Influence of continuous application of inorganic nutrients to a Maize–Wheat rotation on soil...

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Original article

Influence of continuous application of inorganic nutrients toa Maize–Wheat rotation on soil enzyme activity and grainquality in a rainfed Indian soil

Supradip Saha*, K.A. Gopinath, Banshi Lal Mina, Hari Shankar Gupta

Vivekananda Institute of Hill Agriculture (Indian Council of Agricultural Research),

Line Estate, Hawalbagh, Almora, Uttarakhand 263 601, India

a r t i c l e i n f o

Article history:

Received 3 July 2008

Received in revised form

22 August 2008

Accepted 20 September 2008

Published online 14 October 2008

Keywords:

Long-term experiment

Maize–wheat system

Grain quality

Soil enzymes

Yield

* Corresponding author. Fax: þ91 5962 23153E-mail address: [email protected] (

1164-5563/$ – see front matter ª 2008 Elsevidoi:10.1016/j.ejsobi.2008.09.009

a b s t r a c t

To explore long-term impact of organic and inorganic fertilizers on soil health and grain

quality, we monitored the enzyme activities and chemical properties of soil; and chemical

composition of grain from eight treatments at an experimental field site established in

1996. There were eight treatments applied to both wheat and maize seasons: a control; four

inorganic fertilizers, that is, nitrogen and phosphorus (NP), nitrogen and potassium (NK),

phosphorous and potassium (PK) and nitrogen, phosphorus and potassium (NPK); farm

yard manure alone (FYM) and addition of FYM at two different doses (100 and 50% of

recommendation) to NPK that is, NPKþ FYM and 1⁄2 NPKþ FYM. After 11 years of the

experiment the NPKþ FYM and 1⁄2 NPKþ FYM treatments had the highest yields, about

5 Mg maize ha�1 and 2 Mg wheat ha�1 with about 2 and 0.5 Mg ha�1, respectively more than

the NPK treatments. The dehydrogeanse activity of soils increased significantly in FYM and1⁄2 NPKþ FYM. Except urease all other enzymatic activities were increased in those treat-

ments, which received manure. Urease activity was higher in mineral-N applied plots.

Grain protein content of both maize and wheat was highest in mineral fertilized plots. Test

weight also increased significantly on application of mineral fertilizer. Plots treated with

half dose of recommended mineral fertilizer along with FYM were higher in urease,

phosphomono and diesterase activities than that of NPKþ FYM treated plots. Long-term

application of inorganic nutrients along with FYM improved grain mineral composition

and yield. Inhibition of few enzymatic activities were also observed upon application of

inorganic nutrients either alone or in combination.

ª 2008 Elsevier Masson SAS. All rights reserved.

1. Introduction production. Instead, concerns about the impacts of agricul-

Agriculture has transformed from single-goal to multi-goal

management systems. Today optimization of yield is not the

sole criterion for assessing the success of agricultural

9.S. Saha).er Masson SAS. All rights

tural activities on soil fertility, water resources, and environ-

mental safety are now included in assessments of best

management practices [42]. Sustaining the agricultural

production in the era of global climate change maintaining

reserved.

mailto:[email protected]

http://www.elsevier.com/locate/ejsobi

Table 1 – Details of experimental treatments.

Treatment Maize Wheat

N (kg ha�1) P (kg ha�1) K (kg ha�1) FYMa (Mg ha�1) N (kg ha�1) P (kg ha�1) K (kg ha�1) FYMa (Mg ha�1)

Control 0 0 0 0 0 0 0 0

NPK 90 26 33.2 0 60 13 16.6 0

NP 90 26 0 0 60 13 0 0

NK 90 0 33.2 0 60 0 16.6 0

PK 0 26 33.2 0 0 13 16.6 0

FYM 0 0 0 5 0 0 0 51⁄2 NPKþ FYM 45 13 16.6 5 30 6.5 8.3 5

NPKþ FYM 90 26 33.2 5 60 13 16.6 5

a Dry weight basis.


other related aspects is a new challenge. A crucial task is to

quantify the simultaneous impacts of management practices

on crop yield, soil carbon storage, nutrient leaching and soil

health. Agricultural practices that improve soil quality and

agricultural sustainability have been receiving more attention

by researchers and farmers [12]. Inorganic fertilizer, especially

N, P and K, not only maintain, but also can directly or indirectly

change chemical, physical and biological properties of soil.

These changes, in long term, are believed to have significant

influences on the quality and productive capacity of soils.

