Discussion
161
Plants contain several chemicals by birth and purpose of the
presence of these chemicals is defending the plant from pest attack.
Each plant species consist of different chemicals and role of these
chemicals in protecting plant from pest and diseases is well established
since a long time. We investigated the chemicals present in brinjal,
Solanum melongena as plant’s static defense and also confirmed their
biological activity. Extraction, isolation, identification and
characterization study of S. melongena revealed the presence of several
chemicals such as, phenolic and alkaloid compounds and our
experimental results indicated their role in acting against certain pests
and diseases . Among the different solvents used for the extraction of
phytochemicals from shade-dried leaf and fruit of S. melongena,
Methanol proved to be efficient solvent, which could extract phenolic
compound, caffeic acid methyl ester (CME) and alkaloid compounds
solamargine and solasonine. The isolated compounds were evaluated for
thie pesticidal potential against certain agricultural and stored product
pests and were shown to be efficient pest control chemicals. This
indicates the static defense role of above chemicals CME, solamargine
and solasonine isolated from S. melongena plant.
The leaf and fruit extracts of S. melongena and its chromatographic
fractions showed conspicuous antifeedant and growth inhibitory effects
against the larvae of L. orbonalis, S. litura and A. janata. The behaviour of
the insects showed that the larvae occasionally sampled the treated food,
Discussion
162
which suggests reduced feeding of treated food or its rejection. The
methanol eluted fraction from fruit extract of S. melongena showed
excellent feeding deterrent activity against the test larvae. It is evident
from the results for the first time that pest repellent activity of extracts
from the well-known brinjal plant are ideal candidates for a natural
method of pest-control.
Several investigators have reported the antifeedant activity of
botanicals against S. litura (Ulrichs et al., 2008, Sreelatha et al., 2009)
and A. janata (Devanand and Usha Rani, 2008). Pavunraj et al. (2011)
have reported that leaf extract of Pergularia daemia (Forssk) Choiv. and
its column eluted ethyl acetate fraction exhibit good antifeedant activity
against Helicoverpa armigera (Hub.) and S. litura.
Amongst the chemical defensive strategies developed by the plant,
leaf phenolics generated due to insect feeding play a major role in
controlling the herbivore damage. It was interesting to study the effects
of different phenolic acids on the growth and development of feeding
herbivore. The results of feeding deterrent bioassays showed significant
reduction in feeding by the test larvae in choice experiments with food
containing CME than the test phenolic standards CA and CGA. Ding et
al. (2011) have reported the accumulation of induced phenyl propanoids
especially ferulic and p-coumaric acids, in response to Sitodiplosis
nonagriodies (Gehin) (Diptera: Cecidomyiidae) feeding on wheat tissues.
Discussion
163
According to Harborne (1988) the induction of plant phenolics due
to insect feeding makes unpalatable to herbivorous insects. In the
present study it is confirmed that S. melongena fruits containing
hydroxycinnamic acid derivative, CME was responsible for feeding
inhibition activity against the test larvae. The isolated glycol alkaloids
solasonine and solamargine from the fruits of S. melongena, failed to
show the antifeedant activity in test insects at all concentrations tested
by leaf disc assay. These compounds have not shown the potential for
controlling the lepidopteran larvae by feeding inhibition. Along with
antifeedant activity, the methanol extracts of S. melongena were potent
growth inhibitors to L. orbonalis, S. litura and A. janata tested. These
extracts were quite effective in reducing growth of three lepidopteran
larvae in the oral feeding bioassay.
The growth inhibition activities of the extracts of several Meliaceae
plants such as A. indica (Agrawal and Mall, 1988), Melia azedarach (Al-
Sharook et al., 1991), M. toosendan (Chen et al., 1995) and Aglaia species
(Koul et al., 1997) have been extensively evaluated on several insect
pests. Ethyl acetate extract from Syzygium lineare Wall (Myrtaceae)
(Jeyasankar et al., 2010) and methanol extract of M. dubia (Meliaceae)
(Koul et al., 2000) have also showed growth inhibitory activity against S.
litura. In the present experiments also the larval growth inhibitory
activity of methanol eluted fractions from S. melongena plants was
observed. Predominantly, fruit extract of S. melongena eluted with
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164
methanol produced most potent growth inhibitor against L. orbonalis, S.
litura and A. janata by oral feeding assay as compared to leaf extract. The
findings of the study also revealed that crude extracts as well as solvent
eluted chromatographic fractions were toxic to the test insects when
evaluated by contact method.
Janprasert et al. (1993) have reported that the isolated fractions
and compounds from Aglaia odorata have feeding inhibition and growth
regulating activities against S. littoralis. In addition to feeding deterrent
activity, CME and solasonine also exhibited growth inhibitory activity
against the test larvae, S. litura, A. janata and L. orbonalis. A number of
workers in their respective studies have shown that plant derived
compounds act as an effective feeding deterrents and growth inhibitors
against many insect species. Such as Sreelatha et al. (2010) reported
that a new benzil derivative extracted from Derris scandens Benth
(Leguminosae) exhibited antifeedant and growth inhibitory efficacy
against A. janata larvae when tested by oral feeding method. Likewise the
isolated compounds khayanolide B from the stem bark of Khaya
senegalensis (Desr) A Juss (Meliaceae) (EI-Aswad et al., 2003), and
extracts and drimanes of Drimys winteri J.R. Forster et G. Forster
(Winteraceae) (Zapata et al., 2009) exhibited most potent feeding
deterrent and growth inhibitory activities against the cotton leaf worm, S.
littoralis.
