MASS PRODUCTION OF ENTOMOPATHOGENIC FUNGI

Success of any microbial control programmes depends on production of sufficient

quantity of inoculum for field application. Efficient production technologies have been
developed for laboratory and commercial use of entomopathogenic fungi. Most of the
entomopathogenic fungi are facultative pathogens and can be mass produced in synthetic,
semisynthetic or natural media containing suitable nutrient source. Selection of strains of
fungi having high virulence, good growth and sporulation is considered important in
mass culturing. The spore yield depends upon the type of medium used to culture the
fungus (Roberts and Yendol, 1971). A number of successful commercial products based
on conidia / spores of fungal pathogens have already been developed and sold (Jenkins et
al., 1998).

1. Production system
The method of production vary according to the use either for laboratory testing
or large scale field use. For bioassays, isolate screening and maintenance of fungal
pathogens can be cultured in agar slants in glass tubes or 250 ml Erlenmayer flask.
Autoclavable polypropylene plastic bags or wide mouthed jars or fermenters are widely
used for large scale field application or commercial use. Most of them are grown as
surface culture on solid or liquid (broth) media. Some of the entopathogenic fungi are
dimorphic and when grown in submerged shake flask cultures produce yeast like cells
instead of mycelium. These cells are called blastospores and can be mass produced
quickly. However, blastospores are short lived than conidia. In submerged culture a few
fungal pathogen produce mycelia or conidia.

2. Media used production and maintenance of fungal pathogens

Culturing can be done in variety of media containing sugar and salts as sources of
carbon and nitrogen respectively. Specific media have been identified for culturing
different types of fungi (Table 1).
Table 1. Synthetic/Semisynthetic media used for culturing entomopathogenic fungi

Medium Composition Fungal pathogen

 2

1. Sabouraud’s dextrose agar Dextrose 40g; peptone 10g; Beauveria bassiana

with yeast extract (SDAY) yeast extract 2g; agar 15g; B. brongniartii
distilled water 1000 ml
Verticillum lecanii
2. Subouraud’s maltose agar Maltose 40g ; peptone 10g;; Nomuraea rileyi
with yeast extract yeast extract 5g; agar 15g; Zoophthora radicans
(SMAY) distilled water 1000 ml
Paecilomyces farinosus
3. Potato dextrose agar Peeled potato 200g; B. bassiana
(PDA) dextrose 20g; agar 15g; V. lecanii
distilled water 1000 ml
4. Emerson’s YpSs agar Soluble starch 15g; K2HPO4 Metarhizium anisopliae
1g, Mg SO4, 7H2O 0.5g, M. flavoviride
yeast extract 4g, agar 15g,
distilled water 1000 ml
5. Nutrient agar Beef extract 3.0g; peptone Paecilomyces
5.0 g, NaCl 5g; agar 15g; fumosoroseus
distilled water 1000 ml
6. Oat meal agar Oats 100g, agar 15g, P. fumosoroseus
ditilled water 1000 ml
7. Czapek - Dox agar (CDA) Sodium nitrate 2g; K2HPO4 B. bassiana
1g, MgSO4 0.5g; KCl 0.5g,
FeSO4 0.01g; sucrose 30g;
agar 15g; distilled water
1000 ml

2.1. Natural media

Different types of cereals, pulses, brans and vegetables have been reported to be
suitable for mass production of entomopathogenic fungi (Table 2).

Table 2. Natural media for culturing entomopathogenic fungi

Medium Fungal pathogen

1. Sorghum grains Verticillium lecanii (Easwaramoorthy and Jayaraj,
1977); Paecilomyces farinosus (Gopalakrishnan et
al., 1999); Beauveria bassiana (Devaprasad, 1989)
2. Rice Nomuraea rileyi (Silva and Loch, 1987)
…Contd.,
 3

Medium Fungal pathogen

3. Carrot B. bassiana (Sivasankaran et al., 1990);
Metarhizium anisopliae (Sundarababu, 1980)
4. Crushed sorghum grains with N. rileyi (Vimala Devi, 1994)
yeast extract
5. Jack seeds V. lecanii (Easwaramoorthy and Jayaraj, 1977)
6. Cooked rice B. bassiana ; M. anisopliae (Jenkins et al., 1998)
7. Wheat bran B. bassiana (Rombach et al., 1988)
8. Oats M. anisopliae (Villacorta, 1976)
9. Coconut water M. anisopliae (Chandrika Mohan, 2001)
10. Cassava chips + rice bran + M. anisopliae (Chandrika Mohan, 2001)
fish meal

3. Isolation of fungal pathogens

3.1. Collection of cadavers
Mycosed insects are collected individually using fine forceps in glass tubes or
screw cap vials and brought to laboratory. If the fungas has sporulated cadavers can be
stored in the fridge till the isolation of the pathogen. The cadavers without external
growth of fungus can be kept at 20 - 25oC in an environment with high humidity
(eg.moistened filter paper in sterile Petridish). This will induce the growth of the fungus
and sporulation.

3.2. Isolation
The spores / condia on the surface of the cadavers can be transferred aseptically
using sterile inoculation needle to appropriate agar medium containing antibiotics in a
laminar hood. Another isolation method is cutting of cadavers into small bits after
surface sterilization in 70 per cent alcohol or 0.1 per cent mercuric chloride and
transferring bits to agar medium in a laminar hood. The cadavers can also be
homogenized in sterile distilled water and plated on appropriate agar medium.

3.2. Purification
It has to be ensured that after isolation, fungus is free from contaminant
microorganisms and represent a single genotype. Purification is done by single hyphal
isolation or single spore/conidia isolation method.
 4

3.2.1. Single hyphal isolation

The mycelium or conidial suspension is placed centrally in a Petridish containing
agar medium containing low nutrients. The fungas will grow and spread in the form of
thin hyphal strands. The margin of the colony should be examinated under low power
microscope, a bit of agar bearing single hyphal tip is removed using sterile inoculation
needle and aseptically transferred onto appropriate agar medium in slants. The hyphal bit
will grow into a mycelial colony.

3.2.1. Single spore/conidia isolation

The fungal spores/conidia are suspended in sterile distilled water and diluted
serially in sterile test tubes. The diluted suspension is spread onto a relatively transparent
agar medium (12-15 ml) in Petridishes and incubated at 20-25oC. The Petridishes
periodically examined under the microscope and single germinating spores are marked.
Then each of the germinating spore is removed aseptically and transferred on to
appropriate medium in Petridishes or slant tube.

4.Culturing of fungal pathogens

4.1. Preparation of synthetic media
For preparing solid agar media like SDAY, SMAY, Emerson’s YpSs agar,
Nutrient agar and CDA half the quantity of water is used to dissolve the chemicals by
slight heating / boiling. The agar is melted in the other half of water by slow heating and
constant stirring. The melted agar is added to the other half of medium containing
chemicals and mixed thoroughly. Usually pH is adjusted to 6.5 – 6.8 before mixing with
agar. The medium is then dispensed in test tubes (5 ml/tube) or 250 ml Erlenmeyor flask
(50 – 100 ml/flask). The mouth of the test tube or flask is plugged with non absorbent
cotton, covered with paper and autoclaved at 15 PSI for 20 min.

The tubes are allowed to cool in slanted position for getting agar slants. The flask
is allowed to stand until they can be held by hand and the medium is poured asceptically
into sterile Petridishes to produce agar plates.
 5

4.2. Preparation of semi-synthetic medium

4.2.1. Potato dextrose agar
Washed and peeled potato is made into thin chips, boiled in 500 ml of water and
filtered through muslin cloth. To the extract weighed quantity of glucose is added and
mixed. The agar is melted in another half of water and mixed with potato extract
containing glucose. The volume of the medium is made upto to one litre and dispensed
in test tubes or Erlenmeyer flasks and autoclaved as described in section 4.1.

4.2.2. Oat meal agar

The oats is first cooked in 500 ml of water and filtered through muslin. The agar
is melted in another 500 ml of water, added to the extract of oats and final volume is
made upto one litre. Dispensing and autoclaving are done as described earlier.
Some of the synthetic or semisynthetic media are commercially available. The
required quantity of the media is added to distilled water, if necessary boiled to dissolve
the ingrediants and autoclaved according to manufacturer’s instructions.

4.3. Preparation of natural medium

4.3.1. Sorghum medium
A quantity of 100g of sorghum grains is thoroughly washed, cooked in 500 ml of
distilled water and extract is strained through muslin. Extract is dispensed in Erlenmeyer
flasks at the rate of 100 ml/flask and autoclaved. If solid medium is needed agar can be
added.

4.3.2. Carrot medium I

A quantity of 100g of carrot is washed well in tap water, cut into small bits and
cooked in 500 ml distilled water. The extract was filtered through muslin cloth dispensed
in 250 ml Erlenmeyer flask at the rate 100 ml/ flask and autoclaved. If solid medium is
needed of agar can be added.

4.3.3. Carrot medium II

The cut pieces of carrot are transferred to 250 ml Erlenmeyer flasks containing
100 ml of water at the rate of 40 g per flask. Autoclaving is done as described elsewhere.
 6

5. Inoculation
Inoculation should always be carried out in a laminar flow hood. The working
area inside the hood must be disinfected with 70 per cent alcohol before starting the
inoculation. A Bunsen burner is kept inside the hood for flaming the loop of inoculation
needle.
i. Remove the cotton plug of test tube or Erlenmeyer flask containing culture
medium/fungal culture and heat the mouth over the flame in Bunsen burner.

ii. Sterilize the inoculation needle by heating over the flame till it becomes red rot

iii. Cool the needle and introduce it into the tube/flask containing mother fungal
cultures. Pick up the spores and hyphal bodies and inoculate by drawing it lightly
over the surface of fresh medium in slants/flasks/Petridishes.

iv. Flame the mouth of the flasks or tubes again and recap

v. Incubate the culture at 25-27oC in an incubator. It is desirable to keep the flask or

tube inside a humidified chamber.

6. Culture maintenance
Normally sporulation will occur within 7-10 days and subculturing can be done at
this stage. Continuous subculturing on artificial media can result in attenuation of
virulence and affect sporulation. Virulence can be restored after a single or several
passages through the insect host.

7. Storage
The conidia, blastospores and mycelia can be stored at 4oC upto several weeks or
even months depending on the species, strain and media used. Slant cultures can be
stored using mineral oil or distilled water (Humber, 1997). For long term storage the
spores/condia can be stored on sterile anhydrous silica gel crystals at -20oC. Fungal
spores stored in slica gel may remain viable for more than ten years.

8. Large scale production for commercial use

Efficient mass production technologies have been developed for a few fungal
pathogens. Production efficiency was highest in the case of B. bassiana (Wraight and
 7

Carruthers, 1999). In South and Central America, Europe and Asia M. anisopliae and
B. bassiana were produced commercially using cooked rice or other grains in trays or
autoclavable plastic bags or glass jars. The average yield was 1-5 x 109 spores g of
substrate (Alves and Pereira, 1989; Aregger, 1992).

Industrial production of fungal pathogens is generally based on diphasic or two

stage system in which fungal mycelium or hyphal bodies (blastospores) are produced in
liquid culture either in shake flasks or fermenters and transferred to solid substrate for
production of conidia (Jenkins et al., 1998).

REFERENCES

Alves, S.B.and Pereira, R.M. 1989. Production of Metarhizium anisopliae (Metsch)

Sorok and Beauveria bassiana (Bals.). Vaill in plastic trays. Ecossistema, 14:
188-192.

Aregger, E. 1992. Conidia production of the fungus Beauveria brogniartii on barley and
quality evaluation during storage at 2oC. J. Invertebr. Pathol., 59: 2-10.

Chandrika Mohan. 2001. Techniques in microbial control of the Rhinoceros beetle

Oryctes rhinoceros. In: Microbial control of crop pests. Eds. Rabindra, R.J.,
Kennedy, J.S., Sathiah, S., Rajasekaran, B. and Srinivasan, M.R. Tamil Nadu
Agricultural University, Coimbatore, India. pp.271-274.

Devaprasad, V. 1989. Studies on certain entomopathogens of Heliothis armigera Hb.

and Spodoptera litura F. and their interaction with botanicals. Ph.D. Thesis.
Tamil Nadu Agric. Univ., Coimbatore. p.172.

Easwaramoorthy, S. and Jayaraj, S. 1977. Effect of temperature, pH and media on the

growth of the fungus Cephalosporium lecanii. J. Invertebr. Pathol., 29: 399-400.

Gopalakrishnan, C., Anusuya, D. and Narayanan, K. 1999. In vitro production of conidia

of entomopathogenic fungus Paecilomyces farinosus (Holmskiold) Brown and
Smith. Entomon., 24: 389-392.

Humber, R.A. 1997. Fungi: Preservation of cultures. In: Biological Techniques Manual
of Techniques in Insect Pathology. Ed. Lacy, L.A. Academic Press, London.
pp.269-279.

Jenkins, N.E., Heviefo, G., Langewal, J., Cherry, A.J. and Lomer, C.J. 1998.
Development of mass production technology for aerial conida for use as
mycopesticides. Biocontrol News and Information, 19: 21-31.
 8

Robersts, D.W. and Yendol, W.G. 1971. Use of fungi for microbial control of insects.
In: Microbial control of insects and mites. Eds. Burges, H.D. and Hussey, N.W.
Academic Press, New York. pp.125-149.

Rombach, M.C., Aguda, R.M. and Roberts, D.W. 1988. Storing dry Beauveria bassiana
dry mycelium. IRRN., 13: 37-38.

Silva, L.D.A. and Loch, L.C. 1987. Sporulation of the entomopathogenic fungus
Nomuraea rileyi (Farlow) Somson on polished rice grain media. Anais da
Sociedade Entomolgica do Brasil, 16: 213-222.

Sivasankaran, P., Easwaramoorthy, S. and David, H. 1990. Pathogenicity and host range
of Beauveria bassiana, a fungal pathogen of Chilo infuscatellus Snell. J. Biol.
Control, 4: 48-51.

Sundara Babu, P.C. 1980. Studies on the pathogenicity of Metarhizium anisopliae

(Metschanikoff) Sorokin var. major Tulloch in Oryctes rhinoceros (L.). Ph.D.
Thesis. Tamil Nadu Agric. Univ., Coimbatore, India. p.151.

Villacorta. 1976. Tecniques for mass culture of the entomophagous fungus, Metorhizium
anisopliae (Metsch.) in granular form. Ann. Soc. Entomol., Brasil, 5: 102-104.

Vimala Devi, P.S. 1994. Conidia production of the entomopathogenic fungus Nomuraea
rileyi and its evaluation for the control of Spdoptera litura (Fab.) on Ricinus
communis. J. Invertebr. Pathol., 63: 145-150.

Wraight, S.P. and Carruthers, R.I. 1999. Production, delivery and use of myco
insecticides for control of insect pests on field crops. In: Methods in
Biotechnology Vol.5. Biopesticides: Use and Delivery. Eds. Hall, F.R. and
Menn, J.J. Humana Press Inc. Totowa, New Jersy, pp.233-269.
 GENETIC IMPROVEMENT OF BACULOVIRUSES FOR INSECT PEST
MANAGEMENT

Microbial insecticides are considered to be potential alternatives to chemical

insecticides since they are ecofriendly and safe to non target organisms and do not pose
any hazards to human beings. Baculoviruses particularly the nuclear polyhedrosis and
granulosis viruses can play an important role in integrated pest management and
sustainable agriculture. Through intensive research in the past couple of decades, several
of these viruses have been developed as microbial insecticides for the management of
important crop pests in India and other countries. However due to their specificity and
slow action, they have not been used to the extent they should have been. Recent
advances in molecular biology and biotechnology have paved the way for genetically
improving these useful organisms either by non recombinant or recombinant DNA
techniques.
Genetic diversity allows the selection of strains of viruses with improved
virulence and persistence. Nuclear polyhedrosis viruses with increased virulence to
Helicoverpa armigera, Spodoptera litura, Hyphantrea cunea and Spodoptera exigua
have been selected. A strain of NPV of Neodiprion sertifer with increased virulence was
selected from the first larvae to die after inoculation. Selection of a strain of virus with a
higher rate of vertical transmission in S. exigua has also been demonstrated. A strain of
Cydia pomonella granulosis virus 5.6 times more resistant to UV light than the original
isolate and remained infective twice as long in the field was also selected. Serial passage
in a heterologous host as in the case of AcMNPV in P. xylostella larvae and GmNPV
through larvae of Manduca sexta larvae as well as in vitro passage as in HaNPV through
Sf 21 AE cell line are also known to increase the virulence of viruses.
Recent advances in in vitro cloning techniques have enabled the production of
mutant as well as hybrid viruses with greater pest control potential. Molecular techniques
have enabled the deletion of ecdysteroid-UDP glucosyl transferase (EGT) gene from
viruses like HaNPV resulting in deletion mutants which kill the insects faster. The most
phenomenal development however is the cloning of foreign genes into NPV to enhance
their speed of kill. Several pesticidal genes including the scorpion venom gene from the
Australian scorpion Andractonus australis have been cloned into NPV. The scorpion
venom gene (AaIT) was cloned into an EGT gene- delected NPV of Autographa
californica. In field experiments with cotton and tobacco, successful control of Heliothis
virescens with the recombinant virus was demonstrated. Similarly, the insect-specific
neurotoxin gene Tox 34 from the itch mite Pyemotes tritici has been cloned into EGT-
gene delected Helicoverpa zea NPV which killed the H. zea larvae faster than did the
wild type virus. The safety of these genetically engineered viruses to several non target
organisms has already been established. The future of microbial control using
baculoviruses depends very much on genetically improved viruses which can kill insect
pests faster and persist in the field for a longer period than the wild type viruses.

1
 1. Introduction
India is bestowed with a rich diversity of baculoviruses like the nuclear
polyhedrosis and granulosis viruses infecting several agriculturally important insect pests.
Many of these viruses have good potential in pest management and a few have been
developed into microbial pesticides. Several field trials have demonstrated the usefulness
of these viral pesticides in the management of notorious pests like the gram pod borer
Helicoverpa armigera (Rabindra et al 1989 ; Muthiah and Rabindra 1991 ; Muthuswamy
et al 1993 ; Rabindra et al 1986 ; Dhandapani et al 1993 ; Rabindra et al 1991) , the
tobacco cut worm Spodoptera litura (Jayaraj et al 1980 ; Ramakrishnan et al 1981 ;
Santharam and Balasubramanian 1980 ; Santharam et al 1978), the red hairy caterpillar
Amsacta albistriga (Chandramohan and Kumarasamy 1979 ; Rabindra and
Balasubramaniam 1980), the diamond back moth Plutella xylostella (Rabindra unpub.
data) and the sugarcane shoot borer Chilo infuscatellus (Easwaramoorthy and
Santhalaxmi 1988 ; Parameswaran et al 1991 and 1992).
Despite the demonstration of the potential, the baculoviral formulations have not
found widespread use in pest management. The major limitations are their relatively slow
speed of kill, narrow host range and poor persistence in the field. While specificity
confers safety, it leaves the baculoviruses powerless against pest complexes. Poor
persistence in the field necessitates more frequent applications increasing the cost of
treatment. Recent advances in molecular techniques have shown that baculoviruses can
be improved genetically for virulence, persistence, host range, speed of kill and stability
in storage.
2. Genetic improvement of baculoviruses
An understanding of the molecular biology of baculoviruses and development of
molecular tools have enabled the development of genetically improved or engineered
virus capable of competing with Bacillus thuringiensis and even some chemical
insecticides in their efficacy and speed of kill.
2.1. Cloning and production of virulent insect viruses
The development by Hink and Vail (1973) of a suitable method for plaquing
NPVs using TN-368 cell line from Trichoplusia ni enabled the isolation and purification
of new virus strains. Brown and Faulkner (1977) developed a better plaque system by
replacing neutral red with 2 – (P-iodophenyl)-3-(p.nitrophenyl)-5-phenyltetrazolium
chloride. Similar plaque assay systems have been developed by Dougherty (1980) and
Yamada and Maramorosch (1981) using agarose overlays, which provide for better
isolation of progeny virus, and to obtain samples from individual clones. Restriction
endonuclease analysis can be combined with the plaque method for isolating more
virulent strains of NPV.
2.2. Selection of strains with greater virulence
Variants of baculoviruses with heritable variations in virulence and host range
arise spontaneously in nature. Viral strains collected from different geographic areas
differ in biological activity and their genetic variations can be brought out by restriction
endonuclease analysis. Smirnoff (1961) selected a strain of an NPV of increased
virulence to Neodiprion sertifer from the first larvae to die after inoculation. Variants of

2
baculoviruses may also arise when a virus infects an alternate host. An isolate of
Choristoneura fumiferana NPV was fed to the neonate cabbage looper larvae and the
wax moth larvae. After passage, the virus was able to infect new hosts (Stairs et al 1981).
Hukuhara (1968) observed that a tetragonal strain of a nuclear polyhedrosis virus was
more virulent than a hexahedron strain to Hyphantria cunea and in addition, multiplied
more rapidly. Shapiro and Ignoffo (1970), Somasekar et al (1993) and Rabindra et al
(1998) studied several geographic isolates of NPV of Heliothis spp. and H. armigera with
distinct restriction profiles and identified strains with greater virulence to the host insects.
Recently, we have isolated a strain of NPV of S. litura (SlNPV) which is nearly 100
times more virulent than the standard Coimbatore strain of SlNPV to early instar S. litura
larvae. It was interesting to see that this virus also had a distinctly different restriction
pattern for Pst I, Hind III, Bam HI and EcorI.
Stairs (1990) found that serial passage of NPV of Galleria mellonella NPV
through larvae of Manduca sexta increased the pathogenicity of the virus to M. sexta
neonate larvae. Kondo et al (1994) isolated two strains of morphologically distinct NPV
from larvae of Spodoptera exigua. One was cuboidal and the other icosahedral.
Variations in the restriction endonuclease patterns were observed between the two strains.
The cuboidal strain of SeNPV could infect larvae of S. litura and P. xylostella also where
as the icosahedral strain was infective only to larvae of S. exigua. The icosahedral strain
however was approximately 12 times more virulent than the cuboidal strain to S. exigua.
(Rabindra 1992) studying the comparative virulence of several isolates of HaNPV
reported that an isolate from the Nilgiris in Tamil Nadu was the most virulent.
2.3. Selection for vertical transmission
Fuxa et al (1992) selected a strain of virus with a higher rate of vertical
transmission in S. exigua. When fifth instar of Spodoptera frugiperda were fed the
median lethal concentration of the selected virus, the survivors transmitted NPV to 24%
of their progeny, compared with 14% with the wild viral isolate. The selected NPV killed
58% of the infected progeny insects compared with 39% with the wild isolate (Fuxa et al
1992). Fuxa and Richter (1990) proposed that the selected NPV could be released to
improve microbial control of S. frugiperda in host plants such as corn, because transport
of the virus by adult insects must help to initiate viral epizootics in relatively tall plants
that are not easily contaminated by NPV in the soil. Little is known about the virulence of
vertically transmitted NPV or the rate of vertical transmission over several host
generations.
2.4. UV Resistance
A strain of Cydia pomonella GV which was 5.6 times more resistant to UV light
than the original isolate and remained infective for twice as long in the field was selected
by Brassel and Benz (1979). Such investigations in our agroecosystem are bound to yield
fruitful results as there is a greater biodiversity under topical conditions.
2.5. In vitro passage in cell lines
Tompkins et al (1998) found that when the NPV of H. armigera was passaged
repeatedly through (SF 21) AE cell lines, the virulence of virus to neonate larvae of T.ni
was significantly increased.

3
 2.6.Mutants of Baculoviruses
A direct application of genetics to viral insecticides is to generate stable variants
of baculoviruses which are more effective in the field. Wood et al (1981) could isolate a
mutant strain of AcMNPV designated HOB which produced a large number of occlusion
bodies in infected cells and which had higher virulence in insects than the parent strain.
Mutants can be generated in tissue culture by growing the wild type virus in the presence
of a chemical mutagen. The spruce budworm virus was grown in the presence of the
mutagen nitrosomethyl guanidine and the surviving virus was cloned (Ireland, 1982). Of
the 34 plaques isolated and examined, one isolate, CfNTG 29 was more virulent than the
standard.
McClintock and Reichelderfer (1985) reported an almost 100-fold increase in the
virulence of AcMNPV to S. frugiperda when the virus was treated in vivo in larvae of
T.ni with 3-methylcholanthrene. This strain was also highly pathogenic to larvae of T. ni.
2.7.Deletion Mutants
Deletion of ecdysteroid UDP – glucosyl transferase (EGT) gene in NPV (O’Reilly
and Miller, 1989; 1991) as well as GV (Crook and Winstanley 1996) has been shown to
increase the speed of kill by interfering with metamorphosis and moulting. The EGT
gene has recently been identified in several insect viruses including H. armigera SNPV
(Chen et al 1997).
2.8.In vivo recombination
In vivo recombination has been carried out by mixedly infecting G. mellonella
larvae with two closely related baculoviruses (AcMNPV and GmMNPV). REN analyses
of pooled virus DNA extracted from occlusion bodies harvested after five in vivo
passages in the wax moth showed that recombinants were present (Croizier and Quiot
1981). Genetic recombination between viruses with similar but not identical REN
patterns occurs in vitro and gene reassortment has been demonstrated (Summers et al
1980). In vivo recombination between two strains of NPV of S. exigua has been reported
but no enhancement in virulence was observed (Munoz et al 1997). Recombinants with
desirable attributes like enhanced virulence, desirable host range, UV resistance, and
increased environmental persistence may be selected and further improved upon.
2.9.Construction of Hybrid virus in vitro
A virus which can kill a pest complex comprising of two or three insects would be
a very useful tool. Using in vitro techniques involving BmX and Ha L93 cell lines, Safo
(1997) produced a hybrid virus between the NPVs of Bombyx mori and H. cunea which
had a wide host range including the smaller tea tortrix Adoxophyes sp. and the diamond
back moth P. xylostella. The production of a hybrid of AcMNPV DNA and Bam Hl-
digested BmNPV DNA in SF 21 cells by Mori et al (1992) demonstrated the possibility
of generating hybrid viruses in tissue culture for use in pest management programmes.
Such an approach may be useful in developing baculoviruses for controlling pest
complexes in crops like cotton, pulses and vegetables in India.

4
 2.10.Serial passage in heterologous host
Hirsch and Beek (1997) serially passed a wild type AcMNPV through third instar
P. xylostella larvae 20 times and obtained a variant that was 15 times more virulent to
second instar P. xylostella larvae than the parental isolate. Restriction endonuclease
analysis showed marked differences between passaged and wild type AcMNPV. Electron
microscopic observations showed that occlusion bodies of passaged AcMNPV had a
greater number of virions and fewer nucleocapsids per virion than the wild type
AcMNPV. Also, singly enveloped nucleocapsids derived from passaged and wild type
AcMNPV were equally potent against P. xylostella. NPV of G. mellonella a genotypic
variant of AcMNPV is also infective to P. xylostella and it is possible to increase its
virulence to P. xylostella by serial passage.
3. Genetic engineering of baculoviruses to increase their insecticidal activity
Even though baculoviruses have been investigated and utilised as insecticides for
more than 20 years, their widespread use and acceptance has never been achieved due to
the very slow kill which is characteristic of wild-type viruses. A long term perspective of
microbial control with baculoviruses however would be to use recombinant baculoviruses
which can kill insects more efficiently at shorter time. The baculovirus genome is
amenable for genetic engineering and the knowledge of the molecular biology of these
viruses has enabled cloning of several proteins of insecticidal value into NPV. The
proteins are either toxins or disrupters of larval development. They are toxins, Juvenile
hormone esterase, PTTH, mellitin, trehalase, fungal insecticidal protease, scorpion and
mite toxins. Some of the genetically engineered viruses and the effects are given in
Table.1.
Cyanamid company have recently cloned the scorpion venom gene (AaIT) into
the ecdysteroid-UDP-glucosyl transferase (EGT) gene-deleted NPV of A. californica
(Gard 1997). In field experiments with cotton and tobacco, successful control of Heliothis
virescens with the recombinant virus was demonstrated. Similarly the insect-specific
neurotoxin gene Tox 34 from the itch mite Pyemotes tritici has been cloned into the
EGT-deleted HzNPV (Popham et al 1997). This recombinant virus killed H. zea larvae
faster than did the wild type virus. The deletion of EGT gene resulted in early
degeneration of malpighian tabules one of reasons for the increased speed of kill (Flipsen
et al 1995).
3.1. The Enhancin gene
The T.ni GV produces a protease termed as enhancin which increased the
susceptibility of insects to NPV by breaking down the peritrophic membrane. Such an
enhancing factor has also been identified in HaGV and PuGV (Pseudoletia unipuncta
GV). The genes encoding for the enhancins of TnGV (Hashimoto et al 1991), PuGV and
HaGV (Roelvink et al 1995) have been cloned and sequenced. Lepore et al (1996)
produced a recombinant AcMNPV expressing the TnGV enhancin gene; but its utility in
enhancing the activity of the recombinant NPV is yet to be demonstrated. Leaf powder
from transgenic tobacco expressing enhancin when fed to M. sexta and S. frugiperda
larvae however increased the susceptibility to NPV. Enhancin therefore appears to be of
potential in genetic manipulations for enhancing the insecticidal activity of baculoviruses.

5
 3.2. Usefulness of recombinant viruses in IPM
Synergistic interactions were found in H. virescens when the recombinant AcNPV
(AaIT) was combined with cypermethrin and methomyl. No such synergism was found in
combination with the wild type AcMNPV. A pyrethroid resistant strain of H. virescens
was more sensitive to the recombinant virus compared with a susceptible strain
(McCutchen et al 1997). These findings indicate the usefulness of the recombinant
viruses in integrated pest management programmes.
3.3. Safety of genetically engineered virus
The insect specificity of AaIT (Zlotkin et al 1985, 1991, 1993) coupled with the
baculovirus specificity confer a two-fold safety to non target species. The safety of
AcMNPV (AaIT) to several non target species has already been demonstrated (Possee et
al 1993; McNitt et al 1995; McCutchen et al 1996). The safety to numerous insect orders
as well as spiders and earth worm was also seen (Gard 1997). The possibility of
engineering AcMNPV with certain deletions to reduce the field persistence has been
demonstrated (Mans and Vlak 1994). The Lymantria dispar NPV has also been
engineered for non persistence and in field experiments with this virus which was also
engineered with a β. galactosidase marker gene, it was found that the recombinant virus
did not persist in the environment (D’amico et al 1999).
3.4. Problems associated with genetically engineered virus
Recombinant viruses would undoubtedly prove to be successful biopesticides.
However recombinant baculoviruses produce only a fraction of the virus yield within an
insect compared with a wild type baculovirus, thus eliminating in – vivo insect production
as a viable economic option. Instead, commercial production will need to be undertaken
in-vitro utilising optimised insect cell lines grown in large-scale bioreactors. It is believed
that in-vitro production technology for baculoviruses is currently at about the 250 litre
bioreactor level. In order to attain the necessary economic scale for a successful
commercial product, reliable production will ultimately need to be carried out in large
reactors of 16,000 litre capacity and above.
The second dimension is that the regulatory and clearing agency involved in
approval of commercial field use of viruses would feel more comfortable approving a
wild type virus or genetically improved virus developed by non-recombinant rather than
recombinant methods, for example the EGT deletion mutant NPV. Lastly, inspite of
improved insecticidal activity, genetically engineered virus may still find it hard to
compete with synthetic chemical pesticides with quick knockdown effect.
4. Future Programme
Future programmes should focus on development of viral pesticides with
increased speed of kill and persistence, in vitro production of viruses in insect cells at
costs comparable with in vivo production and on intensive educational activity creating
awareness among the regulatory agencies as well as farming community on the
ecological and environmental benefits of using biorational baculovirus insecticides.
Research and development activities in India should focus on identification of
strains of naturally occurring baculoviruses with increased virulence and UV tolerance

6
and genetically improve such promising strains. There is a need for inter institutional
collaboration in order to develop improved viral pesticides for use in IPM and to reduce
the pressure on chemical insecticide use. Commercial biopesticide producers need to be
trained on economic production of baculoviruses as well as quality control. This would
enhance their capability in commercial scale production of quality viral pesticides.
In spite of the enormous potential, the market share of biopesticides continues to
be insignificant. In India, microbial pesticides of good quality are either not available for
the farmers or a few which are available are too expensive. Scientists, industry as well as
government agencies involved in pest management should work together to ensure
production and supply of quality microbial pesticide formulations with good field
persistence and storage stability on a cost-effective basis. Government development
departments and voluntary agencies can play a significant role in educating and training
the farmers in the proper use of microbial pesticides.

7
Table 1. Development of genetically engineered baculoviruses with increased speed
of kill
% increase
Virus Foreign gene Host insect in speed of Reference
kill
Toxin from
Bm NPV B. mori 40 Maeda et al (1991)
A.australis
LT50
Diuretic hormone
B. mori reduced by Maeda (1989)
from Manduca sexta
one day
Toxin 34 from
HzNPV H. zea 47 Popham et al (1995)
P.tritici
Hv JHE with GLY Trichoplusia ni 16 Bonning et al (1995)
AcMNPV for Ser201 Heliothis
substitution virescens 29
Spodoptera O’Reilly and Miller
EGT 22
frugiperda (1991)
Hz PBAN Trichoplusia ni 26 Ma et al (1998)
Toxin 34 from H. virescens 33 Watkins et al (1997)
Pymotes tritici (Pt)
T. ni 56
Toxin 21 A from Pt T. ni 49 Tomalski et al (1993)
Neurotoxin from S. frugiperda 43 Prikhodko et al (1996)
spider angelenopsis
aperta (µ-Aga-IV)
Mag 4 (µ-Aga-IV) + S. frugiperda 37
Mellitin from Apis
mellifera)
T. ni Not Merryweather et al
Bt toxin gene
significant (1990)
Truncated Cry S. exigua Not Martens et al (1995)
1A (b) significant
Maize mito T. ni 40 Korth and Levings
chondrial gene URF (1993)
13
Chitinase gene from S. frugiperda 23 Gopalakrishnan et al
Manduca sexta (1995)

8
Table 1. Development of genetically engineered baculoviruses with increased speed
of kill (continued)
% increase
Virus Foreign gene Host insect in speed of Reference
kill
Neurotoxin Sh I T. ni 37 Prikhod’ko et al
from sea anemone 40 (1996)
S. furgiperda
Stichodactyla
helianthus
T. ni 20 Hughes et al (1997)
Toxin from spider
S. exigua 18
Tegenaria agrestis
H. virescens 19
Latro insecto toxin H. virescens 12 Watkins et al (1997)
from the Black 12
T. ni
widow spider

Human C myc S. frugiperda 28 Lee et al (1997)

antisense gene

9
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877-887

15
 BIOPESTICIDES

1. Introduction
The steady increase in human population, decline in productivity and shrinking
land area are exerting a severe pressure on Indian Agriculture. Intensive agriculture
involving high fertilizer responsive high yielding varieties and monocropping have
resulted in the out break of several pest and disease problems in crops. Though the pest,
disease and weed problems are considered as old as agriculture itself, the intensification
of agriculture has led to drastic increase in problems. Annually, we have been loosing
nearly 50 per cent of our production due to these limiting factors.

Chemicalisation of agriculture has become non sustainable and is responsible for

fall in the productivity of agricultural crops. Widespread use of chemical pesticides has
resulted in development of resistance in insects, resurgence of sucking pests, destruction
of beneficial fauna in addition to several toxic hazards due to large scale manufacture and
handling of chemical pesticides.

In the first major accident involving pesticides in India, more than 100 people
died in Kerala in 1958 due to consumption of wheat flour and sugar contaminated with
parathion leakage during shipment from Bombay to Cochin. More than 3000 people died
by inhaling vapours of methyl isocyanate that leaked from carbaryl manufacturing plant
in Bhopal in 1985. More than 30,000 people were disabled to varying degrees. The
surviving population is still expressing teratogenic, mutagenic, carcinogenic and other
effects involving vital body organs. The recently reported large scale death of peacocks in
Tamil Nadu and Rajasthan due to pesticide exposure in agro ecosystem is only an
indicator of the extent of pollution of our agricultural environment.

