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Studies on plant alpha amylase inhibitor and its role in pest management
Ph.D Thesis. Pankaj R. Gavit. (2014) North Maharashtra University, Jalgaon 1
1.0 Summary:
This chapter begins with the overview of present agricultural scenario of India
highlighting the factors limiting the agricultural productively including damage
caused by insect pests. Various control measures to arrest these pests including use
of traditional methods, chemical pesticides and other approaches are discussed.
Merits and demerits of synthetic pesticide and need of alternative strategies for
effective pest management is dealt with including the role of genetic engineering in
it. It also discusses insect digestive enzymes as possible targets of pest management
using genetic engineering techniques. Alpha-amylase inhibitor occur in many plants
as part of the natural defense mechanism and could be an important tool in pest
management as by inhibiting the α-amylases, they deprived the insect from primary
energy source. The different classes of α-amylase inhibitors and a brief review on
present status of research on them have been given. The chapter ends with
description of two target pests, Callosobruchus chinensis and Helicoverpa
armigera, and plants screened for source of α-AI. The specific aims and objective of
present study are given at end of the chapter.
********
Studies on plant alpha amylase inhibitor and its role in pest management
Ph.D Thesis. Pankaj R. Gavit. (2014) North Maharashtra University, Jalgaon 2
1.1 Human and Agriculture:
Human kind put its first foot towards civilization by developing the skills of
growing and cultivating plants which were useful for survival; at the same time
domesticating animals for various purposes. Ultimately, agriculture and allied
professions proved very valuable to mankind in fulfilling basic needs like food, clothes
and shelter. As the civilization progressed, humans realized the importance of nutrition
for their development and health. Today, we, as a result of advances in science, are able
to identify a number of important macro and micronutrients required for satisfying
energy need of the body and maintaining the well-being in terms of health.
All these nutrients at the end support the survival of all life forms and also
maintain the ecosystems on the earth. As per the reports of Food and Agriculture
Organization (FAO), cereals and legumes together contribute 70% of human food; rest
of the 30% comes from animal sources (Mandal and Mandal, 2000).To feed our
population, which is estimated to be approximately 1.25 billion presently, agriculture is
contributing vitally by producing nearly 250 million metric tons of food grains per year.
Providing food to the ever increasing population is going to be a great challenge in near
future (Pimentel and Wilson, 2004) and to sustain this challenge, India has to increase
the food grain production by developing high yielding crop varieties and minimizing the
losses due to biotic as well as abiotic stresses.
1.2 Factors limiting the agricultural productivity:
Many climatic and ecological factors, listed below, limit the production of crops
(Salunke, 2006).
i) Monsoon dependence
ii) Slow seed replacement
iii) Improper harvest and storage facilities
Studies on plant alpha amylase inhibitor and its role in pest management
Ph.D Thesis. Pankaj R. Gavit. (2014) North Maharashtra University, Jalgaon 3
iv) Little or no use of fertilizers, insecticides/pesticides, micronutrients,
irrigation strategies etc.
v) Overlooked efforts for technology transfer related to development of high
yielding crop varieties and
vi) Heavy losses due to pre as well as post-harvest infestation by microbial
and insect pest
Insect pests are the major competitors with humans for resources generated by
agriculture, and are favored by monocultures (Oerke and Dehne, 2004). The damage
caused by these organisms is one of the most important factors responsible for the
reduced productivity of any crop plant species (Cramer, 1967; Pimentel, 1976; Metcalf,
1996). Losses can occur on the field (pre-harvest) and during storage (post-harvest)
(Oerke, 2006). Both pre- and post-harvest insect pests along with pathogens like fungi,
bacteria and viruses are responsible for severe crop losses ultimately affecting the GDP
of the nation. Insect pests (pre- and post-harvest) are the major contributors for the heavy
economic losses all over the world. Losses in agriculture due to pest attack are estimated
to be around 40% globally, with the small scale farmers being the hardest hit. Accurate
estimates of agricultural losses caused by insects are difficult to obtain because the
damage caused by these organisms depends on a number of factors related to
environmental conditions, the plant species being cultivated, socio-economic conditions
of farmers and the level of technology used. Table 1.1 shows the estimated crop losses
in India due to insect pests.
Studies on plant alpha amylase inhibitor and its role in pest management
Ph.D Thesis. Pankaj R. Gavit. (2014) North Maharashtra University, Jalgaon 4
1.1: Estimated crop losses caused by insect pests to major agriculture crops in India
Crop
Actual
Production*
(Million tonnes)
Approximate estimated
loss in yield Hypothetical
production in the
absence of losses
(million tonnes)
Monetary value
of estimated
losses (Million
Rs)*
Percentage Total
(Million
tonnes)
Cotton 44.03 30 18.9 62.9 339660
Rice 96.7 25 32.2 128.9 240138
Maize 19 20 4.8 23.8 29450
Sugarcane 348.2 20 87.1 435.3 70667
Rapeseed
mustard
5.8 20 1.5 7.3 26100
Groundnut 9.2 15 1.6 10.8 25165
Other oilseeds 14.7 15 2.6 17.3 35851
Pulses 14.8 15 2.6 17.4 43551
Coarse cereals 17.9 10 2.0 19.9 11933
Wheat 78.6 5 4.1 82.7 41368
Total/Average 17.5 863884
*Production and monetary value of estimated losses based on minimum support
price (MSP) fixed by Government of India for 2007-08. Adapted from
Anonymous (2010)
Studies on plant alpha amylase inhibitor and its role in pest management
Ph.D Thesis. Pankaj R. Gavit. (2014) North Maharashtra University, Jalgaon 5
1.3 Crop damage/productivity loss with special reference to diversity
in insects:
Insecta is the largest and most diverse class in the animal kingdom. They are the
most adaptable life forms occurring and inhabiting a variety of environmental conditions
such as forests, deserts, swamps, oceans and even in very harsh surroundings like crude
petroleum. Their number is unbelievably high covering 85% of the known animal
species. Animal taxonomists assume that only one fifth of all insects have been classified
and named so far. They are one of the oldest inhabitants of the earth and existed since
Paleozoic era (Salunke, 2006). The success of insect survival and adaptation can be
attributed to their characteristics like i) small size; ii) short generation intervals; iii) small
or less need for oxygen; iv) very high egg laying potential etc. Though, insects are
natural invaders of standing crops since long ago; the above mentioned remarkable
qualities have helped them to find their ecological niche even in food grain storages
(FAO, 2006).
More than 10,000 species of insects, 30,000 species of weeds, 1000 species of
nematodes and 100,000 diseases which are caused by fungi, viruses, bacteria and other
microorganisms damage most of the crop plants of the world (Hall 1995; Dhaliwal et al.
2007). However, less than 10 per cent of the total identified pest species are generally
considered as major pests. The severity of pest problems has been changing with the
developments in agricultural technology and modifications of farming practices. The
changing scenario of insect pest problems in agriculture as a consequence of green
revolution has been well documented (Dhaliwal et al., 1985; Singh and Dhaliwal, 1991;
Dhaliwal and Arora, 1993; Arora and Dhaliwal, 1996; Dhaliwal et al., 2002; Singh et
al., 2002; Puri and Mote, 2003; Kumar, 2005; Dhaliwal and Arora, 2006; Dhaliwal and
Koul, 2010). Harbivorous habit of the insects is known to be responsible for destruction
of nearly one third of the world’s total annual crop production. In developing countries
Studies on plant alpha amylase inhibitor and its role in pest management
Ph.D Thesis. Pankaj R. Gavit. (2014) North Maharashtra University, Jalgaon 6
like India, the problem of competition from insects becomes even complicated as we
look at increase in population rate in comparison with the food production. Quantitative
assessment of crop losses becomes difficult because of the year to year variability in
infestation. Infestations of stored food grains could even lead to complete loss and if;
such high levels of losses continue to take place because of poor threshing, cleaning,
drying and storage facilities; the farmers and ultimately the consumers could come
across the food security concerns. On this background it can be affirmed that addressing
the issue of insects of agricultural importance (pre and post-harvest pests) is need of the
hour.
