar in the four practices up to 105110 DAS, ranging between 0.11 (in NTR-CTW)
and 1.52 (in NTR-NTW) g pNA g1 h1 (Figure 4). With the maturity stage of wheat (after
110 DAS), glycine-aminopeptidase activities begin to rise. The increase was most rapid under
CTR-NTW, where it rose from 0.47 to 18.72 g pNA g1 h1 in a five-day interval. The rise
was most gradual under NTR-NTW. Two-way ANOVA indicated that tillage practices, DAS and
their interaction had significant effects on activities of all the extracellular enzymes (Figures 3
and 4).

516

D. Pandey et al.

Figure 3. Activities of -glucosidase, cellobiohydrolase, alkaline phosphatase and polyphenol oxidase in soil under
different tillage practices. Values are mean 1 SE (n = 9). Results of two-way ANOVA are presented in the box.
A = days after sowing; T = tillage practice. CTR-CTW: tillage before transplanting rice and sowing wheat; NTR-CTW:
no tillage before sowing of rice and tillage before sowing of wheat; CTR-NTW: tillage before transplanting rice but no
tillage before sowing of wheat; NTR-NTW: no tillage before sowing of both rice and wheat; pNP: p-nitrophenol; PPG:
purpurogallin.

Figure 4. Activities of glycine-aminopeptidase and urease in soil under different tillage practices. Values are mean 1
SE (n = 9). Results of two-way ANOVA are presented in the box. A = days after sowing, T = tillage practice.
CTR-CTW: tillage before transplanting rice and sowing wheat; NTR-CTW: no tillage before sowing of rice and tillage
before sowing of wheat; CTR-NTW: tillage before transplanting rice but no tillage before sowing of wheat; NTR-NTW:
no tillage before sowing of both rice and wheat; pNA: p-nitroaniline.

Chemistry and Ecology

4.
4.1.

517

Discussion
Microbial biomass C and N

The temporal trends of microbial biomass C and N appeared to be controlled jointly by crop
developmental stages and environmental conditions. It is widely reported that adoption of no
tillage leads to increment in microbial biomass in soil.[3436] In the present study also, microbial
biomass C as well as N increased under NTR-CTW, CTR-NTW and NTR-NTW as compared
with CTR-CTW. Incorporation of crop residues associated with no tillage practice increases the
amount of organic substrates and nutrients in the soil and hence major soil nutrients increased
to the greatest degree under NTR-NTW followed by CTR-NTW and NTR-CTW compared with
CTR-CTW (Table 1). Under enhanced nutrient availability, microbial growth and population are
also expected to increase. It has also been demonstrated that tillage activities disrupt microbial
communities in soil, particularly the fungal hyphae.[35] Therefore, microbial biomass under
CTR-CTW was the lowest among the four tillage practices.
Among different developmental stages, lowest microbial biomass C and N during the late
vegetative phases could be due to low Ts during this stage. Though urea top dressing was
expected to foster better microbial biomass during 4555 DAS, low Ts values during this period
might have acted as a limiting factor for microbial proliferation. Various studies demonstrate that
microbial biomass is sensitive to changes in different environmental conditions in soil, particularly moisture and temperature.[22,23,26] In case of agricultural soils also, a direct relationship
between microbial biomass and Ts has been observed,[18,37,38] which is attributed to increase
in substrate availability due to mineralisation alongside more favourable environmental conditions for microbial multiplication. Increase in temperature helps in releasing substrates, which
are physically protected in soil aggregates and are inaccessible to microbes.[18,27] In many
cases, however, soil moisture becomes limiting at higher temperatures and microbial biomass
decreases.[27]
Regarding the practices in which tillage was done for either crop, that is, NTR-CTW and
CTR-NTW, timing of tilling and nature of the residue left on soil appeared to affect microbial
biomass significantly. CTR-NTW represented a system in which residues of rice plants were left
in the field and the soil was not disturbed during wheat cultivation. The freshly added residues of
rice crop increased the microbial biomass C and N under CTR-NTW than NTR-CTW in which
residues of wheat crop was added during the rice cultivation period while no significant plant
residue was present during wheat cultivation. The quality of residues of the two crops could
also affect microbial biomass responses. In a priming and incubation study, Thiessen et al. [39]
also observed that the higher temperature and application of organic matter increased microbial
biomass, and the effect of organic matter application was much higher during the first few days
of application. During the later stages of crop development, gradual rise in Ts provided better
opportunity for utilising the favourable conditions for microbes and for them to multiply under all
the tillage practices. These conditions match well with the environmental conditions during the
present study. Under CTR-CTW, microbial biomass showed relatively low temporal variability,
which could be due to low nutrient accumulation in soil under this practice.

