International Journal of Farming and Allied Sciences
Available online at www.ijfas.com
©2013 IJFAS Journal-2013-2-S2/1337-1342
ISSN 2322-4134 ©2013 IJFAS
Organic Fertilizers Role on Antioxidant Enzymes in
Rice (Oryza sativa L.)
Morteza Siavoshi1* and Shankar L. Laware2
1. Assistant Professor, Department of Agricultural Science, Payame Noor University, I. R. of Iran
2. Associate Professor, Department of Botany, Fergusson College, Pune, India
Corresponding author: Morteza Siavoshi
ABSTRACT: In order to study organic fertilizers role on antioxidant enzymes in rice, an experiment was
carried out in 2010 and 2011, in randomized block design based on 4 replications. Cow manure, poultry
manure, rice straw and husk were used for formulation of organic fertilizers. The treatments of organic
fertilizers CM, PM, CMR, PMR, and CPMR were used alone at 4t/ha in five treatments. CPMR 2t was
used with ½ RDF (N-50, P-25, K-25 kg/ha) at one level and RDF (N=100, P=50, K=50 kg/ha) alone at
one level. The plants without treatments considered as control. Antioxidant enzymes and Grain yield
were significantly increased in all the treatments over control. The maximum grain yield (4776.52 kg/ha)
was noted in plants treated with CPMR 2t +½ RDF. An increase in the grain yield at the abovementioned
treatments was may be due to the increase of antioxidant enzymes which protected plant from the
stresses.
Keywords: Antioxidant enzymes, Organic fertilizers, Rice, Grain yield
INTRODUCTION
Enzymes are vitally important to the existence of life itself. Enzymes are part of the multi-component
defense system and are involved in defense reactions of plants against pathogens and every kind of stress factors.
Peroxidases are involved in several physiological and biochemical processes such as cell growth and expansion
(Lin and Kao, 1999), differentiation and development, auxin catabolism (Mansouri et al., 1999), lignifications
(Sitbon et al., 1999), as well as abiotic and biotic stress responses (Medina et al., 1999). Peroxidases can form a
new cell wall through biosynthesis. In the large family of peroxidases, the process of detoxification of H 2O2 is
particularly important (Ranieri et al., 2000). In the cell wall, POXs are present in soluble forms. The cell wall
stiffening has been attributed mainly to peroxidases whose activity can be detected using the enzymatic assay
mixture (Herbette et al., 2003). Peroxidases (POXs) are the most important part of the multiple plant defense
system and are mostly synthesized in the chloroplasts (Gabara et al., 2003). Catalase (CAT) is an antioxidant
enzyme, which effectively remove the excess H2O2, and give protection to membranes. Superoxide dismutase
enzymes (SODs) act as antioxidants and protect cellular components from being oxidized by reactive oxygen
species. SOD enzyme family maintains normal physiological conditions and deal with stress.
(Ahmed et al., 2010) reported that application of organic manures and biofertilizers increase Catalase and
Peroxidase activities in sorghum. This result was also supported by (Abd El-Ghany, 2007). According to them
these responses may be attributed as an attempt of the plant to overcome the adverse conditions of some
elements required for growth and development of sorghum plants. The increments in these enzymes helped the
plant to destroy H2O2 available in normal or abnormal conditions and maintained the ascorbate pool which in turn
led to elevate the plant tolerance and maintained best growth.
Intl J Farm & Alli Sci. Vol., 2 (S2): 1337-1342, 2013
According to (Trinchera et al., 2008) chemical fertilization is able to supply greater amounts of nitrogen to plants in
a brief period to improve better metabolism without needing for additional activity of the enzymes, however, in case
of plants receiving the organic fertilizers, may face a condition of slowly releasing the available nitrogen. This slow
release of N may be considered as adverse environmental conditions due to nutrient deficiency where the role of
the antioxidant enzymes support plants to become more tolerant against the proposed disturbances in different
plant physiological process. Hence in present investigation it was planned to analyze organic constituents and
assay antioxidant enzymes in rice under different types of organic fertilizers, without or with RDF and compare
these with RDF.
