Harmful effects of Fungicide Treatment on Wheat (Triticum aestivum L.) Seedlings

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International Research Journal of Environment Sciences________________________________ ISSN 2319–1414
Vol. 2(8), 1-5, August (2013)
Int. Res. J. Environment Sci.
Harmful effects of Fungicide Treatment on Wheat (Triticum aestivum L.)
Seedlings
Rangwala Tasneem, Bafna Angurbala and Maheshwari R.S.
Department of Biochemistry, Govt. Holkar Science College, Indore-452017, MP, INDIA
Available online at: www.isca.in
Received 13th June 2013, revised 21st June 2013, accepted 20th July 2013
Abstract
Fungicides, weedicides, insecticides etc. are all pesticides used for protection of plant. A fungicide is a group of pesticide
that controls fungal disease by either inhibiting or killing the fungus that causes the disease. Fungicides are generally used
for increasing yield of crop but they can induce biochemical changes in seedlings which may not be beneficial. Therefore
the study was conducted to know the harmful effect of carbendazim which is a benziimidazole group fungicide on
biochemical parameters of wheat seedlings. The biochemical parameters studied were protein, carbohydrate, total
chlorophyll, chlorophyll a, chlorophyll b and alkaline protease activity. Five different concentration of fungicide were used
viz. 500, 1000, 1500, 2000 and 2500 mg/l. Protein, carbohydrate, total chlorophyll, chlorophyll a, and chlorophyll b were
found to decrease with increase in concentration of fungicide. However alkaline protease activity was increased with
increase in concentration of fungicide. Maximum decrease in protein (– 62.49%), carbohydrate (– 62.5%), total
chlorophyll (-57.44%), chlorophyll a (-47.89%) and chlorophyll b (-63.44%) and maximum increase in alkaline protease
activity (152%) was obtained at highest studied concentration of fungicide i.e. 2500 mg/l. From the present study it was
concluded that presence of fungicide exert harmful effect on the biochemical parameters of wheat seedlings.
Keywords: Fungicide -carbendazim, wheat seedlings, alkaline protease activity,
Introduction
Germination of seed and early seedling growth are considered
the most critical phases for establishment of any species of
plant1. Agriculture research till now has been primarily
concerned how yield can be increased by the use of fertilizers,
pesticides, irrigation etc. Pesticide form an essential part of the
crop production technology that makes it possible for the farmer
to feed the ever growing population2. Fungicides, herbicides,
weedicides, insecticides etc. are all pesticides used in plant
protection. Fungicide is is used to controls fungal disease by
killing the fungus that causes the disease. Various pesticides are
often employed during the maturation of seeds on the parent
plant. Such treatment can influence the subsequent
germinability of the seed crop. The external morphology of
development is often marked by biochemical changes of seed
reserves and enzymes of the internal tissues and is considered as
markers of growth and development. During seed germination
and seedling growth, the seed reserve gets hydrolyzed and a
change in the cellular and organellar constituents such as
proteins, lipids and carbohydrates takes place. However, the rate
of change varies from crop to crop and species to species.
The effect of fungicide on the growth and metabolism of crop
plants whether beneficial or detrimental is the subject of interest
to the scientist since long ago. Hence the present study was
aimed at a detail evaluation of the biochemical behavior during
germination in wheat seedlings under influence of fungicide.
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The fungicide used in the study was carbendazim having trade
name Bavistin because i. This fungicide is used widely for
agriculture, ii. It is broad spectrum fungicide therefore is applied
to control wide range of fungal diseases.
Material and Methods
Seeds of wheat of uniform size were selected and surface
sterilized with 0.1% solution of mercuric chloride for 5min to
avoid any fungal growth, followed by washing for 4-5 times
with distilled water3. The seeds were then placed in Petri dishes
which were lined with Whatman No. 1 filter paper moistened
with distilled water for 24 hours in dark for germination. The
germinated seeds were then grown in presence of five different
concentration of fungicide i.e. 500, 1000, 1500, 2000, 2500 mg/l
for seven days. After seven days various biochemical
parameters were estimated using seedlings and are as follows:
Biochemical parameters: Total protein: Total protein was
estimated using biuret reagent4. The method is based on the
principle that the amide groups of protein form a purple colour
complex with copper ions in the presence of alkaline solution.