4.2.

Enzyme activities

The amount of glucose generated during decomposition of residues during early vegetative
stages probably could not address the microbial demand as the crop reached the late vegetative
stage. Also, it is observed that during early vegetative stages, root exudation rate remains high,
which provides simpler substrates to the microbes,[40] but during the late vegetative stages, root

518

D. Pandey et al.

exudation decreases thereby decreasing the availability of simple molecules. Therefore, as the
glucose was utilised, more -glucosidase was secreted. Expected increases in microbial activity
upon N application might also have induced -glucosidase.[6] This could be a reason that peak
activities were observed during top dressings with urea. tursova et al. [41] also observed that
-glucosidase activities responded to N availability in different types of soils.
Regarding the effect of tillage practices, it was interesting to note that -glucosidase activities
were the highest under NTR-CTW, a practice intermediate of the conventional CTR-CTW and
completely no tillage NTR-NTW. Under NTR-CTW, residues of wheat crop increased the nutrients and organic matter in the soil. Second, the organic material was mixed with the soil through
tillage before sowing of wheat. These conditions could have favoured better -glucosidase activities. Gianfreda and Ruggiero [42] also made similar observations that the enzyme activities in
soil are affected by the presence of plant cover, nature of the residues and degree of residue
mixing with the soil.
The increasing trend of cellobiohydrolase activities until the crop maturity can be attributed
to increase in microbial demand of carbohydrates as previously available substrates produced by
decomposition of crop residues were utilised. It was reflected that N application was creating
sudden C demand, which was fulfilled through induction of -glucosidase and cellobiohydrolase activities. Regarding the lowest cellobiohydrolase activities under CTR-CTW during early
stages and under NTR-NTW during maturity stage, it could be due to differences in microbial
demand and availability of specific substrates under different tillage practices. Keeping in mind
the properties of the two practices, it may be suggested that during the initial stages of crop
development, cellobiohydrolase activity depended on amount of organic matter present in the
soil. In later stages, on the other hand, mixing of residues might have become more important in
stimulating cellobiohydrolase activities.
As discussed, the top dressing at the start of the reproductive stage apparently created C
demand, hence it is probable that polyphenol oxidase was also induced along with -glucosidase
and cellobiohydrolase. Higher activities of -glucosidase under NTR-CTW also support this
probability. The trends of polyphenol oxidase activity in the present study followed the general observation of an inverse relation between organic C content and polyphenol oxidase.[43]
Other hydrolytic enzymes, namely, alkaline phosphatase, urease and glycine-aminopeptidase,
also responded to the tillage practices with increased activities under NTR-CTW, CTR-NTW
and NTR-NTW with respect to CTR-CTW. Similar responses have also been reported from
long-term no tillage practices.[44] Trend of alkaline phosphatase reflected that this enzyme is
controlled primarily by P availability [43] and hence was induced only when the applied P as
fertiliser could not meet the microbial demand.
High urease activity during the initial stages of crop development could be due to high N
demand of plants for rapid vegetative growth and tillering under availability of ample substrate
added during basal dressing. After this, urease activities remained low and constant and even
urea application did not increase its activity, indicating that N demand of plants and microbes
was satisfied by previous urea applications. Small increases observed during the maturity phase
under all the practices reflected that N demand began to rise as previous available N was utilised
by the plants. Glycine-aminopeptidase activity during maturity stages also followed the trend of
urease, indicating that N demand regulated activities of both the enzymes. Though urease and
glycine-aminopeptidase carry out N transformations, their activities have been found to be linked
significantly with availability of other nutrients, mainly C in soil.[45] This happens because
application or organic residues may dilute N as well as P availability, besides activating microbial
biomass and activities.[5,45] Hence activities of enzymes involved in N and P mineralisation
enhance with reduction in tillage intensity.
The results obtained in the present study supported the hypothesis that enzyme activities will
increase as tillage frequency is reduced and will be the maximum under NTR-NTW. Polyphenol

Chemistry and Ecology

519

oxidase was the only enzyme that declined as the frequency of tillage reduced, being the
minimum under NTR-NTW.
4.3.

