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3 MATERIALS AND METHODS
MATERIALS AND METHODS
In the present study experiments have been conducted to find out the efficacy of
four microbial biocontrol agents to control the C. capsici infecting Curcuma longa L.
1. Isolation of the Pathogen
The infected leaves of Curcuma longa were collected from turmeric growing
fields. They were surface sterilized with 0.2% mercuric chloride solution for one
minute, washed thoroughly in sterile distilled Nater four times and the infected portions
of the leaves were cut into small bits and placed on PDA (potato dextrose agar) medium
with 50 ppm of streptomycin and incubated in BOD incubator at 28 i 0.2'C.
1.1. Identification
The pathogens were maintained in BOD incubator at optimum temperature
(28 F 0.2'C). Cultural characters and morphology of conidia and setae were recorded.
36
ldentification of the fungi was done on the basis of reports of Ramakrishnan (1954),
Sundararaman ( I 925).
1.2. Maintenance of the Pathogen
The pathogens Colislorr~chum cups~ci (Syd.) Butler & Bisby were purified by
single-conidia isolatron method. The purrfied culture was stored in the slants of Potato
Dextrose Agar. The pathogens were inoculated in the turmeric leaves at least once in
six months to confirm their virulence.
2. Biocontrol Agents
Pure culture and talc based formulations of Trichodermo viride Pers. Fr. (T v.),
T hurzianum Rifai. (T.h.), Gliociadium virens Miller, Giddens, Foster (G v . ) and
Preudomonus ,fluorescens ( P J ) were obtained from MIS. Rom Vijay Biootectl Pvt.
Ltd., Pondicherry. The chemical fungicide bavistin (0.1%) was used for comparison.
2.1. Quality of the Formulations
2.2. Preparation of Biocontrol Agents
The culture filtrates from the 7Ih day old culture of the above mentioned
biocontrol agents were prepared in different concentrations using sterile distilled
water as Trichoderma viride (0.5, 1 .O, 1.5, 2.0, 2.5 and 3.0%), T harzianum (0.5, 1 .O,
1.5, 2.0, 2.5 and 3.0%), Gliocladium virens (0.5, 1.0, 1.5, 2.0, 2.5 and 3.0%) and
Pseudomonasfluorescens (0.5, 1.0, 1.5, 2.0, 2.5 and 3.0%), filtered through double-
layered cheese cloth and used for in-vitro experiments.
3. In Vitro Study
3.1. Conidial Germination Study
The culture filtrates of the four biocontrol agents were prepared in different
concentrations using sterile distilled water (T viride = 0.5, 1.0, 1.5, 2.0, 2.5 and
3.0%; T harzianum = 0.5, 1.0, I .5, 2.0, 2.5 and 3.0%; Gliocladium virens = 0.5,
1.0, 1.5. 2.0, 2.5 and 3.0% and P~eudomonasfluorescens = 0.5, 1 .O, 1.5, 2.0, 2.5
and 3.0%) and filtered through double-layered cheese cloth. The chemical
fungicide bavistin (0.1%) was used for comparison. Conidial suspension of
C capszci was added to the different concentrations of the biocontrol agents and
bavistin, so as to make the final count adjusted to 8000-12000 conidialml with
the help of haemocytometer. Conidial germination studies were carried out in
cavity slides. Triplicate slides were maintained for each concentration. For
control, conidial suspension was added to the sterile distilled water. The slides
were incubated in moist chamber at 30°C and conidial germination was observed
after 24 h. The percentage of inhibition over control was calculated by the
formula of Vincent (1927).
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C-T I=-x100 C
Where, I = Inhibition over control
C = % o f germination in control
T = % of germination in treated
3.2. Dual Culture Technique
The mycoparasitic action of the antagonistic organisms (T viride, T harzianum,
G virens and P. .fluorescens) was evaluated against C, capsici in laboratory by the
dual culture technique. Discs (9 mm) of the fungal antagonists as well as the pathogen
were cut with the help of sterilized cork borer from the edge of 7 days old culture and
then placed apart on solidified PDA medium. For the P Juorescens-treatments the
PDA plates were first inoculated with the pathogen disc on the first day, followed by
the streaking of the prepared cultured filtrate of the P, fluorescens at one end of the
petriplate on second day. Triplicates were maintained for each treatment. The plates
were incubated (26 2 2'C) for 7 days. lnhibition of mycelial growth of C. cups~c~ by
each antagonist mas recorded on the basis of radial growth in dual culture and
compared with that of the control (having only C capsici disc) plate.
3.3. Poisoned Plate Technique
The radial growth of the mycelium of C capsici was measured by poisoned plate
technique. After the sterilization of petriplates (9 cm), PDA medium, cork borers and
other glass wares in an autoclave at 121 .SPC for 15 min with 15 lbisq inch pressure, the
prepared culture filtrates of the biocontrol agents (7-day old culture) in different
concentrations were added through a sietz filter to the warm PDA medium separately.
39
The plates were inoculated by placing 9 mm discs cut from the growing tip of 7 days old
culture plates of C capsici. All this was done under the laminar flow chamber. PDA
plates without any antifungal agent served as conWol. The control and treated plates were
maintained in triplicates. The inoculated plates were sealed with parafilm and incubated
in BOD incubator at 28 f 0.2'C. The radial growth of the pathogen was measured in cm
along the radial line of the mycelial growth in the pertiplates after 7Lh day of treatment.
3.4. Determination of Optimum Inhibitory Concentration
The optimum inhibitory concenuation (01C) of different biocontrol agents was
selected based on the results of conidial germination and mycelial growh parameter.
The OIC (minimum concentration of the antagonistic organisms at which maximum
~nhibition in the pathogen growth was noted) of different biocontrol agents were
selected and used for further experiments.
3.5. Cell Wall Degrading Enzyme Assays
3.5.1. Extraction Procedure
For pectinoiytic enzyme production the pathogens were grown in Czapek's
broth, supplemented with pectin as carbon source replacing sucrose. Similarly for
cellulolytic enzymes microcrystalline cellulose and carboxy methyl cellulose were
used.
To 50 ml sterilized Czapek's liquid media in a 250 ml Erlenmeyer conical
flask, the culture filtrate of T viride, T harzianum, G virens and P, fluorescens
in their OIC were amended to the media separately. Similarly Bavistin was added
to the broth at the concentration of 0.1%. The two discs of 9 mm were cut with
the help o f a cork borer from the growing tip of the 7 days old culture of
C capsici respectively. They were inoculated in each flask and iricubated in the
BOD incubator at 28 i: O.Z°C for 7 days. The control and treated flasks were all
maintained in triplicates. After incubation, the fungal mat and the liquid media
were separated bq double layered Whatman No.1 filter paper placed in Buchner
funnel under suction by vacuum pump. The filtrates were further centrifuged in a
high speed, cooling centrifuge at 5000 rpm for 10 min and the supernatant was
used as the enzyme source.