2. What are the alternatives?

The drawbacks of chemical pesticides, particularly development of resistance to

pesticides and fall in productivity of crops partly due to the high cost of chemical
pesticides and insecticide induced resurgence of pests forced the farmers to look for
alternate pest control strategies. Maintaining crop habitat diversity, adoption of various
cultural and behavioural methods of pest management and growing of resistant varieties
have been tried. Biopesticides using the natural enemies of crop pests, diseases and
nematodes have become attractive tools in IPM in view of their ecofriendly nature
(Jayaraj et al., 1994).

3. Why Biopesticides?

Despite the slow action and specificity, biopesticides like pathogens, parasitoids,
predators and antagonistic organisms have become invaluable components in agricultural
IPM system in view of high level of specificity, safety and sustainability. In addition to
the natural biocontrol operating in many crop habitats, applied biocontrol can bring about
a successful suppression of crop pests, diseases and nematodes without disruption of the
ecosystem. The high level of human safety, stability of control and renewable nature,
make biopesticides very attractive candidates for pest management (Jayaraj et al., 1994).

4. Scope and implementation

The role of biopesticides has expanded in crop and forest protection with the
discovery of new agents, genetic improvement, application option and compatibility with
other interactions. Biological control emerged as a scientific discipline in the later part of
19th century in India (Jayaraj et al., 1994). Biopesticides find extensive use in the
following contexts.

(a) Where the value of a crop is too low and extension advice inadequate to permit the
use of pesticides or other control methods are expensive. Here, the strategy of
introduction, which if successful, involves no recurrent costs, may be particularly
appropriate. In small-scale farming and subsistence agriculture, farmers may not be
able to afford the pesticides used by large-scale commercial growers. Then biological
control may provide essential control for small farmers and less expensive control for
the large farmers who may contribute to the cost of implementation.

(b) Where overuse of insecticides has created pesticide resistance so high that other
control methods become necessary. In this context, conservation, coupled with a
reduction in pesticide use, may result in effective natural control, which may be
further enhanced by augmentation and introduction. The collapse of the cotton
industry due to over use of insecticides is a case in point.
(c) Elsewhere in cropping systems where biological methods are not currently part of an
already adequate pest management programme, biological control may be usefully
introduced, if it is economical and compatible with existing methods. The difficulty in
the use of a specific biopesticide to control only one of a complex of pests on a crop,
when chemical control must be continued for other pests, is that the chemicals may
reduce the effect of the agent on the target pest. Thus, the best systems for biological
control are those with one, or at most a few, major pests (Wood-McKenzie, 1995).

Of late, scientists are focussing attention on integrated biological control strategy

which is in described as the combination of approaches viz., classical, inundative and
conservative methods and they are viewed as powerful tools in pest management.

5. Biopesticides for Insect Pest Management

5.1. Entomophagous insects

The work on biological control in India commenced with the introduction of the
coccinellid, Cryptolaemus montrouzieri from Australia in coffee in the Lower Pulney
Hills for the control of coffee green scale Coccus viridis (Mayne, 1953). Though not
successful against C. viridis, it established as a common predator of mealy bugs (Rao et
al., 1971). Later, was the introduction of vedalia beetle, Rodolia cardinalis for the control
of cottony cushion scale Icerya purchasi in the first quarter of last century in the Nilgiris
(Rao, et al., 1971). Since then, exploitation of exotic entomophagous insects has been
encouraged as a form of biological control. Some of the notable success achieved in this
direction in India is presented (Table 1).

Many explorative surveys were conducted for the identification of native fauna
inimical to crop pests since 1950s. These efforts brought out the diversity of natural
enemies (Table 2 and 3). In many instances natural enemies were found to decimate the
whole population of specific crop pests (David et al., 1986; Pawar, 1979; Singh,
1993a,b).

5.1.1. Conservation of natural enemies

The rate of success of natural enemies has been primary consideration in pest
management programmes. Some of the important conservation measures adopted were
selective use of chemicals that do not kill the natural enemies (Table 4), creation of
favourable environment for survival and multiplication of natural enemies, judicious
selection of crop mix that facilitate colonization and providing refugia and alternate food
(Hokkanen, 1993). In this process, our scientists are able to regenerate the kind of
biodiversity that can subsidize the sustainability of agroecosystems.

5.1.2. Augmentation

The use of laboratory bred natural enemies to increase the numbers and species
composition in crop habitat has gained momentum due to our successes in mass breeding
programmes (Table 5). These laboratory bred natural enemies were introduced in annual
crops where the load of resident species were already low or in environments not
favourable for natural enemy establishment. In our country, successful experimentation
with entomophagous arthropods for crop pests control has been demonstrated in different
crops. On experimental basis, in 14,000 ha of sugarcane, sorghum and cotton
Trichogramma parasitoids were released to check lepidopterans during 1993 in India
(Hassan, 1993). The establishment of biocontrol agents production facilities both under
governmental and private efforts has enabled year round production and availability of
species for field augmentation (Jayaraj et al., 1994). Currently, atleast a dozen
commercial facilities are mass producing entomophagous arthropods in India (Abdul
Kareem, 1999).

In developing natural enemies for field colonization, several issues have been
addressed and they fall under three categories.

Host and natural enemy rearing procedures

Genetic issues and

Quality issues

In addition, economics involved in the rearing major natural enemies is receiving

wider attention. It may be more cost – effective to rear and release a natural enemy that is
less effective in terms of field performance by inundation, if it can be reared for a much
lower cost per individual, than to release a very effective natural enemy that is expensive
to rear. This adds a dimension in manipulation of rate of release, species composition and
numbers. For example, in cotton ecosystem frequent inundative releases of
Trichogramma spp. and one or two inoculative release of Chrysopa against eggs of
bollworm complex is advocated because the breeding procedures are cost-effective for
the former and expensive in the latter case.

5.1.3. Status in different crops

5.1.3.1. Paddy

Inundative releases of exotic T. japonicum at 50,000 ha-1 during egg laying period
of rice stem borer reduced borer damage and increased crop yield (Manjunath, 1991). It
has been found that 7-9 releases of T. chilonis and T. japonicum @ 1,00,000/ha, starting
at 30 days after transplanting proved as effective as the standard insecticide treatment for
the control of stem borer and leaf folder in Pubjab (Brar, 2000). Inundative releases of T.
japonicum at 10 adults/m2, 4-8 times during the season reduced leaf folder damage by 12
to 60 per cent. The presence of 3 predatory spiders/hill checked the population of brown
plant hopper and white backed plant hopper. Releases of mirid bug @ 100 bugs or 50-75
eggs/m2 at 10-day intervals have been found effective for the control of brown plant
hopper (Singh and Dhaliwal, 1994).

5.1.3.2. Sugarcane

As early as in 1930, mass releases of T. chilonis were initiated against shoot borer
around Mandra sugar factory zone. The releases of egg parasitoids were found useful for
the control of stem and root borers in erstwhile Madras State, Bombay province, Bihar
and Orissa. During recent years, releases of T.chilonis have been found effective for the
management of various shoot and stalk borers in different parts of the country. The
parasitoid releases @ 1,25,000 ha-1 are recommended against shoot borer in Andhra
Pradesh. Weekly releases at 1,25,000 parasitic wasps per ha from 4th to 11th week stage of
the crop provided effective control of internode borer, Chilo saccariphagus indicus in
Tamil Nadu. The release of 12 ml of adult parasitoids (3,00,000) per ha in six split doses
effectively checked internode borer infestation in an area of about 500 ha. Similarly, stalk
borer and Gurdaspur borer have been controlled in parts of sub-tropical India by large
scale releases of T. chilonis.
 During 1988 to 1999, fifty nine small scale field trials were conducted over an
area of 664 ha in Punjab. T. chilonis releases were done @ 50,000/ha at 7-10 day
intervals. The incidence of stalk borer C. auricilius was 11.7 per cent in the released
fields as compared to 32.1 per cent in control. The sequential releases of T. chilonis (12
releases @ 10,000/ha at 10 day intervals) and Cotesia flavipes (6 releases @ 10,000/ha at
10 day intervals) during July to October proved very effective for the control of stalk
borer. For the control of shoot borer, C. infuscatellus, 6-9 releases of T. chilonis during
April to June at 10 day intervals @ 50,000/ha proved effective (Brar, 2000)

In addition to egg parasitoids, field releases of the larval parasitoid, Isotima

javensis exercised effective control of top borer in Tamil Nadu and Karnataka. Releases
of Sturmiopsis inferens at 312 gravid females per ha provided effective control of C.
infuscatellus in coastal areas of Tamil Nadu.

Releases of Epiricania melanoleuca egg masses @ 4-5 lakh and 4000 to 5000
viable cocoons per ha were found effective for the control of sugarcane pyrilla in parts of
Andhra Pradesh, Kerala, karnataka and Madhya Pradesh where this parasitoid was not
found naturally. Lower rates (325 viable cocoons per ha) were found effective in areas
where E. melanoleuca occurred naturally (Varma and Tiwari, 1994). Redistribution of
field collected cocoons of nymphal and adult parasitoid, E. melanoleuca @ 5000
cocoons/ha proved effective for the control of pyrilla in Punjab.

5.1.3.3. Cotton

A number of promising natural enemies have been found attacking cotton pests in
the country. However, large scale use of insecticides has reduced the population of most
of these natural enemies to insignificant levels. In the fields where biocontrol was
practiced, 25 natural enemies were recorded as compared to only 2 in the fields sprayed
with insecticides. A number of exotic parasitoids like Chelonus blackburni from spotted
bollworm and Bracon kirkpatricki and T. brasiliense from pink bollworm have been
recovered in the States of Maharashtra, Karnataka, Tamil Nadu, Gujarat, Haryana and
Punjab.
 Releases of Trichogramma spp. at 1,50,000 parasitized eggs ha-1 at weekly
intervals have proved promising for bollworm control. Sucking pests could be checked
by releasing chrysopids at 1 lakh per ha at fortnightly intervals (Singh, 1994a).

5.1.3.4. Horticultural Crops

The initial work on the introduction of natural enemies was mainly directed
against pests of fruit trees. Augmentative and inoculative release of two exotic
parasitoids, Encarsia perniciosi and Aphytis sp. have given promising resuls for the
suppression of San Jose scale on apples. The Russian strain of E. perniciosi proved
effective in Himachal Pradesh, while Chinese and American strains proved better in Uttar
Pradesh. Aphelinus mali was found effective against woolly apple aphid in Kullu Valley
but subsequent releases in Shimla Hills, Chaubattia, Conoor and Shillong were not
successful.

Similarly, releases of exotic parasitoid, Leptomastix dactylopii have proved

effective for the control of citrus mealy bug, Planococcus citri in Karnataka. Mass
rearing and release of C. montrouzieri has been found effective against citrus mealy bug,
grape mealy bug, guava scale and other scales in a number of plantations (Singh, 1994b).

A number of indigenous natural enemies have also proved effective. These

include predators, Scymnus epius, Scymnus sp. against mealy bugs; parasitoids,
Cephaleta brunniventris and Anicetus ceylonensis against scale insect; Drephenococcus
chiton, Tetrastichus radiatus against citrus psylids; and Anagyrus dactylopii against
grape mealy bug (Tandon, 1994).

In case of vegetable pests, exotic tachinid parasitoid, Eucelatoria bryani has

established on tomato fruit borer, Helicoverpa armigera. Inundative releases of egg
parasitoid, T. brasiliense @ 4000 adults ha-1 week-1 for six weeks suppressed the attack
of fruit borer on tomato. Four exotic parasitoids, Orgilus jenniae, Chelonus kellieae, C.
blackburni and A. subandinus introduced against potato tuber moth.

The indigenous parasitoid, A. plutellae effectively checked the population of

diamondback moth on cabbage in Gujarat and Karnataka under favourable environmental
conditions. In the hill regions of Tamil Nadu, Diadegma semiclausum exercised 70 per
cent parasitism of diamondback moth (Singh, 1994b).
5.1.3.5. Plantation Crops

Inundative release of parasitoids, Goniozus nephantidis and Bracon brevicornis

at 3000 and 4500 per ha, respectively, has given encouraging results for the management
of Opisina arenosella on coconut. Exotic predatory bug, Platymeris laevicollis has
shown considerable promise against another coconut pest, Oryctes rhinoceros (Singh,
1996).

5.1.4. Genetic improvement of Parasitoids and predators

Genetic improvement of arthropod natural enemies can be approached from

different aspects. Entomophagous arthropods may be improved for climatic tolerance,
sex ratio, host finding ability, host preference, increased host range, increased pesticide
resistance etc.

5.1.4.1. Increasing genetic diversity

The importance of increasing the genetic diversity was first recognized by

Clausen (1936). Natural enemies are not constant in their abilities and adaptations
throughout their natural range of occurrence. Therefore, we can make use of these
differences and select an effective strain well adapted to the new environment.

5.1.4.2. Artificial selective breeding

The other methods of improving the beneficial organisms for use in biocontrol is
through artificial selective breeding for increasing fitness to the conditions of the new
environment. In this case, discerned weakness in adaptation is improved upon in the
laboratory by artificial selection before colonization.

Wilkes (1947) was able to influence the temperature preference of certain strains
of the parasitoid, Dhalbominus fuscipennis for oviposition; one strain was selected at 9°C
while the other 25°C. Later, Wilkes was able to improve his laboratory strain of D.
fuscipennis by doubling the mean number of progeny per female, decreasing the number
of sterile males produced from 35 to 2% and reducing the variability in development,
oviposition, and adult life span. While the work on genetic improvement of natural
enemies is at faster pace in other countries, the records are scanty in our country. The
only documented evidence is that of the development of a high temperature tolerant strain
of T. chilonis after 48 generations of rearing at high temperature (Anon., 2000).

Selection for resistance to pesticides, lack of diapause or enhanced temperature

tolerance have been successful, although most projects involved selection for resistance
to pesticides (Hoy, 1990a). Pesticide-resistant predators and parasitoids have been
evaluated in the field and are being implemented in several IPM programmes (Hoy,
1990a). Genetic improvement has proved to be practical and cost-effective when the
trait(s) limiting efficacy can be identified, the improved strain retains its fitness and
methods for implementation have been developed (Headley and Hoy, 1987).

Most cases of genetic improvement involve improvement for pesticide resistance,

though a few attempts have also been made to improve for other traits such as fecundity
(Table 6). Singificant development has been made in the development of pesticide
tolerant predatory mites. Strains selected for resistance to permethrin and carbaryl
retained their resistance to OP insecticides despite the fact that progeny were not
subjected to selection with OP insecticides. The resistant strains spread within the
released trees, multiplied, overwintered, survived pesticide applications in the field, and
had measurable impacts on spider mite population (Beckendorf and Hoy, 1985). In India
the only documented evidence is that of Trichogramma. An endosulfan resistant strain of
T. chilonis termed ‘endogram’ has been developed at PDBC, Bangalore through artificial
selection. This strain is tolerant to 2.5 ml/l of endosulfan. Now it is being selected for
resistance to monocrotophos and fenvalerate for multiple insecticide tolerance (Anon.,
2000).

5.1.4.3. Hybridization

Hybridization has been attempted as a method for improving vigour or fitness

through heterosis. Hoy (1975) found that a hybrid strain of gypsy moth parasite Apanteles
melanoscelus was easier to mass rear than the three strains from which it was derived.
Patil et al. (1997) studied the heterosis in a set of diallele crosses involving six diverse
population of C. carnea for 14 biological traits and found high levels of heterosis for
feeding potential followed by pupal duration, adult emergence and fecundity. This
finding suggests that efficient hybrid strains of C. carnea can be developed.
5.1.4.4. Recombinant DNA technology

Untill recently, genetic improvement of arthropod natural enemies was achieved

by traditional genetic methods: artificial selection, or hybridization of different strains to
achieve heterosis (Hoy, 1990b). Beckendorf and Hoy (1985) suggested that recombinant
DNA techniques could make genetic improvement of arthropod natural enemies more
efficient and less expensive, because once a gene has been cloned it can be inserted into a
number of beneficial species. If the manipulated populations need not be reared for long
periods of time, there is less likelihood that laboratory selection and inbreeding will
occur. One of the significant benefits of recombinant DNA techniques may be that it will
be easier to maintain ‘quality’ in transgenic arthropods.

5.2. Microbial Insecticides

5.2.1. Global scenario

The market for microbial pesticides is growing though it represents less than 1%
of the total crop protection market most of which is accounted for by Bt (Bacillus
thuringiensis)-based products. The estimated market for bioinsecticides in 1995 was US
$380 million including $3-4 million for viruses. By 2000, the market for bioinsecticides
was predicted to increase by $116-141 million (Georgis, 1997). Of the current microbial
insecticides available, the success story has undoubtedly been Bt which dominates the
current market. Bt has seen tremendous genetic improvement through biotechnology and
at the global level, Bt’s are claimed to be increasingly cost-effective which has made
them more competitive with chemical insecticides.

Insect viruses particularly of the family Baculoviridae have great potential for
development as microbial insecticides. The first commercial viral insecticide Elcar®, the
Heliothis zea nuclear polyhedrosis virus was registered by Sandoz with the EPA for use
on crops like corn, cotton and soybean. Examples of other commercial products include
the Spodoptera exigua NPV (Biosys) and the Lymantria dispar NPV (USDA/
Cyanamid). The Cydia pomonella granulosis virus (GV) has seen considerable
development towards commercialization. Madex and Granupom are two commercial
products of this virus registered for commercial production in Europe. A world survey of
virus control of insect pests has summarised the progress made on the use of
baculoviruses mostly NPV and GV in 11 different zoogeographical regions of the world
(Entwistle, 1998).

Entomopathogenic fungi particularly Beauveria and Metarhizium spp. are of

considerable importance because of their ability to infect a wide range of insects like
aphids, white flies, leaf miners, weevils, grass hoppers and cockroaches. Bayer AG
developed B10 1020 a strain of M. anisopliae for the control of the black vine weevil
Otiorhynchus sulcatus, but the production was discontinued because the market value
was considered inadequate (Reinecke et al., 1990). Despite this, products based on
Beauveria and Metarhizium have probably the highest business potential for smaller
companies due to the large number of available strains against a wide range of insect
pests. Verticillium lecanii has been extensively used for whitefly and aphid control in
glass houses but is disadvantaged by requiring a relative humitity of 95-100% to be
effective. Hirsutella thompsoni a fungus showing great promise in the control of the
citrus rust mite was registered in the U.S. as early as 1981 as Mycar® by Abbott.
Nomuraea rileyi is another promising fungus effective against several lepidopteran pests.

Entomopathogenic nematodes particularly of the genera Steinernema and

Heterorhabditis characterized by their symbiotic association with bacteria of the genera
Xenorhabdus and Photorhabdus have been found promising particularly against soil
insects. The high cost of production however limits the use of these nematodes in cash
crops such as cranberry and pot plants. Although mass production can be done on insect
larvae, commercial production is done using either Beddings method or Liquid
Fermentation.

5.2.2. Indian Scenario

Among the different microbial agents developed and tested, bacteria, viruses,
protozoans and fungi are considered promising agents to control insect pests. Information
on these pathogens in pest control is available in surplus in India (Table 7).

5.2.2.1. Insect viruses

Work on insect viruses in India was initiated as early as 1968 with the report of
nuclear polyhedrosis virus (NPV) from H. armigera (Patel et al., 1968) a pest of national
importance and S. litura (Ramakrishnan et al., 1969) a polyphagous pest attacking
several crops. Since then studies on insect viruses have progressed rapidly and several
viruses were reported to occur in insect pests, most of them from the order lepidoptera.
These comprise of NPV, granulosis viruses (GV) and cytoplasmic polyhedrosis viruses
(CPV). Recently a GV from the diamond back moth Plutella xylostella (Rabindra et al.,
1996) was reported.

5.2.2.1.1. Development of NPV

Of the different types of viruses, the NPV has the greatest potential since they are
more virulent and kill the insects much faster than the GV and CPV. The NPV of H.
armigera (HaNPV) has been studied very extensively to evaluate its efficacy as a viral
pesticide. HaNPV a single embedded virion type has a high virulence against H.
armigera (Rabindra and Subramanian, 1974) and the techniques of mass production,
formulation and field use have been developed (Rabindra et al., 1990). The virus at a
dose of 1.5-3 x 1012 polyhedral occlusion bodies (POB)/ha effectively controlled H.
armigera on crops like chickpea (Rabindra et al., 1989), pigeonpea (Muthiah and
Rabindra, 1991), groundnut (Muthuwami et al., 1993), sunflower (Rabindra et al., 1986),
sorghum (Dhandapani et al., 1993), cotton (Dhandapani et al., 1987) and tomato (Mistry
et al., 1984). Rabindra and Jayaraj, (1988) used several adjuvants to increase the efficacy
of the virus. Aqueous leaf extract of Vitex negundo also increased the efficacy of the
virus in the field (Rabindra et al., 1991). Several new strains of NPV of H. armigera
(Somasekar et al., 1993; Rabindra et al., 1997), S. litura, A. albistriga (Rabindra et al.,
1997), and P. xylostella (Rabindra et al., 1997), some with increased virulence have been
isolated.

S. litura a polyphagous defoliator pest has been controlled with NPV on cotton
(Jayaraj et al., 1980), tobacco (Ramakrishnan et al., 1981), banana (Santharam et al.,
1978), blackgram; (Krishnaiah et al., 1984), and cauliflower (Chaudhri and
Ramakrishnan,1980). A wettable powder formulation of the virus was found effective in
reducing the damage to ground nut plants (Sachithandam et al., 1989).

The red hairy caterpillar A. albistriga a gregarious seasonal pest causing extensive
damage to groundnut can be controlled with NPV (Chandramohan and Kumarasamy,
1979) and Rabindra and Balasubramanian, (1980) could induce an epizotic of the viral
disease in field populations of the pest resulting in long term control of the pest.

5.2.2.1.2. Development of granulosis virus

A granulosis virus was found to control the sugarcane shoot borer C. infuscatellus
effectively (Easwaramoorthy and Santhalaxmi, 1988). The virus was found to be safe to
the egg parasitoids T. chilonis and T.japonicum (Easwaramoorthy and Jayaraj, 1987). A
granulosis virus was reported from the rice leaf roller Cnaphalocrocis melinalis from
Kerala.

5.2.2.1.3. Baculovirus of Oryctes rhinoceros

Attempts made to utilize the baculovirus for the suppression of the coconut
rhinoceros beetle O. rhinoceros has given encouraging results. Release of infected
beetles spread the disease to subsequent generation of beetles and larvae in breeding sites
(Mohan et al., 1983)..

5.2.2.2. Bacillus thuringiensis

The spore forming, crystalliferous and ubiquitous bacterium B. thuringiensis (Bt)

is one of the earliest microbial insecticides to be commercially produced world wide. In
India, this bacterial insecticide has been used in the management of several insect pests
notably, P. xylostella larvae on cruciferous vegetables (Asokan et al., 1996). Other insect
pests controlled by Bt are H. armigera on pigeonpea (Putambekar et al., 1997), S. litura
on cabbage (Datta and Sharma, 1997) and fruit borers of okra (Satpathy and Panda,
1997). There are several reports however, indicating the ineffectiveness of Bt
preparations particularly against the noctuids. Delfin was ineffective in controlling H.
armigera on chickpea (Srinivasan et al., 1994).Therefore, it is important that new strains
of Bt with increased host spectrum and efficacy are developed. A few indigenously
prepared Bt formulations are not as effective as the imported preparations. There is an
urgent need to isolate indigenous Bt strains with high pesticidal activity, develop
techniques of mass production and stable formulations and patent them. Since insects can
develop resistance to the Bt toxin, this bacterial insecticide should be used judiciously
(Gelernter, 1997).
5.2.2.3. Fungal pathogens

Fungal pathogens particularly B. bassiana, M. anisopliae, V. lecanii and N. rileyi

have been found to be promising in the control of several agricultural pests. B. bassiana
and M. amisopliae were found effective against S. litura (Gopalakrishnan and Narayanan,
1989), the sweet potato weevil Cylas formicarius (Khader Khan et al., 1990), the termite
Odontotermis brunneus (Khader Khan et al., 1993), and O. obesus (Khader Khan et al.,
1993), M. anisopliae when applied to manure pit suppressed the population of O.
rhinoceros larvae (Sundara Babu et al., 1983). V. lecanii effectively controlled the coffee
green bug C. viridis in coffee plantations and addition of glycerol as a humectant
increased the efficacy of the fungus (Easwaramoorthy and Jayaraj, 1978).

N. rileyi active against several lepidopteran insects was found to occur in

epizootic form on H. armigera (Gopalakrishnan and Narayanan, 1989), S. litura
(Gopalakrishnan and Narayanan, 1988) and S. exigua (Phadke et al. 1978) suppressing
the pests under field conditions. Spray application on castor was effective against S.
litura (Vimaladevi, 1994). Soil application of the fungus gave good control of S. litura
(Vimaladevi, 1995). While N. rileyi killed A. janata larvae, it was not pathogenic to
Telenomus proditor an egg parasitoid of A. janata (Phadke and Rao, 1978). These reports
clearly indicate the scope for entomopathogenic fungus for pest control in India.

When one single method in insect control is usually not sufficient for an entire
pest management programme combinations may produce acceptable levels of control
without upsetting ecological relationships of other animals in the area. The interactions
studied and proved positive are presented in the Table 8.

5.2.2.4. Prospects for microbials in India

Many entomopathogens found effective under different agroclimatic zones on

various crop pests are now being developed commercially by many private producers in
our country. Puri et al (1997) catalogued the availability of biopesticides (Table 9). Of
these biopesticides, the baculoviruses particularly NPVs have tremendous scope for
development as microbial pesticides for the management of H. armigera and S. litura on
crops like cotton, gram and groundnut. The GV of P. xylostella is another promising
candidate. However, commercial availability of quality formulations is the immediate
need (Kennedy et al., 1999). Multinational companies who have the production
capabilities are not interested in view of the limited market potential. A few indigenous
small producers who ventured into commercial production of NPV of H. armigera and S.
litura did not succeed in producing quality virus. Most of the samples produced by
private entrepreneurs tested at the Department of Entomology, Tamil Nadu Agricultural
University had extremely low virus content if not no virus at all. Many had unacceptable
levels of spores of microsporidians. NPV production is done in vivo in respective host
larvae and hence producers should employ properly trained manpower for production of
host insects as well as the virus.

Most of the Bt products now being sold in India are very expensive since they are
imported. One or two products developed indigenously in India are not as effective as the
imported ones. A few institutions in India including the TNAU and the BARC have
isolated indigenous Bt strains which are as potent as the standard HD1. There is ample
scope for development of techniques of indigenous production and formulation of native
B.t. Species of entomopathogenic fungi like Metarhizium, Beauveria, Verticillium and
Nomuraea isolated from the different agroecosystems in India have shown promise in
pest control. We should genetically improve these strains and develop techniques of mass
production and stable formulations. India with the rich biodiversity of organisms, has
tremendous potential for development of native strains of microbials well adapted to the
Indian subcontinent.

The Indian Council of Agricultural Research (ICAR) and the Department of

Biotechnology of the Government of India (GOI) are extending ample support for
research and development of microbials in India for pest management. The GOI should
extend further support for indigenous commercial production of microbial pesticides by
encouraging private entrepreneurs within the constitutional frame work.

Educational programmes in institutions particularly agricultural universities

should give special emphasis on development of biorational pest control technologies like
microbial control. Conventional universities and national institutes should undertake
basic supportive research so that the innovations could be integrated into applied research
programmes in agricultural universities. There is a need to develop inter-institutional
collaborations in order to pool all the resources available in different institutions and
develop microbial pest control technologies which would ultimately reduce the pressure
on chemical insecticide use.

6. Biopesticides for the management of crop diseases

Fungicide resistance among the pathogens has led to the development of

microbial biocontrol agents and they have proved to be an effective and an attractive
alternative to obviate the deficiencies realised through the exclusive reliance on
chemicals..
6.1. Biocontrol of plant diseases
Antagonists like Pseudomonas fluorescens, Bacillus subtilis, Trichoderma viride
and T.harziamum were used to manage seed and soil-borne diseases of pulses, oilseeds,
vegetables, and paddy.The antagonists were delivered through seed coating and soil
application (Table 10).
Table 10. Biocontrol of plant diseases

Pathogen or Method of
Antagonist Host Reference
disease application
B. subtilis M. Phaseolina Pulses Seed coating Jeyarajan et al. (1994)
Chickpea, Vidhyasekaran and
P. fluorescens F. o. f.sp.ciceri Seed treatment
Pigeonpea Muthamilan (1995)
Nandakumar et al.
R. solani (Rice Seed and soil
P. fluorescens Paddy (1998) Rabindran and
sheath blight) application
Vidhyasekaran(1996)
Pythium Seed and soil Ramamurthy et al.
P. fluorescens Vegetables
damping off application (1999)

Pulses Grain Jeyarajan and

M. phaseolina legumes and Seed treatment Ramakrishnan (1991)
Trichodermia oilseeds Jeyarajan et al. (1991)
viride
Black gram,
Green gram, Seed treatment Jeyarajan et al. (1994)
Groundnut
Muthamilan and
T. harzianum S. rolfsii Groundnut Seed treatment
Jeyarajan (1996)

Trichoderma F. udum Pigeonpea Seed treatment Nakkeeran and

spp. M. phaseolina Renukadevi (1997)
Trichodermia M. phaseolina Nakkeeran et al.
Pigeonpea Seed treatment
viride F. udum (1996)
Trichoderma Sankar and Jeyarajan
M. phaseolina Sesame Seed treatment
spp. (1996)
Raguchander et al.
T. viride M. phaseolina Mungbean Seed treatment
(1993)
Trichoderma Raguchander et al.
M. phaseolina Soybean Seed treatment
spp. (1998)
Soil Raguchander et al.
T. viride M. phaseolina Mungbean
application (1997)

6.2. Improving bioefficacy

Bio-efficacy of biocontrol agents could be improved by developing a antagonist
mixture comprising a consortium of biocontrol agents with different modes of action or
by improving the antagonist through genetic engineering.

6.2.1. Strain improvement

Strains of T. viride were improved for their ability to produce antibiotics, and
hydrolytic enzymes. Mutants of T.viride namely M3 and M8 isolates produced maximum
volatile antibiotic substances under in vitro conditions as evidenced by the reduction in
the growth of M.phaseolina (Dinakaran,1997).

a. Enzymes

Lytic enzymes play a vital role in mycoparasitism and lead to death of the
pathogenic propaglues. Cell wall degrading enzymes like β–1,3-glucanases, chitinases,
proteinases and lipases are produced during interaction of both pathogen and leads to
lysis.

b. β-1,3 glucanases

Activity of ß-1,3-glucanase by the strain M2 and M1 was found to be 127.2 and

122.8 nmoles of glucose equivalent /min/mlof the dialysed culture filterate of T.viride.
These strains recorded the lowest root rot ncidence in black gram (Table 11).
 Table-11: ß-1,3-glucanase activity of T.viride strains
ß-1,3-glucanase activity
Strains
(nmolesof equivalent /min/ml)
Parent 96.21
M1 122.8
M2 127.29
M3 162.82
M6 44.41

(Source : Dinakaran,1997)
c. Chitinases
Relationship between mycolytic enzymes, chitinases and β-1,3 glucanases
produced by mycoparasitic fungi and their signficance in fungel cell wall lysis and
degradation have been well established. Treatment of Pseudomonas fluorescens in rice
cultivar IR50 led to the induction of systemic resistance against R.solani, as a result of
increase in the activity of chitinase and peroxidas (Table 12). Two chitinase isoforms
were induced. Among them 35 kDa isoform was a major determinant of induced
resistance (Nandakumar, 1998).
Table-12: Activity of chitinase in P.fluorescens treated paddy plants
Chitinase activity after different Without challenge With challenge
periods of inoculation inoculation inoculation
Control PF1 Control PF1
0 28 58 28 60
24 29 60 40 78
48 31 62 42 88
96 33 66 41 120

(Source : Nandakumar, 1998)

6.2.2. Antagonist mixtures
Development of mixtures for biocontrol of plant diseases (BCPD) could
overcome the shortfalls of individual antagonists. Antagonist mixtures have the
following advantages.
i. It may broaden the spectrum of activity
ii. It may enhance the efficacy and reliability
iii. Allows combination of various biocontrol agents without resorting to genetic
engineering.
 The antagonists used to develop mixture should not be detrimental to each other.
The candidates of the antagonist mixture should have either mutualism or commensalism
or neutralism. Application of mixtures of P.fluorescens to rice seedlings through
combined soil, root dip, foliar spray,and seed application reduced the sheath blight
incidence (Table 13) to a greater extent than individual application of the antagonist
strain (Nandakumar,1998).
Table 13. Effect of mixture of Pseudomonas fluorescens (PF 1 + FP 7) on sheath
blight and yield in Paddy

Percent Percent
Application methods PDI reduction Yield (t/ha) increase
over control over control
19.56b
ST 36.68 4.76 21.74
(26.24)
17.78c
ST + RD 42.44 4.83 23.53
(24.94)
16.89c
ST + RD + FS 45.32 4.8 22.76
(24.26)
14.22d
ST + RD + FS + SA 53.97 5.32 36.06
(22.15)
30.89a
Control - 3.91 -
(33.76)

ST = Seed Treatment, RD = Root Dipping, FS = Foliar spray, SA = Soil Application

(Source : Nandakumar,1998)
6.2.3 Genetic improvement

Though biocontrol is an acceptable green approach, the present day bio-products

can be further improved to obtain greater levels of disease reduction. The improvement of
the existing strains was performed through the alteration of the genome makeup of the
native strains using physical and chemical mutations. Through these technique improved
strains of T.viride with high rhizosphere competence, competitive saprophytic ability,
enhanced activity of β-1, 3 glucanase, chitinase coupled with high level of antagonism
index, increased shelf life and broad spectrum of action against several plant pathogens
have been developed. Strains like MG6, MNT7 and UV10 were effective in the
management of root rot of cotton, green gram, and damping-off of chilly and tomato
(Nakkeeran, 2001). The results are presented in Table 14 and 15. These strains were
found to have broad spectrum of action against R.solani, Macrophomina phaseolina,
Sclerottium rolfsii, Fusarium udum and Pythium ultimum (Renukadevi and
Nakkeeran,2000).

Table 14. Effect of T. viride mutants on green gram root rot (Vr-Co-6)
and grain yield
Germination Root rot DMP Yield
Mutants
(%) (%) (g/plant) Kg/ha
5.29
MG6 (10g/Kg) 100.00 8.2d 733d
(13.29)d
6.97
MNT7 (10g/Kg) 98.0 7.0c 709d
(15.31)d
15.66
UV10 (10g/Kg) 98.0 6.8c 656c
(23.31)c
19.97
Tv.Wd(10g/Kg) 98.0 5.8b 620b
(26.54)b
16.99
CBZ (2g/Kg) 100.0 5.0a 640bc
(24.34)bc
43.98
Control 98.0 4.8a 462a
(40.93)a
LSD (5%) NS 7.21 0.26 44

(Source : Renukadevi and Nakkeeran (2000)

Table 15.Effect of T. viride mutants on the incidence of cotton root rot

(Vr. LRA-5166) and seed-cotton yield
Germination 80 DAS DMP Yield
Mutants
(%) (%) (g/Plant) (Kg/ha)
15.24
MG6 (10g/Kg) 100.00 80.80 846d
(22.98)f
17.64
MNT7 (10g/Kg) 100.0 82.60 900e
(24.84)e
20.65
UV10g/(Kg) 98.0 71.00 800c
(27.03)d
24.32
Tv.Wd(10g/Kg) 100.0 64.40 723b
(29.55)c
28.63
CBZ (2g/Kg) 100.0 36.60 726b
(32.35)b
45.32
Control 98.0 30.00 640a
(42.31)a
LSD (5%) - 6.80 35

(Source : Renukadevi and Nakkeeran 2000)

6.2.4 Formulations
The Tamil Nadu Agricultural University, Coimbatore, has developed the
technology of mass production of talc based formulation of T. viride, and P. fluorescens
for seed treatment. Annually it produces this formulation to cover 20,000 ha. The annual
requirement of Trichoderma has been estimated as 5,000 tonnes to cover 50 per cent area
in India The first commercial seed treatment formulation of biocontrol agent in India was
developed by Jeyarajan et al. (1994). T. viride was grown in molasses-yeast medium for
10 days in flasks. Then 2:51 of the culture was inoculated to 501 of sterilized molasses
yeast medium in fermentor. It was incubated for 10 days with 4-8 hr aeration / day. The
fungal biomass and the broth were mixed with 100 kg talc (super white) powder and 500
g of carboxy methyl cellulose as a sticker. It was dried in shade for 72 hr and packed in
alkathene bags. The initial population of Trichoderma in the produce was 300 x 106
cfu/g. The product should contain a minimum of 20 x 106 cfu/g at the time of use. This
is applied at the rate of 4 g per kg of seed. The shelf life was 4 months

6.2.4.1. Improving formulation characteristics

Renganathan et al., (1995) found that gypsum was a good substitute for talc but
was much cheaper than talc. Nakkeeran and Jeyarajan (1996) tested two industrial waste
namely precipitated silica and calcium silicate in the place of talc and found them to give
a population of 99, 104 x 106 cfu/g respectively compared to 143 x 106 cfu/g in talc
substrate after 4 months of storage. These two substrates were also much cheaper than
talc. The following substrates were also found to support the growth of T. viride and T.
harzianum (Kousalya and Jeyarajan, 1988). Farm yard manure, gobar gas slurry, press
mud, paddy chaff, rice bran and groundnut shell.

