Effect of Allelochemicals from Leaf Leachates of Gmelina arborea on Inhibition of Some Essential Seed Germination Enzymes in Green Gram, Red Gram, Black Gram, and Chickpea


Madhan Shankar, R.; Veeralakshmi, S.; Sirajunnisa, A.Razack.; Rajendran, R.

International Scholarly Research Notices 2014: 108682

2014


The present work focused on identification of allelochemicals from the leaf leachates of Gmelina arborea and analyzing its influence on the germination of red gram, green gram, black gram, and chickpea in terms of the levels of some important germination enzymes like acid phosphatase, catalase, peroxidase, and amylase. The study showed that allelopathic effects were more predominant in chickpea with 80% followed by red gram, green gram, and black gram where the inhibition ranged between 26% and 75%. The vigor index in the seed lots is also considerably reduced. Total chlorophyll content was also reduced by allelopathic effect in all treated seeds ranging between 0.7 and 7.5 μg/g dry weight. The effect of allelochemicals drastically reduced the relative water content in red gram followed by the other seed lots. The total protein content varied considerably in the control and the treated seed lots. Allelochemicals inhibited the expression and activity of the enzymes required for efficient germination. The present study also threw limelight on the effective use of this tree, wherein planting this tree amidst pulse related herb plantations could affect the growth of the economically viable plants, but this tree can very well adapt to diversified soil conditions and rainfall zones.

Hindawi
Publishing
Corporation
International
Scholarly
Research
Notices
Volume
2014,
Article
ID
108682,
7
pages
http://adoi.org/10.1155/2014/108682
Research
Article
Effect
of
Allelochemicals
from
Leaf
Leachates
of
Gmelina
arborea
on
Inhibition
of
Some
Essential
Seed
Germination
Enzymes
in
Green
Gram,
Red
Gram,
Black
Gram,
and
Chickpea
Ramakrishnan
Madhan
Shankar,'
Shanmugham
Veeralakshmi,'
Abdul
Razack
Sirajunnisa,
2
and
Ramasamy
Rajendran
3
'
Department
of
Biotechnology,
PSG
College
of
Arts
and
Science,
Coimbatore,
Tamil
Nadu
641014,
India
2
Department
of
Chemical
Engineering
Annamalai
University,
Annamalai
Nagar,
Tamil
Nadu
608002,
India
3
Department
of
Microbiology,
PSG
College
of
Arts
and
Science,
Coimbatore,
Tamil
Nadu
641014,
India
Correspondence
should
be
addressed
to
Ramakrishnan
Madhan
Shankar;
Received
6
March
2014;
Accepted
21
June
2014;
Published
1
September
2014
Academic
Editor:
Giuseppe
Maurizio
Campo
Copyright
CO
2014
Ramakrishnan
Madhan
Shankar
et
al.
This
is
an
open
access
article
distributed
under
the
Creative
Commons
Attribution
License,
which
permits
unrestricted
use,
distribution,
and
reproduction
in
any
medium,
provided
the
original
work
is
properly
cited.
The
present
work
focused
on
identification
of
allelochemicals
from
the
leaf
leachates
of
Gmelina
arborea
and
analyzing
its
influence
on
the
germination
of
red
gram,
green
gram,
black
gram,
and
chickpea
in
terms
of
the
levels
of
some
important
germination
enzymes
like
acid
phosphatase,
catalase,
peroxidase,
and
amylase.
The
study
showed
that
allelopathic
effects
were
more
predominant
in
chickpea
with
80%
followed
by
red
gram,
green
gram,
and
black
gram
where
the
inhibition
ranged
between
26%
and
75%.
The
vigor
index
in
the
seed
lots
is
also
considerably
reduced.
Total
chlorophyll
content
was
also
reduced
by
allelopathic
effect
in
all
treated
seeds
ranging
between
0.7
and
7.5
pg/g
dry
weight.
The
effect
of
allelochemicals
drastically
reduced
the
relative
water
content
in
red
gram
followed
by
the
other
seed
lots.
The
total
protein
content
varied
considerably
in
the
control
and
the
treated
seed
lots.