The intensity of purple colour complex so produced is measured
at 520 nm colorimetrically.
Total carbohydrate: Total carbohydrate was determined using
anthrone reagent5. Carbohydrate from the seedling was
extracted by boiling it with HCl. Then it was neutralized by
adding sodium bicarbonate. Then the extract was used for
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International Research Journal of Environment Sciences______________________________________________ ISSN 2319–1414
Vol. 2(8), 1-5, August (2013)
Int. Res. J. Environment Sci.
determination of carbohydrate by adding anthrone reagent and
absorbance was measured at 630 nm.
Chlorophyl: Chlorophyll was estimated using 80% acetone and
absorbance of the solution was read at 663 and 645 nm in
colorimeter6. Total chlorophyll, chlorophyll a and chlorophyll b
was calculated from the following formulaChlorophyll (a) in mg/g tissue= 12.7(A663) – 2.69(A645) x
Chlorophyll (b) in mg/g tissue= 22.9(A645) – 4.68(A663) x
Total chlorophyll (mg per g tissue) = 20.2(A645) + 8.02(A663) x
Alkaline protease: The tissue was extracted in phosphate buffer
of molarity 0.05 and pH 8.0. The activity of enzyme was
measured by determining the extent of hydrolysis of caesin as
substrate followed by determination of unaltered caesin by
Lowry's method of protein estimation. The specific activity was
expressed as umoles of tyrosine equivalents liberated/ h/mg
protein7.
Statistical Analysis: Laboratory characteristics of studied
parameters were expressed as mean ± standard deviation (SD).
ANOVA is used to compare these data between treated
seedlings and control seedlings. P values < 0.05 were found to
be significant.
Results and Discussion
Protein content of wheat seedling in control was 124.41 ± 5.83
mg/g. Protein content of wheat seedlings at 500, 1000, 1500,
2000 and 2500 mg/l was found to be 95.83 ± 3.81, 62.5 ± 4.33,
50.83 ± 2.75, 57.5 ± 1.5 and 46.66 ± 2.88 mg/g respectively as
shown in table no.1. The % decrease in protein content at 500,
1000, 1500, 2000 and 2500 mg/l concentration of fungicide was
-22.97%, -49.76%, -51.90%, -53.78% and -62.49% respectively
as compared to control. The highest % decrease in protein
content was – 62.49% which corresponds to 2500 mg/l of
fungicide as shown in table- 2.
As shown in table-1 carbohydrate content of wheat seedling in
control was 14.4 ± 2.92 %mg Carbohydrate content of wheat
seedlings at 500, 1000, 1500, 2000 and 2500 mg/l was found to
be 10.9 ± 0.15, 9.4 ± 0.26, 8.36 ± 0.15, 5.9 ± 0.1 and 5.4 ± 0.2
%mg respectively. The % decrease in carbohydrate content at
500, 1000, 1500, 2000 and 2500 mg/l concentration of fungicide
was -24.3%, -34.72%, -41.94%, -59.02% and -62.5%
respectively as compared to control. The highest % decrease in
carbohydrate content was – 62.5% which corresponds to 2500
mg/l of fungicide.
Total chlorophyll content of wheat seedling in control was 0.47
± 0.01 mg/g as shown in table-1.Total chlorophyll content of
wheat seedlings at 500, 1000, 1500, 2000 and 2500 mg/l was
found to be 0.35 ± 0.008, 0.30 ± 0.012, 0.27 ± 0.002, 0.23 ±
0.002 and 0.20 ± 0.006 mg/ g respectively. The % decrease in
total chlorophyll content at 500, 1000, 1500, 2000 and 2500
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mg/l concentration of fungicide was -25.53%, -36.17%, 42.55%, -51.06% and -57.44% respectively as compared to
control. Thus among the entire studied concentration highest %
decrease (57.44%) in total chlorophyll was observed at 2500
mg/l concentration of fungicide.