3.5.2. Estimation of Pectinolytic Enzymes
I . Poly methyl Esterase (PME)
End product estimation
2 Polymethyl Galacturonase (Pectin Depolyme~ase) (PG)
\'iscosity assay
End product estimation
3. Assay of Polygalacturonate trans eliminase (PTE)
Viscosity assay
End product estimation
3.5.2.1. Polymethyl Esterase (EC 3.1.1.11)
The enzyme hydrolyses pectin to methanol and pectic acid. Increase in free
carboxyl groups was monitored in a Control Dynamics pH meter. The PME was
assayed by the titration method of Muse er a1 (1972) with modification.
4 1
Reagent
1.5 g of pectin in 100 ml of 0.2 M NaCl
0.02 N NaOH
Substrate Preparation: 1.5 g of pectin dissolved in 100 mi of 0.2 M NaCl was
blended with the help of the polytron homogenizer, then passed through two layers of
cheese cloth and pH was adjusted to 7.
Method
To 3 ml of enzyme, 10 mi of 1.5% pectin substrate was added and pH of this
reaction mixture was immediately adjusted to 7 . After 24 h of incubation at 28 +_
0.2"C, pH of the reaction mixture was measured in control dynamics pH meter and
the solution was titrated back to pH 7 with 0.02 N NaOH. Control was maintained
with boiled enzyme as enzyme source. The activity was expressed as specific activity
units (SAU). One unit = pml of 0.02 N NaOH required to maintain pH 7/h.
3.5.2.2. Polymethyl Galacturonase Activity (PMG) (EC. 3.2.1.6.7)
The activity of the Endo-PMG was assayed by measuring the reduction in the
viscosity of the substrate caused by the enzyme. The activity of exo-PMG was
assayed by measuring the mono galacturonic units and the activity was expressed as
SAU. (Mahadevan and Sridhar, 1986).
Reagent
O Acetic acid acetate buffer pH 5.2
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Substrate preparation: Ig of pectin was dissolved in I00 ml of acetate buffer, pH
5.2, heated to S&60°C in a water bath and mixed with the help of a polytron
homogeniser (blender) and then passed through the two layered cheese-cloth. The pH
was adjusted to 5.2 using 1N HCI or IN NaOH. Few drops of toluene was added to
the substrate and stored at 4OC.
Viscosity Assay
To 4 ml of the substrate, I ml of the buffer and 2 ml of the enzyme was pipetted
out into the Ostwald Viscometer-150. Suction was applied through the large arm of
the viscometer to mix the contents and the suction was also applied to the small arm
and to determine the viscosity of the mixture (i.e. zero time). The efflux time of the
reaction mixture at ex'ery 30 min intervals for 3 h was measured and the percentage
loss in viscosity was calculated by the formula.
Where V = percent loss in viscosity
To = flow time of reaction mixture at 0 minute
.ll = f l o ~ time of reaction mixture at a particular time interval
T, = flow time of distilled water
3.5.2.2. I Assay of Exo-PMG
The activity was exo-PMG was assayed by measuring the monomeric
galacturonic acids released by the enzyme by catalysing the pectin degradation. The
results were expressed as specific activity units.
43
Reagents
9 Dinitrosalicylate reagent: l g of 3,5 Dinitrosalicyiate, 30 g of sodium
potassium tartarate and 1.6 g of sodium hydroxide were dissolved in 80 ml
df distilled water and made up to 100 ml.
Sodium aceiate-acetic acid buffer. pH 5.2.
-3 Standard maltose: lnig/mi solution.
Method
From the three hour incubated reaction mixture, 2.0 ml aliquots were pipetted
out. To this 2 mi of DNS reagent was added and heated in boiling water bath for 10
minutes. Then cooled and diluted with 10 ml of distilled water. The orange red colour
was read at 575 nm. Control was maintained with boiled enzyme reaction mixture.
The enzyme activity was expressed as specific activity units. One unit represents yg
of maltose releasedh.
3.5.2.3. Pectin Trans Eliminase (PTE) (EC. 4.2.2.10)
The enzyme PTE cleaves pectin either randomly (Endo) or terminally (Exo),
thereby reducing viscosity of substrate and produces TBA reacting substances. Endo-
PTE activity was determined by measuring ;he loss in viscosity of reaction mixture and
Exo-PTE by determining the production of TBA reacting substances (Mahadevan and
Sridhar, 1986).
Reagent
Boric acid - borax buffer, pH 8.7
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Substrate preparation: 1% of Pectin was prepared in boric acid-borax buffer. The
mixture was kept at 5 M O 0 C in the water bath and then blended with the help of the
polytron homogeniser. It was then passed through two layered cheese cloth and pH
was adjusted to 8.7.
Viscosity Assay
Viscosity loss was determined with the Ostwald Viscometer I50 at intervals of
30 minutes starting from 0 to 180 min after preparing the reaction mixture.
To 4 ml of the substrate, I mi of the buffer and 2 ml of the enzyme was
added and were pipetted into the Ostwald Viscometer 150. The efflux time of the
mixture was measured at every 30 rnin interval for 3 h and the reduction in
viscosity was expressed as percentage loss in viscosity and calculated by the
formula as given in Endo-PMG.
3.5.2.3.1. Estimation of TEA Reacting Substances
Reagent
3 ml of the reaction mixture incubated for 3 h was pipetted out into a 25 ml test
tube, 10 ml of 0.01 M TBA and 5 ml of 0.5N HCI was added and placed in a boiling
water bath for 60 min. This was cooled under mnning tap water and the volume of the
45
solution was adjusted to 18 ml with distilled water. The absorbance of the supernatant
was measured between 480 and 580 nm. The maximum absorbance of the solution
was observed at 547 nm. Enzyme-substrate mixture drawn at zero time incubation and
boiled enzyme was used as blank. The activity was expressed in specific activity
units. One unit represents changes in the absorbance of 0.001ih.
3.5.3. Estimation of Cellulolytic Enzymes
C capsici produces 1,4-P-Exo-glucanase and 4-P-Endo-glucanase when grown
in Czapek's broth.
