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CHAPTER 2
Materials and
Methods
Materials and Methods
32
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%
Materials and Methods
36
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,
Materials and Methods
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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
Materials and Methods
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
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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
Materials and Methods
41
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
Materials and Methods
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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.
Materials and Methods
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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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46
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).
Materials and Methods
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Fig. 2.8: Isolation and identification of active phenolic compound,
caffeic acid methyl ester in brinjal fruit extract
Materials and Methods
50
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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51
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
Materials and Methods
54
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 -
Materials and Methods
57
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
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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
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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
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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)
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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)
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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
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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
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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).
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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
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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
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2. Defensive enzymes
Treated (Infested leaf and fruit) Control (Un-infested leaf and fruit)
24, 48, 72 and 96 hr of insect infestation
3. Phenolic compounds
Treated (Infested leaf and fruit) Control (Un-infested leaf and fruit)
24, 48, 72 and 96 hr of insect infestation
4. Leaf and fruit volatile compounds
Treated (Infested leaf and fruit) Control (Un-infested leaf and fruit)
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.
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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
Extraction and assay
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
Extraction and assay
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).
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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
Extraction and assay
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
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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)
Extraction and assay
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)
Extraction and assay
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).
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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)
Extraction and assay
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.
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2.6.3.4 Superoxide dismutase (SOD)
Extraction and assay
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.
5. The absorbance was measured at 560 nm.
2.6.3.5 Poly phenol oxidase (PPO)
Extraction and assay
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.
4. The absorbance was measured at 470 nm.
2.6.4 Induction of phenolic compounds
Extraction and assay
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
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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.
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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
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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.
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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
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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
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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
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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
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(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
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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
Materials and Methods
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
Materials and Methods
83
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).
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