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165
It is reported that plant phenolic compounds play a crucial role in
plant defense against herbivore attack (Schoonhoven et al., 2005). For
example, when isoquercitrin was added to the diet of the budworm, H.
virescens larval growth was reduced to 90% (Hedin et al., 1983). Rutin is
major trichome component of tomato (Solanaceae), which on adding to
the diets of fruitworms, H. zea inhibits larval growth (Isman and Duffey,
1982). The reduction in larval growth is proposed to result from the
alkylation of amino acids or proteins by o-quinones and subsequent
reduction in the nutritive quality of foliage. The presence of derivative
form of caffeic acid as caffeic acid methyl ester (CME) in the fruits of S.
melongena as well as solasonine showed larval growth inhibition against
test lepidopteran pests. In the present study a significant increase in
growth inhibitory responses has been observed when CME and
solasonine were present in the food of test larvae. However, no such
increase was observed with solamargine.
The results on insect development revealed that the crude extract
from S. melongena fruits, its methanol eluted fraction and the isolated
pure compounds, CME and solasonine disrupted developmental cycle of
larvae by reducing the pupal weight, formation of pupal-adult
intermediates and/or causing pupal mortality. A reduction in pupal
weight and emergence of deformed adults suggested that CME and
solasonine interfere with the mechanisms of the normal development of
the insects, which are hormonally regulated. This type of delayed
Discussion
166
development and appearance of malformations have also been reported
from azadirachtin treated S. litura (Rao and Subrahmanyam, 1987),
plumbagin treated H. armigera (Krishnayya and Rao, 1995) and a new
benzil derivative extracted from Scandens Benth (Leguminosae) treated
A. janata larvae (Sreelatha et al., 2010).
During insect development the shedding of the cuticle known as
moulting or ecdysis occurs. Moulting affects the entire body wall as well
as all internal parts that are formed as invaginations of the wall.
Collectively all changes that involve growth, moulting and development
are known as morphogenesis. Similar results were obtained when last-
instar larvae of S. litura, S. mauritia, Ephestia kuehniella Zell. and
Manduca sexta were subjected to azadirachtin (Jagannadh and Nair,
1992).
In the present study, the normal development of larvae of three
major lepidopteran pests, L. orbonalis, S. litura and A. janata treated with
isolated fractions was arrested, which led to malformation of pupae and
adults. The regulation in growth and development was also observed
when CME and solasonine were provided through oral ingestion. It
indicated the larval intolerance to the treatments. Adult abnormality was
significantly increased with the increase in concentration of test products
and days of exposure. The reduction in the overall growth might be due
to the disturbed digestive physiology and other metabolic activities of the
larvae after ingesting treated food. This type of disturbances not only
Discussion
167
reduces pupal weight but also results in poor growth, development and
production of pupal deformities and finally poor emergence of adults.
Similar to present findings, Deborah et al. (2001) have also reported that
extracts of Trichilia americana exhibit growth reduction in S. litura.
The proteolytic activity of three lepidopteran larvae and their
sensitivity to crude extracts and their isolated compounds, CME,
solamargine and solasonine have been studied. The chemical
solamargine was found least responsive then others. Houseman et al.
(1989) stated that the extracts from the digestive tracts of insects from
many families, particularly those of lepidoptera contains serine
proteases. These serine proteases are responsible for protein digestion
and consequently for the supply of amino acids needed for the
development. Serine proteases, as a group of digestive enzymes, have
also been detected in guts of other Spodoptera species (Jongsma et al.,
1996). The serine classes of proteinases such as trypsin, chymotrypsin
and elastase which belong to a common protein superfamily are
responsible for initial digestion of proteins in the gut of plant herbivores
(Garcia-Olmedo et al., 1987). These proteinases involved in cleavage of
polypeptide chains into short peptides which are then cleaved by
exopeptidases to generate amnio acids, the end products of protein
digestion (Lawrence and Koundal, 2002).
In the present study, the regulation of proteases in the midgut of L.
orbonalis, S. litura and A. janata was recorded, which could be targeted
Discussion
168
with natural products for active insect control. Midgut trypsin,
chymotrypsin and elastase showed diverse level of susceptibility towards
CME and solamargine extracted from the fruit of S. melongena plant and
other test standard compounds. Trypsin has been found to be the
predominant and most active protease enzyme in lepidopterous larvae.
Similar to present observation lower activities of chymotrypsin and
elastase have also been reported by Broadway and Duffey (1986). In this
study, the interesting results were recorded in midgut extracts of L.
orbonalis, S. litura and A. janata with the presence of CME and
solamargine in their food. Gut proteases like trypsin, chymotrypsin and
elastase activities were significantly inhibited by the isolated compounds
from S. melongena, CME and solamargine. This leads to the growth
retardation in the test larvae due to reduction in protein metabolism.
The larvae fed on food containing phenolic compounds, CGA and
solasonine showed higher proteolytic activity in midguts as compared to
those fed with CME, solamargine and solvent applied control. The poly-
phytophagous insects, adapt easily to exogenous protease inhibitor
chemical in their food. Since these insects generally have complex
digestive biochemistries and compensate for the loss of proteolytic
activity by either increasing affected proteases or by expressing novel
proteases insensitive to the ingested protein inhibitors (Broadway, 1997;
Gatehouse et al., 1997; Brousseau et al., 1999). From the results, it was
noticed that the hyper-production of proteases in response to ingested
Discussion
169
compounds leads to an extra load on the insect for essential amino acids,
resulting in retardation of insect growth. It may be stated that the
phenolic compounds, CME and CG and alkaloids, solamargine and
solasonone taken as oral ingestion have the potential to regulate the gut
proteolytic activities of three lepidopteran pests L. orbonalis, S. litura and
A. janata by oral ingestion.
The experiments with S. melongena fruit crude extracts,
chromatographic fractions, and the isolated pure compounds indicated
the toxic potential of the S. melongena fruit chemicals against stored
grain insects. Particularly, the isolated compound, CME exhibited greater
insecticidal activity in a short duration of time to all the test insects and
indicated the potential of this pure compound rather than other alkaloid
compounds, solasonine and solamargine, isolated from the fruit extract
of S. melongena. Among the test insects, R. dominica showed slight
tolerance to the crude methanol extracts of the fruits and also to column
fractions. The increased tolerance of R. dominica toward the plant
extracts treatments were also reported previously (Usha Rani et al.,
2011). Bioactivity of phytochemical compounds against stored product
pests depends upon several factors such as the chemical composition of
the crude extracts and varied susceptibility of target species. For
example, neem (Azadirachta indica A. Juss) seed extracts are reported to
demonstrate a remarkable insecticidal activity against nearly 200 species
of insects (Lowery and Isman, 1995). Some plant derived materials are
Discussion
170
highly effective against insecticide resistant strains of insect pests
(Arnason et al., 1989; Ahn et al., 1997).