1.3.1 Insects and pre-harvest losses:
Apart from the abiotic stresses; losses in crop plants are due to insect infestation,
weeds or diseases affecting the crop plants when it is still in the field. A number of fly
maggots, aphids, cut worms and caterpillars act as stem, and pod borers and leaf miners
causing damages during various stages of standing crops (Atwal 1976; Pradhan, 1992).
1.3.2 Insects and post-harvest losses:
After harvesting the crop from field it needs to be properly stored as stored crops
serve as an open invitation to any and all kinds of biological agents to feed on them.
Because of extreme adaptabilities and dynamism that insects are equipped with, they
can easily increase their natural niche and attack the stored agricultural products.
Improper storage conditions and preventive measures lead to substantial loss in the
quality as well as quantity of the stored grains resulting in the decline in economic gains.
In developing countries, problem of infestation worsens if the crop is stored in old
granaries which favor the infestation to move to and fro from storage sites.
Moreover, repeated use of same bins without proper hygienic conditions helps to
continue the chain of infestation. It is also observed that traditional pest control methods
Studies on plant alpha amylase inhibitor and its role in pest management
Ph.D Thesis. Pankaj R. Gavit. (2014) North Maharashtra University, Jalgaon 7
are no more efficient, resulting in survival of the pests. If infestation survives, there is
every chance that the insects can resume feeding at any time and product destruction
continues (ICIPE, 2006).
Seed respiration is known to cause depletion of nutrients over the time and if it
combines with attack by insects, deterioration of crop quality speeds up. The temperature
rises due to respiration and insect activities. Generation of heat may lead to moisture
condensation within and surrounding the grain mass which encourages further microbial
growth and provides favorable conditions for insect infestation (Imura and Sinha, 1989;
Piergovanni et al., 1993; Kadlag et al., 1995).
There are around one thousand species of insects known to be associated with
stored food crops throughout the world, 60% of which belongs to the Coleoptera
(beetles) and 8-9% to Lepidoptera (moths and butterflies) (FAO, 2006). The larvae of
Lepidoptera do all the damage whereas, in case of Coleoptera larvae and adults both are
known to feed on stored grains and do the damage (ICIPE, 2006). Post-harvest insect
pest can broadly be divided in to primary pests and secondary pests. Primary pests are
those which prefer feeding on undamaged grains, able to cause distinctive damage while
completing their development in single grain. To be precise they are adapted to feed on
a narrow range of commodities and thus are very selective in their behavior of
oviposition. On the other hand, secondary pests attack on previously damaged grains
and cause non distinctive damage. These pests complete their life cycle within grains
but never undergo development within single grain. As the secondary pest prefers a
wide range of commodities they are also not selective in their egg laying behavior.
The members of Genus Callosobruchus are well known as primary post-harvest pests
and genus Tribolium are referred to as major secondary pest.
Studies on plant alpha amylase inhibitor and its role in pest management
Ph.D Thesis. Pankaj R. Gavit. (2014) North Maharashtra University, Jalgaon 8
1.4 Traditional pest control methods:
The traditional methods provide cheap and feasible ways of minimizing the
losses due to insect pests. The hygiene is the key when administration of the pests is
concerned, some of the essential practices that a farmer needs to follow if the fight
against pests to be won are i) cleaning of bean and granaries, ii) avoiding mixing of
infected grains with healthy grains, iii) burning crop residues after harvest, iv) sealing
cracks and holes in muddy structures and v) any other practice which ensures proper and
clean storage. Some of the traditional methods of pest control are discussed below
1.4.1 Drying:
Use of bush dryers, light fire or solar dryer underneath the crops help to reduce
the water content and could also kill different stages of insect’s life cycle. Use of 50 kg
capacity solar heater eradicated infestations of C. maculates from cowpea seed has been
employed in Cameroon, it was found that temperatures up to 85°C did adversely affect
seed germination (Ntoukam et al., 1997). Mechanical removals of insects, infested
grains or crops are some of the suggestible methods to farmers. Shaking, restacking and
winnowing the grains can also lead to disturbance of insects and greatly reduce their
activities (ICIPE, 2006).
1.4.2 Use of sunlight:
Exposure to sunlight or exposure followed by sieving is a well-known method
followed by farmers of Asia and sub-Saharan Africa (Lale and Satawa, 1996).In this
method, grains are spread on dark papers or black polyethylene sheets and left exposed
to sunlight for at least 7 hrs. Grains are then sieved using a 5mm sieve. This process
could be repeated every 3-4 weeks depending on the size of production and availability
of labour. This method proved to be quite effective in reducing bruchid infestation with
Studies on plant alpha amylase inhibitor and its role in pest management
Ph.D Thesis. Pankaj R. Gavit. (2014) North Maharashtra University, Jalgaon 9
no, or minimal, effect on grain quality or germination (Lale and Sastawa, 1996; Songa
and Rono, 1998; Lale and Ajayi, 2001). In Costa Rica, Leal and Zeledon (1994) showed
that a periodic sieving of stored maize helped to decrease the population of adult pests
up to 99% in 24 weeks.
1.4.3 Inert Dusts:
Some traditionally known materials are often added to the product during
storage, which contribute to the reduction of pest’s activity (Arthur, 1996).Inert dust is
one of such materials; it is added in variable amounts to the stored products. Friction of
dust particle with insects’ cuticle leads to desiccation and could hamper the
developmental stages (Arthur, 1996). Hydrophobic amorphous silica dusts have been
found to be very effective in controlling C. chinensis (Arthur, 1996). A similar effect
has also been achieved through treatment with wood ash, collected from burnt tree wood
or a farmers stove. Some farmers may also add fine sand to hinder the pest activity, in
which the high proportion of quartz causes damage to the sensitive cuticle of the newly
hatched larvae (Kroschel and Koch, 1996). In an experiment in India, pre-treatment of
V. radiata seeds with inert clay resulted in 100% adult mortality of C. chinensis within
24 hours. Seeds maintained over 80% germination for up to 12 months of storage under
ambient conditions (Babu et al., 1989). Botanical insect deterrent or seed protectants
may also be applied to products by some farmers with varied degrees of success. Neem
powder, tulsi leaves (Ocimum spp.) or black pepper (Piper spp.) showed some positive
results in limiting insect infestation.
Studies on plant alpha amylase inhibitor and its role in pest management
Ph.D Thesis. Pankaj R. Gavit. (2014) North Maharashtra University, Jalgaon 10
1.5 Synthetic pesticides: The current scenario:
Pesticides are agrochemicals required to combat the attack of various pest(s) on
agricultural and horticultural crops. Due to the significant increase in the human
population, and the consequent increase in the amounts of food and grains produced,
many small scale farmers adopted the use of synthetic pesticides as a means of pest
control. Pesticides fall into three major classes’ viz. insecticides, fungicides and
herbicides, which also include rodenticides, nematicides, molluscicides and acaricides.
Currently, pesticides worth over US $ 30 billion are applied to crops all over the world.
France, USA, Germany, Great Britain and Switzerland together contribute almost 3/4th
of this amount. Though, India is the 12th largest producer of synthetic pesticides, usage
of pesticides is approximately 570 g/ha compared to about 2500 g/ha in USA (Dave,
1996). Organophosphates, synthetic pyrethroids and organochloranes are the major
synthetic/chemical pesticides apparently in use (Figure 1.1). In India, about 50% of the
total synthetic pesticides used are applied on cotton and almost 17% on rice (Gahukar,
1997).
1.6 Prevalent strategies for pest management during storage:
1.6.1 Fumigation:
Fumigants are low molecular weight chemicals, highly toxic and volatile, that
are used during storage to kill all insect stages residing in the produce. Fumigation is a
widely used method all over the world on small as well as large storage scale. The
method is applied at the farm level in gas-tight granaries or silos, under gas-tight sheets
carefully covering the product or at large scale storage as in warehouses. Fumigants are
commercially available in solid, liquid or gaseous state. Phosphine (PH3), for example,
is a formulated fumigant commercially available either as tablets, pellets, bags or
Studies on plant alpha amylase inhibitor and its role in pest management
Ph.D Thesis. Pankaj R. Gavit. (2014) North Maharashtra University, Jalgaon 11
Figure 1.1: Profiles of utilization of various types of pesticides
0
10
20
30
40
50
60
World India
Pes
tici
des
use
d (
%)
Organophosphates Synthetic pyrethroids
Carbamates Organochloranes
Biopesticides Rest
Studies on plant alpha amylase inhibitor and its role in pest management
Ph.D Thesis. Pankaj R. Gavit. (2014) North Maharashtra University, Jalgaon 12
plates (Bell and Wilson, 1995). Methyl bromide (CH3Br), on the other hand, is in
gaseous form and packed in a liquid state in pressurized steel bottles. At temperature
above 40C it takes a gaseous state, thus, once the container is opened, the gas is released
and starts acting as a fumigant. The two compounds are the most widespread fumigants
in use (ICIPE, 2006).
