Soil & Tillage Research 136 (2014) 51–60

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Soil & Tillage Research

journal homepage: www.elsevier.com/locate/still

Effects of conventional tillage and no tillage permutations on

extracellular soil enzyme activities and microbial biomass
under rice cultivation
Divya Pandey a, Madhoolika Agrawal b,*, Jitendra Singh Bohra c
a
Laboratory of Air Pollution & Global Climate Change, Department of Botany, Banaras Hindu University, Varanasi 221005, India
b
Department of Botany, Banaras Hindu University, Varanasi 221005, India
c
Department of Agronomy Institute of Agriculture Sciences, Banaras Hindu University, Varanasi, India

A R T I C L E I N F O A B S T R A C T

Article history: Extracellular soil enzymes hold a cardinal position in nutrient dynamics by regulating bioavailability of
Received 27 May 2013 elements, and hence are linked with soil health. The present study aimed to analyze the responses of
Received in revised form 24 September 2013 extracellular enzymes involved in mineralization of carbon (b-D-glucosidase (BG), cellobiohydrolase
Accepted 28 September 2013
(CBH), polyphenol oxidase (PPO)), nitrogen (urease (UR), glycine-amino peptidase (GAP)) and
phosphorous (alkaline phosphatase (ALP)) under four permutations of conventional tillage and no
Keywords: tillage under rice–wheat system in eastern Indo-gangetic plains during rice cultivation period. The
Extracellular soil enzymes
permutations were: tillage before sowing/transplantation of each crop (RCT–WCT), tillage before
Conventional tillage
transplantation of rice and no tillage before sowing of wheat (RCT–WNT), tillage before sowing wheat
No tillage
Microbial biomass and no tillage before sowing of rice (RNT–WCT) and no tillage before sowing of each crop (RNT–WNT).
Rice cultivation Microbial biomass carbon and nitrogen and activities of BG, CBH, ALP and UR increased with reduction in
tillage frequency, becoming the highest under RNT–WNT and the lowest under RCT–WCT. Principal
component analyses (PCA) condensed the variables in to two components, apparently described by soil
temperature and moisture content under all the tillage permutations. Most of the enzymes and soil
properties identified to be associated under PCA followed linear relationships. Under RCT–WCT, CBH, UR
and ALP were related with BG. Different orders of residue incorporation and tillage under RCT–WNT,
RNT–WCT and RNT–WNT masked these relations. Results indicated that reduction in tillage frequency
made the soil healthier. Relationship of BG with other enzymes appeared as a probable indicator to
reflect deviations from the conventional cultivation practice in the study region.
ß 2013 Elsevier B.V. All rights reserved.

1. Introduction transformations of carbon, nitrogen, phosphorous and sulphur at

fundamental level of nutrient cycling, these parameters have been
Soil being an essential resource to biosphere and human beings, proposed as sensitive indicators for soil quality and health than
is also amongst the most complex and least understood systems. physico-chemical properties (Dick, 1997; Sinsabaugh et al., 2008;
While its role in affecting crop productivity is widely studied, our Hartmann et al., 2009; Finkenbein et al., 2012). These indicators
understanding over biochemical controls on elemental dynamics become even more significant for assessing the performance of
in soil, still needs improvement. Plant growth depends positively different management practices in cultivated soils aiming to
on nutrients which are released during reactions catalyzed by improve soil fertility.
enzymes secreted into the soil by microbes and plants (Kibble- Among different resource conserving practices, no tillage is
white et al., 2008; Karaca et al., 2011). Because microbial becoming popular among farmers of India, particularly in those
population interacts closely with the soil environment and areas of Indo-Gangetic plains, which are facing deterioration in soil
extracellular enzyme activities (EEAs) play crucial role in fertility due to extensive tillage operations (Ladha et al., 2003).
Apart from low economic input, no tillage practice has added
benefits of nutrient conservation and minimizing soil erosion (Lal,
2004). However, complete elimination of tillage under many cases
* Corresponding author. Tel.: +91 542 2368156; fax: +91 542 368174.
is considered practically unfeasible due to increased compaction,
E-mail addresses: [email protected] (D. Pandey),
[email protected], [email protected] (M. Agrawal), reduced aeration and increased incidences of weed infestation
[email protected] (J.S. Bohra). (Pandey et al., 2012). Therefore, frequency and timing of tillage and

0167-1987/$ – see front matter ß 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.still.2013.09.013
52 D. Pandey et al. / Soil & Tillage Research 136 (2014) 51–60