PCA and relationships between extracellular enzymes

PCA condensed the soil properties and enzyme activities on two PCs, which could explain more
than 55% of the total variance (65.85% in CTR-CTW, 61.67% in NTR-CTW, 67.03% in CTRNTW and 57.95% in NTR-NTW) (Figure 5). However, none of the parameter was loaded heavily
on any single PC, except cellobiohydrolase.
Under all the practices, glycine-aminopeptidase and Ts were associated. Laboratory experiments have shown that at lower temperatures, enzyme activities become more sensitive to
temperature [26,27] but in field conditions, response of enzyme activities vary widely due to

Figure 5. Loading plots for soil properties and extracellular enzyme activities on principal components (PC). Ts
= soil temperature; GWC = gravimetric water content; ORP = oxido-reduction potential; CTR-CTW = conventional
tillage before transplantation of rice and sowing of wheat; NTR-CTW = no tillage before sowing of rice and conventional tillage before sowing of wheat; CTR-NTW = conventional tillage before transplantation of rice and no tillage
before sowing of wheat; NTR-NTW = no tillage before sowing of both rice and wheat.

BG

UR

NS

NS

2.40x + 78.24**
1.42x + 52.04**

NS

ALP

CBH

520

Table 2. Equations describing relationships (y = f (x)) between different extracellular enzyme activities and soil properties.
GAP

PPO

x
Ts

GWC

BG

CTR-CTW
NTR-CTW
CTR-NTW

1.77x 4.27***
NS

NS

NS

NTR-NTW
CTR-CTW
NTR-CTW

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

CTR-NTW
NTR-NTW
CTR-CTW

1.13exp(0.06x)*
1.11x 4.33**

NTR-CTW

ALP

NTR-CTW
CTR-NTW
NTR-NTW
CTR-CTW
NTR-CTW
CTR-NTW
NTR-NTW
CTR-CTW
NTR-CTW
CTR-NTW
NTR-NTW

NS

0.002x 0.04*
0.0014x 0.016*

0.03x + 1.56*
NS

1.72 0.82x +
0.001x2 *

NS

NS

NS

NS

0.002x 0.03*

NS
NS

NS
NS

0.004exp(0.244x)*
1.44x 23.77*
0.60x 0.95***
0.51x 7.83***
NS

0.05x2
3.86x + 7.50*

NS
NS
NS
NS
NS
NS

NS

NS

NS

NS

NS

NS

NS

NS

3.64 + 10.90x***

NS

NS

NS

1.0x + 56.46*
0.004x3 0.44x2
+ 14.44x 107.96*
NS

0.90x + 64.72*
39.14 350.29x*
NS

38.93 304.21x**

NS

34.24 + 4.10 103 x


.12 104 x2
+ 8.912 105 x3 **
NS

NS

33.55 264.23x**
NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

1.94 + 8.97x***
0.01 + 2.22x***

22.21x + 3.004*

NS

NS

NS

NS

NS

177.19x2 17.97x
+ 38.77*

NS

10.02 0.82x
+ 0.02x2 **
NS

NS

NS
NS

Notes: Ts = soil temperature; GWC = gravimetric water content; BG = -glucosidase; UR = urease; ALP = alkaline phosphatase; CBH = cellobiohydrolase; GAP = glycine-aminopeptidase; CTRCTW = conventional tillage before transplantation of rice and sowing of wheat; NTR-CTW = no tillage before sowing of rice and conventional tillage before sowing of wheat; CTR-NTW = conventional
tillage before transplantation of rice and no tillage before sowing of wheat; NTR-NTW = no tillage before sowing of both rice and wheat. NS not significant. PPO and Ts had no significant relationship with any
other soil parameter or extracellular enzyme activity.
*p < .05.
**p < .01.
***p < .001.