1.6.2 Dusting:
Dusting along with fumigation are the most commonly used chemical methods
among small-scale farmers (Rai et al., 1987). Dusting, is an easily applied method and
can be implemented with very simple tools such as small perforated metal cans or jute
bags. For small amounts of grains, dust can be mixed with grains using a shovel. Dust
should be mixed thoroughly and distributed evenly all over the produce. Dusters can
also be used as a surface treatment to treat the bags, sacks or the whole granary. For
larger amounts of grains or when storing maize cobs, a "sandwich method" is applied,
whereby dust is spread lightly inside the granary, covering the bottom and walls with a
thin layer, then the produce is entered in to make a layer of 20 cm, followed by another
layer of dust, and so on until the granary is full. The most commonly used insecticide
dusts among farmers belong to two main groups of chemicals: (1) organophosphorus
compounds, such as chlorpyrifos-methyl, fenitrothion, malathion, methacrifos and
pirimiphos-methyl, and (2) pyrethroids, such as cyfluthrin, deltamethrin, fenvalerate and
permethrin (ICIPE, 2006).
1.7 Merits and demerits of synthetic/chemical pesticides:
The synthetic/chemical pesticides are characterized by (i) rapid knock down
effect, (ii) target specificity, and (iii) relatively high residual toxicity. On the other hand,
their repeated use has resulted in (i) disruption of biological control by natural enemies,
Studies on plant alpha amylase inhibitor and its role in pest management
Ph.D Thesis. Pankaj R. Gavit. (2014) North Maharashtra University, Jalgaon 13
(ii) caused development of resistance in pests (Varma and Dubey, 1999), (iii) led to
resurgence of stored product insect pests, (iv) left undesirable effects on non-target
organisms and (v) fostered environmental and human health concerns.
As per the WHOs estimations around 3,50,000 people are killed worldwide and
3 million persons are affected annually as a direct result of pesticide poisoning
(Ellenhorn et al., 1997; WHO 2003). In developing countries inappropriate handling of
such toxicants is widespread and problem of human toxicity due to inadequate
information about this method is considered as a drawback regarding this industry
(ICIPE, 2006). Development of resistance from insects against fumigants is another
problem with the use of fumigants. The problem started as a result of improper
application of these chemicals in the form of incorrect doses, fumigation in non gas-tight
containers or insufficient exposure time. Therefore, fumigation has been highly
discouraged at a small-scale level. Moreover, the use of methyl bromide has been
strongly restricted in industrialized countries because of its ozone-depleting potential.
However, fumigation still remains the most widely used method as an essential large
scale post-harvest practice (ICIPE, 2006).
1.8 Need of alternative strategies:
The increased concern of public about human safety in terms of erratic supply
and prohibitive costs associated with the synthetic/chemical insecticides, resistant
strains/insects, and toxic residues on food and feed, has opened the avenues to search
for alternative methods to combat the attack of various insect pests at different stages of
crop production and storage. Stringent Integrated Pest Management strategies and high
cost involved in research and development has also hindered the process of technology
development in the field of synthetic pesticides (Arthur, 1996).
Studies on plant alpha amylase inhibitor and its role in pest management
Ph.D Thesis. Pankaj R. Gavit. (2014) North Maharashtra University, Jalgaon 14
1.8.1 The alternative methods:
Consumers are now aware of the danger in the use of chemical pesticides to
protect crop products. The world-wide trend to minimize the application of toxic
substances on food products; have led scientists to seek less dangerous alternatives. The
search for other alternatives to pesticides is still on, with the hope that, one day a
competitive and economic method, or an integrated group of methods that can be widely
applicable will emerge. Many trials in different countries using various methods in place
of synthetic pesticides have been conducted and reported to be successful. These
different methods might provide potential alternatives for the wide use of pesticides.
Though their application is still rather limited, however, an intensive amount of research
is carried out to facilitate the use of each method, and to achieve a plausible degree of
integration among the different methods (ICIPE, 2006).
1.8.2 The use of alternative fumigants:
The use of carbon dioxide as a fumigant to replace methyl bromide in the control
of insects and mites damaging stored products has been tried (Newton et al., 1993). Use
of carbon dioxide rich atmospheres showed promising results in disinfesting food
commodities in small storage facilities (Krishnamurthy et al.,1993). A relatively new
technique used by the Indonesian National Logistic Agency (Bulog) for milled rice is to
seal bag sticks into large plastic enclosures flushed with CO2 (Hodges and Surendro,
1996).
The use of "Biogas" as a fumigant, with methane and CO2 as its main
components, may achieve good results in the control of stored pests. Subramanya et al.
(1994) showed that biogas significantly reduced infestations and loss in stored pigeon
pea infested with C. chinensis. In one study, 100% mortality of S. oryzae, R. dominica,
T. granarium and T. castaneum after six days' exposure to biogas in PVC bins was
Studies on plant alpha amylase inhibitor and its role in pest management
Ph.D Thesis. Pankaj R. Gavit. (2014) North Maharashtra University, Jalgaon 15
recorded (ICIPE, 2006). Another method for the control of insects in industrial premises
was developed, where a Gas Operated Liquid Dispensing system was used to mix
separate sources of CO2and insecticide concentrate. The system, given the name
Turbocide GOLD, produces a fine insecticidal aerosol that was reported to give excellent
control of T. castaneum, T. confusum and L. serricorne (ICIPE, 2006).
1.8.3 Temperature:
Temperature is a crucial environmental factor that influences the development
of insects. There is always a minimum, optimum and a maximum range of temperature
in which insects can survive. Insects differ in their tolerance to either low or high
temperatures. However, most stored product pests would follow the same pattern of
survival under a different range of temperatures. As temperature approaches zero, insect
development, activity and movement decline to a minimum. Gradual increase in
temperature will increase insect activity up to a certain range that differs among different
species. Further increase in temperature above the optimum range will lead to increase
in insect mortality and crashing of the population (ICIPE, 2006).
The use of high temperature is a well-known technique to control stored product
pests. For example, temperatures of above 400C are lethal for most stored food pests
(ICIPE, 2006). Adult emergence of S. cerealella, S.oryzae and R. dominica can be totally
suppressed after exposing their pupae to 450C for 72 hours (Sharma et al., 1997).
However, low temperature treatment of grains may also provide a degree of control.
Cold treatment, in combination with drying, is more useful for protecting grain from
attack and deterioration than for disinfestation (ICIPE, 2006). In the USA, a prototype
grain chiller was tested to determine its efficacy as a pest management tool in stored
popcorn. Fewer P. interpunctella were trapped in the chilled aeration bins compared to
the traditionally managed popcorn bins. Costs of chilled aerated (0.11 cents/kg) were
Studies on plant alpha amylase inhibitor and its role in pest management
Ph.D Thesis. Pankaj R. Gavit. (2014) North Maharashtra University, Jalgaon 16
competitive with the costs of conventional pest management practices such as
fumigation and ambient aeration (Mason et al., 1997).
1.8.4 Biological control:
Biological control could provide a safe and useful alternative for the control of
crop pests. However, the use of biological control against stored product pests is still
limited, though recently gaining ground due to increasing health concerns (ICIPE, 2006).
Some promising trials against certain post-harvest pests have been conducted and found
to be useful for controlling stored product insects through the use of natural enemies
(ICIPE, 2006). McGaugheyet et al., (1987) reviewed the use of the entomopathogenic
bacterium Bacillus thuringiensis against pests of stored grain and seed. B. thuringiensis
proved to be ideally suited for use on stored grain and seeds, being compatible with other
protectants and available in different formulations for convenient application. In bulk
stores, dressing a 10 cm deep surface layer with B. thuringiensis at 125 mg/kg controlled
both P. interpunctella and Ephestia cautella. B. thuringiensis retained its activity for up
to 2 years in stored grains, where it was not exposed to ultraviolet radiation, but P.
interpunctella developed resistance levels of over 100-fold in 15 generations on a B.
thuringiensis-treated diet in the laboratory (ICIPE, 2006). Kroschel and Koch (1996)
treated potato tubers with a mixture of B. thuringiensis and fine sand. Good results were
obtained against the potato tuber moth P. operculella. While Raman et al. (1987) showed
that a dust formulation of B. thuringiensis applied to potato tubers was most effective.