6.3. Methods of application

Biocontrol agents can be applied to various plant parts like seed, root, foliage,
inflorescens and also the soil. For this purpose several methods have been developed
effectively and enhanced the yield considerably (Nakkeeran et al. 1995, Nakkeeran and
Renukadevi, 1997).
6.3.1. Solid Matrix Priming
It is a process in which moistened seeds are mixed with an organic carrier and the
moisture content of the mixture is brought to a level just below that required for seed
sprouting. Solid matrix priming can further enhance the efficacy of antagonist in treated
seeds. Trichoderma grow on seed surface during priming process and increase in
numbers and other microbes may not easily dislodge it. Slurry of Trichoderma is first
added to seeds followed by addition of lignite and water. Trichoderma required less
moisture for growth. The primed seeds were incubated for 4 days at 80-100% RH.
During this period Trichoderma colonises seed and sporulation was evident. In Pythium
ultimum infested soil the stand was increased to 70-80% as against 10% in control. In
tomato seed the population of Trichoderma increased by ten fold due to solid matrix
priming

6.3.2. Seed

Antagonist are delivered to the spermosphere by dry seed treatment or wet seed
treatment . The seeds are treated with Trichodertma viride at the rate of 4g/kg of pulses,
oilseedsand cotton (Jeyarajan et al.,1994)

6.3.3. Soil
For effective management of soil borne disease, a high population of the
antagonist in the soil is necessary. Adams (1990) defined effeciency of biocontrol agents
as the ratio of number of propagules of mycoparasites required to obtain disease control
to the typical inoculum density of a plant pathogen. Therefore attempts were made to
develop suitable delivery system to soil. For controlling R. solani, Trichoderma
population of 5 x 106 was required for each propagule of R. solani. Addition of wheat
bran based inoculum to soil gave 80 per cent reduction of root rot over control in
chickpea and bean. Incidence of urdbean root rot was reduced by 91.3 per cent by adding
T.viride + T. harzianum to soil (Kousalya and Jeyarajan, 1988). Delivering T. harzianum
through soil, during sowing increased the per centage of survival of peanut (90%), while
in control none of the plants survived (Muthamilan and Jeyarajan, 1996).
6.3.4. Foliar spray
The biocontrol organisms can be introduced only once (Inoculative) or repeatedly
applied (Inundative). Four applications of a conidial suspension of Ampelomyces
quisqualis (12 x 104 conidia/ml) at weekly intervals to cucumber leaves suppressed
conidial production and viability of powdery mildew. After the application of biotic
agent as foliar spray, mildew colonies turned dull black with abundant pycnidia of A.
quisqualis.

The efficacy of biocontrol agents for foliar diseases is greatly affected by

fluctuation of microclimate. Phyllosphere is subjected to diurnal and nocturnal, cyclic
and non-cyclic variation in temperature, relative humidity, dew, rain, wind and radiation.
Hence water potential of phylloplane microbes will be varying constantly. It will also
vary between leaves or the periphery of the canopy and on sheltered leaves. Higher
relative humidity could be observed in the shaded, dense region of the plant than that of
peripheral leaves. The dew formation is greater in centre and periphery.

The concentration of nutrients like amino acid, organic acids and sugars exuded
through stomata, lenticels, hydathodes and wounds varies highly. It affects the efficacy
and survival of antatognist in phylloplane (Andrews, 1992). When Pseudomonas is
applied to beet leaves, it actively competed for amino acids on the leaf surface and
inhibited spore germination of Botrytis cinerea, Cladosporium herbarum and Phoma
betae (Blakeman and Brodie, 1977).

Application of B. subtilis to bean leaves decreased incidence of bean rust

(Uromyces phaseoli) by 75% equivalent to weekly treatments with the fungicide
mancozeb (Baker et al., 1983). A peptide containing metabolite produced by the
bacterium reduced uredospore germination, prevented normal germ tube development
and caused cytoplasmic abnormalities in the uredospore. The yeast Tilletiopis minor,
Stephanoascus flocculosus and S. rugulosus, have been found to colonize the inactivate
cucumber powdery mildew, Sphaerotheca fuliginea (Jarvis et al., 1989).

6.3.5. Twine Coating

Gadoury et al. (1991) developed a novel method of applying pycnidia of the
mycoparasite Ampelomyces quisqualis to grapes to control Uncinula necator cotton twine
was saturated with malt extract agar and dipped in pycnidial suspension. The twine was
suspended in the trellis above grapevines, when shoots were 15 cm long. Rain splashed
the conidia onto leaves, upto 3 months.

6.3.6. Cut Stump Application

Ricard, (1981) developed a special shears to apply T. viride simultaneously with
prunning to protect fruit trees against silver leaf disease caused by Chondrostereum
purpureum. The shears have a reservoir for suspension of T. viride. Each prunning cut
was covered by spore suspension.

6.3.7. Hive insert

Thomson et al. (1992) developed an innovative method of application of bio-
control agent right in the infection court at the exact time of susceptibility. Erwinia
amylovora causing fire blight of apple infects through flower and develops extensively on
stigma. Colonisation by antagonist at the critical juncture is necessary to prevent flower
infection. Since flowers do not open simultaneously the bio-control agent P. fluresocens
has to be applied to flowers repeatedly to protect the stigma. Nectar seeking insects like
Aphis mellifera can be used to deliver P. fluorescens to stigma. Bees deposit the bacteria
on the flowers soon after opening due to their foraging habits. Thus repeated application
could be avoided. Botrytis cinerea (fruit rot of strawberry) was effectively controlled by
T. virens through honeybees which acted as vector. Peng et al. (1992) developed a novel
and more efficient method of applying inoculum of T. virens on talc and corn meal (5 x
108 cfug/g) which was placed inside the been hive in a dispensor. Bees acquired the
conidia on their legs and bodies as they crawled. Each bee had a spore load of 88 to 1800
x 103. Each flower got 1.6 to 27 x 103 conidia of antagonist. It suppressed B. cinerea on
stamens (54%) and petals (47%) and controlled fruit rot.

6.3.8 Fruit spray

Pseudomonas syringae (10% wettable powder) in the modified packing line was
sprayed at the rate of 10 g/l over apple fruit to control blue and grey mold of apple. The
population of antagonist increased in the wounds more than 10 fold during 3 months in
storage (Janisiew icz and Jeffers, 1997).
6.3.9. Fruit Dip
Dipping of grapes in Pichia guilliermondii and Hanseniaspora uvarum (yeasts)
cell suspension three days before harvest markedly suppressed Rhizopus rot for one to
two weeks (Sutton and Peng, 1993).

6.4. Effect of seed treatment with biocontrol agents on soil-borne diseases

6.4.1 Trichoderma viride

Soil-borne diseases caused by Rhizoctonia solani, Macrophpomina phaseolina,

and Fusarium spp. causes high percentage of mortality of the plants and consequently
great yield losses in rain-fed crops especially in pulses and oilseeds. Treatment of
sunflower seeds with T.viride at the rate of 4g/Kg reduced the root ro incidence to 18.15
as against 37.1% in control. It increased the yield by 21% (Table-16).

Table 16. Efficacy of antagonists on charcoal rot of Sunflower

Treatment Root rot (%) Yield (Kg/Ha)
T.viride 18.1 940
T.harzianum 24.1 885
T.hamatum 26.2 840
T.pseudokoningii 24 790
T.longibrachiatum 20.5 810
Gliocladium virens 24.7 915
B.subtilis 21.8 905
Carbendazim 21.0 940
Control 37.1 775
CD (P=0.05) 3.5 90

(Source : Jeyarajan et al.,1994)

Seed treatment of sesamum with T.viride @ 4g/Kg reduced the root rot incidence
to 12.8% and increased the root, shoot length and oil content .It recorded an yield of
677Kg/ha as against 301 Kg/ha in control (Table 17). Results of the field demonstrations
conducted in the farmer’s fields in groundnut, sesamum, urdbean, and chickpea are
presented in Table 18.
 Table 17.: Effect of antagonist on root rot, growth and yield of sesamum
Root Oil
Root rot Shoot length Yield
Treatments length content
% (cm) (Kg/ha)
(cm) (%)
T.viride 12.8 17.1 122.3 677 48.6
T.longibrachiatum 14.8 10.9 109.7 603 47.5
G.virens 13.5 11.2 121.1 658 48.6
B.subtilis 15.6 10.0 102.5 568 47.5
Carbendazim 29.6 9.2 102.0 453 45.1
Control 60.0 8.5 94.8 301 44.1
CD.(P=0.05) 2.8 0.4 4.6 21 0.5

(Source : Sankar and Jeyarajan,1996)

Table: 18. Efficacy of Trichoderma viride formulation in farmers field.

Crop % Root rot incidence Yield (Kg/ha)

T.viride Control T.viride Control
Groundnut 2.1 9.3 1467 1242

Sesamum 4.4 13.7 792 670

Urdbean 4.0 12.5 518 418

Chickpea 2.5 14.9 655 515

(Source : Ramakrishnan et al.,1994)

6.4.2. Pseudomonas fluorescens

Seed treatment of Pseudomonas fluorescens (10 g/kg) in brinjal and tomato was
effective against the incidence of damping off and wilt diseases under field conditions.
Formulation of Pseudomonas fluorescens prepared by mixing talc, gypsum and zinc was
superior in controlling the above diseases than talc based formulation alone (Table 19).
Combined application of Pseudomonas fluorescens mxitures through seed treatment, root
dip, foliar spray and soil application was effective in reducing the incidence of sheath
blight under field conditions. In addition it also increased the productive tillers and yield
(Table 13).
Table 19. Effect of Pseudomonas fluorescens against damping off and wilt diseases of
vegetables
Brinjal Tomato
Damp Yield Yield
Treatments Wilt Damping Wilt
ing off (Tonnes/ (Tonnes/
(%) off (%) (%)
(%) ha) ha)
Talc + Gypsum
45.5b 13.0ab 22.7 37.1b 10.0a 22.8
(without chitin)
Talc + Gypsum (with
32.7a 13.3ab 23.8 35.1b 9.3a 23.5
chitin)
Talc + Gypsum + Zinc
33.5a 12.7a 23.5 35.9b 9.7a 23.8
(with chitin)
Coc 45.3b 13.0a 20.4 28.8a 9.0a 22.3

Control 63.3c 53.6c 17.1 68.2c 47.3b 18.4

(Source : Ramamurthy,2000)
7. Biopesticides for the management of phytonematodes

Of the several microorganisms known, the rhizosphere bacteria fluorescent

Pseudomonas (Oostendorp and Sikora 1989), opportunistic fungus, Trichoderma
(Parvathareddy and Nagesh, 1998) and beneficial fungi, VAM (Umesh et al., 1988) are
considered as potential bioagents in the management of plant parasitic nematodes. All
these nematode antagonists are available as bioagents commercially at Tamil Nadu
Agricultural University, Coimbatore. The biocontrol potential of commercially available
nematicides with Tamil Nadu Agricultural University is evaluated extensively for the
management of important nematodes affecting different crops. The dosage and method
of application of bioagents are standardized in the management of nematodes. Further
the combined application of biopesticides is also evaluated for their effectiveness.. The
biocontrol agents viz., Pseudomonas fluorescens, Trichoderma viride and VAM tested for
their bioefficacy against different nematodes affecting major crops under glasshouse/field
conditions are given below.

7.1. Rice

Field experiments were conducted during kuruvai and navarai seasons on rice
varieties ADT 36 and ASD 18 for the management of rice nematodes. The results of the
 experiments showed that the seed treatment with commercially available Pseudomonas
fluorescens strain Pf-1 at 10g/kg seed followed by the foliar application of P. fluorescens
(Pf-1) at 0.2% (1 kg/ha) at 45, 55 and 65 DAT checked both rice root nematode
(Hirschmanniella gracilis) and the white tip nematode (Aphelenchoides besseyi).

The treatment registered significant reduction in rice root nematode population in

seedlings (82%), at 45 days after planting (82.2%) and at harvest (86.9%) in addition to
percentage of chaffiness (56.5%) caused by white tip nematode. The reduction in
nematode population resulted in the improvement of shoot (10.3%) and root weight
(5.9%) of seedling, increase in tiller number (8.0%) and grain yield (12%) compared to
untreated. The effect of P. fluorescens as seed treatment coupled with foliar application
of the same and the current recommendation of spraying of monocrotophos 36 EC at
1000 ml/ha at boot leaf stage were on par but superior over the treatmental effect of seed
treatment with P. fluorescens alone in the control of white tip nematode. The treatment
with P. fluorescens costs Rs.558 per hectare and gives an additional income of Rs.3374.
The cost benefit ratio worked out to be 1:6.0 (Table 20).

Table 20. Field evaluation of P. fluorescens for the management of rice nematodes

Nematode population Plant growth

Chaffi
H. gracilis/2g root Seedling
-ness
Grain C:B
Treatment At due to Shoot Root
Nursery At 45 Tiller yield Ratio
har- A. weight weight
DAT No. / ha
vest besseyi (gm) (gm)
(kg)
Seed treatment
with
P. fluorescens (10
5.8 12.3 36.3 6.06 2.24 1.8 9.4 6244
g /kg) + Foliar 1:6.0
(82.0) (82.2) (86.9) (56.4) (10.3) (5.9) (8.0) (12.1)
appli-cation at
0.2% at 45, 55
and 65 DAT
Seed treatment
with
P.fluorescens (10
g /kg) + Foliar 5.8 11.8 27.0 5.63 2.24 1.8 9.2 6187
1:4.5
application of (82.0) (82.9) (89.9) (59.5) (10.3) (5.9) (5.7) (11.1)
Monocrotophos
36 EC @ 1000
ml/ha.
Seed treatment
with P. 5.8 14.6 52.0 10.29 2.24 1.8 9.1 5619
1:4.2
fluorescens (10 g (82.0) (76.2) (81.2) (26.0) (10.3) (5.9) (4.5) (0.9)
/kg) alone
Untreated 33.3 69.1 276.8 13.90 2.03 1.7 8.7 5569 -
CD (P=0.05) 6.16 7.7 24.5 0.96 0.09 0.06 0.23 140 -

Figures in parentheses are percent increase/decrease over control.

7.2. Millets
Seed treatment with Pseudomonas fluorescens @ 10 g/kg reduced parasitization
by the root lesion nematode, Pratylenchus zeae on sorghum and maize and increased the
yield significantly. The reduction in soil population of the lesion nematode in sorghum
and maize was 58.24%, 61.65% respectively, 90 days after sowing. The treatment
increased the yield by 98.98% in sorghum and 68.79% in maize (Table 21).
Table 21. Management of lesion nematode of maize and sorghum
a. Maize

Grain yield per plot (kg) Nematode population (in

Treatments
7.2 m2 200g soil)
Pseudomonas fluorescens 2.654
191.13
seed treatment 10g per kg (3866.11 kg/ha)
(-61.65%)
seed (68.79%+)
1.570
Control 198.37
(2180.56 kg/ha)
CD:0.05 1.56 12.84

b. Sorghum

Pseudomonas fluorescens .868

180.75
seed treatment 10g per kg (1205.56 kg/ha)
(-58.24%)
seed (98.98%+)
Control .437 432.87
(606.94 kg/ha)
CD:0.05 55.98 13.00

Two field trials were conducted to evaluate the effect of VAM along with and
without biofertilizers viz., Azospirillum and phosphobacterium for the control of lesion
nematode, Pratylenchus zeae on maize. Application of VAM culture (Glomus
fasciculatum) @ 2.5 kg/cent (1 spore/g) in the main field along with seed treatment and
main field application of Azospirillum and Phosphobacterium at the recommended
dosage, (i) 10 packets (2000g)/ha of Azospirillum or Phosphobacterium to be
incorporated in the soil while preparing main field, (ii) Seeds to be treated with 3 packets
(600g)/ha Azospirillum or Phosphobacterium before sowing in the field) gave the best
result by suppressing the lesion nematode population and increasing the yield of grains.
The final nematode population in plots which received the combined inoculation of VAM
+ Azospirillum + Phosphobacterium was found to be reduced by 48.5 percent in soil with
the yield increase of 50 per cent.

The cost of treatment for VAM + Azospirillum + Phosphobacterium treatment per

hectare was Rs.5265 and the additional income of Rs. 6150 due to the treatment which
resulted in the cost benefit ratio of 1:1.2 (Table 22).

Table 22. Management of P. zeae with bioagents in maize

Cost
Final Grain Grain Additional
of
nematode yield yield income
Per cent treat- C:B
Treatment population per / per / over
decrease ment ratio
/ 200 ml plot ha control
/ha
soil (kg) (kg) (Rs.)
(Rs.)
VAM +
Azospirillum + 239 48.5 3.69 3690 6150 5265 1:1.2
Phosphobacterium

Control 464 - 2.46 2460 - - -

Two field trials, were conducted to evaluate the efficacy of VAM in nursery
along with and without Azospirillum and Phosphobacterium for the control of reniform
nematode, Rotylenchulus reniformis on ragi. Application of VAM culture (Glomus
fasciculatum) (1 spore/g) @ 100g/m2 in the nursery along with seed treatment and
seedling treatment with Azospirillum and Phosphobacterium at the recommended dosage,
(i) Seed treatment of Azospirillum or Phosphobacterium @ 3 packets/ha (600g/ha), (ii)
Application of 10 packets/ha (2000g) of Azospirillum or Phosphobacterium after mixing
with 25 kg of soil and 25 kg FYM before transplanting in the main field, (iii) Seedling
root-dip with Azospirillum or Phosphobacterium 5 packets (1000g)/ha for 15.30 mts.
before transplanting), gave the best results by suppressing the reniform nematode
population and increasing the yield of grains. The final nematode population in plots
planted with VAM + Azospirillum + Phosphoboacterium treated seedlings was found
reduced by 59.2 percent-age in soil while the yield increased by 68.8 per cent. The cost of
treatment for VAM + Azospirillum + Phosphobacterium treatment per hectare was
Rs.500 and the additional income due to this treatment was Rs.4400, cost benefit ratio
being 1:8.8 (Table 23).

Table 23. Management of R. reniformis with bioagents in ragi

Cost
Final Grain Grain Additional
of
nematode yield yield income
Per cent treat- C:B
Treatment population per / per / over
decrease ment ratio
/ 200 ml. plot ha control
/ha
Soil (kg) (kg) (Rs.)
(Rs.)
VAM +
Azospirillum + 149 59.2 2.7 2700 4400 500 1:8.8
Phosphobacterium

Control 365 - 1.6 1600 - - -

7.3. Blackgram

Soil application of Pseudomonas fluorescens or Trichoderma viride @ 2.5 kg/ha

singly or in combination at the time of sowing is effective to minimize the root
population of Heterodera cajani to the extent of 18.81 to 38.14 per cent. These
treatments increased the grain yield by 21.79 to 42.88%. The cost benefit ratio was
1:3.55, 1:3.79 and 1:3.92 respectively for the above treatments (Table 24).

Table 24. Management of Heterodera cajani in blackgram

Final
Cost
No. of nematode Grain Addit-
of
cysts/ populatio yield Grain ional
S. treat- C:B
Treatments plant n (eggs kg/plot yield income
No. ment ratio
(45 and (4 x 3.15 kg/ha / ha
/ha
DAS) Juveniles m2) Rs.
Rs.
/g soil)
1. Pseudomonas
fluorescens 19.50 19.63 0.531
421.00 380 1350 1:3.55
(Pf) (2.5 (-18.81) (-24.50) (+21.79)
kg/ha)
 2. Trichoderma
18.63 21.13 0.537
viride (T.v.) 426.00 380 1440 1:3.79
(-23.18) (-18.73) (+23.17)
(2.5 kg/ha)
3. 15.00 17.00 0.623
Pf + Tv 494.00 680 2664 1:3.92
(-38.14) (-34.62) (+42.88)
4. Control 24.25 26.00 0.436 346.00 - - -
C.D.(P=0.05
3.23 3.08 0.04
%)

Figures in parentheses are % decrease or increase over control ; INP = >2 eggs and
juveniles/g soil.
7.4. Redgram
Soil application of Pseudomonas fluorescens or Trichoderma viride alone or in
combination at 2.5 kg/ha, at the time of sowing was found to reduce Heterodera cajani
population in roots significantly by 24.59 – 35.79 per cent at 45 days after sowing. These
treatments increased the grain yield from 35.78 to 48.87 per cent. The Cost:benefit ratio
was 1:8.94, 1:8.62 and 1:6.83 respectively for the treatments given in the Table 25.
Table 25. Management of Heterodera cajani in redgram

Final
Cost Addit-
No. of nematode Grain
of ional
cysts/ populatio yield Grain
S. treat- incom C:B
Treatments plant n (eggs kg/plot yield
No. ment/ e ratio
(45 and (4x3.15 kg/ha
ha /ha
DAS) juveniles m2)
Rs. Rs.
/g soil)
1. Pseudomonas
fluorescens 34.5 28.75 0.903
717.00 380 3400 1:8.94
(Pf) (2.5 (-24.59) (-30.72) (+35.78)
kg/ha)
2. Trichoderma
34.25 28.75 0.894
viride (Tv) 710.00 380 3275 1:8.62
(-25.14) (-30.72) (+34.43)
(2.5 kg/ha)
3. Pf + Tv 29.38 24.25 0.990
786.00 680 4645 1:6.83
(-35.79) (-41.57) (+48.87)
4. Control 45.75 41.50 0.665 528.00 - - -
C.D. (p=0.05) 2.992 2.865 0.071

Figures in parentheses are % decrease or increase over control; INP = > 3 eggs and
juveniles/g soil
7.5.Greengram
Soil application of Pseudomonas fluorescens or Trichoderma viride at 2.5 kg/ha,
alone or in combination at the time of sowing was found to significantly reduce
Heterodera cajani population in roots by 15.76 – 29.04 per cent at 45 days after sowing.
These treatments increased the grain yield by 30.27 – 36.30 per cent. The cost:benefit
ratio was 1:8.34, 1:8.81 and 1:5.58 respectively for the above bioagents (Table 26).
Table 26. Management of Heterodera cajani in greengram

Final
Cost Addit-
No. of nematode Grain
of ional
cysts/ populatio yield Grain
S. treat- incom C:B
Treatments plant n (eggs kg/plot yield
No. ment e ratio
(45 and (4x3.15 kg/ha
/ha /ha
DAS) juveniles m2)
Rs. Rs.
/g soil)
1. Pseudomonas
fluorescens 25.38 22.75 0.951
755.00 380 3170 1:8.34
(Pf) (2.5 (-15.76) (-20.53) (+30.27)
kg/ha)
2. Trichoderma
25.13 24.25 0.964
viride (Tv) 765.00 380 3350 1:8.81
(-16.59) (-15.29) (+32.05)
(2.5 kg/ha)
3. Pf + Tv 21.38 19.75 0.995
790.00 680 3800 1:5.58
(-29.04) (-31.02) (+36.30)
4. Control 30.13 28.63 0.730 579.00 - - -
C.D. (p=0.05) 3.576 4.919 0.067

Figures in parentheses are % decrease or increase over control; INP = > 3 eggs and
juveniles/g soil

7.6. Castor

Pseudomonas fluorescens as seed treatment in castor @ 20g/kg seed is effective

in the biomanagement of Rotylenchulus reniformis. Considering the cost of chemical,
safety to environment and cost benefit ratio, P. fluorescens is the best biopesticide
(Table 27).
 Table 27. Management of reniform nematode in castor using biocontrol agents

Nematode population Seed

Sl.
Treatments At yield/ C:B
No. 45 DAS
termination pt (gm)
Pseudomonas fluorescens 20g/kg
1. 880.25 677.25 2036.50 1:2.6
seed (ST)
Trichoderma viride 4g/kg seed
2. 1142.50 746.75 924.00 1:0.6
(ST)
3. Carbofuran 1.0 kg ai/ha 539.00 1249.00 2408.75 1:2.6
4. T1 + T2 771.25 850.50 1155.00 1:1
5. T1 + T3 641.00 653.75 2232.50 1:2.3
6. T2 + T3 704.25 985.50 1592.50 1:1.4
7. Control 1220.00 1619.50 622.50 -
CD at 0.05% 182.71 237.93 575.31 -

The commercially viable biocontrol agents and their current status were
furnished in Table 9. In conclusion the results obtained here demonstrated the higher
antagonistic potential of all the three bioagents against different nematodes affecting
major crops growing in Tamil Nadu as reported by earlier workers (Parvathareddy et al.,
1995; Shanthi and Sikakumar, 1995; Mani et al., 1998; Ramakrishnan et al., 1998;
Sankaranarayanan et al., 1998).

8. Biopesticides in sericulture

Sericulture is one of the rural agro based industries and provides employment to
nearly 12.4 lakh families in India. Tamil Nadu is one of the traditional sericulture states
with an annual raw silk production of 1000 MT. Mulberry the sole food plant for
mulberry silkworm, Bombyx mori, is raised as irrigated, semi irrigated and rain fed crop
in Tamil Nadu. Mulberry crop is attacked by more than 300 arthropod pests
(Narayanaswamy et al., 1996). Availability of the host plant throughout the year due to
phased pruning and introduction of newer high yielding varieties like VI and Viswa (DD)
favours the pests to multiply in space and time. Use of chemical insecticides in mulberry
ecosystem has limited scope due to toxicity to the silkworms. Therefore biological
control has a greater role to play in mulberry ecosystem.
8.1. Key pests of mulberry in Tamil Nadu

Even though several pests damage the mulberry plants, only the pink mealy bug
Maconellicoccus hirsutus and leaf webber Glyphodes pulverulentalis cause economic
damage.

8.1. 1. Pink mealy bug, Maconellicoccus

Young ones and adults remain inside the unopened apical buds and desap the
plant. Affected apical portion exhibits crinkling, distortion and crowding of young shoots.
The symptom is popularly known as “Tukra” or bush top. Affected leaves are unfit for
young age worm (Chawki) rearing.

Experiment conducted in different sericulture belts of Tamil Nadu showed severe

incidence of Tukra in Erode district with 63.12 per cent damage. In other districts, it
causes an average shoot damage of 19.5 per cent with higher intensity of damage during
post monsoon seasons (Dhahira Beevi, 1989). In Coimbatore, the pest causes an average
leaf yield loss of 3000 kg/ha (Palanidurai, 1996).

Management of mealy bug in mulberry ecosystem should be aimed by

superimposing different pest management components over biological method. In South
India, pink mealy bug is affected by more than 10 natural enemies (Mani, 1986).
Coccinellid predators are more important because of their wide adaptability, easy
culturability and higher predatory potential.

Classical biological control using the Australian lady bird beetle, C. montrouzieri
against pink mealy bug in mulberry was documented by several workers (Tirumala Rao
and Leela David, 1958 ; Mani, 1986 ; Dhahirabeevi, 1989 ;Dandin et al., 2000). Field
release of C. montrouzieri @ 800 beeltes/ha was effective against pink mealy bug
(Dandin et.al., 2000). Scymnus coccivora a native coccinellid predator was found
effective against PMB, due to its smaller size and high searching ability to go deep in to
the tender buds in search of pests besides possessing thermal tolerance. Grub consumes
nearly 340 eggs or 70 nymphs or 10 adults female (Palanidurai, 1996).

S. coccivora population was found throughout the year in Coimbatore with

maximum population during July. Release of S. Coccivora @ 800 beetles/ha significantly
reduced the mealy bug population. The difference was apparent from 10th day after
release. Fifty days after predator release, pest population in biocontrol plot recorded 10
bugs as against 250 bugs/five plants in control plot (Palanidurai, 1996).

8.1.2. Mulberry leaf webber, Glyphodes pulverulentalis (Pyralidae : Lepidoptera)

Mulberry leaf webber is an introduced pest from Karnataka (Siddegowda et.al.,

1995). Damage to young shoots makes the tender leaves unfit for young age worms
(chawki) rearing. Besides the direct damage, the pest can also act as a vector for
silkworm diseases like IFV and pebrine (ESCAP, 1990).

Damage to shoots in different districts of Tamil Nadu ranged from 4.74 to 97.8
per cent with a mean damage of 41.87 per cent. Leaf webber inflicts damage to mulberry
shoots from July to January months with subsequent decline in population (Sathyaseelan,
1999). Damage level of 41 per cent in shoots caused an yield reduction of 9 kg of
cocoons/100 DFLS.

Application of insecticides in shoots may hamper the chawki rearing. Association

of natural enemies with different stages of Glyphodes was documented (Sathyaseelan,
1999; Gandhi Gracy, 2000). Parasitoids Apanteles sp., Bracon brevicornis, Phanerotoma,
Eriborus and Elasmus were found associated with larval stages of Glyphodes. Field
release of Bracon brevicornis@ 2 adults/plant significantly reduced shoot damage by
31.21 per cent with 45.16 per cent reduction in larval population (Gandhi Gracy, 2000).
Among the predators, spiders play a signficant role in regulating larval population. Four
spider, Tetragnatha, Oxyopes Peucetia and Olios were found promising in regulating
egg and larval population of Glyphodes. Oxyopes salticus preyed on all the larval stages
of Glyphodes. Prey consumption rate per day depends on the larval stage of the
Glyphodes. The prey consumption rate per day depends on the larval stage of the
Glyphodes. The prey consumption rates per day for different larval stages of host were
6.73, 4.75, 4.30, 3.75 and 1.50. Data generated on the ecology of hunting spiders
indicated that conservative biocontrol may play a greater role in suppressing the leaf
webber population.
8.1.3. Silkworm Uzifly, Exorista bombycis

Mulberry silkworm is affected by Uzifly Exorista bombycis.It is a tachinid

endoparasitoid. Uzifly is prevalent in all the sericulture areas of Tamil Nadu. Early stage
attack on silkworm leads to death of the larva. Late stage attack of uzifly on silkworm,
allows the host to spin cocoon. But emergence of uzifly from silkworm cocoon makes it
unfit for reeling. The pest was prevalent in all the months with higher level of damage
during winter months (Jayaprakash, 1996).

Revenue loss to sericulturists per rearing was estimated to be Rs. 800 per 100 dfls
(Anon., 1999). E. Bombycis was regulated by 18 hyperparasitoids in the families of
Eulophidae, Encyrtidae, Diapridae and Chalcidae (Pradipkumar et.al.,1993). Among
them, Nesolynx thymus and Tetrastichus howardii have higher searching ability, high
intraspecific competitive ability than other groups and can tolerate disinfectants like
formalin and bleaching powder under rearing room condition (Chandramohan and
Manjula, 1999).

Adoption of bio intensive management practices by inundative release of

N.thymus @ 1 lakh parasites/100 dfls in conjunction with other methods reduced the
uzifly incidence from 17.0 to 7.2 per cent with an increased yield of 25.63 kg/100 dfls
(Anon., 1999).

Future thrusts

Pest and disease problems are many and newer problems arise following intensive
cultivation of many crops and destruction of habitats. Sustainable crop production is of
paramount importance to feed the fast-growing human population. In order to obtain
maximum benefits from biocontrol technology, the gaps and issues should be analysed so
that the sound polices can be evolved to promote the concept.

1. Extensive surveys for isolating and identifying naturally occurring biocontrol

agents in various agro-ecosystems should be made.

2. Even the avilable biocontrol agents are not produced adequately for supply to
farmers. Creation of awareness among the farmers and extension personnel
and development of entrepreneurship are important issues.
3. To encourage the entrepreneurs to take up to commercial production of
biopesticides, large number of pilot plants should be established. The
scientists involved in the programme should provide help in technology
transfer, consultancy, training, quality control, trouble shooting and market
development.

4. Some of the biocontrol agents are affected by various kinds of environmental

stresses like UV light, temperature, moisture, pH etc., Adequate efforts have
not been made to exploit biotechnology and genetic engineering concepts to
develop more efficient strains of biocontrol agents.

5. For successful biocontrol of pests and diseases, these agents should be used at
the appropriate time during the cropping season along with the various
components of IPM.

