Final Practical Manual ELEC 230-converted.pdf

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PRACTICAL MANUAL
ELEC 230 (2+1) – BIOPESTICIDES AND BIOFERTILIZERS
COURSE TEACHER
Mr. S. Srinivasnaik, M.Sc. Ag. (Ento.)
Assistant Professor
COMPILED BY
Mr. S. Srinivasnaik, M.Sc. Ag. (Ento.)
Assistant Professor
Dr. P. Swarna Sree, M.Sc. Ag., Ph.D
Professor & Head (Ento.)
DEPARTMENT OF ENTOMOLOGY
AGRICULTURAL COLLEGE, POLASA, JAGTIAL-505 529
PROFESSOR JAYASHANKAR TELANGANA STATE
AGRICULTURAL UNIVERSITY

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C E R T I F I C A T E
Certified that this is a bonafide record of work done by
Mr./Ms.……...............………..……………ID. No.………...........… of II Year
of B.Sc.Ag during the academic year ……….........………and semester
of …………….......…for the course ELEC 230 Biopesticides and Biofertilizers
with credit hours 3 (2+1).
External Examiner Course Teacher

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INDEX
S.No. Title of the exercise
Page
No.
Date of
Experiment
Date of
Submission
Signature
1. Terms, concepts, rules and
regulations followed in
biopesticide laboratory
2.
Isolation and purification of
entomopathogenic viruses and
their production.
3.
entomopathogenic bacteria and
its production.
4.
entomo pathogenic fungi (epf)
and its production
5.
Isolation of other antagonistic
organisms and their production
6.
Field visit to explore natural
infections & epizootics along
with study of symptomology
under field conditions
7. Identification of important
botanicals & quality control
protocols for bio pesticides
8.
Visit to bio pesticides
production unit

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Ex. No: 01
TERMS, CONCEPTS, RULES AND REGULATIONS FOLLOWED
IN BIOPESTICIDE LABORATORY
Date:
AIM
The student should get acquainted with the terms, concepts, rules and regulations
followed in the biopesticide laboratory.
TERMINOLOGY
Entomopathogen/ Insect pathogen: Entomopathogens are infectious agents,
microorganisms that invade and reproduce in an insect and spread to infect other insects. Eg:
Fungi, bacteria, actinomycetes and nematodes etc.
Insect pathology: Insect pathology is the study of anything that goes wrong [i.e., disease
(“lack of ease”)] with an insect.
Infectivity: Ability of microorganism to enter the body of a susceptible insect and produce an
infection
Pathogenicity: The quality or state or being pathogenic, the potential or ability to produce
disease
Virulence: The disease producing power of an organism, the degree of pathogenicity within
a group or species
Dosage: A minimal number of infective propagules needed to pass through the portal of entry
for infection to occur in an insect
Sign: Physical or structural abnormality in an insect as a result of infection. Eg:
Abnormalities in the morphology or structure such as colour, malformed appendages or body
segments, fragility of the integument, etc.
Symptom: Functional and behavioural abnormality in an insect as a result of infection. E.g:
Abnormal movement, abnormal response to stimuli, digestive disturbances (vomiting or
diarrhoea), inability to mate, etc.
Syndrome: It refers to a system complex or a particular combination or sequence of signs
and symptoms (Group of characteristic signs and symptoms)
Course of infection: It is the time from when the entomopathogen infects/enters the host
until its death
Incubation period: It is the time from when the entomopathogen infects/enters the host until
the development of signs and/or symptoms
Acute infection: Acute infections are of short duration and usually result in the death of the
host (i.e., the period of lethal infection is short).