Nosema sp. can also be used for the control of certain pests. One example is the use of
spores against L. serricorne (ICIPE, 2006). Insect death is caused by severe damage to
gut epithelial tissues and fat bodies.
Studies on plant alpha amylase inhibitor and its role in pest management
Ph.D Thesis. Pankaj R. Gavit. (2014) North Maharashtra University, Jalgaon 17
1.8.5 Microbial pesticides:
A very long back, Agostino Maria Bassi isolated a fungus, Beuveria bassiana,
from silkworm. He also showed that the disease could be artificially transferred to
different species of insects. Micro-organisms themselves or their metabolites are known
to exert pathogenic effect by infecting the insect or contaminating its food (Burges and
Jones, 1998). Of the microbial pathogens registered as pesticides worldwide, 36 species
were registered to control animal pests (insects, mites and nematodes) including 11
viruses, 9 bacteria, 9 nematodes, 6 fungi and 1 microsporidia(Flexner and Belvanis,
1998).Similarly, 15 fungi and 5 bacteria, were registered to control plant diseases
(Copping, 1998).
1.8.6 Irradiation:
The use of doses of about 0.2-0.5 Kilogray (kGy) provides another alternative
method for the control of pests in stores(ICIPE, 2006). Treatment of radiation and
carbon dioxide in combination produced a higher mortality in T. confusum than did
either treatment alone (Omar et al., 1988). This method has the advantage of leaving no
residues in the product, though it might not be feasible due to the high costs involved in
application. Some other efforts involved the use of microwaves against stored crop pests.
A special microwave system was made with a variable speed conveyor belt and
employed for insect control in stored milled rice. Results indicated that Cryptolestes
pusillus and T. confusum were killed economically with microwaves (Langlinais, 1989).
1.8.7 Pheromones and trapping:
Pheromones are exocrine secretions used by insects for finding mates,
aggregation, alarming, tracking or trailing (Varma and Dubey, 1999). Pheromones,
being behaviour controlling chemicals or semi-chemicals, give another promising option
to chemical/synthetic pesticides and find important role in insect control within
Integrated Pest Management (IPM) (Suckling and Karg, 1998).
Studies on plant alpha amylase inhibitor and its role in pest management
Ph.D Thesis. Pankaj R. Gavit. (2014) North Maharashtra University, Jalgaon 18
1.8.8 Cultivars / Plant breeding:
There is a wealth of information regarding the selection of resistant plants
through intensive breeding programmes (ICIPE, 2006). Though host plant resistance is
a promising strategy in pest control, insect populations are able to develop biotypes that
can attack formerly resistant varieties, and there is evidence that improved varieties tend
to perform poorly under low input conditions (ICIPE, 2006). However, this strategy may
result, along with other control methods, in a significant degree of pest population
regulation.
1.8.9 Transgenic varieties / Use of transgenic plants:
Molecular biology and genetic engineering have provided us many tools to
manipulate and develop novel transgenic crops for eco-friendly and sustainable
agriculture practices. The use of transgenic plants is currently gaining ground in different
parts of the world. Bt can be introduced to plant tissues and serve as protectants against
infestation by certain pests. Therefore, now it is possible to develop crops, which are
resistant to a particular pesticide or a pest (Shade et al., 1994; Schroeder et al., 1995).
Some of the features of these two concepts are discussed below.
1.8.10 Genetic engineering of crops for pesticide resistance:
The herbicide resistance in plants is being attempted by either overproducing the
target site of an herbicide in a plant or incorporating genes for enzymes of microbial
origin, which are capable of hydrolyzing a herbicide before it reaches its designated
target site in a plant. Thus, a plant could be ‘immunized’, while herbicide selectivity
destroys the herbs competing for soil nutrition under field conditions. Another sound
strategy is to replace the target site of an herbicide by a mutant target site, capable of
binding the herbicide. Appreciable success has been reported in engineering the
herbicide resistance, in a wide variety of plants, using these three strategies (Schroeder
et al., 1995; Hammond-Kosack and Jones, 1997). If engineering resistance to pesticides
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Ph.D Thesis. Pankaj R. Gavit. (2014) North Maharashtra University, Jalgaon 19
is not possible due to practical problems in the modification of genomics, engineering
the resistance to a pest(s) appears feasible.
1.8.11 Genetic engineering of crops for insect resistance:
Various proteinaceous pesticidal molecules were found to be effective against
wide range of agriculturally important pests, which are relatively innocuous to human
beings and environment (Ranjekar et al., 2003). Therefore, it is desirable to review the
efficacy of various insecticidal proteins for the development of insect-resistant
transgenic crops as an important alternative strategy. It involves the use of following
strategies (i) Bt toxin, (ii) plant lectins, (iii) plant proteinase inhibitors and (iv) -
amylase inhibitor encoding genes.
1.8.12 Insecticidal crystal proteins (ICPs) of B. thuringiensis:
ICPs have acquired worldwide acceptability as an eco-friendly biopesticide
(Ranjekar et al., 2003). The spray formulations of Bt ICPs have their own limitations
such as (i) short half-life, (ii) low residual toxicity and (iii) inability to reach burrowing
insects. To overcome these disadvantages, it was desirable to express the genes encoding
ICPs in plant itself, at a level sufficient to kill the insect (Ranjekar et al., 2003). Though,
initially level of expression of Bt genes was very low, in the last decade sufficient
progress was made to modify the cry genes. These genes have conferred resistance
against boll worm and stem borers when expressed in cotton and rice, respectively
(Wilhite et al., 2000a). Similarly, few other crops such as tomato, potato, corn, tobacco
and soybean were transformed with the modified Bt genes (Ranjekar et al., 2003). In a
storage bioassay in Belgium, selected potato line tubers carrying the B. thuringiensis Cry
IAb6 insecticidal crystal protein gene gave 100% control of P. operculella damage
(Jansens et al., 1995).
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1.8.13 Plant lectins:
Lectins are carbohydrate-binding proteins capable of agglutinating cells and
binding glycans of glycoproteins, glycolipids or their polysaccharides (Czapla, 1997).
Lectins have been classified into three major groups based on their structure:
merolectins, hololectins and chimerolectins. The lectins in nature (i) may function as
storage proteins, (ii) have been identified as signal molecules of Azolla-Anabaena
symbiosis, (iii) may stimulate pollen germination in Liliumlongoflorum, (iv) are
involved in plant-symbiont attachment and (v) plant defense (Sengupta et al., 1997).
Plant lectins have been found to be toxic to viruses, bacteria, fungi, insects and higher
animals. The mechanism of toxicity is based on the specific binding of the glyco-
conjugates in the gut of the insect. Three possible interactions include binding of lectins
to (i) the chitin in peritrophic membrane, (ii) glycoconjugates exposed on the epithelial
cells along the digestive tract and (iii) glycosylated digestive enzymes interfering with
nutrient uptake (Sengupta et al., 1997). A lectin from Galanthus nivalis, when expressed
in transgenic tobacco and potato was found to be toxic to aphids and the tomato moth,
Lacanobia oleracea (Ranjekar et al., 2003). The effect of another lectin on larval
survival to adulthood in C. maculatus (cowpea weevil) has also been studied (Czapla,
1997).
1.9 Insect digestive enzymes: Potential targets of pest management:
Insects obtain their nutritional requirements by utilizing food from environment
and proper digestion of ingested food. Digestion following ingestion of food from
various origins is a process by which food molecules (macromolecules such as
carbohydrates, lipids and proteins) are broken down into smaller molecules to be
absorbed by cells in the gut tissue. The ingested foods by insects are polymers such as
starch, proteins and lipids that are digested in three phases. Primary digestion is the
dispersion and reduction in molecular size of the polymers leading to oligomers. During
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intermediate digestion, a further reduction is made, producing smaller molecules
(dimers), and then dimers become monomers as the final step. This process is perfectly
controlled by digestive enzymes that depend on their site of activity in the insect gut.