The pure compound (CME) isolated from S. melongena fruit
material appeared to be highly active giving significant percentage of
mortality of all the four major stored product pests tested, irrespective of
the species difference. This may be due to the easy penetration of
isolated pure chemical into the insect cuticles, when compared with
crude and bio-guided column fractions. In contact method, the test
concentration and exposure time played an important role in producing
the lethal effects. Insect mortality and the duration of the exposure were
found directly proportional in all the treatments of pure compounds. The
concentrations employed also played a major role in determining the
efficacy of CME. Though, the compound, CME at the lower concentration
failed to exhibit toxic symptoms 24 hr after treatment, however, the
percentage mortality has been enhanced with increased duration of
exposure to the compound (up to 72 hr). The pure compound showed
significant mortality to S. oryzae and C. chinensis with the lowest
concentration after 72 hr of treatment compared to other test insects, R.
domonica and T. castaneum. Thus results demonstrated that lower
concentrations with longer exposure time may cause higher mortality
comparable to higher concentrations. It is concluded that an adequate
exposure time is crucial for the effectiveness of CME because the insect
movement increases the cuticular contact with the compound. The test
Discussion
171
isolated compounds, solasonine and solamargine failed to show the
effective toxicity to all the test insects at concentrations tested in both
contact and fumigation mode.
In the fumigant study, the pure compounds extracted were more
effective than crude extracts against all the tested insects in sealed
containers with in 48 hr after treatment. However, crude extracts of S.
melongena showed significant mortality against four major stored pests
after 72 hr of treatment by fumigation method in a delayed mode. In case
of column fractions (methanol) also the considerable fumigant action was
caused by the presence of concentrated levels of fumigant molecules only
after 72 hr of treatment. Owing to the insufficient amounts of volatiles
released from all the test compounds at 24 hr, less mortality was caused
to the test insects. After 48 hr of fumigant action, the containers were
saturated with the chemicals and no increased mortality was found with
the test compounds. Thus contact application appears to be superior
over the other method employed as a lesser quantity of the test material
was required to achieve a 100% kill. Also it appears that the chemical
penetration in to insect body through oral or cuticular route is higher in
this method of application. Form this study it was established that the
test compounds were more effective in contact mode than vapour mode
toxicity.
The author has not come any reference so far reporting the efficacy
of brinjal plant extracts against the stored grain pests. Therefore, it
Discussion
172
appears that this is the first report on the insecticidal activity of S.
melongena and its isolated compound CME against stored product insect
pests. Thus the product based on S. melongena fruit extract or its active
compound, CME may have potential to control the destructive stored
grain insects. It has been well recognized that some plant extracted
insecticidal compounds may be developed into products suitable for pest
control, because they are selective to insects and have no or little
harmful effect against non-target organisms or the environment
(Schmutterer, 1992; Isman, 2000). Although the results of the present
study were promising with regard to the use of isolated compounds from
fruit extracts of S. melongena for the protection of stored grain products,
further investigations are required before using them as grain
protectants.
Feeding deterrent effects of S. melongena leaf and fruit crude
extracts and its chromatographic fractions were studied using flour disc
bioassay method against four stored grain pests. An important feeding
deterrent effect was observed for fruit extract from S. melongena at the
highest concentration tested. Whereas, leaf extract was not effective in
controlling the test stored grain pests, S. oryzae, T. castaneum, R.
dominica and C. chinensis through reduction in feeding. Amongst the
eluted chromatographic fractions, methanol fraction significantly
reduced feeding of treated food against all the test insects. Methanol
extraction of fruits of S. melongena showed good feeding deterrent
Discussion
173
activity against stored product pests. Many reports are available to
support the above data, for example, Park et al. (1997) screened the
methanol extracts from 77 oriental medicinal plant species belonging to
42 families for their larvicidal and antifeedant activities against different
insect pests. Liu et al. (2007b) also reported control of T. castaneum and
Sitophilus zeamais Motschulsky using Chinese medicinal plants in his
research work. Pascual-Villalobos and Robledo (1999) screened 57 plant
species from 21 different botanical families from southeastern Spain for
anti-insect activity using the stored-grain pest T. castaneum as test
insect. Likewise Deborah et al. (2001) screened crude methanol extracts
of 39 plant samples from 6 species of Trichilia collected in Costa Rica for
growth inhibition activity using S. litura. Not only this, according to
Simmonds (2000) about 6250 species of plants have been screened since
1985 for various insecticidal activities.
A diverse range of allelochemic compounds of low molecular
weights are present in plants and play an important role against insects.
The feeding deterrence activities of isolated compounds, CME, solasonine
and solamargine from S. melongena also tested against four stored grain
pests. The insect feeding was significantly inhibited in all treatments with
CME after 7 days of treatment. Whereas, the normal feeding activity was
recorded in food containing test alkaloids, solasonine and solamargine
and solvent (control). It was found that C. chinensis was more susceptible
to all the test extracts tested than other insects. The reduction in growth
Discussion
174
rate of S. oryzae, T. castaneum, R. dominica and C. chinensis possibly
may be due to feeding deterrent and contact toxic effects of test
compounds. Similar mode of actions has been reported by Sackett et al.
