CHAPTER 2 Mat and Methods - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/19374/8/08_chapter...

CHAPTER 2

Materials and

Methods

Materials and Methods

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2.1 MODEL PLANT MATERIAL AND ITS SIGNIFICANCE

2.1.1 Brinjal, Solanum melongena L. (Solanaceae)

Vegetables are an important source of minerals, vitamins and

plant proteins in human diets. Their cultivation is one of the more

important dynamic and major branches of agriculture. In rural areas,

small farmers prefer to cultivate vegetable crops for their livelihood,

because they fetch a good booty within a short period of time. Now big

farmers are also switching over to vegetable cultivation in their farm

houses, as it is becoming an important source of income. At the same

time the net return from the vegetable cultivation is declining day by day

due to heavy input of chemical pesticides and fertilizers to sustain good

production levels. The frequent applications of these synthetic

compounds are a cause of concern due to their deleterious effect on

human health and environment. India is producing about 98 million

tonnes of vegetables from an area of around 6.0 million ha. Among the

vegetable crops, brinjal is more popular and profitable. The added

advantage is that it is also grown as a ratoon crop.

Brinjal, S. melongena is a member of potato family, also known as

nightshade family plant. It has various synonyms like eggplant,

aubergine, melanzana, garden egg, patlican and guinea squash; and

cultivated under tropical and temperate parts of the world (Fig. 2.1). It is

a good source of vitamin B6, C, K, thiamin, and pantothenic acid, and

minerals such as magnesium, phosphorous, potassium, manganese,


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copper and dietary fiber. Amongst the insect pests of crop, brinjal shoot

and fruit borer, Leucinodes orbonalis (Guen.) (Pyralidae: Lepidoptera) is

the most destructive one. Its larvae damage all above ground parts of the

plant (leaves, apical shoots, stem, flower buds, flowers and fruits)

causing considerable yield losses. To cope up with the damage caused by

L. orbonalis, farmers resort to frequent sprays of toxic chemical

pesticides to kill the pest larvae instantly. Such extensive use of

pesticides cuts into profitability of farmers, makes brinjal more expensive

to consumers, poses health hazards and causes environmental pollution,

besides leading to establishment of resistant pest populations. In the

present study brinjal was used as a model system to investigate the

static and induced defense mechanisms in it against a herbivore and to

explore the use of concerned constituent(s) of the plant under IPM

approach to control L. orbonalis and other lepidopteran and coleopteran

pests. This will naturally curtail the cost of crop cultivation, storage loss

of food grains and related health hazard risks.

Plant Fruits

Fig. 2.1: Brinjal, Solanum melongena L.


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2.1.2 Maintenance of experimental brinjal plants

The seedlings of brinjal (variety, Bhagyamathi) were collected from

the nursery of Vegetable Section, Acharya N. G. Ranga Agricultural

University (ANGRAU), Rajendra Nagar, Hyderabad (A.P.). The healthy

seedlings were planted in the 4 x 5 m plots spacing at a distance of 30 x

60 cm at Indian Institute of Chemical Technology (IICT), Hyderabad farm

for experimentation. The normal recommended packages of practices

were followed to raise the crop. No botanical/chemical pesticide

(insecticide, fungicide or herbicide) or plant growth regulator were

applied on the crop.

2.2 MAINTENANCE OF INSECT CULTURES

2.2.1 Leucinodes orbonalis (Guen.) (Pyralidae: Lepidoptera)

The moth of L. orbonalis is small 18-24 mm in wing span and

having brownish and red markings on the whitish forewings. The males

and females are identically patterned. Its larva is the damaging stage

that bores into the leaf petiole and tender shoot in the early stage and

causes ‘dead heart’. Later it bores into flower bud, flower and developing

fruit resulting in bud loss and making fruit unfit for further development

and for human consumption (Fig. 2.2).


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Fig. 2.2: Brinjal shoot and fruit borer, Leucinodes orbonalis (Guen.)

The infested brinjal fruits were collected from the field and L.

orbonails larvae were segregated. These larvae were reared on cut brinjal

fruits till pupation under laboratory conditions 16L: 8D photo period, 28

± 2º C temp. and 65 ± 5% RH. The pupae were collected in Petri dishes

and placed inside nylon mesh cages (45 x 45 x 45 cm). The emerged

adults were allowed pairing in clean circular glass jars (15 x 25 cm) lined

inside by rough purple paper. The jars were covered with fine muslin

cloth and tightened with rubber bands. The adults were fed on 10%


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honey solution. On hatching, neonate larvae were reared on the artificial

diet containing dried brinjal fruit powder (Talekar et al., 1999).

2.2.1.1 Composition of artificial diet

A. Vitamins stock solution

Ingredients Quantity

Nicotinic acid 0.152 g

Calcium pantothenate 0.152 g

Riboflavin 0.191 g

Aneurine hydrochloride 0.095 g

Pyridoxine hydrochloride 0.095 g

Folic acid 0.095 g

D-Biotin 0.076 g

Cyano cobalamine 0.003 g

Water 50.00 ml

B. Fraction A

Ingredients Quantity

Chickpea flour 64.125 g

L-ascorbic acid 1.002 g

Sorbic acid 0.640 g

Methyl-4-hydroxy benzoate 1.067 g

Aureomycin 2.470 g

Yeast 10.260 g

Formaldehyde (40%) 0.86 ml

Vitamin stock solution 3.42 ml

Water 96.20 ml

Brinjal fruit powder 18.00 g

C. Fraction B

Agar-agar 20.00 g

Water 1000.00 ml


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2.2.1.2 Preparation of artificial diet

Young tender un-infested brinjal fruits were collected from the field

and washed thoroughly with tap water. The fruits were cut into thin

slices (2-3 mm thick) and dried under direct sunlight or in an oven set at

60 °C temp. for 48-72 hr. The dried slices were grinded to a very fine

powder. The powder in a tightly sealed container was stored in a

refrigerator, if not used immediately.

Fresh stock solution of vitamins was prepared each time. To

prepare diet, 1000 ml of distilled water was taken in a stainless steel

container and 20 g agar-agar added to it. The mixture was allowed to boil

slowly and stirred intermittently. When solution became clear, it was

allowed to cool to about 55 °C temp. and referred as Fraction B. In the

meantime, the ingredients of Fraction A were mixed in a large blender.

The cooled molten agar solution (Fraction B) was added to it and blended

the mixture thoroughly for about a min. The diet was poured instantly

into small plastic cups (4 x 5 cm) or glass Petri dishes (12.5 cm dia.) and

left to set undisturbed for 10 min. The surplus diet was refrigerated for

later use.

2.2.2 Spodoptera litura (Fab.) (Noctuidae: Lepidoptera)

Tobacco caterpillar, S. litura is 15-20 mm in size, 30-38 mm in

wing span and light brown in colour with dark spotted wings. It feeds

voraciously on the leaves of tobacco, castor, lettuce, cabbage, cauliflower,


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chillies, beetroot, peanuts, cotton, etc. It is believed to have potentially

high economic impact in terms of its direct crop damage (Fig. 2.3).

Fig. 2.3: Tobacco caterpillar, Spodoptera litura (Fab.)

