Download Effect of Systemic Fungicide on Nucleic Acid, Amino Acid and

Hamid and Gul
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Pakhtunkhwa J. Life Sci. Volume 01, Issue 02, 2013, P 70-79
Effect of Systemic Fungicide on Nucleic Acid, Amino Acid and Phenol Content of
Cicer Arietinum
Neelofer Hamid1, Humaira Gul2*
1
2
Department of Botany, University of Karachi.
Department of Botany, Abdul Wali Khan University, Mardan
*Corresponding Author: [email protected]
ABSTRACT
Effect of different concentrations of fungicide Topsin-M was observed on nucleic acid, amino acid and
phenols of Cicer arietinum. Foliar application of fungicide was performed at 15 days old seedling and
at fruiting stage. Nucleic acid (DNA and RNA) decreased while amino acid and phenols increased over
control. Amino acids such as glycine, asparagines, alanine, methionine, phenyl.alanine were found in
both control and treated plants. While cystein, proline, tryptophane and valine were observed in
appreciable amount in treated samples as compare to control after first and second spray. Reduction in
nucleic acid, changes in amino acids and increase in total phenolic contents were significantly
increased at high doses of fungicides after second spray.
Key Words: Cicer Arietinum, Topsin-M, Foliar Application, Amino acids, Nucleic acid, Phenolic
contents.
their source of production up to the areas of
Introduction
Agricultural practices, observed through
growth and deposition of yield material (grains,
the physiological aspect, aim at maximizing the
fruits, etc.). The diseases also deviates the
photosynthetic efficiency of cultures
photoassimilates
and
toward
unproductive
of
fungicidal
growth
and
plant
defense
reactions
and
directing photoassimilates toward the formation
consumptions
of grains and other yield factors, instead of
metabolism,
other
consumptions.
respiration of lesioned tissue, which can be
Cultivated plants are often subject to a variety
considerably greater consumers of resources
of toxic substances leading to important yield
than respiration in unaffected tissues. The
reductions [7]. The infections caused by fungi
attack by phytopathogens thus presents a strong
impair the efficiency of the cultures, reducing
impact on several of the plant’s physiological
the tissue area for photosynthetic activity and
processes, all of which are relevant for
inhibiting the translocation of assimilates, from
production and quality, being that efficiency
unproductive
energy
70
Hamid and Gul
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Pakhtunkhwa J. Life Sci. Volume 01, Issue 02, 2013, P 70-79
functional
nodulation and respiration, photosynthesis,
disturbances in the plant. Therefore, the most
disruption of cell membrane and reduction in
important
total protein and carbohydrate content of the
with
fungicide
prevents
contribution
pyraclostrobin
these
provided
molecule
to
by
the
agriculture
various plant species [18].
is
The present investigation was held on
derived from its wide range fungicidal activity
Chick pea (Cicer arietinum) plant to examine
[2].
Fungicides are those chemicals which
the effect of systemic fungicide Topsin-M, on
control the infection of the pathogens. Topsin-
amino acid, nucleic acid (DNA and RNA) and
M [1, 2 di –{3- methyl carbonyl 2- thioureido}
phenolic contents.
benzene] is a systemic fungicide in which the
active ingredient is thiophanate – methyl. It
Materials and Methods
belongs to the chemical family benzimidazole
The present investigation was conducted
effective against wide range of foliar disease. It
in the field on Cicer arietinum at Department of
is used against the control of diseases affecting
Botany, University of Karachi. Experiment was
legumes,
malvaceous
and
designed as completely randomized block. In
Although
these
the field 12 rows each of 6m2 were prepared for
fungicides are used against diseases but some
study. Three replicates for each treatment
time their use also disturbs the metabolic
including control were used. Seeds were
processes [17] and indiscriminate use of the
surface sterilized with 1% HgCl2 and sown in
systemic fungicides could causes direct and
rows with distance of 30 cm. plants were
indirect problems and causes phyto toxic effect
irrigated twice a week. Different solutions of
on the host [11, 16]. It is presumed that the
Pesticide (Topsin-M) i.e. 1000ppm, 1500ppm
metabolic
and 2000ppm were prepared and sprayed on
solonaceous
cucurbits,
crops
changes
[22].
