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Molecular Microbiology Research (Online) 2013, Vol.3 No.3 20-29
ISSN 1027-5595
http://mmr.sophiapublisher.com
27
3.3.6. Assay of super oxide dismutase (SOD) activity
The enzyme extracts were prepared by homogenizing
1 g tissue of the leaf in 2 ml of 0.2 m citrate phosphate
buffer (pH 6.5) at 4
℃
. The homogenate was
centrifuged at 15000 rpm at 4
℃
for 30 min. The
supernatant served as enzyme source and SOD
activity was determined as its ability to inhibit the
photochemical reduction of NBT (Giannospolitis and
Ries, 1977). The assay mixture (3 ml) contained
50mM sodium phosphate buffer (pH 7.8, 13 mM
methionine, 75µM NBT, 2 µM riboflavin, 0.1mM
EDTA 100 µl of the enzyme extract and the riboflavin
was added at the end. Tubes were shaken and placed
under a 40-W fluorescent at 25
℃
. The reaction was
initiated and terminated by turning the light on and off
respectively. The absorbance at 560 nm was measured
against identical non illuminated in parallel to the
sample tubes for blank. Each extract was substracted
from the blank and multiplied by 100 to obtain the
percentage inhibition of NBT-photoreaction. The SOD
activity was expressed in SOD units g
-1
tissue (50 per
cent NBT inhibition=1 unit) (Belid et al., 1993)
3.3.7. Assay of phenol activity
One gram of the leaf samples was ground in a pestle
and mortar in 10 ml of 80 per cent methanol. The
homogenate was centrifuged at 10,000 rpm for 20
minutes. The supernatant was evaporated to dryness
and the residue was dissolved in five ml of distilled
water. From this, 0.2 ml was taken and the volume
was made up to three ml with distilled water. To that
0.25 ml of (1N) Folin-Ciocalteau reagent was added.
After three minutes, one ml of 20 per cent sodium
carbonate was added and mixed thoroughly. Thus the
tubes were placed in boiling water for one minute and
cooled. The absorbance was measured at 725 nm
against a reagent blank. The phenol activity was
expressed as µg of catechol g
-1
of plant tissue (Zieslin
and Ben-Zaken, 1993).
3.3.8. Activity gel electrophoresis
3.3.8.1. Peroxidase (PO)
To study the expression pattern of different isoforms
of peroxidases in different treatments activity gel
electrophoresis was carried out. One gram of physic
nut leaf tissue was homogenized in 2 ml of 0.01 M
potassium phosphate buffer (pH 7.0), centrifuged at
10000 rpm for 15 min at 4
℃
and the supernatant was
used as enzyme source. For native anionic
polyacrylamide gel electrophoresis resolving gel of 8
per cent acrylamide concentration and stacking gel of
4 per cent acrylamide concentration were prepared.
After electrophoresis the gels were incubated in a
solution containing 0.15 per cent benzidine in 6 per
cent NH4Cl for 30 min in darkness. Then a few drops
of 30 per cent H
2
O
2
were added with constant shaking
until the appearance of bands. After staining the gel
was washed with distilled water and photographed
(Sindhu et al., 1984).
3.3.8.2. Polyphenol oxidase (PPO)
One gram of leaf tissues were homogenized in 2 ml of
0.01 M potassium phosphate buffer (pH 7.0) centrifuged
at 10000 rpm for 15 min at 480C and the supernatant
was used as an enzyme source. After native
electrophoresis the gel was equilibrated for 30 min in
0.1 per cent p-phenylene diamine in 0.1 M potassium
phosphate buffer (pH 7.0) followed by 10 mM
catechol in the same buffer. The addition of catechol
was followed by a gentle shaking which resulted in
the appearance of dark brown discrete enzyme bands.
After staining the gel was washed with distilled water
and photographed (Jayaraman et al., 1984).
3.4. Statistical analysis
The data were statistically analyzed using the
IRRISTAT version 92 developed by the International
Rice Research Institute Biometrics unit, the
Philippines (Gomez and Gomez, 1984). Prior to
statistical analysis of variance (ANOVA) the
percentage values of the disease index were arcsine
transformed. Data were subjected to analysis of
variance (ANOVA) at two significant levels (P < 0.05
and P < 0.01) and means were compared by Duncan’s
Multiple Range Test (DMRT).
Reference
Belid F.I.B., Pike S.M. Novacky A.J., and Seghal O.P., 1993, Lipid
peroxidation and super oxide production in cowpea (
Vigna unguiculata
)
leaves infected with tobacco ring spot virus or southern bean mosaic
virus, Physiological and Molecular Plant pathology, 43: 109-119
http://dx.doi.org/10.1006/pmpp.1993.1044
Benhamou N., Belanger R.R., and Paulitz T.C., 1996, Induction of
differential host responses by
Pseudomonas fluorescens
in
Ri-T-DNA-transformed pea roots after challenge with
Fusarium