3.5.3.1. Measurement of 1,CpExo-Glucanase (C3 (EC. 3.2.1.9.1)
Tbe activity of C, produced by C. cap sic^ was assayed by measuring the
reducing sugars released from microcrystalline cellulose and the activity was
expressed in SAUs. Exo-P-1,4-glucanase activity was measured by estimating the
reducing sugars released by the breakdow of avicel with anthrone reagent
(Mahadevan and Sridhar, 1986).
Reagents
Sodium acetate-acetic acid buffer, pH 5.0
1% Avicel (Microcrystalline cellulose) suspended in buffer.
Q Orcinol reagent: l g of orcinol dissolved in 50 ml of distilled water.
Gradually 20 ml of 67% H2S04 was added on ice. The volume was raised to
100 ml with distilled water.
46
Anthrone reagent: 200 mg of anthrone was dissolved in 100 ml of cold
concentrated H2SOa
Method
To 1 ml of enzyme source, I ml of buffer and 0.5 ml of substrate were
added in a test tube and incubated at room temperature for 2 h. The reaction
mixture was mixed well with vortex mixer at regular interval of 30 minutes. At
the end of the reaction the volume of the reaction mixture was adjusted to 5 ml
with distilled water. The tubes were centrifuged for 15 min at 2000 g to deposit
the residual avicel cellulose. Soluble sugar in the supernatant was measured
with the orcinol reagent. Two ml of the above supernatant, 3 ml of orcinol
reagent was taken in the test tubes and 10 ml of anthrone reagent was added on
ice. The tubes were mixed well with the help of vortex mixture and heated in a
water bath at 80°C exactly for 20 minutes and immediately cooled under
running tap water. The colour developed was read at 485 nm in Systronics
Spectrophotometer. A blank was prepared with 2% H2S04 instead of orcinol.
Control was maintained with boiled enzyme reaction mixture and w ~ t h zero time
reaction mixture.
3.5.3.2. Measurement of 1,rl-PEndo-Glucanase (CJ (EC. 3.2.1.4)
C, cleaves carboxyl methylcellulose randomly (endo-C,) and terminally (exo-
C,). The activity of endo-C, was assayed by the viscosity loss caused by enzyme in
the substrate CMC.
47
Endo-P-l,4-glucanase (C , ) activity was determined by measuring the viscosity
loss in reaction mixture (Mahadevan and Sridhar, 1986) and by estimating the
reducing sugars released by the enzyme sources in the same reaction mixture (Wang
e l a1 , 1997).
Reagent
Sodium acetate-acetic acid buffer, pH 5.2
Substrate preparation: 0.5 g of CMC (carboxyl methyl cellulose) was dissolved
in 100 ml of sodium acetate-acetic acid buffer with pH 5.2 and kept in water bath at
50-60°C for 5-10 min. then the mixture is blended with the help of polytron
homogeniser. The substrate was filtered in two layered cheese cloth and this was
stored at 4'C with a layer of toluene.
Viscosity Measurement
Ostwald viscometer 150 size was used to determine the viscosity loss of
cellulose substrate.
4 ml of carboxyl methyl cellulose, I ml of the buffer and 2 rnl of enzyme was
pipetted out into the viscometer. The contents were mixed by drawing air gently
through the large arm of the viscometer. Suction was applied to the small aim and the
efflux time of the mixture was determined at every 30 min interval for 3 h incubation.
The percentage loss in viscosity was calculated by employing the formula of the
viscosity assay of Endo-PMG.
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3.5.3.2.1. Assay of Exo Endo-f5f,CGlucanase (CJ
Exo activity of endo-P-1,4-glucanase activity was measured by the estimation of
reducing sugar released by the breakdown of CMC with dinitro salicyclic acid reagent
as in the case of Exo-PMG.
3.5.3.3. Estimation of Cellobiase
The amount of reducing sugars released from cellobiose by cellobiase is used to
assay the enzyme.
Reagent
Q Sodium acetate-acetic acid buffer, pH 5.8
0 : 5 mM cellobiose
Method
1.5 ml of the buffer, 2.5 ml of 5 mM cellobiose and 1 ml of the enzyme was
taken in a test tube and incubated at 30°C for 2 h. The reaction was tenninated by
placing the test tube in a boiling water bath for 10 min. 'The amount of glucose
liberated by the enzyme using DNS (dinitro salicyclic acid) reagent was measured at
575 nm in Systronic Spectrophotorneter. Glucose was used as standard.
4. In vivo Study
4.1. Field Location and Layout
A field experiment was conducted in sandy loamy soil adjacent to
Pondicherry University campus. The plot size was 4 x 5 rn2 and a standard spacing
49
of 30 x 20 cm was adopted for turmeric plantation. Triplicate plots were
maintained for each treatment and they were arranged in randomized block design.
Cultural practice and application of fertilizers was done following the
recommended package of practices (Anonymous, 1991). No other pesticide or
insecticide sprays were given other than the treatments. The healthy plots were
placed far away from the treatment plots in order to prevent secondary (air borne)
infection by the pathogen.
4.2. Experimental Design
The mother rhizomes of mutant CO-1 variety were soaked in 01C of the
biocontrol agentsibavistin (0.1%) separately for 24 h before plantation. 90-day-
old turmeric plants were used for inoculation. The plants were sprayed with
C, capsici culture spray containing 7-12 x lo4 sporeslml. The young leaves
were injured with the help of surgical needles, and spraying was done on the
leaves at early morning. Control plants were sprayed with the same volume of - sterile distilled water. All the plants were immediately covered with polythene
bags sprinkled with sterile water on the inner side to maintain high humidity and
kept undisturbed for 24 h. The 1'' biocontrol agentslbavistin spray was given
after 24 h (Table 1). The leaves were collected on 1 0 ' ~ day after I s ' spray
of biocontrol agentibavistin for biochemical estimations and enzyme assay.
The second biocontrol agentibavistin spray was given 15 days after the 1"
biocontrol agentibavistin spray. Then the leaves collected on 1 0 ' ~ day after 2"d
spray was used for analysis. Periodical observations on disease development
were done.
Seven days after the 1" biocontrol agent!bavistin spray, the leaves
(treatments and infected plots) were observed for the disease development. The
pathogen were re-isolated from the infected area of the inoculated leaves and
compared with the original isolate
101 - l ( l 0 DAT)
, 106 - 20d -
4.3. Disease Intensity Study
1 116
Twenty five plants in each plot per treatment were selected at random for
recording the data on leaf spot incidence. The intensity of leaf spot disease infection
was categorised into five groups based on the number of disease spots per leaf namely
-
(iij Light I 1 spot
- / I1 (10 DAT) 1
(i) Nil
/ (iii) Medium 1 2-5 mots. I
Free of disease spots.