(2007) by using furoquinoline alkaloids against S. litura and Trichoplusia
ni (Hubner) as a dietary supplement. On comparison of isolated phenolic
compound, CME with other two isolated glycol alkaloid compounds,
solasonine and solamargine for their effect on insect feeding; it was
observed that the glycol alkaloids treated food was consumed by test
insects without any repellent effect. Hence, it may be stated that these
alkaloid compounds have no adverse effect on S. oryzae, T. castaneum, R.
dominica and C. chinensis up to 7 days of feeding. Since CME treated
food was found less damaged by insect feeding, it may be stated that it is
one such allelochemic compound that showed promising results in
feeding deterrence assays. Further studies on mode of action of CME
interfering nutritional metabolism of the test insects are required to
exploit this molecule in the management of stored product insect pests.
The findings thus suggest that CME being a feeding deterrent may serve
as a starting point for developing an effective insect control agent.
The crude leaf extracts and their eluted fractions of S. melongena
at higher concentrations have shown excellent repellent effects against S.
oryzae, T. castaneum, R. dominica and C. chinensis in the present study.
It is reported that the essential oils and their constituent monoterpenoids
act as neurotoxins against insects (Keane and Ryan 1999; Enan, 2001;
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175
Kim et al., 2003). Obeng-Ofori et al. (1997) found that the monoterpenoid
1.8-cineole was highly repellent and toxic against several stored product
pests such as Sitophilus granarius L., S. zeamais, Tribolium confusum
Jacquelin du Val and Prostephanus truncatus Horn. A number of herbs
and their isolated compounds, when being studied for pharmaceutical
uses, it was found that they might not be responsible for the insecticidal
or feeding-deterrent activity against S. zeamais and T. castaneum (Liu et
al., 2007b). Although the insecticidal mode of action of the plant extracts
used in the present study has not been determined, it may be pointed
out that leaf and fruit extracts of S. melongena are likely to act as
neurotoxins presumably owing to their high phenolic contents.
It is highly advantageous when plant extract possess repellent
activity, as it prevents the pest approach to damage the commodity. The
repellent effects of phytochemicals on stored product pests depend on
several factors amongst which the chemical composition of the crude
extracts and insect susceptibility towards the test compounds are the
main. Since fruit extract from S. melongena exhibited greater repellent
effects against the test insects, it demonstrates a scientific rationale for
the incorporation of the fruit powder of plants in to grain protection
practices of commodities in the South Indian communities. Additionally,
this research provides a scientific basis for extracting and applying
phytochemicals from S. melongena for stored product protection in the
South Indian region. Moreover, provided with a proper formulation and
Discussion
176
dosage, crude extracts may be exploited for use against insect infestation
at the small scale farmers’ level, since they may be more economical,
effective and less cumbersome than application of dried foliage and their
isolated compounds. The plant chemicals tested at lower concentrations
have also shown antifeedant activity to some extent against the test
insects. This may be an added advantage that the residues left to sub
lethal doses after the application of test products may cause the feeding
deterrence leading to slow or ill development of the insect. Though, the
development of resistance by pests and vectors against botanicals has
not been reported so far. The studies will be required to ascertain the
possibilities of resistance development to these isolated compounds.
Interestingly, present studies showed varying toxic properties of
chloroform, ethyl acetate, hexane and methanol extracts of S. melongena
plant. Amongst them methanol extract showed high level of toxicity
against adults of stored grain insects S. oryzae, T. castaneum, R.
dominica and C. chinensis. The toxic activity of extracts with other
solvents was comparatively low. This differential type of toxicities may be
due to the nature of the compounds and their solubility in the solvents.
Suppressing progeny production of stored product pests after contact
with treated commodities with toxic substances is of great importance, as
this would de-facilitate invading adults from establishing a sustainable
population.
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177
The present results indicate that S. oryzae, R. dominica, T. castaneum
and C. chinensis are all susceptible to test chemicals which limit their
progeny under laboratory conditions. This is a frequent finding in studies
assessing the efficacy of plant extracts in mediating the mortality and
suppression of progeny production by stored product pests (Huang et al.,
1997; Paneru et al., 1997; Obeng-Ofori et al., 1998). Belmain et al. (
2001) reported that Cassia sophera L. leaf powder mixed with different
commodities at 1% and 5% concentrations significantly reduced F1
emergence of S. zeamais, C. maculatus and R. dominica in laboratory
experiments, although such treatments did not affect progeny production
of P. truncates. For long term protection of stored grains, the suppression
of progeny production is more important than mortality (Athanassiou et
al., 2004). The present findings suggest that progeny production of test
insect species is suppressed in grains treated with products extracted
from the fruits of S. melongena. Inhibition of adult emergence is an
important phenomenon while evaluating potential of botanicals. It is
found that the crude extracts and chromatographic fractions and also
isolated pure compound, CME may be used successfully as grain
protectants against stored product pests. In contact mode of evaluation
CME was found more effective in inhibiting the adult emergence fed on
treated grains than isolated alkaloids. Previously several compounds
have been shown to be highly effective to suppress the insect emergence,
leading to grain protection with progeny control against most of the
Discussion
178
major stored product pests (Athanassiou et al., 2004; Athanassiou et al.,
2005; Vayias et al., 2006; Daglish and Nayak, 2006; Stathers et al.,
2008).
It has been well recognized that some plant-derived insect control
agents could be incorporated into an Integrated Pest Management
strategy, since these agents are selective to pests, have no or little
harmful action against non-target organisms and the environment and
demonstrate a differential mode of action against pests (Arnason et al.,
1989; Schmutterer, 1992; Hedin et al., 1997). The results of the present
study indicated that the brinjal fruit extract has vital components toxic
in nature. The major advantage of the plant materials tested in the
present study is their high volatility, which is a desirable characteristic
for insecticidal preparations acting as fumigants for the control of stored
product pests (Coats et al., 1991; Konstantopoulou et al., 1992;
Regnault- Roger and Hamraoui, 1995; Ahn et al., 1998). Based on the
results of this study, plant extracts of S. melongena and its isolated pure
phenolic compound, CME and two alkaloid compounds, solasonine and
somargine can be considered as potent insecticides, antifeedants and
progeny production inhibitors. Hence they become suitable for the
control of pests in stored commodities.