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Chronic infection: Chronic infections are of long duration and the hosts may or may not die
Latent infection: Latent infections in insects have been detected primarily with viruses. In
such cases, the term latent or occult viral infection is used, and the virus is referred to as an
occult virus and not as a latent virus
Epizootiology: It deals with epizootic and enzootic levels of animal disease. Epizootic is
defined as an outbreak of disease in which there is an unusually large number of cases,
whereas an enzootic refers to a low level of disease that is constantly present in a population
CONCEPTS
Robert Koch’s postulates
One of the basic tenets in pathology for establishing the etiological or causal agent of
a disease involving microorganisms is the application of Koch’s postulates. Robert Koch
(1843-1910), a German physician who is considered one of the founders of microbiology,
made brilliant discoveries on the causal agents of anthrax, tuberculosis, and cholera through
the application of postulates that bear his name:
1. The suspected pathogen must be found associated with the disease in all the diseased
insects examined. The organism must be isolated from the diseased insect and grown
in pure culture on nutrient media and its characteristics described (non-obligate
parasites) or in a susceptible host (obligate parasites), and its appearance and effects
recorded.
2. When a healthy insect, of the same species or variety, is inoculated with this culture, it
must produce the disease and show the characteristic symptoms.
3. The organism must be re-isolated from the inoculated insect and must be shown to be
the same pathogen as the original. If all the above steps have been followed and
proved true, then the isolated pathogen is identified as the organism responsible for
the disease.
Diagnosis
Diagnosis is a fundamental branch of insect pathology which involves the process by
which one disease is distinguished from another. The identification of the etiological or
causal agent alone is not diagnosis, but only one of a series of steps in the operation to
determine the cause of the disease. To conduct a proper diagnosis, a study has to be made of
the etiology, symptomatology, pathogenesis, pathology and epizootiology of the disease. The
importance of diagnosis in insect pathology lies in the fact that one must know the nature of
the disease and what ails or has killed an insect before the disease can be properly studied,
controlled, or suppressed, used as a microbial control measure, its potential for natural spread
determined, or its role in the ecological life of an insect species ascertained.

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RULES AND REGULATIONS
• Always wear apron before entering the laboratory for, protecting clothes from
contamination or accidental discoloration by staining solutions
• Before and after each laboratory period, clean your work bench with a disinfectant
• Never eat or drink in the laboratory
• Never place pencils, labels or any other material in your mouth.
• If a live culture is spilled, cover the area with a disinfectant such as 0.1% mercuric
chloride for 15 min and then clean it.
• Be careful of lab burners and put them off when not in use.
• The waste paper and contaminated glassware should be kept in waste basket trays.
• Wash your hands with soap and water before leaving the lab.
• Broth culture must never be pipetted with mouth.
• Keep aseptic culture tubes in an upright position in a rack or basket.
• Materials such as stains, pipettes must be returned to original location after use.
• Familiarize yourself in advance with the exercise to be performed.
• Label all plates, cultures and tubes before starting.
• Before execution of work, read over the exercise to be done and calculate the
requirement and plan.
• Each lab class will begin with a short introduction period. Don't begin work until you
have received instructions.
• Ask questions when you don't understand any method.
• Properly record all experimental details and observations and keep your note book up
to date.
• Clean the bench with a clean cloth or a piece of cotton, after completion of exercise
Exercise:
1) Understand the terms and concepts and know about important scientists
3) Observe the different instruments and understand their principle of working and utility

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Identify the following scientists and write their contributions ?

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Identify the following instruments and write their utility?

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Ex. No: 02 ISOLATION AND PURIFICATION OF
ENTOMOPATHOGENIC VIRUSES AND THEIR PRODUCTION
Date:
================================================
Aim: To aquitaine the isolation and purification methodology of entomopathogenic virus.
In India, Helicoverpa armigera is of major importance damaging a wide variety of
food, fibre, oilseed, fodder and horticultural crops. The nuclear polyhedrosis virus of H.
armigera (HaNPV) is currently used for the management of H. armigera on chickpea, cotton,
pigeon pea, tomato and sunflower. Mass production of Nuclear Polyhedrosis Virus (NPV) on
commercial scale is restricted to in vivo procedures in host larvae which are obtained by
✓ Field collection from cotton, pigeon pea and chickpea
✓ Mass culturing in the laboratory in semisynthetic diet
Some small scale producers use field – collected larvae for mass production of NPV in spite
of the following constraints.
1. Collection of a large number of larvae in optimum stage (late IV / early V instars) is
time-consuming and can be expensive in terms of labour and transportation costs.
2. Wild populations of insects may carry disease causing organisms like
microsporidians, cytoplasmic polyhedrosis virus, stunt virus and fungal pathogens
which will affect both virus production and quality.
3. Introduction of wild strains of NPV resulting in quality control problems.
4. Transportation of a large number of larvae with cannibalistic behaviour will be a
difficult task.
5. Parasitized larvae collected from the field will die prematurely yielding small quantity
of virus.
Rearing of larvae in the natural host plant will involve frequent change of food at least
once a day during the incubation period of 5-9 days increasing the handling time and
hence the cost. In order to reduce the cost, field collected larvae are released into semi
synthetic diet treated with virus inoculum. Mass culturing of insects in semi synthetic diet
involves high level of expertise, hygiene and cleanliness