The major digestive enzymes in the midgut of insects consist of amylases, glycosidases,
lipases and proteases that are similar in their hydrolytic nature. α-Amylases (α-1,4-
glucan-4-glucanohydrolases; EC 3.2.1.1) are the hydrolytic enzymes that catalyze the
hydrolysis of α-D-(1,4)-glucan linkages in glycogen and other related carbohydrates.
Glycosidases (EC 3.2) catalyze cleavage of internal bonds in polysaccharides and
hydrolyze oligosaccharides as well as disaccharides. Lipases (triacylglycerol-acyl-
hydrolase EC 3.1.1.3), which catalyse the hydrolysis of fatty acid ester bonds, are widely
distributed among animals, plants and microorganisms. The most characteristic property
of lipases is their activity on substrates at the interface between the aqueous and the lipid
phase. Peptidases (peptide hydrolases, EC 3.4) act on peptide bonds and include
proteinases (endopeptidases, EC 3.4.21–24) and exopeptidases (EC 3.2.4.11–19) (Terra
and Ferreira, 2005).
Since synthetic insecticide lead to various problems in ecosystem and
agricultural production, trends are to focus on digestive physiology of insects to control
them. There are numerous studies showing effect of specific inhibitors, especially
amylases and proteases, on digestive enzymes which suppress their activity and overall
disruption of digestive process. Many of these inhibitors are now used for incorporating
resistance in transgenic plants. In fact, creating resistant varieties using a
biotechnological approach has led to the development of insect-resistant transgenic
plants through the transfer of several insect resistance genes to suppress insect growth.
It is mandatory to study insect pests’ digestive enzymes in order to develop
biotechnological processes to provide resistant host plants. The digestive enzymes of
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Ph.D Thesis. Pankaj R. Gavit. (2014) North Maharashtra University, Jalgaon 22
insects could be a target to overcome their feeding ability so their developmental
secretion and site of activity in the insect gut needs specific attention.
1.9.1 Proteinase inhibitors (PIs):
Plants are known to contain an array of proteinase inhibitors, which are induced
in response to herbivore attack (Green and Ryan, 1972). These PIs are constitutively
expressed at high levels in storage organs and seeds. PIs block the midgut proteinases of
insects which adversely affects the protein digestion. This inhibition leads to reduced
nutrition and retarded growth of insects (Jouanin et al., 1998). PIs of Leguminosae,
Graminae and Solanaceae have been extensively studied (Harsulkar et al., 1998).
Depending on the midgut pH, insect endoproteinases are grouped into four main
categories: (i) cysteine (or thiol) proteinases (pH 2-5), (ii) aspartyl (or carboxyl)
proteinases (pH 2-5), (iii) serine proteinases (pH 7-9) and (iv) metallo-proteinases
(Girard et al.,1998a, 1998b, 1998c). Insects of class Coleoptera predominantly use
cysteine proteinases, while those of Diptera and Lepidoptera use serine proteinases
(Wilhite et al., 2000a). Coleopteran insects have demonstrated an ability to utilize serine
and aspartyl proteinases in exceptional cases (Wilhite et al., 2000b). Therefore, the use
of multiple PIs for insect control is necessitated. Of utmost importance is the fact that
Coleopteran larvae are difficult to reach with classical insecticides as they bore inside
the grain.
Soybean is known to contain Bowman-Birk type trypsin inhibitors, also called
as Soybean Kunitz Trypsin Inhibitor (SKTI) or serine proteinase inhibitor (Nandi et al.,
1999). These inhibitors are known to confer resistance against brown plant hopper
(Nilaparvata lugens Stal.), a major pest of rice (Lee et al., 1999) and cotton boll worm
(H. armigera), a Lepidopteran polyphagous pest of major crop plants like cotton, tomato,
tobacco, chickpea and pigeon pea (Harsulkar et al., 1998; Nandi et al., 1999).
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Ph.D Thesis. Pankaj R. Gavit. (2014) North Maharashtra University, Jalgaon 23
1.9.2 α-Amylase inhibitors:
α-Amylases (a-1,4-glucan-4-glucanohydrolases) are widespread hydrolytic
enzymes found in microorganisms, animals and plants. They catalyze the initial
hydrolyses of α-1,4-linked sugar polymers, such as starch and glycogen, into shorter
oligosaccharides, an important step towards transforming sugar polymers into single
units that can be assimilated by the organism.
α-Amylases from insects and mammals have been characterized from
biochemical, molecular and structural point of view in considerable details (Qian et al.,
1993; Grossi-de-Sa¯ and Chrispeels, 1997; Strobl et al., 1998). These widely distributed
molecules are the most important digestive enzymes of many insects that feed
exclusively on seed products during larval and/or adult life. When the action of the
amylases is inhibited, nutrition of the organism is impaired causing shortage of energy.
α-Amylase inhibitors occur in many plants as part of the natural defense
mechanisms. They are particularly abundant in cereals (Abe et al., 1993; Feng et al.,
1996; Yamagata et al., 1998; Franco et al., 2000) and legumes (Marshall and Lauda,
1975; Ishimoto et al., 1996; Grossi-de-Sa et al., 1997). Research into α-amylase
inhibitors is relevant with respect to several aspects of human health: from diagnosis of
pancreatic hyperamylasemia disorders (O’Donnell et al., 1977; Turcotte et al., 1994) to
control of diabetes, obesity and hyperlipdaemia (Bischoff et al., 1994) and nutritional
and toxicological aspects of foods. In addition, amylase inhibitors are of great interest
as potentially important tools of natural and engineered resistance against pests in
transgenic plants (Chrispeels et al., 1998; Gatehouse and Gatehouse, 1998; Valencia et
al., 2000).
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Ph.D Thesis. Pankaj R. Gavit. (2014) North Maharashtra University, Jalgaon 24
1.10 Classification of proteinaceous α-amylases inhibitors:
Proteinaceous α-amylases inhibitors isolated from several plant sources exhibit
a remarkable structural and functional diversity. Some α-amylases inhibitors exhibit
high affinity for both mammalian and insect α-amylases while other recognize either
insect or mammalian α-amylases
Based on similarities in amino acid sequences and 3D structures, Richardson
(1990) has proposed that α-amylases inhibitors can be classified into six classes. The
alpha amylase inhibitor from Streptomyces constitutes the seventh class (Table 1.2).
1.10.1 Lectin like α-amylase inhibitor:
This class includes the three isoforms designated as αAI-1, αAI-2 and αAI-3
isolated from white, red and black kidney beans, respectively (Wilcox and Whitaker,
1984; Ho et al., 1994 and Lee et al., 2002). These three iso-inhibitors are encoded by
two different alleles and only αAI-1 inhibits both mammalian and insect α-amylases
whereas αAI-2 inhibits different insect α-amylases but does not inhibit mammalian
amylases (Lee et al., 2002). αAI-3, the third isoform, is a single chain α- amylase
inhibitor like protein that is completely inactive towards all α-amylases tested (Mirkov
et al., 1994). The enzyme inhibitor complex formation for this class of α-amylase
inhibitor is dependent on time, pH and concentration (Grossi de sa et al., 1997, Le Berre
Anton et al., 1997).