6. The cost of biological control of pests can be optimised by proper rate and
timing of the release of the biological control agents. Information on pest
ecology and monitoring through insect sex pheromone technology should be
collected.
 Table 1. Important exotic natural enemies of pests established/used in India
Enemy Origin Pest Crop Result Reference
Planococcus spp. and Citrus, guava, Singh (1978) Mani and
Cryptolaemus
Australia other mealy bugs, custard apple, S Thontadarya (1988), Mani et al.
montrouzieri
shield scale grapes (1990)
Citrus, coffee, Krishnamoorthy and Singh
Leptomastix dactylopii West Indies Planococcus citri E
guava, ornamentals (1987), Nagarkatti et al. (1992)

Platymeris laevicollis Zanzibar Oryctes rhinoceros Coconut E Antony et al. (1979)

Rodolia cardinalis USA Icerya purchasi Citrus, casuarina S Rao and Kamath (1966)

Telenomus alecto Colombia Borer Sugarcane E Rao et al. (1971)

T. remus New Guinea Spodoptera litura Tobacco S Nagarkatti and Jayanth (1981)

South
Trichogramma Helicoverpa armigera Cotton R Nagarkatti and Singh (1989)
America

T. pretiosum USA H. armigera Tomato Nagarkatti and Singh (1989)

R – Recovered, S – Success, E – Established

 Table 2. Potential parasitoids for the management of crop pests
Crop Pest Natural enemies Reference
Telenomus dignoides, Trichogramma japonicum, Rao, 1972; Israel and
Scirpophaga incertulas
Tetrastichus schoenobii, Isotima javensis Padmanabhan, 1976
Cnaphalocrosis medinalis Macrocentrus spp. Rao et al., 1970

Rice Anagrus sp., Oligosita sp. Manjunath, 1991

Leaf hoppers / Plant hoppers
Chilo infuscatellus
Chilo sacchariphagus indicus
Sugarcane Scirpophaga excerptalis
Pyrilla perpusilla
Melanaspis glomerata
Corn Chilo partellus
Gonatocerus sp., Paracentrobia sp. Bentur and Kalode, 1985
Haplogonatopus sp., Echthrodelphox fairchildii,
Manjunath, et al., 1978
Elenhus sp.
Trichogramma sp. Telenomus sp., Cotesia flavipes,
Sturmiopsis inferens
Trichogramma spp. Cotesia flavipes
Telenomus beneficiens, Cotesia flavipes, Isotima David et.al., 1986, Pawar,
javensis 1979, Singh, 1993b
Oencyrtus papilionis, Tetrasticha pyrillae,
Epiricania melanoleuca
Adelencyrtus mayurai, Botryoideclava bharatiya,
Chandy, 1955; Nair; 1988,
Trichogramma chilonis, Cotesia flavipes
Subba Rao et al., 1969
Stenobracon deesae Isaac, 1941; Saxena, 1972

S. nicevelli Saxena, 1972

Trichogramma spp., T. achaeae, T. chilonis Thontadarya and Jai Rao, 1978

Cotton Earias spp.
Trichogrammatoidea sp. Sekon and Verma, 1983
 Rogas aligarhensis Thontadarya and Jai Rao, 1978

Bracon greeni, Brachymeria nephantidis Sekhon and Verma, 1983

Apanteles angaleti Sekhon and Verma, 1983

Pectinophora gosspiella Goniozus sp., Apanteles pectinophorae,
Sundarmurthy, 1990
Microbracon gelechidiphaga, Chelonus sp.
Cotton Trichogramma chilonis, Campoletis chloridae,
Helicoverpa armigera Carcelia illota, Goniophthalamus halli, Patel, 1980
Palexoristea laxa
Telenomus rowani, Chelonus formosanus,
Spodoptera litura Chari and Rao, 1990
Apanteles, agricanus and Peribaea abater
Aphis gossypii Aphelinus sp., Aphidencyrtus sp. Aphidius Singh, 1994a

Bemisia tabaci Encarsia transvena, Eretmocerus mundus Singh, 1994a

Chalikwar et al., 1984; Sathe
Exelastis atomosa Cotesia orientalis, Diadegma sp.
and Nikam 1985.
Pulses
Peter and Balasubramaniam,
Ophiomyia phaseoli Sphegigaster brunneicornis
1984
Stenomeisus japonicus, Sympiesis sp., Tetrastichus
Aproaerema modicella Sekharappa et al., 1990
sp. Chelonus sp.
Amsacta albistriga Telenomus manolus Sundaramurthy et al., 1976

Groundnut Spodoptera litura Telenomus spp. Singh, 1993a

Trichogramma chilonis, T. achaea, Telenomus sp.,
Patel and Yadav, 1979
Trissolcus sp.
Achaea janata
Rai and Jayaramaiah, 1978;
Microplitis maculipennis, Euplectus sp.
Murthy, 1989
 Apanteles obliquae, A. ruiclsus Shetgar et al. 1990
Sunflower Spilosoma obliqua
Carcelia illota, Exorista xanthaspis Patel and Talati, 1987

Trathala flavoorbitalis, Apanteles sp. Kalra, 1989

Sesamum Antigastra catalaunalis
Diadegma sp. Patel and Bhalani, 1990
Apanteles taragamae, Bracon hebetor,
Coconut Opisina arenosella Brachymeria nosatoi, B. nephantidis, Pillai, 1985
Xanthopimpla punctata, X. nana nana
 Table 3. Potential predators for the management of crop pests
Crop Pest Predator Reference
Cyrtorhinus lividipennis,
Manjunath, 1991; Samal and
Rice Coccinella arcuata, Micraspis Hoppers
Misra, 1975
discolor, Microvelia atrolineata
Brumoides suturalis, Serangium parcesetosum,
Bemisia tabaci Anonymous, 1983
Scymnus sp.
Cotton
Chrysoperla carnea, Mallada boninensis, Geocoris
Puri, 1991
bicolor, Orius spp.
Aproaerema modicella Chlaenius sp. Shanower and Ranga, 1990
Pulses
Cheilomenes sexmalulata, Coccinella
Aphis craccivora Upadhyay and Vyas, 1985
septempunctata
Sunflower Spilosoma obliqua Chrysoperla carnea Singh, 1993a

Opisina arenosella
Coconut
Oryctes rhinoceros
Parena nigrolineata, Calleida splendidula Nikam and Sathe, 1983
Santalus parallelus, Scarites sp., Harpalus sp.
Pillai, 1985
Pheropsophus sp., Agryphus sp.
 Table 4. Pesticides Found Relatively Safe to Natural enemies
Crop Name of the Pest Natural enemy Safe pesticides identified
Brown plant hopper Cyrtorhinus lividipennis Phosphamidon, phosalone, carbofuran
Lycosa pseudoannulata Carbofuran, Phosphamidon
Nymphs of homoptera and Coccinella rependa, Micraspis Fenitrothion, oxydemeton methyl,
larvae of lepidoptera discolour Chlorpyriphos
Phosalone, endosulfan, fenthion, diazinon,
Gall midge Platygaster oryzae
phorate
Rice
Yellow stem borer T. japonicum Endosulfan, lindane
Endosulfan, lindane, deltamethrin, diazinon,
monocrotophos, oxydemeton methyl,
T. chilonis
Leaf folder deltamethrin, phosphamidon, dicofol,
phosalone, fenvalerate, cypermethrin
T. japonicum Endosulfan, lindane
Endosulfan, lindane, deltamethrin, diazinon,
monocrotophos, oxydemeton methyl,
Corn Tissue borers T. chilonis
phosphamidon, dicofol, phosalone, fenvalerate,
cypermethrin
Gall midge Tetrastichus sp. Phosalone, endosulfan
Sorghum
Tissue borer T. chilonis Endosulfan, lindane, deltamethrin
Aphids Aphidencyrtus aphidivorus Endosulfan
Endosulfan, quinalphos, malathion, dimethoate,
Epiricania melanoleuca Tetrastichus pyrillae
vamidothion, sevimol
Scale insects Adelencyrtus mayurai Endosulfan, malathion
Sugarcane Endosulfan, lindane, deltamethrin, diazinon,
monocrotophos, oxydemeton methyl,
Tissue borers Trichogramma chilonis
phosphamidon, dicofol, phosalone, fenvalerate,
cypermethrin
T. japonicum Endosulfan, lindane
 Aphelinus gossypii Fenvalerate, phosalone
Aphids
Cheilomenes sexmaculata Neem derivatives
Endosulfan, fenvalerate, dimethoate,
Aphid, leaf hopper C. sexmaculata
monocrotophos, carbaryl, deltamethrin
Phosphamidon, phosalone, zineb, sulphur,
Apanteles angaleti
dicofol, deltamethrin, fenvalerate, carbaryl
Phosalone, dicofol, zineb, sulphur, deltamethrin,
Bracon kirkpatricki
permethrin, cypermethrin
Fenvalerate, deltamethrin, permethrin,
Chelonus blackburni diflubenzuron, phosalone, fenpropathrin,
endosulfan, fenvalerate, carbamate
Mancozeb, dicofol, phosalone, monocrotophos,
Trichogramma achaeae fenvalerate, oxydemeton methyl, permethrin,
Bollworms
deltamethrin
Mancozeb, dicofol, phosalone, monocrotophos,
deltamethrin, fenvalerate, endosulfan,
T. brasiliensis
Cotton fenpropathrin, cypermethrin, dichlorovos, neem
oil
Cypermethrin, zineb, sulphur, dicofol,
Eucelatoria bryani
phosalone
Carcelia illota Phosalone, dicofol
Rhogas aligarhensis Permethrin
Endosulfan, dicofol, monocrotophos, phosalone,
oxydemeton methyl, cypermethrin,
Chrysoperla carnea
phosphamidon, dimethoate, fenvalerate,
sulphur, zineb, carbaryl
Dichlorovos, cypermethrin, fenvalerate,
Aphid, leaf hopper, bollworms endosulfan, oxydemeton methyl, dicofol,
metalaxyl + mancozeb, fosetyl-AI, mancozeb,
Mallada boninensis carbendazim, tridimefon, chlorothalonil,
tridemorph, dinocap, captan, thiophenate
methyl, sulphur, copper oxychloride, ziram,
Bordeaux mixure, fluvalinate
 Lepidopterans Andrallus spinidens Phosalone, malathion
Chlorpyriphos, phosalone, acephate, endosulfan,
Pulses
Helicoverpa armigera Campoletis chlorideae carbaryl, phosalone, monocrotophos,
oxydemeton methyl, fenthion
Aphidius sp. Endosulfan, phosalone, oxydemeton methyl
Phosphamidon, endosulfan, oxydemeton,
methyl, phosalone, thiometon, endrin,
C. septempunctata oxydemeton, methyl alcoholic extracts of Melia
Oilseeds Aphid
azadarach drupes and Acorus calamus flag
rhizome, fenvalerate
Endosulfan, phosalone, oxydemeton methyl,
C. rependa
phorate, carbofuran, disulfotan, dimethoate
Dichlorovos, endosulfan, chlorpyriphos,
Bracon brevicornis
methomyl
Coconut caterpillar
Goniozus nephantidis Phosalone, endosulfan
Trichospilus pupivora Trichlorfon, diazinon
Phosalone, permethrin, deltamethrin,
fenvalerate, cypermethrin, carbendazim,
sulphur, mancozeb, zineb, captan,
Diamondback moth Cotesia plutellae chlorothalonil, fluvalinate, carbaryl, acephate,
Coconut oxydemeton, methyl, neem seed kernel extract
2%, metalaxyl + mancozeb, chlorothalonil,
copper oxychloride
Oxydemeton methyl, deltamethrin,
Chilli aphids Aphelinus sp. fenvalerate, sulphur, mancozeb, zineb,
chlorothalonil, captan, carbendazim
Okra mites Amblyseius tetranychivorus Sulphur, zineb
Hyposoter didymator Monocrotophos, phosalone
Trichogramma brasiliensis Dicofol, fenvalerate

(Source : Modified after Singh, 1994c and 1996)

 Table 28. Commercially viable bioagents for the management of Nematodes

Bioagent Commercial name Country Nematode species Current status Reference / Source
Arthrobotrys Royal 300® UK Ditylenchus Not available Cayrol et al., 1978.
robusta (NTF) myceliophagus
A. superba (NTF) Royal 350® UK Meloidogyne sp. Not available Cayrol and
Frankowski, 1979.
Paecilomyces Biocon Phillippines Meloidogyne spp. Not available Stirling, 1991
lilacinus (EPF)
Bioact UK Meloidogyne spp. Available Stirling, 1991
Pseudomonas Pf 1 TNAU, Coimbatore, Meloidogyne spp. Available
fluorescens (PGPR) India Heterodera cajani,
Hirschmanniella
gracilis,
Aphelenchoides
Department of Plant
besseyi,
Pathology
Pratylenchus zeae,
Rotylenchulus
reniformis
Trichoderma viride - TNAU, Coimbatore, H. cajani, Available
(NAF) India R. reniformis
Glomus mosseae VAM TNAU, Coimbatore, R. reniformis Available Department of
(VAM) India Microbiology

NTF = Nematode trapping fungus

EPF = Egg parasitic fungus
VAM = VAM fungus
NAF = Nematode antagonistic fungus
PGPR = Plant growth promoting rhizobacteria
 Table 5. Laboratory multiplication of important natural enemies in India
Natural enemy
Parasitoids
Apanteles angaleti
Bracon brevicornis
B. hebetor
B. kirkpatricki
Goniozus nephantidis
Chelonus blackburni
Copidosoma koehleri
Elasmus nephantidis
Leptomastix dactylopii
Tetrastichus israeli
Trichogramma spp.
Telenomus remus
Trichospilus pupivora
Predators
Adonia variegata
Brumoides suturalis
Chrysoperla carnea
Cryptolaemus montrouzieri
Mallada spp.
Menochilus sexmaculatus
Micraspis discolor
Scymnus spp.
(Source : Gautam 1994; Singh, 1994b)
Target pest
Pink bollworm
Coconut black headed caterpillar
Cotton boll worms
Cotton pink boll worm
Coconut black headed caterpillar
Cotton bollworms and potato tubermoth
Potato tubermoth
Coconut black headed caterpillar
Coffee mealybugs
Coconut black headed caterpillar
Lepidopterans
Tobacco caterpillar
Coconut black headed caterpillar
Aphids, thrips
Aphids, psyllids, mites, mealybugs
Aphids, insect eggs and larvae
Mealybugs
Mealybugs, aphids, mites
Aphids, psyllids, mealybugs
Aphids
Aphids, mealy bugs
 Table 6. Genetic improvement of parasitoids and predators

Natural enemy Host Target pest Reference

Dhalbominus fuscipennis Pine sand flies Increased fecundity Wilkes (1947)

Macrocentrus ancylvorus Oriental fruit DDT resistance Pielou and

moth Glasser (1952)

Typhlodromus pyri Apple red spider Cypermethrin Solomon and

mite resistance Fitzgerald (1993)

Trichogramma chilonis Eggs of several Endosulfan Singh (1994c)

lepidopteran resistance

Anisopteromalus Sitophilus oryzae Malathion resistance Baker and

calandrae Weaver (1993)

Aphytis melinus Aonidiella Carbaryl resistance Spollen and Hoy

aurantii (1992)

Predators

Chryosoperla carnea Carbaryl resistance Cardwell and

Hoy (1986)
 Table 7. Insect pathogens recorded in India

Pathogen Pest
Helicoverpa armigera, Spodoptera litura,
S. exigua, S. mauritia, A. moorei,
Agrotis ipsilon, A. segetum,Anadevidia peponis,
Chrysodeixis chalcites, Trichoplusia ni,
Thysanoplusia orichalcea,Spilarctia obliqua,

Nuclear Polyhedrosis Viruses Adisura atkinsoni, Lymantria obfuscata,

Plutella xylostella, Hellula undalis,
Corcyra cephalonica, Mythimna separata,
Galleria mellonella, Phthorimaea operculella,
Dasychira mendosa, Antheraea mylitta,
Pericallia ricini

Cnaphalocrocis medinalis,P. ricini,

Achaea janata, A. ipsilon, A. segetum, A. biconica,

Granulosis viruses P. operculella, S. litura,

G. mellonella, T. orichalcea,T. ni, P. xylostella,
C. cephalonica, Chilo infuscatellus

Non occluded virus Oryctes rhinoceros

H. armigera, S. obliqua,
Cytoplasmic polyhedrosis virus Sphenarches anisodactylus, Maruca testulalis,
Papilio demoleus, Euproctis fraterna
Asco virus H. armigera, S. litura
Pox virus H. armigera, Amsacta moorei
Rickettsiae like organism (RLO) S. litura, Achaea janata
Plutella xylosella
Crocidolomia binotalis
Pieris brassicae
Helicoverpa armigera
Bacteria Spodoptera litura, H. armigera
S. litura
Athalia lugens proxima
Papilio demoleus
Achaea janata

Bacteria
Fungus
Beauveria bassiana
Metarhzium anisopliae
Verticillium lecanii
V. aphidicola
Nomuraea rileyi
Hirsutella spp.
Erynia neoaphidis
Paecilomyes
Protozoa
Vairimorpha sp.
Farinocystis triboli
Nosema sp.
Mattesia dispora
Bacillidium sp.
Microsporidium sp.
Tetrahymena sp.
(Source : Puri et al.,1997)
H. armigera, Amsacta moorei
Achaea janata
H. armigera, amsacta moorei
Acigona steniellus
Earias vittella
Spoladea (=Hymenia) recurvalis
Phthorimaea operculella
Helicoverpa armigera, Spodoptera litura, Chilo
partellus, Scirpophaga incertulas, Myllocerus sp.,
Lymantria obfuscata
Pyrilla perpusilla, Oryctes, rhinoceros, Holotrichia
consanguinea
Coccus viridis, Archips termias, Empoasca sp.,
Aphis gossypii, Tetranychus sp.
Aphis spiraecola, Lipaphis erysimi, Brevicoryne
brassicae, Myzus persicae
H. armigera, S. litura, Trichoplusia spp.
Nilaparvata lugens, Tetranychus sp.
L. erysimi
Bemisia tabaci, Aleurocanthus woglumi farinosus
H. armigera, S. litura, S. exigua
Tribolium castaneum
Anadevidia (=Plusia) peponis, Spilarctia obliqua,
Adisura atkinsoni, crocidolomia binotalis
Ephestia cautella
Thrips flavus
S. litura, S. exigua, Utetheisa pulchella
Chilo partellus
 Table 8. Interaction matrix for five commonly used entomopathogens
Factors 1 2 3 4 5
Nuclear Polyhedrosis Viruses • •
Bacillus thuringiensis • •
Verticillium lecanii
Beauveria bassiana • •
Metarhizium anisopliae •
Paecilomyces spp. •
Other Natural Enemies
Other viruses •
Protozoa •
Nematodes • •
Predators • •
Parasitoids • •
Chemical Pesticides
Insecticides • • • • •
Fungicides •
Herbicides •
Insect Growth Regulators • • •
Natural Products
Oils •
Neem • •
Plant metabolites •
Host Plant effects
Species • • •
Cultivar • •
Surface factors •
Plant extracts •
Cultural Control
Soil type • •
Soil Condition •
Fertilizer •
Irrigation • •
Environmental Conditions
Temperature • • • • •
Humidity • •
Irradiation • • • •
Rainfall • •
Formulation • • • • •
Adjuvants • •
Application Technology • • • • •
1. Nuclear Polyhedrosis Virus ; 2. Bacillus thuringiensis; 3. Beauveria bassiana;
2. 4. Verticillium lecanii; 5. Metarhizium anisopliae
(Source : Dent , 1997)
 Table 9. Commercially available biopesticides in India

Biopesticide Crop Pest

Cotton Boll worms
Brinjal Fruit borer
Tomato Fruit borer
Okra Fruit borer
Bacillus thuringiensis var.kurstaki
Cabbage DBM, Semilooper
Cotton Spodoptera
Pulses Pod borer
Rice Stem borer
Sugarcane Top shoot borer, shoot borer
Granulosis viruses
Shoot borer
Cotton
NPV of Helicoverpa armigera Pulses Helicoverpa
Vegetables
Cotton
Castor
NPV of Spodoptera Groundnut Spodoptera
Tobacco
Tomato
Castor
NPV of Amsacta albistriga Red Hairy caterpillar
Groundnut
Baculovirus oryctes Coconut Rhinoceros beetle
Coffee Berry borer
Beauveria bassiana
Sugarcane Shootborer
Rice Borers
Metarhizum anisopliae Sugarcane Pyrilla
Groundnut White grubs
Cotton Helicoverpa
Pulses Spodoptera
Nomuraea rileyi
Groundnut Spodoptera
Castor Semilooper, looper

Contd…
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SCIENTIFIC WORKERS CONFERENCE 2001
THEME PAPER

BIOPESTICIDES

by

R. J. Rabindra, G. Ramakrishnan, G. Rajendran,

A. Subramanian, Sabitha Doraisamy, N. Sathiah, J. S. Kennedy,
T. Raguchander, S. Nakkeeran, S. Subramanian,
N. Ramakrishnan and N. Chandramohan

Centre for Plant Protection Studies

Tamil Nadu Agricultural University
Coimbatore – 641 003
 TECHNIQUES IN ESTABLISHMENT AND MAINTENANCE OF
INSECT CELL LINES

1. Introduction

Insect cell lines have become a diverse and important system for cloning and
production of virulent insect viruses and novel proteins of medical importance using
baculoviruses expression vector systems. Insect cell lines have been established from
about 70 species of insects belonging to the orders Lepidoptera, Diptera, Orthoptera,
Hemiptera, Coleoptera and Hymenoptera (Hink et al., 1985).

2. Establishment of insect cell lines

Cell lines can be established from various tissues (explants) viz., embryos,
haemocytes, fat bodies, imaginal discs, pupal and adult ovaries and larval gonads (Hink
and Ignoffo, 1970; Goodwin, 1975; Linn, 1989; Pant et al., 1998 and Mitsuhasi, 1998).
Different types of media for culturing cell lines are available commercially either as
powders or liquids. The commonly used media are TNM-FH, TC 100, IPL 41 etc.

3. Materials needed
3.1. Equipment
Laminar flowhood, cooled incubator, oven, pH meter, osmometer, inverted
phasecontrast microscope, water purification system (MilliQ, Millipore), dissecting
microscope, peristaltic pump and ultrasonic washer.

3.2. Glassware
Pipettes 5 and 10 ml, beakers 100, 250 and 500 ml, embryocups, glass rods,
20 ml syringe with leur lock, Pasteur pipettes.

3.3. Other materials

Micro dissecting scissors, fine forceps, tissue culture flask, wax dissecting dish,
wire guaze 100 mesh, insect pins, rubber policeman, 22 µ filter, face mask.

3.4. Media components

 2

TNM-FH medium (Sigma), sodium bicarbonate, sodium chloride (Nacl), sodium

hydroxide (NaoH), streptomycin sulphate, Penicillin, Foetal bovine serum (Sigma Cat.
No.F 3018), Hank’s balanced salt solution (Filter sterilized).

4.1. Preparation of glassware and other materials for cell culture work
Pipettes, beakers, embryo cups, wire guaze, glass rods, scissors, forceps and other
materials are washed in tap water and rinsed in distilled water followed by Milli-Q-water
or deionised water. After drying for one hour in a hot air oven, they (except pipettes) are
wrapped in aluminium foil and sterilized in a hot air oven at 2500C for 2h. Pipettes
(plugged with cotton at the neck), wax dishes, Milli-Q-water and silicone rubber
policeman are sterilized by autoclaving at 1200C at 20 PSI for 30 min. Laminar hood is
sterilized by swabbing the inner surface with ethyl alcohol 70.0% 30 min. prior to use.

4.2. Preparation of medium

TNM-FH powdered medium is dissolved in tissue culture grade Milli-Q-water at
the rate of 5.12 g per 100 ml and the contents are gently stirred until dissolved. Then
35 mg of sodium bicarbonate is added and mixed well. The pH of the medium is
adjusted to 6.2 with I N sodium hydroxide. Then 10,380 units of penicillin G sodium
salt, 50 mg of gentamycin and 10 ml of foetal bovine serum (FBS) are added. The
medium is sterilized by positive pressure filtration through a 0.22 µ millipore membrane
filter using a peristaltic pump. The medium is collected in sterile bottle and stored in a
refrigerator at 2-60C. All the above steps are to be done in a sterile laminar hood.

5. Establishment of cell lines from different explants

5.1. Embryos
The egg masses within 24 h of hatching collected from the laboratory - reared
healthy colonies are surface - sterilized by immersing in 0.2 per cent sodium hypochlorite
for two minutes and rinsed in distilled water. They are then collected in a sterile muslin
cloth, transferred to sterile laminar hood and immersed in 70 per cent ethyl alcohol for
2-3 min. followed by rinsing twice in sterile Milli-Q-water.

Embryo culturing procedure described by Lynn (1989) can be followed. The

surface - sterilized eggs are crushed along with a few drops of TNM-FH medium
 3

containing cysteine (60 mg per 100 ml) through a sterile stainless steel guaze using a
sterile silicone rubber policeman. Cysteine is added to prevent melanization of the
medium. The explants are washed down by adding a few more drops of medium into
sterile embryo cup and transferred into tissue culture flasks (25 cm2) containing five ml
of TNM-FH medium. The flasks are incubated at 27.50C in a cooled incubator.
Whenever cell attachment is seen, the medium in such flasks are changed once in
7-10 days.
5.2. Pupal ovaries and testes
Six to seven day old pupae are immersed in 70 per cent ethyl alcohol for two
minutes and rinsed twice in sterile Milli-Q-water in a sterile hood. The pupae are placed
on a sterile dissecting dish containing sterile Hank’s balanced salt solution (HBSS) and
fixed firmly on the dish using sterile stainless steel pins at both ends. The pupae are cut
open in the mid dorsal line and the ovaries/testes transferred to embryo cups containing
sterile HBSS. The unwanted tissues like fat bodies, malpighian tubules and trachea
attached to the gonads are removed and the gonads alone transferred to another embryo
cup containing a few drops of TNM-FH medium. The gonads are minced with sterile
micro dissecting scissors and the explants transferred to culture flask containing five ml
of medium. They are incubated in a cooled incubator at 27.50C.
5.3. Larval and pupal fat bodies
Fat bodies are dissected out from larvae and pupae of H. armigera and S. litura
aseptically and transferred to sterile HBSS. The adhering tracheoles are removed and
only the fat body lobes transferred to embryo cups containing sterile TNM-FH medium.
The explants are then minced repeatedly with the help of micro scissors and transferred to
tissue culture flasks containing five ml of medium. The flasks are kept in the incubator
at 27.50C.
5.4. Larval haemocytes
The mouth and rectum of final instar larvae are ligatured to prevent regurgitation
and excretion and surface sterilized with 70 per cent ethyl alcohol for two minutes and
rinsed twice with Milli-Q-water. Proleg of the larvae is cut and the haemolymph is
collected in an embryo cup containing medium with cysteine. The blood cells are
transferred to culture flask containing five ml of TNM-FH medium containing cysteine.
The flasks are kept in the incubator at 27.5oC.
 4

6. Maintenance and subculturing

The primary and established cultures are maintained in cooled incubator at
27.50C. Once in 5-7 days, four ml of the spent medium is removed and replaced with
fresh medium in primary cultures. For the established cell lines, subculturing is done at
the confluence stage. In the case of attached cell line, about one ml of spent medium is
retained and the remaining is removed. Then four ml of fresh medium is added and the
cell are scraped with a silicone rubber policeman. Then five ml more of fresh medium is
added, thoroughly mixed and split into two culture flasks. The subculturing is repeated
at every confluence stage.
References
Goodwin, R.H. 1975. Insect cell cultures : Improved media and methods for initiating
attached cell lines from the lepidoptera. In vitro 11: 369-378.

Hink, W.F. 1970. Established insect cell lines from the cabbage looper Trichoplusia ni.
Nature 226:466-467.

Hink, W.F. and Ignoffo, C.M. 1970. Establishment of a new cell line (IMC-HZ-1) from
ovaries of cotton bollworm moths, Heliothis zea (Boddie). Exp. Cell Res.,
60: 307-309.

Hink, W.F. and Ralph, D.A. and Joplin, K.H. 1985. Metabolism and characterisation of
insect cell cultures. In: Comprehensive Insect Physiology, Biochemistry and
Pharmacology. Eds. G.A. Kerkut and L.I. Gilbert. Pergamon Press, New York.
pp.547-570.

Linn, D.E. 1989. Methods for development of cell lines from insects. J. Tissue Cult.
Methods. 12:23-31.

Mitsuhashi, J. 1998. Cell culture. In: Insect Viruses and Pest Management. Eds.
F.R.Hunter-Fujita, P.F. Entwistle, H.F. Evans and N.E. Crook. John Wiley and
Sons, Chichester. pp.485-517.

Pant, U., Athavale, S.S., Basu, A. and Banerjee, K. 1998. A new cell line established
from pupal ovaries of Spodoptera litura F. (Lepidoptera : Noctuidae). Indian J.
Expt. Biol. 36: 195-198.
 GENETIC IMPROVEMENT OF PARASITOIDS AND PREDATORS

Biological control involving the use of parasitoids and predators is an

important element in sustainable crop production. Entomophagous insects play a key role
in natural as well as applied biocontrol programme forming an important component in
pest management programmes. Biological control of the cottony cushion scales and the
cassava mealy bugs are examples of successful cases of biological control. The role of
the egg parasitoids Trichogramma spp. in the management of several agricultural pests
like the sugarcane internode borer, larval and pupal parasitoids in the management of the
coconut black headed caterpillar and the green lacewings in the control of soft bodied
insects are well recognized. In addition, several parasitoids and predators are found to
attack important agricultural pests under natural conditions. In spite of this, pests cause
appreciable damage to crops necessitating the adoption of other control tools including
chemical pesticides.

Some of the factors which govern the efficiency of an entomophage are

i. Specificity
ii. Host searching ability
iii. Dispersal ability
iv. Ability to survive under extreme environmental conditions
v. Reproductive rate and
vi. Favourable sex ratio
Through genetic improvement, the field performance of entomophages can be
improved

Genetic improvement of Natural enemies

Genetic improvement of arthropod natural enemies can be approached from

different aspects.

1
1. Increasing genetic diversity

The importance of increasing the genetic diversity was first recognized by

Clausen (1936). Natural enemies are not constant in their abilities and adaptations
throughout their natural range of occurrence. Therefore we can make use of these
differences and select an effective strain well adapted to the new environment.

2. Artificial selective breeding

The other methods of improving the beneficial organisms for use in biocontrol is
through artificial selective breeding for increasing fitness to the conditions of the new
environment. In this case discerned weakness in adaptation is improved upon in the
laboratory by artificial selection before colonization. Entomophages may be selected for
climatic tolerance, improved sex ratio, host finding ability, host preference, and increased
insecticide resistance.

Wilkes (1947) was able to influence the temperature preference of certain strains
of the parasitoid, Dhalbominus fuscipennis for oviposition; one strain selected 9°C while
the other 25°C. Later Wilkes was able to improve his laboratory strain of D. fuscipennis
by doubling the mean number of progeny per female, decreasing the number of sterile
males produced from 35 to 2% and reducing the variability in development, oviposition,
and adult life span.

Selection for resistance to pesticides, lack of diapause or enhanced temperature

tolerance have been successful, although most projects involved selection for resistance
to pesticides (Hoy, 1990b). Pesticide-resistant predators and parasitoids have been
evaluated in the field and are being implemented in several IPM programmes (Hoy,
1990b). Genetic improvement has proved to be practical and cost-effective when the
trait(s) limiting efficacy can be identified, the improved strain retains its fitness and
methods for implementation have been developed (Headley and Hoy, 1987).

Traits primarily determined by single major genes, such as pesticide resistance,

are most appropriate for manipulation at this time because methods for manipulating and
stabilizing traits that are determined by complex genetic mechanisms are not yet
available. Genetic improvement can be useful: (i.) when the natural enemy is known to be

2
a potentially effective biological control agent except for one limiting factor; (ii.) the
limiting trait is primarily influenced by a single major gene; (iii.) the gene can be
obtained by selection, mutagenesis or cloning; (iv.) the manipulated strain is fit and
effective, and (v.) the released strain can be maintained by some form of reproductive
isolation. Typically, applications of pesticides are employed to reduce populations of
susceptible natural enemies, allowing the resistant population to establish and persist.
Alternatively, genetically manipulated strains can be released into greenhouses, or in new
geographic regions, so that the population is maintained in isolation with the desired trait
intact. It is not clear whether genetically manipulated natural enemy strains can
interbreed with native populations and if field selection will result in a new ‘hybrid’
population that carries the introduced attribute (Hoy, 1990a).

Most cases of genetic improvement involve improvement for pesticide resistance,

though a few attempts have also been made to improve for other traits such as fecundity
(Table 1). Singificant development has been made in the development of pesticide
tolerant predatory mites (Fig. 1). Strains selected for resistance to permethrin and
carbaryl retained their resistance to OP insecticides despite the fact that progeny were not
subjected to selection with OP insecticides. The resistant strains spread within the
released trees, multiplied, overwintered, survived pesticide applications in the field, and
had measurable impacts on spider mite population (Beckendorf and Hoy, 1985).

Hybridisation

Hybridisation has been attempted as a method for improving vigour or fitness

through heterosis. Hoy (1975) found that a hybrid strain of gypsy moth parasite Apanteles
melanoscelus was easier to mass rear than the three strains from which it was derived.
Patil et al. (1997) studied the heterosis in a set of diallele crosses involving six diverse
population of Chyrysoperla carnea for 14 biological traits and found high levels of
heterosis for feeding potential followed by pupal duration, adult emergence and
fecundity. This finding suggests that efficient hybrid strains of C. carnea can be
developed.

3
Recombinant DNA technology

Untill recently, genetic improvement of arthropod natural enemies was achieved

by traditional genetic methods: artificial selection, or hybridization of different strains to
achieve heterosis (Hoy, 1990a). Beckendorf and Hoy (1985) suggested that recombinant
DNA techniques could make genetic improvement of arthropod natural enemies more
efficient and less expensive, because once a gene has been cloned it can be inserted into a
number of beneficial species. If the manipulated populations need not be reared for long
periods of time, there is less likelihood that laboratory selection and inbreeding will
occur. One of the significant benefits of recombinant DNA techniques may be that it will
be easier to maintain ‘quality’ in transgenic arthropods.

Steps involved

Genetic improvement by recombinant DNA techniques involves several steps

(Fig.2). A successful outcome generally requires that we have a thorough knowledge of
the biology, ecology and behaviour of the target species. Identifying one or more specific
traits which, if altered, would potentially achieve the goals of the project is a critically
important first step. Next, suitable genes must be identified and cloned and appropriate
regulatory sequences must be identified so that the inserted gene will be expressed at
appropriate levels in the correct tissues and at a relevant time. Some potential methods
for stable transformation of arthropods is given in Table 2.

Inserting cloned DNA into pests or beneficial arthropods could be accomblished

by several different techniques (Table 2). The effects of the inserted DNA could be
transient and short-term or stable and long-term. If the inserted DNA is incorporated into
the chromosomes in the cells that give rise to the germ line, the foreign genetic material
should be transmitted faithfully and indefinitely to successive generations. Cloned DNA
can be isolated from the same or other species, and it is technically feasible to insert
genes from microorganisms into arthropods and have the DNA transcribed and translated.
However, DNA coding sequences isolated from microorganisms must have promoters
(controlling elements) and other regulatory DNA sequences derived from a higher
organism attached so that the gene will be expressed in arthropods. These regulatory

4
sequences determine when a gene will be transcribed, at what level, in what tissues and
how long the messenger ribonucleic acid (RNA) can be used for translation.

Germ line transformation methods

a) P element vectors

P transposable element was genetically manipulated to serve as a vector to carry

exogenous genes into the chromosomes of germ-line cells (Rubin and Spradling, 1982;
Spradling and Rubin, 1982). Since the pioneering research of Rubin and Spradling with
DrosophilaI (1982), P element vectors have been investigated as possible vectors for
other arthropods. P element-mediated transposition may be limited to Drosophila species
(Handler and O’Brochta, 1991), because there is no firm evidence that integration of any
exogenous DNA in an insect outside the genus Drosophila has been P-element-mediated.
As a result, a variety of other methods for avhieving transformation have been considered
and evaluated.

b) Microinjection

Microinjecting DNA carried in P-element vectors into Drosophila eggs is a well-

developed technique (Santamaria, 1986). Presnail and Hoy (1992) found that eggs of the
phytoseiid predator Metaseiulus occidentalis were extremely difficult to dechorionate and
dehydrate and that the needle tip had to be modified. It appears that Drosophila
microinjection methodology will have to be adapted empirically to each insect species
and will not be feasible with all. Variables to consider include whether to dechorionate or
not, whether to dehydrate and for how long, at what age/stage to inject, what holding
conditions to implement after injection and what size and shape of needle to use. It may
be possible to microinject exogenous DNA into insect embryos without using any
transposable-element vector with stable transformation occurring at a low rate (Walker,
1989). Early preblastoderm eggs present within adult females of the predatory mite
Metaseiulus occidentalis were microinjected by inserting a needle through the cuticle of
gravid females. This technique, called ‘maternal microinjection’, resulted in relatively
high levels of survival and stable transformation without the aid of a transposase-
producing helper plasmid (Presnail and Hoy, 1992). Presnail and Hoy (1996) have

5
demonstrated the successful microinjection of DNA of an endoparasitoid Cardiochiles
diaphaniae.

c) Soaking eggs in DNA solution

Exogenous DNA was introduced into Drosophila embryos by soaking them in

DNA solutions after dechorionation (Walker, 1989). However, the method was not much
used because of law uptake (<2%), variable phenotypes and the difficulty in establishing
stably transformed lines of flies. Most experiments used total genomic DNA, and Walker
(1989) speculated that soaking embryos in specific cloned gene sequences could produce
higher rates of stable transformation. Whether this method can be used for other
arthropod species remains to be determined. The ‘gene gun’ has been used successfully to
transform major crop plants, yeast and cultured cells. Its use with arthropod eggs is
limited, but Balderelli and Lengyel (1990) obtained transient expression of DNA in
Drosophila embryos. The authors suggest this method may, with some modification, be
suitable for stable germ-line transformation. This technique may be particularly useful for
species that deposit large numbers of eggs. It would not be advantageous for species such
as parasitic Hymenoptera, which deposit their eggs into the body of their insect host,
because obtaining large numbers of eggs by dissection would be extremely tedious.

d) Transplanting nuclei and cells

Zalockar (1981) reported methods for injecting and transplanting nuclei and pole
cells into eggs of Crosophila. Thus, it might be possible to genetically transform insect
cells in cell culture, isolate the nuclei and transplant them into the region where the germ-
line cells (pole cells) will develop in embryos. Cultured insect cells can be induced to
take up exogenous DNA by electroporation, liposomes, laser micropuncture and several
types of microinjection, and the transformed cells can be used to evaluate the expression
of genes and promoters (Walker, 1980; Fallon, 1991).

Potentially useful Resistance genes

Potentially useful genes that confer resistance to several pesticides are given in
Table 3.