D. Pandey et al.

UR

CTR-NTW
NTR-NTW
CTR-CTW

CBH

0.001x2 0.043x
+ 0.481**

NS

0.06x 0.52*

Chemistry and Ecology

521

different activity ranges of responsible microbes, properties of soil matrix and conditions of substrate availabilities.[40] The curve estimation revealed a positive linear relationship between Ts
and glycine-aminopeptidase under all the tillage practices except under CTR-CTW in which
there was exponential increase in glycine-aminopeptidase activity with Ts. This indicated that
under CTR-CTW, glycine-aminopeptidase activity might be limited primarily due to low temperature. -Glucosidase and urease were also found to be associated with ORP under CTR-CTW
and NTR-CTW, with GWC under NTR-NTW or with GWC and ORP under CTR-NTW (Table
2). The nature of the relationship between -glucosidase and urease was linear under CTRCTW and NTR-NTW, which could be due to synchronised demand of C and N by the microbes.
Their association with GWC or ORP indicated that it was not only nutrient demand which
directly affected the activities of these two enzymes, but also the environmental variables which
might have modified the responses. Previous studies have also reported that both urease and
-glucosidase are highly sensitive to temperature, water content, pH and presence of toxins,
but the relative dominance of such factors depends on specific environmental conditions of the
system.[10]
Under CTR-CTW, cellobiohydrolase and -glucosidase were loaded on the same PC. The linear equation describing the relationship between the two also supported this observation. Under
NTR-CTW and CTR-NTW, alkaline phosphatase increased with increasing temperature (Table
2). Although alkaline phosphatase is observed to be sensitive to Ts in the present study, the
strongest driver of this enzyme is suggested to be the P stress.[10] It is probable that alkaline phosphatase increased with decrease in P availability with crop development, and Ts also
increased from sowing (in November) to harvest (in April); therefore, the two appeared to be
associated.
Tillage practice-specific associations were found between alkaline phosphatase and polyphenol oxidase under CTR-CTW, cellobiohydrolase and GWC under NTR-CTW and alkaline
phosphatase and cellobiohydrolase under NTR-NTW. Relationships between -glucosidase and
cellobiohydrolase were significant under all the practices except CTR-NTW, but their natures
were different. This indicated that -glucosidase and cellobiohydrolase being involved in C mineralisation acted simultaneously but behaved differently under different tillage practices. There
was no consistent association identified for polyphenol oxidase either through PCA or through
curve estimation. Polyphenol oxidase was associated with alkaline phosphatase positively under
CTR-CTW and NTR-NTW. Under CTR-CTW, -glucosidase was related significantly with the
activities of cellobiohydrolase, urease and alkaline phosphatase. In the rest of the practices, most
of these relationships were masked and none of the enzymes was found to be related with glucosidase significantly under CTR-NTW. Except -glucosidase, cellobiohydrolase was not
found to be related consistently with any of the soil properties or enzyme activities.
In light of the PCA and curve estimation results, our hypothesis that relationships between
different enzyme activities and dependence of different enzymes on environmental factors will
remain unaffected under different tillage practices was not supported.

5.

Conclusion

Activities of extracellular soil enzymes were controlled by soil temperature and moisture, but
the nature of relationships between them varied among different tillage practices. This suggested
that the environmental factors and tillage managements interacted in complex ways to influence
such relationships. Overall, the reduction in tillage frequency improved the soil quality in terms
of microbial biomass and enzyme activities involved in nutrient regulation under CTR-NTW,
NTR-CTW and NTR-NTW as compared with the conventional CTR-CTW. More field studies

522

D. Pandey et al.

are, however, required to explore the dependency of different enzyme activities on environmental
factors and synchrony between their expressions under diverse agricultural practices as well as
natural ecosystems.

Acknowledgements
Divya Pandey is thankful to the Council for Scientific and Industrial Research (CSIR), New Delhi, and National Academy
of Sciences, India, for research fellowships.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
Authors acknowledge University Grants Commission (UGC), New Delhi, for financial support through research project.

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