Allelochemicals
inhibited
the
expression
and
activity
of
the
enzymes
required
for
efficient
germination.
The
present
study
also
threw
limelight
on
the
effective
use
of
this
tree,
wherein
planting
this
tree
amidst
pulse
related
herb
plantations
could
affect
the
growth
of
the
economically
viable
plants,
but
this
tree
can
very
well
adapt
to
diversified
soil
conditions
and
rainfall
zones.
1.
Introduction
Allelopathy
is
the
negative
effect
of
chemicals
released
by
one
plant
species
on
the
growth
and
reproduction
of
another
[1].
Allelopathy
can
affect
many
aspects
of
plant
ecology,
including
occurrence,
growth,
and
plant
succession,
the
structure
of
plant
communities,
dominance,
diversity,
and
plant
productivity.
These
effects
can
be
either
positive
or
negative.
There
are
four
main
classes
of
chemical
inter-
actions,
antibiotics
(microorganisms
to
microorganism),
kolines
(plants
to
plants),
marasmins
(microorganisms
to
plants),
and
phytoncides
(plants
to
microorganisms).
In
allelopathic
terms,
a
chemical
is
"conveyed"
to
a
"receiver"
which
can
either
be
"impaired"
or
"assisted"
[2].
Interaction
between
soil
organisms
and
plants
is
important
in
allelopathy.
The
activity
of
nitrogen
fixing
bacteria
could
be
affected
by
allelopathy.
Allelochemicals
representing
numerous
chem-
ical
groups
have
been
isolated
from
over
30
families
of
terrestrial
and
aquatic
plants.
Some
Indian
plants
possess-
ing
allelochemicals
are
Pongamia
glabra
(Karani),
Mathuca
indica
(Molarah),
Schleicheria
teijuga
(Kusum),
and
Celastrus
paniculatus
(Malkanguni).
Allelochemicals
can
cause
oxidative
stress
in
the
target
plants.
Therefore,
allelochemicals
activate
the
antioxidant
mechanism.
Autotoxicity
occurs
when
plant
species
releases
toxic
chemicals
that
inhibit
germination
and
growth
of
same
plant
species
[3].
Allelopathy
has
also
been
observed
in
several
tree
species.
Leucaena
leucocephala,
the
tree
promoted
2
International
Scholarly
Research
Notices
for
revegetation
of
soil,
contains
a
toxic
nonprotein
amino
acid
in
the
leaves
which
inhibits
growth
of
others
but
not
its
own
seedlings.
Mimosine
is
the
allelochemical
that
is
released
from
this
tree
[4].
Gmelina
arborea
belongs
to
the
family
Verbenaceae.
It
is
commonly
called
Gmelina
and
white
beech
(English),
Melinaphocal
(Spanish),
Gamar
in
Bangladesh,
Melina
gam-
bar
in
India,
Gmelina
in
Indonesia,
Yemane
in
Philippines,
and
Soh
in
Thailand
[5].
It
has
an
extensive
geographical
distribution
in
the
Indian
continent.
Gmelina
arborea
is
a
medium-sized
deciduous
tree,
up
to
40
m
in
length
and
140
cm
in
diameter,
but
it
is
usually
smaller
than
other
trees.
It
can
show
in
a
wide
range
of
diameter
zones
ranging
from
50
m
to
4500
m
MSL
and
in
regions
of
annual
rain
fall
ranging
from
50
mm
to
9500
mm
[6].
Shankar
et
al.
[7]
analyzed
allelochemicals
effect
of
Gmelina
arborea
on
Vigna
mungo
and
Vigna
radiata.
The
presence
of
allelopathic
compounds
such
as
polyphenols
and
terpenoids
was
analysed
by
TLC
and
GC-MS.
The
extract
inhibited
the
proteolytic
enzyme
important
for
seed
germination.
The
extract
inhibited
the
germination,
seedling
growth,
and
total
protein
content
of
both
test
crops.