MATERIALS AND METHODS
This investigation was carried out at Baykola Research Center, Neka, Mazandaran, Iran during years 2010 and
2011. The experimental farm is geographically situated at 36°, 60'N latitude and 53°, 13'E longitude at an altitude
of 4 m above mean sea level.
Formulation of organic fertilizers:
Cow manure, poultry manure, rice straw and husk were used for formulation of organic fertilizers. These
materials were mixed in required proportions and composted for about 40 days. After completion of composting,
these were sieved thorough 2 mm mesh and then analysed for their nutrients. The proportions of raw materials
used are given in Table1.
Table 1. Raw materials used in formulation of organic fertilizers.
Sr. No.
1
2
3
4
5
Formulation
(Abbreviated Name)
CM
CMR
PM
PMR
CPMR
Ingredients
Cow Manure (kg)
1900
1500
500
Poultry Manure
(kg)
1900
1500
900
Rice Straw
(kg)
400
400
500
Rice husk
(kg)
100
100
100
100
100
Total
2000
2000
2000
2000
2000
(1) CM: Cow manure+ rice husk,
(2) CMR: Cow manure+ rice straw and husk,
(3) PM: poultry manure+ rice husk, (4) PMR: poultry manure+ rice straw and husk,
(5) CPMR: Cow manure+ poultry manure+ rice straw and husk
Treatments
Organic fertilizers CM, PM, CMR, PMR, and CPMR were used alone at 4t/ha in five treatments. Half dose of
CPMR was used with half dose of RDF (N-50, P-25, K-25 kg/ha) at one level and RDF (N=100, P=50, K=50 kg/ha)
alone at one level. The plants without treatments considered as control. The recommended chemical fertilizer dose
for rice is N-100: P-50: K-50. This combination was termed as RDF and used in one treatment. Based on RDF
amounts the NPK values were calculated from the organic fertilizers analysis and 4 t/ha dose was decided based
on NPK content of CMR, PMR and CPMR and similar volume (i.e. 4 t/ha) of CM and PM organic fertilizers was
st
used for treatment. Nitrogen in the form of urea was used three times during growth season (1 dose at the time of
nd
rd
transplanting, 2 dose at tillering time and 3 dose at the time of flowering). Hand weeding was done after 3 weeks
of transplanting. The pests and diseases were controlled by application of insecticides and tricycasol was used for
rice blast.
Table 2. Details of the field experiment.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
Season
Crop
Variety
Plot size
Crop duration
Date of sowing
Date of transplanting
Date of harvesting
Design
Number of replications
May-August
Oryza sativa L.
Local Tarom
5×2m
90- 95 days
20. 04. 2010-11
24. 05. 2010-11
20-25.08.2010-11
RCBD
4
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Intl J Farm & Alli Sci. Vol., 2 (S2): 1337-1342, 2013
Estimation of antioxidant enzyme activities
Enzymes like peroxidase (POX), Catalase (CAT) and Superoxide dismutase (SOD) were extracted in suitable
phosphate buffer from fully matured and freshly cut flag leaves of control and treated plants. Properly diluted crude
extracts were assayed for aforesaid enzymes by following standard protocols.
Assay of enzyme Peroxidase (E.C. 1.11.1.7)
Fresh and physiologically active flag leaves of fertilizer treated and control plants were collected and cut into
small pieces. Accurately 100 mg of leaf samples were homogenized in pre chilled mortar and pestle, in 5 ml of 0.1
0
M phosphate buffer (pH 7.0). The extract was centrifuged at 4 C in cooling centrifuge at 15000×g for 10 minutes
and the supernatant was used as source of enzymes.