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Production procedure
The NPV of H. armigera is propagated in early fifth instar larvae. The virus is
multiplied in a facility away from the host culture laboratory. The dose of the inoculum used
is 5 x 105
polyhedral occlusion bodies (POB) in 10-l
suspension. The virus is applied on to the
semisynthetic diet (lacking formaldehyde) dispensed previously in 5 ml glass vials. A blunt
end polished glass rod (6 mm) is used to distribute the suspension containing the virus
uniformly over the diet surface. Early fifth instar stage of larvae are released singly into the
glass vials after inoculation and plugged with cotton and incubated at a constant temperature
of 25o
C in a laboratory incubator. When the larvae exhausted the feed, fresh untreated diet is
provided. The larvae are observed for the development of virosis and the cadavers collected
carefully from individual bottles starting from fifth day. Approximately, 200 cadavers are
collected per sterile cheese cup (300 ml) and the contents are frozen immediately. Depending
upon need, cadavers are removed from the refrigerator and thawed very rapidly by agitation
in water.
Processing of NPV
The method of processing of NPV requires greater care to avoid losses during
processing. The cadavers are brought to normal room temperature by repeatedly thawing the
container with cadaver under running tap water. The cadavers are homogenized in sterile ice
cold distilled water at the ratio 1: 2.5 (w/v) in a blender or precooled all glass pestle and
mortar. The homogenate is filtered through double layered muslin and repeatedly washed
with distilled water. The ratio of water to be used for this purpose is 1: 7.5-12.5 (w/v) for the
original weight of the cadaver processed. The left over mat on the muslin is discarded and the
filtrate can be semi-purified by differential centrifugation. The filtrate is centrifuged for 30-60
sec. at 500 rpm to remove debris. The supernatant is next centrifuged for 20 min at 5,000 rpm.
Then the pellet containing the polyhedral occlusion bodies (POB) is suspended in sterile distilled
water and washed three times by centrifuging the pellet in distilled water at low rpm followed by
centrifugation at high rpm. The pellet finally collected is suspended in distilled water and made
up to a known volume, which is necessary to calculate the strength of the POB in the purified
suspension.
Exercise
1. Isolate entomopathogenic virus from insect cadaver and purify the virus collected.

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Ex. No: 03
ISOLATION AND PURIFICATION OF ENTOMOPATHOGENIC
BACTERIA AND ITS PRODUCTION
Date:
Aim: To aquitaine isolation methodology for entomopathogenic bacteria collected from
different sources and its mass production.
Isolation from soil
• Each sample is divided into 2 to 4g lots and each lot is added to screw-capped tubes
containing 10 ml sterile water.
• Each tube is vortexed and proceeded with heat treatment and plating
Isolation from insect cadavers
• Insect cadavers are placed into tubes containing 1 ml of sterile water per 0.2 to 0.4 g
of insect.
• Sample is homogenized (addition of Tween 80 to 0.5% may aid the homogenization), then
proceeded with heat treatment and plating
Heat treatment and plating
• The samples are heated in a water bath at 80 ºC for 10 min, and then allowed to chill
rapidly on ice. This step kills most vegetative cells of Bacilli and non spore forming
bacteria, thereby enriching for spores of Bacillus species (due to their heat-resistant
nature).
• After allowing the solid content of the tubes to settle, 100 µl of each of the heated
sample and dilutions of the heated sample (usually 10-1
and 10-2
, exclusive for
Bacillus) is plated onto a Petri dish containing a growth medium (MBS
medium/Nutrient Agar ) and incubated for 24 h at 30ºC to allow for bacterial growth.
• Plates are examined for bacterial growth. Using a ﬁne sterile loop, each colony is
transferred to 10 ml growth media in sterile tubes and shake at 250 rpm on an orbital
shaker for 48 h at 30º C.

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Mass culturing techniques
The growth of most commonly used entomopathogenic bacteria, B. thuringiensis and
B. sphaericus is typically done at 30 ºC and UG medium has provided reliable and
reproducible growth, sporulation and production of parasporal bodies in both cases.
Preparation of a 10-ml preculture
• From a stock or a colony from a fresh plate, is inoculated into the tube containing 10
ml UG medium to serve as a preculture.
• After inoculation, culture is incubated on a shaker for 48 h at 30ºC and then observed
for sporulation. After sporulation occurs, the preculture is heat-treated at 80 ºC for 10
minutes to kill vegetative cells. Heat treatment allows for a more consistent growth of
the new culture. This preculture will be used to inoculate the cultures for large scale
production.
Harvesting of spores/crystals
• Erlenmeyer flask (1 litre) containing 100 ml UG medium is subjected to sterilization
at 121ºC for 15 minutes. After sterilization, glucose is added to give 1%final
concentration. [Glucose stock solution of 10% that has been filter sterilized should be
used]
• Flask is inoculated with ~ 0.5 ml of a preculture and incubated at 30º C with orbital
agitation for 48 to 72 h until cell lysis is complete. Culture should be checked under a
phase-contrast microscope to monitor cell lysis, the sporulation rate and presence of
parasporal crystal proteins.
• Then culture is subjected to centrifugation at 5000 rpm for 15 to 20 minutes.
Supernatant is decanted and pellet of spores and crystals is collected
• The pellet of spores and crystals is resuspended with 0.5 M NaCl for 15 min to avoid
exoprotease activity.
• The resuspended spore crystal mix is centrifuged at 5000 rpm for 15 to 20 min. Again
the pellet is resuspended in distilled or deionized water and centrifugation is repeated.
• Finally, the pellet is resuspended in a water volume identical to the initial culture (i.e.
200 ml). This material can be used in bioassays to determine target insects or pellet
of spores/ crystals is freeze dried for long term storage
Exercise
1) Isolate entomopathogenic bacteria from insect cadaver and soil and culture it.