(iv) Heavy / 5-10 spots.
(v) Very heavy I more than 10 spots
The disease intensity was given numerical for statistical analysis (nil = 0; light =
1; medium = 2; heavy = 3 and very heavy = 4).
51 002660
4.4. Electrolytic Leakage
For in-vtvo study one g of leaf tissue from each treatment was cut into discs of 9
mm diameter and washed in double distilled water. These discs were tied in a cheese
cloth and dipped in 40 ml of double distilled water. The conduct:vity of bathing
solution was recorded on control-dynamics conductivity bridge after 5 h. Results were
expressed in milli Siemens (mS) per cm2 of bathing solution.
4.5. Photosynthetic Pigments
4.5.1. Estimation of Total Chlorophyll Conten t
The total chlorophyll content of the leaves was estimated according to the
method of Moran and Porath (1980) using the formulae suggested by lnskeep and
Bloom (1985). Fresh leaf discs of about 50 mg were cut and placed in a test tube
containing 10 ml of' DMF and stored for 24 h at 4°C. The coloured supernatant was
used for chlorophyll and carotenoid estimation. By reading the absorbance at 647 nm
and 666 nm in the spectrophotometer with DMF as blank, the total chlorophyll
content was calcuiuted using the following formulae.
Total chlorophyll (mg g-l fw) = (17'9 + (8'08 V 1OOOxwxa
Where
A -Absorbance at specific wavelength (nm)
w - Fresh weight of the sample (mg)
V -Volume of the sample (ml)
a - Length of the light path in the cell (1 cm)
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4.5.2. Estimation of Carotenoid Content
The carotenoid content was determined using the method of Ikan (1969).
Absorbance values of the leaf extracts were determined at 480,647 and 666 nm.
Corrected 0.D = f (0.1 14 X A666) - (0.638 X AGd7)
1 1 Total carotenoids (mg g.' fw) = Corrected 0.D.x - x - 100 w
4.6. Extractions and Assay of Photosynthetic Enzymes
4.6.1. Extraction Procedure
All extractions were performed at 4'C. The leaves (10 g) were
homogenized with 50 volumes of 100 mM Tris-HCI (pH 7.5) containing 5 mM
DTT, 10 mM MgC12, 1 mM EDTA, 5 mM magnesium acetate and 1.5% PVP-40.
The homogenate was squeezed through four layers of cheese cloth and
centrifuged at 10,000 g for 10 min. The solution was tiltered off to remove the
cellulose and washed thrice with extraction medium. The protein was
precipitated with 75 % (wiv) ammonium sulphate and spun at 30,000 g for
30 min and the precipitate was dissolved in 50 nlM Tris-HC1 buffer (pH
7.8) containing 1 mM DTT and 2 mM EDTA. The preparation was applied
to a column of Sephadex G-25, equilibrated with 10 mM Tris-HCI (pH 8.0)
which contained 1 mM DTT, 10 mM NaHCO,, 20 rnM MgC12 and 0.2 mM
NADPH. The elutes were collected at room temperature. Protein content was
53
estimated according to the method of Furlong er 01. (1973) with BSA as
standard.
4.6.1.1. RuBP Carboxylase
The extraction of the enzyme was performed at 4'C. The leaf blades (10
g) were homogenized with 50 ml of 100 mM Tris HCI buffer (pH 7.8)
containing 5 mM DTT, 10 mM MgC12, I mM EDTA, 5 m M magnesium acetate
and 1.5 % PVP-40. The homogenate was squeezed through 4 layers of cheese
cloth and then centrifuged at 10,000 g for 10 min. The solution was filtered
off to remove the cellulose and washed thrice with the extraction medium.
The protein was precipitated with 75% (wlv) ammonium sulphate and
spun at 30,000 g for 30 min and the precipitate was dissolved in 50 mM
Tris-HC1 buffer (pH 8.0) which contained 1 mM DTT, 10 mM NaHC03, 20
mM MgC12 and 0.2 mM NADPH. The elutes were collected at room
temperature.
RuBP Carboxylase activities were assayed at 30°C by the incorporation
of I4c02 into acid stable products (Lorimer el a!., 1977). The radioactivity
was measured in liquid scintillation counter. The activity was expressed in
mol mg-' h-'.
4.6.1.2. Sucrose Phosphate Synthase (SPS. EC 2.4.1.14)
The enzyme Sucrose phosphate synthase (SPS) was assayed by the method of
Sinha rr a1 (1 997).
Reagents
9 Extraction Buffer: The buffer contained 100 mM HEPES, 5 mM
magnesium chloride, 1 mM ethylene diamine tetraacetic acid, 25 mM P-
Mercaptoethanol, I mM Phenyl methyl sulphonyl fluoride and 0.02 %
Triton-X 100 at pH 7.4.
4 Assay buffer for Vmax activity: The buffer contained 50 mM HEPES, 15
mM magnesium chloride, 4 mM fructose-6-phosphate, 5 mM UDP glucose
and 20 mM glucose-6-phosphate at pH 7.5.
Assay buffer for Vlim activity: The buffer contained 50 mM HEPES,
15 mM magnesium chloride, 5 mM UDPG, 2 mM fructose-6-phosphate,
10 mM glucose-6-phosphate and IOmM potassium di-hydrogen phosphate
at pH 7.5.
Anthrone Reagent (0.14 %): 40 ml of concentrated sulphuric acid was
added to 10 ml of distilled water and later 70 mg of anthrone was
dissolved.
9 Potassium hydroxide (30%): 30 g of potassium hydroxide was dissolved in
100 mi of distilled water.
Extraction
The leaf material was homogenized in 10 volumes of the extraction buffer
with the help of mortar and pestle. The homogenate was filtered through muslin
cloth and the filtrate was centrifuged at 13,000 g for I0 minutes. The supernatant
was desalted on a Sephadex G-25 column, equilibrated with the extraction buffer
without Triton-X 100. The elute was centrifuged and supernatant was stored for at
4°C the assay.
Enzyme Assay
To 50 pl of the enzyme extract, 100 p1 of the assay buffer was added and
incubated at 25°C for 20 minutes. Then the reaction was terminated by adding 100 p1
of 30% potassium hydroxide. The tubes were placed on a boiling water bath for
10 minutes to destroy the unreacted fructose-6-phosphate. After cooling, 1.0 ml of
anthrone reagent was added. The tubes were incubated at 40°C for 20 minutes on a
water bath. The absorbance of the solution was read at 620 nm. For the 'control', the
reaction was terminated at '0' minute with 30 % potassium hydroxide. The above
reaction was done with assay buffers for both Vmax and Vlim activities. The activity
of sucrose phosphate synthase was determined using the standard curve obtained with
known concentrations of sucrose ranging between 10 to 50 fig. The activity was
expressed as p moles g' h".