The
present
study
focused
on
identification
of
the
alle-
lochemicals
by
HPTLC,
determination
of
the
effects
of
the
allelochemicals
on
the
germination
of
black
gram,
green
gram,
red
gram,
and
chickpea,
and
analysis
of
allelopathic
effects
by
conventional
pot
experiments.
The
study
also
elaborated
on
determination
of
the
activity
of
peroxidase,
acid
phosphatase,
amylase,
and
catalase
on
various
stages
of
germination
and
correlating
the
loss
of
activity
to
the
detrimental
effects
of
the
allelochemicals
present
in
the
leaf
leachates
of
Gmelina
arborea.
2.
Materials
and
Methods
2.1.
Source
and
Preparation
of
Leaf
Leachate.
Fresh
leaves
and
leaf
litters of
Gmelina
arborea
were
collected
from
a
six-year-
old
tree
and
stored
in
polyethene
bags
in
a
moisture-free
atmosphere.
The
seeds
and
plant
were
identified
from
the
Institute
of
Forest
Genetics
and
Tree
breeding,
Coimbatore,
Tamil
Nadu,
India.
The
leaves
were
dried
in
partial
shade
and
stored
for
the
study.
A
quantity
of
50
g
of
fresh
leaf
litter
was
soaked
in
159
mL
distilled
water
for
24
hours
at
25°C.
The
leachate
was
collected
and
stored
at
4°C
for
further
use.
Harrington
suggested
that
agricultural
seeds
retain
their
longevity
in
terms
of
germination
efficiency
if
stored
between
0
and
50°C;
bringing
down
the
temperature
by
every
5
degrees,
the
life
of
the
seed
doubles.
In
the
present
study,
since
germination
index
assessment
is
the
hallmark,
retention
of
inherent
viability
is
essential.
Further,
orthodox
seeds
would
require
lower
temperatures
and
storage
at
subzero
temperatures
[8].
2.2.
Source
of
Seed
for
Bioassays.
Vigna
mungo
(Green
gram
Co
7),
Vigna
radiata
(Black
gram
Co
5),
Cajanas
cajan
(Red
gram
Co
6),
and
Cicer
arietinium
(Chickpea
Co
4)
required
for
the
seed
bioassay
were
procured
from
Department
of
Seed
Science
and
Technology,
Tamil
Nadu
Agriculture
University,
Coimbatore,
Tamil
Nadu,
India.
The
seeds
were
stored
in
airtight
containers,
away
from
moisture
at
4°C.
2.3.
Phytochemical
Analysis
for
Phenolics.
Ferric
chloride
test:
2
mL
of
water
was
added
to
1
mL
of
each
extract.
Two
to
three
drops
of
10%
ferric
chloride
solution
were
added
and
development
of
green
color
was
to
be
observed.
Liebermann's
test:
1
mL
of
20%
sulphuric
acid
was
added
to
1
mL
of
each
extract
followed
by
addition
of
few
drops
of1%
sodium
nitrate
solution
and
the
tubes
were
observed
for
the
formation
of
red
color,
which
on
dilution
and
in
alkaline
condition
with
sodium
hydroxide
turn
blue.
2.4.
HPTLC
Analysis
of
Leaf
Leachate.
The
aqueous
extract
of
leaf
sample
was
centrifuged
and
the
supernatant
was
collected,
which
was
used
as
test
solution
for
HPTLC
analysis.
A
volume
of
2µL
of
the
test
solution
and
3µL
of
standard
solution
was
loaded
as
6
mm
band
length
in
the
3
x
10
silica
gel
60F
254
TLC
plate
using
Hamilton
syringe
and
CAMAG
LINOMAT
5
instrument.
The
samples'
loaded
plate
was
placed
in
TLC
twin
trough
developing
chamber
(after
being
saturated
with
solvent
vapor)
and
the
plate
was
developed
in
the
respective
mobile
phase
(toluene-acetone-formic
acid
(4.5
:
4.5
:1))
up
to
90
mm.
The
developed
plate
was
dried
using
hot
air
to
evaporate
solvents
from
the
plate.
The
plate
was,
then,
kept
in
photodocumentation
chamber
(CAMAG
REPROSTAR
3)
to
capture
the
images
under
white
light,
UV
254
nm
and
UV
366
nm.
