1.0 Summaryshodhganga.inflibnet.ac.in/bitstream/10603/27507/11/11_chapter 1.p… · Pankaj R. Gavit. (2014) North Maharashtra University, ... 5.8 20 1.5 7.3 26100 Groundnut 9.2 15

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.

********



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



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.



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)



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



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



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.



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



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.



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



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



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,



(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).



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



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



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.



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).



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



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).



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



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



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).



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).



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).



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)



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



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 -



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-



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



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).



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.,



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



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).



Photoplate 1.1:Chronological developmental stages in the life cycle of

Callosobruchus chinensis.



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



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



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



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,



Photoplate 1.2 : Chronological developmental stages in the life cycle of Helicoverpa

armigera.



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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1.0 Summaryshodhganga.inflibnet.ac.in/bitstream/10603/27507/11/11_chapter 1.p… · Pankaj R. Gavit. (2014) North Maharashtra University, ... 5.8 20 1.5 7.3 26100 Groundnut 9.2 15