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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~chumcups~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 30C and conidial germination was observed after 24 h. The percentage of inhibition over control was calculated by the formula of Vincent (1927).
38

I=-x100 C

C-T

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 by were incubated (26 2 2'C) for 7 days. lnhibition of mycelial growth of C. c u p s ~ c ~ 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.SPCfor 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 7Lhday 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 ~nhibitionin 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.ZC 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. 41

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

Substrate preparation: Ig of pectin was dissolved in I00 ml of acetate buffer, pH 5.2, heated to S&60C 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.IAssay 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. EndoPTE 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

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 0.5NHCI 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% H2S04was 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 80C 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 (exoC,). 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 a , 1997). 1

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
50-60C 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

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
0:

Sodium acetate-acetic acid buffer, pH 5.8 5 m M 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 30C 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
49
x

5 rn2 and a standard spacing

of 30

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-dayold 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 Is' 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 ' ~ after 2"d day 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

106 116

l ( l 0 DAT)
20d

I1 (10 DAT)

4.3. Disease Intensity Study


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

(i) Nil (iij Light

Free of disease spots.

I 1 spot
1 2-5
mots.

/ (iii) Medium
(iv) Heavy
(v) Very heavy

/ 5-10 spots.

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 C o n t e n 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 4C. 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 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

(8'08 1OOOxwxa
+

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 =

(0.1 14 X A666) (0.638 -

AGd7)

1 Total carotenoids (mg g.' fw) = Corrected 0.D.x 1 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 30C 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 I 0 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 4C the assay.

Enzyme Assay
To 50 pl of the enzyme extract, 100 p1 of the assay buffer was added and incubated at 25C 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 40C 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.ff.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 peroxidefree 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 1 0 % 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 D N A
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 4C 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 4C 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 70C 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 4C.
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 90C 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 R V N g 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) 1 g of sodium citrate and

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 100C 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


.:* Method
The tissues were weighed and cut into pieces of 1-2 cm each and then transferred to a blender. Chilled Acetone (-20C) 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

Phosphate buffer, pH 6.6.

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 4C for 1Q-15 min in a mortar and pestle. The extract was centrifuged at 2000 g for 20 min at 4C 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

.:.
Method

Hydrogen peroxide: 2% Hydrogen peroxide was prepared by adding 2 ml of 6%hydrogen peroxide in 4 ml of distilled water.

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 heatkilled 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


water.

1 mg Catechol dissolved in 100 ml of distilled

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 m l 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 4C.
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 glycine.
curie

prepared for

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 m of the 80% ethanol extract 0.1 ml of 30% aqueous KOH was added and l 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 40C 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, g zinc 2 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 350C. 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
1

- TitrevaluexNormalityof HCIxNitrogen factor 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).