Soil enzyme activities are important for the transformation

of nutrients, required for plant growth and they depends on

crop rotation, amendments, tillage and agricultural manage-

ment [6,12,19,23].

Although some researchers commented that soil organic

matter was not a plant growth requirement in-and-off itself

[31], almost all have realized its ecological significance in over-

coming constraints to crop growth, particularly, in the areas of

nutrient supply, nutrient and moisture relation, soil structural

stability and detoxification [36]. Information is also needed to

assess the contribution of the organic matter in maintaining soil

microbial community and which in turn, related to specific

enzymatic reactions, i.e. to biochemical processes involved in

different nutrient cycling for assessing sustainability.

There is scant information on enzyme activities in the sub-

temperate condition and we are unaware of information

available about the enzyme activities of soils in North western

Himalayan region. This study investigated enzyme activities

known to play critical roles in organic matter decomposition

and mineralization of C, N, and P nutrients in soils, and that

are reported to be sensitive to changes in soil quality due to

fertilizer application [4,6,7,15,16]. Information regarding the

changes in quality attributed due to long-term fertilizer

treatment is also limited.

We hypothesized that long-term application of C through

manure and N, P through fertilizers will have distinct effects

on the activities of dehydrogenase, cellulase, invertase,

protease, urease, acid and alkaline phosphatase which in turn

will depict the efficiency of C, N and P cycle. Further more, we

hypothesized that the changes in soil chemical attributes will

have definite role in grain mineral constituents, mainly, N, P

and K contents. For this reason, soil enzymatic and chemical

properties, grain yield and quality were compared in soils

from a long-term field experiment with eight different

treatments.

2. Materials and methods

2.1. Experimental treatments and crop management

The experiment included two crops per year, maize (June–

Sept) and wheat (Oct–May), and commenced with the maize

crop in 1996. Eight treatments (summarized in Table 1) were

applied to each crop in each year. The experiment was con-

ducted at the experimental farm of Vivekananda Institute of

Hill Agriculture, located in the Indian Himalayan region at

Hawalbagh (29� 360N and 79�400E with 1250 m above mean sea

level), in the state of Uttarakhand, India. The climate is sub-

temperate, characterised by moderate summer (May–June),

extreme winter (Dec–Jan) and general dryness, except during

the southwest monsoon season (June–Sept). Weekly rainfall,

average air and soil temperature during the experimental

period are shown on Fig. 1.

Treatments were arranged in a randomized complete block

design with three replications. The plot size was 10.8 m2. The

fertilizer application rates used in both the crops were those

recommended for Uttarakhand hills (Table 1) [37]. The sources

of fertilizers were urea for N, single super-phosphate for P,

and muriate of potash for K. In both crops, half of the N and

full dose of P and K were applied before sowing. Remaining

half of the N was applied as a top dressing 30–35 days after

sowing in maize and during January in wheat, coinciding with

winter rains. The applied FYM, analysed every five year, had

370 g moisture kg�1 and contained 7.1–7.5 g N kg�1, 2.1–

2.4 g P kg�1 and 5.3–5.8 g K kg�1 on oven-dry weight basis. FYM

was thoroughly incorporated into 15 cm surface soil three

weeks before sowing of each crop.

The maize variety VL Sankul Makka 16 was used till 2004

and thereafter VL Sankul Makka 11 was grown. For wheat, the

variety used was VL 616 till 2003–2004 and VL 829 was grown in

the subsequent years. Both crops were grown under rainfed

conditions in all the years. For weed control, atrazine @

1.25 kg a.i. ha�1 and isoproturon @ 1.0 kg a.i. ha�1 was applied

to maize and wheat respectively.

2.2. Sampling and handling of soil

Fresh samples were taken after harvest of maize in October,

2006 for being analysed for their enzyme activities. Random

cores were taken from each plot with 5-cm diameter tube

0

10

20

30

40

50

July

, 96

Jan,

97

Jul,

97

Jan,

98

Jul,

98

Jan,

99

July

, 99

Jan,

00

Jul,

00

Jan,

01

July

, 01

Jan,

02

Jul,

02

Jan,

03

Jul,

03

Jan,

04

Jul,

04

Jan,

05

Jul,

05

Jan,

06

Jul,

06Se

p, 0

6Se

p, 0

6

July

, 96

Jan,

97

Jul,

97

Jan,

98

Jul,

98

Jan,

99

July

, 99

Jan,

00

Jul,

00

Jan,

01

July

, 01

Jan,

02

Jul,

02

Jan,

03

Jul,

03

Jan,

04

Jul,

04

Jan,

05

Jul,

05

Jan,

06

Jul,

06

Ave

rage

air

tem

pera

ture

(°C

)

0

10

20

30

40

Soil temperature-m

id day (°C)

Avg. air temp. Soil temp.

