44
3 MATERIALS AND METHODS

Prot Okoli

Embed Size (px)

DESCRIPTION

protokoli

Citation preview

Page 1: Prot Okoli

3 MATERIALS AND METHODS

Page 2: Prot Okoli

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

Page 3: Prot Okoli

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

Page 4: Prot Okoli

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).

38

Page 5: Prot Okoli

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

Page 6: Prot Okoli

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

Page 7: Prot Okoli

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

Page 8: Prot Okoli

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

42

Page 9: Prot Okoli

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

Page 10: Prot Okoli

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

44

Page 11: Prot Okoli

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

Page 12: Prot Okoli

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

Page 13: Prot Okoli

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

Page 14: Prot Okoli

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.

48

Page 15: Prot Okoli

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

Page 16: Prot Okoli

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.

Page 17: Prot Okoli

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

Page 18: Prot Okoli

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)

52

Page 19: Prot Okoli

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

Page 20: Prot Okoli

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).

Page 21: Prot Okoli

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

Page 22: Prot Okoli

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),

Page 23: Prot Okoli

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

Page 24: Prot Okoli

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.

Page 25: Prot Okoli

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

Page 26: Prot Okoli

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

Page 27: Prot Okoli

':' 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

Page 28: Prot Okoli

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

Page 29: Prot Okoli

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

Page 30: Prot Okoli

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

Page 31: Prot Okoli

.:. 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

Page 32: Prot Okoli

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

Page 33: Prot Okoli

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).

Page 34: Prot Okoli

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

Page 35: Prot Okoli

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

Page 36: Prot Okoli

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

Page 37: Prot Okoli

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

Page 38: Prot Okoli

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

Page 39: Prot Okoli

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

Page 40: Prot Okoli

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

Page 41: Prot Okoli

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

Page 42: Prot Okoli

*:' 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

Page 43: Prot Okoli

%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

Page 44: Prot Okoli

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).