The
developed
plate
was
sprayed
with
a
spray
reagent
(fast
blue
B
reagent)
and
dried
at
120°C
in
hot
air
oven.
The
plate
was
photodocumented
under
day
light
and
UV
366
nm
using
the
same
photodocumentation
chamber.
Before
derivatization,
the
plate
was
fixed
in
scanner
stage
and
scanning
was
done
at
254
nm.
The
peak
table,
peak
display,
and
peak
densitogram
were
noted.
2.5.
Seed
Bioassays.
A
quantity
of
50
g
of
soil
sample
was
taken
in
pots
of
depth
of
3
inches
and
five
seeds
were
sown
in
each
pot.
Triplicates
were
maintained;
one
seed
was
taken
from
each
part
of
the
replicate
and
ground
in
3
mL
of
0.2
M
phosphate
buffer
(pH
7.0)
and
centrifuged
at
5000
rpm
for
10
minutes;
the
supernatant
was
collected
and
used
for
biochemical
analysis.
Similarly,
four
replicates
containing
five
seeds
in
each
pot
were
maintained
to
calculate
the
total
chlorophyll
content,
percentage
of
germination,
germination
index,
vigour
index,
root
length,
and
shoot
length.
The
number
of
seeds
germinating
was
counted
everyday
up
to
the
sixth
day
and
the
percentage
of
germination
was
calculated
by
the
formula
Germination%
=
I
t
*
100,
(1)
where
n
is
the
number
of
seeds
germinated
and
t
is
the
total
number
of
seeds.
The
germination
index
was
calculated
according
to
Wiese
and
Binning
[9]
using
the
formula
GI
=
a
'
(2)
where
n
is
the
number
of
seedlings
emerging
on
day
"d" and
d
is
the
day
after
planting.
The
seed
vigour
index
was
calculated
International
Scholarly
Research
Notices
3
by
multiplying
germination
percentage
and
seedling
length
(cm).
The
root
length,
shoot
length,
and
seedling
length
were
measured
using
a
grading
scale
on
sixth
day
after
germination.
The
whole
plant
was
weighed
after
germination,
which
constituted
the
fresh
weight.
The
whole
plant
was
wrapped
in
aluminium
foil
and
placed
inside
an
oven
for
48
hat
45
°
C.
The
dry
weight
was
measured.
The
relative
water
content
was
calculated
at
sixth
day
after
germination
by
Relative
water
content
(%)
Dry
weight
(3)
=
Fresh
weight
-
Fresh
weight
The
total
protein
content
present
in
control
seeds
and
treated
seed
lots
was
estimated
by
Bradford's
Method
(1976)
[10].
2.6.
Total
Chlorophyll
Content.
A
quantity
of
0.5
g
fresh
weight
of
green
tissue
from
both
leaf
and
stem
was
weighed.
The
tissue
was
ground
in
10
mL
of
95%
ethanol.
The
contents
were
centrifuged
at
5000
rpm
for
10
minutes
and
the
super-
natant
was
collected.
The
absorbance
was
read
at
both
647
nm
and
664
nm
against
reagent
blank.
Total
chlorophyll
content
was
checked
by
Einhelling
method
[11],
using
the
formulae
Chlorophyll
a
=
13.19
(ABS
at
664
nm)
-
2.57
(ABS
at
647
nm)
yg/g
dry
weight,
Chlorophyll
b
=
22.10
(ABS
at
647
nm)
(4)
-
5.26
(ABS
at
664
nm)
yg/g
dry
weight,
Total
chlorophyll
=
7.93
(ABS
at
664
nm)
+
19.53
(ABS
at
647
nm)
yg/g
dry
weight.
2.7
Enzyme
Assays
2.71.
Catalase
Assay.
The
catalase
assay
was
carried
out
using
the
method
of
Chance
and
Maehly
[12].
A
volume
of
1.9
mL
distilled
water,
1
mL
of
0.059
M
hydrogen
peroxide,
and
0.1
mL
of
diluted
seed
extract
was
added.