induced
by
systemic
fungicides effects the host metabolism [5]. It
plant twice.
has been suggested that plants sprayed with
Spray-1=
chemical pesticide suffer from chemical stress
seedlings.
and phenolic compound produced as a result of
Spray-2= At fruiting stage.
stress may act as a protective compounds
Leaf samples were collected after each spray
against pest and diseases [8, 18]. Phyto toxin in
for the analysis of nucleic acid, amino acid and
the
phenols as:
form
responsible
of
for
phenolic
limiting
compounds
cell
are
division,
71
at 15 days after emergence of
Hamid and Gul
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Pakhtunkhwa J. Life Sci. Volume 01, Issue 02, 2013, P 70-79
Extraction and Estimation of Nucleic Acid:
rpm and supernatant was cooled. To the residue
RNA and DNA and DNA were determined by
0.5 ml of 0.5N PCA was added and centrifuged
the method of [14].
for 5 minutes. The supernatant was pooled with
For the extraction, 0.5 gm plant material
the previous one and treated for DNA
was crushed in mortar with 0.5 PCA (chilled)
estimation.
and kept for 30 minutes at room temperature
and then centrifuged at 1000 rpm for 5 minutes.
Reagents:
The supernatant was discarded. The residue
1- Orcinol reagent: 1 gm FeCl3 dissolved
was again centrifuged in 1 ml of 0.2N PCA
and made upto 1 L with extra pure HCl.
(chilled) at 1000 rpm for 5 minutes. The
Just before use dissolve orcinol in FeCl3
supernatant was again discarded and residue
solution) 1 mg/ml of FeCl3 solution).
was again centrifuged in 1 ml of ethanol: ether
2- Diphenylamine Reagent: 27.5 ml of
(1:1) at 1000 rpm for 5 minutes. Supernatant
concentrated H2SO4 made up to 1 L
was again discarded.
with glacial acetic acid. Just before use
add DPA in the acid solution in the ratio
Removal of RNA: 1 ml of 0.3N KOH was
of 10ml/ml.
added to the residue and kept for 1 hour at 37
Estimation:
o
C. After incubation 2 drops of 70% PCA was
RNA: For the estimation of RNA to 1 ml of
added and then 1 ml of 1N PCA (chilled) was
supernatant 4 ml of orcinol reagent was added.
added and allowed to stand for 10 minutes and
The resultant solution was heated in boiling
centrifuged at 1000 rpm for 10 minutes.
water bath for 20 minutes and then cooled. For
Supernatant was pooled for the estimation of
reagent blank 1 ml of 0.5N PCA was taken and
RNA. To the residue 0.5ml of 0.5 N PCA
treated in the same way. Optical density was
(chilled) was added and centrifuged for 3
recorded at 600nm against reagent blank.
minutes at 1000 rpm. Supernatant was pooled
DNA: To 1.5 ml 4.5 ml of DPA reagent was
with previous one for RNA estimation.
added and heated in boiling water bath for 20
minutes. The solution was cooled. Blank was
Removal of DNA: To the remaining residue 1
prepared by taking 1.5 ml of 0.5N PCA treated
ml of 0.5N PCA (chilled) was added and kept
in the same way. Optical density was recorded
in boiling water bath for 10 minutes and the
at 600nm against reagent blank.
cooled. Centrifuged it for 5 minutes at 1000
72
Hamid and Gul
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Pakhtunkhwa J. Life Sci. Volume 01, Issue 02, 2013, P 70-79
Amino Acid Estimation by Harbon (1987): I
Determination
gm of plant material was cut into small pieces
Estimation of phenolic content is based on
and placed in a conical flask. 10 ml of 80%
Folin-Ciocalteu method (1927) as modified by
ethanol was added and boiled on water bath for
[24].