The peroxidase activity was assayed by (Vidyasekharan and Durairaj, 1973) method. The assay mixture of 3
ml contained 1.8 ml of 0.1 M phosphate buffer (pH 7.0), 1 ml freshly prepared 10 mM Guaicol, 0.1 ml enzyme
extract and 0.1 ml of 12.3 mM H2O2. Initial optical density was read at 436 nm and then increase in optical density
was noted at the intervals of 30 seconds on UV-visible spectrophotometer (Shimadzu- 1700). The enzyme activity
-1
was expressed as units g fresh weight.
Catalase (EC. 1.11.1.6)
Fresh and physiologically active flag leaves of fertilizer treated and control plants were collected and cut into
small pieces. Accurately 100 mg of leaf samples were homogenized in pre chilled mortar and pestle, in 5 ml of 0.06
0
M phosphate buffer (pH 7.0). The extract was centrifuged at 4 C in cooling centrifuge at 15000×g for 10 minutes
and the supernatant was used as source of enzymes.
The catalase activity was calculated by using (Maxwell and Bateman ,1967) method. The reaction mixture
contains 2.95 ml of 0.06 M phosphate buffer, 10% (w/v) H2O2 and 0.05 ml enzyme extract. The decrease in
absorbance was measured at 240 nm by using UV-visible spectrophotometer (Shimadzu-1700). The residual H2O2
-1
concentration was calculated using extinction coefficient 0.036 µM ml . The enzyme activity was expressed as
-1
units g fresh weight.
Superoxide dismutase estimation (EC. 1. 15. 1. 3)
Superoxide dismutase (SOD), a metal containing enzyme plays a vital role in scavenging superoxide radical.
The Superoxide dismutase activity was calculated by using (Sadasivam and Manickam ,1996). Crude enzyme
extract 100 µL was mixed with a 3 ml reaction cocktail containing: 50Mm potassium phosphate buffer (pH 7.8), 13
mM methionine, 2µM riboflavin, 0.1 mM EDTA and 75 µM NBT. Final volume of reaction mixture was made equal
by adding distilled water. A blank was set without enzyme and NBT to calibrate the spectrophotometer and
another control was set with NBT but without enzyme as reference control. The reaction tubes were exposed to
400 W bulbs (4×100 W bulbs) for 15 minutes and immediately absorbance was taken at 560 nm. The percent
inhibition was calculated. The 50% inhibition of the reaction between riboflavin and NBT in the presence of
-1
methionine is taken as 1 unit of SOD activity and the enzyme activity is expressed as units g fresh tissue.
Grain yield (kg/ha)
Samples from 1 M² area from each plot were used for Grain yield.
RESULTS AND DISCUSSION
-1
Peroxidase (units g )
The results pertaining to effect of organic fertilizers, combination of chemical fertilizer with organic fertilizers
and recommended dose of NPK on leaf peroxidase content in rice is given in table 3. Among the treatments the
half dose of CPMR+ RDF, RDF and CPMR organic fertilizer show results at par with each other and better than
CM, CMR, PM and PMR in both the years and even in pooled means.
-1
Catalase (units g )
The results pertaining to effect of organic fertilizers, combination of chemical fertilizer with organic fertilizers
and recommended dose of NPK on leaf Catalase content in rice is given in table 3. Among the treatments the half
dose of CPMR+ RDF, CPMR organic fertilizer and PMR show results at par with each other and better than CM,
CMR, PM and RDF alone in both the years and even in pooled means.
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Intl J Farm & Alli Sci. Vol., 2 (S2): 1337-1342, 2013
-1
Super oxide dismutase (units g )
The results pertaining to effect of organic fertilizers, combination of chemical fertilizer with organic fertilizers
and recommended dose of NPK on leaf super oxide dismutase (SOD) activity in rice is given in table 3. Among the
treatments the half dose of CPMR+ RDF, CPMR organic fertilizer and RDF show results at par with each other and
better than CM, CMR, PM and PMR in both the years and even in pooled means.
Table. 3. Effect of different levels organic fertilizer, recommended dose of chemical fertilizer and combination of organic and
-1
-1
chemical fertilizers on rice leaf Peroxidase content (units g ), Catalase content (units g ), Super Oxide Dismutase content
-1
(units g ) and grain yield in rice (O. sativa).