For the practical use of these natural extracts and their active
ingredients as novel grain protectants, further research is required as far
as safety issues for human health are concerned. Other areas requiring
Discussion
179
attention are development of cost effective formulations with improved
and persistent efficacy, anti-feedant potency and stability against the
stored product pests along with organoleptic qualities of treated
commodity.
The Environmental Protection Agency in 1994 set up a separate
branch to evaluate and facilitate the registration of such products under
the Biopesticides & Pollution Division (BPPD) to help and speed up the
registration processes. Active principles from many plants have been
recognized, isolated, purified and formulated as insecticides.
In the study, it was observed that insect damage to brinjal plants
has induced changes in primary or nutritive compounds and subsequent
quantitative changes in the plant biochemical and enzymatic levels. In
most of the insect wounded plants or due to herbivory, there has been
increase in the levels of biochemicals such as phenols, carbohydrates,
proteins and amino acids. An induction in oxidative enzymes peroxidase
(POD), catalase (CAT) and superoxide dismutase (SOD) activities and
increase in other enzymes phenylalanine ammonia lyase (PAL) and
polyphenol oxidase (PPO) level has also been observed. An obvious
difference in several biochemical parameters of L. orbonalis infested
plants was found in comparison to un-infested brinjal plants. The plant
responses mainly depended on the type of insect damage in fruits, as
this is related to the severity of plant damage. Fruit borers that mainly
drill into the fruit cause considerably higher changes in the plants as
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180
compared to other wounded or damaged plant parts. The increased levels
of carbohydrates in S. melongena infested plants suggest their role in
plant defense mechanism by inducing the signaling pathways in plants.
The induced defense may be direct or indirect.
The studies of Watanabe and Kitagawa (2000) showed that plant
hopper, Nilaparvata lugens infestation causes physiological changes that
reduces photosynthesis or alter the translocation of photosynthates.
Translocation may cause reduction in carbohydrate content in L.
orbonalis damaged S. melongena plants. Studies of Flinn et al. (1990)
revealed that potato leafhopper, Empoasca fabae damage increased total
non-structural carbohydrates of alfalfa from 49 to 465% over the un-
damaged plants. N. lugens infestation caused an increase in the free
amino acids and a decrease in the soluble protein (Sogawa, 1971). The
present data on protein and amino acid content confirm the above
observation. This decrease in nutrient content may induces several
changes in plants, which finally makes plants less palatable for insects
and in turn their development held up.
An increase in POD activity is rather a common phenomenon of
induced plant responses in all plant systems. The increase of the POD
activity in herbivore infested plants can be attributed to the fact that
these are the key enzymes that participate in several plant cell wall
building processes (Chittoor et al., 1999). Huckelvohen et al. (1999) and
Bestwick et al. (1998) had described that the activation of POD might
Discussion
181
result in production of hydrogen peroxide and might involve in
hypersensitive cell death. The final products of such enzymatic activities
would be considered anti nutritive because they cannot be effectively
digested and assimilated by herbivore insects (Constabel, 1999). The
same results were observed in L. orbonalis infested brinjal plants after 72
hr of infestation. The increased CAT activity in L. orbonalis infested
plants seems to be related to the plant response to the extent of feeding
plant cell damage. Bi and Felton (1995) observed similar results with H.
zea feeding on soybean plant. Chen et al., (1993) reported that the
increased levels of CAT is known to be involved in increasing the cell wall
resistance and acting as local signals involved in induction of defense
genes. Most lepidopteran insects cause extensive damage to plant tissues
while feeding, whereas this was not so common due to the homopteran
feeding. Normally, the homopteran insects feed from the contents of
vascular tissues by inserting a stylet between the overlying cells, thus
limiting cell damage and due to this reason the minimize induction of a
wounding response in plants are observed. The decrease in the activity of
CAT in L. orbonalis damaged brinjal plants was however, similar to that
observed in Russian wheat aphid infestation (Mohase and Van der
Westhuizen, 2002).
There was a diminutive change observed in the SOD activity due to
biotic stress induced by L. orbonalis in leaf and fruit extracts of brinjal in
comparison to control or undamaged plants. L. orbonalis feeding damage
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182
causes an increase in the SOD activity in brinjal plants. Increased SOD
enzyme activity has been shown to interfere with insect feeding, growth
and development, facilitate microbial infection and finally cause death
(Shapiro et al., 1987; Wang et al., 1996). Though L. orbonalis larvae are
internal feeders and feed inside the brinjal fruits, the induction of
increase in oxidative enzyme activity was found in the fruits as well as in
leaves of infested brinjal plants. The induction of the enzyme might also
be involved in the biosynthetic pathway promoting the induced volatile
emission, which has been observed in earlier studies (Usha Rani et al.,
2007). In earlier studies on pathogen infested groundnut and pest
infested castor plants, changes in oxidative enzymes were also observed
(Usha Rani and Jyothsna, 2009; Jyothsna et al., 2009).
There was an enhanced activity of the defensive enzymes, PAL and
PPO in both the leaf and fruit extracts of L. orbonalis damaged plants
compared to undamaged (Control) plants after 24 to 96 hr after
treatment. The first committed enzyme in the phenylpropanoid and
flavonoid pathways involved in biosynthesis of phytoalexins, lignins and
salicylic acid associated with disease resistance expression has been
discussed by Mauch-Mani and Slusarenko (1996). Activation of PAL in A.
densiflorus was reported to directly affect accumulation of secondary
toxic compounds, such as phytoalexins, phenolic compounds that might
be released through root exudates and inhibit fungal spore germination
and growth (Chenyang et. al., 2001). Similar results were recorded in L.
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183
orbonalis infested brinjal plants after 48 to 72 hr of infestation. The
damaged plants showed significant induction in PAL and PPO enzyme
activities in both fruit and leaf extracts. PAL is considered as the key
enzyme in phenols biosynthesis, since it catalyses the first reaction in
the general pathway of phenylpropanoid biosynthesis, which includes the
formation of flavonoids and hydroxycinnamic acids. The induction of PAL
activity in L. orbonalis damaged leaves and fruits of brinjal plants
indicated that PAL enhancement may be due to ROS generation, which
occurs as primary reaction in response to biotic stress caused by L.
orbonalis larvae. Induced activity of PAL, the key enzyme in
phenylpropanoid pathway indicated the biosynthesis of phenolic
compounds in damaged plants. The induction in biosynthesis of phenolic
compounds in plants have an impact on the herbivore feeding behaviour
that leads to the reduction in further damage caused by L. orbonalis in
brinjal plant system.
