26

Chapter - 3

MATERIAL AND METHODS

Root-knot nematode, Meloidogyne incognita (Kofoid and White, 1919)

Chitwood (1949) is considered to be a representative of the plant parasitic

nematodes and its infestations are easily recognized by the characteristic root

galls produced. The flask shaped swollen adult female nematodes which are

sedentary in habit, incapable of movement within roots are harboured within

root galls. The male appears as a slender eel worm with bluntly rounded tail.

Nematode has six stages in the life cycle, the egg, four larval stages and the

adult. One cycle may be completed in a few days or may require several

months depending upon of environmental conditions and susceptible hosts.

The host plant selected for the present study was Vigna mungo, which is

susceptible to root-knot nematode infection. The seeds were procured from

Tamilnadu Agricultural College and Research Institute, Killikulam, Tirunelveli

district.

Preparation of Sand - Soil Mixture

River sand, garden soil and red soil mixed in the ratio of 4:1:1 were

selected for the rearing of plants in the laboratory, because this mixture is

favourable for penetration of nematode larvae and the better growth of the root

system. This mixture was sieved to remove coarse particles.

27

Sterilization of Sand and Soil Mixture

Following the method of Fred and Wakesman (1928) the sand soil

mixture was sterilized in an autoclave at 20 lbs pressure for two hours to

destroy various organisms including bacteria, fungi, nematodes etc. After

sterilization, the mixture was aerated overnight and transferred to a plastic

container to prevent contamination by dust.

Seedling Culture

The seeds of V.mungo were surface sterilized with 0.1% mercuric

chloride solution and rinsed for 5 minutes in sterile distilled water 6 times

before sowing. Five seeds were sown in sterilized pots containing 1.5kg

sterilized sand - soil mixture. Seven days after germination, seedlings were

thinned to a stand per pot, ensuring they were all of uniform growth and vigour.

The seedlings were allowed to grow up to 2 leaves and a bud stage. After this

they were ready for inoculation.

Source of Inoculam

Matured egg masses of M.incognita were isolated from the plant,

Acalypha indica L as primary inoculum. The egg masses were surface

sterilized with 0.01% sodium hypochlorite and transferred to brass gauze filters

overlaid with mull cloth. The gauze assembly was lowered in a small plastic

vial (1” × 1”) and kept in contact with a small quantity of water at the bottom

for hatching. Freshly hatched juveniles were collected and used for inoculation.

28

Inoculation of the Pathogen

Each seedling was inoculated with 2000 second stage larva by pipetting

more or less evenly into four holes dug around the seedling in the soil of the

pots to facilitate infection from all sides around the roots. The holes were then

covered with thin film of sterilized sand and watered very lightly. All the plants

such as control-uninfected, infected-untreated and infected-treated plants were

watered with nutrient solution once a week till the harvesting period (30 days).

Type of Nutrient Solution

Nutrient solution formulated by Arnon and Hogland (1940) was

employed for present culture experiment.

KNO3 - 1.02 gm/ litre

Ca(NO3)2 - 0.492 gm/ litre

NH4H2PO4 - 0.230 gm/ litre

MgSO4 . 7H2O - 0.490 gm/ litre

FeSO4 (0.5%) - 0.6 ml

C4H6O6 (0.4%) - 0.6 ml

H3BO3 - 2.86 mg

Mncl2 4H2O - 1.89 mg

Cu SO4 . 5H2O - 0.8 mg

Zn SO4 . 7H2O - 0.22 mg

29

H2 MO4 . 4H2O - 0.09 mg

PH of the nutrient solution - 4.5 to 6.0

Preparation of Pongamia pinnata Leaf Powder

Fresh leaves of P.pinnata without galls were collected, washed well in

running tap water and then with sterile distilled water. They were air dried

under shade at room temperature for about 10 to 12 days. The dried leaves

were powdered in mixer grinder and meshed.