Treatments
(Per hectare
Control
RDF (NPK)
100:50:50 kg
CM
4t
CMR 4t
PM
4t
PMR 4t
CPMR 4t
CPMR 2t +
½ RDF
CD (0.05)
CD (0.01)
Peroxidase
(units g-1)
Pooled PIOC
33.21
0.00
Catalase
(units g-1)
Pooled PIOC
320.37 0.00
Super Oxide Dismutase
(units g-1)
Pooled
PIOC
41.51
0.00
Grain yield
(Kg/ha)
Mean
PIOC
3943.90 0.00
47.30
42.43
390.37
21.85
56.35
35.75
4582.32
16.19
37.68
40.53
41.54
44.08
45.78
13.46
22.03
25.08
32.73
37.85
338.77
347.46
361.19
413.39
420.42
5.74
8.46
12.74
29.04
31.23
46.22
51.96
53.54
55.55
57.18
11.35
25.17
28.98
33.82
37.75
4044.92
4135.21
4377.07
4594.59
4699.31
2.56
4.85
10.98
16.50
19.15
50.86
53.15
447.73
39.75
58.94
41.99
4776.52
21.11
1.30
2.29
-
11.40
20.13
-
1.24
2.19
-
96.36
170.13
_
_
PIOC: Percent increase over control
Grain yield (kg/ha)
The organic fertilizers treatments had significant effect on the grain yield. The results in table 3 clearly indicate
that application of organic fertilizers increased the grain yield significantly in all the treatments over control. Among
the treatments half dose of CPMR+ half dose of RDF, CPMR and PMR show results at par with each other and
significantly better than CM, CMR, PM and RDF alone in both the years and even in pooled means.
Under normal conditions, plants posses scavenging systems that keep reactive oxygen species below
damaging levels (Larson, 1988). When plants are subjected to environmental stresses such as drought, salinity,
heat, chilling and mineral deficiency, a variety of toxic oxygen species (TOS) such as superoxide, hydrogen
peroxide, hydroxyl radicals and singlet oxygen have been noted (He et al., 2011; Gorji et al., 2011). The balance
between production of reactive oxygen species and the quenching activity of the antioxidants is considered
essential to avoid oxidative damage in plants (Negm et al., 2003). To prevent or alleviate injuries from activated
oxygen species (AOS), plants have evolved several mechanisms that include scavenging by natural antioxidants
and enzymatic antioxidants system such as superoxide dismutase, catalase and peroxidases (Scandalios, 1993).
Cooperation among these enzymes is essential for the effective protection from AOS e.g., superoxide dismutases
(SODs) react with superoxide radicals to produce O 2 and H2O2 whereas catalase and/or peroxidase convert H 2O2
to O2 and water (Seebba and Vitagliano, 1998).
Plants face numerous stresses in their life and the oxidative state of plants plays a pivotal role in plant defence
against such stresses. Induction of various antioxidant enzymes and other defensive compounds is a common
phenomenon in plants in response to different biotic and abiotic stresses (Omidi, 2010). This response occurs both
in the plant organs originally attacked (local response) and in non-attacked organs (systemic response) (Metraux et
al., 2002). Plants protect themselves from cytotoxic effects of aforesaid ROS with the help of antioxidant enzymes
such as Peroxidase (POX), Polyphenol Oxidase (PPO), Catalase (CAT) and Superoxide Dismutase (SOD) induced
in plants in response to the stress (He et al., 2011). Induced oxidative response enables the plants to cope with
various kinds of stresses and allows them to be phenotypically plastic. It makes the plant more unpredictable for
stress causing agents due to the variations in defence constituents of the plant (War et al., 2011a, b).