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Ph.D Thesis. Pankaj R. Gavit. (2014) North Maharashtra University, Jalgaon 25
Table 1.2: Classification of proteinaceous α-amylase inhibitors
Inhibitor type Source α-Amylase
target
Amino acids
/ s-s bonds
Ki (M) Abbreviations
Legume lectin
Like
Common bean Mammalian,
insect, fungi
240-250 / 5 30 x 10 -12 αAI1
Knottin like Amaranth Insect. 32 / 3 Strong AAI
ϒ – Purothionin
like
Sorghum Insect,
Mammalian
47-48 / 5 Strong SIa1, SIa2, SIa3
Cereal like Barley, wheat,
ragi, rye
Mammalian,
insect,
bacterial
124-160 / 5 0.1 x 10 -9 RATI (RBI),
0.19, 0.28
(WMAI-1),
0.53, WRP26,
BMAI-1
Kunitz type Barley, wheat,
rice
Cereal, insect. 176-181 / 1-2 0.22 x 10 -9 BASI, WASI,
RASI
Taumatin like Maize Insect, fungi 173-235 / 5-8 - -
Microbial Streptomyces Mammalian,
bacterial
74-76 9 x 10 -12 Haim, Paim,
Tendamistat
(HOE 467)
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1.10.2 Knottin type α-amylase inhibitor:
This class of α-amylase inhibitor includes the smallest known natural
proteinaceous α-amylase inhibitor isolated from a Mexican crop plant, Amaranth
(Amaranthus hyochondriacus). It comprises of 32 amino acid residues and 3 disulphide
bonds (Chagolla-Lopez, 1994). The name, knottin type, is derived from the observation
of the presence of a knottin fold made up of 3 anti parallel β strands and a characteristic
disulphide topology (Carugo et al., 2001, Martins et al., 2001). This α-AI is an ideal
candidate in the development of insect resistant transgenic plants since it specifically
inhibits insect α-amylases and not mammalian α-amylase (Chagolla-Lopez, 1994).
1.10.3 Cereal type of α-amylase inhibitor:
This class of inhibitors include the large protein family from cereal seeds
containing 120-160 amino acid residues and five disulphide bonds inhibiting both
mammalian and insect α-amylases (Buonocore et al., 1977; Campos and Richardson,
1983; Lyons et al., 1987; Garcia-Moroto et al., 1991; Kusaba-Nakayama, 2000). These
are also referred to as the CM proteins due to their appearance in the chloroform –
methanol extracts. This family of α-amylase inhibitors is encoded by multiple genes
(Franco et al., 2000). With their different sequences providing a wide array of inhibitor
specificity (Kusaba-Nakayama; 2000, Garcia-Casada et al., 1994). It shows a wide
spectrum of activity against α-amylases from birds, mammals, insects and Bacilli.
1.10.4 Kunitz type α-amylases inhibitor:
This type of α-amylases inhibitor is present in cereals such as barley, wheat and
rice (Garcia-Olmedo et al., 1992; Otsuba and Richardson; 1992; Gvozdeva et al., 1993).
The bifunctional barley amylase subtilisin inhibitor (BASI) is the most well
characterized inhibitor in this class of α-amylases inhibitors. Initially a subtilisin
inhibitor was found to be present in barley, which showed a close sequence similarity to
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Ph.D Thesis. Pankaj R. Gavit. (2014) North Maharashtra University, Jalgaon 27
the Kunitz soybean trypsin inhibitor. Subsequently this barley protein was identified in
complex with an endogenous α-amylases suggesting an additional α-amylases inhibitory
activity and hence named BASI (Mundy et al., 1984). Homologous proteins with 92%
and 58% sequence identity are present in wheat (WASI) (Kadziolo et al., 1998) and rice
(RASI), respectively (Garcia-Olmedo et al., 1992).
BASI is involved in regulating degradation of seed carbohydrate, preventing
endogenous α-amylase 2 (AMY 2) from hydrolyzing starch and thus preventing
premature sprouting (Yi-Hung Lin et al., 2006). BASI specifically inhibits barley AMY2
with Ki= 0.22 nM (Abe et al., 1993). It also protects seeds from exogenous proteinase
and α-amylases produced by pests and pathogens (Mundy et al., 1984). The structural
features of BASI include 181 amino acids with two disulphide bonds and a β trefoil
topology (Svendsen et al., 1986; Valle et al., 1998; Bruix et al., 1993). This class of
inhibitors prevents substrate access by binding strongly through H-bond, van der Waal’s
forces and salt bridges to the A and B domain near the catalytic site of the enzyme rather
than by binding to the actual catalytic site.
1.10.5 Thaumatin like α-amylase inhibitors:
This class of α-amylases inhibitors is so called since they closely resemble
thaumatin, a seed protein present in the fruit of Thaumatococcus danielli (Bruix et al.,
1993; Schmioler-O’ Rourke et al., 2001). Zeamatin, the α-amylases inhibitor from maize
(Zea mays) is the most well studied protein from this group. It has bifunctional activity
against α-amylases as well as trypsin. The surface of the protein is rich in arginine and
lysine residues (Batalia et al., 1996). Zeamatin is active against α-amylases of Tribolium
casteneum, Sitophilus zeamays, Rhizopherta dominica and trypsin from pocine pancreas
(Blanco-Labra et al., 1980; Schmioler-O’ Rourke et al., 2001). Zeamatin can bind to β -
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1, 3- glucan present in the fungal cells and thus shows anti-fungal activity (Trudel et al.,
1998, Selitrennikoff et al., 2000).
1.10.6 -Purothionin like α-amylase inhibitor ( -Thionins):
This class comprises relatively small proteins containing 47-48 amino acid
residues. These proteins are rich in sulphur. SIα1, SIα2 and SIα3, the three isoforms of
α-amylase inhibitor from sorghum (Sorghum bicolor) are well characterized. These
isoforms contain four disulphide bridges and have 42-87% sequence identity (Nitti et
al., 1995). They belong to the ϒ-thionin superfamily of distantly related proteins, of
which several members are involved in plant defense through a wide variety of
mechanisms (Franco et al., 2000). They strongly inhibit α-amylases from insects such
as locusts and cockroaches, poorly inhibit the fungal and human salivary amylases and
do not inhibit α-amylases from porcine pancreas, barley and Bacillus species (Wilson et
al., 2000).
1.10.7 Microbial α-amylase inhibitors (Streptomyces inhibitors):
Tendamistat, Haim, Paim and related proteins constitute a family of small
proteins of approximately 75 amino acid residues which have been purified from
different Streptomyces species. These inhibitors possess about 30% sequence identity
and show conserved disulphide topology. They act on α-amylases from animals,
Streptomyces species and Bacillus species (Murao et al., 1980; Murao et al., 1983;
Verstesy et al., 1984; Hoffman et al., 1985; Wiegand et al., 1995).
1.11 Present status of research on α- amylase inhibitors:
There is a great diversity of α-AIs from different source and they are reported to
inhibit digestive enzymes of mammals, insects and fungi (Diaset al., 2005). Alpha AIs
found in wheat, barley and Indian finger millets have shown excellent inhibition of alpha
amylase from insect particularly of the order Coleoptera (Farias et al.,2007). Some α-
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Ph.D Thesis. Pankaj R. Gavit. (2014) North Maharashtra University, Jalgaon 29
AIs show strict enzyme specificity and recognize one out of several closely related iso-
enzymes or enzymes of different species (Franco et al., 2000).
Leguminous seeds are rich sources of α-AIs especially of lectin-like (Payan,
2004). Two different α-AI variants of lectin like were purified from different varieties
of Phaseolus valgaris. Though they have high degree of amino acid sequence similarity,
their inhibitor specificities are different (Grossi-de-Sa and Chrispeels, 1997). The first
variant, known as α-AI1, inhibits α-amylase from porcine pancreas, Callosobruchus
maculatus and Callosobruchus chinensis, the second variant, α-AI2, shows inhibition
against amylase from Zabrotes subfasciatus (Grossi-de-Sa et al., 1997).
The Kunitz types α-AIs are commonly found in cereals such as barley and rice
(Micheelsen et al., 2008). Among them, bifunctional α-amylase/subtilisin inhibitor have
been implicated in plant defense and regulation of α-amylase. Recently, Alves et al.,
(2009) have reported four novel Kunitz like α-AIs from seeds of Brazilion species,
Delonix regia with significant inhibitory activity against α- amylase of two coleopteran
insects.
Alpha amylase inhibitor from the Maxican crop plant, Amaranth is the smallest
known natural proteinaceous inhibitor. It has 32 amino acids with 3 disulfide bridges
and belongs to knottin- like family of α-AIs. It has been shown to strongly inhibit the
α-amylase activity of Tribolium castaneum and P. truncatus (Chagolla-Lopez et al.,
1994). Similarly, 3 isoforms of α-AI from sorghum containing 47 amino acid and 4
disulfide bridges and belonging to γ–thionin super family showed strong inhibition of
insect amylase (Bloch and Richardson, 1991). Zaumatin, a thaumatin- like α-AI from
maize was found to be specific for insect α-amylase (Franco et al., 2002).