0

40

80

120

160

200

Rai

nfal

l (m

m)

A

B

Fig. 1 – Weekly total rainfall (A) and average air temperature and mid-day soil temperature measured at 15 cm depth (B)

during the total experimental period.

e u r o p e a n j o u r n a l o f s o i l b i o l o g y 4 4 ( 2 0 0 8 ) 5 2 1 – 5 3 1 523

auger and bulked. The moist soil samples were sieved

(2 mm) after removing plant material and roots. Half of soil

samples were air-dried and stored at room temperature

until analysed for their chemical properties. 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 sieved fresh soils (2 mm) were immediately trans-

ferred to the laboratory for biochemical analysis. Soil samples

were kept at 4 �C in plastic bags for a few days to stabilise the

microbiological activity disturbed and then analysed within 2

weeks. Dry soil samples were rewetted and analysed for their

dehydrogeanase activity after wheat crop (2005–2006). All

microbial results reported are means of three replicates and

are expressed on a moisture-free basis. Moisture content was

determined after drying at 105 �C for 24 h.

2.3. Chemical and biochemical analyses

The pH of soil was analysed in 1:2.5 soil:water suspension [17],

available K by 1 N NH4OAc using a flame photometer [17], total

C and N by dry combustion [26] with a CHN analyzer (Foss-

Heraeus, Germany), oxidizable SOC as reported by Walkley

and Black [44], Kjeldahl-N by FOSS Tecator (Model 2200), and

available P by the method of Olsen et al. [27].

Soil dehydrogenase activity was estimated by reducing

2,3,5-triphenyltetrazolium chloride [8]. Soil (5 g) sample was

mixed with 50 mg CaCO3 and 1 ml 3% (w/v) 2,3,5-triphenylte-

trazolium chloride (TTC) and incubated for 24 h at 37� 1 �C.

The 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 glucose

equivalent after mixing soil sample (10 g) with 15 ml

acetate buffer (2 M, pH 5.5) and 15 ml 0.7% (w/v) carboxy

methyl cellulose and incubated for 24 h at 50� 1 �C [32].

Glucose equivalent was estimated following the DNS

method [24].

Protease activity was assayed by determining the tyrosine

released when 1 g oven-dry equivalent of field-moist soil

Table 2 – Soil pH, organic matter, C-to-N ratio, available N, P, K of 0–15 cm soil affected by differential fertilizer treatments.

Fertilization pH C-to-N ratio Organic matter (g kg�1) Available N (mg kg�1) Olsen-P (mg kg�1) Available K (mg kg�1)

Control 6.5 15.6 10.5 91 4.8 61.0

NPK 5.8 14.5 11.6 120 8.5 96.4

NP 5.5 13.1 12.4 144 8.5 87.5

NK 5.4 12.5 12.4 151 5.9 100.2

PK 6.3 13.2 13.0 100 8.5 90.0

FYM 6.9 15.1 18.7 96 7.4 94.01⁄2 NPKþ FYM 6.5 14.5 15.2 112 8.0 83.2

NPKþ FYM 6.7 13.6 18.7 110 8.2 94.4


sample was incubated with 5 ml 50 mM tris buffer (pH 8.1) and

5 ml of 2% Na-caseinate at 50� 1 �C for 2 h [20]. The residual

substrate was precipitated with 0.92 M trichloroacetic acid;

the aromatic amino acids released were extracted and

measured colorimetrically using Folin–Ciocalteu reagent at

700 nm. Protease activity was expressed as mg tyrosine

produced g�1 soil 2 h�1.

Urease activity was measured following the method

developed by Tabatabai and Bremner [39]. Soil (5 g) was

incubated with 5 ml of 0.05 M THAM buffer (pH 9.0) and 1 ml of

0. M of urea solution at 37� 1 �C for 2 h. Excess urea was

extracted with KCl-PMA solution and estimated colorimetri-

cally at 527 nm.