The
decrease
in
absorbance
was
recorded
at
240
nm
for
2-3
minutes
and
calculated
at
240
nm/min
from
the
initial
(45
seconds)
linear
portion
of
the
curve.
The
enzyme
activity
was
further
calculated
using
the
following
relationship:
Units/mg
=
240/
min
*
1000
*
mg
enzyme/mL
reaction
mixture.
43.6
(5)
2.72.
Determination
of
Acid
Phosphatase.
Estimation
of
acid
phosphatase
was
done
according
to
Malik
and
Singh
[13].
The
reaction
mixture
consisted
of
0.5
mL
of
substrate
solution
(50
mg
of
p-nitrophenyl
phosphate
was
dissolved
in
10
mL
distilled
water).
The
pH
was
adjusted
to
4.8
with
0.5
N
NaOH
and
0.1
mL
of
suitably
diluted
seed
extract.
The
mixture
was
incubated
at
35°C
for
30
min.
The
reaction
was
stopped
by
adding
2.4
mL
of
0.1N
NaOH.
A
volume
of
0.2
to
1
mL
(4
to
20
mM)
of
the
standard
(69.75
mg
of
p-nitrophenol
dissolved
in
5
mL
distilled
water
to
give
a
final
concentration
of
100
mM)
was
diluted
to
3
mL
with
0.1N
NaOH.
The
absorbance
was
recorded
at
410
nm
against
reagent
blank.
Specific
activity
was
expressed as
moles
of
p-nitrophenol
were
released
per
minute
per
mg
of
protein.
2.73.
Estimation
of
Amylase.
Amylase
in
the
sample
was
estimated
according
to
the
technique
of
Bernfeld
[14].
A
volume
of
1
mL
of
1%
starch
solution
was
taken
and
1
mL
of
properly
diluted
seed
extract
was
added
to
it.
1
mg/mL
of
maltose
was
used
as
a
standard.
The
contents
were
incubated
at
27°C
for
15
minutes.
The
reaction
was
stopped
by
the
addition
of
2
mL
of
dinitrosalicylic
acid
reagent
(1
g
of
dinitrosalicylic
acid,
200
mg
of
crystalline
phenol,
and
50
mg
sodium
sulphite
in
100
mL
1%
NaOH).
The
solution
was
heated
in
a
boiling
water
bath
for
5
minutes.
A
volume
of
1
mL
of
40%
potassium
sodium
tartrate
was
added
and
cooled
under
running
tap
water.
The
absorbance
was
read
at
560
nm
against
reagent
blank.
One
unit
of
amylase
was
expressed
as
mg
of
maltose
released
during
5-minute incubation
with
1%
starch
solution
at
27°C.
2.74.
Peroxidase
Assay.
Peroxidase
was
checked
using
Shan-
non
et
al.
method
[15].
The
assay
mixture
containing
2.5
mL
of
phosphate
buffer,
0.2
mL
of
suitably
diluted
seed
extract,
and
0.1
mL
of
o-dianisidine
(50
mg
of
o-dianisidine
was
dissolved
in
50
mL
of
methanol)
was
incubated
at
28°C
in
a
water
bath
for
2
minutes.
The
reaction
commenced
by
adding
0.2
mL
of
H
2
0
2
(0.6%)
and
left
for
5
minutes.
The
absorbance
was
recorded
at
430
nm
against
reagent
blank.
3.
Results
and
Discussion
The
leaf
leachate
of
Gmelina
arborea
was
subjected
to
phy-
tochemical
analysis
that
showed
a
strong
positive
result
for
the
presence
of
phenols
followed
by
moderate
indication
for
alkaloids.
The
aqueous
leaf
leachate
was
also
subjected
to
HPTLC
analysis
and
results
are
shown
in
Figure
1.
Orange
brown
colored
zone
at
day
light
was
observed
after
derivatiza-
tion
which
confirmed
the
presence
of
phenolics
having
an
RF
value
of
0.51.
The
standard
quercetin
was
used
to
compare
the
unknown
sample.
Quercetin
had
an
RF
value
of
0.58.