6
Regulatory signals

Whether a coding region is transcribed and translated in a specific tissue is

determined by a number of regulatory sequences in the DNA, including promoters and
enhancers. The stability of messenger RNA is influenced by polyadenylation [poly (A)]
signals at the 3’ end of the RNA, which can influence the amount of protein produced. It
is crucial to obtain expression of the inserted gene at appropriate times, levels and tissues.
Another factor that may be important in maintaining the inserted DNA in the transgenic
line over time is the presence of origins of replication (DePamphilis, 1993). If exogenous
DNA is inserted into a region of the chromosome far from a site where an origin of
replication occurs naturally, the exogenous DNA could be lost over time because it is not
replicated (Benbow et al., 1992). The heat-shock (hsp 70) promoter from Drosophila is
commonly used as an inducible promoter. It is the strongest promoter known in
Drosophila and appears to function in all cells. Heat-shock proteins are present in all
organisms subjected to high temperatures and, while the number of these proteins varies
from organism to organism, all produce a 70 kDa protein. It is likely that the Drosophila
hsp70 promoter can be used whenever an inducible promoter is required that will
function in all cells.

Other commonly used regulatory sequences from Drosophila are the actin 5C
promoter, the α1-tubulin promoter and the metallothionein (Mtn) promoter.
Identification, cloning or genetic modification of promoters and other regulatory
sequences may increase the precision with which desired proteins are transcribed and
expressed in transgenic arthropods. Research to understand the structure and function of
regulatory sequences for use in transgenic arthropods should have high priority.

Identifying Transformants

Identifying transformed individuals could be achieved by using a pesticide

resistance gene, such as the opd gene as the selectable marker. Another option is to use
the neomycin (neo) antibiotic resistance gene, which functions in both Drosophila and
mosquitoes and is less likely to provoke concern about risks of releasing transgenic
arthropods into the environment. Another marker is the β-galactosidase gene (lacZ)
isolated from E. coli and regulated by the Drosophila hsp70 promoter, which has been

7
expressed in both Drosophila and the phytoseiid predator Metaseiulus occidentalis
(Presnail and Hoy, 1992). If an appropriate selectable marker is not available, identifying
transformed lines can be accomplished with PCR and subsequent analysis by Southern
blot hybridization or an immunological procedure.

Risk assessment

Protocols for evaluating the risks associated with releasing genetically

transformed parasitoids and predators do not exist. But the following issues should be
considered.

a) Possible alteration of host range or host preference

b) Possible alteration of geographical range

c) Stability of genetic alteration: It is important that the pesticide resistance gene

can not be transmitted to a pest species.

Conclusion

For many years, genetic manipulation of arthropod natural enemies was

considered to be impractical for pest-management programmes (Hoy, 1976), and this
limited the resources devoted to this tactic. The demonstration that a laboratory-selected
strain of predatory mite could provide cost-effective control of spider mites in an
agricultural crop (Headley and Hoy, 1987) provided an impetus to this research tactic in
biological control. It is important to demonstrate that a transgenic beneficial arthropod
can be effective in regulating pest populations in the field and have no negative impacts
on the environment. Until this has been achieved, adequate resources and funding will be
difficult to obtain because it is considered to be high-risk research. One factor hindering
progress in the genetic manipulation of beneficial arthropods is the lack of a ‘universal’
transformation system. The availability of a transformation method that would provide a
rapid and general system of a transformation method for introducing exogenous DNA
into species for which little genetic information is available would revolutionize the
genetic engineering of arthropods (Presnail and Hoy, 1992a).

8
 Table 1. Genetic improvement of parasitoids and predators

Parasitoids Host Target pest Reference

Dhalbominus fuscipennis Pine sand flies Increased fecundity Wilkes (1947)

Macrocentrus ancylvorus Oriental fruit DDT resistance Pielou and

moth Glasser (1952)
Metasieulus occidentalis Two spotted Permethrin and Hoy (1984)
spider mites carbaryl resistance
Typhlodromus pyri apple red spider Cypermethrin Solomon and
mite resistance Fitzgerald (1993)
Trichogramma chilonis Eggs of several Endosulfan Singh (1994)
lepidopteran resistance
Anisopteromalus Sitophilus oryzae Malathion resistance Baker and
calandrae Weaver (1993)
Aphytis melinus Aonidiella Carbaryl resistance Spollen and Hoy
aurantii (1992)
Predators

Chryosoperla carnea Carbaryl resistance Cardwell and

Hoy (1986)

9
 Table 2. Some potential methods for stably transforming arthropods other than
Drosophila

Technique Example(s) available Reference(s)

DNA delivered by Transient expression only in Balderelli and Lengyel
microprojectiles Drosophila embryos (1990)

Electroporation Transient transformation of Kamdar et al. (1992)

Drosophila only

Maternal microinjection Metaseiulus occidentalis Presnail and Hoy

(1992)

Microinjection of eggs Three mosquito species; P Morris et al. (1989)

element apparently not functional

P-element vectors Drosophila species only Handler and

O’Brochta (1991)

Soaking dechorionated eggs Drosophila Walker (1989)

in DNA solution

Sperm as vectors of DNA Lucilia cuprina Apis mellifera Atkinson et al. (1991)
(DNA bound externally only?)

Transplant nuclei and cells Drosophild Zalokar (1981)

Transposable-emelemtn None at this time, but Tes are Michaille et al. (1990)
(TE) vectors from target known from several, including Besansky (1990)
species Anopheles gambiae and Bombyx Robertson (1993)
mori

10
 Table 3. Some cloned resistance genes possibly useful for genetic manipulation of
beneficial arthropods

Gene (abbreviation)
Source(s) Refernce(s)
resistance

Acetylcholinesterase (Ace) D. melanogaster Fournier et al. (1989)

(pesticide resistance) Anopheles stephensi Hoffmann et al. (1992)

γ-Aminobutyric acid A D. melanogaster Ffrench-Constant et al.

(GABAA) (dieldrin resistance) (1993)

Cytochrome P450-B1 (DDT D. melanogaster Waters et al. (1992)

resistance)

Esterase β1 amplification core Culex species Mouches et al. (1990)

(organophosphate resistance)

Glutathione-S-Transferase D. melanogaster Toung et al. (1990)

(Dmgst 1-1) (DDT resistance)

Glutathione-S-Transferase Musca domestica Fournier et al. (1992)

(MdGST 1-1)
(organophosphate resistance)

Parathion hydrolase (opd) Pseudomonas diminuta Serdar et al. (1989 )

(parathion, paraoxon Flavobacterium sp. Dumas et al. (1990)
resistance)

11
 Fig. 1. Outline of a genetic improvement project with Metaseiulus occidentalis

Potentially effective predator

Determine traits needing improvement

Adequate population sampling Production of genetic variability

Efficient rearing and maintenance

Selection programme

Lab, greenhouse or field cage Genetic analysis of Traits

evaluations of efficacy

Specific predator-prey models

Determine field release strategy

General predator Prey models

Release and field efficacy evaluations

Cost analysis Programe implementation

12
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Besansky, N.J. (1990) A retrotransposable element from the mosquito Anopheles
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Cardwell, G.E.E. and Hoy, M.A. (1986) Genetic improvement of common green
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Headley, J.C. and Hoy, M.A. (1987) Benefit/cost analysis of an integrated mite
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15
 57. In vitro Replication of Baculoviruses

1. Introduction

In vitro production of insect virus in cell lines has got several advantages. Cell
cultured viruses are free from undesirable viral or microbial contamination, secondary
biotypes and insect debris. In insect cell line’ virus production can be monitored easily.
It is easy to maintain cell lines continuously for virus production. Large scale production
is possible using fermentors/bioreactors. Baculovirus replication in insect cell line has
been reported for several nuclear polyhedrosis viruses (Goodwin et al., 1976, McIntosh
and Ignoffo, 1981 and Lynn et al., 1983).

2. Preparation of virus inoculum

Fourth instar larvae are inoculated with the homologous NPV by allowing them to
feed on diet surface contaminated with 106 polyhedra per bottle. After three days of
inoculation, the larvae are washed in sterile distilled water and the haemolymph is drawn
by cutting the prolegs and collected in 10ml of TNM-FH medium containing cysteine to
prevent melanization. The medium is then centrifuged at 780 x g for 10 min to sediment
haemocytes and passed through a 0.45 µm millipore membrane filter under a laminar
hood.

3. Challenging of cell lines

At log phase of the cell line, the spent medium is removed and 2.5 ml of medium
containing virus inoculum is added to the cells and an adsorption time of two hours is
allowed with periodical rocking of flasks at an interval of 10 min. The medium is then
removed, the cells washed twice gently with sterile Hanks balanced salt solution
(HBSS). Finally, after removing the HBSS completely, 5 ml of TNM-FH medium
containing 10 per cent fetal bovine serum is added and incubated at 27.50C. The cells
were observed daily for virus infection under a microscope. Virus can be harvested after
five to six days.

4. Identification of in vitro - cultured viruses

 The cell cultured virus can be subjected to DNA analysis using restriction
endonucleases. The methodology is described elsewhere. The DNA profiles of cell
cultured virus and in vivo produced virus can be compared. Similarity in the DNA
profiles of both viruses will indicate that in cell line the virus has not undergone any
recombination or mutation.

5. Biological activity of in vitro - cultured viruses

The efficacy of cell cultured viruses has to be tested by conducting bioassays.
The virus infected cells are harvested 10 days after inoculation and stored at -200C.
Before processing, the cells are thawed out and sonicated in ice bath using a Branson
Sonifier 450 to disrupt the cells and release the polyhedra. The cell debris are removed
by centrifugation of 200 x g for one minute. The supernatant is again centrifuged at
780 x g for five minutes to pellet the virus. Then the polyhedra are suspended in distilled
water and the concentration assessed with the help of a haemocytometer under phase
contrast microscope.

The virus harvested from the cell line can be bioassayed to compare their
biological activity with that of the in vitro produced virus. A standard diet surface
contamination method is followed starting with a dose of 3.5x105 polyhedral occlusion
bodies (POB) ml-1. Seven-fold serial dilutions are made to give five doses each.
Chickpea seed based-semi-synthetic diet is dispensed into sterile five ml glass vials and
allowed to cool down to room temperature under a laminar hood. After 2-3h, an aliquot
of 10 µl of the different viral dilutions are applied on the diet and spread uniformly over
the entire diet surface using a four mm glass rod with rounded tip. After 15 min., one
larva of second instar is introduced into each vial and plugged with sterile cotton. Thirty
five to forty larvae are used in each dose. A similar set of larvae without virus treatment
is maintained as control. Observations on the larval mortality is recorded daily for ten
days and data are subjected to probit analysis. LC50 values of the two viruses is then
compared.
References
Godwin, R.H., Adams, J.R., Vaughn, J.L. and Louloudes, S.J. 1976. Invertebrate tissue
culture techniques and in vitro baculovirus host range. Canada Proc. Ist Int. Coll.
Invertebr. Pathol., Kingston, Canada, pp.94-98.

Lynn, D.E., Miller, S.G. and Oberlander, H. 1983. Nuclear polyhedrosis virus replication
in epithelial cell cultures of lepidoptera. J. Invertebr. Pathol., 42: 424-426.

McIntosh, A.H. and Ignoffo, C.M. 1981. Replication and infectivity of single embedded
nuclear polyhedrosis virus, Baculovirus heliothis, in homologous cell lines. J.
Invertebr. Pathol., 37: 258-264.
 MASS CULTURE OF HOST INSECTS

The maintenance of insect colonies has become vital to modern pest

management. The science and art of insect rearing has increased in
complexity and sophistication as the need for insects of known traits has
grown phenomenally. Today scientists are able to modify characters in the
laboratory principally by selection. The development of artificial diets,
assurance of diet ingredient quality and new container designs had major
impact on the development of rearing procedures. Experience has shown that
as the insect concentration increases in a production system there is always a
risk of contamination, disease incidence and related problems. Considering
these, the objective of the programmes in any insectary has become to
produce insects of acceptable quality that conforms to a predetermined
standard or specifications by a unique combination of procedures,
equipment, space and environmental parameters.
General requirements
The basic requirements in a production system will depend on the
types of insect species being reared in the system. There is always a
commonality in the use of materials in multiple species rearing systems.
Hence a systematic approach is needed in the use of materials. To achieve
this end the desired characteristics must be known.
Containerization
The development in the technology of insect rearing as it moved
forward from the natural diet to artificial diet containerization became very
standardized. An effective container must protect the food; present the food
to insect in an acceptable manner; provide proper surfaces and atmosphere
for the insect and confine and separate cannibalistic insects and permit easy
handling cleaning and storing. Currently, metal, fiberglass and
thermoplastics are in use in many of the lepidopterous rearing systems.

ARTIFICIAL DIETS
They have relieved the researchers from the maintenance of expensive
green house facilities and host plants for phytophagous insects. The
continual advancements in artificial diets have made it possible for the
maintenance of many additional species of phytophagous insects and also
resulted in the consistent and economical production of vigorous competitive
insects.
Standardization of the insect diet is important because the
characteristics of the reared insect depend on nutrition. It is always essential
to apply quality standards to the formulation of standard diets. The diet shall
include many variables: protein, carbohydrate, lipid minerals, vitamins,
microbial inhibitors and secondary substances. Also the pH, moisture
content, texture, consistency are considered important.
Sanitation and decontamination
Often the stocks of insects reared are found to harbour pathogens. Due
to inefficient protocols the rearing environment frequently gets gross
microbial contamination. Inadequate understanding on the part of the
workers contributes to the malady. Therefore, strict sanitation measures are
needed to minimize or eliminate microorganisms in the rearing facility. The
rearing procedure for any insect has to be evolved along with sound
sanitation procedures. Sensible use of various sterilization procedures viz.,
heat, steam and chemical methods have to be followed.

Elements of Rearing
Establishment of Laboratory Colony
The success of the rearing programme depends upon obtaining a
disease-free and a genetically efficient and uniform parental stock. The
preferred stage from the field for starting the colony is gravid female,
which can be collected at a light trap. This method offers a better chance
of avoiding diseases and parasitoids. However, in practice, it is a
difficult proposition to collect too many numbers with uniformity.
Therefore, A starter colony of H. armigera can be established by field
collection of grown-up larvae from chickpea, pigeonpea, cotton,
groundnut, sunflower, bhendi, etc so that a continuous supply of adult
moths of both sexes will be available for the breeder stock. The colonies
established from collections made from different crops and areas have
to be kept isolated and examined for the incidence of protozoan
pathogens Vairimorpha. Mother moth examination has to be done to
eliminate the pathogen if incidence is noticed .
Production of Host insects
During the last two decades much effort has been expended on
developing suitable diets and containers to house larval stages of H.
armigera. Many of the research laboratories depend on the semi-
synthetic diet developed by Shorey and Hale (1965).
Maintenance of Colony
i. Adult handling
The pupae from primary parental colony are kept in a 30x30-cm adult
emergence cage for eclosion. Daily an adult feed solution containing 10%
sucrose fortified with vitamins is provided in the cage for adults. Ten pairs
of healthy adults are transferred to oviposition chamber. The oviposition
chamber consists of a 30x20cm plastic bucket. The mouth of the unit is
covered with muslin cloth which serves as a oviposition substrate. Daily the
oviposition substrate is removed and replaced with fresh ones. Adult feed is
also changed daily. To improve sanitation, the adults which are weak are
removed.
ii. Egg handling
The muslin clothes are collected and labeled properly. They are kept
in an atmosphere of higher saturation. This is facilitated by keeping them
inside a humidifying chamber. We operate an inexpensive humidifying
chamber for our production purposes. It consists of a plastic bucket with a
lid. Sodium hypochlorite 0.25% (v/v) is poured inside the bucket upto 2-cm
height level. The egg laden muslin cloths are kept inside a clear plastic
container (20x10-cm) and allowed to float on the solution. The lid is firmly
placed over the bucket. Twenty-four hours after the collection of egg-cloth,
and further incubation, the eggs are watched for development of embryo.
Eggs showing distinct germ band are suitable for surface sterilisation. This is
done to eliminate the contaminant microbes. Surface sterilisation is achieved
by treatment in formaldehyde.

The egg-laden clothing is submerged in a 10% formaldehyde solution

for 10 minutes. After the expiry of time, the clothes are washed in tap water
for 20 min to eliminate the traces of formaldehyde. The cloths are shade
dried and kept in sterile plastic cups inside a sterile humidifying chamber for
eclosion.
iii. Larval handling
The larvae are supported by a semi-synthetic diet. It is prepared by
blending and cooling with the following ingredients:

Chickpea seeds :.100g Wesson salt mix : 7.2 g

Agar agar :12.8 g Streptomycin sulphate : 40 mg
Yeast : 30 g Vitamin supplement : 2 ml
Methyl-para-hydroxy benzoate :2g Formaldehyde 40% : 1ml
Sorbic acid :1g Carbendazim : 500mg
Ascorbic acid : 3.2 g Water : 720 ml

Chickpea seeds (previously soaked for 6-8 h in water) is boiled in water (300
ml) and transferred to a blender. Wesson's salt mixture, sorbic acid and
methyl-para-hydroxy benzoate and yeast, is added while blending.
Simultaneously, agar is boiled in the remaining portion of water (360 ml),
cooled to 60°C and then finally added to the above mix. After thorough
blending for 2-3 min. the remaining fractions of the chemicals have to be
added one after another. The hot liquid depending upon the need is dispensed
into rearing trays and glass vials for rearing early and older stages of the
larvae respectively. After solidification the containers and vials are kept in
insect proof area for subsequent use the next day.

In the rearing trays newly hatched larvae are transferred and confined
properly with the help of orgami tissues reinforcement beneath the lid. The
trays are kept inverted to facilitate the positively photropic and negatively
geotaxic larvae to feed unhindered on the diet. When the larvae reach second
instar premoult stage they are transferred to glass vials (5ml) and plugged
with cotton.

iv. Pupal handling

The prepupae of uniform size are collected and transferred to diet
trays filled up to 1/3rd level with cotton wool for pupation. Pupae are
removed from the culture and washed in sodium hypochlorite 0.25%
solution and kept in adult emergence cage. The production cycle is repeated.

The culturing is a labour intensive process and involves handling of

live stages. Commonly we encounter development of fungus, bacteria in the
diet used for mass and culture. Vairimorpha and NPV epizootic also occur.
These can be overcome by proper sanitation, restriction of personnel inflow
into the rearing area.

Guidelines for production flow maintenance

The following important guidelines should be observed during the
production process (Sathiah and Jayaraj, 1996).

Continuously examine each step in production for ways to reduce the

time and labour input.
Maintain good hygiene and cleanliness and monitor microbial
contamination.
Keep biological records of the insects.
Check quality of the insect frequently.

Preparation of Schedules
 It is often important to draw schedules for daily routine work.
Without unifying a set of steps together in order, the return for current
investment made cannot be harnessed. The following sequence of work
has to be attended in order to maximise the productivity:

1. Prepare the facility for use.

2. Sanitize the rearing areas.
3. Prepare adult feed.
4. Prepare semi-synthetic diet and cool.
5. Release the freshly hatched larvae into the trays.
6. Sanitize the implantation area.
7. Move the rearing trays to holding rooms.
8. Harvest, surface sterilize and incubate eggs.
9. Provide adult feed.
10. Collect adults, sex, pair and allow for oviposition,
11. Harvest pupae, surface sterilize and keep in adult emergence
cages.
12. Sanitize the adult holding room.
13. Sort out trays for host culture and subsequent recycling and
for virus production.
14. Prepare materials for wash up operation.
15. Surface sterilize the materials appropriately.
16. Record production, quality etc.
17. Close up operations

EFERENCES
SATHIAH, N. and S. JAYARAJ. (1996). Technology for Mass Production
of Helicoverpa armigera and its NPV : A Constraint Analysis. Paper
Presented at the National Seminar on "Advancement in the Production
and Use of Biocontrol Agents held on 11 October 1996, Entomology
Research Institute, Loyola College, Madras
SHOREY H. H and R. L. HALE.(1965). Mass reraring of the larvae noctuid
species on simple artificial medium. J. Econ. Entomol., 58: 522-524.
 MASS PRODUCTION AND STANDARDIZATION OF NUCLEAR
POLYHEDROSIS VIRUSES AND GRANULOSIS VIRUSES

The use of viral pathogens of insects in most agricultural crops is

inundative and does not utilize their full epizootic potential, but takes
advantage of their virulence and specificity (Payne, 1982). Their efficacy,
specificity and production of secondary inoculum make baculoviruses
attractive alternatives to broad spectrum insecticides and ideal components
of IPM systems. The most successful commercial baculovirus in terms of
total area covered is the NPV of the velvet bean caterpillar, Anticarsia
gemmatalis, a major pest of soybean in Brazil (approximately 1 million ha of
soybean is treated annually)(Wood and Granados, 1991; Moscardi and Sosa-
Gomez, 2000 ).

In India, Helicoverpa armigera NPV, Spodoptera litura NPV and

Chilo infuscatellus GV are being used in management of these pests. The
use of baculoviruses for insect control within the IPM context is expected to
increase in the coming years, particularly in developing countries where the
cost of imported insecticides is high and that of labour is lower, in vivo
production could provide both a viable means of producing large quantities
of virus and a source of employment (Lacey et al., 2001).

Methods of mass production of NPVs of Helicoverpa armigera,

Spodoptera litura and Amsacta albistriga and GV of Chilo infuscatellus are
explained in following paragraphs. The enumeration and standardization
procedures of NPV and GV are also explained.

I. Mass production of baculoviruses

a) Helicoverpa armigera NPV (HaNPV)

Baculoviruses are obligate pathogens having a high degree of host

specificity and hence can be multiplied only in their respective host insects.
The viruses can also be multiplied in vitro on insect cell lines but the media
used for cell production are costly and the production systems are out of our
reach (Evans and Shapiro, 1997; Kennedy and Sathiah, 2001). Research on
large-scale use of cell lines that could reduce cost of production is warranted
(Weiss and Vaughn, 1986; Weiss et al., 1994; Granados and McKenna,
1995). Hence HaNPV is mass multiplied in vivo on larva of H. armigera.
The following are the steps involved in in vivo mass production of HaNPV.

Host insect multiplication

1. First the host insect H. armigera has to be multiplied in the laboratory on

artificial diet following standard procedures available for this purpose
(Sathiah et al., 2001; Rabindra, 1973) and a disease-free laboratory
culture of the host insect must be established.
2. Ninety percent of this laboratory-reared insect can be used for mass
production of virus while 10% of insects may be retained for continuing
the disease-free host insect production.
3. Standard semi-synthetic diet (minus Formalin) may be half filled in 5 ml
vials.

Virus inoculation and harvest

1. Nucleus HaNPV viral preparation may be obtained from standard

laboratories and diluted to required concentration.
2. Viral suspension having 5 x 107 POB/ml (Sathiah, 2001) may be
prepared.
3. Ten µl of the viral suspension may be dropped on the surface of the diet
in 5 ml vials and spread with the blunt end of a glass rod.
4. Early fifth instar larvae (8-9 days old larvae -head capsule measurement
1.6 mm) may be transferred using a fine forceps to the virus-
contaminated diet and plugged tightly with cotton. The stage of larvae is
highly crucial for maximizing virus production, as the larvae of lesser age
will yield lower POB/larvae while very late stage larvae when released
may escape from disease
5. The larvae are allowed to feed on the contaminated diet by incubating the
vials at 25°C.
6. Infected larvae when moribund or dead are collected from 5th day after
inoculation and frozen.

Extraction and purification of virus

1. For extraction, the larvae are macerated in ten times volume of distilled
water
2. The homogenate is then filtered through double layer muslin cloth.
3. The filtrate is centrifuged at 500 rpm for half a minute to remove larger
contaminants (Discard the pellet)
4. The supernatant is spun at 5000 rpm for 15 minutes to pellet the virus
5. The supernatant is discarded and the pellet containing virus particles is
re-suspended in small volume of water and stored at refrigerated
condition (4o C).
6. One virus killed larva can yield 2 x 109 POB
7. Hence approximately 750 infected caterpillars are required to get 1.5 x
1012 POB which is the dose needed to cover one hectare
8. Enumeration of the POB can be done following procedure that will be
explained in later part of this paper

Caution

1. Larvae dying within 3 days should not be collected as these could be

dying due to bacterial infection and may not contain POB at all.
2. Mother moth examination should be done periodically as in the case of
silkworm to prevent infection by microsporidian (Vairimorpha)
3. Insects infected with this microsporidian should not be used for mass
culture
b) Spodoptera litura NPV

The procedure explained for the mass multiplication of HaNPV may

as well be used for multiplying SlNPV also.
1. The host insect (Spodoptera litura) may be mass multiplied either on
natural host (castor leaves) or on artificial diet.
2. The virus may be propagated by contaminating the artificial diet in 5 ml
vials and allowing early fifth instar larvae to feed individually on
contaminated diet
3. Alternatively, the virus can be propagated by providing castor (Ricinus
communis) leaves dipped in viral suspension.
4. In the latter method, the leaves are dipped in viral suspension (5 x 107
POB/ml) and shade-dried.
5. The starved early fifth instar S. litura larvae (7 - 9 days old larvae with
head capsule measurement of around 1.4mm) are provided with the
contaminated leaves.
6. On the second day also virus treated leaves are provided
7. Subsequently untreated leaves may be provided.
8. The infected larvae showing characteristic symptom after 4-5 days may
be collected and frozen.
9. Extraction and purification procedures are same as in case of HaNPV.
c) Amsacta albistriga NPV

Mass culturing of the host insect, Red hairy caterpillar of groundnut,

Amsacta albistriga has been difficult due to diapause in pupal stage of this
insect. Hence field collection of 10 day old larvae is recommended. The
following steps may be followed in propagation of the virus.

1. Ten day old larvae (4th instar) measuring 2.5 - 3 cm may be field
collected and starved overnight
2. Virus suspension of Amsacta albistriga may be prepared at 1 x 106 POB/
ml concentration.
3. Calotropis leaves are collected from field and the wax coating is
removed with cotton wool and the leaves are dipped in viral suspension
and shade-dried.
4. The starved larvae are provided with the contaminated Calotropis leaves.
5. On the second day also virus treated leaves are provided
6. Subsequently untreated leaves may be provided.
7. Larvae start dying after day 5 of inoculation. Infected larvae show
pinkish white colouration on the ventral surface of the body due to
accumulation of polyhedra. After death the skin ruptures and the liquified
body content oozes out.
8. Collect the dead (virosed) caterpillars and freeze.
9. Follow the steps mentioned under HaNPV for extraction of virus.

d) Chilo infuscatellus Granulosis virus (GV)

i) Propagation of virus

1. Third or 4th instar field collected larvae are suitable for multiplication of
the virus.
2. A virus suspension containing 107 - 108 Inclusion bodies (IB)/ ml of
water is prepared
3. The larvae are fed with a drop of virus suspension through a pin head or
the head of larvae dipped in virus suspension.
4. The virus fed larvae are reared on sugarcane shoot bits at the rate of 3-5
per plastic container (7.7 x 6.4 cm dia). The plastic containers are
provided with filter paper for absorption of excess moisture and three
pieces of sugarcane shoot bits split open at one end. The shoot bits and
filter paper are changed on alternate days.
5. The infected larvae begin to show symptoms in 5-8 days.
6. The larvae start dying from 8 days upto 20 days and stored in refrigerator
at minus 20°C.

ii) Extraction and Purification of virus

1. Larvae are macerated in distilled water and filtered through double

layered muslin cloth to remove debris.
2. The filtrate is spun at 500 rpm for 3 min and the sediment containing
larger debris are removed.
3. The supernatant is centrifuged at 10,000 rpm for 30 min to sediment the
virus.
4. The supernatant is discarded and pellet re-suspended in small volume of
water which gives a fairly pure virus preparation.
5. The virus may be stored in a refrigerator at 4-5°C in distilled water in a
amber coloured bottle. The virus can be stored for three or more years
without apparent loss in infectivity.

II. Enumeration and Standardization of NPV and GV

Use of appropriate dose of the virus is highly essential for successful

control of pests. Hence is necessary to estimate the concentration of
polyhedral occlusion bodies in the virus suspension before it is applied on
the crop. Most of the crop pests are successfully managed with a dose of 1.5
to 3 x 1012 POB/ ha. It has been found that a full grown larva of H. armigera
or S.litura yields on an average 2 x 109 POB (Rabindra, 2001).

The concentration of the polyhedra can be estimated with the help of a

improved Neubauer haemocytometer under light microscopy.
Haemocytometer is a thick glass slide having fine ruled grid of squares of
defined dimensions, which is visible under a compound microscope (Fig 1.).
In the haemocytometer 1x1mm area is divided in 25 equal large squares
each measuring 0.2 x 0.2 mm (area 0.04 mm2). Each of this larger square is
divided into 16 smaller squares each measuring 0.05 x 0.05 mm (area 0.0025
mm2). There will be totally 400 small squares within 1 mm2 area.When a
thick cover slip is placed over the grid lines in a depression, a fixed depth
(usually 0.1 mm) is created, and the depression holds a known volume
(multiplying area by depth) of liquid. The following steps may be adopted
during enumeration of POB using a haemocytometer.
 1 mm 0.2 mm

0.05mm

0.05mm

Fig. 1. View of grid lines of a haemocytometer under compound microscope

1. Dilution of the virus suspension with known quantity of water will be

required before counting the POBs. Serial dilution technique may be
followed (1µl made upto 10µl, then 1µl of this suspension again made
upto 10µl and so on). A digital micropipette may be used for this
purpose. Dilution factor will be calculated as follows
1µl of viral suspension made upto 10 µl - Dilution factor 10
1µl of the above dilution made upto 10 µl- Dilution factor 100
1µl of the above dilution made upto 10 µl- Dilution factor 1000
5 µl of the above dilution made upto 10 µl - Dilution factor 2000
and so on

2. The haemocytometer and the cover slip are wiped with ethanol using a
clean tissue paper.
3. Haemocytometer is placed on a clean flat surface and then the cover slip
is placed exactly over the depression provided in the counting chamber.
4. The cover slip is press down firmly (not too hard as the cover slip will
break).
5. Ten µl of diluted virus suspension is pipetted out into the edge of the
cover slip so that the liquid is taken up and the chamber is filled
completely. Over flooding of the chamber may be avoided.
6. The slide may be allowed to stand for 20 min to reduce the amount of
Brownian movement of the POBs. Counting may be carried out
 preferably in a air-conditioned room or in saturated atmosphere as
increase in room temperature may cause evaporation of liquid from
haemocytometer, thus giving erroneous results.
7. Then it is examined under a compound microscope preferably, a phase
contrast microscope. The number of polyhedra in each of the small
squares (0.0025 mm2 are) from at least five large squares (total 80 small
squares) is counted.
8. The polyhedra within each smaller square and those touching the top and
left sides are counted. Those touching bottom and right sides are not
counted.
9. If there are more than 5 polyhedra within a small square, counting may
be difficult in which case the virus suspension may be diluted 10 fold and
enumeration done.
10. Since the dimensions of the grid and its depth are known the
concentration of the virus suspension in terms of number of POB/ml is
calculated as follows.

Calculation
Number of polyhedra per ml = D× X
Where
D= dilution factor
N×K
X =total number of polyhedra counted
N=number of small squares counted
K=volume above one small square in cm3

K = area of one small square in mm2 x depth of the chamber in mm x

1/1000
= 0.0025 mm2 x 0.1mm x 1/1000 (for converting 1mm3 to 1cm3)
= 0.00025 mm3 x 1/1000
= 0.00000025
HenceK = 2.5 x 10-7 cm3
Worked example
The viral preparation is diluted 1000 fold and we observe 345 POB in 80
small squares, to calculate the number of POB per ml of undiluted sample
In this case D = 1000, X = 345, N= 80 and K = 2.5 x 10-7

D× X 1000 × 345 3.45 × 105

= = = 1.72 × 1010
N×K 80 × 2.5 × 10 −7 2 × 10 −5

So there are 1.72 x 1010 Polyhedra per ml of undiluted sample

 Enumeration of inclusion bodies (IB) of GV can be done by using a
counting chamber having a depth of only 0.02mm instead of 0.1mm. The
inclusion bodies of GV can be observed and counted under a dark field
microscope.

REFERENCES:

EVANS, H and SHAPIRO, M. (1997). Viruses. In: Manual of Techniques in

Insect Pathology (Lacey eds) Academic press Limited, pp 17 -53.
GRANADOS, R. R., and McKENNA, K. A. (1995). Insect cell culture
methods and their use in virus research. In “Baculovirus Expression
Systems and Biopesticides” (M. L. Shuler, H. A. Wood, R. R.
Granados, and D. A. Hammer, Eds.), pp. 13–40. Wiley–Liss, New
York.
KENNEDY, J.S. and SATHIAH, N. (2001).Technology for the mass
production of Helicoverpa armigera nuclear polyhedrosis virus
(HaNPV). In: Microbial control of crop pests. Eds. R.J.Rabindra., J.S.
Kennedy, N.Sathiah, B.Rajasekaran, and M.R.Srinivasan. Graphic
skill publishers. pp 144 - 147.
LACEY,L.A., FRUTOS, R.. KAYA, H.K. and VAIL, P. (2001). Insect
Pathogens as biological control agents: do they have a future?
Biological Control 21: 230 - 248
PAYNE, C. C. (1982). Insect viruses as control agents. Parasitology 84, 35–
77.
RABINDRA, R.J. (1973). Studies on the polyhedrosis of three species of
Lepidoptera. M.Sc (Ag.) Thesis submitted to Tamil Nadu Agric.
Univ., Coimbatore 123 p
RABINDRA, R.J. (2001).Emerging trends in microbial control of crop prsts
In: Microbial control of crop pests. Eds. R.J.Rabindra., J.S. Kennedy,
N.Sathiah, B.Rajasekaran, and M.R.Srinivasan. Graphic skill
publishers. pp 1 - 17.
SATHIAH, N., KENNEDY, J.S and RABINDRA, R.J (2001).Mass
production of the gram pod borer Helicoverpa armigera (Hubner). In:
Microbial control of crop pests. Eds. R.J.Rabindra., J.S. Kennedy,
N.Sathiah, B.Rajasekaran, and M.R.Srinivasan. Graphic skill
publishers. pp 134 - 143.
WEISS, S. A., and VAUGHN, J. L. (1986). Cell culture methods for large-
scale propagation of Baculoviruses. In “The Biology of
Baculoviruses.” Vol. II “Practical Application for Insect Control” (R.
R. Granados and B. A. Federici, Eds.), pp. 63–87. CRC Press, Boca
Raton, FL.
WEISS, S. A., DUNLOP, B. F., GEORGIS, R., THOMAS, D. W., Vail, P.
V., HOFFMANN, D. F., and MANNING, J. S. (1994). Production of
baculo-viruses on industrial scale. Proc. VIth Int. Colloq. Invertebr.
Pathol., pp. 440–446.
WOOD, H.A. and GRANADOS, R.R. (1991). Genetically engineered
baculoviruses as agents for pest control. Ann.Rev.Microbiol., 45: 69-
87
 MASS PRODUCTION OF GRANULOSIS VIRUSES

The intensified agricultural production systems have greatly solved the food crisis
due to the ever-increasing human population, but has also initiated the vicious cycle of
chemical pesticides. The harmful effects of chemical pesticides on man and his
environment have intensified the search for ecofriendly alternatives to chemical
pesticides. Insect viruses have emerged as one of the alternatives to chemical pesticides.
Insect viruses predominantly consist of two groups belonging to family Baculoviridae,
namely Nuclear Polyhedrosis Virus (NPV) and Granulosis virus (GV).

Natural occurrence of granulosis viruses

Next to NPV, GV are important insect viruses. Several granulosis viruses have
been reported from insect pests like Agrotis ipsilon, A. segetum, A. bicornica, Cydia
pomonella, Achaea janata, Cnaphalocrosis medinalis, Phthorimaea operculella,
Spodoptera litura, S. litorallis, Galleria melonella, Plutella xylostella Chilo infuscatellus,
C. sacchariphagus indicus, Corcyra cephalonica, Helicoverpa armigera etc. (Singh,
1999). However only few have been used successfully in the field.