Both acid and alkaline phosphatase activities were assayed

using 1 g 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

[38]. After incubation for 1 h at 37� 1 �C 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 g�1 soil h�1. Phos-

phodiesterase activity was measured as reported by Browman

and Tabatabai [5]. In this case substrate used was 0.005 M bis-

p-nitrophenyl phosphate and concentration of p-nitrophenol

was measured.

0

10

20

30

40

50

Control NPK NP NK

mic

rogr

am T

PF

pro

duce

d g-1

h-1

After Wheat

After Maize

cdc

aab

bca

ab bc

Fig. 2 – Dehydrogenase activity in soils of different treatments

letter are not significantly different (P < 0.05). Error bars represent

2.4. Grain analysis and crop yield

Maize and wheat grains were harvested at maturity and dried

naturally for further processing. The dried grains were stored

at room temperature for three months prior to analysis. The

samples were analysed in laboratory for chemical parameters

after tri-acid digestion. Samples were taken using the quar-

tering procedure [21]. Protein content was determined as

described by AOAC method using formula N� 6.25 and

N� 5.83 for maize and wheat respectively [1]. Phosphorus was

estimated photometrically via development of phospho-

molybdate complex, as described by Taussky and Shorr, [41].

Potassium was determined by flame photometer.

Whole plots were harvested in May and October each year

for wheat and maize respectively. Grain yield was deter-

mined at 14% moisture content. Thousand kernel weight was

calculated as the mean weight of three sets of 100 grains per

plot.

2.5. Statistical analyses

Each sample was analysed in triplicate and the values were

then averaged. Data were assessed by Duncan’s multiple

range tests [10] with a probability P� 0.05. 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.

PK FYM 1/2NPK+FYM

NPK+FYM

dc

h

f

gg

e

f

after wheat and maize crop season. Bars sharing the same

standard deviation.

0.0 50.0 100.0 150.0 200.0 250.0 300.0

Control

NPK

NP

NK

PK

FYM

1/2 NPK+FYM

NPK+FYM

mg glucose eq. g-1 h-1/mg glucose eq. g-1 h-1

Cellulase

Invertase

bc

a

ab

d

cde

bcbc

bc

bc

a

a

ee

cd

ab

Fig. 3 – Cellulase and invertase activity in soils of different treatments. Bars sharing the same letter are not significantly

different (P < 0.05). Error bars represent standard deviation.


3. Results

3.1. Chemical properties of soil

The pH values in the 0–15 cm surface soil ranged from 5.4 to

6.9, with that of the untreated control soil around pH 6.5

(Table 2). Manure application increased soil pH significantly

(P< 0.05), while chemical fertilizer application decreased soil

pH (Table 2). The C-to-N ratio was lowest in NP treatment

and highest in FYM treatment (Table 2). Considerable

difference was detected in SOC and N content among

samples from different sampling times. In the surface 0–

15 cm, soil oxidizable carbon was less in different fertilized

treatment than FYM treated plots. Application of FYM alone

0 50 100

Control

NPK

NP

NK

PK

FYM

1/2 NPK+FYM

NPK+FYM

µg urea hydrolysed g-1 h

ca

b

b

a

a

a

b

a

c

c

Fig. 4 – Urease and protease activity in soils of different treatme

(P < 0.05). Error bars represent standard deviation.

increased the C content in surface soil. Addition of FYM at

10 Mg ha�1 year�1 along with NPK for 11 years increased 62

per cent organic C content than NPK alone.

Available N content also varied among the treatments

(Table 2). The Kjeldahl-N content of soils was higher in

mineral fertilized soils than others. It follows the order of

NPK<NP<NK. Depletion of available P in soil occurred in NK

treatment. NPKþ FYM and 1⁄2 NPKþ FYM helped to maintain

available P in the soil. Soil K content was increased by most of

the mineral fertilized treatments as compared to the control.

3.2. Soil enzymatic activities

Application of manure increased dehydrogenase activity,

which is an index of microbial activity of soil (Fig. 2). The

150 200 250 300

-1/µg tyrosine produces g-1 2h-1

UreaseProtease

d

d

d

d

e

nts. Bars sharing the same letter are not significantly different


enzymatic activities NP, NK and NPK treated soil were

significantly lower than that of control. Application of FYM in

soil enhanced soil dehydrogenase activity from 9.7 and 11.4 mg

TPF produced g�1 h�1 in control to 34.4 and 40.4 mg TPF

produced g�1 h�1 in FYM treatment after maize and wheat

crop respectively.