Preparation of Extracts for Phytochemical Screening

Hot Maceration Method using Soxlet Apparatus

The powder (100g) was extracted successively with hexane, benzene,

acetone and methanol, each 250 ml in a soxlet apparatus. The process lasted 24

hours. All the extracts were evaporated on a water bath and finally dried in

vacuum. The residues obtained were used for screening the nematicidal

activity.

Laboratory Screening Test for Selection of Solvent

From the residues of each organic solvent extract different

concentrations viz., 2500, 3000, 3500 ppm were prepared with Tween 80

solutions as solubilizer and was made up to 100 ml with distilled water. 0.2ml

of water suspension containing 100 number of second stage larvae of

M.incognita was placed into cavity blocks. To this 5ml of 2500, 3000 and 3500

30

ppm concentration of each solvent were added separately and tested for larval

mortality. A control was maintained with distilled water at room temperature.

Each treatment was replicated 5 times. A check was also maintained with

Tween 80 and distilled water. The larval mortality was recorded every 3 hours

upto 24 hours with stereoscopic microscope. Among the solvents tested,

methanol solvent extract showed maximum larval mortality at 3500 ppm.

Hence the methanol solvent extract was selected. Based on larval mortality test

and phytotoxic effect of the methanol leaf extract, 3 different concentrations

namely 2500, 3000 and 3500 ppm were selected for the present study.

Experimental Design

In the present study, earthern pots were selected for culture experiment.

150 plants were raised and maintained under green house condition. Of them,

30 plants were left uninoculated and used as control and another 30 plants were

kept for inoculated-untreated. The remaining plants were used in three

experimental sets having 30 plants for each treatment viz., 2500, 3000 and

3500 ppm concentration of methanol leaf extract of P.pinnata. The pots were

arranged in completely randomized block design.

Solvent Extract Treatment for the Host Plant

The experimental pots were treated with 10ml of 2500, 3000 and

3500 ppm of methanol leaf extract by soil drenching method (Osman and

Viglierchio, 1988). 10 ml of extract was applied for each plant 3 days before

inoculation as pre treatment and 3 days after inoculation as post treatment.

31

Manner of Recording (Alam et al., 1980)

Thirty days after inoculation, the plants were depotted and washed with

tap water to remove sand particles, rinsed again with distilled water and

blotted. For evaluation, the plants were separated into root and shoot and

weighed as quickly as possible to reduce water loss. The roots and shoots were

then separated and dried in a hot air oven at 50 + 50C for a week to obtain a

constant weight. These dried samples were ground to 60 mesh powder

separately. These dried powders were stored in refrigerator and used for

biochemical studies.

Histopathological Studies

Collection of Specimens

The plant root sample was collected from experimental plants cultivated

in pots.

Anatomical Studies

The required samples of root were cut into bits of 1 to 2cm in length and

immediately fixed in FAA (Formalin- 5ml + Acetic acid- 5ml + 70% Ethyl

alcohol-90 ml). The root samples were left in preservative for 24 hours; then

the materials were washed in water and processed further.

Dehydration, Infiltration and Sectioning

32

Dehydration of the specimens were carried out employing tertiary butyl

alcohol series (Sass, 1940). After total dehydration, infiltration of paraffin wax

was carried out by gradual addition of paraffin shavings (Emerck paraffin wax

with 58oC - 60

oC melting point) till super saturation was achieved. After

transferring the material to pure melted paraffin wax twice, the materials were

embedded in paraffin blocks.

Transverse sections of 10 to 12 µm thickness were prepared with Rotary

microtome. Paraffin ribbon with section was mounted on slides smeared with

Haupt’s adhesive. Dewaxing of the section was done using customary

procedures (Johansen, 1940).

The sections were stained with toluidine blue as per the method

published by O’Brien et al., (1964). Some sections were stained with safranin

and fast green as counter stain.

Photomicrographs

Microscopic descriptions of tissues are supplemented with micrographs

wherever necessary. Photographs of different magnifications were taken with

Nickon Labphot 2 Microscopic unit. For normal observations bright field was

used. Magnifications of the figures are indicated by the scale bars.