no tillage practices are needed to be studied under diverse 2824) sequence was established in June 2003, at Agriculture
cropping systems. Rice–wheat system covers 13.5 million ha in farm of Banaras Hindu University, India. The site is located in the
south-east Asia, the largest part of which falls in India (Timsina and eastern Indo-Gangetic plains at 25.148N, 82.038E and 76.19 m
Connor, 2001). While agronomists are assessing economic turn- above mean sea level. The experimental soil was inceptisol with
outs under different permutations of tillage and no tillage, it is sandy loam texture. Under conventional tillage, soil was
essential to understand the behavior of biochemical properties and ploughed up to 15 cm depth during land preparation for sowing
their controls for strategic evaluation of soil quality. of wheat or transplantation of rice. Under this practice,
Studies conducted under different climatic regimes have shown preceding crop was harvested manually, removing the above
that these EEAs respond to even the slightest modifications in ground biomass. In case of no tillage, only ripened ears of the
environmental conditions and management practices like organic preceding crop was harvested while rest of the plant part
and mineral fertilization (Garcı́a-Ruiz et al., 2008), tillage (>20 cm) was left standing behind. Sowing of the next crop was
operations (Alvear et al., 2005), and herbicides and pesticides done by placing seeds into the holes drilled between the
applications (Seghers et al., 2003; Gianfreda and Rao, 2011). BG is standing stubbles. The four permutations of tillage practices
found to be useful as a soil quality indicator for management were: (i) conventional tilling before transplantation/sowing of
effects, organic matter stabilization, and physicochemical changes each crop (RCT–WCT), (ii) no tilling before sowing of rice and
(Bandick and Dick, 1999; Ndiaye et al., 2000). Class of enzymes conventional tilling before sowing of wheat (RNT–WCT), (iii)
called phosphatases are significantly correlated to phosphorous conventional tilling before transplantation of rice and no tilling
stress, and are also good indicators of soil fertility (Mudge et al., before sowing of wheat (RCT–WNT) and (iv) no tilling before
2002). Enzymes involved in protein decomposition are known to sowing of each crop (RNT–WNT). The present study was
control nitrogen mineralization, but their interaction with soil conducted during rice cultivation period of 2010 (July–October
environment is still unclear. It is suggested that proteases 2010). Average soil properties at inception of the tillage
including GAP might be playing crucial role in maintaining soil experiment in 2003 and before the transplanting of rice in
health (Das and Varma, 2011). UR on the other hand has received 2010 is presented in Table 1.
lot of attention because it hydrolyses urea, which is the most Under RNT–WCT and RNT–WNT, sowing of rice (Oryza sativa
common nitrogen fertilizer applied in cultivated soils. UR activity cultivar PHB-71) was done on the same date when seeds were
responds to organic matter content, soil depth, fertilizer applica- soaked for nursery raising. Under RCT–WCT and RCT–WNT, 21
tions, heavy metals, and environmental factors such as tempera- days old seedlings were transplanted on puddled bed. Urea was
ture (Tabatabai, 1977; Yang et al., 2006). applied at a rate of 150 kg N ha1 in three splits, half at the time
With above considerations, the present study was undertaken in of transplantation/sowing and the rest in two equal splits at
rice–wheat system subjected to different permutations of conven- critical tillerring and panical initiation stages. Phosphorous and
tional tillage and no tillage to understand the changes in microbial potassium were applied each at the rate of 75 kg ha1 as P2O5
biomass carbon and nitrogen and extracellular enzymes participat- and K2O, respectively at the time of sowing/transplantation.
ing in carbon, nitrogen and phosphorous mineralization during rice
cultivation period. The extracellular enzymes, b-glucosidase (BG), 2.2. Soil sampling and analyses
cellobiohydrolase (CBH), polyphenol oxidase (PPO), glycine amino-
paptidase (GAP), urease (UR), and alkaline phosphatase (ALP), Measurement of gravimetric water content (GWC), oxidor-
widely used in soil quality assessments were quantified, and their eduction potential (ORP), soil temperature (Ts) and EEAs was
relationships were calculated for better understanding of soil initiated on the day of transplantation and continued up to the
biochemistry and identification of probable indicators to reflect harvesting. It was tried to maintain an interval of five days
changes in soil conditions under different tillage permutations. between two successive measurements. ORP was measured with
the help of double junction ORP Tester 10 (Eutech Instruments,
2. Materials and methods Mumbai, India). The electrode was inserted 15 cm deep into the
soil and was left undisturbed for approximately five minutes till
2.1. Experimental set up ORP value displayed on the instrument stopped became
constant. The measurements were done at ten randomly
Field experiment involving four permutations of convention- selected sites per treatment. When soil was dry enough to
al and no tillage practices in rice (cv PHB-71)–wheat (cv HD prevent good contact between soil particles and electrode, as

Table 1
Soil properties at time of sowing/transplanting rice in 2003 and in 2010 (mean  1SE). Different letters in a row indicate that means of 2010 are different from each other in
accordance with Duncan’s test (p < 0.5).

2003 2010

RCT–WCT RCT–WCT RNT–WCT RCT–WNT RNT–WNT

Texture Sandy loam Sandy loam Sandy loam Sandy loam Sandy loam
Sand (%) 59.51 62.58 63.73 63.21 60.47
Silt (%) 14.19 13.64 13.76 11.12 17.23
Clay (%) 26.30 23.78 22.51 25.67 22.30
pH 7.20  0.01 7.21  0.01ns 7.21  0.07ns 7.20  0.10ns 7.22  0.04ns
Dry bulk density (g cm3) 1.44  0.01 1.47  0.01c 1.49  0.01c 1.53  0.10b 2.55  0.01a
Soil organic C (mg g1) 3.33  0.02 3.32  0.02c 6.21  0.02b 7.82  0.06b 9.91  0.09a
Total N (mg g1) 0.32  0.02 0.38  0.03b 0.62  0.02a 0.53  0.04a 0.58  0.03a
NO3-N (mg g1) 0.11  0.01 0.14  0.01c 0.17  0.01c 0.16  0.01c 0.14  0.01c
NH4+-N(mg g1) 0.19  0.01 0.22  0.01c 0.38  0.01c 0.38  0.01c 0.40  0.01c
Total P (mg g1) 2.45  0.05 2.44  0.07d 2.65  0.06 c 2.71  0.04 b 2.96  0.03a
Available P (mg g1) 1.23  .07 1.05  0.04c 1.43  0.08b 1.46  0.06b 1.87  0.05a