The egg masses of S. litura were obtained from the Entomology

Division, Directorate of Oilseeds Research (DOR), Hyderabad. The culture

was further maintained on castor leaves (Ricinus communis L.) at IICT,

Hyderabad under laboratory conditions 16L: 8D photo period, 28 ± 2º C

temp. and 65 ± 5% RH. The egg masses which turned blackish were

placed over castor leaves in 12.5 cm dia. Petri dishes for hatching. The

newly hatched neonate larvae were carefully transferred with fine camel

hair brush over fresh castor leaves in glass jars (15 cm x 25 cm). The jars


39

were covered with muslin cloth and tightened with rubber bands. The

castor leaves and jars were changed daily in the morning till pre

pupation stage. For pupation, the jars were also provided with moist fine

sieved soil at the base. The adults emerged on the same day were allowed

to pair in nylon mesh cages (45 cm x 45 cm x 45 cm). The cages were

provided with 10% honey solution as adult food and castor leaves along

with folded green paper strips for oviposition. The egg masses were kept

age wise in 12.5 cm dia. Petri dishes for hatching and reared as stated

above.

2.2.3 Achaea janata (Linn.) (Noctuidae: Lepidoptera)

A. janata is known as semi looper due to its locomotion pattern. It

is about 15 mm long, 37-50 mm in wing span and greyish-brown in

colour with wavy lines on the fore wings (Fig. 2.4). Castor and croton are

its preferred hosts. Other occasional hosts include banana, cabbage,

pomegranate, rose, sugarcane, mustard, tomato, citrus, mango, etc. The

third instar caterpillars of A. janata were obtained from the DOR,

Hyderabad. The culture was further maintained on castor leaves at IICT,

Hyderabad under laboratory conditions 16L: 8D photo period, 28 ± 2º C

temp. and 65 ± 5% RH. The larvae were released over castor leaves in

glass jars (15 cm x 25 cm). The jars were covered with muslin cloth and

tightened with rubber bands. The castor leaves and jars were changed

daily in the morning till pre pupation stage. For pupation the jars were

also provided with moist fine sieved soil at the base. The adults emerged


40

on the same day were allowed to pair in nylon mesh cages (45 cm x 45

cm x 45 cm). The cages were provided with 10% honey solution as adult

food and castor leaves along with folded green paper strips for

oviposition. The eggs were kept age wise in 12.5 cm dia. Petri dishes for

hatching. The newly hatched neonate larvae were carefully transferred

with fine camel hair brush over fresh castor leaves in glass jars (15 cm x

25 cm) and reared as stated above.

Fig. 2.4: Semi looper, Achaea janata L.

2.2.4 Sitophilus oryzae Linn. (Curculionidae: Coleoptera)

The rice weevil, S. oryzae is a small about 2-3 mm long, stout in

appearance, dark brown in colour having four light reddish or yellowish

spots on elytra (Fig. 2.5). They attack wheat, corn, oats, rye, barley,

sorghum, buckwheat, rice, dried beans, cashew nuts, etc. It is regularly


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being reared at IICT, Hyderabad. Fifty numbers of 1-2 days old adult

pairs were isolated from the mother culture in clean glass jar (15 cm x 25

cm) containing 500 g whole wheat (Triticum aestivum L.). The jars were

covered with muslin cloth, tightened with rubber bands and left

undisturbed for a period of about 7-10 days under laboratory conditions,

15L: 9D photoperiod, 28 ± 2° C temp. and 65 ± 5% RH for mating and

oviposition. The adults were separated out thereafter, and food with eggs

was transferred to different culturing jars (20 cm x 30 cm) containing

fresh food for further development of life stages. These jars were held as

stated above and observed periodically. The adult beetles of subsequent

progenies (6-8 days old) were used for experimentation.

2.2.5 Tribolium castaneum (Herbst) (Tenebrionidae: Coleoptera)

The red flour beetle, T. castaneum is a small about 2.5-3.5 mm

long, reddish brown and flat beetle (Fig. 2.5). It attacks grains, seeds,

vegetable powders, dry fruits, oil cakes, nuts, museum specimens, etc. It

is regularly being reared at IICT, Hyderabad. Fifty numbers of 1-2 days

old adult pairs were isolated from the mother culture in clean glass jar

(15 cm x 25 cm) containing 500 g coarsely crushed/powdered rice (Oryza

sativa L.) and wheat flour (T. aestivum) enriched with 5% yeast. The jars

were covered with muslin cloth, tightened with rubber bands and left

undisturbed for a period of about 7-10 days under laboratory conditions,

15L: 9D photoperiod, 28 ± 2° C temp. and 65 ± 5% RH for mating and

oviposition. The adults were separated out thereafter, and food with eggs


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was transferred to different culturing jars (20 cm x 30 cm) containing

fresh food for further development of life stages. These jars were held as

stated above and observed periodically. The adult beetles of subsequent

progenies (6-8 days old) were used for experimentation.

2.2.6 Rhyzopertha dominica Fabr. (Bostrichidae: Coleoptera)

R. dominica is one of the smallest and most important grain-

infesting beetles. It is about 3 mm long, polished dark brown or black in

colour, strong flyer and capable to bore directly into the maize grains

with powerful jaws (Fig. 2.5). It is regularly being reared at IICT,

Hyderabad. Fifty numbers of 1-2 days old adult pairs were isolated from

the mother culture in clean glass jar (15 cm x 25 cm) containing 500 g

whole wheat (T. aestivum). The jars were covered with muslin cloth,

tightened with rubber bands and left undisturbed for a period of about 7-

10 days under laboratory conditions, 15L: 9D photoperiod, 28 ± 2° C

temp. and 65 ± 5% RH for mating and oviposition. The adults were

separated out thereafter, and food with eggs was transferred to different

culturing jars (20 cm x 30 cm) containing fresh food for further

development of life stages. These jars were held as stated above and

observed periodically. The adult beetles of subsequent progenies (6-8

days old) were used for experimentation.

2.2.7 Callosobruchus chinensis Linn. (Bruchidae: Coleoptera)

C. chinensis attacks pulses, cotton, sorghum and maize seeds in

storage. Besides Callosobruchus species are known to attack pigeon pea


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pods in the fields (Fig. 2.5). It is regularly being reared at IICT,

Hyderabad. Fifty numbers of 1-2 days old adult pairs were isolated from

the mother culture in clean glass jar (15 cm x 25 cm) containing 500 g

green gram (Phaseolus mungo L.). The jars were covered with muslin

cloth, tightened with rubber bands and left undisturbed for a period of

about 10 days under laboratory conditions, 15L: 9D photoperiod, 28 ± 2°

C temp. and 65 ± 5% RH for mating and oviposition. The adults were

separated out thereafter, and food with eggs was transferred to different

culturing jars (20 cm x 30 cm) containing fresh food for further

development of life stages. These jars were held as stated above and

observed periodically. The adult beetles of subsequent progenies (4-6

days old) were used for experimentation.

2.2.8 Trichogramma chilonis Ishii (Trichogrammatidae:

Hymenoptera)

The tiny pale yellow wasp, T. chilonis <1 mm long is a lepidopteran

egg parasitoid. For rearing, Corcyra cephalonica (Stainton) eggs

parasitized by it were obtained from the Project Directorate of Biological

Control (PDBC), Bangaluru (Karnataka). The emergence of adult wasp

started after eight days of host eggs parasitization (Fig. 2.6). These

emerged adults were collected every day, paired and transferred to glass

tubes (2.5 cm dia. × 14 cm long) containing 10% honey solution for

feeding and strips of C. cephalonica eggs for oviposition. The tubes were

held under the laboratory conditions (15L: 9D photoperiod, 26 ± 2º C


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temp. and 70 ± 5% RH) till adult emergence. For experimentation three

days old T. chilonis females were used. The details of C. cephalonica

rearing are given under stored grain pests.