Basic characteristics of granulosis virus

The GV mainly multiplies in the cell nucleus and the cytoplasm of the fat body,
and to a very little extent in the tracheal matrix and the epidermis. The general symptoms
of the granulosis virus infection are appearance of milky white colour, especially on the
ventral surface and the integument often become mottled, loss of appetite, sluggishness
and cessation of feeding. (Huger, 1963). Alteration in the sex ratio, pupal and adult
deformation and reduced fecundity are other symptoms observed with granulosis virus
infection (Easwaramoorthy and Jayaraj, 1989; Melamed and Raccah, 1989).
Based on the period between the infection and the logarithmic OB increase, which
decide the speed of kill, GV can be classified into two classes. They are viruses with
relatively short period similar to that of NPV (e.g. Pieris brassicae GV, Cydia pomonella
GV, P. xylostella GV, C. infuscatellus GV etc.) and those where the period between the
infection and the logarithmic OB increase is abnormally long (eg. Agrotis segetum GV, S.
litura GV, S. littoralis GV, H. armigera GV etc.) (Hunter et al., 1998). Mostly members
belonging to the former class have been used successfully at field level. Some of the
basic characteristics in the production point of view are, long incubation period for most
of the GV and intact cadavers unlike with NPV where the larvae on death becomes
liquefied.

Field efficacy of granulosis virus

Due to the slow action of the granulosis viruses, only very few of them have been
successfully used at field level. Some of the granulosis viruses, which have been used
successfully are, C. pomonella GV in many western countries for the control of codling
moth on apples and pears, Erinnyis ello GV in Brazil against cassava horn worm, P.
xylostella GV against diamond backmoth on crucifers, Agrotis segetum GV and leaf
roller, Adoxophyes orana GV (Crook, 1994). In India, Sugarcane shoot borer, Chilo
infuscatellus GV is used on a large scale (Easwaramoorthy and Santhanalakshmi, 1988)
in Tamil Nadu. Apart from this field efficacy of P. xylostella GV (Sairabanu, 2000) and
Helicoverpa armigera GV (Kuppuswamy, 1994) have also been studied in India.
Laboratory studies with other granulosis viruses like Spodoptera litura GV (Narayanan,
1985), Achaea janata GV (Singaravelan, 1999) etc. have been carried out.

Mass production of granulosis viruses

Granulosis viruses are widely mass multiplied in their homologous hosts, with
exceptions like the C. pomonella GV which has been multiplied in Cryptophlebia
leucotreta, an alternate host (Reiser et al., 1983). In vitro mass production has not been
reported widely because of the high cost involved in this method and the problem of scale
up of the production process.

Chilo infuscatellus GV (CiGV)

The shoot borer GV is mass-produced only in field collected larvae, as artificial
and semi-synthetic diet has not been developed for mass-culture of this insect. larvae are
collected from the field in 30 – 90 days old sugarcane crop and are reared on shoot bits
till they reach the optimum stage. Third and fourth instar larvae are suitable for
multiplication of the virus. The larvae are fed with virus suspension (107 – 108 IB/ml) by
pinhead feeding method or by larval head dip method. The treated larvae are
continuously reared on sugarcane shoot bits at the rate of 3 – 4 per plastic container (7.7 x
6.4cm dia). The plastic containers are lined with filter paper to absorb excess moisture.
The filter paper and the shoot bits are changed on alternate days. The infected larvae are
collected from 8 – 20 days as and when they die and stored in refrigerator at -20°C.

P. xylostella GV (PxGV)
The granulosis virus of P. xylostella is mass multiplied on laboratory reared
larvae, which are raised on cauliflower leaves. Early third instar larvae fed with a virus
dosage of 350. 0 OB/mm2 has been found to be optimum for the mass production of
PxGV. Larvae of the optimum size are allowed to feed on leaf discs (3.3cm dia) treated
with the GV suspension of 24µl (12µl on either side of the lamina) containing 1x108
OB/ml. The inoculated larvae are transferred to untreated leaves after 24h and they are
incubated at 25°C. Virosed larvae are collected from 5th day after treatment and stored at
-20°C.

S. litura GV (SlGV)
Mass production of granulosis virus of S. litura is carried out on laboratory reared
larvae maintained on semi-synthetic diet. The optimum stage of larvae and dose of the
inoculum are late fourth instar and 1.25 x105 OB/mm2 respectively. Treatment of the
larvae is carried out by diet surface contamination method. Semi-synthetic diet taken in 5
ml glassvial is contaminated with the GV by dispensing 10µl of virus suspension
containing (3.0 x109 OB/ml) and spread uniformly over the entire surface. Late fourth
instar larvae are allowed to feed on the contaminated diet. Treated larvae are incubated at
25°C and cadavers are collected from the 12th day onwards and stored at -20°C.

H. armigera GV (HaGV)
Mass production of H. armigera GV is similar to the S. litura GV. The optimum
stage of larvae and the dose of inoculum are fourth instar and 1 x 107 OB/ larvae.
Treatment of the larva is carried out by diet surface contamination method. Virosed
larvae are collected from 10 – 12 days after treatment and stored at -20°C.
Semi-purification of granulosis virus
The infected larvae are macerated in distilled water and the suspension is filtered
through a double layered muslin cloth to remove the tissue debris if any. The resultant
suspension is subjected to centrifugation at 750 – 1000 rpm for 3 minutes and the
supernatant is collected. Centrifuge the supernatant at 10,000 rpm for 30 minutes to
sediment the virus. The supernatant is discarded and the pellet is resuspended in a known
volume of water and virus concentration in the suspension is enumerated.

Practical Exercise
Mass production of granulosis virus of H. armigera and S. litura
Objective
To mass-produce the granulosis virus of H. armigera and S. litura.
Materials required
1) fourth instar larvae of H. armigera and S. litura
2) granulosis virus of H. armigera (1 x 109 OB/ml), granulosis virus of S. litura
(3.00 x 109 OB/ml)
3) Five ml glass vials with semi-synthetic diet lacking formalin.
4) Finnipipettes to dispense the viral suspension
5) Cotton rolls to plug the vials
6) Blunt ended glass rod to spread the virus over the diet surface
Procedures
Ten microlitres of the above virus concentration is taken using a finnipipette and
dispensed into the 5 ml glass vials with semi-synthetic diet and spread uniformly with a
blunt ended glass rod. Fourth instar larvae of S. litura and H. armigera is allowed to feed
on the diet contaminated with their homologous virus and the vials are secured with
cotton plugs. The treated larvae are incubated at 25°C and are provided with fresh diet as
and when it is exhausted. The virosed larvae are collected as and when they die. The
larvae begin to die from 10th day onwards. The virus yield per larvae is enumerated using
a haemocytometer (Weber Scientific Ltd., England) with 0.02mm depth of counting
chamber.
References
Crook, N.E.1994. Baculoviruses: Granulosis virus. In: Encyclopidia of virology. Vol.I
(Webster, R.G. and Granoff, A. eds.). pp. 127 – 130. Academic Press. New
York.

Easwaramoorthy, S and Jayaraj, S.1989. Vertical transmission of granulosis virus of

sugarcane shoot borer, Chilo infuscatellus Snell. Trop. Pest Manage.,35: 352 –
353.

Huger, A. 1963. Granulosis virus of insects. In: Insect Pathology (Steinhaus, E.A.
ed.) Vol.I. pp. 531 – 575. Academic Press, New York.

Hunter, F. R., Entwistle, P.F., Evans, H.F. and Crook, N.E. 1998. Insect Viruses and Pest
Management. John wiley and sons, New York. 599p

Kuppuswamy, S.1994. Investigations on the granulosis virus disease of Heliothis

armigera (Hubner) and its use in microbial control. Unpub. M.Sc (Ag.). thesis
Tamil Nadu Agricultural University, Coimbatore.

Melamed, M. V. and Raccah, B. 1989. The trans-stadial and vertical transmission of a

granulosis virus from the corn borer, Sesamia nonagroides. J. Invertebr.
Pathol., 33: 259 –264.

Narayanan, K. 1985. Susceptibility of Spodoptera litura (F.) to a granulosis virus. Curr.

Sci. 54: 1288 – 1289.

Reiser, M., Groner, A. and Sunder, A. 1983. Cryptophlebia leucotreta a promising

alternate host for mass production of the Cydia pomonella granulosis virus
(CpGV) for biological pest control. Zeitschrift for Pflanzenkrankheiten
Undpflanzenschutz, 100: 586 – 598.

Sairabanu, B.2000. Microbial control of Plutella xylostella (Linn.) (Lepidoptera:

Plutellidae). Unpub.Ph.D thesis. Tamil Nadu Agricultural University,
Coimbatore.

Singaravelan, R. and Ramakrishnan, G.1998. Characterization of granulosis virus from

castor semilooper, Achaea janata L.J. Invertebr. Pathol.,71: 227 – 235.

Singh, S. P.1999. Microbial Control in India-Problems and Perspectives. In: Emerging

trends in Microbial control of crop pests. (Rabindra, R. J., Santharam, G.,
Sathiah, N.S. and Kennedy, J.S. eds.) Proc. of the summer school held on
emerging trends in microbial control of crop pests. Tamil Nadu agricultural
university, Coimbatore. 1999. pp.36 - 78.
 MASS PRODUCTION OF SOME ENTOMOPHAGOUS INSECTS

The green lacewing, Chrysoperla carnea, Stephens is a successful

predator on many species of aphids, caterpillar eggs and several soft bodied sap
feeders, mites and eggs of lepidopterans. For successful mass production a
continuous supply of the eggs of rice moth Corcyra cephalonica is required. The
multiplication of Corcyra is as follows:

Culturing of Corcyra cephalonica

The rice moth C. cephalonica is cultured in broken grains of pearl millet or

sorghum or maize in plastic round basins. Heat sterilized broken grain is taken
at 2.5 kg per plastic basin (30 cm dia) to which groundnut kernel powder is added
at 100 g and yeast powder added at 5 g (16 tablets). To prevent bacterial
infection in the food medium streptomycin sulphate 0.05% spray is given at 10-20
ml/basin using a hand sprayer or atomizer. Sulphur W.P. is added at 5 gm/basin
to prevent storage mite. Then nucleus Corcyra eggs are added at 0.5 l/basin
containing 2.5 kg of grain medium (Wajnberg and Hassar, 1994).

After uniform mixing of the contents of the basin, it is covered with kada
cloth (0.25 mt) and secured by rubber band. The young Corcyra larvae that
hatch out from the eggs in 3-4 days fed on the medium, constructing webbing.
The adult Corcyra moths start emerging from the medium from 30-35 days
onwards and continued to emerge upto 90 days after inoculation of eggs due to
staggered development of larvae in the medium.

The adult moths rest on the inner surface of the cloth cover. They are collected in
morning hours using glass specimen tubes (14 x 2.5 cm) or a specially designed modified
vacuum aspirator (TNAU model). The moth collection is effectively done by keeping the
basins inside a mosquito net house so that escape of moths is prevented. The adult moths
are then transferred into a specially designed mating drum made of G.I. sheets with wire
mesh bottom. Adult moths are provided with honey solution (50%) added with Vitamin
E as feed (one capsule / 20 ml of 50% honey) to boost the vigour of the adults and to get
higher quantity of healthy eggs. The adult food is given by dipping cotton swab and
allowing them to hang inside the drum with a thread.

Daily, fresh moths are collected and allowed into a fresh mating drum
which is cleaned and dried under sun earlier. Corcyra eggs are loosely laid by
moths and they are collected through the wire mesh bottom on a receiving
container with funnel set up or enamel tray. Eggs were collected daily,
th
continuously for 4 days from each drum. On 5 day it is vacated and cleaned. A
sheet of blotting paper is spread of the tray or in the funnel set up. It retained
most of the moth scales and body fragments while the eggs easily rolled out
during cleaning.

The eggs thus obtained are cleaned with the help of plastic sieves of
different meshes. One cc (or) ml of Corccyra eggs approximately contains 18000
eggs. About 100 pairs of Corcyra moths (50% female) produce 1.5 cc of eggs
during its egg laying period 4 days. From each Corcyra rearing basin an average
of 2500 moths emerge. Hence from each basic 18-20 cc of eggs are obtained
during the period of 90 days. After 90 days the contents of the basin are
discarded and the basic is washed, disinfected with 2% formalin solution and
dried thoroughly before reusing.

Materials required for Corcyra rearing (King and Leppla, 1984)

• Broken bajra (Pearl millet) grain

• Mosquito net (6’x6’x’6’)
• Plastic basins (30 cm dia)
• Specimen tubes (glass 15 x 2.5 cm)
• Kada cloth, Aspirator (TNAU model), Yeast tablets
• Rubber bands and twines
• Moth scale separator (TNAU model)
• Groundnut kernel, sieves and filters (plastic)
• Sulphur W.P. Streptomycin sulphate
• Home milling machine, vacuum pump
• Exhaust fan / Ceiling fan
• Measuring cylinder 10 ml, 50 ml, 100 ml
• G.I. mating drums (25 x 25 cm)
• Formaldehyde 40%, enamel tray
• Hand sprayer or hand atomizer

Blotting paper sheets, Honey

• Camel hair brush, Vitamin E capsules

• Shoe brush, absorbing cotton

Management of Tribolium in Corcyra laboratory

In any laboratory culturing of Corcyra cephalonica, the red flour beetle,

Tribolium castaneum is causing serious problems by competing for food resulting
in reduction of Corcyra moth production drastically. Keep flour traps viz., trays
containing 250 gm of wheat flour/trap added with 0.5% brewers yeast. The trays
are kept open in different spots in between the Corcyra trays. The beetles
attracted to the traps are sieved and discarded on alternate days. The grubs (or)
pupae developed if any in the flour are also removed.

Chrysoperla mass culture

Larval rearing

It is done in GI round basins (28 cm dia) at 250 larvae/basin covered with

kada cloth. The eggs of Corcyra cephalonica are given as feeding material for
the larvae in the laboratory. For rearing 500 Chrysoperla larvae the total quantity
of Corcyra eggs required is 25.0 c.c. at the rate of 5.0 c.c./feeding for 5 feedings
in alternate days. The Chrysoperla larvae pupate into round white coloured
silken cocoons in 10 days. The cocoons are collected with fine brush and
transferred into 1 lit plastic containers with wire mesh window for emergence of
adults. From the coccons, pale green coloured adults with transparent lace like
wings emerge in 9-10 days (Singh, 1995).
Adult raring

The adults are collected daily and transferred to pneumatic glass troughs
or G.I. round troughs (30 cm x 12 cm). Before allowing the adults, the rearing
troughs are wrapped inside with brown sheet which act as egg receiving card.
About 250 adults (60% females) are allowed into each trough and covered with
white nylon or georgette cloth secured by rubber band. On the cloth outside
three bits of foam sponge (2 sq.in) dipped in water are kept. Besides an artificial
protein rich diet is provided in semisolid paste form in three spots on the cloth
outside. This diet consisted of one part of yeast powder, one part of fructose,
one part of honey and one part of Proteinex. Water is mixed to make it as paste.
The adults fed the food and lay eggs on the brown sheet. The adults are
collected daily and allowed into fresh rearing troughs with fresh food. From the
old troughs, the brown paper sheets along with Chrysoperla eggs is removed.

Storage and destalking of eggs

The brown paper sheet kept inside the adult rearing troughs contain large
number of eggs each laid on a stalk or pedicel. As such the sheet is stored at
100C in B.O.D. incubator or refrigerator for about 21 days. When the eggs are
required for culturing or for field release the egg sheet is kept at room
temperature for a day and the eggs turned into brown and hatched on 3rd day.
The first instar larvae are either taken for culture tray for recycling or for field
release.

Field release of Chrysoperla

The first instar larvae of Chrysoperla are released in horticultural crops at

50,000 to 1,00,000/ha for 3-5 times at 10 days interval to control aphids, whitefly,
Spodoptera, Helicoverpa, pink bollworm, thrips and mites. The larvae are taken
in plastic containers containing a small quantity (1-2 c.c) of Corcyra eggs and
loose paper strips. The paper strips along with larvae sticking on them are
dropped in the field at random while walking across the fields (Ridgeway and
Vinson, 1977).

Storage of Chrysoperla eggs

Storage is done by keeping destalked eggs after sodium hypochloride egg

wash (at 0.1% for 1 minute) at 100C in B.O.D. incubator adjusted for constant
temperature. Daily a sample of 10 destalked eggs can be taken and kept outside
and the per cent hatchability can be checked. This method is best for storage.

Cryptolaemus montrouzieri Mulsant (Coleoptera:Coccinellidae) rearing

The predator, Cryptolaemus montrouzieri is native of Australia. In 1892 it

was introduced into California for the control of citrus mealybugs. Following the
success the beetle it was introduced into India in 1898 by New Port. It has given
effective control of mealybugs in fruit crop like citrus, grapes, guava etc., To
release the predator in large number in the field, mass culture of Cryptolaemus is
a pre-requisite. Cryptolaemus is easily cultured on a large scale on the mealy
bugs in the laboratory.

Production procedures
Mealy bug production
In the large scale production of mealy bugs, potato sprouts or ripe
pumpkins have been utilised. The mealybug Planococcus citri (Risso) is better
adapted to the insectary programme than the other mealy bug species by reason
of its shorter life cycle, higher fecundity, and suitability as host for
Chryptolaemus.

Potato sprouts

The use of potato sprouts as an insectary host for mealybug culture has undergone
several modifications since reported first. Planting trays made of red wood (45x15x10
cm) are used for this purpose. Approximately tiny tubers of potato, three months after
harvest or when sprouts begin to appear, are ready for planting. Whole potatoes are used
and 25 to 30 tubers are placed about ½” layer of sandy silt soil in the tray and covered
with slightly moist soil.
These trays are kept in racks in the production room and watered.
Temperature of 70 - 740F appears to be optimum for facilitating sprout growth.
The time from planting until infesting with the mealybugs is usually 21 days in
summer and 10 days in winter. Stock from one mealybug tray is sufficient to
infest 20-25 trays of sprouts.
Pumpkin

The culture of mealybugs on pumpkin is another common method. The pumpkins

are selected with ridges and grooves with a small stalk which makes handling very easy.
They are cleaned with water to get rid of any dust on them. To prevent rotting, pumpkins
are treated with 0.1% Benlate (1 g/lit.). The wounds, if any on the pumpkin, are plugged
with paraffin wax. Ovisacs of the mealybug are placed over the pumpkin for about 48
hrs. The infested pumpkins are kept on a plastic stand in wooden cages, with glass
sliding in the front and cloth on other sides. In due course, crawlers emerge from
ovisacs, settle on all sides of pumpkin and develop into fully mature mealybugs in 30-40
days. This technique of propagating mealybugs culture on pumpkin fruit has been
standardized.

Cryptolaemus production

When mealybugs are 8 days old, a stock of 15-20 females of

Cryptolaemus is placed in each tray in the dark room at 74-76 F. Their eggs are
deposited liberally on the sprouts and wooden trays. Twelve days later, the
ovipositing adults are removed from the tray. Burlap strips are attached to the
fronts of racks to accommodate pupating Cryptolaemus and the room is against
darkened. 18 days after removal of sting stock, the emerging adults are collected
into plastic vials by means of a broad flat scoop funnel which is attached to the
open top of the vial. Each plastic vial contains 100 beetles.

In about 20-25 days after the infestation with the mealybug, Cryptolaemus
adults are released into the cage through its sleeve. The adult beetles, besides
feeding on the mealybugs, lay their eggs singly or in groups of 4-12 in the
ovisacs of female mealybugs. If the females are not fully developed, eggs are
laid anywhere near the mealybugs. The grubs are visible in about a week’s time.
Initially, they feed on the eggs of mealybugs or smaller nymphs. As they grow,
they start feeding on all stages of the mealybugs. Cannibalism is observed when
the mealybug population is low. The older grubs feed upon young grubs. The
fully developed grubs pupate on the pumpkin or anywhere inside the breeding
cage.

The first beetle emerges in 30 days time from the date of exposure of the
mealybugs to the beetles. The beetles continue to emerge for another 5-10 days. The
beetles are collected in glass vials using an aspirator. Each breeding cage yields 100-200
beetles. They are fed with honey solution (50%) or honey agar in the laboratory. In
about 10-15 days, when the adult beetles complete the mating and preoviposition, they
are ready for field release.

The production cost of 100 beetles has been estimated to be about Rs.150/-

The beetles are also fed on diet containing agar powder ( 1 gm), sugar (20
gm), honey (40 cc) and water (100 cc). The adult beetle diet is prepared by
boiling sugar in 70 cc of water, adding 1 gm agar, diluting 40 cc honey in 30 cc of
water and adding to the sugar and agar mixture when it comes to boiling point.
The hot liquid diet is kept on small while plastic cards in the form of droplets
which get solidified on cooling. Such cards containing adult diet can be fed not
only to C. montrouzieri but also to many other species of coccinellids. From each
cage about 175 beetles are obtained. The emergence of the beetles is
completed within 10 days. The cost of production of 100 larvae at 1977 price
index was Rs.6/- and at 1992 Rs.60/-. The cost of production of 100 beetles at
1977 price index was Rs.10/- and at 1999 price index Rs.150/-

Beetles can also be reared on Corcyra cephalonica eggs but empty

ovisacs of P. citri are to be kept for inducing egg laying by the beetles. The
beetles are also multiplied on semisynthetic diet which is still in the process of
further refinement.

Precautions
All due precautions should be taken to avoid scarcity of food for the grubs to
avoid cannibalism by grubs.
 All the pumpkins showing sign of rotting should be properly incinerated.

References

Kind, E.G. and N.C. Leppla. 1984. Advances and challenges in insect rearing.
Agric. Res. Service, U.S.D.A., New Orieans, La., p.306.
Ridgway, R.L. and S.B. Vinson. 1977. Biological Control by Augmentation of
Natural Enemies. Plenum Press, New York. p.480.
Singh, S.P. 1995. Technology for Production of Natural Enemies. Project
Directorate of Biological Control, Bangalore, India. p.221.
Wajnberg, E. and S.A. Hassan. 1994. Biological Control with Egg Parasitoids. CAB
International, Wallingford, UK. p.265.
 Biotechnology – Over view
I. Plant Breeding (in Maryland - 100 genetic companies)
A. Traditional method of improving plants - selective breeding
1. Selection of desired characteristics: yield, palatability, resistance to
disease and insects, aesthetic characters
2. Hybridization - bringing together desired genes (by controlled mating)
from two or more individuals; results in a combination of desired characters
in the offspring - a hybrid
a. farmers would save seed from the best plants to grow next year
B. Green Revolution - The example of successful crop improvement (dwarf, high
yielding wheat varieties)
1. Norman Borlaug, awarded the Nobel Prize in 1970 for his role in the Green
Revolution
2. Borlaug is remains concerned about the future noting in particular the
international agribusiness control of genetic material
C. Limitations
1. Can only use genes from within one species or several closely related
species or wild species, becoming a limitation due to loss of genetic
diversity
2. Takes many years to develop an improved variety
II. Recombinant DNA [Important Illustration] and the new Genetics: Genetic
Engineering
A. Introduction
1. In the early 1970's a Moratorium on a certain type of research was called
by those doing the work
a. legislation was introduced before the US congress which would require
congressional approval for these experiments
b. involves a newly developed technology of gene manipulation [Important
Illustration]
c. experiments were deferred for 18 months so that an assessment could be
made regarding the potential danger of the research
2. Technique: ability to construct new combinations of DNA molecules, which
do not exist naturally= Recombinant DNA. Remember the Central Dogma of
Molecular Biology?
III. Combines two different technologies: Restriction Enzymes and Bacterial
Plasmids
A. Restriction Enzymes [Important Illustration]
1. The real basis for the recombinant DNA techniques [Important
illustration]: Sequence specific DNases: Recognize short sequences of bases
in DNA, and make a double stranded cut in the DNA molecule
a. function in bacteria- destroy foreign DNA which might enter the cell
1) each bacterium has its own restriction enzymes
b. each enzyme recognizes only one type of sequence
 1) sequence recognized is called a Palindrome
2) reads the same on the two strands in the opposite direction
3) example: G A A T T C
CTTAAG

some enzymes cut straight across, and others make

a staggered cut.

GAATTC
CTTAAG

G AATTC
CTTAA G

Creates fragments of DNA, all with the same

overlapping ends.
2. Value of restriction enzymes
a. each enzyme only recognizes the same palindrome regardless of the
source of DNA
b. so, fragments, from different sources, produced by the same enzyme,
contain the same overlapping ends
c. treat with ligase = permanently joined together = Recombinant DNA
molecule
B. Plasmids
1. Extrachromosomal genetic elements in bacteria
2. Closed circular DNA molecules
3. Replicate independent of the chromosome = many copies/cell
4. Contain genes controlling such things as fertility, and antibiotic
resistance
C. Molecular cloning
1. The usefulness of the technology combines plasmid biology with
restriction enzymes in the following way
a. join a DNA fragment from one source to a bacterial plasmid which has
been cut once with the same restriction enzyme
b. plasmid is now called a vector and carries a foreign piece of DNA
c. recombinant plasmid is reintroduced back into a bacterial cell
d. inside the bacterial cell, the plasmid will carry one or more
antibiotic resistance factors, so that when plated on a medium containing
the antibiotic, only the strain of interest will grow. Plasmid is
replicated and the number increased greatly = MOLECULAR CLONING
IV. Actual gene transfer to plants: - what is a transgenic plant
(see this-process of making transgenic plant - animation of
process)
A. Agrobacterium tumefaciens - a bacterium that causes crown gall disease in
many plants; unique among pathogens because it causes the disease by
transferring bacterial DNA from its plasmid into the plant's chromosome,
 causes tumors
B. Tissue culture - used to regenerate an entire plant from a single cell;
involves growing cells under sterile conditions with different nutrients and
hormones; in some plants you can grow entire new plant from one cell:
C. Plant genetic engineering using Agrobacterium tumefaciens: method to have
one plant species (tobacco - easy to grow, we know a lot about their breeding)
express a gene from a different species
1. Leaf disk method of plant transformation
a. punch out leaf disks
b. incubate with Agrobacterium culture carrying foreign gene
c. place disks on culture medium to induce regeneration and to select for
transgenic plants
d. root regenerated plants
e. every cell in the transgenic plant has the foreign gene
f. these plants pass genes on to their offspring
V. Applications of technology
A. Getting a gene from one species into another and having it expressed
1. Example - bacteria that makes human insulin, insulin extracted from cows,
but gene not human/ now human gene in bacteria to produce insulin
2. Example - tobacco plant that glows in the dark (luciferin gene from
fireflies), transfer of gene not just from one species to another but from
one kingdom to another
B. Examples of application of gene technology in agriculture
1. Herbicide resistant plants
a. in some areas, crops genetically modified for herbicide tolerance could
decrease the amount of herbicide used and allow for no-till agriculture,
which can minimize erosion
1) Roundup - a herbicide that is effective, non-toxic to animals,
short-lived in environment
2) Research groups developed crop plants resistant to Roundup, notably
soybean, corn, tomato, potato, wheat, cotton, alfalfa, etc.
2. Insect resistant plants
a. Bt toxin gene is cloned from bacteria and expressed in plants to
provide resistance from insect without the need for insecticides - data
about Bt toxin
b. currently in crops of corn, corn, cotton
c. current controversies
-effect of pollen from transgenic Bt corn on Monarch butterfly larvae
-one form of Bt corn (StarLink) approved for animal feed entered human
food products
3. Controlled ripening tomato
a. two varieties of tomato now on the market engineered for delayed
ripening
1) one tomato variety has an extra gene - a reverse copy of the gene
responsible for an enzyme that breaks down cell walls; as a result, the
tomato softens more slowly
 2) the other variety has a gene that controls the enzyme necessary for
the production of ethylene, one factor that makes a tomato soft
4. Enhanced resistance to viral diseases
a. Crops bioengineered for pest resistance could increase yield, eliminate
the use of several insecticides now derived from fossil fuel, and reduce
health risks and groundwater contamination from pesticides
1) used Agrobacterium to insert gene from viral protein into plant cells

2) when regenerated, the plant (tobacco) produced this protein and were
more resistant to the virus
3) currently employed in papaya and squash
5. Production of vitamin A in rice
a. "golden rice" -a transgenic rice in which the genes for the production
of vitamin A have been inserted
(news article)
6. Future ideas
VI. Advantages of gene technology
A. Foreign genes (from outside species) can be introduced
B. Potentially faster than traditional plant breeding
C. Specific genes can be transferred; much more control than in traditional
plant breeding
VII. Other current and possible benefits of gene technology
A. Already benefits in human health (human insulin).
B. Genetically engineered fish that grow much faster than wild (growth hormone
gene).
C. Recombinant bovine growth hormone already enables cows to use feed more
efficiently and produce more milk.
D. Gene Therapy: Use this technology of gene transfer to correct genetic
defects; any human diseases known to be due to a single gene defect; use to
correct single gene defect and cure disease
1. Severe Combined Immune Deficiency. Bubble baby. due to a single gene
defect- adenosine deaminase
2. Lesch-Nyhan- severe mental retardation, self destructive behavior. One
brain enzyme missing
3. Cystic Fibrosis- single gene, inhale DNA with normal gene
E. The future of genetic research: One review
X. Pros and cons of genetic engineering
A. Cons- risks and concerns
1. Herbicide-resistance or insect-resistance genes could spread from the
engineered crops to wild relatives and create weeds that are especially
difficult to control
2. Some scientists fear that the USDA does not require sufficient
precautions to prevent the spread of genes from engineered plants to their
wild relatives in field trials
3. Some bioengineered products could wipe out the major exports of some
developing nations
 a. example: a genetically altered bacterium is under development that
produces vanilla flavoring; this could eliminate markets for the vanilla
beans, one of Madagascar's major agricultural products
b. bovine growth hormone too expensive for small dairyman, so can't
compete with big companies
4. Biological control may solve the problem, cheaper and more effectively,
as shown with the work on the cassava mealybug by Dr. Hans Herren, winner of
the 1995 World Food Prize
B. Pros
1. Almost 100 million people are expected to be added to the world's
population each year for the next 30 years
a. some believe that without biotechnology, we won't be able to increase
the availability of affordable basic food.
b. although biotechnology has potential risks, starvation is worse
Other Sites of Interest:
All about Arabidopsis: "Guinea pig of the plant world" by Bryan Ness
Biotechnology and Agriculture
A series of short reviews; excellent summaries!
Access Excellence: WWW based teaching resources sponsored by Genentech, Inc.
Brief review and excellent definitions
Bio Online: Links to biotech information
BioBlox: Biotechnology Resource Center
A primer on molecular genetics but pretty technical
BioEthics: Links to resources on bioethics (little botanical)
Bt toxin links- USDA National Agriculture Library
Code of Conduct for Plant Biotechnology
A discussion of plant breeding, still a bit technical
CIMMYT: Where Norman Borlaug started the Green Revolution
Reviews of books and articles on the Green Revolution
Perils Amidst the Promise: Ecological risks of transgenic crops in a global
market by Jane Rissler & Margaret Mellon -- An excellent review worthy of
serious review
 Restriction Endonuclease Analysis in the Study of Baculoviruses

1. Introduction
The technological advancements in molecular biology has led to the elucidation of
baculovirus genome which is used nowadays in genetic manipulations. In research and
development and commercialisation of baculovirus pesticides the primary requirement is
their clear identity. With DNA analysis, the baculovirus genomes have been characterised
and their relationships have been investigated. The methodology of DNA nucleotide
sequencing is advanced, rapid and gives information on the identity of the virus. DNA
restriction enzymes analysis and hybridization have shown genetic relatedness between
baculoviruses (Knudson and Tinsley, 1978; Smith and Summers, 1978; Vlak, 1980; Vlak
and Groner, 1980) and restriction endonuclease analysis of the viral genome has revealed
that the wild stocks of viruses, in particular, the nuclear polyhedrosis group are often
composed of several genetically different isolates (Lee and Miller, 1978; Knell and
Summers, 1981; Gettig and McCarthy, 1982; Cherry and Summers, 1985). These methods
have proven useful for the comparison of the geographical isolates of baculoviruses from
the same or similar species (Vlak and Groner, 1980). Presently in quality control of
baculoviruses they are beginning to play an useful yet vital role in authentication of the
strain or isolate in production. It is also useful to monitor the genetic changes taking place
under adverse environmental conditions.
2. Restriction endonucleases
The discovery of a variety of restriction endonucleases numbering more than
hundred has revolutionized the research on DNA based technologies. These enzymes
found in prokaryotes synonymously called restriction enzymes recognize specific
nucleotide sequences in the DNA and cut both strands of the DNA within recognition site.
They are site specific. The commonly used enzymes are given in Table 1. Different
enzymes found in different organisms recognize different nucleotide sequences and
therefore cut DNA at different cleavage sites. Apparently, their function is to protect the
bacterium and its genetic material from the invasive effects of foreign DNA. They serve to
cleave the foreign DNA and render them ineffective. These restriction enzymes generally
have three important features (Ignacimuthu, 1995):
a. Restriction enzymes cut in palindromic sequences
b. The cuts are usually not directly opposite to one another, and
c. The enzymes generate DNA fragments with complementary ends.

Table I.
Commonly used restriction endonucleases and their recognition sites

Recognition sequence
Enzyme Source
and cleavage site
 Eco RI Escherichia coli Ry1 3
Hind III Haemophilus influenzae
Bam H1 Bacillus amyloliquefaciens
Pst I Providentia stuarti

3. Principles of restriction endonuclear analysis

The baculovirus genome is circular and double stranded. From the polyhedra or
capsule the DNA is collected after dissolution of the capsule or polyhedra. The DNA is
cleaved at specific sites based on the recognition pattern of the restriction enzyme used.
This generates a number of fragments of varying molecular weight or few copies of the
same, Which are usually measured in terms of kilo base pairs (kbp). The fragments get
separated based on their size in an electrical field. Based on the weight of the cut
fragments they migrate in a medium of agarose gel and create a unique pattern for the
concerned isolate of the virus which is called a finger print. When several isolates are
compared for their diversity or relatedness based on the number of comigrating taxa the
closeness is determined and a cladogram is constructed.