RCT–WCT, tillage before transplanting rice and sowing of wheat; RNT–WCT, no tillage before sowing of rice and tillage before sowing of wheat; RCT–WNT, tillage before
transplanting rice but no tillage before sowing of wheat; RNT–WNT, no tillage before sowing of both rice and wheat.
 D. Pandey et al. / Soil & Tillage Research 136 (2014) 51–60 53

observed during the late maturity stages of the crop, the selected 2.3. Statistical analysis
area on the soil (15 cm 15 cm) was wetted with approximately
250 ml of distilled water before inserting the electrode. The Mean and standard error for all the assessed parameters were
instrument was calibrated to +245 mV with reference Zobbel calculated. To test the difference between means of soil
buffer before every measurement. Ts was measured with a parameters under different tillage permutations in 2010 from
mercury thermometer (1/20 8C). The sensor of the thermometer initial conditions in 2003, t-test was performed. One way
was inserted at 15 cm depth in the soil and temperature was ANOVA with Duncan’s multiple range test (DMRT) was
recorded after two minutes when reading became constant. Ts conducted to test differences between mean MBC and MBN
were also measured at ten sites per tillage permutation selected under different tillage permutations. Two way ANOVA was
randomly. GWC was determined by using moisture sensor EC-05 conducted to analyze the individual and interactive effects of
(Decagon Devices, USA). The sensor was connected with hand tillage permutations and age of plant on EEAs. In order to
held and battery operated datalogger (Li-1400, LICOR Bios- identify the parameters which are most likely to be responsible
ciences, Lincoln, USA). The sensor was inserted in the soil up to for the variances under different treatments of tillage and no
the depth of 15 cm and the moisture content value displayed on tillage and to classify the extracellular soil enzymes and soil
the datalogger was recorded. The sensor was calibrated for the parameters, Principal Component Analysis (PCA) was conducted,
experimental soil. which is an applicable method in the present context. PCA was
For assessing EEAs, soil samples were collected with the help of applied to extract principal components through Varimax
core sampler (diameter 6.5 cm and depth 15 cm) after removing orthogonal rotation. To study the relationships between differ-
loose litter from three sites per treatment. The sampled soils were ent EEAs and soil properties, curve fitting analyses was carried
stored immediately in an insulated box maintained at 4 8C. In out. All statistical analyses were performed with the help of
laboratory, all procedures involved in soil enzyme assays were statistical software SPSS version 10, IBM, USA.
carried out in cold conditions (4 8C) and analyses were completed
the same day. The activities measured were cellobiohydrolase (EC 3. Results and discussion
3.2.1.91) (CBH), b-glucosidase (EC 3.2.1.21) (BG), polyphenol
oxidase (EC 1.10.3.2) (PPO), alkaline phosphatase (EC 3.1.3.1) 3.1. Microbial biomass carbon (MBC) and microbial biomass nitrogen
(ALP), glycine aminopeptidase (EC 3.4.11.-) (GAP) and urease (EC (MBN)
3.5.1.5) (UR). Activities of CBH, BG, ALP, and GAP were assayed
following Allison and Jastrow (2006). PPO activity was determined As presented in Fig. 1, MBC and MBN were the highest under
following the method of Keilin and Mann (1938). Briefly, 10 g fresh RNT–WNT followed by RNT–WCT, RCT–WNT and RCT–WCT. Being
soil was disrupted in cold (4 8C) Tris buffer (pH 7.4) for one an integral part of nutrient cycling, microbial biomass is strongly
minute. The enzyme extract separated through cold centrifugation affected by soil management activities and its increment after
was incubated with respective substrates selected according to the adoption of no tillage practice is a common observation (Doran,
bond hydrolyzed by the corresponding enzyme as presented in 1987; Wright and Hons, 2005). Practicing no tillage brings about
Table 2. The mixtures incubated in an incubator maintained at the two main modifications in the soil system. Firstly, crop residues are
temperature equal to the soil temperature. The product formed retained on the field which increases organic substrate and
was p-nitrophenol (pNP) in case of BG, CBH, and ALP, and p- nutrient availability in the soil. Secondly, disturbance of soil is
nitroaniline (pNA) in case of GAP. Absorbance of the products was reduced which prevents disruption in microbial consortia and soil
measured at 460 nm using spectrophotometer (Visiscan 167, aggregates. Both factors together foster better microbial growth
Systronics, India). In case of PPO activity assay, absorbance of (González-Chávez et al., 2010). Therefore, with respect to the
purpurogallin was measured at 420 nm. UR activity was deter- conventional practice i.e. RCT–WCT, both MBC and MBN increased
mined following Kandeler and Gerber (1988). For this, the enzyme under RCT–WNT, RNT–WCT and RNT–WNT. The temporal trend of
extract in cold tris buffer was incubated with urea at 37 8C. NH4+ MBC and MBN was similar under all the tillage permutations, being
thus released was estimated following salycilate method (Nelson, the maximum during early vegetative stage, and decreasing
2003). All enzyme activities were expressed in terms of the product afterwards. During rapid plant growth in early vegetative stages i.e.
formed g1 dry soil h1. up to 30 days after transplantation (DAT), root exudation rate
Microbial biomass carbon (MBC) and nitrogen (MBN) are remains high thereby providing preferred substrates to rhizo-
indicators of microbial biomass. They were determined following spheric microbes (Singh et al., 2009). Basal application of NPK was
chloroform fumigation and extraction method (Brookes et al., also expected to address microbial demand for these nutrients.
2005). Fresh soil samples were fumigated under chloroform Therefore, microbial biomass was the highest during this period. As
vapors in vacuum desiccators for 24 h. Difference between carbon crop growth advanced, nutrient availability to microbes could have
and ninhydrin reactive nitrogen contents in fumigated and declined due to immobilization in plant and microbial biomass,
unfumigated samples were determined following the methods which could have resulted in limitation of certain nutrients to
of Walkley and Black’s (1947) and Joergensen and Brookes (1990), microbial proliferation. Apart from this, an important change in
respectively. soil conditions occurred when flooding of the field was stopped

Table 2
Extracellular enzymes, their actions and substrate specific reactions.