Phenolics, a group of secondary metabolites, play very important
roles in plants particularly protection against herbivore attack
(Hahlbrock and Scheel, 1989). In regard to insect pests, phenolics act as
digestion inhibitors and antioxidants by producing free radicals (Appel,
1993). According to Ananthakrishnan et al. (1991) increase in total
phenols are considered as elements of induced resistance in hosts
against herbivory. III and IV instar larvae of L. orbonalis voraciously fed
on brinjal fruits and tender shoots and cause extensive damage. By
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184
comparing the increase in level of foliar and fruit phenolics in case of L.
orbonalis damage, it can be stated that the feeding of L. orbonalis induce
higher amount of foliar and fruit phenolics. From the quantitative
analysis of foliar and fruit extracts significant increase in phenolic
compounds was observed with L. orbonalis infested S. melongena plants
after 48 to 72 hr of damage, when compared with undamaged brinjal
plants after same period of time. This increase however, declined after 96
hr indicating a rapid response. It indicated that the brinjal plants’
response to the specific pest, L. orbonalis was more and as a result
higher amount of phenolic acids were accumulated. Phenols are the
metabolic products in phenylpropanoid pathway and induce in response
of plants to herbivore stress, an increase in phenols accumulation
indicated that the secondary metabolisms of leaves and fruits were
enhanced.
Phenolic acids are structurally diverse class of phytochemicals,
which play major roles in plants protection against herbivore attack
(Hahlbrock and Scheel, 1989). Total amount of phenolic acids (presented
as sum of phenolic compounds) was lowest in control plants and
increase was observed after insect’s infestation. A similar result of low
level free phenolics was observed in control soybean plants and increase
in their content after exposure to stress factors (Janas et al., 2002).
The induction or accumulation of higher quantities of phenolics
acids was due to rapid herbivore feeding on brinjal plant. Normally the III
Discussion
185
instar L. orbonalis larvae feeds on brinjal fruits up to pupation and at the
time of voracious feeding by larvae plant responds to the herbivore and
accumulates phenolics in leaves and fruits of the plant. This raid
accumulation impacts on the further feeding by insect herbivore. Jain
and Yadav (2003) have reported that the increase in quantity of total
phenols might be attributed to defense mechanism. The increase in
phenolics in relation to resistance has been reported in Brassica by
Singh (2004). As discussed earlier, higher PPO and PAL activity in brinjal
plant fruits infested by the III instar larvae of L. orbonalis was observed.
This increase in the defensive enzyme activity is associated with the
resistance reaction, which could be due to increased phenolic acid
concentration.
From the analysis of total phenolics, it was observed that increase
in phenolic acids was comparatively higher in case of fruit extracts than
leaf extracts of damaged plants. It is known that fruit borer pests too
induce systemic chemicals in the entire plants on which they feed and
cause changes in the uninfested leaves of the fruit borer infested plants.
Earlier reports indicate that mode of feeding plays an important role in
inducing plant responses (Usha Rani, 2006). The observation recorded
revealed that the changes in brinjal plant were a systemic response, that
the leaves of the fruit borer infested plants also bring changes in leaf
phenolics. But this change was quite low, when compared with the fruit
phenolics. An increase in phenolics, considered a common reaction to
Discussion
186
herbivory (Karban and Baldwin, 1997) has been correlated with its
negative effects on the pest larva (Haukioja and Niemela, 1977). It is
reported by Hori (1973) that Lygus bug feeding on sugar beets results in
increased quinines, which inhibits subsequent bug feeding.
The severe damage in the plant system by herbivore was its
duration of feeding on the plants. The duration of pest feeding also has a
profound impact on phenolic production. Though the duration of feeding
or the extent of damage is same in herbivore infestation, the differences
in the period of infestation and availability of phenolic acids in the leaf
and fruit tissues of brinjal were critically observed in the present study.
Phenolic acid levels in L. orbonalis infested leaf and fruit extracts reached
to maximum level after 72 hr of infestation, which remained above the
control levels for 96 hr of post infestation.
Individual phenolic profiles from L. orbonalis infested fruit extracts
quantified by HPLC analysis suggest that the quantities of chlorogenic,
caffeic, vanillic, cinnamic, chlorobenzoic and synergic acids increased
whereas, coumaric acid decreased in comparison to undamaged fruit
extracts. In case of leaf extracts, quantities of few phenolic acids like
chlorogenic, caffeic, vanillic, coumaric and chlorobenzoic acids were
increased, when compared with undamaged leaf extracts. Unlike fruit
extracts, cinnamic and synergic acids were not noticed in leaf extracts.
The chlorogenic, caffeic and chlorobenzoic acids were quantitatively
increased in brinjal plant up to 72 hr after feeding. Hildebrand et al.
Discussion
187
(1986), Usha Rani and Jyothsna (2009), Jyothsna et al. (2009) and
Felton et al. (1992) have found that increased concentration in phenolic
compounds is according to the extent of tissue damaged by feeding
insects or due to pathogen infection. Interestingly, in case of L. orbonalis
infested plants the presence of caffeic acid was recorded even after 96 hr
after infestation. Among the seven phenolic acids monitored, chlorogenic,
caffeic and chlorobenzoic acid were maximum followed by cinnamic acid.
From the leaf and fruit analysis for phenolic compounds, the major
phenolic acids of brinjal plant exhibited good potency against the test
insects, through reduction in feeding and growth of insect herbivore
leading to less damage in the plant.
HPLC results suggest that caffeic acid is one of the phenolic acids,
which play a major role in biosynthesis of lignin (Boerjan et al., 2003).
