Effect of Crop Cover and Stage of Crop Growth on Soil L-Glutaminase Activity

Abstract

A pot culture experiment was conducted at glass house of Department of Soil Science and Agricultural Chemistry, College of Agriculture, Rajendranagar, Hyderabad. The aim of the present experiment was to study the influence of crop cover and stage of crop growth on soil L- glutaminase activity in an Alfisol and Vertisol. The experiment was under taken with six crops viz., two cereals (Rice, Maize), two legumes (Groundnut, Greengram), one oilseed (Sunflower) and one vegetable (Bhendi) crop. The experiment was conducted in Completely Randomized Block design with three replications along with the uncropped control. The results obtained with regard to the effect of these crops on soil L-glutaminase activity showed that there was an increase in enzyme activity with age of the crop upto 60 DAS and it varied with crops grown. The increased enzyme activity (μg of NH4+ released g-1 soil 4h-1) varied from 5.56 to 12.17 for groundnut, 5.58 to 11.25 for greengram, 5.43 to 10.87 for sunflower, 5.48 to 8.61 for rice, 5.39 to 8.23 maize and 5.31 to 7.92 for bhendi in Vertisol. In Alfisol the L-glutaminase activity (μg of NH4+ released g-1 soil 4h-1) under different crop cover found to vary from 6.72 to 13.59 (groundnut), 6.68 to 12.71 (greengram), 6.63 to 11.96 (sunflower), 6.61 to 10.25 (rice), 6.59 to 9.47 (maize), 6.62 to 9.26 (bhendi). A close perusal of the data indicates that the L-glutaminase activity followed the sequence groundnut > greengram > sunflower > rice > maize > bhendi, in both Alfisol and Vertisol.

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*Corresponding author: E-mail: bhargavnarasimha444@gmail.com;
International Research Journal of Pure & Applied Chemistry
21(24): 334-340, 2020; Article no.IRJPAC.65422
ISSN: 2231-3443, NLM ID: 101647669
Effect of Crop Cover and Stage of Crop Growth on
Soil L-Glutaminase Activity
M. Bhargava Narasimha Yadav1*, G. Padmaja1, T. Anjaiah1
and J. Aruna Kumari1
1Department of Soil Science and Agricultural Chemistry, Professor Jayashankar Telangana State
Agricultural University, Rajendranagar, Hyderabad–500030, India.
Authors’ contributions
This work was carried out in collaboration among all authors. Author MBNY performed the statistical
analysis, wrote the protocol and wrote the first draft of the manuscript. Authors GP and TA managed
the analysis of the study. Author JAK managed the literature searches.
All authors read and approved the final manuscript.
Article Information
DOI: 10.9734/IRJPAC/2020/v21i2430373
Editor(s):
(1) Dr. Wolfgang Linert, Vienna University of Technology, Austria.
Reviewers:
(1) O. P. Bansal, D. S. College, India.
(2) Ashay D. Souza, University of Agricultural Sciences, India.
Complete Peer review History: http://www.sdiarticle4.com/review-history/65422
Received 25 October 2020
Accepted 28 December 2020
Published 31 December 2020
ABSTRACT
A pot culture experiment was conducted at glass house of Department of Soil Science and
Agricultural Chemistry, College of Agriculture, Rajendranagar, Hyderabad. The aim of the present
experiment was to study the influence of crop cover and stage of crop growth on soil L- glutaminase
activity in an Alfisol and Vertisol. The experiment was under taken with six crops viz., two cereals
(Rice, Maize), two legumes (Groundnut, Greengram), one oilseed (Sunflower) and one vegetable
(Bhendi) crop. The experiment was conducted in Completely Randomized Block design with three
replications along with the uncropped control. The results obtained with regard to the effect of these
crops on soil L-glutaminase activity showed that there was an increase in enzyme activity with age
of the crop upto 60 DAS and it varied with crops grown. The increased enzyme activity (μg of NH4+
released g-1 soil 4h-1) varied from 5.56 to 12.17 for groundnut, 5.58 to 11.25 for greengram, 5.43 to
10.87 for sunflower, 5.48 to 8.61 for rice, 5.39 to 8.23 maize and 5.31 to 7.92 for bhendi in Vertisol.
In Alfisol the L-glutaminase activity (μg of NH4+ released g-1 soil 4h-1) under differe
nt crop
cover found to vary from 6.72 to 13.59 (groundnut), 6.68 to 12.71 (greengram), 6.63 to 11.96
(sunflower), 6.61 to 10.25 (rice), 6.59 to 9.47 (maize), 6.62 to 9.26 (bhendi). A close perusal of the
data indicates that the L-glutaminase activity followed the sequence groundnut > greengram >
sunflower > rice > maize > bhendi, in both Alfisol and Vertisol.
Original Research Article