Enzyme Action Substrate Products

b-D-glucosidase Hydrolysis of terminal b-D-glucosidic bond from non reducing end pNP-b-D-glucopyranoside pNP* + glucose
Cellobiohydrolase Hydrolysis of (1–4)- b-D-glucosidic linkages in cellulose releasing glucose dimers pNP-cellobioside pNP + cellobiose
Polyphenol oxidase Oxidation of hydroxyphenol bond Pyrogallol Purpurogallin
Glycine aminopeptidase Cleavage of peptide bond releasing amino acid Glycine p-nitroanilide pNA** + Glycine
Phosphatase Dephosphorylation pNP-phosphate pNP + H3PO4
Urease Hydrolyses of urea Urea CO2 + 2NH3
*
p-nitrophenol.
**
p-nitroanaline.
54 D. Pandey et al. / Soil & Tillage Research 136 (2014) 51–60

RCT-WCT RNT-WCT RCT-WNT RNT-WNT after completion of tillerring stage. This led to a transition from
1.4 anaerobic to aerobic conditions, which was likely to act as a shock
a
1.2 to microbial consortia particularly the anaerobic population. All
b
these factors manifested in the form of gradually declining
MBC (mg g-1)

1.0
c microbial biomass along the crop advancement as reflected by
0.8
the values of MBC and MBN.
a
0.6
a a
b b
0.4 d b b 3.2. Enzyme activities
c c b b
c
0.2
EEAs reflect the cumulative effects of substrate availability,
demand of the product, microbial activity and environmental
1.4
14
a conditions (Allison et al., 2011). In the present study, all the
12
1.2 a enzymes except PPO were generally the highest under RNT–WNT,
MBN (mg g-1)

10
1.0 and the lowest under RCT–WCT among the four permutations.
b
0.88 Reduction in tillage frequency is reported to enhance EEAs due to
0.66
a improvement in microbial biomass, more substrate availability
a a
c b b and reduced soil disturbance (Sinsabaugh et al., 2008). Effect of
0.44 b
c tillage operations on fungal EEAs, which constitute the major part
0.22 c in enzyme pool, is also detrimental due to disruption of mycelia
b
d c bc
0
Early vagatative Late vagatative Reproductive Maturity (van der Wal et al., 2006). Two way ANOVA indicated that tillage
(0-30) DAT (30-60) DAT (60-90) DAT (90-120) DAT permutation and stage of plant had significant individual and
interactive effects on activities of all the enzymes (Figs. 2 and 3).
Fig. 1. Monthly microbial biomass carbon (MBC) and nitrogen (MBN) in different
tillage practices. Bars represent mean  1SE (n = 3). In a particular month, bars with
different letters are significantly different in accordance with Duncan’s multiple range 3.2.1. Activities of b-glucosidase, cellobiohydrolase and polyphenol
test (p < 0.05). RCT–WCT, conventional tillage before transplantation of rice and oxidase
sowing of wheat; RNT–WCT, no tillage before sowing of rice and conventional tillage BG, CBH and PPO are considered to be the key controllers of
before sowing of wheat; RCT–WNT, conventional tillage before transplantation of rice carbon dynamics in soil (Sinsabaugh et al., 2008). BG and CBH act
and no tillage before sowing of wheat; RNT–WNT, no tillage before sowing of both rice
and wheat.
as strong reflectors of carbon mineralization, and hence are often
employed to indicate rate of incorporation of labile carbon in soil
(Kang et al., 1998). PPO on the other hand participates in lignin

RCT-WCT RNT-WCT RCT-WNT RNT-WNT

-D-glucosidase A*** Cellobiohydrolase A***

T** T*** 50
60 AXT*** AXT***
* * 40 g pNP g-1 h-1
g pNP g-1 h-1

40
30

20 20

0 10

Alkaline Phosphatase A*** Polyphenol oxidase A***

1.4 T* T***
AXT*** AXT*** 35
1.2
* **
g PPG g-1 h-1
g pNP g-1 h-1

1.0 30

0.8
25
0.6

0.4 20

0.2
15
0.0
10
0 20 40 60 80 100 120 0 20 40 60 80 100 120 140
Days after transplantation

Fig. 2. Temporal variations in activities of b-D-glucosidase, cellobiohydrolase, alkaline phosphatase and polyphenol oxidase under different tillage practices. Results of three
ways ANOVA is presented inside the box. A, days after transplantation; T, tillage practice. Levels of significance: *p < 0.05, **p < 0.01, ***p < 0.001. RCT–WCT, conventional
tillage before transplantation of rice and sowing of wheat; RNT–WCT, no tillage before sowing of rice and conventional tillage before sowing of wheat; RCT–WNT,
conventional tillage before transplantation of rice and no tillage before sowing of wheat; RNT–WNT, no tillage before sowing of both rice and wheat. pNP, p-nitrophenol; PPG,
purpurogallin.
 D. Pandey et al. / Soil & Tillage Research 136 (2014) 51–60 55