Nematode Development

33

The number of root galls was counted by taking one gram of nematode

infected root from the homogenous mixture and total galls were calculated. Egg

masses were separated carefully from the root system with the help of forceps

and counted with hand lens. The eggs per egg mass were counted by adopting

the method of Reddy et al. (1980). The root sample with egg masses was fixed

in 4% formaldehyde solution for 24 hours. The hardened egg masses were

transferred to a little quantity of 0.1% acid fuchsin in lactophenol on a glass

slide and stained for 15 to 20 seconds by heating over flame. On cooling, the

egg masses were transferred on to a grated slide with a drop of water. A cover

slip was placed over the egg mass and gently pressed. The stained eggs were

separated sufficiently to allow convenient counting under stereoscopic

microscope.

Root Population and Developmental Stages of the Nematode

(Hadisoeganda and Sasser, 1982)

Three plants from each experimental pot except control were plucked

periodically after inoculation of juveniles of M.incognita. Every alternate day

the plants were uprooted carefully by flushing water slowly into the pot to

prevent damage of small branches for the root population and nematode

developmental study till the termination of experiment. The roots were cut into

bits (1-2 cm) and immersed in boiling 0.05% acid fuchsin - lactophenol for 3

minutes, rinsed in tap water and destained in plain lactophenol for 24 hours.

The roots were later pressed between microscopic slides and examined under a

dissecting microscope at x30 for larval population and different stages of the

34

nematode. The deformation of different stages of nematode were recorded and

photographed by using Nikon camera.

Soil Population of Nematode

Soil population was estimated by adopting the method of Chawla and

Prasad (1974). The soil sample was mixed thoroughly and placed 250 ml of the

sample into a basin. Two litres of water was added into the soil of basin 1 and

thoroughly mixed. Now the basin 1 was held steadily for about 10 seconds

immediately after mixing to permit the heavy soil particles to settle and

decanted through a coarse sieve (60 mesh per square inch) into another basin II.

The contents of basin II were mixed carefully by hand to ensure that nematodes

and silt particles on the base of the basin are in suspension. The pan was held

steadily for about 10 seconds after mixing, to permit the heavy silt particles to

settle and decanted the suspension onto a fine sieve (350 mesh per sq. inch)

where the nematodes were retained. This operation was repeated twice. The

nematodes and fine silt on the fine sieve were washed off and poured into a

beaker. These nematode suspensions were poured onto a wire gauze sieve

containing two layers of tissue paper kept in modified Baerman funnel holding

sufficient water to remain in contact with the bottom of the wire gauze. The

funnel was left over night. During this period, the nematodes find the minute

gaps in the tissue paper by random movement and then pass through them and

they were collected at the bottom of the funnel stem, leaving the majority of

fine silt particles on the tissue paper.

35

Evaluation of Host Tolerance Level

The total environmental resistance factor ERF(T) to the increase in

population size was assessed as follows :

ERF(T) = RR / RPI

ERF(S) = population final Total

population final Soil × ERF(T)

ERF(P) = ERF(T) – ERF(S)

where RR = Rate of pathogen’s reproduction / unit weight of the root

RPI = Rate of the population increase of pathogen.

RR = 100 1

root gper female root wt) g f(P

×

÷÷

RPI = days) (30 periodn inoculatiopost P

P - P

i

if

×

Pf is the final nematode population which includes eggs × egg masses +

juviniles in the plant and the soil after 30 days of infection and Pi is the initial

inoculam of nematode. Female per g weight = total number of female required

to produce the Pf based on eggs per egg mass assuming that each egg mass is

the product of one female developed from one juvenile.