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335
Keywords: L-glutaminase; Vertisol; Alfisol; groundnut; greengram.
1. INTRODUCTION
The enzyme L-glutaminase (L-glutamine amido
hydrolase E.C. 3.5.1.2) in soils hydrolyse L-
glutamine to glutamic acid and ammonium, thus
it is important in making the amide form of
nitrogen available to plants. Nitrogen is present
in soil in the organic and inorganic forms. The
organic N has two fractions, they are
hydrolysable fraction (60 to 80%) and non
hydrolysable fraction [1] about 24 to 37 percent
of total N in surface soils could be accounted for
amino nitrogen. Bremner [2] reported that after
acid hydrolysis of humic substances, 7.3 to
12.6% of total nitrogen was in the form of amide
nitrogen. The important enzymes involved in
mineralization of amide forms of nitrogen in the
soils are urease, amidase, glutaminase and
asparaginase.
The presence of crop cover and the type of
plant grown on the soil will have marked effect
on the enzyme activities. Their effect could be
directly through endoenzymes contained in the
plant residues or the extracellular enzymes
secreted by living roots which also may also
make significant contribution to enzyme activity.
This is because cropping systems that have
higher carbon input or that conserve carbon
inputs promotes enzyme activity. Hence crop
cover and stage of crop growth plays an
important role especially on characteristics which
include soil micro biomass, soil
enzymes and soil respiration. Hence the
present experiment was conducted to study the
influence of crop cover, soil L-glutaminase
activity under six crops [3].
2. MATERIALS AND METHODS
A pot culture experiment was conducted at,
College of Agriculture, Rajendranagar,
Hyderabad during the year 2019 with two soils
(Alfisol and Vertisol), six crops viz., two cereals
(Rice, Maize), two legumes (Ground nut,
Greengram), one oilseed crop (Sunflower) and
one vegetable (Bhendi). The experiment was
conducted in Completely Randomized Block
design with three replications along with the
uncropped control. The soil samples were
collected at 10 days interval from 0 DAS to 90
DAS and at harvest and were assayed for L-
glutaminase activity. The activity of L-
glutaminase was assayed by Frankenberger
and Tabatabai [4] and the rate of NH4+
released was quantified by the modified
Indophenol method as given by Dorich and
Nelson [5].
2.1 REAGENTS
2.1.1 THAM buffer (0.1 M)
It was prepared by dissolving 12.28 g of THAM
(Tris hydroxy methyl amino methane) in about
800 mL of distilled water and adjusting the pH
by adding 0.1N HCl or 0.1N NaOH to the desired
pH and then makes up the volume to 1 litre with
water.
2.1.2 L-Glutamine (0.125M)
The solution was prepared by dissolving 18.25g
of L- glutamine in 1 litre of THAM buffer of
desired pH.
2.1.3 Potassium chloride (2.5M)-silver
sulphate (100ppm) KCl-Ag2SO4
solution
It was prepared by dissolving 100 mg of Ag2SO4
in 700 mL of distilled water and dissolving 186.4
g of KCl and diluting the solution to one litre with
distilled water.
2.1.4 Ethylene diamine tetra acetic acid
(EDTA) 6%
This was prepared by dissolving 6 g of EDTA in
distilled water and made up to 100 mL. The pH
of the solution was adjusted to 7 with diluted
0.1 N NaOH.
2.1.5 Phenol-nitroprusside
7 g of Phenol and 34 mg of sodium-
nitroprusside was dissolved in distilled water and
diluted to 100 mL with distilled water.
2.1.6 Buffered hypochlorite
This was prepared by dissolving 14.8 g of
NaOH and 49.8 g of Na2HPO4 in 400 mL of
distilled water, adding 400 mL of NaOCl (4-
5%), adjusting the pH to 11.8 and the volume
was made up to 1 litre.
2.1.7 Standard Ammonium Solution
Primary stock solution of 100 μg mL-1 of
ammonium was prepared by dissolving 0.4717 g