4.6.1.3. Superoxide Dismutase (SOD, EC 1.15.1.1)
SOD was determined by the method of Beauchamp and Fridovich (1971)
as modified by Dhindsa and Matowe (1981), which measures the inhibition in
the photochemical reduction of nitroblue tetrazolium. In the spectrophometric
assay. 1 ml reaction mixture contained 50 mM phosphate buffer (pH 7.8),
0.1 m M EDTA, 13 mM methionine, 75 pM nitroblue tetrazolium (NTB),
2 p M riboflavin and 100 p1 of the enzyme supernatant. Riboflavin was
added at last and the reaction was initiated by placing the tubes under two
15-W fluorescent lamps. The reaction was terminated after 10 min by
removal from the light source. Non-illuminated and illuminated
reactions without supernatant served as callbration standards. Reaction
product was measured at 560 nm. 'Thc volumc of the supernatant
corresponding to 50% inhibition o f the reaction was assigned as one enzyme
unit.
4.6.f.4. Catalase (CAT, EC f . f f .7 .6)
Modified method of Luck (1974) was employed for the assay of CAT.
To 50 p1 of the enzyme extract, 3 ml of hydrogen peroxide-phosphate buffer
(pH 7.0) was added. The time required for decrease in absorbance from 0.45
to 0.40 was noted. The enzyme solution, which contained hydrogen peroxide-
free phosphate buffer was used a s control. The activity was expressed as units.
The change in the absorbance o f 0.001lminlml of enzyme was assigned as one
unit.
4.7. Extraction for Protein and Nucleic Acids Estimation
4.7.1. Extraction Method
Nucleic acids and protein were extracted by the method of Shneider
(1945). 500 mg of fresh leaf materials from treatment was weighed; to this
5 ml of 10% cold trichloro acetic acid was added on ice. This was homogenised
with a polytron homogeniser and then allowed to stand for 30 min and
57
supernatant was discarded. To the pellet, 3 ml of 10% cold TCA was added
and mixed thoroughly in a cyclomixer. It was centrifuged at 2500 rpm for
10 min and supernatant was discarded. To the pellet 3 ml of isopropanol
was added and mixed thoroughly in a cyclomixer. This was centrifuged at
2500 rpm for 10 min. The supernatant was discarded. lsopropanol washing was
repeated thrice To the precipitate, 5 ml of 5% PCA was added mixed and
heated in a boiling water bath for 15 min. The tubes were centrifuged at
3000 rpm for 25 min and the supernatant was used for the estimation of DNA
and RNA.
The residual pellet was dissolved in a 5 ml of 0.1 N NaOH and
centrifuged at 3000 rpm for 10 min. The supernatant was used for protein
estimation.
4.7.1.1. Estimation of DNA
Method of Burton (1956) was used for the DNA estimation.
Reagents
Diphenylamine Reagent: 1.5 g diphenylamine was dissolved in 100 ml of
acetic acid. To this, 1.5 ml of conc. H2S04 was added and stored at 4°C in
dark coloured bottle. To every 20 ml of the reagent, 0.1 ml of 1.6%
aqueous acetaldehyde was added just before use to induce the colour
development.
4. Acetaldehyde solution: Redistilled acetaldehyde at a concentration of
1.26% was prepared as an aqueous solution and stored at 4OC.
Perchloric acid - PCA: 5 % PCA was prepared by dissolving 5 ml of 73%
PCA in 68 ml of distilled water.
'3 DNA Standard: 2 mg of Calf thymus DNA was dissolved in 10 ml of 5 %
PCA to prepare stock solution and stored at 4°C when not in use. Standard
curve was prepared by using calf thymus DNA.
Method
To 1.5 ml of PCA extract, 3 ml of diphenylamine reagent was added. The tubes
were kept in a water bath and maintained at 70°C for 20 min and then cooled. The
colour development was read at 600 nm on a Systronics Spectrophotometer. A
Standard calibration curve was prepared by using known concentrations of calf
thymus DNA. The DNA content was expressed in mg DNAig fresh weight of the leaf
tissue.
4.7.1.2. Estimation of RNA
RNA was estimated by the method of Rawal et oi (1977)
Reagents
Reagent A: 1 g of orcinol dissolved in 100 ml of distilled water was stored
in a refrigerator at 4°C.
8 Reagent B: Conc. HC1
0 : Reagent C: FeCI, 6H20, 10% solution.
8 Orcinol Reagent: To the 10 ml of reagent A, 40 ml of reagent B and I ml of
reagent C were added. Orcinol reagent was freshly prepared at the time of
use.
RNA Standard: Standard curve was prepared by using known concentration
of purified RNA.
Method
To the 0.5 ml nucleic acid fractions, 3 ml of orcinol reagent was added and
heated on a water bath for 20 min at 90°C and then cooled. The colour development
was read at 665 m. A standard calibration curve was prepared by using k n o w
concentrations of purified RNA. The RNA content was expressed in mg RVNg fresh
weight of the leaf tissue.
4.7.1.3. Estimation o f Protein
Protein was estimated by the modified method of Lowry (Furlong el 0 1 , 1973).
Reagents
8 Reagent A: 0.5 g copper sulphate (CuSO1,51-1~0) and 1 g of sodium citrate
dissolved in 100 ml of disrilled water.
Q Reagent B: 20 g of sodium carbonate (Na2CO3) and 4 g sodium hydroxide
were dissolved in I litre water.
Reagent C: To 50 ml reagent B, 1 ml reagent A was added.
60
':' Reagent D: Folin-ciocalteau reagent was prepared by adding equal volume
of distilled water to the commercial reagevt.
Reagents C and D were prepared fresh at the time of use
3 Protein Standard: Standard curve of protein was prepared by using known
concentrations of Uovinc Scrum Albumin (BSA).
Method
To 0.5 ml of the protein extract, 2.5 ml of reagent C was added and the mixture
was incubated for 8-10 min and then 0.25 ml of reagent D was added. The mixture
was incubated for another 20-30 min. The colour developed was read at a wavelength
of 610 nm. The protein standard was estimated by using BSA as a stock solution.
Protein content was expressed in mg of proteinlg of fresh leaf tissue.