RCT-WCT RNT-WCT RCT-WNT RNT-WNT

Urease A*** Glycine aminopeptidase A***

T*** T* 1.4
0.10
AXT*** AXT***
1.2
0.08

g pNA g-1 h-1

mg NH4+ g-1 h-1
1.0
0.06 0.8

0.04 0.6

0.4
0.02
0.2
0.00
0.0

0 20 40 60 80 100 120 0 20 40 60 80 100 120 140

Days after transplantation

Fig. 3. Temporal variations in activities of urease and glycine aminopeptidase under different tillage practices. Results of three ways ANOVA is presented inside the box. A,
days after transplantation, T, tillage practice. Levels of significance: *p < 0.05, **p < 0.01, ***p < 0.001. RCT–WCT, conventional tillage before transplantation of rice and
sowing of wheat; RNT–WCT, no tillage before sowing of rice and conventional tillage before sowing of wheat; RCT–WNT, conventional tillage before transplantation of rice
and no tillage before sowing of wheat; RNT–WNT, no tillage before sowing of both rice and wheat. pNA, p-nitroaniline.

degradation, and therefore is found to be linked with recalcitrant of organic and conventionally managed olive farms further
carbon fraction (Shi, 2011). observed that BG was increased under organic amendments. This
The trend of BG observed under different tillage permutations is effect, however, is not necessarily true for certain cellulolytic
presented in Fig. 2. Peak activities of BG were observed during 5–10 enzymes, as higher activities of certain enzymes may depend on
DAT. The second, but relatively smaller peak appeared at 45–55 distribution of organic matter in soil matrix (Kandeler and Bohm,
DAT, after which BG activities declined and remained low 1996).
throughout the maturity stage. Highest and lowest activities were Contrary to BG, CBH followed different temporal trends under
recorded under RNT–WNT (53.83 mg pNP g1 h1) and RCT–WCT different tillage permutations. Since the date of transplantation
(5.02 mg pNP g1 h1), respectively. CBH activity under RNT–WNT CBH activities either increased as under RNT–WNT, or remained
remained nearly constant (18.85–20.00 mg pNP g1 h1) up to 60 nearly constant as under RCT–WNT with advancement in crop
DAT and then increased sharply (Fig. 2). Among rest of the growth. Under RCT–WCT and RNT–WCT, CBH activity decreased
permutations, increase in CBH activity was observed only under up to 15–20 DAT, afterwards which it remained nearly constant
RCT–WNT after 110 DAT (Fig. 2). Activities of both the enzymes (BG throughout the crop period. Such differences in temporal trends
and CBH) were highest under RNT–WNT and the lowest under clearly indicated that activity of CBH was highly sensitive to
RCT–WCT on most of the monitoring dates. different soil conditions in the four permutations during different
BG controls the release of glucose which is an important phases of crop development. This was also indicated by the two
source of energy to microbes. High microbial biomass during way ANOVA as presented in Fig. 2.
early stages of crop development as indicated by the trends of Activity of CBH has been found to be inhibited under presence
MBC and MBN (Fig. 1) could have created glucose demand thus of high nitrogen concentration (Fan et al., 2012). It is possible that
stimulating BG activity. As microbial biomass declined with crop application of urea as a basal dose led to inhibition of CBH and
development, BG activity also decreased. Wang and Lu (2006) hence its activity declined for a short term under RCT–WCT and
observed that in paddy soils, water logging inhibited the activity RNT–WCT. Under RNT–WNT and RCT–WNT, CBH activities were
of BG. But in the present study, BG activities under all the tillage already low and hence, further declination was not observed.
permutations were the highest during the vegetative stages Constant CBH activity under RCT–WCT, WCT-WNT and RNT–WCT,
when field was flooded. This difference can be due to presence of indicated that carbon mineralization was taking place at nearly a
rice plants, which were absent in the experiment of Wang and Lu fixed rate. Under RNT–WNT however, organic residues were
(2006). Since, rice plants conduit atmospheric oxygen to the soil, present in abundance and microbial activity was also high.
it was likely to prevent inhibition of BG, which otherwise could Therefore, as soon as the inhibitory effect of nitrogen was reduced
have occurred. However, there was a dip in BG activity during by immobilization in plant and microbial tissues, CBH activities
25–35 DAT under all the permutations but to a greater extent increased. Rise in CBH activity after tillering stage could have been
under RCT–WCT, and RNT–WCT. additionally promoted by senescence of some tillers, which led to
Tillage permutations also affected BG activity, and on most of increase in cellulosic substrates. It is reported that activity of CBH
the monitoring dates it was the highest under RNT–WNT and the is influenced by microbial community, particularly the relative
lowest under RCT–WCT. In case of RCT–WNT and RNT–WCT abundances of bacterial and fungal populations, which may get
however, there were no clear differences. It is widely reported that altered under different fertilizer and organic matter status in the
activities of most of the hydrolytic enzymes including BG are soil (Štursová et al., 2012). Lynd et al. (2002) demonstrated that
increased when reduction in tillage intensity improve soil organic aerobic and anaerobic microbes adopt different strategies for
matter content and microbial biomass (Lagomarsino et al., 2009; cellulose decomposition. While anaerobes are less active in
Roldán et al., 2005). Presence of residues of previous crop (wheat) cellulose degradation, aerobic bacteria secrete extracellular
could also increase BG activity in RNT–WCT and RNT–WNT, as enzymes for the same. The trend of CBH indicated that minerali-
evidenced in earlier studies conducted under similar conditions zation rate of carbon increased under this permutation as crop
(Allison and Vitousek, 2005). Debosz et al. (1999) also showed that stages advanced.
organic matter incorporation in soil stimulated BG activity in Debosz et al. (1999) observed nearly similar trends in BG and
agricultural soils. Garcı́a-Ruiz (2008), in their analyses of 18 pairs CBH activities in soil as was the case under RCT–WCT of the present
56 D. Pandey et al. / Soil & Tillage Research 136 (2014) 51–60