10 minutes. Then the material was kept
Reagent A:
overnight. It was centrifuged twice at 4000
Folin-Ciocalteu reagent 1:9 ratio (1ml Folin-
rpm. Supernatant was taken in another tube and
Ciocalteu reagent + 9 ml of distilled water).
the volume of the supernatant was reduced upto
Reagent B:
1 ml at 40 oC. After that 1 ml of 50% ethanol
Saturated solution of sodium bicarbonate.
of
Phenolic
Compounds:
1 gm sample was plunged into 1N hot
was added to the reduced extract to make the
HCl with the result that the tissues were killed
volume upto 2 ml.
a
immediately. After that tissues were crushed in
chromatogram and dried at room temperature.
a mortar using 10 ml of 1N HCl. The crushed
The chromatographic tank was saturated by the
material was taken in a tube and boiled for
solvent which was used for the estimation of
about half an hour on water bath. Then it was
amino acids. The chromatogram was put into
filtered and the filtered was left out in vacuum
the tank carrying the solvent n-butanol, acetic
over CaCl2 in a desiccator’s room temperature
acid and water at ration of 120:30:50 for 7
until drying.
Plant
material
was
loaded
on
hours (Harbor, 1973). After that chromatogram
0.5 ml of pure ethanol was added to the
was dried at room temperature the spraying was
dried extract. After 5 minutes 0.1 ml of extract
done on the chromatogram with 0.2% ninhydrin
was taken in another tube and 0.5 ml Folin-
in acetone. After spraying the chromatogram
Ciocalteu reagent (1:9) and 5 ml of distilled
was immediately put into heating chamber at 80
water was added to the extract. Tube was
o
C for about 10 minutes. Amino acids appeared
shaken for 3 minutes and then 1 ml of saturated
as coloured spots. All the spots were marked
NaHCO3 solution was added. Tube was shaken
and examined under ultraviolet light. Rf values
again and then incubated about half an hour at
were calculated by taking the distance traveled
26 oC. Optical density was recorded at 660nm
by solute and the distance traveled by solvent.
against reagent blank. For reagent blank, 0.1 ml
For the identification Rf values of unknown
of distilled water was taken as treated in the
aminoacids were compared with the standard
same manner as the phenol solution. Amount of
map of known amino acids.
73
Hamid and Gul
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Pakhtunkhwa J. Life Sci. Volume 01, Issue 02, 2013, P 70-79
total phenolic content (ug/ml) was calculated by
has been suggested that the plants treated with
using standard curve.
agrochmicals such as systemic fungicides suffer
from the chemical stress [18] and these
phenolic content which produced due to stress
Results and Discussion
act
Nucleic acid (DNA and RNA), amino
as
a
protective
compounds
against
acids and phenolic contents are affected by
pathogens and other pests [8].
fungicide Topsin-M in the treated plant at high
Table 1. Effect of different concentrations of
concentration as compare to control. It has been
systemic fungicide Topsin-M on different
reported
amino
that
stress
condition
causes
acids
of
Cicer
abnormalities in the biochemical pathway due
to which toxic phenolic compounds like
flavones are formed [13].
Phenolic
content
are
significantly
(P<0.001) increased in the treated plants with
respect to concentration as compare to control
(Fig. 1) that indicated the stress condition
developed by the use of agrochemicals [21]. It
SPRAY-1
Amino Acids
Control
1000 ppm
1500 ppm
2000 ppm
Cystein
-
-
+
++
Aspartate
-
-
-
-
Threonine
-
-
+
-
Glycine
+
-
+
+
Glutamate
-
-
-
-
Arginine
-
-
-
-
Tyrosine
-
-
-
-
Proline
-
+
+
+
Asparagine
+
+
++
+
Tryptophane
-
++
+
++
++
+
-
+
-
+
++
++
+++
+
++
+
+
+
-
+
Alanine
Methionine
Phenylalanine
Leucine
74
arietinum.