Particular attention has been focused on the lectin-like inhibitors present in the
common bean P. vulgarisseeds, which have been shown to have toxic effects to several
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Ph.D Thesis. Pankaj R. Gavit. (2014) North Maharashtra University, Jalgaon 30
insect pests (Ishimoto and Kitamura, 1989; Huesing et al., 1991a; Ishimoto and
Chrispeels, 1996; Grossi-de-Sa¯ et al.,1997). The effect of the bean amylase inhibitors
on the amylases of different organisms was well determined not only by enzymatic
activity, but also in feeding assay experiments (Ishimoto and Kitamura, 1989; Ishimoto
et al., 1996; Grossi-de-Sa¯ et al., 1997). Complete resistance against bruchids, the pea
weevil (Bruchus pisorum), the cowpea weevil (C. maculatus) and the adzuki bean weevil
(C. chinensis), was found in transgenic pea and adzuki bean seeds expressing the
inhibitor, α-AI-1, of the domesticated common bean P. vulgaris(Shade et al., 1994;
Schroeder et al., 1995; Ishimoto et al., 1996; Chrispeels, 1996; Morton et al., 2000). The
transgenic grains showed minimal effects on mammalian digestive system (Pusztai et
al., 1999) suggesting that these proteins can be safely introduced into food plants.
In Phaseolus seeds, α-AI is a member of a protein family that includes two other
defense proteins, the phyto-hemagglutinin (PHA) and arcelins (Arc) (Chrispeels and
Raikhel, 1991). The members of these plant defense proteins are encoded by tightly
linked genes in the P. vulgaris genome and their deduced amino acid sequences are
highly homologous (45–85% identical) (Chrispeels and Raikhel, 1991; Nodari et al.,
1993; Mirkov et al., 1994). It is likely that these genes family originated from a common
ancestral gene through both duplication and divergence (Osborn et al., 1986; Kornegay
et al., 1993; Nodari et al., 1993). The proteins display insecticidal activities and protect
seeds against different predators through different mechanisms. PHA is a lectin that
binds to the glycans on the glycoproteins of the intestinal epithelium of animals and act
as a mitogen. In contrast, arcelins are suggested to bind to the peritrophic membrane of
the insect gut interfering with nutrient absorption and causing rupture of gut membranes
(Paes et al., 2000). As mentioned, α-AI inhibits the activity of some mammalian and
insect a-amylases (Grossi-de-Sa¯ et al., 1997).
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Ph.D Thesis. Pankaj R. Gavit. (2014) North Maharashtra University, Jalgaon 31
Phaseolus genus contain at least four phenotypes of α-AIs (α-AI-1, α-AI-2, α-
AI-3 and the null type) with alleles encoding the inhibitors being co-dominant and with
the presence of inhibitors dominant in relation to its absence (Suzuki and Ishimoto,
1999). Of particular interest is the specificity of the two isoforms α-AI-1 and α-AI-2
towards different α-amylases. α-AI-1, found in most cultivated common bean varieties,
inhibits mammalian α-amylases such as porcine pancreatic amylase (PPA) and the insect
larval α-amylases of C. chinensis, C. maculatusand B.pisorum, but is not active against
the a-amylase of the Mexican bean weevil (Zabrotes subfasciatus), which is an
important storage pest of the common bean (Grossi-de-Sa¯ and Chrispeels, 1997). The
second variant, α-AI-2, which shares 78% amino acid homology with α-AI-1, is found
in few wild accessions of common beans and specifically inhibits the Z. subfasciatus
larval α-amylase, ZSA, (Ishimoto and Kitamura, 1992, 1993; Suzuki et al., 1993; Grossi-
de-Sa¯ and Chrispeels, 1997; Grossi-de-Sa¯ et al., 1997). α-AI-2 can also weakly (40%)
inhibit the pea bruchid α-amylase, thus delaying the maturation of the larvae (Morton et
al., 2000).
This inhibitor provides an excellent example of the co-evolution of insect
digestive enzymes and plant defense proteins. Different species of bruchids are found
all over the world. For example, while the two main bruchids species of common bean,
Z. subfasciatus and Acanthoscelides obtectus, have evolved in the Americas and are able
to feed on all cultivated varieties of the common bean, the pea and cowpea weevil have
evolved in the Old World and are not able to consume the common bean. They thrive on
cowpeas, mung beans, and other Eastern Hemisphere legumes. The amylase inhibitor
found in the common bean does not inhibit the amylase of the Mexican bean weevil, but
completely inhibits the amylases of the pea and cowpea weevils (Grossi-de-Sa¯ et al.,
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1997; Chrispeels et al., 1998), which suggest a co-evolution between insect and their
food source.
The toxic effect of α-AI-1 toward bruchid pests is assumed to be caused by
inhibition of digestive amylases. Its failure, however, to affect Z. subfasciatus has two
possible explanations: either the amylase of the insect is not inhibited by α-AI-1, or the
insect has an intestinal serine proteinase that is able to digest the inhibitor (Ishimoto et
al., 1996; Silva et al., 2001a). Recent studies implicated the presence of the inhibitor in
the insect’s diet with the induction of new amylase activities and inhibition of the
constitutive larval Z. subfasciatus α-amylase by α-AI-1 when starch granules were used
as substrate (Silva et al., 1999, 2001b).
1.12 Target Pests:
The two target pests chosen for evaluating the activity of isolated α- amylase
inhibitor were
i) Callosobruchus chinensis (Pulse beetle), an important primary post-
harvest pest of grain legumes in our country and
ii) Helicoverpa armigera (American boll worm), an economically important
polyphagous pre harvest pest of cotton and leguminous crops in our
country.
A brief description of these two pests along with their life cycles are described
in the following pages
1.12.1 Callosobruchus chinensis L., (Coleoptera: Bruchidae) (Pulse
beetle):
Pulse beetle is cosmopolitan in distribution. It attacks a number of bean of
various species and has the ability to alternatively attack other pulse crops. C. chinensis
is known to be originated in tropical Asia but currently has occupied all over the tropics
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and subtropics. Mostly, it attacks green grams, chickpeas and cowpeas (Ahmed et al
2003; ICIPE 2006). Adult beetles are brownish in color with black marks and possess
long straightened antennae with a total body length of 2-4 mm having a very short life
span lasting only for 12 days and do not feed (ICIPE, 2006). The same insect behaves in
two forms; i) flying (active form) and ii) flightless (common form). The active flying
form colonizes cowpea fields, but in storage conditions the normal flight less form keeps
reproducing till the storage ends and then after the flying form reappears to disperse to
new locations (Atwal, 1976).
1.12.2 Life cycle of Callosobruchus chinensis:
The life cycle of C. chinensis goes through four different life stages (Photoplate
1.1). The beetles breed freely from March to November and hibernate at the larval stage
during winter. During January to April, adults start appearing and start to copulate
immediately after emergence. The females lay small oval shaped and scale like eggs just
after a day glued to seed surface or to the pods. C. chinensis can lay 1 to 8 eggs per grain
and undergo metamorphosis to develop in to adults in separate chambers. C. chinensis
are known to lay 34 to 113 eggs at a rate of 1 to 37 per day (Atwal, 1976). The larvae
tunnel inside the seeds where the entire development takes place. In early stages, the
larvae appear whitish, with a light brown ting on head, to become creamy white later.
The mature larvae are 6 to 7 mm long, feed inside the grain and cause damage to pulses
(Hozomi and Miyataka, 2005).
Studies on plant alpha amylase inhibitor and its role in pest management
Ph.D Thesis. Pankaj R. Gavit. (2014) North Maharashtra University, Jalgaon 34
Photoplate 1.1:Chronological developmental stages in the life cycle of
Callosobruchus chinensis.
Studies on plant alpha amylase inhibitor and its role in pest management
Ph.D Thesis. Pankaj R. Gavit. (2014) North Maharashtra University, Jalgaon 35
C. chinensis causes substantial economic loss during the storage of pulses (Shu
et al., 1996). Severe infestation by C. chinensis greatly reduce the quality and
germination potential to under 20% within just four months and up to 100 % within 6
months of storage (Seck et al., 1996; Aslam, 2004). February to August is the period of
maximum damage because all the developmental stages appear simultaneously. The
damaged grain does not remain fit for human as well as animal consumption (Aslam,
2004). Partially infested grains provide favorable conditions for fungal attacks and emits
very bad odor (Ahmad et al., 2003).