study. It is proposed that the complex of enzymes carrying out while ALP activities were similar in the rest. This could be due to
decomposition of organic matter keeps on changing because some the reason that under RNT–WNT, microbial demand as well as
enzymes depend on fresh litter input, plant growth, root exudates substrate availability and microbial biomass were high. This was in
quantity and quality, while others may depend primarily on agreement with the observations made by Shi et al. (2013).
specific environmental conditions (Dick, 1997). Increase in Maximum and minimum activities were observed in RCT–WNT
cellulose content is shown to promote activities of cellulases, (1.19 mg pNP g1 h1) and RNT–WNT (0.01 mg pNP g1 h1), re-
including BG and CBH (Geisseler and Horwath, 2009) and this was spectively.
also followed in the present study as highest activities of both the
enzymes was observed under continuous absence of tillage 3.2.3. Urease and glycine amino peptidase
practice (RNT–WNT) which harbor cellulose rich plant residues Temporal variations in UR and GAP activities are illustrated in
to the greatest extent. Fig. 3. Like ALP, UR activities were also low during initial stages of
PPO activity declined gradually since the day of transplantation crop development, but increased gradually reaching maximum by
in all the permutations (Fig. 2). By late vegetative stages (40–50 35–40 DAT, corresponding to early elongation phase (maximum in
DAT), it again started increasing, attaining peaks during 65–70 RCT–WNT 0.11 mg NH4+ h1 g1). High dose of urea and initial
DAT. During vegetative phase (0–40 DAT), PPO activity was usually flooding which expectedly hydrolyzed urea readily to NH4+, could
the lowest under RCT–WCT, but after 60 DAT, it became the highest have been responsible for low UR activities during this stage. As
under RCT–WCT among the four permutations. demand of NH4+ increased, UR activity also increased under all the
Extracellular PPO is secreted by plants, bacteria and fungi to tillage permutations. UR was suppressed following top dressing at
degrade lignin and phenols, generally to mitigate phenol and metal 30 DAT due to increased nitrogen availability in the soil.
toxicity or as an antimicrobial agent. PPO activity requires aerobic Afterwards, UR activities subsided and came down to nearly
conditions and can be inhibited by lack of oxygen. Therefore, PPO constant values, which were similar in all the permutations. At 60
activities under all the tillage permutations were low during the DAT, top dressing with urea did not affect UR activities
tilerring stage (0–45 DAT) because field was flooded and hence soil considerably. Presence of enough nitrogen in soil from previous
was anaerobic. After tillering stage when floodwater was removed fertilizer applications might be a reason behind this trend.
and soil gained aerobicity, PPO activities also increased under all GAP activities kept on fluctuating throughout the rice cultiva-
the tillage permutations. Our observations however, do not comply tion period, except sudden dips following urea applications (Fig. 3).
with Saiya-Cork et al. (2001) and Waldrop et al. (2004) who Allison and Vitousek (2005) also reported fall in GAP after NH4+
demonstrated that nitrogen application may inhibit PPO produc- application in soil. This suggested that nitrogen demand was the
tion in the soil. Since PPO is suggested to act on complex polymers chief regulator of both the enzymes. With multiple sources of
thus releasing nitrogen in usable form, its elevated secretion under carbon and nitrogen in the soil, Geisseler and Horwath (2008) also
nitrogen stress is usually observed (Carreiro et al., 2000). However, found that enzymes participating in protein degradation are
in the present study, it was unlikely that nitrogen stress could controlled mainly by the concentration of nitrogen substrate and
reach to an extent that PPO would be secreted in its response. available nitrogen. GAP activity was the highest under RNT–WCT
Among the tillage permutations, PPO activities followed trend during 0–40 DAT but afterwards its activity became highest under
different from that of BG and CBH. It was the maximum under RCT– RCT–WNT. This was contrary to the trends of other enzymes,
WCT and minimum under RNT–WNT on most of the monitoring where highest activities were observed either under RNT–WNT or
days. This complied with Sinsabaugh et al. (2008) who reviewed RCT–WCT because of their dependencies on organic matter
EAAs at global scale and concluded that PPO activity tends to vary content. RCT–WNT and RNT–WCT represent the systems, which
inversely with organic matter content in the soil. Increase in PPO were rich in organic matter content with respect to the RCT–WCT,
activity often indicates rapid carbon loss as it helps in decomposi- but the organic matter was mixed to a better degree as compared
tion of complex and otherwise less active organic compounds. to RNT–WNT due to tillage during one crop. Therefore, organic
Behavior of PPO however, remains largely unpredictable as its matter present in the soil might be more easily available to the
secretion is also guided by presence of toxic components in the soil. microbes due to better distribution.
If the residues which are added under no tillage practice could be
acted upon easily by BG and CBH, activity of PPO might not be 3.3. Principal component analysis and relationships between
required. extracellular enzymes