4 . Protocol for restriction endonuclease analysis of baculoviruses (NPV and GV)

4.1. Extraction and dissolution of virus
a. Place one larva in microtube and add distilled water up to 1 ml. Squash gently with
pestle or pipette tip and vortex for 10 sec. Aliquot if necessary (Skin may be removed
carefully with the tip of a pipette, but avoid contaminating outside the tube. If
semipurified virus is used, begin at step (6).
b. Centrifuge on fast spin for maximum 3 sec.
c. Transfer supernatant carefully with filter tip to clean microtube (Heavily virosed larvae
may pellet some NPV at this point on top of the insect debris layer. This can also be
removed with the supernatant).
d. Centrifuge for 2 min. at 10,000 rpm (10 min. for GV) and possibly higher speed.
e. Remove and discard supernatant (The pellet of NPV will be seen as a light coloured
area and any dark layer above it can be discarded with the supernatant. It is possible to
gently loosen the top dark debris layer with the tip of a pipette to assist removal).
f. Add 750 µl distilled water. Vortex to resuspend pellet.
g. Centrifuge for 2 min at 10,000 (10 min for GV).
h. Remove supernatant and add 120 µl distilled water. Vortex to resuspend pellet.
i. Add 25 µl 0.5 M EDTA* + 3 µl of proteinase K (20 mg/ml). Incubate for 1½ h at 37°C
(* EDTA to disrupt enzymes, proteinase K to disrupt DNA from host).
j. Centrifuge for 1 min at 10,000 rpm to Add 75 µl (approx. half valume) of 1 M Na2CO3
and incubate for 15 min at 37°C (The pH of the suspension should be >9.3 for
dissolution to take place and at least 10 for CPV. When this happens, the liquid
 becomes clear instead of milky. If this does not occur, add another 30 µl Na2CO3).
k. Add 25 µl of 10% SDS* and incubate for 30 min at 37°C. (* Warm mildly if SDS
undissolved, in a water bath or dryblock).
l. pellet undissolved polyhedra or any remaining debris. Remove supernatant to a clean
tube (At this point some of the liquid in the tube can be very viscous. This should be
retained with the rest of the supernatant).
4.2. Phenol extraction of DNA
a. Add an equal volume of tris saturated phenol*. Put the pipette tip through the tris layer
to the liquid phenol layer at the bottom to pipette up the phenol (* Handle phenol
carefully and wash off immediately if comes in contact with skin).
b. Agitate gently for at least 5 minutes.
c. Centrifuge at 10000 rpm for 5 min. This will result in a viral DNA layer at the top, phenol
at the bottom and usually a visible white protein interface.
d. Carefully remove the upper phase to a clean microtube, taking care not to disturb the
interface (If the tip of the pipette is cut diagonally to produce a larger opening this will
help to prevent a surge of liquid dragging the interface upwards).
e. Repeat the procedure in steps 1 to 4 using an equal volume of 25:24:1, tris saturated
phenol: chloroform: isoamyl alcohol. If the interface is still not clear, this step should be
repeated.
f. Repeat steps 1 to 4 again, but this time using an equal volume of 24: 1, chloroform:
isoamyl alcohol (Tips should not be cut for this step).
g. The DNA is now in solution and ready for dialysis or ethanol precipitation.
4.3. Dialysis
a. To prepare a microtube for use in dialysis, cut the cap with its hinge from the tube. Cut
the top 5 mm off the tube and discard the lower section.
b. Prepare 30 x 30 mm pieces of dialysis membrane by soaking tubing in x 1 tris - acetate
buffer for 5 min. and slitting down both sides (The membrane must be handled carefully
to avoid too much contamination with salts, etc. from the skin, but any gloves used
should not have powder on the outside).
c. Pipette the extracted DNA (no more than 250 J.1l) into the microtube cap, as prepared
above.
d. Lay a single piece of dialysis membrane across the cap and carefully fit the 5 mm
section of tubing over this, enclosing the DNA. Push down evenly to avoid tearing the
membrane (There will usually be some trapped air bubbles. This does not matter).
e. Place the cap assembly into a large (600-1000 ml) beaker of tris-acetate buffer solution
with the membrane uppermost. Submerge to ensure contact of the membrane with the
buffer and while submerged, carefully turn the assembly over to float with the cap
 uppermost and membrane below.
f. Dialyse at. 4°C for at least 36 h, changing the buffer three times.
g. Remove the assembly from the beaker using forceps and dab the membrane surface
very gently with a tissue.
h. Nick the membrane with a scalpel, using a fresh one for each sample and pipette out
the DNA into a clean labelled microtube. Discard used scalpel (It is only necessary to
make a very smart cut).
i. The DNA is now ready for digestion with restriction endonuclease enzymes. Store at
4°C.
A . Digestion
a. Pipette 25 µl of DNA solution into a clean microtube (Note I ).
b. Take enzymes and their buffers from the freezer and place on ice.
c. Add x 10 enzyme buffer to the DNA. The amount required will be equal to 1/10 of the
final volume in the tube, i.e. DNA + buffer + enzyme.
d. Add a quantity of R.E. enzyme equal to ½ th volume of buffer used, i.e. 1.5 µl (Hold the
enzyme container carefully by the top only and return to the ice immediately to the
freezer as soon as all samples have been completed) (To ensure the buffer and
enzyme are well mixed, pipette directly into DNA solution). (Note 2).

e. Stroke the side of the tube gently to mix.

f. Incubate at 370C for 2 to 4 h.
g. At the end of digestion and stopping mix equal to 1/10 of the volume in the tube, i.e. 3
µl. Stroke the side of the tube to mix (Note 3).
h. The sample is now ready for electrophoresis and should be stored at 4°C until needed.
4.5. ElectroPhoresis
a. Make a 0.6% gel by adding 250 ml of x 1 tris-acetate buffer to 1.5 9 of agarose
(molecular biology grade) in a 500 ml conical flask (This makes a gel 200 x 200 x 10
mm. A 100 mm x 75 mm gel will need approx. 70 ml of agarose/buffer solution).
b. Bring to the boil in a water bath or microwave and boil gently until all the grains have
dissolved. Stir occasionally during heating (Do not use microwave at maximum power
or the liquid will "Volcano").
c. Cool slightly, stirring or "swirling" occasionally to prevent differential cooling and then
add 50 µl of 1 mg/m ( or 5µl of 10 mg/m) ethidium bromide. This will bind to the DNA
and make it visible under UV light (CARE-powerful mutagen! Use gloves when
handling) (Note 4).
d. Wipe the plastic gel mould with ethanol and make the end walls by fastening adhesive
 tape along the two open sides, sticking it to the base and to the end of the side walls
(Sellotape, masking tape or labelling tape can be used).
e. Cool the gel to between 55 and 60°C, still stirring, then pour into the mould ensuring
that there are no air bubbles. Arty bubbles or loose particles can be carefully moved to
the sides of the mould with a pipette tip.
f. Immediately place the required comb in position at one end of the gel to create wells
(Make sure the comb is clean or it may tear the gel when it is removed).
g. Allow to set for 30 min. to 1 h then gently remove the comb and sellotape. Use as soon
as possible to prevent drying (Use gloves when handling the gel and mould because
ethidium bromide has been added).
h. Pour sufficient x I tris-acetate buffer into the tank to cover the flat base.
i. Place the mould with the gel on it into the tank, making sure it slots in correctly and with
the wells at the negative end. Top up with buffer to cover the gel by about 3-5 mm.
j. Using a very fine pipette tip carefully place digested DNA into the wells. The sample
should be released slowly from the pipette when the tip is in the buffer and just above
the well. The sample will sink to the bottom of the well. Discard the tip (Note 5).
k. Into one well pipette the molecular weight marker, eg. digested lambda DNA or 2 µl of
a 1 kb ladder (Either of these will provide a reference scale for determining the size of
fragments in each band. Reduce the amount if a small gel is being used).
l. Replace the lid and conriect to the power supply.
m. If the Iarge tank is being used, set the power level to 35 V and 50 MA and run
overnight. The small tank can be run at a higher voltage for about 5 h.
n. The furthest extent of sample movement through the gel will be indicated by the
position of blue dye. Do not allow the dye to run beyond the end of the gel or some
bands may be lost.
o. Switch off the power supply and remove the mould + gel from the tank. Gloves must be
worn.
p. Place the assembly on a UV transilluminator to see if the bands are visible (Wear UV
protection goggles or visor when viewing the bands).
q. If more staining is needed, prepare a solution of 0.8% ethidium bromide in x 1 tris-
acetate buffer. Slide the gel very carefully from the mould Into this solution and leave
for 30 min. preferably on a rocking table. Replace the gel on the mould to return to the
transilluminator (Gloves!) (Note 6).
r. Slide the gel from the mould onto the UV transilluminator surface and view through
protective eye shields or photograph as required.
s. To Photograph, place the hood carefully over the gel and put the Polaroid camera on
the top. For a large gel, aperture the time for a Polaroid photograph should be 5.6 for 2
min. For a small gel, using the small hood, the settings will be 5.6 for 10s (Note 7).
4.6. Notes
a. The amount of DNA sample to be used depends on the concentration of DNA in the
dialysed solution. This can be estimated to some extent by the size of the pellet
recovered in extraction step 5.
b. Alternative method of adding RE enzyme. If the DNA solution is quite concentrated and
several samples are to be digested with the same enzyme, a "Master mix" of the
enzyme can be made. For each DNA sample you will need: 2.9 µl buffer, 1.5 µl
enzyme (In practice is always best to make a mix for one more sample than you need
because of pipetting errors). Put 25 µl of each DNA solution into separate tubes.
Pipette 4.4 µI of mix into each tube and continue from stage 5 of digestion procedure.
c. Stopping mix: EDT A stops the reaction, Ficoll increases the density in order to hold it in
the wells and bromo phenol blue marks the samples in the well and through the gel.
See Solutions section for quantities.
d. Ethidium bromide is not essential at this stage, but it is occasionally useful to be able to
see the progress of the DNA fragments through the gel. If not used now the gel will
need to be stained after electrophoresis for 30 min in 0.8% ethidium bromide intris-
acetate buffer, i.e. 800 µl of 1 mg/ml ethidium bromide in 1 litre of buffer.
e. Liquid placed in the wells should come just below the top of the gel surface to prevent
it "streaming" over the edge of a well. Too much DNA will result in streaked or non-
discrete bands. 25 µl can be loaded into each of the 22 wells in a large gel.
f. If the gel has been stained, it is best to remove excess ethidium bromide solution by
placing in a container of distilled water and agitating gently for a few minutes. This
prevents background glare on the photograph.
g. Two different films can be used. 665 will give you a negative for enlarging as well as a
positive print. 667 produces a positive only, but gives good resolution in a ¼ sec.
exposure time. 665 needs an exposure time of at lest 2 min. to visualise the weaker
bands. Too much exposure to UV can cause degeneration of the DNA.
4.7. Solutions
a. Tris-acetate buffer : Tris-acetate EDT A buffer, pH 8.3 usually supplied as x 10
liquid or powder to be made up to x 10.
b. 0.5 M EDTA : Use a heated magnetic stirrer. This solution may
recrystalize, but.will redissolve on heating
c. Proteinase K : Supplied as a powder. Make up at 20 mg/ml.
d. 1 M Na2CO3 : Should be made up fresh at least each week.
e. 10% SDS : Sodium dodecyl sulphate made up at 10% (w/v) with glass
distilled water.
f. Tris-saturated : Make up fresh as required and keep in dark. Half fill a
phenol McCartney bottle with tris buffer. Add phenol and shake to
dissolve. The phenol will form a layer at the bottom and is
drawn off with a fine pipette. Use a fume cupboard and
gloves. Discard unused sat. phenol via safety officer.
Keep phenol in freezer. 8- hydroxyquinoline can be added
to a final volume of 0.1 %, which will extend the time it can
be kept before the phenol darkens and becomes
unusable.
g. Chloroform/isoamyl : Make up at 24: I. The isoamyl alcohol prevents foaming.
alcohol
h. Enzymes and : Each enzyme is supplied with its appropriate buffer. A
buffers chart is available to indicate the correct combinations to
use. Keep at -20°C.
i. Stopping mix : 25% Ficoll (w/v), 0.1 M EDTA, 0.25% bromophenol blue
(w/v).
j. I kb ladder : 20 µl ladder, 20 µl stopping mix, 80 µl x 1 tris buffer
5. References
Cherry, C. L. and M. D. Summers. 1985. Genotypic variation among wild isolates of two nuclear
polyhedrosis viruses isolated from Spodoptera littoralis. Journal of Invertebrate
Pathology, 46: 289-295.
Gettig, R. R. and W. J. McCarihy. 1 982. Genotypic variation among wild isolates of Heliothis spp.
nuclear polyhedrosis viruses from different geographical regions. Virology, 117: 242-245.
Ignacimuthu, S. 1995. Basic biotechnology. Tata McGraw Hill. New Delhi, India. 317 p.
Knell, J. D. and M. D. Summers. 1981. Investigation on the genetic heterogeneity in wild isolates of
Spodoptera frugiperda nuclear polyhedrosis virus by restriction endonuclease analysis of
plaque-purified variants. Virology, 112: 190-197.
Knudson, D. L., and M. D. Summers. 1974. Replication of a nuclear polyhedrosis virus in a
continuous cell culture of Spodoptera frugiperda: purification, assay of infectivity and
growth characteristics. Journal of Virology, 14: 934-944.
Lee, H. H. and L. K. Miller, 1978. Isolates of genotypic variants of Autogarpha californica nuclear
polybedrosis virus. Journal of Virology, 27: 754- 767.
Smith, G. E. and M. D. Summers. 1978. Analysis of baculovirus genomes with restriction
endonucleases. Virology, 123: 393-406.
Vlak, J.M. 1980. Restriction endonucleases: A new tool in baculovirus identification. In: Integrated
control of insect pests in Netherlands. Pudoc. Wageningen, pp.265-270.
Vlak, J.M. and A. Groner. 1980. Identification of two nuclear polyhedrosis viruses from the cabbage
moth, Mamestra brassicae (L.) (Lepidoptera: Noctuidae). Journal of Invertebrate
Pathology.
 Role of infochemicals in biological control of insects

Infochemicals are defined in a tritrophic context as chemicals that convey

information eliciting behavioural or physiological responses. Tritrophic interactions
involve interactions between three trophic levels, which include the plant, the pest
(herbivore) and the natural enemy (carnivore). Every living green plant has insect
herbivores that attack it. However, despite this plant communities still retain a green
appearance. This is attributed to ability of plants defend themselves effectively against
most herbivores and natural enemies suppress herbivore populations to very low levels
and have an overriding impact on herbivores thereby releasing the plants from herbivore
attack. In the past ecological research concerning plant defence against herbivores
focussed primarily on the first point, investigating interactions between two trophic
levels, the plant and the pest. In the 1980's research started to consider the role of all three
trophic levels in insect-plant interactions. Consequently current ecological studies take a
more holistic approach, with complex interactive studies considering the role of both
plant defence and natural enemy impact on insect-plant relations.
Plants have direct and indirect defence mechanisms, which they use against their
herbivore attackers. Direct defence includes physical and chemical strategies. Indirect
defence mechanisms bypass the second trophic level (herbivores) by promoting the
effectiveness of the third (natural enemies), through the provision of alternative food
sources (e.g. nectar and pollen) and shelter, or by the attraction of natural enemies to the
plant after herbivore infestation. There are strong links between direct defence (host plant
resistance) and indirect defence (biological control), and these links can be mediated by a
variety of physical and chemical plant factors. Indirect plant defence, where the plant
benefits from the natural enemies of herbivores, essentially involves tritrophic
interactions. The chemicals released by the plant in response to attack by a herbivore may
influence the foraging activity of parasitoids and predators. Natural enemies make use of
these infochemicals to detect their herbivore hosts. Tritrophic interactions focus on the
abilities of natural enemies to use plant traits to locate insects, besides emphasizing on
the ecological and evolutionary implications of the food chain. In long or short-range
locations of their insect hosts natural enemies use the host plant traits which influence
their ability to detect and utilize insect hosts, so that phytochemicals enhance attack by
natural enemies. Insect damage-induced chemical and physical responses of plants
influence the biology of parasites so that a phytochemical connection becomes
established at different trophic levels. The release rates of infochemicals tend to differ
altering the quality of the host insects to the parasitoids. Chemical, tactile or visual cues
enable the natural enemies to distinguish between infested and non-infested plants.

Influence of direct defence on natural enemies

Natural enemies of herbivores benefit the plants by reducing herbivore pressure.
Plants may, in turn, influence carnivores by making herbivores more vulnerable to them
(Price, 1986). But this positive influence does not occur in every situation; plant
resistance may adversely affect natural enemies of herbivores. Price (1986) has
tentatively classified the interactions between the three trophic levels (Plant, herbivore
and carnivore) as semiochemically mediated, chemically mediated and physically
mediated interactions. Some of the research works related to the infochemical mediated
interactions are tabulated below:
Plants Mediate Host/Prey Accessibility Obrycki et al., 1983; Obrycki, 1986; Schuster
and Calderon, 1986; Kauffman and Kennedy,
1989; Kareiva and Sahakian, 1990; Grevstad
and Klepetka, 1992; Farrar et al., 1994;
Weisser, 1995; Vohland, 1996; Sutterlin and
van Lenteren, 1997
Plants Provide Host/Prey Finding Cues Nordlund et al., 1988; Lewis and Martin, 1990;
Ma et al., 1992; Powell and Wright, 1992; Wa¨
ckers and Lewis, 1994; Dicke, 1994; Godfray,
1994; Whitman and Nordlund, 1994; Turlings
et al., 1995; Takabayashi and Dicke, 1996;
Drukker et al., 1995; Potting et al., 1995;
Geervliet et al., 1996; Bertschy et al., 1997;
Sullivan et al., 1997; Powell et al.,1998.
Volatiles influencing parasitoid foraging Elzen et al., 1983, 1984; Vinson et al.,
behaviour 1987;Navasero and Elzen, 1989; Martin et al.,
1990; Turlings et al., 1991a,b, 1995; Udayagiri
and Jones, 1992, 1993; McCall et al., 1993;
Agelopoulos and Keller, 1994a,b, Ngi-song et
al., 1996
The role of plant chemical signals in the Flint et al., 1979, Greany and Hagen, 1981,
orientation of other predator species toward Spadbery, 1973, Ponsonby and Copland,1995,
their prey Wyatt et al.,1993.

Parasitoids are also able to learn cues that Arthur, 1966; Weseloh, 1972, 1986; Wardle,
are consistently associated with the 1990; Wardle and Borden, 1990; Ma et al.,
presence of their hosts 1992; Wackers, 1994; Wackers and Lewis,
1994.

Influence of intrinsic and extrinsic factors Nettles,1979; Elzen et al., 1985, 1986; van
on quantity and quality of plant signals Emden, 1986; Dicke et al., 1990a; Turlings et
al., 1993b; Takabayashi et al., 1994, 1995;
Loughrin et al., 1995; Du et al., 1996; Rapusas
et al., 1996
Plants Influence Host/Prey Suitability Barbosa et al., 1982; Duffey and Bloem,
1986;Duffey et al., 1986; Barbosa, 1988; van
Emden, 1995;Kester and Barbosa, 1991
Plants Mediate Host/Prey Availability Feeny, 1976; Moran and Hamilton, 1980; Price
et al., 1980; Price, 1986; Osier et al.,
1996,Benrey and Denno, 1997
Influence on natural enemy longevity and Leius, 1963; McMurtry and Scriven, 1965;
fecundity Syme, 1975, 1977; DeLima and Leigh, 1984;
Hagley and Barber, 1992; Idris and Grafius,
1995; Olson and Nechols, 1995; White et al.,
1995; Taylor and Foster, 1996; Baggen and
Gurr, 1998
Extra floral nectaies influencing natural Rogers, 1985; Schuster and Calderon, 1986).
enemies Whitman, 1994. Lewis and Takasu, 1990;
Stapel et al., 1997. Lewis et al., 1998.

Influence of transgenic plants on natural Hilbeck et al., 1998, Koziel et al., 1993 Dogan
enemies et al.,1996

Induced defenses as signals to natural enemies

Volatiles released by damaged plants after insect feeding tend to increase the
efficiency of natural enemies and plants producing such alarm responses attract more
parasitoids and predators. A whole suite of chemicals are released by damaged Brassica
oleracea such as isothiocyanates, which are hydrolysis products of glucosinolates,
undamaged plants emitting them in smaller amounts as also several
terpenoids.Intraspecific host plant variation that influences parasitoid success has been
evident in both agricultural and natural systems and varietal differences in crop plants are
known to influence parasitoid preference due to change in the nature of volatiles so that
the role of plant phenotypic variation becomes important in the behavioural diversity of
parasitoids. It is this intricate interaction between plants and natural enemies such as
parasitoids that acts as a driving force leading to the production of adequate signals
affecting the behaviour of natural enemies in a positive way. The parasitoids make use of
these volatiles to identify the vicinity of host-infested plants, in particular the orientation
of the larval parasitoids to the frass of host larvae, since they are in a position to identify
the damaged plants from a distance. Foraging parasitoids like Microplitis croceipes
would be exposed to a wide range of volatile blends both from host plant and insect
complexes in its environment. Learning by parasitoids enables behavioural variability,
recognizing the odour of hosts as well as their exact site and environment. On the basis of
information acquired immediately upon emergence from host puparium and during the
first oviposition, they learn more in accordance with odours acquired by the host.
Divergent views exist regarding the role of induced defenses on herbivores, providing
reliable signals to natural enemies indicating the presence of host insects. The view that
plant volatiles have evolved to serve tritrophic communication is supported by evidence
that release of green leaf volatiles increased after insect herbivore attack with many
parasitoids and predators being attracted to the damaged host. Localized induction tends
to increase the activity of phytophagous insects within the plant enabling easy detection
by natural enemies. Induction of allelochemicals may prolong development of host insect
larvae making them more susceptible to the natural enemies or as in many cases
allelochemicals may simultaneously reduce growth and attract more natural enemies.
Indirect effects of induced chemicals relate to their ability to prolong development of
insect larvae, enhancing the possibility of natural enemy attraction. Constitutive variation
in phytochemistry as well as induced responses in plants after insect attack form the basis
for phytochemical involvement with the third trophic level.
Manipulating Plant Attributes to Enhance Biological Control
Plants and insect herbivores have long been competing in an evolutionary race in
which plants evolve to reduce consumption, while herbivores evolve to increase it. As a
consequence, plants have developed a number of direct chemical and morphological
defenses that limit herbivore attack (Rosenthal and Berenbaum, 1991). Direct chemical
defenses include production of toxins, repellents, and digestibility reducers, while
morphological defenses include trichomes, spines, waxes, and tough foliage. The value of
these defenses was realized early in agriculture, and production practices based on the
selection and use of different herbivore-resistant crop varieties have frequently been
implemented. Furthermore, with the progress of genetic engineering it is now possible to
directly manipulate these defenses by inserting new genes into crop plants (Gasser and
Fraley, 1989). In addition to these direct defenses against herbivores, plants also benefit
from indirect defenses provided by parasitoids and predators that use herbivores as hosts
or prey. Plants are not passive in the interplay between entomophagous arthropods and
herbivores. Rather, they actually mediate many of the interactions and thereby increase or
decrease the intensity of protection received. Chemical and morphological plant attributes
can directly influence survival, fecundity, and foraging success of natural enemies on
hosts or prey. These traits can also have indirect effects by affecting qualities of an
herbivore that in turn affect the physiology, behavior, or development of natural enemies.
Plant breeding and biological control have mostly been parallel but independent pest
management practices in the past (Thomas and Waage, 1996). Studies are revealing
substantial interactions between plant traits conferring herbivore resistance and biological
control agents (Bergman and Tingey, 1979; Boethel and Eikenbary, 1986; Shepard and
Dahlman, 1988; Hare, 1992; Panda and Khush, 1995; Thomas and Waage, 1996; Gould,
1998), thereby emphasizing the importance of managing plant attributes from a tritrophic
perspective. Therefore, special emphasis should now be placed on breeding crop plants
with natural enemy-enhancing traits. Detailed understanding of biochemical, molecular
and physiological processes that are involved in the production of herbivore-induced
plant volatiles may allow us to genetically modify plants to produce herbivore-induced
signals more quickly and at lower herbivore infestation levels.
Conclusion
Blends of plant infochemicals and herbivore-induced volatiles vary with plant
species, plant cultivars, plant developmental stage, herbivore species and herbivore
developmental stage. For an efficient and sustainable use of infochemical mediated plant
signaling in biological control, both large-scale and long-term field studies are necessary.
Although laboratory studies are useful in understanding the role of synomones in
interactions among plants, herbivores, and natural enemies, they may not always be
reliable predictors of real natural enemy activity in the field. Furthermore, laboratory
studies rarely consider interactions between different natural enemies, even though
biological control is a complex process often involving different species of predators and
parasitoids with different ecological characteristics. Also, long-term field studies are
necessary to better understand the influence of naturally produced plant attractants on
pest and natural enemy population dynamics. Finally, the relevance of each attribute for
biological control depends not only on the nature of the plant, herbivores, and beneficial
arthropods considered but also on the characteristics of the relationships among these
trophic levels.

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Syrphidae). J. Econ. Entomol. 88,1171–1176.
Whitman, D. W. 1994. Plant bodyguards: Mutualistic interactions between plants and the
third trophic level. In ‘‘Functional Dynamics of Phytophagous Insects’’ (T. N.
Ananthakrishnan, Ed.), pp. 133–159. Oxford and IBH Publishing, New Delhi,
India.

Whitman, D., and Nordlund, D. A. 1994. Plant chemicals and the location of herbivorous
arthropods by their natural enemies. In‘‘Functional Dynamics of Phytophagous
Insects’’ (T. N. Anan-thakrishnan,ed.), pp. 207–248. Oxford and IBH Publishing,
New Delhi, India.

Wyatt, T. D., Phillips, A. D. G., and Gregoire, J. C. 1993. Turbulence, trees and
semiochemicals: Wind-tunnel orientation of the predator, Rhizophagus grantis, to
its barkbeetle prey, Dendroctonus micans. Physiol. Entomol. 18, 204–210.
 INTSTRUMENTATION AND TECHNIQUES IN BIOTECHOLOGICAL RESEARCH WITH
SPECIAL REFERENCE TO ENTOMOLOGY

1. Introduction
Human beings have been biologically minded for a very long time, since man could never
have become civilized without being aware of the world of living things. Biology has been a
descriptive science in the usual sense, however, for only a few centuries. Nevertheless biology
has developed as analytical science only after developments in the physical sciences.
The earliest biologists were descriptive scientists, concerned with naming and describing
organisms. The invention of the microscope permitted the examination of smaller units, but the
approach of the biologists remained about the same; that is, they still described what they saw.
In time, it became possible to consider more abstract relationships. Changes that take place over
a period of time, as in the growth of an animal or plant, or as in the various physiological
processes within an individual, obviously require a rather different kind of observation. If a
biologist is to explain the inter-relationships of the activities of various organs in an animal, a
higher level of intellectual activity is required than if he merely describes their structure.
The experimental approach is especially valuable in these highly abstract phases of
biology. In fact, about the year 1625 William Harvey used some of the first experiments
in biology to demonstrate the continuous circulation of the blood. At about the same
time, van Helmont’s experiment showed that plants do not take all their food from the
soil. About a century later Stephen Hales performed many experiments on the pressures
and the movement of liquids within plants and animals. His books, Vegetable Staticks
(1727) and Haemastaticks (1732), demonstrate the ingenuity of man, as well as providing
very interesting reading. Physicists at the time were measuring pressure by observing the
height to which a liquid would rise in a tube. Hales applied similar observations in
biology. In one heroic experiment, he measured the blood pressure of a horse by
attaching a long glass tube to one of the large arteries in the horse’s neck. Fortunately
this method of measuring blood pressure never became popular among the physicians. It
is enlightening to read a modern laboratory manual for plant physiology, to compare it
with Vegetable Staticks, and to note how many of the usual experiments in present-day
courses were actually designed by Stephen Hales (Orster and Pollister, 1956).
2. Some important Techniques
Some of the Important Techniques used in entomological research are listed in
Table-1
2.1. Centrifuge
Centrifuging Techniques is the process designed to separate materials of different density
from each other by virtue of centrifugal force. Since the
centrifugal force is similar in its effects to gravity, most things that can be separated in a
centrifuge would eventually settle because of gravity, but a very long time might be required.
The centrifuge allows us to hasten this effect by applying a larger force. In laboratory
centrifugation, at least one of the components to be separated is a liquid. The other might be
solid particles, another liquid, or, rarely, even bubbles of gas. So many other kinds of mixtures
must be separated in the laboratory routine, that a centrifuge is used almost daily. In addition to
its use in preparing materials, the centrifuge is a valuable analytical tool.
Table - 1 Important Techniques in Entomological Research
Instrument Application in Entomology
Compound Microscopy h To observe small Arthropods and classify them properly.

Phase Contrast Microscopy
Electron Microscopy
Centrifuges
Colorimetery and
Spectrophotometry
Chromatography
Isotopic tracer Techniques
Electrophoresis
Spray drying and Freeze drying
Microtomy
Electroantennogram
h To study the functional ecology of various append ages of small
Arthropods.
h To study parts of the body of the insects when they are
alive.
h To observe histopathological changes when an insect is in
fected by microbials.
h To study the damages in the plants caused by insects.
h Diagnosis of insect diseases.
h To study the feeding mechanism of minute Arthropods like mites.
h Study of insect pathogens.
h To separate proteins, nucleic acids and other macro
molecules for various bio chemical studies.
h To separate larger particles including viruses, ribosomes and other
cellular particulates isolated from cells that can be used to modify
the life systems of the insects.
h To semi purify insect pathogens.
h To find out the presence of
heavy metals and other toxic contaminants present in the insect
body that could be indicators for the level of pollution in the
environment.
h To assess the pesticidal residue in soil, plants, water
and various agricultural products.
h To estimate various biochemicals like sugars, proteins, amino acids,
etc. to study the metabolism of insects.
h To assess pesticidal residues very precisely.
h To study metabolic pathway in insects.
h To study the movement of inecticides in insect body and
to study the mode of action ofinsecticides.
h To identify various proteins, enzymes and esterases that would help
to study the reaction of insects to various insecticides.
h To make DNA finger prints to evaluate the occurrence of different
geographical isolates in insect population as well as their disease
causing organisms.
h To formulate the biopesticides which contains
particulates of living materials.
h To make thin sections of insect body for observation under
microscope.
h To study the maycetomes.
h To screen semio-chemicals.

2.2. Microscopy
If any technique that should be given credit for contributing more than any other to the
development of biology as a science, that credit should go to the compound microscopy. The
history of detailed observation of living things parallels the development of magnification. Every
improvement in the art of combining lenses made it possible to see smaller structural units. First
the cells and later the minute structures inside cells were discovered. It is now possible to
photograph biological structures even as small as single virus particles.
Although the microscope has contributed to biology, biology has contributed to microscope
as well. The need for better magnifying systems with which to examine parts of organisms has
been the most important stimulus for the opticians.
2.2.1. Compound microscopy
 The compound microscope is so called because it contains two sets of lenses: an objective
lens, which produces an image, and an eyepiece or ocular, which further magnifies the image.
The magnification available is the product of the magnifications produced by the separate lens
units. The objective and ocular systems of the compound microscope are elaborate
combinations of individual components fitted together according to a formula which is intended to
provide the best possible view of the object being examined.
2.2.2. Phase contrast microscopy
Some biological materials and some parts of cells, especially when alive, offer so little
contrast in density, refractive index, or color that they are difficult to study with an ordinary
microscope. Some of these can be studied more effectively through the techniques of phase
microscopy.
In ordinary “bright field” microscope, high resolution depends upon
diffraction of light and the fringes that appear on the edges of structures. The diffracted light must
be caught by the objective lens, and the image is formed from the interference pattern of the
direct or axial light and the diffracted light. If there is sufficient contrast (difference in absorption,
reflection, or refraction) at these edges, the image is sharp and objects are easily recognizable.
Sometimes when the contrast is high, the bright or dark interference bands around small objects
are so obvious that they become annoying. If the contrast is insufficient, the diffracted rays will
be weak, the direct rays will seem too bright, and the object will be difficult or impossible to see.
Phase contrast is basically a means of balancing the direct and the diffracted rays to increase the
visibility of the interference fringes.
5.2.3. Electron microscope
The electron microscope employs a beam of electrons instead of a beam of light. These
electrons, which are produced by a heated filament, can be focused into a beam by an
electrostatic or a magnetic field. The electrons behave as if they had a frequency and a
wavelength, but this wavelength is much shorter than the wavelengths of visible light. The
‘optical’ parts of the electron microscope are analogous to those in the light microscope,
consisting of an electrostatic or magnetic “objective lens” and a similar projector lens. The
electron beam passes through the object, where electrons are either transmitted or scattered in
various directions, depending on the nature of the material in the object. The transmitted
electrons are brought to focus on a photographic plate in a pattern corresponding to regions of
high transmission or high scattering in the object. Since the wavelength of the electrons is short,
the resolution is greater than that available in the light microscope.
Biological materials offer difficulties in electron microscopy, but the
solution to these problems has permitted pictures showing exceedingly fine details of structure.
Most recent biology books contain excellent examples. Even though these show great detail,
much is lost in the printing process; the original photographs are truly magnificent.
The electron beam must operate in a vacuum, which means that the
biological material must be dry (and therefore dead). Exceedingly thin layers of material must be
used, and usually, since biological materials are relatively ineffective in electron scattering,
atoms of metal are sputtered to increase contrast.
2.3. Colorimetery
The colorimetric procedure, one of the most common methods used in analytical
chemistry today, finds application in biology also. The method depends upon those
physical principles which are related to the colour of various substances. In its simplest
form, colorimetry measures the amount of material by measuring the intensity of its
colour. The greater the concentration, the more highly colored the solution. An
extension and refinement of the technique is commonly given the term
spectrophotometry. Spectrophotometry also can be used to determine concentrations, but
has the added advantage that it can be used to identify materials and measure rates of
reactions. Any material that has colour, or more properly, any material that absorbs
radiant energy in the visible region, in the ultraviolet, or in the infrared, is adaptable to
measurement by this procedure.
In order to determine the concentration of a colored material, it is
necessary to measure the amount of light actually absorbed by that material. The greatest
response is obtained if measurements are made with light of a colour strongly absorbed
by the molecules. The typical colorimetric instruments isolate a band of wavelengths in
the vicinity of this maximum absorption, by means of a system of colored glass filters or
by the so-called interference filters. For routine measurements these filter systems are
extremely convenient and rapid to use. One merely places the pure solvent in the light
path and measures the amount of light striking the photocell. This measurement is often
used to establish an electrical zero point. The solvent is then replaced by the colored
solution and the diminution in light intensity is noted. The concentration of the solution
and the reduction in the light transmitted through it are related, as will be described later.
Such an instrument would be called a colorimeter.
2.4. Spectrophotometry
Spectrophotometers depend upon a monochromator which uses a prism or a diffraction
grating to resolve a beam of white light into its spectrum. The spectrum is allowed to fall upon an
adjustable slit which allows light of only one colour to pass through.
Many models of spectrophotometers are available on the commercial
market. They differ chiefly in the quality of the optical system and in the
electrical measuring system. The smaller and less expensive instruments
typically use a direct reading galvanometer of some sort. The deflection of this matter is
proportional to the output of the photocell.
2.5. Chromatography
2.5.1. Paper chromatography
In paper chromatography, the solid material is ordinary filter paper. Various combinations of
solvent can be used to separate different kinds of mixtures. If solution, a paper would allow a
more rapid determination than would the preparation of a column. The chief disadvantage of the
paper.
2.2. Thin layer chromatography
A technique which combines the advantages of column and paper
chromatography is the separation on thin layer plates. Originally, and still very commonly, a
slurry of a solid adsorbing material, with a binding agent if needed, is spread as a thin film on the
surface of a glass plate. In one common procedure, for example, a slurry of specially prepared
silica gel, with CaSO4 as a binder, is spread on glass plates of 20 cm2. Spreading devices are
used to spread the slurry in a film of uniform thickness. The plates are dried, usually in an oven,
and then can be handled in as much the same way as paper is used in ascending
chromatography. After the separation, spots can be recovered by scraping from the glass.
Separation on thin layer plates is accomplished very rapidly. With some mixtures a paper
chromatogram may require 24 hours to develop properly. With thin layer chromatography, the
same job may be finished in less than an hour. Another advantage is that somewhat larger
quantities of the mixture can be separated than on paper.
2.5.3. Gas chromatography
A major development is gas chromatography, a separation of materials in the vapour phase.
A tube is packed with a solid material which is then coated with a selected solvent. In another
system, the solvent forms a thin coating on the inner walls of a long, very fine capillary tube. In
either case, a mixture of gases is allowed to pass through the tube. Those components
appearing later as the individual gases emerge they are detected by a device that measures
thermal conductivity or some similar physical property. The amount of each component is
recorded electrically.
2.6. Isotopic tracer technique
Probably the single most valuable technique to become available to the biologist in recent
years is the isotopic tracer method. Increased understandings in a number of important areas
can be attributed directly to these materials, which came into general use shortly after the end of
World War II.
Several kinds of problems, otherwise insolvable, are experimentally easy if tracers are used.
If a material moves from one place to another within an organism but several different pathways
are possible, the tracer can identify the pathway taken. For example, mineral ions move from the
roots where they are absorbed upward to the leaves of plants. They might move through either
xylem or phloem; the proper application of tracer experimentation tells which tissue is the actual
path. In an animal, a certain material might move from place to place through blood or lymph, and
a tracer could be used here also.
2.7. Electrophoresis
If an electrical field is imposed across a liquid containing charged
particles, these particles will migrate in the liquid. Negatively charged particles migrate towards
the positive electrode; positively charged particles migrate towards the negative electrode. This
behavior is the basis of an important method, known as electrophoresis, for separating materials.
Proteins were the first and still the most important group of compounds to be studied.
Proteins are readily separated by electrophoresis because of the nature of the protein
molecule. It is composed of a large number of amino acids, some of which possess side
chains having an acidic-COOH group, others of which contain basic groups of one kind
or another. The dissociation of these groups depends upon pH. At low pH, the carboxyl
group will be associated (-COOH), and basic groups, such as-NH2, will carry an
additional proton (-NH3+). At high pH, the carboxyl groups dissociate (-COO-), as do the
basic groups. The net charge on the molecule thus depends strongly on pH. At some pH
value, the isoelectric point, intermediate between the extremes, positive and negative
charges balance each other.
Paper or gel electrophoresis is more commonly used today. Better resolution is available,
because the components move in relatively discrete zones when the buffer is stabilised by the
addition of the solid supporting phase. Mixing by convection is avoided.
2.7.1. Paper electrophoresis
A strip of filter paper, moistened with buffer solution, is suspended between two reservoirs of
buffer solution, with one end dipping into each solution. Electrodes are placed in the buffer
reservoirs, usually physically separated from direct contact with the paper in order to avoid
complications from possible electrode reactions. A small amount of the mixture to be separated
is applied near the center of the paper, the voltage is applied, the buffer solution conducts a
current, and the components move in one direction or the other, at rates determined by their
physical properties.
2.7.2. Gel electrophoresis
The apparatus for electrophoresis on starch gels is similar in many respects to that for paper
electrophoresis. Specially prepared starch is cast in slabs impregnated with buffer. Buffer
solution is carried to the ends of the slab and electrical connection is established by means of
paper wicks or some similar device.
 Electrophoresis on starch gels, and on paper as well can be adapted to a number of classes
of compounds other than proteins. Amino acids, organic acids, some carbohydrates, etc. might
be accommodated by the technique. If the molecules are not naturally charged, it is often
possible to modify them chemically, or to form complexes which are charged.
Electrophoresis on polyacrylamide gels yields very high resolution of proteins. The
polyacrylamide is polymerized inside small glass tubes. The tubes are then arranged upright,
with the bottom end in one reservoir of buffer, the top connected to another reservoir. Small
samples of proteins are separated electrophoretically, and stained to determine their positions.
2.8. Eelectro antennogram
The attraction of insect pests towards a particular plant is mediated by volatile
chemicals secreted by the later. Electro antennogram is a novel equipment to analyse the
movement of insects towards the source of such semio-chemicals. Minute electrodes are
fixed at the base of the antennae where the olfactory receptors are located. The
movement is monitored in a computer. This equipment will help to screen different kinds
of pheromones, kairomones and other semio-chemicals.
2.9. Microtomy
Instrumentation is but one phase of the development that have led to modern microtomy.
There has also been a parallel evolution of elaborate ancillary methods for preparing biological
material for the cutting process. For the most part these, latter do not involve physical
techniques; but a minimum knowledge of them is necessary if one is to understand all phases of
the physical aspects of the process of preparing a specimen for microscopical study.
Early plant histology was worked out with the uncorrected compound microscope on material
cut with a machine in which the twig or stem was mounted on a screw, which could be raised
through a table, across which razor was drawn by hand (the hand microtome). The full potentiality
of the achromatic microscope, invented in 1809, only became realized after the middle of the
century, when the hand microtome was improved by supporting the knife rigidly on the heavy
block that could be pulled along polished ways to slice the tissue (the sliding microtome).
Discoveries in cytology and embryology owe much to the convenience and precision of a later
development, the rotary microtome, in which the turn of a flywheel automatically provides the
cutting motion and advances the tissue to yield hundreds of successive slices that in thickness
are nearly perfect replicates.
2.10. Gene delivery Technique
The ability to introduce foreign genes into plants, such that they are stably expressed and
transmitted from generation to generation has revolutionized plant biology. During the past one
decade the success in designing insect-resistant crop plants through gene transfer has been
impressive.
Currently there are several approaches to the generation of stably transformed plants, and
the approach adopted varies according to the aims of the project.
The transformation technique that, for the cereals at least, has perhaps received the most
attention in recent years is microprojectile bombardment. The aim is to penetrate cells or tissues
with accelerated metal microspheres which are coated with DNA. Such microspheres, of
tungsten or gold (used because of their high densities), and of diameters of 1-4mm, have been
shown to penetrate cells when accelerated at high velocities, and the DNA can be released and
expressed transiently or can be stably integrated into chromosomes. The DNA-coated
microspheres are placed on a macroprojectile which is accelerated towards the tissue following
an explosive discharge (using for example, a blank gunpowder charge, an electric discharge that
rapidly evaporate water in a confined space, or under pressure from high-pressure helium, as
used in the commercially available Du Pont biolistic apparatus). The accelerated macroprojectile
hits a stopping plate and microprojectiles are propelled onwards and penetrate the tissue
depositing the DNA as they go. Microprojectile bombardment has been successfully used to
transform tissues that show good morphogenetic responses in a wide range of species including
tobacco, soybean, rice, barely and wheat.
2.11. Formulation Techniques
2.11.1. Spray drying
When the liquid droplets come into contact with the hot gas, they quickly reach a temperature
slightly above the wet-bulb temperature of the gas. The surface liquid is quickly evaporated, and
a tough shell of solids may form in its place. As drying proceeds, the liquid in the interior of the
droplet must diffuse through this shell. The diffusion of the liquid occurs at a much slower rate
than does the transfer of heat through the shell to the interior of the droplet. The resultant
buildup of heat causes the liquid below the shell to evaporate at a far greater rate than it can
diffuse to the surface. The internal pressure causes the droplet to swell, and the shell becomes
thinner, allowing faster diffusion. If the shell is non-elastic or impermeable, it ruptures, producing
either fragments or bud-like forms on the original sphere. Thus, spray-dried material consists of
intact spheres, spheres with buds, ruptured hollow spheres, or sphere fragments.
Spray driers differ from most other driers in that they can handle only fluid materials such as
solutions, slurries, and thin pastes. The fluid is dispersed as fine droplets into a moving stream of
hot gas, where they evaporate rapidly before reaching the wall of the drying chamber. The
product dries into a fine powder, which is carried by the gas current and gravity flow into a
collection system.
2.11.2. Freeze drying
Freeze drying depends on the phenomenon of sublimation, whereby water passes directly
from the solid state (ice) to the vapour state without passing through the liquid state.
Freeze dryers are composed of four basic components: (1) a chamber for vacuum drying, (2)
a vacuum source, (3) a heat source, and (4) a vapour-removal system. The chamber for vacuum
drying is generally designed for batch operation and thus can be compared to the vacuum shelf
dryer. Special inlet and outlet mechanisms have been designed in some drying chambers to
achieve continuous drying operation. Vacuum is achieved by pumps, steam ejectors, or a
combination of the two. Heat is provided by conduction or radiation, or by both. Three different
methods for the removal of water vapour are employed: condensers, desiccants, and pumps.
The water vapour is removed from the drying chamber and condensed in the form of a thin layer
of ice on a heat-transfer surface in the condenser. The ice is removed intermittently by melting it
with a heated fluid that is circulated through the condenser, or in the case of continuous
operation, by means of scraper blades. Liquid or solid desiccants are often employed in the initial
vapor removal to enhance the efficiency of the pumps removing the water vapor.
2.12. DNA technique by polymerase chain reaction
The application of DNA techniques to study genetic variations by polymerase chain reaction
(PCR) and others led to the unexpected discovery of large number of transposable elements
which can be used as a tool to impart desirable traits like tolerance to pesticide and adverse
climatic conditions in natural enemies. In PCR, the small amount of DNA bits extracted from an
insect is annealed and hybridized by subjecting to thermal cycling. Thus large quantities of DNA
can be artificially produced. By subjecting further to electrophoresis, genomic variation in
different geographical populations can be assessed. It is also possible to mark the desirable
gene for further gene transfer.
3. Suggested references for further reading
Crampton, J.M. and Eggleston E., 1992. Insect Molecular Science. Academic Press, London. 270 p.
Lachman, L., Lieberman, H.A., and Kanig, J.L. 1991. The Theory and
Practice of Industrial Pharmacy. Varghese Publishing House, Bombay, 902 p.
Orster, G. and Pollister, A.W., 1956. Physical Techniques in Biological Research. Volume I to V. Academic Press
Inc., New York
Van Norman, R.W. 1971. Experimental Biology. Prentice-Hall, Inc., Englewood cliffs, New Jersey. 269 p.
 The Sterile Insect Release Method and
Other Genetic Control Strategies
INDUCED STERILITY IN INSECTS