The highest cellulase activity was found in NPKþ FYM

treated soil (240 mg glucose eq. g�1 h�1), followed by FYM

(195 mg glucose eq. g�1 h�1) treated soil (Fig. 3). Invertase

activity was little affected by treatments and highest value

was found in NPKþ FYM treated soil (69 mg glucose

eq. g�1 h�1), followed by control soil. The FYM treated soil

showed the lowest invertase activity (46 mg glucose

eq. g�1 h�1).

Unlike the other enzyme activities, urease activity was

higher in soils treated with mineral fertilizer (Fig. 4). Highest

value was found in NK (259 mg urea hydrolysed g�1 h�1) fol-

lowed by NP (237 mg urea hydrolysed g�1 h�1) and NPK

(227 mg urea hydrolysed g�1 h�1) treated soil. Protease activity

was higher in manure applied soil with or without mineral

fertilizer. The activity was least in control and is at par with

NK treatment. Among mineral fertilized treatments, PK was

found best.

Highest acid phosphatase activity was found in1⁄2 NPKþ FYM treatment and was significantly more than

NPKþ FYM (Fig. 5). Similar trends were also observed in case

of alkaline phosphatase and phosphodiesterase activity.

Mineral fertilizer treated soil was least and FYM was highest

in this enzyme activity.

A correlation matrix (Table 3) shows some significant

relationships among the enzyme activities studied. There was

positive correlation (r¼ 0.810; P< 0.01) between SOC and

dehydrogeanse activity among treatments. Total organic

carbon was correlated with the entire assayed enzyme except

urease, invertase and acid phosphatase. Dehydrogenase

activity showed a strong positive correlation with phospho-

mono and di-esterases. Soil pH was correlated with most of

0

200

400

600

800

1000

Control NPK NP NK

mic

rogr

am p

-nir

ophe

nol g

-1 h

-1

AcP

AkP

PDE

a

aaaa

b

b

b

a

a

aa

Fig. 5 – Acid, alkaline monoesterases and diesterase activity of

are not significantly different (P < 0.05). Error bars represent stand

the enzymes. Acid phosphatase shared a strong correlation

with phosphodiesterase (r¼ 0.830, P< 0.01).

3.3. Yield

Weather conditions are summarized in Fig. 1. The quantity

and distribution of rainfall were highly variable during the

total experimentation years, which affected the yield of maize

and wheat. Grain yields of maize and wheat were affected by

the different fertilizer treatments (Table 4). Eleven years

average maize grain was highest in NPKþ FYM and least in

un-amended control. 1⁄2 NPKþ FYM resulted less grain yield

than NPKþ FYM. On the other hand, it was similar in case of

wheat yield (Table 4). Maize yield was increased by eight fold,

whereas, it was doubled in wheat yield. Application of manure

only also increased grain yield by more than 2.5 fold in maize.

The yield increase was more prominent in maize than in

wheat.

3.4. Grain quality

Grain protein content was more in those treatments which

received N only through mineral fertilizer (Table 5). The trend

in P and K in maize and wheat grains was almost similar.

Grain protein content was highest in NP and least in FYM

treatment in both the crops. Though protein content was

detected less in NPKþ FYM treatment than NPK alone,

total protein yield was highest in the former treatment.

Thousands grain weight of maize was highest in NPKþ FYM

followed by 1⁄2 NPKþ FYM. On the other hand, in wheat it was

highest in PK treatment followed by NPKþ FYM treatment.

Figs. 6 and 7 summarize the composition of maize and wheat

grain, respectively, grown under different treatments. Grain P

and K content was highest in NPKþ FYM treatment and least

in control and NPK. Manure application increased the P and K

content in grain when applied along with different doses of

mineral fertilizer.

PK FYM 1/2NPK+FYM

NPK+FYM

bc

c

c

d

d

e

ef

d d

d

soils with different treatments. Bars sharing the same letter

ard deviation.

Table 3 – Linear correlation coefficient between soil chemical properties, enzyme activities and grain quality (*, ** indicate P < 0.05, P < 0.01 respectively. TOC [ Totalorganic carbon; N [ Kjeldahl N; AP [ Available phosphorous; AK [ Avaialble potassium; DA [ Dehydrogenase; Cell. [ Cellulase; Inv [ Invertase; Ure [ Urease;Pro [ Protease; AcP [ Acid phosphatase; AkP [ Alkaline Phosphatase; PDE [ phosphodiesterase; MN [ Maize N; MP [ Maize P; MK [ Maize K; WN [ Wheat N;WP [ Wheat P; WK [ Wheat K).