1.12.3 Helicoverpa armigera (Hubner) (Pod borer)(Lipidoptera:
Noctuidae):
Members of Helicoverpa species are distributed word wide and are considered
as highly polyphagous agricultural pests (Grover and Pental, 2003). As it is a serious
pest of cotton in USA, it is also called as American bollworm. In India, it causes serious
damage to cotton, sorghum and other Rabi crops, especially red gram and Bengal gram
(Pradhan 1992, Gujar et al., 2000). Larvae of H. armigera are voracious foliar feeders
as early instars and later shift to the developing seeds, fruits, or bolls. In its adult stage
this insect is a stoutly built moth with olive green to reddish brown wings. Females lay
eggs on the flowering and fruiting structures of the crops, where voracious larval feeding
leads to substantial economic loss (Harsulkar et al., 1999). This insect is a very
dangerous pest as it i) possesses extreme fecundity, ii) can host over 180 different plant
species, iv) can undergo diapause during adverse conditions and iv) has the ability to
migrate over long distances (Manjunath et al., 1989; Cunnigham et al., 1999; Shanower
and Romeis, 1999). H. armigera has evolved a high degree of resistance to
organophosphate and pyrethroid insecticides making the matter even worst (Armes et
al., 1996). H. armigera larvae prefer to feed and develop on the reproductive structures
Studies on plant alpha amylase inhibitor and its role in pest management
Ph.D Thesis. Pankaj R. Gavit. (2014) North Maharashtra University, Jalgaon 36
of crops which are rich in nitrogen making them extremely damaging (Fitt, 1989). These
structures are often the part of the crop that is harvested (King, 1994). Depending on the
crop, bollworm induced damage can range from 50 to 90 percent of the yield (Reed and
Pawar, 1982, Sehgal and Ujagir, 1990).
A detailed account of developmental stages and life cycle is given below (King,
1994).
Eggs: The eggs are spherical in shape having approximately a diameter of 5 mm,
initially they are white in color and then get darken to grayish brown prior to eclosion.
Vertical ridges of alternating length are sculptured on the eggs and have a smooth apical
area that contains micropyle.
Larvae: First instar larvae are characterized by a black to brown head capsule
and a yellowish-white body with a spotted appearance as a result of sclerotized setae,
tubercle bases and spiracles. As the growth progresses, larval color darkens with
successive molts for the 6 instar stages which is typically observed for H. armigera.
Larval coloration may vary considerably due to diet content. The final instar larval size
could range from 3.5 – 4.2 cm in length.
Pupae: Size of the pupa ranges from 14-22 mm long and 4.5-6.5 mm in width at
the widest point and are a dark brown color with a smooth surface.
Adult: The adult moth demonstrates the characters of a typical Noctuid. The
stout bodied moth has a wing span range of 35-40 mm and the body length range of 18-
19 mm. The coloration varies from dull greenish yellow to olive gray or light brown and
females are darker than males.
1.12.4 Life Cycle : Helicoverpa Armigera:
The reproductive structures of plants are the eggs laying sites and depending
upon temperature their incubation take place for approximately 3-14 days (Pearson
Studies on plant alpha amylase inhibitor and its role in pest management
Ph.D Thesis. Pankaj R. Gavit. (2014) North Maharashtra University, Jalgaon 37
1958; Fitt, 1991; Shanower and Romeis, 1999). Newly hatched larvae eat the egg sheath
and then start its search for a good site to feed (King, 1994). Flowers, buds, bolls and
leaves are the preferred feeding sites. Fluctuation in the number of larval instars and the
duration of the larval period is always observed as a function of change in the host plant
and temperature (Shanower and Romeis, 1999). Larval period of this insect before
pupation lasts for 12-32 days and typically involves 5-7 instars (Reed, 1965; Rajagopal
and Channa Basavanna, 1982) the prepupal stage lasts for 1-4 days. Pupation occurs in
the ground between 1.2 inches (3 cm) and 7.1 inches (18 cm) (King,1994). If the insect
is not in diapauses, the pupal period takes 10-14 days (Shanower and Romeis, 1999). If
in diapause, the pupal period can take several months to complete. Adult moths emerge
from just after dark to midnight and crawl onto a plant or vertical substrate where their
wings dry (King, 1994). Moths feed on nectar, females release sex pheromones and
mating occurs approximately 4 days after emergence (Pearson, 1958; Hardwick, 1965;
Ramaswamy, 1990). After a pre-oviposition period of 1-4 days, females begin to
oviposit in the reproductive structures of the crop (Jayaraj, 1982; Fitt, 1989). Generally,
oviposition occurs after dark and females can lay up to 3,000 eggs each (Shanower and
Romeis, 1999).
H. armigera is an extremely well adapted to agro-ecosystems and can exhibit up
to 11 generations a year under good conditions (Shanower and Romeis, 1999). By
exhibiting overlapping generations in the field this insect very cleverly compounds the
problem of control.
The bollworm has evolved 2 major strategies for adapting to adverse conditions
i) by virtue of excellent migratory abilities it can fly up to 155 miles (250 km) in search
of a viable food source. (McCaffery et al.,1989) and ii) the dynamic nature helps in
withstanding adverse conditions in terms of temperature by entering in to facultative
Studies on plant alpha amylase inhibitor and its role in pest management
Ph.D Thesis. Pankaj R. Gavit. (2014) North Maharashtra University, Jalgaon 38
diapauses. These smart qualities of H. armigera allow it to survive until environmental
conditions improve. A pictorial representation of chronology of events involved in life
cycle is given Photo plate 1.2.
1.12.5 Plants screened for the source of alpha amylase inhibitor:
The following plants (their different parts) belonging to family Amaranthaceae
were screened for determining the good source of alpha amylase inhibitor.
(i) Amaranthus panicultus linn; (Rajgira),
(ii) Achyranthus aspera,
(iii) Celosia argentae,
(iv) Amaranthus tricolor,
(v) Amaranthus spinosa and
(vi) Amaranthus sessilis
The Amaranth (or pigweed) family is a large group of dicotyledonous flowering
plants known as the Amaranthaceae. It is divided into two subfamilies: the
Amaranthoideae and the Gomphrenoideae, based on certain morphological
characteristics of their flowers. It is a relatively large family, having about 65 genera and
900 species. Most of the 900 species of Amaranthaceae are native to tropical and
subtropical regions of Africa, Central America, and South America. The number of
Amaranthaceae species decline as one approaches the northern and southern temperate
zones. The species in this family are mostly annual or perennial herbs, although a few
species are shrubs or small trees as well. Many species of Amaranthaceae are considered
weeds, since they invade areas such as agricultural fields and roadsides.
` Plants from the Amaranthaceae family are used in indigenous system of medicine for
their antiarthritic, antifertility, laxative, abortifacient, anthelmintic, aphrodisiac, antiviral,
Studies on plant alpha amylase inhibitor and its role in pest management
Ph.D Thesis. Pankaj R. Gavit. (2014) North Maharashtra University, Jalgaon 39
Photoplate 1.2 : Chronological developmental stages in the life cycle of Helicoverpa
armigera.
Studies on plant alpha amylase inhibitor and its role in pest management
Ph.D Thesis. Pankaj R. Gavit. (2014) North Maharashtra University, Jalgaon 40
antispasmodic, antihypertensive, anticoagulant, diuretic and antitumour activities. They
are used to treat cough, renal dropsy, fistula, scrofula, skin rash, nasal infection, chronic
malaria, impotence, fever, asthma, amenorrhea, piles, abdominal cramps and snake
bites. Furthermore, some of the members from this family have important active
components and phytochemicals such as rutin which is a strong antioxidant compound
and saponins (Singh, 2009).
With this back ground, the aims and objectives of the present study were:
1.13 Aims and objectives:
i) To screen a few members of family Amarantheceae for the presence of
α-AIs
ii) Isolation, purification and characterization of α-amylase inhibitor from
plants/plant part showing maximum activity.
iii) To study its insecticidal properties against target pests (C. chinensis and
H. armigera).
The materials used and methods followed for achieving the above mentioned objectives
are described in the next chapter.
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