Biochemical Studies

Estimation of Sugar (Seifter et al., 1950)

36

10 mg of dried materials of root and shoot were separately digested for

10 minutes in test tubes with 1 ml of 30% potassium hydroxide in a boiling

water bath at 100oC. The digested material was cooled and 1 ml of absolute

alcohol was added to each tubes. These tubes were then placed in hot water

bath for 5 to 10 minutes. Then the contents in the tubes were centrifuged for 10

minutes. The supernatant solution was discarded and the sedimented thin layer

of sugar was redissloved in 2 ml of distilled water. The samples were cooled by

immersing in ice cold water (5oC), and 4 ml of anthrone reagent was added

(0.2% anthrone in concentrated sulphuric acid). The reactants were mixed by

swirling the tube. The tubes were then transferred to a boiling water bath

maintained at 100oC and kept for 10 minutes. The developed colour was read in

a systronics UV-VIS 118 spectrophotometer at 625 nm.

Estimation of Protein (Lowry et al., 1951)

100 mg of dried root and shoot powdered samples were ground well

separately with 15 ml of phosphate buffer (0.2M, pH 7.2). The extracts in the

tubes were centrifuged for 10 minutes at 3000 rpm. The lower layer was

discarded and the supernatant was taken. To the supernatant, an equal amount

of cold 5% TCA was added. The tubes were left for 30 minutes in an ice bath.

The precipitated protein was taken and again centrifuged. The supernatant was

discarded and the pellet was dissolved in 25 ml of 0.2 N sodium hydroxide.

From this, 1 ml was taken and mixed with 4 ml of alkaline copper reagent. It

was shaken well and was allowed to stand for 10 minutes at room temperature.

Then 0.1 ml of 1: 1 folin phenol reagent was added and mixed well. After 30

37

minutes the optical density was read at 650 nm using systronics UV-VIS 118

spectrophotometer.

Estimation of Lipid (Bragdon, 1951)

10 mg of oven dried samples of root and shoot were placed in separate

dry mortars and ground thoroughly with sufficient amount of chloroform. This

was allowed to stand for 48 hours for extraction. Each tube was then

centrifuged and the chloroform extract was taken in another tube and

evaporated to dryness. 3 ml of potassium dichromate reagent (2% potassium

dichromate in concentrated sulphuric acid) was added to the sample tube. The

tube was shaken well and diluted with equal amount of distilled water. Blank

was prepared by adding 3 ml of distilled water to equal amount of reagent in a

clean empty tube. The colour developed was read in systronics UV-VIS 118

spectrophotometer at 626 nm.

Extraction and Estimation of Phenols

Extraction (Biehn et al., 1968)

100 mg of oven dried samples of root and shoot were taken in separate

tubes. They were placed in boiling ethanol for 15 minutes. After cooling, the

liquid was decanted and the sample was ground in fresh 80% ethanol. The

slurry was boiled for 15 minutes. Immediately the slurry and the decanted

liquid were combined and filtered using Buchner funnel.

Estimation (Bray and Thorpe, 1954)

38

1 ml of the above alchoholic extract was pipetted into a graduated 25 ml

tube and 1 ml of folin phenol reagent was added followed by 2 ml of 20%

sodium carbonate solution. The tube was immediately shaken and heated in a

boiling water bath for exactly one minute. The solution was cooled and diluted

to 25 ml with distilled water. After 30 minutes, the developed blue colour was

read in spectrophotometer at 650 nm along with a blank prepared in the same

way, using 1 ml of distilled water instead of alcoholic extract.

Estimation of Starch (Jayaraman, 1981)

500 mg of fresh root and shoot were ground well separately with 2 ml of

80% acetone. The solutions were centrifuged for five minutes. The green

coloured supernatant in the tubes were discarded and again nearly 3 ml of

acetone was added to the precipitate and shaken well. This solution was again

centrifuged and the supernatant was discarded. The above procedure was

repeated 2 to 3 times till all chlorophyll pigments were removed. In each time

the precipitate was retained. After removing all chlorophyll pigments, a known

volume of distilled water was added and boiled for a few minutes. The boiled

residue was centrifuged and total starch was estimated in the supernatant. 0.5

ml of supernatant was taken, to this 0.1 ml of iodine reagent (3 gms of iodine

and 1.5 gms of potassium iodide in 100 ml of distilled water) was added. Then

the volume was made up to 5 ml with distilled water. The contents were

thoroughly mixed and absorbance was read in systronics UV.VIS 118

spectrophotometer at 600 nm. Starch was served as standard.