3.2.2. Alkaline phosphatase PCA was used to extract latent factors for classification of EEAs
Up to 40 DAT, ALP activity in all the tillage permutations was and assessed soil variables. Equations describing relationships
low and remained temporally constant (Fig. 2). Afterwards, it between EEAs and soil parameters obtained through curve fitting
started increasing gradually up to 60 DAT after which ALP activities analyses are presented in Table 3. Two principal components (PC)
followed a fluctuating temporal trend till crop maturity and were retained in all the tillage permutations, which could explain
harvest. This enzyme is recognized as a reliable indicator of more than 50% of the total variance (56.44% in RCT–WCT, 55.94% in
phosphorous stress (Speir and Ross, 1978). A signal of phosphorous RNT–WCT, 65.35% in RCT–WCT and 65.35% in RNT–WNT). The
deficiency in the soil triggers its secretion from plant roots and loading plots obtained were different for the four tillage
microbes in order to solubilize and remobilize bound phosphate permutations thus indicating different controls over the expres-
(Mudge et al., 2002). This explains low ALP activity at initial stages sion of EEAs (Fig. 4). Ts and GWC were loaded heavily on
of crop development since phosphorous was added to the soil at orthogonal PCs in all the tillage permutations, which showed that
basal dressing. Inhibition of ALP in waterlogged soils is also these two factors were mutually independent. On the other hand,
reported (Wang and Lu, 2006). Net effect of the two could have led GWC and ORP were found to be loaded on same PC, but in opposite
to very low ALP activities up to 40 DAT in the present study. It is directions, thus revealing that increase in GWC caused ORP to
also reported that nitrogen fertilization triggers phosphorous decrease, thus making soil conditions more reducing.
demand and hence ALP activity (Allison et al., 2006). This could be Among the four tillage permutations, loading plot for RCT–WCT
the reason that ALP activities increased following urea applications was relatively different from the rest. Under this permutation,
as top dressing in the present study. Among the four permutations, there were two clusters, one containing enzymes related to carbon
RNT–WNT witnessed the highest ALP activity on most on the dates, mineralization (BG and CBH) and the other with enzymes
 D. Pandey et al. / Soil & Tillage Research 136 (2014) 51–60 57

Table 3
Equations describing relationships (y = f(x)) between different extracellular enzyme activities and soil properties.

y!

x# BG UR ALP CBH GAP

ORP RCT–WCT 105x2 + 0.26x + 19.50*** NS NS NS NS

RNT–WCT NS 105x + 0.01* NS 0.04x + 20.75* 4  104x + 20.78

RCT–WNT NS NS NS NS NS

RNT–WNT NS NS NS NS NS

GWC RCT–WCT 0.262x + 28.73*** NS NS 0.86x + 36.26*** NS

RNT–WCT NS (0.02x)0.63 NS NS NS

RCT–WNT NS NS NS NS NS

2 3 2 *
RNT–WNT NS (4.22  10 x  410.88x + NS 0.46x + 11.53 (0.91x3 + 1.75x2
1.22x + 0.01)  103*  0.85x + 0.30)  102*

BG RCT–WCT 4  105x + 0.03* 0.16x + 0.70* 0.18x + 11.14** NS

RNT–WCT NS NS NS NS

RCT–WNT NS NS NS 0.19x  0.35*

RNT–WNT NS NS NS NS

UR RCT–WCT NS 0.07x + 11.07* 13.58x + 0.25*

RNT–WCT NS (846.7x3 + 84.1x2 NS
 2.1x + 0.03)  103***
RCT–WNT NS NS NS

3 2
RNT–WNT NS (1.89x + 2.94x NS
 1.09x + 0.03)  102**

ALP RCT–WCT NS NS

RNT–WCT 35.82x + 6.88*** NS

RCT–WNT NS NS

RNT–WNT NS NS

CBH RCT–WCT NS

RNT–WCT NS

RCT–WNT NS

RNT–WNT NS

Ts, soil temperature; GWC, gravimetric water content; BG, b-glucosidase; UR, urease; ALP, alkaline phosphatase; CBH, cellobiohydrolase; GAP, glycine amino peptidase; RCT–
WCT, conventional tillage before transplantation of rice and sowing of wheat; RNT–WCT, no tillage before sowing of rice and conventional tillage before sowing of wheat;
RCT–WNT, conventional tillage before transplantation of rice and no tillage before sowing of wheat; RNT–WNT, no tillage before sowing of both rice and wheat. Levels of
significance. NS, not significant.
*
p < 0.05.
**
p < 0.01.
***
p < 0.001.