Fig. 2.5: Stored grain pests. (1) Sitophilus oryzae L. (2) Tribolium

castaneum (Herbst) (3) Rhyzopertha dominica (F.) (4)

Callosobruchus chinensis (L.)

2.3 STATIC DEFENSE MECHANISM IN BRINJAL PLANTS

To find different secondary metabolite chemicals present in the

Brinjal plants as static defense, the whole leaf extracts of the S.

melongena plants were taken and exposed to certain inscet species with

an intention of knowing its pesticidal capacity.


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2.3.1 Preparation and extraction of plant material

The fresh brinjal leaves and fruits were collected from the field and

washed with distilled water to remove dust and other contaminants. The

clean leaves and fruits were dried in an oven at 30 ºC until all the

moisture content was evaporated. The dried material approximately 500 g

was milled to 4.0 mm particle size in an electric grinder. The ground

leaves and fruits were subjected to extraction in soxhlet apparatus using

1200 ml AR grade acetone as solvent. The extraction continued to 15-18

hr and the solvent was evaporated under reduced pressure in a rotary

vacuum evaporator (Heidolph Laborota 4000) at 40-45 oC. The yield of

extraction has been shown in (Fig. 2.7). The extracts were diluted in

acetone to get 500 mg/ml (w/v) concentration and denoted as ‘crude

extract’ which was employed in all the experiments.


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Fig. 2.6: Parasitization of host egg and development of

Trichogramma chilonis Ishii

2.3.2 Fractionation by chromatography

The crude extract of brinjal leaves and fruits was chromatographed

on a silica gel column (4 cm dia., 50 cm length) with chloroform [(100%)

(Fraction 1)], ethyl acetate [(100%) (Fraction 2)] and methanol [(100%)

(Fraction 3)] as eluents. Each eluted material was further concentrated

using a rotary vacuum evaporator to remove excess solvent and stored at

-20 ºC for further bioassay studies (Fig. 2.7).


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Fig. 2.7: Extraction of brinjal leaves and fruits and fractionation of

crude extract

2.3.3 Extraction and isolation of phenolic compound

Dried and powdered brinjal fruits (500 g) were extracted with

methanol in a soxhlet apparatus for 25-30 hr. The resulting methanol

extract was evaporated to dryness under reduced pressure to afford

syrupy residue (40.610 g). A portion of methanol extract (5 g) was

subjected to column chromatography in a silica gel column (60-120 mm

mesh, 60 x 4 cm) and eluted with CHCl3/MeOH (80:20) in the increasing


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order of polarities to afford three fractions. Fraction 3 was found more

active than Fraction 1 and 2. The Fraction 3 was, therefore, further

subjected to column chromatography and eluted with CHCl3/MeOH

(95:5) to give 0.090 g active compound. This active compound was

identified as -1, caffeic acid methyl ester (CME) by using analytical

techniques UV, IR and NMR (Fig. 2.8).

2.3.4 Extraction and isolation of two alkaloid compounds

Dried and powdered brinjal fruits (500 g) were extracted with

methanol in a soxhlet apparatus for 25-30 hr. The resulting methanol

extract was evaporated to dryness under reduced pressure to afford

syrupy residue (40.610 g), which was partitioned between 10% acetic

acid and toluene-ether (1:1). After addition of NH3 to the aqueous layer,

the latter was extracted with CHCl3:MeOH (70:30). After evaporation of

the organic solvents in rotary evaporator, residue (3 g) was subjected to

chromatography over silica gel (100-200 mm mesh) and eluted with

CHCl3:MeOH:H2O (70:25:5) to obtain two compounds alkaloid-1 (225 mg)

and alkaloid-2 (212 mg) (Fig. 2.9).


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Fig. 2.8: Isolation and identification of active phenolic compound,

caffeic acid methyl ester in brinjal fruit extract


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Identification of alkaloids, solasonine and solamargine based on extensive UV, IR, NMR and ESI-MS studies.

Fig. 2.9: Isolation and identification of active alkaloid compounds,

solasonine and solamargine in brinjal fruit extract.

500 g of S. melongena fruit powder

Soxhlet extraction with

Methanol up to 25-30 h at 59º C

Concentrated under reduced

pressure using rotavapor

40 .61 g of fruit extract

After addition of NH3 to the

aqueous layer, the latter was

extracted with CHCl3 and

MeOH (70:30)

Alkaloid work -up

3 g of residue

Column Chromatography

Eluted with CHCl3:MeOH: H2O

Alkaloid – 1 (255 mg)

Solasonine

Alkaloid – 2 (212 mg)

Solamargine


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2.4 BIOLOGICAL STUDIES AGAINST AGRICULTURAL

FIELD CROP PESTS

2.4.1 Antifeedant assay

Antifeedant activity of test compounds, crude extract, purified

chromatographic fractions and pure compounds CME, solasonine and

solamargine were assessed against S. litura, A. janata and L. orbonalis.

The experiments were conducted according to the classical no-choice

leaf-disc method (Fig. 2.10) against S. litura and A. janata (Devanand and

Usha Rani 2011). Circular leaf discs of 3.5 cm dia. (≈ 10 cm² leaf area)

were cut from fresh castor leaves. The test compounds were dissolved in

acetone to get different concentrations viz., 5, 10, 15, 20 and 25 mg/mL

of crude extracts; 0.5, 1.25, 2.5, 3.75, 5.0 mg/mL of chromatographic

fractions; and 0.005, 0.0125, 0.025, 0.0375, 0.05 mg/mL of pure

compounds. Two mL of each concentration was applied on the upper

(ventral) surface of the leaf discs with the aid of a glass atomizer. Thus

the compound deposited on the leaves was equal to 10, 20, 30, 40 and

50 mg of crude extract/10 cm² area; 1.0, 2.5, 5.0, 7.5 and 10.0 mg of

chromatographic fractions/10 cm² area; and 0.01, 0.025, 0.05, 0.075,

and 0.10 mg pure compounds/10 cm² area. Azadirachtin standard 95%

(Sigma-Aldrich) was taken as the active control for comparison and

dissolved in acetone to get 0.0015, 0.003, 0.0045, 0.006, 0.0075 mg/mL

of azadirachtin active ingredient. The application of two mL of each


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concentration per leaf disc will be equal to deposition of 0.003, 0.006,

0.009, 0.012, 0.015 mg/10 cm² area. The leaf discs were treated with the

same volume of acetone alone, which served as control treatment. The

solvent was allowed to evaporate by air drying briefly for a couple of

minutes and the discs were placed inside glass Petri dishes (9 cm dia.)

lined with moist absorbent tissue paper at the base. For studies against

L. orbonalis thin layer of artificial diet of 3.5 cm dia. (≈ 10 cm² surface

area) was treated.

In each Petri dish, a single pre starved healthy III instar larva of

each test insect was released separately for assessing the feeding

deterrent activity of the test compounds. Progress of the consumption of

the leaf area was measured at 6, 12 and 24 hr in each treatment. Areas

of untreated and treated leaf discs consumed were measured using AM-

300 leaf area meter (ADC, Bioscientific Limited, England, UK). The

antifeedant index was then calculated as (C-T)/(C +T) x 100, where C is

consumption of control discs and T is consumption of treated discs

(Belles et al., 1985). For each dose, 30 experimental sets were assayed.