39

Estimation of Ascorbic Acid (Jayaraman, 1981)

25 mg of oven dried root and shoot were placed in a separate mortars

and ground thoroughly with sufficient amount of 5% metaphosphoric acid and

10% acetic acid solution (15 g of metaphosphoric acid dissolved in a mixture

of 40 ml glacial acetic acid and 450 ml of distilled water). A small amount of

Norit was added to the solution taken in test tubes and shaken vigorously. A

separate tube without the material was taken and the same amounts of reagents

mentioned above were added to make a blank solution. Then the mixture was

centrifuged and the supernatant was taken for analysis. To 4 ml of the

supernatant 1 drop of 10% thiourea solution and 1 ml of 2, 4 - dinitro-phenyl

hydrazine reagent (2g of reagent in 100 ml of 9 N sulphuric acid) were added.

The tubes were placed in a hot water bath at 37oC for 3 hours. All the tubes

were cooled by immersing in ice cold water and 5 ml of 85% sulphuric acid (to

100 ml distilled water, 900 ml of concentrated sulphuric acid) was added in

dropwise. The developed colour was read in systronics UV-VIS 118

spectrophotometer at 625 nm against a reagent blank.

Measurement of Oxygen Uptake

Apparatus

Oxygen consumption was measured by pressure sensitive manometers

described by Umbreit et al (1957). In brief the apparatus consisted of glass

40

bottles of 10 ml capacity, used as respiration chambers. These were connected

to ‘U’ tube manometers calibrated to read accurately upto 1 mm. The

manometers tubes were filled with Brodie’s fluid. The fluid used was made up

of 46 gm sodium chloride, 5 gm sodium glycocholate, 400 mg Evan’s blue and

a few drops of thymol solution as a preservative, in 1 litre of distilled water.

One end of the manometer was open and other end was connected to the

respiration chamber at the beginning of the experiment, but remained closed

while the measurement of respiration was being made. Small vials of different

sizes in keeping with the size of the respiration chamber were placed inside the

respiration chambers in the center. Control (blank) manometer of identical

capacity was used in conjunction with the manometer in every experimental

run. From the control, corrections for fluctuations caused by atmospheric

changes caused by temperature oscillations in the bath were effected to the

actual readings obtained from the manometer. The above setup was found to be

easy to assemble and operate, especially in the context and nature of the present

work.

100 mg of fresh root and shoot tissues were suspended in 3 ml of 0.1 M

phosphate buffer (pH 6.8) in 10 ml Warburg manometer flasks. 0.2 ml of 10%

potassium hydroxide and a folded filter paper were kept in the central wall.

This soaked filter paper is meant for absorbing carbon-dioxide released by the

plant during respiration. The flasks were equilibrated for 5 minutes by shaking.

The fluid level in the closed arm was raised depending on the reactions in the

flask. The increase in the fluid level was noted after a period of every 10

41

minutes for half an hour and the amount of oxygen consumed by the tissues in

terms of µl/gm was calculated by multiplying the values by the flask constants,

k of the corresponding flask.

The flask constant was defined by the equation

K = 0

fg

P

VT

273V +

where,

K = Flask constant

Vg = Volume of gaseous phase in flask and connecting tube

Vf = Volume of fluid in the flask

T = Temperature in absolute scale

α = Solubility of evolved or consumed gas in the fluid in the flask (in mg gas

/ ml fluid) at one atmospheric pressure and at temperature ‘T’ (0.026)

P0 = Standard pressure in terms of manometric fluid (10005.808)

Determination of Calorific Values (Karzinkin and Tarkovskaya, 1964)

20 mg of the dried sample of root and shoot were taken in separate

clean, dry, round bottom flasks. 3 ml of 5% potassium iodate and 20 ml of

concentrated sulphuric acid were added to each flask. Then the flask was

connected to a reflux condenser and heated for 1 hour on an electronic heating

mantle without shaking. Oxidation commences immediately on heating and the

lilac coloured free iodine is released. After one hour, the flask was cooled and

42

removed from the reflux condenser. The cooled solution (greenish yellow in

colour) was diluted with 50 ml of distilled water by pouring it carefully along

the sides. Free iodine was liberated in the process, making the solution pinkish

orange. Again the solution was heated on the mantle using low heat until the

colour and smell of iodine disappeared. The solution was then cooled and made

upto 250 ml with distilled water. 10 ml of 10% potassium iodide was added.