participating in nitrogen mineralization (UR and GAP). Such a Similar reasons can be proposed behind tendency of UR and
grouping was absent in rest of the permutations. RCT–WCT GAP to appear in the same group, which were found to follow a
represented a system in which nutrients were mixed homo- linear relationship under RCT–WCT. Fig. 4 shows that UR and
geneously in soil, and residue accumulation was low. Because BG GAP appeared to be controlled by the same factor and also by Ts.
and CBH participate in cellulose degradation, it is probable that UR and GAP, being linked with nitrogen mineralization are
microbial demand induced secretion of both the enzymes, and reported to be guided by nitrogen demand by plants and
hence there was coherence in their expressions. Regression microbes. Since urea was applied to the soil, it was supposed to
analyses indicated that BG and CBH were linearly related. The be the main nitrogen supplier. However, induction of GAP in lieu
results followed the observations of Baldrian et al. (2010), who also of microbial nitrogen demand is expected in light of previous
reported significant positive correlation between CBH and BG. studies (Allison et al., 2011). It has already been demonstrated
These two enzymes had low loading on both the PCs suggesting that temperature and moisture are the important factors
that their activities were not very sensitive to either of GWC or Ts, controlling kinetics of various EEAs including UR (Lai and
which apparently described PC I and PCII, respectively. Regression Tabatabai, 1992; Fraser et al., 2013) and GAP (Koch et al., 2007),
analysis, however, indicated that BG activity declined with but degree of response depends on other environmental factors,
increasing moisture content in soil. microbial activities, soil conditions as well as temperature
58 D. Pandey et al. / Soil & Tillage Research 136 (2014) 51–60

RCT-WCT RNT-WCT
1.0 1.0
BG
UR GAP
Ts

0.5
Ts
0.5
CBH ORP
GWC ORP
GAP
PC 2

PC 2
0.0 UR

GWC PPO
0.0 PPO
-0.5
CBH ALP
ALP BG

-0.5 -1.0
-1.0 -0.5 0.0 0.5 1.0 -1.0 -0.5 0.0 0.5 1.0
PC 1 PC 1

RNT-WNT
RCT-WNT
1.0 1.0 GWC
ORP
PPO
CBH UR

0.5 0.5
BG Ts
ALP
ALP
PC 2

PC 2
0.0 CBH GAP 0.0 GAP
BG
Ts

PPO
-0.5 -0.5
UR
GWC
ORP
-1.0 -1.0
-1.0 -0.5 0.0 0.5 1.0 -1.0 -0.5 0.0 0.5 1.0
PC 1 PC 1

Fig. 4. Loading plots for soil properties and extracellular enzyme activities on principal components (PC). Ts, soil temperature; GWC, gravimetric water content; ORP, oxido-
reduction potential; BG, b-glucosidase; CBH, cellobiohydrolase; PPO, polyphenol oxidase; ALP, alkaline phosphatase; UR, urease; GAP, glycine amino peptidase; RCT–WCT,
conventional tillage before transplantation of rice and sowing of wheat; RNT–WCT, no tillage before sowing of rice and conventional tillage before sowing of wheat; RCT–
WNT, conventional tillage before transplantation of rice and no tillage before sowing of wheat; RNT–WNT, no tillage before sowing of both rice and wheat.

ranges (Koch et al., 2007). In RCT–WNT and RNT–WNT also, Ts Nature of the relationships indicated that UR activity depended on
was found to be a strongest regulator of GAP (Fig. 4). GWC, but the response was not at a constant rate. BG was found to
Curve fitting analyses further showed that under RCT–WCT all be associated with Ts under all the permutations except under
the EEAs except GAP were related linearly with BG. The equation RCT–WCT and with other EEAs like GAP under RCT–WNT, and GAP
describing relationship of BG with UR and ALP showed that and PPO under RNT–WNT. Therefore, it can be suggested that
increase in BG tended to decrease ALP and UR. This relation could under no limitation of substrates, Ts was the main regulator of BG.
be a result of high microbial biomass in the soil during early On the contrary, ALP was found to be loaded on same PC on which
vegetative stages of the crop which required high BG activity to Ts was loaded, but in opposite direction. But, this linkage could
meet carbon demand. However, due to fertilizer application, have appeared because ALP activity increased after transplantation
nitrogen and phosphorous were sufficient to address requirements as discussed in Section 3.2.2. But Ts decreased in accordance with
of microbes and (or) plants with low UR or ALP secretion. The the normal meteorological trend in the study region.
trends reversed after 30–35 DAT, as discussed in Sections 3.2.2 and It was indicated that under permutations having residues of
3.2.3. previous wheat crop (RNT–WCT and RNT–WNT), PPO and GAP
Unlike RCT–WCT, in rest of the permutations, CBH and BG were were associated (Fig. 4). Among all the EEAs, CBH appeared to be
found to fall under different groups, whereas UR and GAP were also the most sensitive extracellular enzyme as it was associated
separated. The different trends reflect changes in soil conditions directly with ORP in RNT–WCT, with GWC in RNT–WNT and
under different tillage permutations during the course of seven inversely with Ts in RCT–WNT. Curve fitting analyses further
years. Previous studies have shown that in case of no tillage, indicated quantitative relationships between CBH and UR under all
distribution of organic matter becomes heterogeneous along the the permutations except under RCT–WNT, although nature of
depth as organic matter tends to stratify in upper horizons (López- relations varied from linear (RCT–WCT) to cubic (RNT–WCT and
Fando and Pardo, 2011). Under such heterogeneous conditions, RNT–WNT).
microbes may adopt different strategies of nutrient acquisition,
thus making the loading plots different from RCT–WCT. 4. Conclusion
In all the permutations except RCT–WCT, UR and GWC were
grouped, suggesting that UR was promoted by water content in Activities of BG, CBH, GAP and ALP were the highest under RNT–
soil. However, equation describing the relationship between UR WNT, while low organic matter content and regular disturbance in
and GWC could be obtained only under RNT–WCT and RNT–WNT. soil under conventional practice led to the lowest activities of these
 D. Pandey et al. / Soil & Tillage Research 136 (2014) 51–60 59

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