4.7. Nitrate Reductase Activity
Nitrate reductase activity was assayed by the method of Jaaorski (1971)
with suitable modifications (Muthuchelian er ai., 1990). Harvested fresh leaves
were washed and cut into 5 mm discs. Leaf bits corresponding to 100 mg fresh
weight were incubated in vials containing 5 ml of incubation medium. The
incubation medium was prepared by mixing 0.1 N KNO, (1 ml), 0.1 M phosphate
buffer of pH 7.5 (3.75 ml), 0.1% of Triton X-100 (0.01 mi) and 1% propanol
(0.25 ml). Incubation was carried out in dark for one hour at room temperature
(28 * 2OC) giving occasional shakings. Aliquots of 0.2 rnl from the incubation
mixture were analysed for nitrite after 60 min. To 0.2 ml of incubation medium,
1.8 ml of distilled water, 1 ml of 3% sulphanilamide in 3 N HCI and 1 ml of
0.02% N-(I-naphthyl) ethylene-diamine dihydrochloride were added in quick
succession. This was incubated for 15 min in darkness for colour development
and absorbance was read at 540 nm with suitable blank in a Systronic
Spectrophotometer. The amount of nitrite formed was expressed as nmoles of
nitrite produced per minute per mg fresh weight using a sodium nitrite standard
curve.
4.8. Proline Estimation
Reagents
4- Aqueous Sulpho salicylic acid (3%): 3 gm of sulphosalicylic acid was
dissolved in I00 ml of distilled water.
Acid Ninhydrin: 1.25 gm of Ninhydrin was dissolved in a warm mixture of
30 ml of glacial acetic acid and 20 ml of 6 M Phosphoric acid with
agitation. The reagent was stable for 24 hours when stored at 4'C.
a:. Standard Proline: 5 mg of proline was dissolved in 10 ml of 0.1 N
Hydrochloric acid.
Extraction
The extraction and estimation of proline was done according to the method
of Bates et a1 (1973). The midribs of a leaf were removed and 500 mg of the leaf
tissue was weighed. It was homogenised with 10 ml of 3% sulphosalicylic acid
in a mortar and pestle. The homogenate was filtered through a Whatrnann No. 2
filter paper. The procedure was repeated with the residue and the filtrates were
pooled.
62
To 2.0 ml of the filtrate, 2.0 ml of acid ninhydrin and 2.0 ml of glacial acetic
acid was added. The tubes were incubated for 1 h at 100°C on a water bath. The tubes
were transferred on ice to terminate the reaction and 4.0 ml of toluene was added and
mixed vigorously for 15-20 seconds. The chromophore containing toluene was
aspirated from the aqueous phase. It was allowed to reach room temperature and the
absorbance measured at 575 run. A reagent blank was maintained. A standard curve
was obtained using a known concentration of authentic proline. The proline content
was expressed as mg of proline per gram fresh weight.
4.9. Cell Wall Degrading Enzymes Assay
4.9.1. Acetone Powder Preparation
Reagents
Q Acetone
*> Diethyl ether
*:. Phosphate buffer, pH 6.6.
Method
The tissues were weighed and cut into pieces of 1-2 cm each and then
transferred to a blender. Chilled Acetone (-20°C) was added to cover the tissues and
then blended at high-speed 12,000 rpm for 3-5 minutes in cold condition with the
help of polytron homogenizer. The resultant slurry was filtered through Buchner
funnel using Whatman No.1 filter paper and the powder was washed with chilled
acetone in the Buchner funnel under suction. Then it is washed again with cold
63
diethyl ether. The powder was dried overnight under room temperature. The powder
was spread on Whatman No.1 filter paper and air-dried for about 1 h. The powder was
stored in containers with tight caps in a freezer.
4.9.1.1. Enzyme Preparation
0.1 g of acetone powder was weighed and ground In 5 ml of Phosphate buffer
(0.1 M pH 7) at 4°C for 1Q-15 min in a mortar and pestle. The extract was centrifuged
at 2000 g for 20 min at 4°C and the supernatant was used as the enzyme source.
4.9.2. Estimation of Pectinolytic Enzymes
Estimation of the pectinolytic enzyme was measured with similar procedure as
mentioned in in-vilro studies.
4.9.3. Estimation of Cellulolytic Enzymes
Estimated of cellulolytic enzymes was measured with similar methods as
mentioned in in-vitro studies.
4.10. Measurement of Peroxidase Activity
Peroxidase activity was measured by the method of Harnpton (1962) method
Reagents
*:. Phosphate buffer (0.05 M) pH 6.5
O Pymgallol (0.001 M): 0.01261 g of pyrogallol was dissolved in 100 ml of
Phosphate buffer.
64
.:. Hydrogen peroxide: 2% Hydrogen peroxide was prepared by adding 2 ml
of 6%hydrogen peroxide in 4 ml of distilled water.
Method
To I ml of 0.001 M pyrogallol, 1.8 ml of distilled water was added in a
cuvette and the absorbance was adjusted to zero at 470 nm. Immediately 0.1 ml of
2% (0.588 M) Hz02 and 0.1 ml of enzyme were added. The contents were mixed
well and placed in Systrenics Spectrophntmeter. The change in the absorbance at
every 30-second interval for 3 minutes was measured. Suitable control with heat-
killed enzyme was maintained.
4.1 1. Measurement of Polyphenol Oxidase Activity
Polyphenol oxidase activity was measured by the method of Mana and Dimond
(1963).
Reagents
0.2M Phosphate buffer pH 7.0
.:+ Catechol (0.1 M): 1.101 1 mg Catechol dissolved in 100 ml of distilled
water.
Method
To 0.5 ml of enzyme, 0.5 ml of the phosphate buffer (pH 7.0) and 1.5 ml of
distilled water were added and the absorbance was adjusted to zero at 495 nm in
Systronics Spectrophotorneter and immediately 0.5 ml of 0.1 M catechol was
added into the cuvene and the changes in the hsorbance at every 30 seconds
intervals up to 3 min was recorded. Control was maintained with heat-killed
enzyme.
4.12. Preparation of Alcohol Extract
Fresh leaves were dried in hot air oven and powdered with the help of
mortar and pestle. Dried leaf powder of about 50 mg was boiled in a water bath
with 10 ml of 80% ethyl alcohol. The homogenate was first cooled and then
centrifuged at 600 rpm for 15 min. The supernatant was saved and made up to
20 ml with 80% ethyl alcohol. This extract was used for quantitative estimation
of carbohydrates, phenols and nitrogen content. The residue was saved for starch
estimation
4.12.1. Estimation of Reducing Sugars
The reducing sugars were estimated by the Nelson's modification of Somogyi's
method (Nelson, 1944).