Hamid and Gul
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Valine
Pakhtunkhwa J. Life Sci. Volume 01, Issue 02, 2013, P 70-79
-
+
+
+
Control
1000 ppm
1500 ppm
2000 ppm
Cystein
+
-
-
+++
Aspartate
+
-
++
+
Threonine
+
-
++
+
Glycine
-
-
+
+++
Glutamate
++
-
-
++
Arginine
+
++
++
+
Tyrosine
+
-
-
+
Proline
-
+
++
-
Asparagine
+
-
+
++
Tryptophane
+
+
++
++
Alanine
+
+
++
+++
Methionine
+
-
+
++
Phenylalanine
+
+
-
++
Leucine
+
-
-
++
Valine
-
+
+
-
SPRAY-2
Phenolic content (ug/mg fr. wt)
Amino Acids
3.5
3
2.5
2
1.5
1
0.5
0
Control
1000
1500
2000
Concentrations of fungicide (ppm)
Spray 1
Spray 2
Figure 1. Effect of different concentrations of Topsin-M on phenolic content of Cicer arietinum.
75
Hamid and Gul
Available online at: www.awkum.edu.pk/PJLS
Pakhtunkhwa J. Life Sci. Volume 01, Issue 02, 2013, P 70-79
Nucleic acids (DNA and RNA) contents
only in appreciable amount in treated samples
were significantly (P< 0.001) reduced with the
as compare to control after first and second
higher concentration (2000 ppm) of the Topsin-
spray. The accumulation of amino acids might
M (Fig. 2). It has been suggested that reduction
be the consequent of protein hydrolysis which
observed in nuclear DNA content in maiz stem
might enter the tricarboxylic acid cycle either
tissues after fungicide treatment and this
pyruvate by diamination or by trans amination
reduction of DNA in maize seedling due to
with alpha ketogluteric acid or oxalo acetic acid
fungicide treatment has been observed to be
[19]. Activation of enzymes involved in amino
concentration
[12].
Certain
acid and amides biosynthesis in plants have
hormones
which
been observed in certain cases resulting in the
effected with the pesticides inhibited protein
increase level of various amino acids and
synthesis in various ways. These compounds
amides [1]. Resistance to the simple protectants
enter the nucleus, precipitate or remove the
fungicides
histone shields and uncover some portion of the
compounds is rare and unimportant [3]. In
DNA code for transcription due to that the
general the development of resistant strain is
RNA content was decreased in the higher doses
not a major problem but resistance to the
of fungicides. It has been suggested that 2,4-D
systemic fungicide is much more important.
decreased number of histone suggesting that the
[15] reported the occurrence of strain of the
herbicides influence protein synthesis by acting
cucumber
at the histone DNA level [6]. Topsin-M at
fuliginea
higher concentration induced greater inhibitory
benomyl. Since then the development of
effect in RNA and DNA levels of the Cicer
resistance to benomyl in various pathogens has
arietinum plant.
been reported [4]. There is evidence that
metabolites
dependent
and
natural
such
mildew
which
as
copper
pathogen
was
highly
and
sulphur
Sphaerotheca
resistant
to
Amino acids which were observed in
resistant strains lack vigour and are unable to
treated and control plants neutral amino acids
compete with sensitive ones on absence of
i.e. Glycine, Aspargine, Alanine, methionine
fungicide [10].
and phenyl alanine were found in both treated
and control plants (Table 1). While cystein,
proline, tryptophane and valine were found
76
Hamid and Gul
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Pakhtunkhwa J. Life Sci. Volume 01, Issue 02, 2013, P 70-79
RNA content (ug/mg fr. wt)
7
6
5
4
3
2
1
0
Control
1000
1500
2000
Concentrations of fungicide (ppm)
Spray 1
Spray 2
DNA content (ug/mg fr. wt)
8
7
6
5
4
3
2
1
0
Control
1000
1500
2000
Concentrations of fungicides (ppm)
Spray 1
Spray 2
Figure 2. Effect of different concentrations of Topsin-M on nucleic acid of Cicer arietinum.
The present investigation suggested that
suggested that criteria regarding for the use of
repeated application of systemic fungicides and
systemic fungicides against disease should be
their high doses are injurious for host plant and
followed
results in decrease on nucleic acid and change
concentration, time of application and the stage
in metabolism. The basis of present finding
of host in order to reduce phytotoxicity.
77
strictly
with
regards
to
their
Hamid and Gul
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Pakhtunkhwa J. Life Sci. Volume 01, Issue 02, 2013, P 70-79
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