Basic HTML Version
Molecular Microbiology Research (Online) 2013, Vol.3 No.3 20-29
ISSN 1027-5595
http://mmr.sophiapublisher.com
26
Pseudomonas
fluorescens
and
Bacillus
EPCO16 were
obtained from the culture collection section,
Department of Plant Pathology, Centre for Plant
Protection Studies, Tamil Nadu Agricultural
University, India.
3.2. Glass house experiments
A pot culture experiment was laid out in completely
randomized design to test the efficacy of fungicide
(Mancozeb), bioagents
Pseudomonas
fluorescens,
Bacillus
EPCO16 and plant oils Lemon grass oil and
Copper sulphate. Potting medium (red soil: Cow dung:
manure at 1:1:1 w/w/w) was autoclaved for 1h for two
consecutive days and filled in pots. The culture of
C.
clavata
was inoculated by wound inoculation method
at leaf surface in all the treated plants. The pathogen
alone inoculated served as control. Four replications
were maintained at each treatment and the pots were
arranged in a randomized manner. The leaf blight
disease incidence of
Curvularia
clavata
was recorded
at 30, 60 and 90 days after planting and expressed as
percentage of disease incidence. All the treatments
were applied as foliar spray viz., Mancozeb 75%WP
@ 0.2%; Foliar spray of
Pseudomonas
fluorescens
(Pf1)
Bacillus
EPCO16 and @ 0.2%; Lemongrass oil
@ 0.1%; Copper sulphate @ 0.1% and Healthy
Control.
3.3. Studies on induction of defence mechanism
3.3.1. Sample collection and enzyme extraction
Six key defence enzymes viz., phenylalanine ammonia
lyase (PAL), peroxidase (PO), polyphenol oxidase
(PPO), β-1, 3 glucanase and super oxide dismutase
(SOD) were estimated. Leaf samples were collected
from the following treatments (T1 - Foliar spray of
Lemongrass oil @ 0.1%; T2 - Foliar spray of Copper
sulphate @ 0.1%; T3 - Foliar spray of
Pseudomonas
fluorescens
(Pf1) @ 0.2%; T4 - Foliar spray of
Bacillus
EPCO16 @ 0.2%; T5 – Inoculated control;
T6 – Healthy Control) at bottom portion of the plant at
two day interval up to nine days.
3.3.2 Assay of phenylalanine ammonia lyase (PAL)
activity
PAL activity was determined as the rate of conversion
of L-phenylalanine to trans-cinnamic acid at 290 nm.
Sample containing 0.4 ml of enzyme extract was
incubated with 0.5 ml of 0.1M borate buffer, pH 8.8
and 0.5 ml of 12 mM L-phenylalanine in the same
buffer for 30 min at 30
℃
. Enzyme activity was
expressed in fresh weight basis as nmol
trans-cinnamic acid min
-1
mg
-1
of sample (Dickerson
et al., 1984).
3.3.3. Assay of peroxidase activity
Fresh plant leaves (1 g) were homogenized in 3 ml of
0.1 M sodium phosphate buffer (pH 7.0) in a
prechilled mortar and pestle. The homogenate was
centrifuged at 18000 rpm at 58
℃
for 15 minutes and
supernatant was used within two to four hours which
served as an enzyme source. To a spectrophotometric
sample cuvette, 3 ml of buffer solution, 0.05 ml
guaiacol solution, 0.1 ml enzyme extract and 0. 03 ml
H
2
O
2
solution were added and mixed well. The
absorbance was recorded at 420 nm using
spectrophotometer. The enzyme activity was
expressed as changes in absorbance min
-1
g
-1
of fresh
tissue (Hammerschmidt and Kuc, 1982).
3.3.4. Assay of polyphenol oxidase (PPO) activity
The polyphenol oxidase activity was determined as
per the procedure given by Mayer et al. (1965). The
reaction mixture consisted of 1.5 ml of 0.1 M sodium
phosphate buffer (pH 6.5) and 200 µl of the enzyme
extract. To start the reaction, 200 µl of 0.01 M
Catechol was added and the activity was expressed as
change in absorbance min
-1
g
-1
of protein.
3.3.5. Assay of β-1, 3-glucanase activity
The enzyme extracts was prepared by homogenizing 1
g tissue of the leaf in 5 ml of 0.05 M sodium acetate
buffer (pH 5.0) at 4
℃
. The homogenate was
centrifuged at 20000 rpm at 4
℃
for 10 min and the
supernatant was used as enzyme source. The crude
extract of 62.5 ml was added to 62.5 ml of laminarin
(4 per cent) and then incubated at 40
℃
for 10 min
and the reaction was stopped by adding 375 ml of
dinitrosalicylic acid and heated for 5 min in a boiling
water bath. The resulting solution was diluted with 4.5
ml distilled water and the absorbance was read at 500
nm. The crude extract preparation with laminarin with
zero time incubation served as blank. The activity was
expressed as µg equivalent of glucose min
-1
g
-1
of
fresh tissue (Kavitha et al., 2005).