Yadav et al.; IRJPAC, 21(24): 334-340, 2020; Article no.IRJPAC.65422
336
of ammonium sulphate in distilled water and
was made up to 100 ml with distilled water.
2.2 Procedure
Soil sample (10 g) was taken in a 150 mL
conical flask and adds 0.4 mL of Toluene, to
which 12 mL of 0.1 M THAM buffer of pH 8 was
added. The flasks were gently swirl to mix the
contents followed by addition of 8 ml of 0.125 M
L-glutamine were added, so that concentration
substrate was 50 mM. The flasks were gently
shaken for few seconds and covered with
polythene paper. Then the contents were
incubated at 37±0.5oC for 4 hours in BOD
incubator. After incubation, reaction was
terminated by addition of 30 mL of 2.5 M KCl -
(100 ppm) Ag2SO4 solution. The contents were
agitated on mechanical shaker for 30 min to
release all NH4+ formed and the suspension
was allowed to settle and filtered. In the
controls the same procedure described above
was followed but the L-glutamine solution was
added after deactivating with KCl-Ag2SO4
reagent. The incubation time of 4 hours was
chosen because, preliminary investigations
carried out various time intervals indicated an
incubation time of 4 hours to be optimum time for
the assay of enzyme during the investigation.
Similarly the amount of 10 grams of soil was
based on a preliminary investigation taking
2,4,6,8,10,12 and 15 grams soil sample for
assaying L-glutaminase activity out of which 10
grams was found to be optimum for assay.
One mL of supernatant from the soil suspension
after incubation with L-glutamine and
deactivation with KCl - Ag2SO4 was transferred
to 25 mL volumetric flask. To this, 1 mL of 6%
EDTA was added followed by addition of 2 mL
of Phenol-nitroprusside and 8 mL of buffered
hypochlorite reagent. The volume was then
made up to the mark, mixed thoroughly by
inverting several times and placed in water bath
for 30 min at 40oC for colour development.
The flasks were removed and brought to room
temperature and the absorbance of blue colo ur
was measured at 636 nm using UV-1800
spectrophotometer. The L-glutaminase activity
was measured with respect to the amount of
NH4+ liberated and expressed as μg of NH4+
released g-1 soil 4h-1.
3. RESULTS AND DISCUSSION
The effect of crop cover and crop growth on L-
glutaminase activity were presented in Table
1and 2. There was an increase in enzyme
activity with age of the crop and it varied with
plant species grown. The activity of L-
glutaminase increased from 0 to 60 DAS which
was coincides with maximum flowering stage of
crops and then decreased upto harvest. The
activity varied for crops grown in Vertisol and
Alfisol. The L-glutaminase activity was
consistently high with groundnut followed by
greengram cover crop. The enzyme activity (µg
of NH4+ released g-1 soil 4h-1) varied from 5.56
to 12.17 for groundnut, 5.58 to 11.25 for
greengram, 5.43 to 10.87 for sunflower, 5.48 to
8.61 for rice, 5.39 to 8.23 maize and 5.31 to 7.92
for bhendi in Vertisol (Table1 and Fig. 1). In
Alfisol the L-glutaminase activity (µg of NH4+
released g-1 soil 4h-1) under different crop
cover (Table 2 and Fig. 2) found to vary from
6.72 to 13.59 (groundnut), 6.68 to 12.71
(greengram), 6.63 to 11.96 (sunflower), 6.61 to
10.25 (rice), 6.59 to 9.47 (maize), 6.62 to 9.26
(bhendi).
The trends indicated that the L-glutaminase
activity was higher in Alfisol as compared to
Vertisol. A close perusal of the data indicates
that the L-glutaminase activity followed the
sequence groundnut > greengram >
sunflower > rice > maize > bhendi. The
significantly higher L-glutaminase activity under
groundnut could be due to continuous growth
and extensive root system and high release of
extracellular enzymes secreted by
groundnut roots thus effecting substrate
concentration in the rhizosphere. In addition,
crop cover increased the biochemical variables
like substrates related to microbial activity and
increased the activity of L-glutaminase due to
increase in carbon turnover and nutrient
availability. The increased L-glutaminase activity
under groundnut and greengram during flowering
might be due to well developed root nodules
where nitrogen fixation occurs. Hence it is
possible that the ammonical nitrogen fixed during
atmospheric nitrogen fixation is reduced to
nitrogen rich organic compounds like glutamine
and this might have a stimulatory effect on L-
glutaminase activity in soil [6].