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Ex. No: 04 ISOLATION AND PURIFICATION OF ENTOMO PATHOGENIC
FUNGI (EPF) AND ITS PRODUCTION.
Date:
Aim: To aquitaine the Isolate procedure of entomopathogenic fungi collected from different
sources and its mass production.
Isolation from insect cadavers
• The cadavers of the insect that appeared to be infected by fungi were collected and
brought to the laboratory and the pathogens can be isolated on specific media.
• To isolate the fungi, mycosed samples collected from the fields is surface sterilized
with four per cent sodium hypochlorite for few seconds and then thoroughly washed
with sterilized double distilled water several times.
• The excess water can be removed by keeping the cadaver in Whatman filter paper no.
1. The cadavers are then cut into small pieces with the help of sterile blade and the
bits are aseptically transferred with sterilized inoculation needle on to sterilized
petridishes containing selective media and incubated at 25±2ºC
• However, if the identity of the fungus is unknown, virtually any medium used for
propagation of entomopathogenic hypocreales can be used. Routinely Sabouraud’s
Maltose Agar enriched with one per cent yeast extract (SMAY) media or Sabouraud
Dextrose Agar with yeast extract (SDAY) supplemented with streptomycin sulphate
(0.08%) is used.
Isolation from soil
Collection of soil samples
• Entomopathogenic fungi are usually heterogeneously distributed in soil, putatively in
or near insect cadavers.
• Hence, during the collection, the depth is usually limited to the top 10 to 15 cm of the
organic and/or a horizon soil zone and the collection tool should be surface-sanitized
between samples to avoid cross contamination.
• Upon collection, the soil samples are usually placed in a cool environment (~5 ºC)
and
• Samples should be processed as quickly as possible, usually within 5 days of
collection

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Dilution spread plating
• Place 10 g of soil into 90 ml of sterile water.
• The sample is then homogenized (stirring the slurry with a magnetic stir bar or on a
mechanical shaker for 20 to 60 min) to release propagules from the soil matrix.
• Following homogenation, the aliquots of 1 ml are spread on to an appropriate medium
using an inclined rotary motion of the petri dish and incubated at an appropriate
temperature (20 to 25 ºC for most taxa) for 3 to 7 days
• Individual colonies can then be transferred to a suitable nutrient medium.
Insect baiting
• The extraction technique facilitates collection of entomopathogens without ever
needing to find a diseased host.
• Entomopathogenic hypocreales are considered to be weak saprotrophs but since they
possess the ability to infect living insects, they can gain access to a living insect
relatively free of competitors. Larvae of the greater wax moth (Galleria mellonella)
are most commonly used insect bait
• Soil samples are placed in containers, the soil is moistened, and larvae (15 nos /
container) are added to the soil and incubated for ~ 14 days
• Larvae are placed on the soil surface, and the soil is agitated or the containers are
gently inverted or shaken periodically to ensure that the larvae remain exposed to the
soil.
• Cadavers are collected at intervals and processed for isolation
Purification and storage of propagules
• Following isolation, it is necessary to ensure that the isolated fungus is free from
contaminant microorganisms and, that it represents a single genotype. The two most
common methods used to achieve genotype purity are the isolation of individual
conidia or the isolation of hyphal tips.
• Hence, after 48 h of isolation, the hyphal tip of fungi is transferred to SMAY and
further purification is achieved by subculturing
• The isolated cultures can be maintained at 25±2ᵒC in an incubator on SMAY media.
The pure stock cultures need to be sub cultured at 15 days interval in petridishes (9
cm diameter). Pure stocks in SMAY slants is held under refrigerated condition (4ºC)
until further use
• The spores from well sporulated cultures can be scraped with sterile blade and
preserved in sterilized 60 per cent glycerol at -40ᵒC for long term storage.