Coumaric, caffeic and ferulic acids are central intermediates of lignin
biosynthesis. Thus the increase in caffeic acid may be correlated with
lignin biosynthesis. Lignin and other phenolics can strengthen cell walls
and therefore can be anti-nutritional (Brodeur-Campbell et al., 2006;
Schroeder et al., 2006). It is interesting to study the effects of different
phenolic acids on the feeding, herbivore growth, mortality and behaviour.
The results of antifeedant and growth inhibitory bioassays
indicated a significant interaction between the plant feeding and
herbivore performance. Herbivory modify the leaf and fruit chemical
structure, which affect the leaf and fruit suitability to pest larvae.
Discussion
188
Ingestion of caffeic acid methyl ester and caffeic acids deterred the
feeding of the test lepidopteran L. orbonalis, S. litura and A. janata. It is
noteworthy that compounds with less toxicity and having antifeedant
properties were increased due to infestation by L. orbonalis larvae.
Summers and Felton (1994) proposed that the induction of oxidative
stress may be an important component of phenolic toxicity in
lepidopteran larvae. Induced accumulation of phenyl propanoids in
response to insects feeding was reported, especially for the ferulic and p-
coumaric acids in wheat tissues as a response to Sitodiplosis mosellana
(Ding et al., 2000). Chlorogenic acid and rutin, major phenolic
constituents inhibit early larval growth of the fruit worm, H. zea when
added to its artificial diet (Isman and Duffey, 1982). When the tomato
fruit worm H. zea or the beet army-worm S. exigua feed on tomato
foliage, a substantial amount of the ingested chlorogenic acid is oxidized
to chlorogenoquinone, a highly reactive electrophile by PPO in the insect
gut.
The reduction in larval growth is proposed to result from the
alkylation of amino acids or proteins by o-quinones and subsequent
reduction in the nutritive quality of foliage. The cinnamic acid derivatives
like chlorogenic acid and rutin represent model phenolics in the study of
plant antiherbivore defense due to their ubiquitous occurrence among
terrestrial plants and well documented toxicity to insect herbivores
(Isman and Duffey, 1982; Harborne, 1991).
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189
The results presented show much higher biosynthesis of phenolic
acids in brinjal plant leaves and fruits after insects feeding. Thus it can
be suggested that feeding of the herbivorous insects induce antibiosis
based on accumulation of the phenolic acids in brinjal plants. The
summing up of results of this study show that the leaf phenolics are
modified quantitatively due to pest feeding and further these enhanced
phenolics have profound effects on feeding herbivore larval performance
and mortality.
Plant synthesized semio-chemicals that act as insect behaviour
modifying chemicals can be used as tools for management of insect pest
populations (Powell and Pickett, 2003). These chemicals are capable of
inducing a variety of responses in plants, including induction of defenses
against pathogens and herbivores (Walters et al., 2002) and modification
of volatile profiles (Dicke, 1999). The modification of the volatile profile of
plants could repel herbivores. In this way the immigration of
lepidopteran insect, L. orbonalis into a brinjal field could be limited.
The results demonstrated that damaged plant leaf and fruit
extracts of S. melongena increase parasitization efficiency of egg
parasitoid T. chilonis. They indicate that the volatile chemicals extracted
from the surface of L. orbonalis infested brinjal leaf (DLC) and fruit (DFC)
cause arrestment of parasitoids in host vicinity and stimulate oviposition
and orientation by T. chilonis. The parasitoid’s stronger response to
damaged leaf and fruit volatile extracts in comparison to undamaged leaf
Discussion
190
(UNDLC) and fruit (UNDFC) volatile extracts indicated that this response
was specific to the volatile chemicals present in L. orbonalis infested leaf
and fruit chemicals. The present findings support previous studies that
have shown the influence of hydrocarbons and terpene compounds on
the behaviour of Trichogramma spp (Jones et al., 1973; Ananthakrishnan
et al., 1991). Potting et al. (1999) stated that parasitoid uses mainly
plant-derived olfactory stimuli in its orientation towards infested plants.
Egg parasitoids locate hosts by using a variety of signals.
Parasitoids use chemical stimuli from their herbivorous hosts or
the host’s food plant during their host searching process (Vinson, 1976;
Vet and Dicke, 1992). Volatile stimuli (synomones) from certain plants
were preferred by Trichogramma pretiosum Riley in an olfactometer and
increased its parasitism rate (Nordlund et al., 1985). Although host-
derived stimuli are reliable indicators of host presence, but they are
difficult to detect at a long distance and are usually used as contact
stimuli. In contrast, plant-derived volatiles are generally more easily
detectable at a long distance (Vet et al., 1991). Many parasitoid species
are known to discriminate in-flight between uninfested plants and host-
infested plants. The evidences are now accumulating in support of
natural enemies that use specific herbivore induced chemical volatiles
emitted by herbivore infested plants (Turlings et al., 1990; Dicke, 1994).
It has been shown that the responsiveness of a number of insect
Discussion
191
parasitoids to volatile odour cues can be increased, if the parasitoid is
allowed to oviposit in the presence of those cues (Turlings et al., 1993).
Few terpene and hydrocarbons are important class of compounds that
were found in the pest damaged leaf and fruit extracts. From the GC-
GCMS analysis of infested leaf (DLC) and fruit (DFC) and un-infested leaf
(UNDLC) and fruit (UNDFC) extracts; few terpene chemicals, α-pinene,
limonene, sabinene hydrate and linalool and few hydrocarbon
compounds such as pentadecane, hexadecane, nonadecane, eicosane,
docosane, tricosane, tetracosane, pentacosane, hexacosane and
octacosane were detected. This finding supports previous work
demonstrating that these compounds existed in pest infested cotton
plants (Loughrin et al., 1995). The monoterpene (E)-β-ocimene emitted
from lima bean leaves after damage by S. littoralis, comprised the
majority (64 to 69%) of the total emissions (Arimura et al., 2007).