Each test was replicated five times (N= 150). For L. orbonalis artificial diet

was weighed at different time intervals and diet consumed was

calculated.


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Normal feeding (Untreated leaf disc) Antifeedant (Treated leaf disc)

Fig. 2.10: Antifeedant activity of test compounds

2.4.2 Topical application

Toxicity of the compounds was determined by topical application

against III instar larvae of S. litura and A. janata following the method of

Devanand and Usha Rani (2011). Different concentrations of each test

compound 0.1, 0.2, 0.3, 0.4 and 0.5 g/mL were made in acetone. A dose

volume of two µL was applied directly on the dorsal side of the larva at

thoracic region (Fig. 2.11) using micro applicator (Hamilton, 600 series,

62RNR). Thus the insects were treated at a dose level of 0.2, 0.4, 0.6, 0.8

and 1.0 mg/larva. Azadirachtin standard taken as the active control for

comparison was dissolved in acetone to get 0.00125, 0.0025, 0.005 and

0.0075 g/mL concentrations. The application of two µL of each

concentration resulted to the dose level of 0.0025, 0.005, 0.010 and

0.015 mg/larva. In the control treatment larvae were treated with equal

volume of acetone only. The treated and untreated S. litura and A. janata

larvae were individually fed on fresh castor leaves daily in glass Petri


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dishes (9 cm dia.). L. orbonalis larvae were also treated in the same way

but fed on artificial diet. Three independent trials were conducted each

replicated four times, leading to overall 12 replications per concentration

of test compounds. In each replication five larvae and total 60 larvae per

treatment were taken. Mortality was recorded after 24 hr of treatment.

Larvae that lost elasticity and showed no response when provoked by

hair brush were considered as dead.

Fig. 2.11: Topical application of compounds

2.4.3 Larval growth inhibitory activity of test compounds

Larval growth inhibitory activity of test compounds along with

standard was studied by oral feeding method (Usha Rani and Devanand

2011). Test compounds were diluted in acetone in such a way that the

crude extracts of leaves and fruits were applied @ 10, 20, 30, 40 and 50

mg/10 cm2, chromatographic fractions @ 1, 2.5, 5, 7.5 10 mg/10 cm2

and pure compounds @ 0.01, 0.025, 0.05, 0.075 and 0.1 mg/10 cm2 on

the upper (ventral) surface of circular leaf discs of 3.5 cm dia. (≈ 10 cm²

leaf area) with the aid of a glass atomizer as described under antifeedant


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assay. Similarly Azadirachtin standard taken as active control was used

@ 0.003, 0.006, 0.009, 0.012, 0.015 mg/10 cm² area. Control discs were

sprayed with acetone alone. The solvent was allowed to evaporate by air

drying briefly for a couple of minutes and the discs were placed in glass

Petri dishes (9 cm dia). The surface treated artificial diet was used for

studies against L. orbonalis. Pre starved III instar larvae of S. litura

(average weight 299 ± 35 mg per larva) and A. janata (average weight 335

± 40 mg per larva) were released separately into each petri dish. Each

test was replicated five times (N= 150). After 36 hr of feeding the larvae

were transferred to the clean Petri dishes containing fresh normal

untreated food. The cleanliness was maintained and fresh food was

provided daily up to 7 days. The larval development was observed

periodically and weight gain was recorded after 7 days of treatment.

Percent growth inhibition was calculated by following the formula

suggested by El-Aswad et al. (2003), which is as under:

Growth inhibition (%) = [(CL-TL) / CL)] x 100

Where CL is the larval weight gained in the control and TL is the

larval weight gained in the treatment. The mean of 12 replicates was

taken to calculate percent mortality with standard error for each

concentration after 7 days of treatment.

2.4.4 Effect of test compounds on pupal and adult development

Another set of experiment was conducted to study the effects of

test compounds on pupal and adult development after oral ingestion by


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the advanced stage larvae. Freshly moulted IV instar larvae of S. litura

and A. janata were allowed to feed on the castor leaves treated with test

compounds at different concentrations of crude extract 40 and 50 mg/10

cm2 area, chromatographic fractions 7.5 and 10 mg/ cm2 and pure

compounds 0.075 and 0.10 mg/10 cm2 area. After moulting, the larvae

were separated out and reared on untreated fresh food up to adult

development. For L. orbonalis artificial diet was used as food. Pupal

weight and percent pupal mortality, adult abnormality and adult

emergence were recorded. For each concentration, 30 experimental sets

were assayed. Each test was replicated five times (N= 150).

2.4.5 Effect of test compounds on larval mid-gut proteases

2.4.5.1 Isolation of protease enzyme

Fourth instar larvae of S. litura and A. janata were allowed to feed

on the circular castor leaf discs of 3.5 cm dia. (≈10 cm² leaf area) sprayed

with test compounds at effective concentrations (crude fruit extract 50

mg/10 cm2, chromatographic fractions at 10 mg/10 cm2 and pure

compounds at 0.1 mg/10 cm2 area) for 12 hr. Leaves sprayed with

acetone served as control. After exposure the larvae were dissected and

their guts were removed over ice cold normal saline and homogenized

immediately in 50 mM Tris-Cl, pH 8.0 [1:1 ratio (w/v)]. The crude gut

homogenate was centrifuged at 14,000 rpm for 10 min at 4 ºC. The clear

supernatant was filtered through a Whatman No.1 filter paper and

transferred to a pre-chilled eppendorf tube. The samples were stored at -


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20 ºC if not used immediately. Protein analysis in the gut contents was

carried out according to the procedure described by Bradford (1976),

with BSA as a standard protein.

The proteolytic activity was measured according to the method

described by Marchetti et al. (1998) with slight modifications using 2%

(w/v) azocasein as a substrate. Typically, the sample (20 µL containing

10-20 µg protein) and 0.1 M Tris buffer, pH 10.0 (500 µL) were pre-

incubated for 5 min at 30 ºC before the addition of 25 µL 2% azocasein

(w/v, in glass-distilled water). After 30 min of incubation, the reaction

was stopped using 400 µL 10% trichloroacetic acid (w/v). Tubes were

kept on ice for 10 min and then centrifuged at 5,000 rpm for 5 min; 500

µL aliquots of the supernatant were withdrawn and mixed in a cuvette

with 500 µL 1 M NaOH and absorbance at 420 nm was determined.

Blanks (test tubes without samples) were run in all cases. One unit of

proteolytic activity is the amount of enzyme that causes the formation of

1 µg of TCA-soluble positive material per min. Each experiment

replicated five times (N= 50). Specific activity of total protease activities

(U) was calculated as:

Absorbance value at 420 nm (test) - Absorbance value at 420 nm (blank)

30 min x mg protein


58

Assays to quantify specific serine protease activities were

conducted using paranitroanalide-conjugated peptide substrates in 96-

well micro-titre plates. Trypsin-like activity was detected using BApNA

(N-benzoyl DL-arginine p-nitroanilide), whereas for chymotrypsin-like

activity SAAPFpNA (N-succinyl-alanine-alanine-pro-phe-p-nitroanilide)

and for elastase-like activity SAAApNA (N-succinyl-alanine-alanine-

alanine-p-nitroanilide) substrates were used. Briefly, 0.1 M Tris buffer,

pH 10.0 (1.35 mL) and the sample (15 µL containing 5-15 µg protein)

were pre-incubated for 5 min at 30 ºC before the addition of 0.2 mL 7.8

mM BApNA/ SAAPFpNA/ SAAApNA (in 13% dimethyl sulfoxide; 1 mM

final concentration) to start the reaction. After 10 min of incubation, the

reaction was stopped with 0.75 mL 30% acetic acid and absorbance was

measured at 410 nm. Assays were carried out in triplicate and

appropriate blanks were run in all cases. The molar extinction coefficient

(M-1 cm-1) for pNA at 410 nm equals to 8800 (Erlanger et al., 1961) was

taken in to account to calculate trypsin, chymotrypsin and elastase

activities (BApNA/ SAAPFpNA/ SAAApNA units/min/mg protein) using

the formula.