Hence
taking
into
account
the
closeness
of
RF
values,
it
could
be
inferred
for
the
presence
of
phenolic
compounds.
Further,
the
FTIR
spectrum
ascertained
the
presence
of
phenolic
compounds
that
revealed
several
peaks
ranging
between
47.50
cm
-1
and
2305.01
cm
-1
.
This
wide
range
of
peaks
could
be
attributed
to
the
presence
of
various
phyto
-
chemical
constituents
in
the
aqueous
leachate
representing
several
bond
stretches.
Phenols
showed
the
characteristic
C=C
stretches
and
C-H
vibration
for
aromatic
residues.
4
600
Track
1,
ID:
sample
A
700
International
Track
2,
ID:
quercetin
standard
Scholarly
Research
Notices
600
500
uercet
500
400
,...,
400
5
..4
300
-
,
4
300
200
200
Quercetin
100
2
1
4
5
6
7
100
0
0
0.00 0.10 0.20
0.30
0.40 0.50 0.60 0.70
0.80
0.90
0
00
0.10 0.20
0.30
0.40
0
50
0.60 0.70
0.80
0.90
RF
RF
(a)
(b)
A
QUER
A
QUER
(c)
(d)
FIGURE
1:
Showing
densitograms
of
leaf
leachate
(a)
and
reference
Quercetin
(b)
and
chromatograms
of
HPTLC
analysis
after
derivatization
under
UV
(c)
and
day
light
(d).
The
strong
OH
stress
band
might
swamp
the
weaker
C-
H
stretches
band
just
above
3000
cm
-1
.
It
is
difficult
to
distinguish
a
phenol
from
an
aryl
alcohol
from
infrared
evidence.
Our
earlier
studies
had
shown
similar
results
where
various
phenolic
compounds
like
3,4,5-hydroxybenzoic
acid,
3-(4-hydroxyphenyl)
prop-2-enoic
acid,
and
4-hydroxy-3-
methoxybenzoic
acid
were
found
to
be
present
in
the
aqueous
leaf
leachate
[7].
Similarly,
several
benzoic
acid
derivatives
had
been
shown
to
possess
potential
allelopathic
effect
on
red
gram
[16].
The
crude
aqueous
leaf
leachates
were
tested
for
their
allelopathic
effects
on
black
gram,
red
gram,
chickpea,
and
green
gram.
The
allelopathic
effects
were
more
predomi-
nantly
seen
in
chickpea
with
80%
inhibition
of
germination
(Table
1).
In
the
other
systems,
namely,
black
gram,
red
gram,
and
green
gram,
the
percentage
of
inhibition
ranged
between
26%
and
75%.
The
results
were
also
consistent
with
earlier
report
where
allelochemicals
from
Eucalyptus
showed
around
75%
inhibition
in
red
gram
[16].
There
was
also
profuse
inhibition
in
terms
of
reduction
in
the
seedling
length.
Though
only
a
few
seeds
in
the
chickpea
seed
lot
germinated,
the
sustenance
of
the
plantlet
was
not
very
stable
as
the
seedling
was
watered
with
the
leachate
even
after
germination.
Hence,
it
could
be
inferred
that
the
allelochemicals
of
Gmelina
arborea
were
not
only
of
inhibitory
type
to
germination
but
also
with
retarded
plantlet
growth
after
germination.
Terzi,
2008,
[17]
had
also
observed
such
kind
of
high
negative
inhibition
where
juglone
on
allelochemicals
from
walnut
leaf
juice
significantly
retarded
the
seedling
growth
of
musk
melon
and
cucumber.
In
the
present
study,
the
vigour
index
observed
in
various
seed
lots
ranged
between
33.2
and
1522
with
a
mean
of
88.3
for
black
gram,
between
124.3
and
1236
with
a
mean
of
687
for
red
gram,
and
between
533.8
and
992
with
a
mean
of
785
for
green
gram
seed
lots.