The flasks were kept in darkness for about 10 minutes. The solution was

titrated against 0.1 N sodium thiosulphate using starch as an indicator until the

blue colour disappeared. The process was done without using sample for the

blank. The calorific content of the tissue expressed in calories /gm was

calculated using the formula given by Vinberg et al., (1934).

Energy value = A

3.38 1000 0.667 b)(a ×××−

where,

‘a’ is the amount of sodium thiosulphate consumed by the blank

‘b’ is the amount of sodium thiosulphate consumed by the sample

‘A’ is the weight of the sample.

The reading denotes the energy values in terms of calorie per gram dry

weight of the sample.

Estimation of Chlorophyll

Chlorophyll contents were estimated by employing Arnon (1949)

method. 500 mg of fresh leaves were homogenized with 2 ml of 80% acetone

in a porcelain mortar and pestle. The homogenate was centrifuged for 10

43

minutes. Only the precipitate was kept in the tube. 2ml of 80% acetone was

added to the precipitate and homogenized. This homogenate was centrifuged

for 10minutes. The supernatant was kept for further analysis. This procedure

was repeated until all chlorophyll pigments were removed. The supernatant

was pooled and made up to 20 ml by adding 80% acetone and the optical

density was read in systronics UV-VIS 118 spectrophotometer at 645 nm.

Calculation

Total chlorophyll (mg /ml)

0.0202 × O.D 608 + 0.00802 × O.D 608

Chlorophyll a (mg /ml)

0.0127 × O.D 608 – 0.00269 × O.D 608

Chlorophyll b (mg /ml)

0.0229 × O.D 608 – 0.00488 × O.D 608

Enzyme Assays

Preparation of Plant Materials for Enzymes Studies (Kannan, 1967)

The root and shoot samples were separated and washed well with

distilled water. They were ground in a chilled mortars using ice cold Butanol :

Benzene : Acetone (2:1:1) mixture and stored in a refrigerator at 4oC overnight.

44

Excess of the solvent was decanted and the residual paste was dried at room

temperature. The dried residual powder, thus obtained, was stored at 4oC.

In the present study, triphenyl tetrazolium chloride (TTC) as an artificial

electron acceptor was employed to determine the activities of the various

enzymes investigated. The method of Kun and Abood (1949) was followed.

During enzymatic reaction, electrons are liberated from the substrate. The

colourless TTC accepts electrons and become reduced into red colour

formazon. The intensity of the reduced red coloured formazon, indicative of the

enzyme activity was matched with artificially reduced formazon standard.

Preparation of Enzyme Standard

100 mg of TTC was reacted with 5 ml of 10% sodium hydroxide to

produce the formazon. The formazon was then dried, weighed and known

concentrations, ranging from 1 to 1000 µg were prepared by diluting in 6ml of

toluene. Standard curve was drawn after reading the colour in UV-VIS

spectrophotometer 118 at 450 nm. Enzyme activities were expressed as mg of

TTC reduced / g dry weight of the tissue.

Determination of Enzyme Activities

Activities of dehydrogenases of alcohol, formate, glucose, glycerol and

succinate, ascorbic oxidase and total endogenous reductases were determined

(Kannan, 1967a).

Alcohol Dehydrogenase (ADH)

45

50 mg of dried material was extracted with 5 ml of 0.02 M sodium

pyrophosphate buffer (pH 8.6). To the entire extract, 1 ml of 0.1 M ethanol

and 1 ml of freshly prepared 0.1% TTC were added and the mixture was

incubated at 45oC for 30 minutes. The developed red colour was extracted with

6 ml of toluene and it was read in UV-VIS spectrophotometer 118 at 450 nm

along with a blank containing 6 ml of toluene alone.