Absorbance value at 410 nm/min x 1000 x volume of reaction mixture

8800 x mg protein in the assay


59

2.5 BIOLOGICAL STUDIES AGAINST STORED GRAIN PESTS

2.5.1 Vapour toxicity

The vapour toxicity of the plant extracts from leaves and fruits

were separately evaluated against stored product pests according to the

method described by Usha Rani et al., (2011) (Fig. 2.12). In brief, small

100 ml capacity plastic airtight containers (5.8 cm dia. x 5 cm height)

were used as fumigation chambers and filled with 30 g of respective

rearing diet of the test insects, T. castaneum, S. oryzae, C. chinensis and

R. dominica. One mL of each test compound was applied individually to a

small ball of absorbent cotton weighing 300 mg which was attached

underneath the aluminum screw cap of each container. Six

concentrations of crude plant extracts (10, 20, 30, 40 and 50 mg/100

mL), five concentrations of each of the chromatographed fraction of

brinjal fruit extract (0.02, 0.04, 0.06, 0.08, and 0.10 mg/ 100 mL) and

five concentrations of purified compounds of brinjal fruit extract (0.01,

0.025, 0.05, 0.075 and 0.10 mg/100 mL) were used to calculate EC50

values. An additional absorbent cotton ball was treated only with

acetone, which served as control for each of the extracts. Twenty

unsexed adults of each species, 2-6 days old, were released in to the

chamber and the container sealed. All tests were carried out at 28 ± 2ºC

temp. and 65 ± 5% RH. Insect mortality was recorded after 24, 48 and 72

hr post treatment after confirmation by probing the body with a slender

camel hair brush. There were five replicates per treatment and the tests


60

were repeated three times on different days each time. The LC50 values

were calculated by Probit analysis for each test insect species (Finney,

1971).

Fig. 2.12: Evaluation of vapour toxicity of test compounds against

stored grain insect pests

2.5.2 Contact toxicity

Contact toxicity tests were carried out following the bioassay

procedure as described by Usha Rani et al., (2011). The samples were

diluted in acetone so as to get 10, 20, 40, 60, 80 and 100 mg/mL

concentration of crude leaf and fruit extracts; 1, 2, 4, 6, 8 and 10 mg/mL

of purified fractions and 0.01, 0.02, 0.04, 0.06, 0.08 and 0.1 Mg/mL of

purified compounds. A volume of one mL of each concentration was

applied on Whatman No. 1 filter paper using micro applicator. The

treated filter paper were kept in Petri dishes. The treated filter paper

were allowed to evaporate for about 15 min after treatment. Acetone

alone was used for control treatment. The adults of each test insect

species were released carefully in the Petri dishes and mortality counts

were made after 24, 48 and 72 hr of exposure.

Cotton ball treated with test compound (Treated) Cotton ball treated with

solvent (Control)


61

Fig. 2.13: Evaluation of contact toxicity of test compounds against

stored grain insect pests by filter paper impregnation

method

2.5.3 Flour disc bioassay

A flour disc bioassay was used to study the feeding deterrent

effects of crude plant extracts, chromatographic fractions and pure

compounds extracted from brinjal fruits against four stored grain insect

pests, T. castaneum, S. oryzae, C. chinensis and R. dominica following the

method of Xie et al. (1996) with slight modifications as suggested by

Huang et al., (2000) (Fig. 2.14). Flour discs (80 ± 7 mg/disk) were

prepared using 200 µL of a stored suspension of wheat flour (T.

castaneum) and green gram powder (C. chinensis) in water (50 g in 100

mL) separately. Test solutions of each compound were prepared in

acetone to apply at different concentrations viz., crude extracts @ 10, 15,

20, 25 and 30 mg/disc; isolated compounds @) 2, 4, 6, 8 and 10 mg/disc

Filter paper treated with solvent (Control)

Filter paper treated with Test compound (Treated)


62

and purified compounds glycol alkaloids @ 0.015, 0.030, 045, 0.060 and

0.075 mg/disc for all the test insects. In control acetone alone was used.

The solvent was allowed to evaporate for 24 hr.

Two flour discs of the same treatment were weighed and placed in

a plastic container (4 cm dia. x 2 cm height) per treatment. Then 10 pre-

weighed larvae of each test insect were released in to separate containers

containing their respective treated discs. A total of 15 replicates were set

up for each compound and control. After 7 days, the flour discs and live

insects were weighed again. Feeding deterrence index was calculated

following Huang et al. (2000) as under:

Feeding deterrence index (FDI) (%) = [(C–T)/C] x 100

Where, C - consumption of control discs

T - consumption of treated discs

Fig. 2.14: Feeding deterrent assay by flour disc method

Feeding deterrent assay by flour disc method


63

2.5.4 Effects on progeny production

The tests were carried out following the bioassay procedure

described by Usha Rani et al., (2011). The tested concentrations 10, 15,

20, 25 and 30 mg in 100 µL/30 g food of each of the leaves and fruits

extracts; 1, 2, 3, 4 and 5 mg in 100 µL/30 g food of chromatographic

fractions and 0.1, 0.25, 0.5, 0.75 and 1.0 mg in 100 µL/30 g food of pure

compounds were made in acetone and applied n respective food material

using a micro applicator. Acetone treated respective food served as

control for each test insect species. The treated food was thoroughly

shaken immediately after treatment till all seeds get uniformly coated.

The solvent was allowed to evaporate for about 15 min after treatment.

After complete evaporation of the solvent, 10 pair of adults of S. oryzae,

R. dominica, T. castaneum and C. chinensis were released in to the jars

(15 x 25 cm) containing their respective food. After 21 days all adults

(dead and live) were removed and kept the jars at laboratory conditions

28 ± 2°C and 65 ± 5% RH for an additional period of 45 days for F1

progeny emergence. At the end of this period, the jars were opened and

the total number of insects counted. The increase or decrease in number

indicates the effect of the compounds on the progeny. Each experiment

was replicated 15 times. The percent progeny inhibition rate was

calculated using the formula of Tapondjou et al. (2005).

Cn – Tn Progeny Inhibition rate (%) = _____________ x 100 Cn


64

Where, Cn = Number of newly emerged insects in the untreated control

Tn = Number of newly emerged insects in the treatment

2.5.5 Repellent effects

The repellent effects of tested crude extracts, purified fractions and

pure compounds were studied using a glass made olfactometer (Stem –12

cm, arms –12 cm, angle 120o) (Fig. 2.15). The detailed description of the

olfactometer has been given by Goyal et al. (2005). Small cotton swabs

(100 mg) were impregnated with 25 µl acetone solutions of the tested

extracts at different concentrations. The impregnated cotton swabs were

inserted in to the distant ends of the Y olfactometer tube. Each single

trial comprised the test sample and acetone impregnated cotton swabs

for comparison. Initially, five insects were released in to the opening stem

of the tube ensuring their random movement inside the olfactometer. The

number of insects that covered a distance of more than ¾ length of side

arm within 15 min of their introduction were classified as positive

responders while the remaining were classified as negative responders.