History

Discoveries in such diverse disciplines of science as radiation biology, chemistry,

animal behavior, molecular biology, genetics and entomology have contributed to our
current ideas concerning the control of insect pests. In 1895 W. K. Röntgen observed and
reported his discovery of X-rays. By 1903 the effects of such rays from radium upon the
reproductive systems and development of insects had been reported by C. Bohn. G. A.
Runner, in 1916, found that low doses of X-rays decreased the reproductive capacity of
the cigarette beetle, while higher doses killed them.

By 1926 H.J. Muller had demonstrated that X-ray radiation will cause heritable
changes in fruit flies (Drosophila melanogaster Meigen). Dominant lethal mutations
were found to be one of the most frequent types of induced mutation. The "partial
sterility" of X-ray treated males was of concern to Muller because this effect reduced the
numbers of visible mutations which could be recovered for a given dose of radiation.

As early as 1937, E. F. Knipling had conceived an approach to insect control in

which the natural reproductive processes of the screwworm fly (Cochliomyia
hominivorax Coquerel) are disrupted by chemical or physical mechanisms thus rendering
the insects sterile (Knipling 1985). These sterile insects are then released into the
environment in very large numbers (10 to 100 times the number of native insects) in
order to mate with the native insects that are present in the environment. A native female
that mates with a sterile male will produce eggs, but the eggs will not hatch (the same
effect will occur for the reciprocal cross). Since there are 10 to 100 more sterile insects in
the population than native insects, most of the crosses are sterile. As time goes on, the
number of native insects decreases and the ratio of sterile to normal insects increases,
thus driving the native population to extinction.

This unique insect control method is now known as the sterile insect technique
(SIT), or the sterile insect release method (SIRM). SIT proved to be an extremely
successful method for control of the screwworm fly when ionizing radiation was used as
the sterilizing source. The success of the screwworm SIT program led to investigations of
the effects of radiation on the reproductive performance of many other economically
important insects.

The use of mutagenic chemicals to sterilize insects for sterile insect release
programs was also considered because this seemed to be an efficient method of causing
sterility (Knipling 1979). For example, chemical sterilants could be added to the rearing
diet, or the chemical could be applied to eggs or pupae at times when they are being
handled in the normal rearing process. However, the use of chemical sterilants is
presently very limited because of fears of environmental contamination by insects
carrying carcinogenic materials. There are also problems with disposing of the diet or
other carriers used to administer the chemosterilant to the insects. Certain types of photo-
chemically reactive dyes (e. g. D&C red number 28) have been demonstrated to have
insecticidal properties but are environmentally safe. There is a challenge for investigators
to discover similar chemicals that could be used as sterilants.

The methods that are presently employed for the successful use of the sterility
principle (Bartlett 1990) have not changed significantly since E. F. Knipling's original
formulation:

1. Techniques that make it possible to produce large numbers of the target insect.
(Rearing component)
2. Techniques that make it possible to sterilize large numbers of the target insect.
(Treatment component)
3. Reasonably competitive insects that can be released after sterilization.
(Competitiveness component)
4. Economical systems for releasing and distributing the insects over the treatment
area. (Release component)
5. Tools that will assess native populations accurately before and after the release of
the treated insects. (Evaluation component)
6. A treatment area large enough (or well-isolated enough) to exclude the possibility
of immigration of inseminated females into the release area. (Re-infestation
component)

There are difficulties encountered when SIT is applied to certain Lepidoptera,

Coleoptera, or to insects (like mosquitoes and houseflies) whose obnoxious behavior
precludes the release of large numbers in the vicinity of people or animals. For example,
a dose of 5500 rad (55 Gy) is sufficient to guarantee 100% sterility of screwworm flies.
However, even after doses exceeding 50,000 rad (500 Gy), some lepidopteran pests will
still produce F1 progeny. Investigators have found that these F1 progeny exhibit levels of
sterility equal to or higher than those of their treated parents. This inherited (or delayed)
sterility has been suggested to be of use in control programs (Anonymous 1993).

The existence of insect species that do not respond to our attempts at sterilization
has led investigators to search for other methods which use genetic principles, but do not
necessarily involve radiation-induced sterility. These other "autocidal" control methods
are discussed in more detail after we discuss the cases where induced sterility has been
used.

Insect Control Programs Using Induced Sterility

The successful use of sterile insects for the eradication of the screwworm from the
United States and Mexico has been detailed in a number of symposia and books. The U.
S. D. A., Animal and Plant Health Inspection Service maintains a home page on the
screwworm eradication program. The screwworm program has had its share of skeptics
and detractors who theorized that the sterile insect release method could never work (see
a discussion of some pro's and con's concerning insect eradication in Cox 1978). In spite
of all the scientific criticism, eradication proceeded quickly across the southern tier of the
United States. The US portion of the screwworm eradication program started in Florida in
1957 and by 1966 all self-sustaining screwworm colonies in the US were eliminated.
However re-infestations from flies migrating from Mexico compromised the program, so
in 1972 a joint United States-Mexico program was initiated. This program enabled
Mexico to be officially declared free of screwworms in 1991, Belize and Guatemala in
1994, and El Salvador in 1995. Honduras is considered technically free of screwworms
since no flies have been detected since January, 1995. The ultimate goal of a proposed
United States-Central America project is to maintain a sterile insect barrier at the Darien
Gap in Panama starting in 1997. This program has saved billions of dollars in livestock
and wildlife loses since its inception.

Although the screwworm eradication program is the most visible and successful
use of SIT, a number of other insect species have been subjected to the release of sterile
insects with varying success. This includes the following pests and areas where programs
have been carried out (this is not an all-inclusive list): (1) screwworm fly /USA, Mexico,
Libya; (2) Mediterranean Fruit Fly (Ceratitis capitata Wiedemann)/USA (California),
Mexico; (3) Melon Fly (Dacus cucurbitae Coquillett)/Japan, Taiwan; (4) Pink Bollworm
(Pectinophora gossypiella Saunders)/USA (California); (5) Tsetse Fly (Glossina
species)/Tanzania, Zimbabwe, Upper Volta; (6) Mosquitoes (various) USA (Florida),
East Africa, Venezuela; (7) Boll Weevil (Anthonomus grandis Boheman)/Southeastern
USA; (8) Mexican Fruit Fly, (Anastrepha ludens Loew)/USA (Texas), Mexico; (9)
Gypsy Moth (Lymantria dispar Linnaeus)/Northeastern USA, Canada; (10) Stable Fly
(Stomoxys calcitrans Linnaeus)/USA (St. Croix, Virgin Islands - experimental); (11)
Horn Fly (Haematobia irritans Linnaeus)/USA (Texas - experimental); (12) Corn
Earworm (Helicoverpa zea Boddie)/USA (St. Croix, VI); (13) Tobacco Hornworm
(Manduca sexta Linnaeus)/USA (St. Croix, VI).

One of the more successful and long-term sterile insect release programs involves
the pink bollworm in the San Joaquin Valley of California (Staten et al. 1993). Over a 24
year period there has been a continuous release of sterile pink bollworm adults during
each day of the cotton growing season. This program was initiated in 1968 to protect the
ca 500,000 ha of cotton in the valley from infestation by the pink bollworm. In other
words, this program was conceived as a barrier against the high populations of pink
bollworms found in Arizona, Southern California, and Northern Mexico during the
decades of 1950 and 1960.

The pink bollworm program has been a cooperative effort of the United States
Department of Agriculture, the California Department of Food and Agriculture and the
California Cotton Pest Control Board. From 1970 to 1991 the pink bollworm rearing
facility in Phoenix produced from 99 million to 826 million dye-marked moths per year.
Most of the moths were radiation sterilized and released from airplanes over cotton
natives in the San Joaquin; however, large numbers of moths have also been provided for
research programs on sterility, insecticide evaluation, and pheromone isolation and
identification.

During the course of this release program, the numbers of native moths captured
in the San Joaquin Valley has varied considerably from year to year (Staten et al. 1993).
Because of careful monitoring of the dye-marked sterile and native populations through
the use of pheromone baited traps, the program was able to quickly adjust both the
patterns of release and numbers of sterile insects released in a given area. Each year
releases are made in sections of cotton which had native moth captures the previous year.
As native pink bollworms appear in trap captures, the release patterns and rates are
adjusted accordingly. In only one year (1990), out of 24 years of the program, have
conventional insecticides been used to control pink bollworm infestations and then on a
very limited basis (two sprays on 280 acres out of 1184 total). No carry-over of this
population from 1990 to 1991 was apparent, and overall numbers of native captures in
1992 were low. To the present time, no other detectable economically damaging
populations of pink bollworm have developed in the San Joaquin Valley.

OTHER AUTOCIDAL INSECT CONTROL TECHNIQUES

Some species of insects have not proved to be good candidates for the sterile
insect technique, so other methods of using the insect to control itself (autocidal control)
have been investigated. The following approaches have been investigated (see Davidson
1974, Pal and Whitten 1974, and Whitten 1985, for some early discussion of the use of
genetics in insect control).

Inherited Sterility

This effect has also been referred to as: delayed sterility, F1 sterility, partial
sterility, etc. This phenomenon is of use in organisms, such as Lepidoptera and
Homoptera, that contain polycentric chromosomes. It involves the transmission of
aberrant chromosomes (usually in the form of translocations) from the released
population to the native population.

This can be accomplished in two ways: 1) by treating a laboratory-reared

population of insects with a dose of radiation which is sufficient to break chromosomes,
but low enough to produce little direct sterility, or 2) by culturing stocks of the target
species which contains homozygous translocations (this is rather difficult and tedious to
accomplish). When the aberrant chromosomes are passed on to the native populations,
the individuals carrying the aberrations in the heterozygous state will show sterility. The
amount of sterility will depend on the number and size of the translocations carried by
each individual. Some advantages of this method include: 1) enhanced reproductive
competitiveness of the partially sterile released individuals compared to the fully
sterilized individuals used in SIRM; 2) the F1 individuals are produced in the native
population, thus freeing rearing facilities for the production of higher numbers of the
primary release organism (Anonymous 1993).
Conditional Lethal Mutations

In this technique, strains of insect are produced (by genetic manipulation)

carrying traits that are detrimental to the species in the native environment, but which are
not detrimental under laboratory conditions. For example, the inability to diapause would
be a conditional lethal in an insect that had to go into diapause to survive a host-free
period, but in the laboratory no diapause would be necessary. Some other types of
conditional lethal traits which could be used are cold-sensitive lethal mutations, high-
temperature-sensitive lethal mutations, inability to fly, lack of sex pheromone production
or response, lack of ability to develop on a particular host, change in protective
coloration, or any other genetic change which could affect an individual's ability to
survive or reproduce in the native. In the past, the introduction of conditional lethals was
dependent upon our ability to identify and isolate induced or naturally occurring
mutations in the target species. Investigators are presently investigating the possibility
that such mutations can be taken from genetically well known organisms, such as D.
melanogaster, and introduced into economically important organisms using new
techniques developed in molecular biology (Cockburn, et al. 1989)

Behavioral Changes

It would be advantageous to cause changes in the specific insect behaviors that

make them undesirable or destructive. For example, in many pest species, a change from
multi-voltine to univoltine development would eliminate the economic destructiveness of
the pest. Such a change is often shown to be genetic in character. If we could learn
enough about insect physiology to be able to identify the genetic determinants behind
such behavior, then we should be able to genetically manipulate the species and release
the new strain in such large numbers that the new trait would become predominant in the
population. Some other such traits would be alterations of host range, time of
development, fecundity, sex-ratio, humidity requirements, pupation sites, etc. We should
also consider traits in insects that would make the insect susceptible to cheaper or
environmentally benign chemicals.

Hybrid Sterility

The evolutionary background of many insect pests leads us to believe that

reproductive barriers may exist between races of the same species in different geographic
areas or between closely related species. Sterile hybrids could be raised in the laboratory
and released into native populations. This phenomenon has been clearly demonstrated in
crosses between Heliothis virescens males and Heliothis subflexa females (Laster et al.
1996) and should be more widely investigated.

Simply Inherited Mutations

Genetic changes in a single gene could lead to decreased fitness in the native if
the gene could be forced into the native population in large numbers. Some examples of
such changes would be recessive lethal mutations, eye color changes (to increase or
decrease light sensitivity), pupal or adult body color changes, antennae, leg, or wing
deformities.

It is obvious that each of the previously mentioned phenomena requires

significant research data on insect genetics, physiology, behavior, and reproduction to
bring any of the techniques to fruition.

GENETIC SEXING TECHNIQUES

In many applications of autocidal control, it would be efficient to separate the

males and females before release. Possible reasons for such separation are to avoid
assortative mating; to avoid any increase in the size of the feral population during a
genetic control procedure; to eliminate females which may be disease vectors, extremely
obnoxious, or which cause damage to produce or livestock. Another reason for using a
genetic sexing procedure is to bring about cost savings in the rearing process if one sex
could be eliminated in the egg stage (pre-zygotic sexing). In this case, twice as many
insects (of one sex) could be produced with a given expenditure for diet and labor.

Thus, removal of females during the rearing process has been a goal in autocidal
control research for many pest insects. The most successful efforts have been with a
mosquito, Anopheles albimanus, the medfly, Ceratitis capitata, and the stable fly,
Stomoxys calcitrans. Two of these approaches demonstrate what can be done. A
combination of mechanical separation of the sexes and genetic manipulation has been
employed in the medfly. Wild-type pupal color is brown. Mutants involving either black
pupae or white pupae have been linked with the sex chromosomes by means of single or
multiple chromosomal translocations. By this means strains are produced in which male
pupae are black and female pupae are brown. The pupae are then separated by electronic
color recognition circuitry in a mechanical or air-driven sorter. Some disadvantages of
this technique are the possible breakdown of the translocation stocks due to crossing-
over, contamination of the strain by wild-type individuals, and partial sterility of the
strain due to the translocation.

If no use can be found for the reared females, then it would be worthwhile to
eliminate them before they consume expensive diet. A number of schemes have been
proposed to accomplish removal of females at the egg or early larval stage. In some
species, alleles resistant to specific toxic chemicals, such as ethyl alcohol, endrin, purine,
potassium sorbate, dieldrin, cyromazine, and propoxur have been selected. Then
translocations between one of the sex-chromosomes (usually the Y-chromosome) and the
chromosome bearing the resistance locus are induced. When the toxic substance is
introduced into the colony, only the sex carrying the resistant allele survives. This
method has been successful for three species of mosquitoes and is being actively
investigated for the medfly.
MOLECULAR GENETIC TECHNIQUES

The demonstration of genetic transformation of D. melanogaster with

transposable vectors (P-elements) raised the hope that similar techniques could be
employed in other insect species. At the least, the demonstration that exogenous DNA
sequences can be introduced into Drosophila germ lines has suggested new approaches
that can be taken with other insect species.

Unfortunately, attempts at using Drosophila P-elements as vectors of exogenous

DNA have not yet been successful in non-drosophilids. Alternative gene vectors will
have to be discovered. Some possibilities for vectors are various insect viruses, such as
baculoviruses, which might form stable transformations. Investigators are also searching
in other insect species for transposable elements analogous to the Drosophila P-element.

Some of the techniques developed in molecular genetics that may be useful in

autocidal control programs for insect pests include: nucleic acid hybridization, restriction
mapping of chromosomes, nucleotide sequencing, cloning DNA sequences, insertion of
recombinant DNA (transformation), and development of monoclonal antibodies.

Obstacles to progress in the use of molecular genetics for insect control are: 1)
lack of a general vector for gene transfer, 2) lack of suitable selective criteria for useful
loci, 3) uncertainty about what genetic modifications will prove most detrimental to a
given species, 4) lack of knowledge of the basic genetic biology of most insect pests.
None of these obstacles is insurmountable.

INTEGRATION INTO PEST MANAGEMENT PROGRAMS

The sterile insect release method and other autocidal control techniques are
completely compatible with other types of insect control that might be used in IPM
programs. In fact, E. F. Knipling has always insisted that autocidal control must be
integrated with other measures in order to be used in the most effective way. The
autocidal control technologies are most efficient and economical when pest populations
are already at low levels. One reason for this is that these techniques generally involve
release of laboratory (or factory) reared insects. Thus, the numbers of insects that can be
reared will determine the size of the area of release. If less native insects are in the release
zone, then a higher release ratio will be attained or a larger release area can be covered.

Any insect control measure that will reduce population densities either before or
during the application of an autocidal control program will enhance the effectiveness of
both procedures. For example, use of biocontrol organisms is generally most effective at
high pest densities, but lose efficiency as pest densities decrease. Thus, a Sterile Insect
Release Program would be a natural adjunct to any type of biological control program.
This same reasoning applies to the use of insecticides to reduce high pest populations and
the use of pheromones in confusion programs. Cultural control methods, which are
generally used to reduce pest populations during their most vulnerable stages during the
year, are completely compatible with the use of autocidal control programs because the
latter programs would generally be used during the time of high reproductive potential of
the target species. It is hard to think of an Integrated Pest Management Program that
could not be enhanced by the addition of an autocidal control technique.

REFERENCES

Anonymous. 1993. Radiation Induced F1 Sterility in Lepidoptera for Area-wide Control.

Proceedings of a Research Coordination Meeting, Phoenix, AZ, International
Atomic Energy Agency, Vienna.

Bartlett, A. C. 1990. Insect sterility, insect genetics, and insect control, pp. 279-287. In
D. Pimentel [ed.], Handbook of Pest Management in Agriculture, Vol. II. CRC
Press, Boca Raton, FL.

Cockburn, A. F., A. J. Howells, and M. J. Whitten. 1989. Recombinant DNA

technology and genetic control of pest insects, pp. 319 -349. In G. E. Russell [ed.]
Genetical and Biochemical Aspects of Invertebrate Crop Pests. Intercept Ltd.,
Andover, Hampshire, UK.

Cox, H. C. 1978. Eradication of plant pests - pro's and con's. Bulletin of the
Entomological Society of America. 24: 35 - 54.

Davidson, G. 1974. Genetic Control of Insect Pests. Academic Press, New York, NY.

Knipling, E. F. 1979. The Basic Principles of Insect Population Suppression and

Management. U. S. Dept. of Agriculture. Agriculture Handbook No. 512.
Washington, D. C.

Knipling, E. F. 1985. Sterile insect technique as a screwworm control measure: The

concept and its development, pp. 4-7. In O. H. Graham [ed.], Symposium on
Eradication of the Screwworm from the United States and Mexico. Misc. Publ.
Entomol. Soc. America 62, College Park, MD.

Laster, M. L., D. D. Hardee, & J. C. Schneider. 1996. Heliothis virescens

(Lepidoptera: Noctuidae) suppression by sterile backcross releases. J. Econ.
Entomol. (in press).

Pal, R. and Whitten, M. J. 1974. The Use of Genetics in Insect Control. American
Elsevier, New York, NY.

Staten, R. T., Rosander, R. W., and Keaveny, D. F. 1993. Genetic control of cotton
insects, pp. 269-283. In Management of Insect Pests: Nuclear and Related
Molecular and Genetic Techniques. (Proc. Symp. Vienna, 1992), International
Atomic Energy Agency, Vienna.
Whitten, M. J. 1985. Conceptual basis for genetic control, pp. 465-528. In G. A. Kerkut
and L. I. Gilbert [eds.], Comprehensive Insect Physiology, Biochemistry and
Pharmacology. Pergamon Press, Oxford.
 TRANSGENIC Bt. PLANTS: RESISTANCE MANAGEMENT STRATEGIES

Introduction

Recent advances in molecular biology and genetic engineering have made

possible the construction of crop plants possessing novel pesticidal genes. The use of
such transgenic plants in pest management would have the following benefits.

1. Provides season long protection

2. Insects are exposed at the most sensitive stage

3. Protection is independent of weather

4. No application costs

5. Protects plant tissue difficult to reach with chemical pesticides

6. Only phytophagous insects are exposed

7. The active principle does not leach into the environment

8. Active factor is biodegradable

9. Factors non-toxic to man and animals can be chosen

10. Consumer acceptability is high since no residue problems are seen

11. Leads to financial savings – an economic advantage to the farmer.

Insect Resistant Plants

Two groups of genes viz., The B.t crystal protein gene as well as the protease
inhibitors have been engineered into plants to confer pest resistance (Table 1). The first
results on insect control by plants engineered with the crystal protein gene were
published in 1987 (Barton et al., 1987; Fischhoff et al., 1987; Vaeck et al., 1987).
Subsequent to large scale field trials, several million acres of B.t. crops (cotton, corn,
maize and potatoes) were planted in the U.S.A. in 1996.
Commercialization of B.t transgenic crops

As of early as 1997, over 70 transgenic crops have been approved for

commercialization in nine countries plus the E.U. Many are approved for growing and
human consumption particularly in the U.S.A. and Canada where as some are for import
and human consumption of the product and over 10 crops are pending approval. Of the
80 crops or so approved or pending approval in eight countries, 21 are B.t. transgenic
crops dominated by corn and followed by potato and cotton. The developing countries
that commercialized transgenic crops are Mexico and South Africa. The commercial B.t.
plants sold by different companies are given in Table 2.

Development of Resistance Management Strategy

As in the case of B.t products, insects can develop resistance to B.t toxin in
transgenic plants also. In order to sustain the long term impact of B.t plants, it is essential
to develop tactics for deployment of insect resistance genes in plants. The issue of
managing the deployment to reduce insect resistance is not unique to transgenics. Four
strategies have been suggested which are not mutually exclusive.

1. Gene Strategies i. Single gene

ii. Multiple gene (e.g. Pyramid)
iii. Chimeric genes

2. Gene Promoter i. Constitutive

ii. Tissue specific
iii. Inducible (e.g. wounding)

3. Gene Expression i. High dose

ii. Low dose
iii. Mixtures

4. Field Tactics i. Uniform single gene

ii. Mixture of genes
iii. Gene rotation
iv. Mosaic planting
v. Refuges (Spatial and temporal)

2
Refugia and Mixtures
The most promising and currently practical strategy is that of using refuges. This
aims at reducing the possibility of long term impact by preventing resistant insects from
mating with other resistant insects thereby preventing the creation of resistant population.
This can be achieved by ensuring that there are always plenty of susceptible insects
nearby for the few resistant ones to mate with.

High dose approaches

The principle is to deploy plants with high levels of expression of the toxin with
the expectation that it would take a long time for insects to overcome the toxin and that
even resistant homozygotes would be killed by the high level of toxin. This approache
has to be incorporated into the refuge approach.

Multiple gene deployment

When combined with refugia and high dose approach, deployment of multiple
gene appears to be a viable complementary strategy. This strategy requires more than one
resistance gene with different modes of action (or binding sites in the case of B.t).

Targeted Expression

A toxin gene is expressed only specifically in a certain vulnerable part of the plant
e.g. stem in the case of maize borer or is expressed both in certain part of the plant as
well as at a particularly critical time in the development of the plant like square formation
in cotton. This strategy would allow plenty of susceptible insects to breed normally thus
increasing their predator and parasitic population while at the same time be prevented
from causing damage in the critical plant parts or life cycles. Deployment strategies
already implemented show that a high dose strategy with refuges has been adopted in
different B.t crop plants.

Future strategies
Two attractive areas are (i.) incorporation of host plant resistance traits into B.t
cotton and (ii.) incorporation of a novel protein that provides effective control of
lepidopteran pests. Introduction of a second non. B.t insecticidal protein may enhance the
stability of pest control potential.

3
 The first generation transgenic plants containing single B.t genes will be followed
by more sophistigated second and third generation plants with greater flexibility for use
in IPM system. These include plants with inducible and tissue specific expression system
as well as multiple genes. Future IPM programme will involve a combination of
genetically engineered and modified B.t microbial products, several types of engineered
plants and traditional B.t products (Marrone, 1993).

4
 Table 1. Transgenic plants with pest resistance

Crop Gene Pest Reference

Cotton Protease inhibitors : B. tabaci Thomas et al. (1995)
Anti Trypsin
Anti-chymotrypsin
Antielastase
(From Manduca sexta)

Cotton B.t. toxin Cry 1 A (b) Boll worm Perlak et al. (1990)
Maize Syn Cry 1 A (b) O. nubilalis
Tomato Cry 1 A (c) Fruit borer Fischhoff et al.
(1987)
Rice (Japonica) Modified Cry 1 A (b) Striped stem borer Fujimoto et al. (1993)
+ C. medinelis
Tobacco Cry 1 A (a) Barton et al. (1987)
Cauliflower Cry 1 A (a) Kumar et al

Rice (Japonica) Proteinase inhibitors II Sesamia inference Duan et al. (1996)

Rice (India) Syn Cry 1 A (b) S. incertulas Wunn et al. (1996)
C. medinalis
C.suppresalis
M. patnalis

Table 2. Commercial B.t. plants

Crop Company Gene Trade Name

Maize Novartis Cry 1 A (b) Maximiser
Maize Monsanto Cry 1 A (b) Yield Gard
Maize Mycogen Cry 1 A (b) Nature Gard
Cotton Monsanto Cry 1 A (c) Boll Gard
Potato Monsanto Cry 1 A (a) New Leaf

5
 Table 3. Minimum Refuge Areas in Different transgenic Crops to prevent the
likelihood of insect resistance

Product Crop Measure

BollgardTM Cotton 25% of unprotected cotton (non-transgenic) as a refuge using

conventional insect controls if desired or 4% of unprotected
cotton (non-transgenic) as a total refuge using no insect
control whatsoever.
Nature GardTM Corn High-dose strategy with levels exceeding the LC 99 for
European corn borer; also propose to plant 5-50% of
unprotected corn (non-transgenic)
New LeafTM Potato 20% of unprotected potatoes (non-transgenic) as a refuge
using conventional insect control if disired
MaximizerTM Corn High-dose strategy with levels exceeding the LC 99 for
European corn borer; also propose to plant 5-50% of
unprotected corn (non-transgenic)
Yield GardTM Corn 25% of unprotected cotn (non-transgenic) as a refuge using no
conventional insect controls whatsoever.

BollgardTM, Yield GardTM and New LeafTM are trademarks of Monsanto, MaximizerTM of
Novartis and Nature GardTM of Mycogen.

6
References

Barton, K., Whitely, H. and Yang, N.S. 1987. Bacillus thuringiensis (delta) endotoxin in
transgenic Nicotiana tabacum provides resistance to lepitopteran insects. Plant
physiology, 85 : 1103-1109.
Duan, X., Li, X., Xue, Q. Abo-E1-Saad, M., Xu, D. and Wu, R. 1996. Transgenic rice
plants harboring an introduced potato proteinase inhibitor II gene are insect
resistant. Nature Biotechnology, 14 :494-498.
Fischhoff, D.A., Bowdish, K.S., Perlak, F.J., Marrine, P.G., McCormick, S.H.,
Neidenmeyer, J.G., Dean, D.A., Kusomo Kretzmer, K., Mayer, E.J., Rochester,
D.E., Rogers, S.E. and Fraey, R.T. 1987. Insect tolerant transgenic tomato plants.
Biotechnology, 5: 807-813.
Hideya Fujimoto, Ximiko Itoh, Mikihiro Yamamoto, Junko Kyozuka and Ko Shimamat,
1993. Insect resistant rice generated by introduction of a modified Endotoxin gene
of Bacillus thuringiensis Bio/Technology, 11: 1151-1155.
Marrone, P.G. 1993. Engineered plants and microbes in Integrated Pest Management
system. In “Advanced Engineered Pesticides” (Leo Kim ed.), pp. 233-248.
Marcel Dekkr, Inc., New York.
McIntosh, S.C., Kishore, G.M., Perlak, F.J., Marrone, P.G., Stone, T.B., Sims, S.R. and
Fuchs, R.L. 1990. Potentiation of Bacillus thuringiensis insecticidal activity by
serine protease inhibitors. J. Agric. Food Chem., 38 : 1145-1152.
Perlak, F.J., Deaton, R.W., Armstrong, T.A., Fuchs, R.L., Sims, S.R., Greenplate, I.T.
and Fischheff, D.A. 1990. Insect Resistant Cotton. Biotechnology, 8 : 939-943.
Thomas, J.C., Adams, D.G., Keppenue, D.V., Wasmann, C.C., Brown, J.K., Kanost,
M.R. and Bohnert, H.J. 1995. Protease inhibitors of Manduca sexta expressed in
transgenic cotton. Plant cell Reports, 14 : 758-762.
Vaeck, M., Reynaerts, A., Hofte, H., Jansons, F., de Beuckeeler, Deen, C., Zabean, M.
Van Montague, M. and Leemans, J. 1987. Transgenic plants protected from insect
attack. Nture, 328: 33-37.