Content of chlorophyll a wheat seedlings was found to be
decreased at all the studied concentrations of fungicide as
compared to control. Chlorophyll a content of wheat seedling in
control was 0.19 ± 0.003 mg/g. As shown in table-1 chlorophyll
a content of wheat seedlings at 500, 1000, 1500, 2000 and 2500
mg/l was found to be 0.147 ± 0.006, 0.167 ± 0.03, 0.12 ± 0.002,
0.111 ± 0.005 and 0.099 ± 0.0005 mg/ g respectively. The %
decrease in chlorophyll a content at 500, 1000, 1500, 2000 and
2500 mg/l concentration of fungicide was -22.63%, -12.10%, 36.84%, -41.57% and -47.89% respectively as compared to
control. Thus among the entire studied concentration highest %
decrease in chlorophyll a content i.e. 47.89% was observed at
2500 mg/l concentration of fungicide.
Chlorophyll b content of wheat seedlings was found to be
decreased at all the studied concentrations of fungicide as
compared to control as shown in table-1. Chlorophyll b content
of wheat seedling in control was 0.279 ± 0.008 mg/g.
Chlorophyll b content of wheat seedlings at 500, 1000, 1500,
2000 and 2500 mg/l was found to be 0.205 ± 0.004, 0.179 ±
0.003, 0.155 ± 0.002, 0.12 ± 0.009 and 0.102 ± 0.004 mg/g
respectively as shown in table-1. The % decrease in chlorophyll
b content at 500, 1000, 1500, 2000 and 2500 mg/l concentration
of fungicide was -26.52%, -35.84%, -44.44%, -56.98% and 63.44% respectively as compared to control. Thus among the
entire studied concentration highest % decrease in content of
chlorophyll b (-63.44%) was observed at 2500 mg/l
concentration of fungicide.
As shown in table- 1 alkaline protease activity of wheat
seedlings at 500, 1000, 1500, 2000 and 2500 mg/l was found to
be 0.250±0.042, 0.267±0.027, 0.274±0.019, 0.355±0.011 and
0.378±0.022 µmoles/h/mg protein respectively. The % increase
in alkaline protease activity at 500, 1000, 1500, 2000 and 2500
mg/l concentration of fungicide was 66.66%, 78%, 82.66%,
136.66% and 152% respectively as compared to control. The
highest % increase in alkaline protease activity was 152% which
corresponds to 2500 mg/l of fungicide.
As seen in the results fungicide caused decrease in protein
content which is significant for wheat seedlings. An osmotic
shock caused by systemic fungicides results in the release of
protein and loss of membrane transport ability in the cells of
leaves. It has been suggested that the toxic substances which
were produced by the use of systemic fungicides inhibits protein
synthesis by binding to the larger ribosomal subunits and thus
induces change in the enzymatic system 8. The results of our
study were in parallel with the work of some workers9. As the
concentration of fungicide gets increased, amount of protein get
gradually decreased10. The results of present study were
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International Research Journal of Environment Sciences______________________________________________ ISSN 2319–1414
Vol. 2(8), 1-5, August (2013)
Int. Res. J. Environment Sci.
contradicted by work of some scientists 11,12. The difference in
result may be due to different species used.
The results of present study demonstrated that carbohydrate
contents decreased with increase in concentration of fungicide.
Reduction in net CO2 assimilation occurred due to changes in
stomatal conductance and intercellular CO2 concentration were
reported in Malus domestica and Cucumis sativus after
application of fungicide13. Use of systemic fungicides caused a
significant decrease in protein and carbohydrate content as
compared to the control14.
Under the influence of fungicide, wheat seedlings exhibited
decrease in the levels of photosynthetic pigments, namely
Chlorophyll a, b and total chlorophyll with increase in
concentration of fungicide, Pigment biosynthesis was found to
be inhibited by the use of systemic fungicide like benomyl. This
type of fungicide causes a considerable reduction in the
chlorophyll a, chlorophyll b, carotenoids, and the total pigments
content in the plants of Helianthus annuus15. The treatment of
Vitis vinifera and Nicotiana tabacum with fungicides also
decreases the chlorophyll and carotenoid content16,17. It was
reported that chlorosis in leaf occurred after use of benomyl
fungicide on Impatiens walleriana, Cucumis sativus, Celosia
plumose, Petunia hybrid, and Lycopersicon esculentum18. Total
chlorophyll content declined consistently with fungicides dose
rates and application days11.