The number of insects present in the control (Nc) and test (Nt) arms of the

‘Y-tube’ were recorded at an intervals of 1, 3, 5, 10 and 15 min. Tests

were carried out at 28 ± 2o C temp. and 60 ± 5% RH. To minimize the

impact of experimental errors, the experiment was repeated 25 times at

different time intervals. Percent repellency was calculated using the

formula of Tapondjou et al. (2005).


65

Nc – Nt Percent Repellency (PR) = ___________ × 100

Nc

Where Nc = number of insects rested at control arm

Nt = number of insects present at test arm

The mean repellency value of each test product was calculated and

assigned to repellency classes (Juliana and Su, 1983) from 0 to V:

Class 0 (PR < 0.1%), Class I (PR = 0.1-20%), Class II (PR = 20.1- 40%),

Class III (PR = 40.1- 60%), Class IV (PR = 60.1- 80%), Class V (PR = 80.1

– 100%).

Fig. 2.15: Y – tube glass olfactometer

2.6 INDUCED DEFENSE MECHANISM IN BRINJAL PLANTS

2.6.1 Plant treatment

Effect of L. orbonalis infestation on primary metabolites or nutritive

compounds and defensive enzymes in brinjal, S. melongena plant system

were studied by conducting experiments on 50-55 days old brinjal plants

Y- tube olfactometer


66

grown under green house conditions. Healthy and well established

brinjal plants at reproductive phase were identified and isolated from the

rest. On these plants, III instar L. orbonalis larvae were released carefully

with a fine camel hair brush on fruits at the rate of single larva per fruit

per plant. The larvae settled on the fruits to feed within 5-10 min. after

their release. These plants were termed as treated plants. The un-

infested brinjal plants were termed as control or normal healthy plants.

All these treated and control plants were maintained uniformly

under the green house conditions during July - September, 2009. Five

plants per replication and 12 replications per treatment were taken (n=

60). At different time intervals of insect releasing, the leaves from infested

and normal healthy plants were collected to study the changes in

induction of plant primary or nutritive compounds, defensive enzymes,

phenolic compounds and volatile compounds due to pest feeding. The

details of study parameter, treatment and interval of brinjal sample

leaves collection has been shown in Table 2.1.

Table: 2.1. Details of treatment and interval of sample leaves

collection

S. No.

Study Parameter

Treatment Sample interval

1. Primary or nutritive compounds

Treated (Infested leaf and fruit) Control (Un-infested leaf and fruit)

24, 48, 72 and 96 hr of insect infestation


67

2. Defensive enzymes



3. Phenolic compounds



4. Leaf and fruit volatile compounds


72 hr of insect infestation

2.6.2 Plant primary or nutritive compounds

Standard methods were employed to estimate amino acids (Moore

and Stein, 1954), carbohydrates (Dubois et al., 1956), proteins (Lowry,

1951) and phenols (Singleton and Rossi, 1965). For extracting the

biochemicals, the leaves were excised at the upper end of petiole followed

by maceration in a tissue homogenizer. The biochemicals estimations

were expressed as µg/g fresh weight (FW).

2.6.2.1 Amino acid estimation

Extraction and assay

1. 0.1 g leaves weighed and homogenized with 10% TCA. Kept the

samples at cool condition for 30 min.

2. After 30 min. of incubation samples were filtered and used for amino

acid estimation.

3. To 0.1 ml of plant leaf extract, 2.0 ml Ninhydrin reagent was added

and boiled for 10 min.


68

4. After 10 min. the samples were cooled and made up to 10 ml with

DH20.

5. Absorbance was measured at 570 nm (Ninhydrin method).

2.6.2.2 Carbohydrate estimation


1. 0.1 g leaves weighed and homogenized with 80% cold ethanol.

2. The samples were centrifuged at 5000 rpm for 10 min. and

supernatant was used as plant extract for carbohydrate estimation.

3. To 0.1 ml of plant extract, 0.5 ml 5% phenol and 2.5 ml sulfuric acid

were added.

4. Stock solution of glucose (1 mg/ml) was prepared and added to all

test tubes and a blank was maintained for the series.

5. Samples were incubated at 30 oC for 20 min.

6. Absorbance was measured at 490 nm (Phenol-Sulphuric acid

method).

2.6.2.3 Protein estimation


1. 0.1 g plant leaf material was weighed and homogenized with 10 ml

cold 80% Acetone.

2. The sample was centrifuged at 10000 rpm at 4 oC for 10 min.

3. The above step was repeated by adding 5 ml 80% acetone.

4. Again centrifuged at 10000 rpm at 4 oC for 10 min.

5. Supernatant was decanted and the pellet was suspended in 2 ml Tris-

HCl buffer 0.1 M (pH-8.0).


69

6. The mixture was shaken for 5 min. and centrifuged at 5000 rpm for 5

min. The supernatant was collected which contain protein.

7. To 0.5 ml of plant extract, 0.5 ml distilled water was added.

8. Stock solution of BSA was prepared and added to all test tubes and a

blank was maintained for the series.

9. 2 ml of Solution D was added to all test tubes.

10. 0.2 ml Folins reagent (1:1) was added to each tube.

11. Sample tubes were incubated for 30 min.

12. Absorbance was measured at 600 nm (Lowry method).

2.6.2.4 Phenols estimation


1. 1 g of leaf material was weighed and 10 ml 80% methanol was added.

2. The mixture was agitated for 15 min. at 70 oC and the supernatant

was used as plant extract for phenol estimation.

3. To 100 µl of the plant extract 1 ml distilled water was added.

4. 250 µl of Folin – Ciocalteu’s reagent (1:1 dilution) was added to all

tubes and 2.5 ml sodium carbonate solution (20%) was added

sequentially in each tube.

5. Soon after vortexing the reaction mixture, the tubes were incubated at

room temperature for 1 h.

6. Absorbance was measured at 765 nm (Folin – Ciocalteu’s method).

2.6.3 Plant defensive enzymes

Standard methods were employed to estimate Peroxidase

(Hammerschmidt et al., 1982), Catalase (Aebi, 1983), Phenylalanine


70

Ammonia Lyase (Dickerson, et al., 1984), Super Oxide Dismutase (Beyer

and Fridovich, 1987) and Polyphenol Oxidase (Thaler et al., 1996). For

measuring all the enzyme activities, the leaf material from the L.

orbonalis infested and the healthy (normal) plants were collected and the

leaf materials were homogenized using preferred buffer solutions.

2.6.3.1 Peroxidase (POX)


1. 1 g of plant leaf material was weighed and homogenized with 0.1M

phosphate buffer (pH – 7.0).

2. Sample was centrifuged at 10000 rpm for 20 min. and collected

the enzyme.

3. To 0.5 ml (1:10 dilution) plant enzyme 1.5 ml 0.05 M pyrogallal was

added.

4. After 10 min. 0.5 ml 1% H2O2 was added.

5. All the sample tubes were incubated in water bath at 25 oC for min.

6. 1 ml of 2.5 N H2SO4 was added to stop the reaction.