TOC N AP AK DA Cell Inv Ure Pro AcP AkP PDE MN MP MK WN WP WK

pH 0.543 �0.840** �0.212 �0.464 0.698* 0.715* 0.187 �0.893** 0.498 0.498 0.682* 0.778* �0.774* 0.466 �0.240 �0.808** 0.122 �0.401

TOC �0.211 0.293 0.392 0.810** 0.735* �0.007 �0.515 0.798* 0.443 0.874** 0.777* �0.421 0.868** 0.366 �0.291 0.744* 0.184

N 0.126 0.506 �0.401 �0.408 �0.085 0.872 �0.489 �0.614 �0.405 �0.629 0.866** �0.160 0.485 0.859** 0.085 0.672*

AP 0.531 0.050 �0.003 �0.204 0.005 0.564 0.284 0.189 0.174 0.051 0.465 0.311 0.254 0.408 0.322

AK 0.039 �0.102 �0.354 0.364 0.354 �0.106 0.127 �0.068 0.213 0.236 0.476 0.428 0.469 0.455

DA 0.573 �0.169 0.621 0.602 0.450 0.973** 0.837** �0.572 0.678* 0.060 �0.493 0.439 �0.111

Cell 0.418 �0.507 0.570 0.354 0.638 0.611 �0.297 0.594 �0.058 �0.280 0.336 �0.168

Inv �0.167 0.001 0.315 �0.041 0.160 0.282 0.183 0.199 0.206 0.167 0.122

Ure �0.647 �0.757* �0.650 �0.877** 0.864– �0.572 0.029 0.847** �0.346 0.275

Pro 0.762* 0.735* 0.807** �0.580 0.747* 0.163 �0.371 0.659 �0.082

AcP 0.573 0.830** �0.545 0.585 0.117 �0.422 0.568 �0.198

AkP 0.900** �0.535 0.77* 0.147 �0.416 0.554 �0.053

PDE �0.687* 0.804** 0.180 �0.592 0.624 �0.099

MN �0.309 0.256 0.958** �0.151 0.471

MP 0.605 �0.212 0.862** 0.431

MK 0.296 0.783* 0.933**

WN �0.027 0.470

WP 0.553

eu

ro

pe

an

jo

ur

na

lo

fs

oil

bio

lo

gy

44

(2

00

8)

52

1–

53

15

27

Table 4 – Average grain yields (1996–2006) of maize and wheat in a maize–wheat long-term experiment.

Treatment Maize Wheat

Grain yield (Mg ha�1) Yield increase (%) Grain yield (Mg ha�1) Yield increase (%)

Control 0.61a� 0.12 – 0.70a� 0.20 –

NPK 2.87d� 0.91 370 1.66bc� 0.37 138

NP 1.68bc� 0.27 176 1.11ab� 0.25 60

NK 1.59bc� 0.17 160 0.93a� 0.25 33

PK 1.23ab� 0.17 102 0.83a� 0.37 18

FYM 2.33cd� 0.55 282 1.03a� 0.27 481⁄2 NPKþ FYM 4.87e� 0.35 697 2.04c� 0.36 192

NPKþ FYM 5.55e� 0.47 808 2.09c� 0.39 199

Values are the mean� SD of three independent determinations; Means with the same letter within a column do not differ significantly (P< 0.05).


4. Discussion

Soil management not only changed C and N content in the

surface soil (0–15 cm), but also in the enzymatic activity. Soil

pH ranged from 5.4 in the NK plots, which is significantly

lower than in all other treatments, to 6.9 in the FYM plots. The

pH of the N treated plots were significantly lower than that of

the control and manured plots. Long-term applications,

especially N, therefore had acidifying effects resulting in

lowering of pH. This result confirms earlier findings that most

N-containing fertilizers tend to acidify soil [2,3]. This is mainly

due to the fact that most fertilizers supply N as NH4þ first,

which upon oxidation released Hþ ions, responsible for acidity

[22]. This result is consistent with our earlier study in same

agroclimatic region, where pH of NK plot was 5.3 and 6.5 in

NPKþ FYM plot [30].

Manure-treated plots were significantly greater in C built

up than fertilized treatments. Recommended dose of fertilizer

application has been shown to result in modest increases in

labile organic matter content. 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. Soil samples from fertilized plots

had lower C-to-N ratios than soil from the unfertilized plots.

The C-to-N ratio was narrowed down most in NK treatment.