Alkaline protease activity which is a marker for degradation of
protein was get increased with increase in concentration of
fungicide. The work of some researchers was not parallal to
results of present study and showed decrease in protease activity
by endosulfan treatment10. At higher concentration of fungicide
the rapid growth of seedlings may be due increased availability
of amino acids for protein synthesis. These amino acids may
result from increased activity of alkaline protease enzyme. Thus,
future investigation should be done in order to produce data
which may be either in favour or against of the above fact.
Conclusion
Although the use of fungicide is considered good for increasing
yield of crops but it adversely affects the biochemical
parameters like protein, carbohydrate and chlorophyll.
Therefore some other means should be used for increasing
productivity or some fungicide should be designed which does
not harm the crop adversely.
Table-1
Showing effect of fungicide on various biochemical parameters of wheat seedlings
Total chlorophyll
Conc. of
Total
Alkaline protease
Total protein
(mg/g)
Chloro a (mg/g)
Chloro b (mg/g)
fungicide
carbohydrate
(µmoles/h/mgprotein)
(mg/g)
in mg/l
(% mg)
0
124.41±5.83
14.4±2.92
0.47±0.01
0.19±0.003
0.279±0.008
0.15±0.06
500
95.83±3.81**
10.9±0.15*
0.35±0.008**
0.147±0.006**
0.205±0.004**
0.250±0.042*
1000
62.5±4.33**
9.4±0.26*
0.30±0.012**
0.167±0.03*
0.179±0.003**
0.267±0.027*
1500
59.83±2.75**
8.36±0.15**
0.27±0.002**
0.12±0.002**
0.155±0.002**
0.274±0.019**
2000
57.5±1.5**
5.9±0.1**
0.23±0.002**
0.111±0.005**
0.12±0.009**
0.355±0.011**
2500
46.66±2.88**
5.4±0.2**
0.20±0.006**
0.099±0.0005**
0.102±0.004**
0.378±0.022**
*=Values are Significant (p<0.05),**= Values are very significant (p <0.01) and ns= not significant (p>0.05).
Table-2
Showing % increase/decrease in studied biochemical parameters of wheat seedlings under influence of fungicide
Conc. of
Total
Total chlorophyll
Alkaline protease
fungicide in Total protein
Chloro a
Chloro b
carbohydrate
activity
mg/l
500
-22.97%
-24.3%
-25.53%
-22.63%
-26.52%
66.66%
1000
-49.76%
-34.72%
-36.17%
-12.10%
-35.84%
78%
1500
-51.90%
-41.94%
-42.55%
-36.84%
-44.44%
82.66%
2000
-53.78%
-59.02%
-51.06%
-41.57%
-56.98%
136.66%
2500
-62.49%
-62.5%
-57.44%
-47.89%
-63.44%
152%
International Science Congress Association
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International Research Journal of Environment Sciences______________________________________________ ISSN 2319–1414
Vol. 2(8), 1-5, August (2013)
Int. Res. J. Environment Sci.
mg%
Effect of fungicide on carbohydrate
16
14
12
10
8
6
4
2
0
carbohydrate
0
500
1000
1500
2000
2500
conc. of fungicide
Figure-1
Showing effect of fungicide on carbohydrate content of wheat seedlings
Effect of fungicide on protein content
140
120
mg/g
100
80
protein
60
40
20
0
0
500
1000
1500
2000
2500
conc. of fungicide
Figure-2
Showing effect of fungicide on protein content of wheat seedlings
Effect of fungicide on chlorophyll content
0.5
mg/g
0.4
Total chlorophyll
0.3
Chlorophyll a
0.2
Chlorophyll b
0.1
0
0
500
1000
1500
2000
2500
conc of fungicide
Figure-3
Showing effect of fungicide on total chlorophyll, chlorophyll a and chlorophyll b of wheat seedlings
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International Research Journal of Environment Sciences______________________________________________ ISSN 2319–1414
Vol. 2(8), 1-5, August (2013)
Int. Res. J. Environment Sci.
µmoles/h/mgprotein
Effect of fungicide on Alkaline protease activity
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
Alkaline protease
0
500
1000
1500
2000
2500
conc. of fungicide
Figure-4
Showing effect of fungicide on alkaline protease activity of wheat seedlings
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