Reagents
Copper tartarate solution (A): 25 g of anhydrous sodium carbonate, 25 g of
sodium potassium tartarate, 20 g of sodium bicarbonate, 200 g of anhydrous
sodium sulphate were dissolved in 800 ml of distilled water, diluted to 1
litre, then filtered and stored in a brown bottle.
66
9 Copper sulphate solution (B): 15 g of copper sulphate was added to 100 ml
of distilled water. One or two drops ofConc.Hz SO4 were added.
O Copper reagent: 25 ml of reagent A and 1 ml of reagent B were
mixed.
6 Arsenomolybdate reagent: To 450 ml of distilled water, 25 g of ammonium
molybdate was dissolved. To this, 21 ml of conc. H2SOo wa? added. To the
above mixture, Three grams of sodium arsenate dissolved in 25 ml of
distilled water was added and incubated at 37'C for 48 h. The reagent was
stored in a glass stoppered brown bottle.
To 1 ml of ethanolic extract. I ml of fresh copper reagent prepared
by mixing copper tartarate and copper sulphate solution (25:1 vlv) was added.
The mixture was heated for 20 min in a boiling water-bath and cooled. One ml
of arsenomolybdate reagent was added and the contents incubated for
15 min. The solution was then diluted to 25 ml with distilled water and the
colour intensity was read at 500 nm in Systronics Spectrophotometer. The
content of the reducing sugar was calculated using the standard graph for
glucose.
4.12.2. Estimation of Total Sugars
The total sugars were estimated by the method proposed by Dubois et 01.
(1956).
Reagents
Anthrone reagent: To 40 ml of distilled water, 100 ml of concentrated
sulphuric acid was added. To 100 ml of the above mixture, 200 mg of
anthrone was added and thoroughly mixed until a golden yellow colour
appear.
Method
Four ml of cold anthrone reagent was added to 1 ml of ethanolic extract.
This mixture was shaken vigorously and boiled for 10 min in a boiling water
bath. After cooling in running tap water, the absorbance was read at 620 nm in
Systronics Spectrophotometer. A standard curve was prepared with known
amounts of glucose.
4.12.3. Estimation of Non-Reducing Sugars
The amount of non-reducing sugars was determined by following the formula
suggested by Loomis and Shu11(1937).
Non-reducing sugars = Total sugars - free reducing sugars x 0.95
4.12.4. Estimation of Ortho Di-hydroxy Phenols
The Ortho Di-hydroxy phenol content was estimated according to the method
proposed by Johnson and Shoal (1952).
68
Reagent
9 Arnow's reagent: Ten g of sodium nitrite and 10 g of sodium molybdate
were mixed in 100 ml of distilled water. The reagent was stored in a brown
bottle.
Method
To 1 ml of alcoholic extract, 1 ml of 0.5 N HC1 and I ml of Arnow's
reagent were added. To this, 2 ml of I N NaOH and 10 ml of distilled water were
added. A pink colour appeared immediately on adding NaOH. The colour
intensity was reduced by diluting it to 25 ml with distilled water and the
absorbance read at 515 nm. The O.D. phenols were calculated using a standard
curve with catechol.
4.12.5. Estimation of Total Phenol
The total phenol content was estimated according to the method of Bray and
Thorpe (1954).
Reagents
8 20 % sodium carbonate: Twenty g of sodium carbonate was mixed with
100 ml of distilled water.
Folin-Ciocalteau reagent: Commercial Folin-Ciocalteau was diluted with
distilled water in 1 :2 ratio.
69
Method
To 1 ml of alcoholic extnct, 1 ml of Folin-Ciocalteau reagent and 2 ml of 20%
sodium carbonate were added and shaken well. The mixture was heated in a boiling
water bath for 1 min and cooled under running tap water. The blue solution was diluted
to 25 ml with distilled water and read at 650 nm in Systronics Spectrophotometer.
Phenols were quantified using catechol as standard.
4.12.6. Estimation of Amino Nitrogen Content
Reagents
.:. Citrate buffer: 21 g of citric acid was dissolved m 200 ml of 1 N NaOH and
the same was made up to 500 rnl with distilled water. The pH was adjusted
to 5.0 by adding 1 N NaOH/HCI.
Ninhydrin reagent:
o Solution A: 800 mg of stannous chloride was dissolved in 500 ml
of Citrate buffer, pH (5.0).
o Solution B: 20 g of ninydrin was dissolved in 50 ml of methyl
cellosolve.
o Solution C: To I rnl of Solution A, 1 ml of solution B was added.
9 Standard: The known quantity of glutamic acid was used as standard.
Method
The pH of the alcoholic extract was adjusted to 7.0 by adding 0.1 N NaOHIHC1.
TO 1 ml of the above extract I ml of ninhydrin reagent was added. Then, it was heated
70
for 20 min and cooled. 5 ml of distilled water was added and the absorbance was
measured at 475 nrn in Systronics Spectrophotometer.
4.22.7. Amino Acid Estimation
The amino acid content was estimated by the method of Moore and Stein (1954)
Reagents
20.5 ml of 0.2 M solution of citric acid and 29.5 ml of 0.2 M solution of
sodium citrate were mixed and diluted to a total volume of 100 ml with
distilled water and the pH was checked in a pH meter.
Citric acid solution (0.2 M): 21.09 g of citric acid was dissolved in 500 ml
of distilled water.
Sodium citrate solution (0.2 M): 29.41 g of sodium citrate was dissolved in
500 ml of distilled water.
Ninhydrin solution: To 500 ml of 0.2 M citrate buffer (pH 5.0) 0.8 g of
stannous chloride was added. Four gram of ninhldrin in 500 ml of methyl
cellosolve was added to the above mixture. The reagent was stored in a
refrigerator at 4°C.
O Diluant solution: Distilled water and n-propanol were equally added.
Method
One ml of ninhydrin solution was added to 0.1 ml of alcoholic extract
and shaken well. To this, 0.9 ml of distilled water was added and the above
71
mixture was heated in a boiling water bath for 20 min and cooled under running
tap water. Five ml of diluant solution was added to the above mixture and
kept for 15 min. The absorbance was read at 570 nm. The amino acid contents o f
the sample were determined with the help of a standard curie prepared for
glycine.
4.12.8. Estimation of Sucrose
The sucrose content was estimated by the method of Van Handel (1968).