Table 5 – Grain protein content and yield, thousand grain weigapplication.

Treatment Maize

Protein Thousand grain weigh

Concentration(g kg�1)

Yield(kg ha�1)

Control 84.1b� 3.7 51.23a� 9.4 117.0a� 4.6

NPK 93.8c� 2.2 269.65d� 88.1 153.8abc� 15.2

NP 97.6c� 2.5 164.74bc� 29.3 141.7ab� 50.1

NK 96.7c� 2.6 153.64bc� 18.8 141.2ab� 5.0

PK 76.1a� 2.7 93.93ab� 14.8 165.2bc� 6.7

FYM 72.8a� 1.7 170.37c� 43.6 179.4bcd� 23.21⁄2 NPKþ FYM 86.5b� 0.9 420.77e� 36.3 214.0d� 7.0

NPKþ FYM 87.6b� 2.3 485.34e� 15.9 191.5cd� 13.3

Values are the mean� SD of three independent determinations; Means wi

Available P in soil was built up due to application of

mineral-P fertilizer along with manure. Whereas, K content in

soil was more in only mineral fertilized soils. Build up of P in

soil is due to maintaining soil pH towards neutral range,

which prevents from P fixation.

Enzyme activities of soil depend on the type of treatment

and the type of enzyme. Dehydrogenase activity was lower in

soils that had received the mineral fertilizers and it was

increased several folds in the presence of manure. This

maximum activity might be linked to more substrate avail-

ability in these soils. This reflects the greater biological

activity in these plots and the stabilization of extracellular

enzymes through complexation with humic substances

[7,9,14,18,25]. This result is in consistent with our previous

study conducted under similar soil and climatic influence [30].

In our earlier study also more than three fold increase in

dehydrogenase activity in FYM treated soils. This result also

suggests that dehydrogenase activity was highly sensitive to

the inhibitory effects associated with mineral fertilizer addi-

tions. Greater dehydrogenase activity of 1⁄2 NPKþ FYM as

compared to NPKþ FYM confirms the negative impact of

mineral fertilizer on the enzyme. Simek et al. [33] reported

that dehydrogenase activity was inhibited by large amount of

fertilizer in a long-term experiment.

Soil invertase catalyses the hydrolysis of sucrose to glucose

and fructose, and is linked to the soil microbial biomass

ht of maize and wheat grown under different fertilizer

Wheat

t (g) Protein Thousand grain weight (g)

Concentration(g kg�1)

Yield(kg ha�1)

74.2b� 0.67 51.68a� 14.5 41.4a� 1.1

87.5d� 0.86 144.98b� 31.3 46.3abcd� 2.4

87.6d� 0.57 97.53a� 21.6 41.8ab� 5.9

87.2d� 0.92 81.08a� 21.1 42.9abc� 3.9

73.0ab� 1.57 60.02a� 26.5 49.7d� 0.5

71.4a� 1.67 73.76a� 19.1 47.9bcd� 1.5

80.9c� 0.29 165.05b� 31.3 45.9abcd� 4.8

81.5c� 1.50 169.88b� 27.8 48.7cd� 3.8

th the same letter within a column do not differ significantly (P< 0.05).

0

5

10

15

20

Control NPK NP NK PK FYM 1/2NPK+FYM

NPK+FYM

Con

cent

rati

on (

g kg

-1)

NPK

c c

c

abc

d

cd

a

dc

a a a

d

bc

d

ab

cd

bc

cd

ab

cd a

b

a

d

Fig. 6 – N, P and K content in maize grain, grown under different treatments. Bars sharing the same letter are not significantly



[13,29]. In our earlier study [30], it was found that FYM did not

contribute significantly to invertase activity. In this experi-

ment also lowest activity of invertase in FYM treated plots

confirms that organic amendment has very little role in the

functioning of invertase activity. Cellulases play an important

role as a group of enzymes in global recycling of the most

abundant natural polymer, cellulose. The rate of cellulase

activity was highest in NPKþ FYM followed by FYM. This is

attributed to addition of more substrate which leads to more

enzymatic activity. This result confirms our earlier study in

soybean–wheat cropping system [30], where application of

FYM improved cellulase activity significantly. Significant

correlation (0.735, P< 0.05) between TOC and cellulase activity

confirms the impact of substrate on enzyme activity.