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Intl J Farm & Alli Sci. Vol., 2 (S2): 1337-1342, 2013
Among the ROS, H202 is a relatively stable, partially reduced form of hydrogen, diffuses freely and thus is an
important factor in generation of defence responses in plants (Boka et al., 2007). A close interaction occurs
between perception of H202 in response to biotic and abiotic stresses in plant systems (Syeed et al., 2011; Idrees et
al., 2011). H202 acts through signal transduction pathways, which leads to the expression of defence genes (Idrees
et al., 2011). Accumulation of H202 instigates a cascade of events that trigger physiological and molecular
responses in plants to defend plants against biotic and abiotic stresses (War et al., 2011a, b).
Catalase is universally present oxidoreductase that decomposes H 2O2 to water and molecular oxygen and it is
one of the key enzymes involved in removal of toxic peroxides (Lin and Kao, 1999). (Sabrina et al., 2011) observed
increase in activities of oxidative enzymes like peroxidase, and Catalase due fertilizers rich in phosphate in wheat
plants; they showed positive correlation of antioxidant enzymes activities with levels of phosphate and MDA
production in wheat plant grown under different levels of NPK fertilizers. (Whereas Malusa et al., 2002) studied role
of phosphate on antioxidative system in bean plant (Phaseolus vulgaris). (Anwarul et al., 2003) observed increase
in peroxidase and polyphenol oxidase in jute leaves due application of cow dung alone and in combination with
NPK as well as NPK along with other minerals. (Amal et al .,2010) reported marked increase in peroxidase and
catalase in sorghum plants treated with bio-fertilizers and compared to organic fertilizers. According to them, these
responses may be attributed as an attempt of the plant to overcome the adverse conditions of some elements
required for growth and development of sorghum plants.
Peroxidase (POX) enzyme showed increased activity with different organic fertilizers, similar observations
were made by (Ahmed et al., 2010) in sunflower with Bio-N-P fertilizer and confirmed by (Kamran et al. ,2006)
who detected an increase in POX activity when used bacterial strains of Azotobocter to inoculate the seeds of
Trticum aestivum. Moreover some investigators recorded increments in peroxidase activity as a result of plant
inoculation with pseudomonas (Yi et al., 1987) on tobacco (Balamuralikrishnan et al., 2005) on sorghum bicolor
and (Sardi et al., 2006) on Phaseolus vulgaris.
In present investigation SOD, POX and CAT activities showed marked increase over control plants. The
increments in these enzymes might have helped the plant to destroy H 2O2 available in normal or abnormal
conditions and maintained the ascorbate pool which in turn led to elevate the plant tolerance and maintained best
growth.
(Kumar et al., 2009) studied the effect of organic fertilizers on rice and observed increase in CAT, POX and
SOD and PPO due to organic fertilizer treatments. According to them increase in enzyme activity may be due to
interaction of available rhizobacteria and micorrhizal fungi with roots of rice fertilized with organic fertilizers.
Mineral fertilization are able to supply control plants greater amounts of nitrogen in a brief period to improve
metabolism with no need of additional antioxidant enzymes, whereas in case of plants that receive the organic
fertilizers face a condition of slow release of available nitrogen. That might be considered as adverse
environmental conditions due to nutrient deficiency where the role of the antioxidant enzymes support plants to
become more tolerant against the proposed disturbances in different plant physiological process. Soil microbes
flourish and compete with plants for N and utilize organic matter for their growth; plants have some mechanism to
avoid interaction of such microbes (Trinchera et al., 2008).
CONCLUSION
In present investigation increase in SOD, POX and CAT can be attributed to (i) interaction of VAM fungi with
rice roots (ii) slow release of N responsible for nitrogen imbalance in initial growth stages and more requirement of
N for growth and multiplication of microbes (iii) production of organic acid, lowering alkalinity and increase in
salinity- explained on the basis of soil EC and pH and (iv) phosphate solubilization by microbes and increase in
phosphate increases AO activity. In a general conclusion 2 t/ha CPMR+½RDF, CPMR and PMR organic fertilizers
treatments supported better plant growth and metabolism and antioxidant enzymes, involved in detoxification ,
hence more productivity in rice better grain qualities were observed in these treatments.
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