Since
the
percentage
of
inhibition
was
too
high
for
chickpea
seed
lot,
decipherable
vigour
index
Seedling
length
(cm)
Germination
percentage
(%)
Control
Treated
Control
Treated
Vigour
index
Control
Treated
Samples
58.45
17.04
92.17
176.53
49.07
266.54
27.84
18.45
30.12
16.38
84.23
71.98
9.33
60.82
45.26
18.97
88.70
79.42
Green
gram
Red
gram
Black
gram
International
Scholarly
Research
Notices
5
TABLE
1:
Effect
of
leaf
leachate
of
Gmelina
arborea
on
germination
of
seeds
and
seedling
growth.
Green
gram
14.46 10.46
80
75
1157
785.2
Red
gram
13.60
10.57
85
65
1156
687.3
Black
gram
18.02
12.18
95
72.5
1712
883
Chickpea
9.85
60
591
TABLE
2:
Relative
water
content
of
leaf
leachate
treated
and
untreated
seed
lots.
Samples
Seedling
fresh
weight
(mg)
Seedling
dry
weight
(mg)
Control
Treated
Control
Treated
Relative
water
content
(%)
Control
Treated
was
not
observed
(Table
1).
The
results
indicated
that
the
leachate
influenced
more
vigour
loss
in
chickpea
followed
by
red
gram,
black
gram,
and
green
gram,
respectively.
Similar
profuse
vigour
loss
was
also
reported
by
Pawar
and
Chavan
[18]
where
the
allelopathic
effects
of
Eucalyptus,
Melia,
Moringa,
and
Parthenium
were
observed
on
wheat,
rice,
millet,
and
sorghum.
The
effect
of
allelochemicals
of
the
leaf
leachate
obtained
from
Gmelina
arborea
also
drastically
reduced
the
relative
water
content
in
red
gram
followed
by
the
other
seed
lots.
The
relative
water
content
had
a
mean
of
71.5%
(Table
2).
Sim-
ilarly,
the
dry
matter
production
also
considerably
reduced
in
red
gram
seed
lots.
This
parameter
was
not
accessible
for
chickpea
seed
lot
where
the
percentage
of
survival
was
almost
nil.
The
total
chlorophyll
content
also
reduced
due
to
allelo-
pathic
effect
in
treated
seeds
of
all
the
seed
lots
ranging
between
0.7
and
7.5
yg/g
dry
weight.
Similarly,
the
total
protein
content
also
varied
between
1.02
and
0.45
in
the
control
and
between
0.25
and
0.44
in
the
allelochemicals
treated
seed
lots
(Figure
2).
Enzymes
are
one
of
the
chief
molecules,
which
are
stimulated
when
a
seed
germinates;
hence
it
would
be
very
appropriate
in
inhibiting
the
enzymes
like
acid
phos-
phatase,
catalase,
peroxidase,
and
amylase
whose
expression
is
thought
to
be
upregulated
during
seed
germination.
In
the
present
study,
activity
of
enzyme
was
found
to
increase
as
days
of
germination
increased;
for
instance,
the
level
of
peroxidase
in
control
red
gram
was
0.76
units
(Figure
3).
This
trend
increased
exponentially
after
the
second
day
of
germination
reaching
a
peak
of
1.97
units
in
control,
after
6th
day
of
germination.
The
inhibition
of
phosphatase
directly
affects
the
energy
metabolism
and
ATP
production
and
contributes
to
the
seed
vigour
and
retardation
is
visible
in
germination.
Senna
et
al.,
2005,
[19]
had
reported
the
favorable
role
of
acid
phosphatase
in
maize
seeds
germination
where
the
activity
of
the
acid
phosphatase
increased
in
24
h
of
onset
of
germi-
nation.
Similarly,
the
levels
of
prolyl
aminopeptidase
and
acid
phosphatase
were
higher
in
germinating
seeds
of
L.
esculenta.
These
enzymes
have
the
potential
role
in
liberation
of
proline
and
other
amino
acids
that
are
released
by
the
enzymatic
Total
protein
content
I
I
I
Green
gram
Red
gram
Black
gram
Chickpea
Control
Treated
FIGURE
2:
Illustrating
the
total
protein
content
of
the
control
and
samples
treated
with
leaf
leachate
of
Gmelina
arborea.
hydrolysis
of
several
seed
storage
proteins.