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337
Table 1. Effect of crop cover on soil l-glutaminase activity in vertisol
Crops
Days after sowing / Days after t
r
ansp
l
anting
L-glutaminase activity ( µg of NH4+ released g-1 soil 4h
-1
)
0
10
20
30
40
50
60
70
80
90
Harv
e
st
M
e
an
Rice 5.48 5.81 6.28 6.81 7.59 8.12 8.61 8.03 7.65 6.58 5.59 6.95
Maize 5.39 5.54 5.91 6.27 7.13 7.78 8.23 7.91 6.83 6.49 5.57 6.64
Groundnut 5.56 6.31 7.97 9.15 10.49 11.47 12.17 11.26 9.13 7.34 5.79 8.76
Greengram 5.58 6.17 7.54 8.47 9.59 10.31 11.25 10.17 8.93 6.95 5.73 8.25
Sunflower 5.43 6.04 7.24 8.11 9.26 9.98 10.87 9.84 8.64 6.62 5.61 7.96
Bhendi 5.31 5.56 5.78 6.01 6.68 7.43 7.92 7.31 6.78 5.91 5.54 6.37
Control 5.42 5.39 5.36 5.38 5.32 5.42 5.43 5.35 5.29 5.27 5.31 5.36
Mean 5.45 5.83 6.58 7.17 8.00 8.62 9.2 8.55 7.60 6.45 5.6
C.D. (5 %)
SE(m) ±
L-glutam
i
nas
e
0.194 0.069
Crop Cov
er
0.154 0.055
L-glutaminase X Crop Cov
er
0.512 0.183
Fig. 1. Effect of crop cover on soil l-glutaminase activity in vertisol
As compared to uncropped control higher
activity of L-glutaminase was observed with
crops. Various factors like rhizosphere effect,
age of crop, nature of crops influenced L-
glutaminase activity in soil. L-glutaminase
activity increases from 0 to 60 days after
sowing which coincides with the active growth
stage of the crop, enhanced root activity and
the release of cellular enzyme in to soil
solution during the active growth phase which
resulted in higher rate of mineralization of
nutrients in the soil. Vandana et al.[7]; Pavani
[8] and Kumari et al. [9] observed the difference
in enzyme activity mainly due to type of
vegetation. The enzymes secreted by plant
roots and microorganisms associated in
rhizosphere release large amount of enzymes
into soil. In addition plant type has been
reported to be dominant factor affecting soil
microbial and L-glutaminase activity [10-11].
Besides, the higher enzyme activity
under crop cover could be due to the

Yadav et al.; IRJPAC, 21(24): 334-340, 2020; Article no.IRJPAC.65422
338
incorporation of organic residues coupled
with greater microbial activity in cover
areas [12]. Thus a large and complex number of
factors during crop growth contribute
to an increased L- glutaminase activity.
In a study conducted by Balezentiene and
Klimas [13] the legume grass mixtures formed
an even cover over the soil during the year
of sowing and improved the micro-biological
properties of the soil. The grasses form better
conditions for organic matter decomposition
and hence, urease and saccharase activities
were found to be highest in the
grass grown soils compared to other crops.
Table 2. Effect of Crop Cover on soil L-glutaminase activity in Alfisol
C
r
ops
Days after sowing / Days after t
r
ansp
l
anting
L-glutaminase activity ( µg of NH4+ released g-1 soil 4h
-1
)
0
10
20
30
40
50
60
70
80
90
Harv
e
st
M
e
an
R
ice
6.61 6.98 7.42 8.23 9.14 9.68 10.25 9.31 8.74 7.56 6.77 8.27
M
aize
6.59 6.91 7.22 7.75 8.38 8.79 9.47 8.51 7.96 7.27 6.72 7.78
Ground nut 6.72 7.48 8.29 9.52 11.11 12.46 13.59 12.23 10.91 8.73 6.96 9.82
Gr
ee
ngram 6.68 7.31 7.99 9.15 10.56 11.76 12.71 11.47 9.86 8.17 6.83 9.29
Sun f
l
ow
e
r 6.63 7.28 7.81 8.86 10.17 11.15 11.96 10.61 9.12 7.87 6.81 8.93
Bh
e
nd
i
6.62 6.84 7.18 7.69 8.27 8.62 9.26 8.23 7.74 7.16 6.71 7.67
Con
t
ro
l
6.65 6.61 6.58 6.56 6.53 6.59 6.56 6.49 6.45 6.44 6.44 6.54
M
ea
n 6.64 7.11 7.5 8.25 9.17 9.86 10.54 9.54 8.63 7.6 6.75
C.D. (5 %)
SE(m) ±
L-glutam
i
nas
e
0.115 0.041
Crop Cov
er
0.092 0.033
L-glutaminase X Crop Cov
er
0.306 0.109
Fig. 2. Effect of crop cover on soil l-glutaminase activity in alfisol