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Mass culturing technique
Most entomopathogenic fungi are easily propagated on deﬁned or semi-deﬁned media
containing suitable nitrogen and carbon sources. In general, as for isolation, SMY broth is
used for mass multiplication purpose but this may vary depending on the biological
requirements of particular fungal isolate.
• 250 ml of SMY broth should be poured in round bottom flask (five hundred ml
capacity) and autoclaved at 121ᵒC for 20 minutes.
• After cooling, 1 ml of spore suspension of fungal isolates inoculated into each flask
separately and kept in orbitrary shaker for three days and incubated at room
temperature for 10 days. After sporulation, the fungal isolates can be ground in a
mixer and filtered through double layered muslin cloth. The suspension then shaken
thoroughly with a drop of Tween 80®
in order to disperse the spores in solution. The
conidial suspension then vortexed for 5 min to produce a homogenous conidial
suspension which can be utilized for field experiments.
Exercise
1) Isolate entomopathogenic fungi from soil and insect cadaver and culture it.

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Ex. No: 05 ISOLATION OF OTHER ANTAGONISTIC ORGANISMS AND
THEIR PRODUCTION
Date:
Aim: Isolation and culturing of the entomopathogenic nematodes through sandwich method.
Entomopathogenic nematodes (EPN) represent a group of soil-inhabiting nematodes that
parasitize a wide range of insects. These nematodes belong to two families: Steinernematidae and
Heterorhabditidae. Until now, more than 70 species have been described in the steinernematidae
and there are about 20 species in the heterorhabditidae. The nematodes have a mutualistic
partnership with enterobacteriaceae bacteria and together they act as a potent insecticidal
complex that kills a wide range of insect species.
Material required:
1. Hand shovel
2. Soil samples
3. Plastic sealable container
4. Small petri dish
5. Larger petri dish
6. Large sealable bottle
Procedure (Sandwich method):
1. Use the hand shovel to collect a sample of soil
2. Fill the small plastic sealable container to about halfway with the soil sample you
collected.
3. Next, drop about 5 or 6 wax worm larvae or rice moth and layer of soil, alternatively
into the plastic container and allow it to incubate at temperature for one week.
4. After the incubation period remove the wax worms from the soil container and place
them in the small petri dish lined with a sheet of filter paper.
5. Moisten the filter paper with a few drops of distilled water.
6. Half-fill the larger petri dish with distilled water and place the smaller petri dish into
it. (This should resemble a moat-like design). Incubate the dishes for one week.
7. After a week the nematodes will have emerged and will be visible in the water of the
larger petri dish. Remove the small petri dish.
8. Pour the nematode solution from the large petri dish into the large sealable bottle.
9. Fill the large sealable bottle with distilled water and refrigerate for one week. This
allows the nematodes to reproduce
Exercise
Isolate entomopathogenic nematodes from different agroecosystems

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Use of nematodes as biological insecticides
Common name Scientific name Crop Efficacious Nematodes
Armyworms Lepidoptera: Noctuidae Vegetables Steinernema carpocapsae, S. feltiae, S. riobrave
Banana moth Opogona sachari Ornamentals Heterorhabditis bacteriophora, S. carpocapsae
Banana root borer Cosmopolites sordidus Banana S. carpocapsae, S. feltiae, S. glaseri
Black cutworm Agrotis ipsilon Turf, vegetables S. carpocapsae
Black vine weevil Otiorhynchus sulcatus Berries, ornamentals
H. bacteriophora, H. downesi, H. marelata, H. megidis, S.
carpocapsae, S. glaseri
Cat flea Ctenocephalides felis Home yard, turf S. carpocapsae
Citrus root weevil Pachnaeus spp. Citrus, ornamentals S. riobrave, H. bacteriophora
Codling moth Cydia pomonella Pome fruit S. carpocapsae, S. feltiae
Corn earworm Helicoverpa zea Vegetables S. carpocapsae, S. feltiae, S. riobrave
Corn rootworm Diabrotica spp. Vegetables H. bacteriophora, S. carpocapsae
Crane fly Diptera: Tipulidae Turf S. carpocapsae
Fungus gnats Diptera: Sciaridae Mushrooms,greenhouse S. feltiae, H. bacteriophora
Large pine weevil Hylobius albietis Forest plantings H. downesi, S. carpocapsae
Leaf miners Liriomyza spp. Vegetables,ornamentals S. carpocapsae, S. feltiae
Mole crickets Scapteriscus spp. Turf S. carpocapsae, S. riobrave, S. carpocapsae
Scarab grubs Coleoptera: Scarabaeidae Turf, ornamentals
H. bacteriophora, S. carpocapsae, S. glaseri, S. scarabaei, H.
zealandica
Shore flies Scatella spp. Ornamentals S. carpocapsae, S. feltiae
Sweet potato weevil Cylas formicarius Sweet potato H. bacteriophora, S. carpocapsae, S. feltiae
At least one scientific study reported 75% suppression of these pests using the nematodes indicated in field or greenhouse experiment.