7. Absorbance was measured at 420 nm.

2.6.3.2 Catalase (CAT)


1. 1 g of plant leaf material was weighed and homogenized with 0.1M

extraction buffer (phosphate buffer pH 7.0 containing 100 mg PVP

and 0.1 M EDTA).


71

2. The sample was centrifuged at 6000 rpm at 4 oC for 5 min. and

collected the plant enzyme.

3. To 2.8 ml of 50 mM phosphate buffer (pH– 7.0), 120 µl of enzyme

extract was added.

4. Next 80 µl of 0.5 M H2O2 was added.

5. The reaction was stopped after 5 min. by adding 2.5N H2SO4.

6. Absorbance was measured at 240 nm and enzyme activity was

calculated as µmol H2O2/min.

2.6.3.3 Phenylalanine ammonia lyase (PAL)


1. 1 g of plant leaf material was weighed and homogenized in 10 ml of

sodium borate buffer (0.1 M, pH – 7.0).

2. Centrifuged for 20 min. at 10000 rpm and collected the enzyme

sample.

3. To 100 µl of plant enzyme 600 µl of 1mM L-Phenylalanine and 500 µl

of 50 mM Tris HCl (PH – 8.8) was added.

4. The sample tubes were incubated for 60 min. at room temperature.

5. By adding 2N HCl the reaction was arrested.

6. The mixture was extracted with 1.5 ml of toluene by vortexing for 30

seconds.

7. Toluene was recovered after centrifugation at 3000 rpm for 5 min.

8. The absorbance was measured at 290 nm.


72

2.6.3.4 Superoxide dismutase (SOD)


1. 1 g of plant leaf material was weighed and homogenized in a solution

containing 5 ml of 50 mM phosphate buffer (pH 7.0) with 1%

polyvinylpyrrolidone.

2. Centrifuged at 15000 g for 10 min. and plant enzyme was collected.

3. To 20 µl enzyme sample 1 ml of reaction mixture was added along

with 10 µl of riboflavin (riboflavin is to be added only in the last).

4. The tubes were illuminated for 7 min. in an aluminum foil lined box

containing 2 x 20 W florescent lamps.


2.6.3.5 Poly phenol oxidase (PPO)


1. 1 g of plant leaf material was weighed and homogenized with 10 ml of

pH – 7.0 Phosphate Buffer (0.1 M) containing 1% polyvinyl

polypyrollidoned with 0.4 ml 10% triton X –100.

2. Centrifuged at 6000 rpm for 15 min.

3. To 30 µl of enzyme extract, 2.8 ml of sodium phosphate buffer pH –

8.0 and 100 µl of 15 mm catechol were added and the volume was

made to 3 ml by addition of buffer.


2.6.4 Induction of phenolic compounds


Leaves and fruits were collected from infested and control plants as

per details given in Table 2.1. One g leaf was weighed and extracted in 20


73

ml of 95% methanol for 3 days under continuous shaking condition. The

solution was filtered and evaporated into dryness by rotavapor. The dried

material was re-suspended in 2 ml of HPLC grade methanol and was

stored at -20 oC until HPLC analysis. The amount of total phenolics in

extracts was estimated according to the Folin-Ciocalteu method

(Singleton and Rossi, 1965). The total phenolic content was compared

with gallic acid as standard and expressed in terms of Gallic Acid

Equivalent (GAE).

The Phenolic acids were analyzed using HPLC according to the

method described by Tuzen and Ozdemir (2003). The separation of

phenolic compounds was accomplished on a Gilson GX-271 semi

preparative HPLC system. The column was C18 (2.5 x 30 cm Gilson

apparatus), and a liquid handler with auto injector was employed. For

phenolic acid analysis a gradient elution programme was applied, and

elution was carried out with solvent A [acetic acid: water (2: 98 v/v)] and

solvent B [acetic acid: acetonitrile: water (2: 30: 68 v/v)] as mobile phase.

Initial condition was programmed as 100% A, 0–5 min.; changed to 100%

B, 25–35 min.; with a flow rate of 1.0 mL/min. and the injection volume

was 200 µl. The signals were detected at 254 nm. Retention times for the

standard compounds and the major peaks in the extract were recorded.

Qualitative and quantitative estimation of phenolic compounds from each

sample were analyzed by comparing retention times obtained from

standard chromatograms of phenolic acids.


74

2.6.5 Volatile infochemicals released due to pest feeding

in brinjal and its impact on pest’s natural enemies

2.6.5.1 Extraction of plant volatile compounds

Fresh brinjal leaves and fruits were collected in the morning hours

as per details given in Table 2.1 and extracted immediately. The leaves

and fruits of the infested and normal brinjal plants of the same variety

were weighed (100 g) separately and dipped in the solvent DCM (100 mL)

for 30 sec. at room temperature. The collected volatile chemicals were

concentrated using nitrogen gas. The solvent trapped the chemicals

present on the plant leaf surface, including emitted volatiles, if any. This

permits the extraction of several chemicals, including n-alkanes, alkyl

esters, free fatty acids, fatty alcohols and terpenoids (Varela and

Bernays, 1988; Usha Rani et al., 2007).

The isolated volatile surface chemicals were bio-assayed in the

laboratory for oviposition and orientation behaviours against mated and

gravid female parasitoid, T. chilonis. The bioassay guided to evaluate the

efficiency of bioactive surface chemicals and in identification of a set of

chemical profile resulted by subjecting them to GC-MASS spectral

analysis. The chemical profile of DCM extracts of the pest-damaged

plants (leaves and fruits) was compared with the profile of the intact

undamaged plants (leaves and fruits).

Several hydrocarbon and terpene chemicals found to occur in the

active compounds were obtained in the synthetic form (Sigma – Aldrich


75

Chemicals Pvt Ltd., India) and assayed in a similar manner for

confirmation.

Gas Chromatography

The GC (Agilent 6890 series) equipped with a HP-1 capillary

column (30.0 m long × 320 µm i.d. × 0.25 µm film thick) and detector

was programmed to increase its oven temperature from 100 °C to 300 °C

at 10 °C/min. Nitrogen at a flow rate of 2 ml/min was used as carrier

gas. Injection temperature and detector temperature were set at 150 °C.

A fixed volume (1 µl) of each fraction was injected into GC for analysis.

Gas Chromatography–Mass Spectrometry

The concentrates of the hexane–ethyl acetate eluate of the extracts

were injected into the GC (Agilent 6890 series) equipped with a 5973N

model mass selective detector (MS). A HP 5 MS (Hewlett Packard, Palo

Alto, CA, USA) capillary column (30 m long × 250 µm i.d. × 0.25 µm film

thick) was used. The column oven temperature was programmed to rise

from 50 °C to 300 °C at 10 °C/min after an initial delay of 2 min. Helium

was used as carrier gas with a flow rate of 1.2 ml/min. The sample (1 µl)

was introduced in split ratio of 10:1 at an injection temperature of 150

°C. The chemical structure of each compound was elucidated by

comparison of the mass spectra with those of authentic chemical

samples or by comparison with data in the mass spectral library of the

Analytical Division, IICT, Hyderabad. Relative amounts were calculated

from the chromatogram peak area.