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339
Plate 1. Assessment of l-glutaminase activity by modified indophenol method
Plate 2. Pot culture experiment
4. CONCLUSION
L-glutaminase activity increases from 0 to 60
days after sowing which coincides with the
active growth stage of the crop, enhanced root
activity and the release of cellular enzyme in to
soil solution during the active growth phase
which resulted in higher rate of mineralization of
nutrients in the soil. The increase in L-
glutaminase activity with the age of crop varied
with the type of crop grown. The enzyme
activity was higher under legume crop cover

Yadav et al.; IRJPAC, 21(24): 334-340, 2020; Article no.IRJPAC.65422
340
conditions. There was a higher activity of L-
glutaminase in Alfisol as compared to all the
crops grown in Vertisol.
ACKNOWLEDGEMENTS
The authors are extremely thankful to Professor
Jayashankar Telangana state Agricultural
University, Rajendranagar, Telangana (India) for
financial help and technical guidance to carry
out this research work.
COMPETING INTERESTS
Authors have declared that no competing
interests exist.
REFERENCES
1. Stevenson FJ. Humus chemistry,
Genesis, Composition and reactions.
John Wiley and Sons. Newyork; 1994.
2. Bremner JM. Exchangable ammonium
nitrate and nitrite of soil analysis, in
methods of soil analysis part-2. American
society of agronomy. 1965;1191-1206.
3. Dick RP. Soil enzyme activities as
indicator of soil quality. In Doran JV,
Coleman DC, Bezdicek DF, Stewart
VA.(eds.) – Defining soil quality for
sustainable environment, Soil Science
Society of America, American Society of
Agriculture, Madison. 1994;107–124.
4. Frankenberger WT Jr, Tabatabai MA. L-
Glutaminase activity of soils. Soil
Biology & Biochemistry. 1991;869-874.
5. Dorich RA, Nelson DW. Direct
colorimetric measurement of ammonium
in potassium chloride extracts of soil.
Soil Science Society of American
Journal. 1983;47(4):833–836.
6. Hamido SA, Kpomblekou AK. Cover crop
and tillage effects on soil enzyme activities
following tomato. Soil & Tillage Research.
2009;105:269–274.
7. Vandana JL, Rao PC, Padmaja G. Effect
of crop cover on soil enzyme activity. The
journal of research ANGRAU.
2012;40(4):1-5.
8. Pavani GL- glutaminase activity in soils–
assay, distribution, kinetics and the effect
of crop cover on its activity M.Sc. Thesis.
Professor Jayashankar Telangan State
Agricultural University, Hyderabad; 2015.
9. Kumari JA, Rao PC, Padmaja G, Reddy
RS, Madhavi M. Effect of crop cover and
stage of crop growth on soil L-
asparaginase, Alkaline and Acid
phosphatase in an alfisol enzyme activity.
The journal of research PJTSAU.
2015;43(4):1-6.
10. Chandra R. Effect of summer crops and
their residue management on the yield of
succeeding wheat and soil properties.
Journal of Indian Society of Soil Science.
2011;59(1):37–42.
11. Rai TN, Yadav J. Influence of organic
and inorganic nutrient sources on soil
enzyme activities. Journal of Indian Society
of Soil Science. 2011;59(1):54–59.
12. Dinesh R, Suryanarayana MA, Chaudhuri
SG, Sheeja TE. Long-term influence of
leguminous cover crops on the
biochemical properties of a sandy clay
loam Fluventic Sulfaquent in a humid
tropical region of India. Soil and tillage
research. 2004;69–77.
13. Balezentiene L, Kilimas E. Variation in
soil microbiological activity in relation to
the yield of fodder galega swards.
Grassland Science in Europe.
2003;8:437-440.
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