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Ex. No:06 FIELD VISIT TO EXPLORE NATURAL INFECTIONS &
EPIZOOTICS ALONG WITH STUDY OF SYMPTOMOLOGY
UNDER FIELD CONDITIONS
Date:
===================================================
Both living and dead invertebrates that are patently infected with entomopathogens can
be found by / in natural terrestrial and aquatic ecosystems, agroecosystems, and in laboratory and
commercial insect colonies. A visual search of habitats of interest or of insect colonies in the
laboratory for insects with a typical appearance is a common means of collecting diseased
specimens.
Recognition of diseased invertebrates in the field
Insects that are patently infected with entomopathogens often manifest following
characteristic symptoms and signs (syndrome) of disease
• Striking color changes, luxuriant growth of the pathogen on the outside of the specimen
• Signs of the pathogen or etiological agent inside the host that are visible through the
cuticle
• Dysentery, peculiar behavior including lack of feeding or unusual position on host plants,
• Tremors, mummification, fragility or hardening of the integument, noticeable difference
in size, aborted molt or pupation
Identification of major entomopathogen groups in field
Fungi
• Insect cadavers are often hardened and mummified due to mycoses.
• Haemocoel filled with hyaline hyphae and spherical to ovoid resting spores
• Thread-like growth of organisms on surface of insect, often becoming powdery (e.g.,
white, green, red, etc.) and sometimes limited to intersegmental areas, or growth
macroscopic and club-like
Bacteria
• Insect cadavers shows distinct change in colouration, usually brown, black or reddish in
coloration and especially in scarabeid grubs, it shows white bluish to blue grayish
discolouration.
• Body tissues may be liquified and often exhibits putrified odour.

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• Various body tissues and cells containing minute, rod shaped cells just visible by light
microscopy, usually exhibit brownian movement and are highly refringent, often
occurring in pairs or chainlike structures. Crystals may or may not be present.
Virus
• Insect cadavers turn orange to green to blue in colouration and dead caterpillars may be
attached to host plant in inverted V-shaped manner hanging by abdominal prolegs.
• Cadavers become flaccid and integument may be very fragile, liquified body tissues filled
with spherical to polyhedral-shaped occlusion bodies (POBs) which may also occur in the
cytoplasm or nuclei of intact cells
• Intestine (gut) abnormally opaque and polyhedral shaped particles only visible with
phase microscopy in cytoplasm of gut cells
Nematodes
• Color of cadaver may be different from natural colouration, usually red or cream to light-
brown in color. Many cadavers shows distinct unshed second stage integument.
• Mass of worm like (elongate and non-segmented) organisms over surface of insect or
inside its body
Protists
• Distinct abnormalities in morphology of insects, unlike in other entomopathogens is not
shown for protists
• Abnormal whitish masses in haemocoel associated with various tissues (e.g., fat body) or
in hemolymph itself, masses composed of round or ovoid to pyriform spores refringent
under phase microscopy
Collection and initial handling of diseased specimens
• In the field, diseased terrestrial insects should be removed from the substrate upon which
they are found, with fine forceps and placed individually, if possible, in clean dry
containers.
• To avoid damaging cadavers that are tightly attached to the substrate, the portion of the
host plant upon which they are fixed should be collected with the insect attached. Minute
insects, such as scales and whiteflies, can also be collected in this manner.
• Once insects are returned to the laboratory, they may be refrigerated, frozen or
maintained in a dried or aqueous state at room temperature depending on biological
requirements.