The results of Lozano et al. (2000) indicated that α-pinene and 2-
decanone were intricately involved in the attraction and location of the
parasitoids, Dendrosoter protuberans and Cheiropachus quadrum towards
their host, Phloeotribus scarabaeoides. The emission of volatile terpenes
after insect feeding and the subsequent attraction of the infesting insect’s
natural enemies have been shown to occur in several plants by Dicke
(1994). Piperitone 6-isopropyl-3-methylcyclohexane-2-one is present in
the floral volatiles of Tanacetum vulgare L. and is responsible for luring
Lobesia botrana (Gabel et al., 1992). Quantitatively these terpene and
Discussion
192
hydrocarbon chemicals significantly induced in higher concentrations in
damaged plants (DLF and DFC) than in un-damaged ones (UNDLC and
UNDFC). Due to the higher concentrations of these chemicals they may
play an important role in the arrestment responses of T. chilonis by
stimulating oviposition on the treated surfaces. It is presumed that the
brinjal plants undergo physiological changes due to the stress of the L.
orbonalis larval feeding that results in the emission of the chemical
signals.
The GC and GC-MS results combined with the observational
outcomes demonstrate clear evidence of the presence of several
supplementary chemicals in the L. orbonalis infested plants. This
suggests that the quantity of monoterpene and long chain hydrocarbons
are crucial for the arrestment and oviposition stimulation of T. chilonis.
Despite the fact that L. orbonalis leads a concealed life style within the
fruits of the brinjal plants, the parasitoids were able to locate and
identify the pest infested plants among several non infested brinjal plants
and surrounding weed plants. This is strong evidence that the brinjal
plants infested with advance staged L. orbonalis larvae emit volatile
chemicals into the surrounding air, which disperses over large distances.
While there is no particular benefit to T. chilonis, which is an
endoparasitoid of the eggs to identify the larval infested plants, they are
able to discriminate and land on plants infested with IV instar stage
Discussion
193
larvae. It is presumed that this may indicate the likely occurrence of
adults or host eggs on nearby plants.
The major compounds identified as attractant and oviposition
stimulants for egg parasitoids are terpenes and hydrocarbons, which
generally serve many functions in insect behaviour. Parasites have
evolved the ability to utilize these volatile terpene and hydrocarbon cues
produced by plants as primary cues in locating their host insects. These
compounds seem to elicit a fixed behavioural pattern exhibited by
antennal searching of a contaminated area, followed by ovipositor
probing in some parasitoids. In the present findings the induction of
quantitatively higher concentrations of α-pinene, limonene and linalool in
L. orbonalis damaged fruit extracts were found. From the results, it was
observed that extracted volatile chemicals from the damaged fruits led to
significantly higher rates of oviposition and orientation responses by egg
parasitoid, T. chilonis than infested leaf extracts. The similar findings
were noted in a number of other studies. The introduction and over
expression of certain terpene synthases genes in to a host as well as a
non host plant under a constitutive promoter can lead to an enhanced
release of specific terpenes that attract parasitoids searching for their
host (Schnee et al., 2006; Chen et al., 2007).
Mainly pentadecane, heptadecane, eicosane, docosane, tricosane,
pentacosane and hexacosane were the major hydrocarbons present
exclusively in both damaged leaf and fruit extracts of S. melongena
Discussion
194
plants. They may be important cues for host location and ovipositional
stimulation in T. chilonis towards host plant. Wackers and Lewis (1994)
studies by using egg parasitoid, T. japonicum in host location behaviour
support the above results. Certain chemicals such as pentadecane,
heptadecane, eicosane, docosane, tetracosane, pentacosane and
hexacosane were isolated and identified from leaf and fruit extracts. They
have shown increase parasitism (oviposition response) by the parasitoid
T. chilonis in both dual and multiple choice tests, when applied to host
eggs. Whereas, the above compounds showed significant orientation
response in both four-choice olfactometer and culture tube bioassays
against T. chilonis. These compounds were assumed to contribute to an
increase oviposition and orientation responses by T. chilonis, because
these hydrocarbon compounds have a high number of carbon atoms.
They might act as contact stimulants, whereas the chemicals with less
than 10 carbon atoms are more volatile and might attract parasitoids to
the near vicinity of the host eggs.
The increased stimulation of oviposition and orientation by the
extracts of L. orbonalis infested plants may be due to the presence of
chemicals in the leaf and fruit extracts that are emitted through leaf and
fruit surfaces. It was observed only in the fruits infested with advance
staged larvae and larval feeding inside the fruit. Because of this, it is
presumed that the quantity of the chemicals released might depend on
the quantity of feeding inside the galleries and possibly the quantity of
Discussion
195
excretion. This prediction is based on the observation that the damaged
fruit and leaf extracts produced greater orientation and host searching
responses followed by high oviposition by T. chilonis in the laboratory
behavioural bioassays (Usha Rani et al., 2007).
On infestation of fruits by L. orbonalis, the induced changes were
observed in both damaged fruits as well as in undamaged leaf surfaces.
This was achieved due to the systemic response of the brinjal plant.
Fatouros et al. (2005) stated that if this systemically induced odour
attracts natural enemies to the vicinity of the plant then local cues
restricted to the oviposition or feeding site can facilitate fine scale
orientation. Hence, it is unlikely that parasitoids use chemical cues
emanating from the both leaf and fruit surfaces as cues in locating the
egg masses of L. orbonalis to oviposit over them.
The results of this study revealed numerous parallels with other
plant species in which the volatile emission physiologically characterized
(Pare and Tumlinson, 1999). T. chilonis can discriminate between odours
emitted from undamaged leaves and fruits and from infested with or
damaged by its host L. orbnalis. Based on these findings, it can be
concluded that brinjal plants under the stress of herbivory, L. orbonalis
emit volatile chemicals through their leaf and fruit surfaces. These
volatile bouquets serve as signals for mated female parasitoids T.
chilonis. Therefore, plant volatiles take on the functional role of
synomones that are sequestered within plant tissue and T. chilonis rely