76

2.6.6 Ovipositional Behaviour

2.6.6.1 Petri-plate method

The efficiency of a test chemical is measured in terms of successful

or increased oviposition on the treated surface. The methodology

suggested by Jones et al. (1973) and modified by Usha Rani et al. (2007)

was adopted for the studies. The host eggs (L. orbonalis) were glued (20

no.) on small white cards and surface treated with the test samples at

the rate 20 and 40 µg/ 20 µL concentrations. The synthetic terpene and

hydrocarbon compounds were used at 200 ppm level. In case of control

only acetone was used. After air drying for few sec., the egg cards (6 no.)

were placed equidistant inside a Petri dish (15.0 cm dia.). Five mated one

day old females T. chilonis, which did not have any experience with the

host herbivore or the host plant associated cues prior to bioassay, were

released at the centre of the Petri dishes carefully. These Petri dishes

were covered by the lids ensuring that there is no escape of the

parasitoid. The parasitoids were allowed to oviposit for one hr, which was

considered sufficient for successful and complete oviposition.

In dual choice experiments, each Petri dish contained alternatively

arranged three treated and three untreated (control) egg cards (Fig. 2.16).

In multiple-choice tests all three samples; control (solvent), healthy plant

extract and infested plant extract were applied on the egg surfaces. These

were placed diagonally opposite to each other in alternating sectors. The

experiments were performed in the laboratory at 28 ± 2 ºC temp., 60 ± 5


77

% RH and under a fluorescent light bank of 1200 Lux. Twelve periodic

observations per day were made for each treatment. After one hr of

oviposition, the parsitoids were removed and egg parasitisation was

observed. The bioassay tests were repeated on three different occasions,

in order to rule out any time-to-time variation. The mean percentage

parasitization of 36 replicates (12 replicates/occasion; 12 x 3= 36) was

used for calculations.

Fig. 2.16: Petri plate assay for ovipositional behaviour

2.6.7 Orientation behaviour

2.6.7.1 Orientation behaviour by four choice Olfactometer

Orientation responses of T. chilonis to test volatile chemicals emitted by

different odour sources were measured in a four-arm olfactometer (Fig.

2.17). Before experimentation air was circulated through an empty water

Petri-plate

Filter paper

Egg cards containing Test insect eggs

Petri-plate


78

bottle before entering the exposure through the four arms. Air passed out

from the chamber through a hole that was covered with a fine mesh to

prevent insects from escaping. The total chamber was constructed of

Plexiglas. Its inner dimensions were 10 mm in height and 93 mm across

at its narrowest width. The top had a central hole (5 mm) closed with a

plug. Air flow in each of the four arms was adjusted with an air flow

meter to 100 ml/min.

Fig. 2.17: Four choice olfactometer

To deliver the odour to one of the four fields of the olfactometer,

the corresponding arm was connected with odour source and tightly

closed with parafilm. All four odour sources were connected to the pure

air. A circular neon light source underneath the olfactometer provided

even illumination of 400 lux in the exposure chamber. The total


79

olfcatometer setup was placed in a temperature controlled room at 28 ± 2

ºC temp., 60 ± 5 % RH. Before tests, it was confirmed that in pure air T.

chilonis behaviour was similar in the four fields of the olfactometer. The

test materials were used at four concentrations 1, 2, 5 and 10 µg/ µL

applied on filter paper. The volatile chemicals start emitting out after 15

min.

Individual parasitoids were introduced through the top hole of the

exposure chamber and placed on the fine mesh. Test observations

started when the parasitoid left the center and lasted for 5 min. If the

parasitoid left the exposure chamber through one of the arms and

entered the odour source for more than 60 sec (a period after which T.

chilonis were never observed returning to the exposure chamber in

preliminary experiments), the experiment was terminated and the

remaining experimental time was counted. After every 10-15

observations, the position of odour fields was changed and the exposure

chamber and the odour sources were washed. The time spent was

measured in each field. All observations were done under controlled

conditions.

2.6.7.2 Orientation behaviour by culture tube method

The parasitoids behaviour towards the volatile chemicals was also

studied in the laboratory using small flat-bottom culture tubes (9 cm

long; 2.5 cm dia.). The test material was applied @ 5 µg and 10 µg/ 2 µL

concentrations as a localized circular spot on a piece of absorbent paper


80

(4 × 0.7 cm). After permitting the solvent to evaporate for 2 min., the

paper strip was hung from the inner side of the screw cap lid. A separate

set up with solvent treatment served as control. One single adult-mated

female was released into each glass tube and the lid was closed.

Observations on landing behaviour, time spent on each treated and

control patch, probing and antennation were recorded for 20 min (Fig.

2.18).

Fig. 2.18: Orientation behaviour by culture tube method

Behaviours were classified on a 0–4 scale, with 0 = no reaction and

no movement; 1 = upward movement towards the paper strip; 2 =

circular movement around the paper strip; 3 = entering or landing on the

CULTURE TUBE ASSAY

Test sample

Trichogramma chilonis released here

Filter paper strip


81

paper strip; and 4 = the parasitoid antennated and probed the chemical

spot. The behaviour of 20 females each time was observed for all the

treatments including control.

2.6.8 Evaluation of synthetic compounds

Terpene and hydrocarbon compounds occur in almost all L.

orbonalis - related material extracts. They are the most abundant

compounds. Therefore, experiments were conducted to test whether

these compounds actually account for the activity of the extraction in

which they were found. The effect of 8 synthetic terpene and 10

hydrocarbon compounds on the parasitization rate was studied by using

the above described method of dual and multiple choice tests. The details

of which are as under:

Terpenes Hydrocarbons

α-pinene Pentadecane

Myrcene Heptadecane

Limonene Eicosane

Carene Docosane

Sabinene hydrate Tricosane

Linalool Tetracosane

Caryophyllene Pentacosane

Farnesene Hexacosane

Octacosane

Tricontane

The mean percentage parasitization was calculated from 36

replicates (20 eggs per replicate). In another set of orientation


82

experiments the above compounds were tested by culture tube and four

choice olfactometer methods. Based on the both behavioural assays

effective compounds were identified, which played an important role in

host location behaviour of T. chilonis. For each treatment 12 observations

were made per day, and all treatments were evaluated simultaneously on

the same day. The experiments were repeated at three occasions to avoid

time-to-time variation.

2.7 STATISTICAL ANALYSIS

Mortality counts were corrected for control mortality as suggested

by Abbott (1925) to the data wherever it was considered necessary.

Statistical analysis of the toxicity data was performed using probit

analysis to determine the LC50and LC95 (Finney 1971). Antifeedant and

growth inhibitory activities were calculated using different concentrations

of each extract. The data was subjected to probit analysis to determine

the EC50 values representing the concentrations that caused 50% feeding

deterrence and growth inhibition along with 95% confidence intervals. All

experimental data were subjected to a t-test to determine differences

between two samples, using the statistical software Sigmastat v3.5. All

experimental data were subjected to a one-way ANOVA to determine

differences between two or more samples, using the statistical software

Sigmastat v3.5. Means were separated using the Tukey’s HSD test at the

5% level. All the statistical analysis was performed and the figures were

plotted using the software Origin (version 8.0). The differences in the


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phenolic acid levels between pest damaged and undamaged brinjal

plants were calculated from HPLC chromatograms and were analyzed

using paired t-test at p<0.05. Statistical differences between two groups

in dual choice tests were evaluated with the paired t-test while multiple-

choice test results were analyzed using one-way ANOVA to identify

significant differences in the various behaviors among the three or more

treatment groups (SigmaStat Ver. 3.5).

CHAPTER 2 Mat and Methods - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/19374/8/08_chapter...

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