Reagents
Anthrone reagent
30%KOH
Method
To 1 ml of the 80% ethanol extract 0.1 ml of 30% aqueous KOH was added and
kept in a boiling water bath for 10 min. The samples were cooled and 3.0 ml of
anthrone reagent was added and kept at 40°C for 10 min. The absorbance was read at
620 nm. Glucose of known concentration was used as standard.
4.12.9. Estimation of Starch
The starch content was estimated according to the method proposed by
McCready er a[. (1950).
72
Reagents
9 Anthrone reagent: Anthrone (200 mg) was dissolved in 100 ml of cold 95%
H2So4.
4. Perchloric acid: To 18 ml of distilled water, 52 ml of commercial perchloric
acid (70%) was added to get 52% perchloric acid.
Extraction
The residue left behind after alcoholic extraction of the leaf materials was
dissolved in 5 ml of 52% perchloric acid (PCA) for 1 h. The mixture was filtered
through Whatman's filter paper (No. 42) and the filtrate was made up to 100 ml with
distilled water.
Method
To 1 ml of the PCA extract, 4 ml -of distilled water and I0 ml of freshly
prepared cold anthrone reagent were added carefully along the side of the tube. The
contents of the tubes were shaken vigorously and heated in s boiling water bath for
7.5 min. The tubes were then cooled immediately in runnlng tap water and shaken
well before reading the colour intensity at 630 nm in Systronics Spectrophotometer.
The starch content was calculated with reference to glucose standard and multiplied
by 0.9.
4.13. Nitrate-Nitrite Estimation
Nitrate-nitrite content was estimated according to the method proposed by
Wooley et a[. (1960). About 50 mg of shade-dried leaf powdered material was
73
boiled for 10 min in 5 ml of distilled water. One ml aqueous extract was added to
9 ml of 20% (viv) acetic acid solution containing 0.2 ppm of CuS04. One gram of
salt mixture as described by Nelson el a1 (1954) was added to each sample. The
salt mixture was made by mixing thoroughly the finely ground chemicals namely,
100 g barium sulphate, 75 g citric acid, 10 g MnS04, 4 g sulphanil,~ ac~d, 2 g zinc
powder, and 2 g I-napthyl amine.
Tubes without extract served as blank. Tubes containing the assay mixture
were shaken at least thrice at 3 min intervals and finally centrifuged at 3000 rpm
for 10 min. Absorbance of the clear supernatant was read at 520 nm against a
reagent blank.
The procedure was repeated for another batch of samples omitting Zn, MnS04,
and CuS04. The second run gave the quantity of nitrite alone present in the sample.
The first value minus the second gave the quantity of nitrate present in the samples.
The amounts of nitrate and nitrite were calculated from standard graphs for potassium
nitrate and sodium nitrite respectively.
4.14. Estimation of Curcumin Content
The cwcumin content was estimated quantitatively followed by the method
proposed by Manjunath el a/. (1 991).
Method
Finely ground turmeric powder (0.1-0.2 g) is extracted by refluxing over a
water-cooled condenser with 40 ml of distilled alcohol for two and half hours. The
74
extract was made to 100 ml with alcohol in a standard volumetric flask. It is then
filtered and an aliquot of 5 ml is .transferred to the flask and the volume made to
100 ml. It is mixed well and the absorbance of this solution is measured at 425 nm
against alcohol blank. Using the absorbance value of a standard solution of curcumin
(0.00025 g1100 ml gives an absorbance of 0.42) the percentage of curcumin was
calculated.
Curcumin (%/wt) = 0.00025 x A,,, 100 x 100 Absorbanceof x weight of sample x 5 the standard
The alcohol used for the extraction should be in the pH range below 6.5.
4.15. Total Nitrogen Content
The nitrogen content of the leaves was estimated according to Kjeldahl method
using the KJEL PLUS System (Pelican).
The method involves three stages:
1. Digestion
2. Distillation
3. Titration
Reagents
.:* Hydrochloric acid (0.1 N): 0.82 ml of concentrated hydrochloric acid was
added to 99.18 ml of distilled water.
9: Digestion activator: 25 g of potassium sulphate, 5 g of copper sulphate,
0.5 g of selenium were mixed.
75
*:' Sodium hydroxide (40%): 40 g of sodium hydroxide was dissolved in
I00 ml of distilled water.
6 Mixed indicator: 30 mg of bromocresol green and 20 mg of methyl red
were dissolved in 40 ml of 90 %ethanol.
4.15.1. Digestion
Leaves from healthy, infected and treated plants were dried and powdered after
removing the midribs. 500 mg of the powder and 3 g of digestion activator were
weighed and added to the digestion tube of the KJEL PLUS Digestion Block System.
To this 10 ml of concentrated sulphuric acid was added. The tuhes were loaded on to
the block and the temperature set at 350°C. The samples were digested for 1 hour.
4.15.2. Distillation
The digestion tube was placed inside the KJEL PLUS DISTIL-M chamber
through the alkali hose. The alkali hose at the back panel was immersed in to the bottle
containing 40 %alkali solution and the volume of the alkali was fixed. The receiver end
of the hose was immersed into a conical flask containing 20 mi of boric acid and 2-3
drops of the mixed indicator. 30 ml of the alkali was added to the digestion tube. The
distillation time was fixed at 6 minutes and the distillation process started.
4.1 5.3. Titration
The solution collected in the conical flask was titrated against 0.1 N
Hydrochloric acid. The titer value was noted. The percentage of Nitrogen was
calculated using the following formula:
76
%of - TitrevaluexNormalityof HCIxNitrogen factor 1 - Weight of thesample
Where Nitrogen factor = 1.401.
The Nitrogen content was expressed as percentage of nitrogen per gram fresh
weight.
4.16. Yield
Rhizomes were harvested from each treatment upon maturity (after 270 days
from the date of sowing). Yield was calculated separately for each treatment and
expressed in terms of t/h.
4.16.1. Yield-Related Parameters
Fifty plants from each treatment were selected randomly for recording the
various morphological characters of the rhizome. The parameters analysed were
given below:
Mother rhizome: Length, girth, numbers of nodes, internodal length, fresh and dry
weight.
Primary rhizome: Number of fingers/plant. length, girth, number of nodes,
internodal length, fresh and dry weight.
Secondary rhizome: Number of fingerslplant, length, girth, number of nodes,
internodal length, fresh and dry weight.
77
4.17. Statistical analysis
The results of the experiments were tested by a multiple range testing
programme. Tukey's multiple range test (TMRT) was applied for the experimental
data at 5% level of significance (Zar, 1984).