Ascorbic Acid Oxidase (AAO)

50 mg of dried material was extracted with 5 ml of 0.2 M citrate

phosphate buffer (pH 5.4). To the entire supernatant, 1 ml of 0.1% ascorbic

acid and 1 ml of 0.1% freshly prepared TTC were added and the mixture was

incubated at 45oC for 30 minutes. The developed red colour was extracted with

6 ml of toluene and it was read in UV-VIS spectrophotometer 118 at 450 nm

along with a blank containing 6 ml of toluene only.

Formate Dehydrogenase (FDH)

50 mg of dried material was extracted with 5 ml of 0.5 M phosphate

buffer. To the entire supernatant, 1ml of 0.5 M sodium formate and 1 ml of

0.1% freshly prepared TTC were added and the mixture was incubated at 45oC

for 30 minutes. The developed red colour was extracted with 6 ml of toluene

and it was read in UV-VIS spectrophotometer 118 at 450 nm along with a

blank containing 6 ml of toluene alone.

Glucose Dehydrogenase (GDH)

46

50 mg of dried enzyme powder was extracted with 5 ml of citrate

phosphate buffer (pH 5.2). To the entire supernatant, 1 ml of 0.1% glucose and

1 ml of 0.1% freshly prepared TTC were added and incubated at 45oC for 30

minutes. The developed red colour was extracted with 6 ml of toluene and it

was read in UV-VIS spectrophotometer 118 at 450 nm along with a blank

containing 6 ml of toluene only.

Glycerol Dehydrogenase (GLDH)

50 mg of dried enzyme powder was extracted with 5 ml of 0.1 M

sodium pyrophosphate buffer (pH 8.6). To the entire supernatant, 1 ml of 1 M

glycerol and 1 ml of 0.1% freshly prepared TTC were added and incubated at

45oC for 30 minutes. The developed colour was extracted with 6 ml of toluene

and it was read in UV-VIS spectrophotometer 118 at 450 nm along with a

blank containing 6 ml of toluene alone.

Succinate Dehydrogenase (SDH)

50 mg of dried material was extracted with 5 ml of 0.1 M sodium

pyrophosphate buffer (pH 7.6). To the entire supernatant, 1 ml of 0.2 M sodium

succinate and 1 ml of 0.1% freshly prepared TTC were added and the mixture

was incubated at 45oC for 30 minutes. The developed red colour was extracted

with 6ml of toluene and it was read in UV-VIS spectrophotometer 118 at 450

nm along with a blank containing 6 ml of toluene only.

Total Endogenous Reductase (TER)

47

50 mg of dried material was extracted with 5 ml of 0.2 M phosphate

buffer (pH 7.4). To this, 1 ml of 0.1% freshly prepared TTC was added and the

mixture was incubated at 45oC for 30 minutes. The developed red colour was

extracted with 6 ml of toluene and it was read in UV-VIS spectrophotometer

118 at 450 nm along with a blank containing 6 ml of toluene alone.

Estimation of ββββ-amylase Activity (Bernfeld, 1955)

100 mg of the fresh material of root and shoot were separately ground

with 1 ml of distilled water in dry mortars. Then the extract taken in separate

tubes were centrifuged for 5 minutes. 1 ml of the supernatant of the extract was

incubated for 3 minutes at 20oC with 1 ml of substrate (1 g of starch in 0.016M

acetate buffer, pH 4.6). The enzyme reaction was interrupted by the addition of

2 ml of 3, 5 - dinitrosalicylic acid (1 g of dinitrosalicyclic acid and 30 g of

Rochelle salt in 20 ml of 2 N NaOH and the solution was made upto 100 ml

with distilled water). The tubes were heated for 5 minutes in boiling water,

cooled and 10 ml of distilled water was added. The coloured solution was read

in UV-VIS spectrophotometer 118 at 623 nm.