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Collection and initial handling of living arthropods to screen diseased individual
• Collection of large number of living insects using standard insect collecting techniques
(traps, sweeping, hand picking, aquatic nets and dippers) with subsequent screening for
diseased individuals in the field or laboratory is another strategy.
• Two major considerations when collecting invertebrates to detect pathogens: avoiding
exposure to UV radiation and excessive heat in the field and in vehicles, and avoiding
death and decomposition of the host during transport.
• Containers for collecting live hosts may include any type of cage that prevents escape,
including plastic vials with pinholes in the lids or with cotton plugs for air circulation and
prevention of condensation.
• The addition of a drying agent, such as silica gel, to the container used for temporary
storage will slow or prevent germination of entomopathogenic fungi and bacteria, and
help eliminate the growth of saprobic fungi on specimens
• Aquatic invertebrates and specimens containing nematodes and certain protistan parasites
should not be allowed to dry.
Collection of patently living insects
• Patently infected living insects should be kept in the medium in which they are found (i.e.
on foliage, in soil or water) and transported to the laboratory and examined as soon as
possible.
• Healthy insects should be also collected for comparative purposes and held separately
under conditions that enable good survival. Where diseased individuals are rare or
infections are unapparent, large numbers of apparently healthy individuals can also be
used for rearing under conditions that are less favorable for survival. Rearing in stressful
conditions may accelerate the incubation period and the development of pathogens that
are present at low levels, occult, or in an eclipse period at the time of collection.
• Stressing insects by crowding, starving, or other conditions may result in an overt infection or
the appearance of other diseases that might not be apparent in the field.
Exercise
1) Visit to the agricultural garden lands and observe the insects affected by
entomopathogens like virus, bacteria, fungi etc. and identify the signs/symptoms
produced by them
2) Bring the mycosed insect and prove the Robert Koch’s postulates

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Ex. No:07 IDENTIFICATION OF IMPORTANT BOTANICALS &
QUALITY CONTROL PROTOCOLS FOR BIO PESTICIDES
Date:
===================================================
Aim: Identify the different botanicals having pesticidal property and understand the quality
protocols of entomopathogens
India is bestowed with 7% of the floral diversity in the world. Among all 2,400 plant
species are reported to have pesticidal properties. Most promising botanical pesticides for use are
present in substances derived from species of the families Meliaceae, Rutaceae, Asteraceae,
Labiatae and Canellaceae.The single most important botanical source of pesticidal compounds is
Azadirachta indica, belongs to family meliaceae.
Important families having pesticidal properties are
S.No. Plant family Number of plants having pesticidal property
1. Meliaceae >500
2. Myrtaceae 72
3. Asteraceae 70
4. Ephorbiaceae 65
5. Leguminosae 60
6. Fabaceae 55
Major botanicals having the pesticidal property
1. Indian neem tree
✓ Neem is native to India and Burma
✓ The active ingradients is a mixture of Azadirachtin, melantriol, salannin, nimbin and
nimbidin and these all belong to group of tetranotriterpenoids.
✓ The main active ingradient that has potential insecticidal activity present in neem is
azadirachtin, which is present in seeds and leaves and it varies from 2-4 mg/g kernal
✓ Azadirachtin has several stereoisomers but so far 7 stereoisomers have been reported viz.,
AZA (A-G). Azadirachtin A constitutes 85% followed by Azadirachtin B almost 14%
✓ Neem has various effects on insects viz., antifeedant action, insect growth regulatory
activity inhibits juvenile hormone synthesis, oviposition deterrent, repellent action,

22
reduction of life span of adults and intermediates are formed giving rise to larval-pupal,
nymphal-adults and pupal-adults intermediates.
2. Rotenone
✓ It is resin derived from roots of leguminous plants Lonchocarpus spp.
(South American plant) and Derris eliptica (Malaysia)
✓ It is a broad spectrum and stomach poison
✓ It effects nerve and muscle cells in insects and sometimes causes insects to stop feeding
✓ It inhibits respiratory metabolism
✓ It is used as dusts containing 0.75-1.5% rotenone and is effective against beetle and
caterpillars
✓ It is extremely toxic to fish
3. Sabadilla
✓ It is an alkaloid found in seeds of tropical lily, Schoenocaulon officinale
(Family:Liliaceae)
✓ The alkaloids mainly ceyadine and veratridine act as nerve poisons
✓ It is a primarily contact poison
✓ Sabadilla is harmful to pollinators and honey bees
5. Ryanodine
✓ It is an alkaloid derived from woody stems of South American shrub, Ryania speciosa
(Family: Flacourtaceae)
✓ It acts as muscular poison by blocking the conversion of ADP to ATP in striated muscles
✓ It acts as slow acting stomach poison and causes insects to stop feeding after they eat it
✓ It is reportedly effective against thrips and worms
✓ It is used as dust (20-40%)
6. Nicotine
✓ Nicotine is obtained from tobacco plants, Nicotiana tobaccum, N. rustica (Family:
Solanaceae)
✓ Activity: Mimics acetylcholine in the nerve synapse causing tremors, loss of coordination
and eventually death.
✓ It is extremely fast acting, causing sever disruption and failure of nervous system
✓ Sold commercially as a fumigant Nicotine or as a dust (Nicotine Suphate)