Matveyeva
et
al.,
2003,
[20]
had
reported
on
several
compounds
like
furosem-
ide,
bumetanide,
and
so
forth,
which
are
inhibitors
of
calcium
cotransporters
to
suppress
the
germination
rate
to
less
than
50%.
There
is
potential
role
of
EMP
enzymes
particularly
dehydrogenases
in
seed
germination
upon
treatment
with
inhibitors.
Further,
the
activity
of
these
enzymes
was
also
not
stable
after
the
plant
has
started
the
photosynthesis.
Similar
trends
were
also
observed
with
the
activity
of
amylase
being
seen
only
in
green
gram
(Figure
4).
This
explains
that
in
every
individual
type
of
seed
different
enzymes
either
singly
or
in
combination
are
responsible
for
an
efficient
germination
and
plantlet
establishment,
all
with
a
common
role
to
play,
namely,
release
of
amino
acids
and
other
small
molecules
from
the
stored
proteins
and
carbohydrates
in
the
seed,
which
could
be
used
for
germination.
4.
Conclusions
The
present
study
showed
that
the
allelopathic
compounds
present
in
the
leaf
leachate
of
Gmelina
arborea
such
as
1.2
-
-
A
E
c2,
0.2
-
0
En
zy
me
ac
t
iv
ity
(un
its
/mg
)
7
-
6
-
5
-
4
-
3
-
2
1
-
0
Catalase
Enzy
me
ac
t
iv
ity
(m
M)
6
International
Scholarly
Research
Notices
1
Green
gram
Red
gram
Black
gram
Chickpea
3.5
3
2.5
2
1.5
0.9
-
0.8
-
0.7
-
0.6
-
0.5
-
0.4-
0.3
-
0.1
-
1
1111
0.1
-
0
Green
gram
Control
Treated
Peroxidase
Acid
phosphatase
Red
gram
Black
gram
Chickpea
Control
Treated
I
1-
0.5
-
0
Green
gram
Red
gram
Control
Treated
Black
gram
Chickpea
FIGURE
3:
Activity
of
various
enzymes
in
germination
of
control
and
samples
treated
with
leaf
leachate
of
Gmelina
arborea.
2.5
Amylase-green
gram
1
2
3
4
5
6
Days
Control
Treated
FIGURE
4:
Activity
of
amylase
observed
in
green
gram.
phenolics
and
4-hydroxy-3-benzoic
acid
inhibited
crucial
enzymes
responsible
for
seed
germination.
Compared
to
the
studied
four
seed
lots,
the
germination
was
inhibited
in
chickpea
predominantly.
The
addition
of
leaf
leachate
to
the
germinating
seed
also
affected
the
seedling
length.
The
relative
water
content
and
total
chlorophyll
content
were
varied
from
81%
to
50%
and
from
97%
to
64%
in
control
and
treated
seed
lots.
The
vigour
index
represents
the
viability
of
seeds.
The
present
study
indicated
that
vigour
index
also
varied
and
it
was
much
skewed
in
black
gram
seed
lots.
The
protein
content
also
got
affected.
The
present
study
also
revealed
that
the
allelochemicals
released
from
Gmelina
arborea
affected
the
germination
and
growth
of
black
gram,
green
gram,
red
gram,
and
chickpea.
Thus,
the
extract
reduced
the
growth
of
economically
important
seeds.
In
the
future
study,
Gmelina
arborea
allelochemicals
could
be
used
in
the
control
of
weeds.
The
present
study
also
threw
limelight
on
the
effective
use
of
this
agroforestry
tree,
wherein
planting
this
tree
amidst
pulse
related
herb
plantations
could
affect
the
growth
of
the
economically
viable
plants.
Potential
plantations
of
Gmelina
arborea
could
be
tried
in
uncultivable
dry
lands
as
this
species
can
very
well
adapt
to
diversified
soil
and
climatic
conditions.
Conflict
of
Interests
The
authors
declare
that
there
is
no
conflict
of
interests
regarding
the
publication
of
this
paper.
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