GC-MS Analysis

The methanol solvent extract of P.pinnata leaves was obtained from the

hot maceration and it was used for GC - MS analysis.

GC-MS was performed with GC clarus 500 Perkin Elmer equipment.

Compounds were separated on Elite-1 capillary column (100% Dimethyl

48

polysiloxane). Samples were injected with a split ratio of 10 : 1 with a flow rate

of helium 1ml per minute (carrier gas). Mass detector – Turbo mass gold –

Perkin Elmer software – Turbo mass 5.1 was used as detector. Other conditions

are oven temperature upto 110oC – 2 minutes hold; upto 280

oC at the rate of

5deg / min-9

minutes hold. Injector temperature was maintained at 250oC.

The constituents were identified after comparison with those available in

the compute library (NIST ver. 2.1) attached to the GC-MS instrument and

reported.

Statistical Analysis

Results obtained have been subjected to the following statistical

analysis:

(i) Standard deviation (S.D)

σ = 1N

x2

−

∑, where x represents the deviation of each score (X) from

the actual mean x and N the total number of observations.

(ii) Student’s ‘t’ test

Student’s ‘t’ test was used to compare two means by applying the

formula:

49

t = 22

21

21

SESE

XX

+

+

where 21 X,X represent the means compared and SE1 and SE2 their

respective standard errors.

Standard error was calculated using the formula:

SE = 1n −

σ

The level of significance for the ‘t’ at corresponding degrees of freedom

(DF = N-2) was read from the probability table given in Zar (1974)

where ‘N’ is the number of scores in both experiments.

(iii) Simple correlation coefficients (r)

The simple correlation co-efficient ‘r’ was determined from the

formula:

r =

( )( )

( ) ( )N

YY

N

XX

N

YXxy

2

2

2

2 ∑∑

∑∑

∑∑∑

−×−

−

(iv) Simple regression

50

The regression equation was computed using the least square method.

The basic formula followed was

Y = a + bX

where Y is the dependant variable

X is the independent variable

a is the intercept on Y

b is the slope

the formulae used to derive the values a and b are

b =

∑

∑2X

xy

a = XbY −

where X and Y denote the means of Y and X, ∑xy and ∑ 2X are

derived as follows:

∑ 2X = ( )

N

XX

2

2 ∑∑ −

∑ 2Y = ( )

N

YY

2

2 ∑∑ −

∑xy = ( )( )

N

YXxy

∑∑∑ −

Capital X and Y and denote the raw scores and small x and y, the deviation

scores.

Analysis of Variance (Downey and Heath, 1970)

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The participation of the total variance due to different experimental

conditions (nematode infections, varied concentrations of methanol leaf extract

of P.pinnata) were carried out following the procedure described by Zar

(1974). Values obtained at the different experimental conditions were tabulated

in different columns. For each column X and X2 were calculated. The sum of

the squares can be obtained using the following equation :

Total SS = ∑ 2X = ∑ 2X – ( )

N

X2∑

Between sum of squares = ∑X2

b =

N

X2∑ –

( )N

X2

T∑

Within sum of squares = ∑ 2x = ∑ 2X – ( )

N

X2

∑

Summing for all three groups ‘with in’ sum of squares can be obtained directly

by subtracting the ‘between’ sum of squares from the total sum of squares.

Mean squares = freedom of degrees ofnumber Respective

squares of Sum

‘F’ value = groups within''for squareMean

groups between''for squareMean

F ratios or values were interpreted by using F table D.11 given in Zar (1974).

v) Critical Difference (Gupta and Kapoor, 1976)

If the treatments show significant effect then it would be relevant to find

out which pair(s) of treatment differ significantly. For this, instead of

calculating Student’s t for different pairs of treatment means, the least

52

significant difference at the given level of significance was calculated. This

least difference is known as critical difference.

C.D for any two treatment means = t0.05 and t0.01 for error d.f × SE n

2

n = number of times a treatment is replicated

SE = mean squares for within group