The activity of assayed enzymes was generally well

correlated with the organic C content because all of these

0

5

10

15

20

Control NPK NP NK

Con

cent

rati

on (

g kg

-1)

abbc

a

aab b

ababcd bcd cd

a

NPK

Fig. 7 – N, P and K content in wheat grain, grown under differen


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

[15,34]. Being the substrate of enzymes, soil organic matter

plays a vital role in protecting soil enzymes since they form

complexes with clay and humus [40].

Our results show that the most of the enzymatic activities

were highest in plots, which received NPK and FYM. In

contrast, urease activity followed different pattern, where

organic manure had very little effect on the activity. Activity of

urease was significantly higher in plots received N fertilizer in

mineral form. This result is in contrast with our earlier result

[23,30]. In another study [12] reported that urease activity

decreased during first 4 h with addition of C and N and then

increased continuously. Variation of urease activity was due

to the extracellular character of this enzyme, which is

PK FYM 1/2NPK+FYM

NPK+FYM

c

cb b bbcd abc dabcd

a a

c

t treatments. Bars sharing the same letter are not significantly


adsorbed and protected by organic complexes. Both intracel-

lular and extracellular forms of urease resulting from micro-

bial activity exist in soil. The decrease of urease observed in

mineral fertilized plots could be related to the crop, a legume

that obtains N from its symbiosis with Rhizobium [23].

The N mineralization potential in soil was characterised 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 is mainly due to an increased growth of the

microbial community and it was supported by the study of

Kandeler et al. [18], which showed that biomass-specific

protease activities of different soils were not related to the

organic matter 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 [7]. In another study, protease activity was

always higher in organic matter amended soil than conven-

tionally managed soil [23].

Since pH of the soil was generally below 7.0, acid phos-

phatase was higher than alkaline phosphatase [11]. Acid

phosphates activity was more in those plots, which received

manure and it was supported by Saha et al. [30] and Haynes

and Swift, [16], who found that acid phosphatase activity of

soils generally decreases in response to fertilizer application.

In our earlier study [30], the influence of FYM or pH was more

pronounced than this study, which may be due to long-term

(33 years) impact. Phosphatases play an important role in P

cycling where organic P is more due to limited biological

mineralization of organic matter as a result of formation of

complexes of organic P with active Al and Fe [43] and the

amount of available P is low. P transformation and cycle also

depend on soil reaction. Acid phosphatase activities in

different treatment were greatly regulated by soil pH. In

general FYM applied to soil have long been employed to

enhance favorable soil conditions in terms of pH and avail-

ability 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 protease (P < 0.01). The significantly greater activities of

alkaline phosphatase in the manure-treated soil were due 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 [28]. The inhibition of phosphatase activities with mineral

fertilizer could be seen as well. Control soils exhibited similar

activities than the mineral fertilized soils except PK. This can

be explained by the inhibition of phosphatase synthesis by

mineral fertilization. In slight contrast to our result, literature

reported that this inhibition was due to mineral-P [4,35].

Alkaline phosphatase was negatively correlated to soil P

concentrations and microbial biomass C and P [45]. Phospho-

diesterases was highest in 1⁄2 NPKþ FYM treatment and was

more than NPKþ FYM. This clearly depicts that mineral

fertilizer had a negative impact on this enzyme.

Maize responded more to applied nutrient sources espe-

cially N than wheat. Yield increase was more prominent in

maize than wheat after 11 years of continuous application of

manures and fertilizers. This is attributed to favorable crop

physiology to draw nutrients. Accumulation of dry matter in

grain was higher in treatments, which received both the

mineral and organic fertilizer. Interestingly, phosphorous and

potassium contributed more in dry matter accumulation in

wheat grain. This may be attributed to important role of

potassium in carbohydrate translocation. Grain protein

content was more in those treatments, where N was applied

only in mineral form. This is due to quick availability of this

nutrient to plants. Plants that received both mineral and

organic fertilizer produced more protein yield per plot rather

than more concentration in grain. Grain phosphorous and

potassium content was more in NPKþ FYM treatment than

others. This may be attributed to continuous availability of

these nutrients, which can be seen in significant correlation of

soil available N and grain N concentration (P < 0.01).

5. Conclusion

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. Farm yard

manure along with recommended fertilizer even at half of

recommended dose increased microbiological activities in

sub-temperate soil. Manure along with reduced dose of

mineral fertilizer not only increased grain yield, but also

enhanced grain quality with significant nutrient build up in

soil. It also maintained a balance in enzymatic activity with

a lesser pollution potential than higher doses of mineral

fertilizers.

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