23
✓ It is commercially avaible as nicotine sulphate 40 % (Black Leaf 40) and manufactured in
India only for export purpose
✓ It acts as contact poison
✓ It is effective against soft bodied sucking insects like thrips, leafhoppers, mealybugs and
leaf miners
7. Pyrethrum
✓ Pyrethrum refers to powdered dried flowers of Chrysanthemum cinerarifolium and
pyrethrins are all toxic constituents of the pyrethrum flowers and pyrethroids are the
synthetic analogues of pyrethrins
✓ Pyrethrum is occupied 80% global botanical insecticide market
✓ Chrysanthemum cinerarifolium is native of Dalmatian mountains, Croatia
✓ Kenya is a largest producer of pyrethrum
✓ Pyrethrins are esters formed by combination of two acids i.e., chrysanthemic acid and
pyrethric acid with three alcohols namely pyrethrolone, cinerolone and jasmolone. These
six active principles together are responsible for toxicity and knockdown action.
S.No. Ester (Pyrethrins) Acid Alcohol
1. Pyrethrin I Chrysanthemic acid Pyrethrolone
2. Pyrethrin II Pyrethric acid Pyrethrolone
3. Cinerin I Chrysanthemic acid Cinerolone
4. Cinerin II Pyrethric acid Cinerolone
5. Jasmolin I Chrysanthemic acid Jasmolone
6. Jasmolin II Pyrethric acid Jasmolone
8. Limonene and linanool
✓ These are citrus peel extracts which cause insect paralysis.
✓ They evaporate quickly in environment and are used to control aphids, mites and fleas

24
Other promising pesticidal plants
S.No. Plant Scientific name Family
Active
principle
Plant
parts
used
Target insect
1
Custard
apple
Annona squamosa
A. reticulata
Annonaceae e Anonaine
Seeds,
bark and
roots
Caterpillars
2 Periwinkle Vinca rosea Apocynaceae Vinblastine All parts
Red cotton
bug
3 Goat weed
Ageratum
conyzoides
Asteraceae
Chromenes:
Prococenes
I&II
Leaves
Antijuvenile
hormones
4 Garlic Allium sativum
Amaryllidaceae
ae
Diallyl
sulfide
Rhizome
Mosquito, Red
cotton bug
5 Plumbago
Plumbago
zeylanica
Plumbagin indica
Plumbaginaceae
e
Plumbagin Root Red cotton
bug
6. Pongame
Pongamia glabra
Pongamia
pinnatata
Fabaceae karinjin Seeds
Red cotton
bug
7
African
marigold
Tagetus erecta Asteraceae
Allyl Iso
thiocyanate
Root IGR
8
Sweet flag Acorus calamus
Acoraceae Beta-asarone Rhizome
Stored grain
pests
9 China berry Melia azedarach Meliaceae
Meliantrol,
Melianone
Seed
kernel
Antifeedant
action against
Locusts
10
Congress
grass
Parthenium
hysterophorus
Asteraceae Parthenin
Leaf
extracts
Tobacco
caterpillar, red
cotton bug
11
Black
pepper
Piper nigrum Piperaceae Piperine seeds
Helicoverpa
armigera
12 Soybean Glycine max Fabaceae Pinitol Pods
Sitophilus
oryzae
Exercise
1. Collect any 15 botanicals having pesticidal property located at your college and prepare the
herbarium sheets

25
Identify the following botanicals, write scientific names along with active ingredient

26
QUALITY CONTROL PROTOCOLS FOR BIO PESTICIDES
1. Quality control protocols in insect viruses
Important factors that contribute to the poor quality of viral biopesticides are
a) Contamination of host culture with unwanted pathogens like microsporidia and saprophytic
fungi.
b) Genetic drift in host and virus during production
c) Presence of bacterial contaminants in the end product
d) Amount of polyhedral occlusion bodies (POB)/ Occlusion bodies (OB) in the end product
Suggested Indian Standards:
I) Viral units/unit of formulated product:
NPV – 1x109
POB/ml or gm
GV – 5x109
OB/ml or gm
II) Contamination: Salmonella, Shigella or Vibrio should be absent. Other microbial
contaminants should not exceed 1x104
counts/ml or gm.
III) Identification of baculoviruses by restriction enzyme analysis and southern blot.
IV) The strain should be indigenous, naturally occurring and not exotic and genetically
modified.
V) Expected standards for NPV against II instar larvae.
Species LC50 POB/mm2
Helicoverpa armigera 0.5
Spodoptera litura 20.0
VI) Shelf life – 18 months
2. Quality control in bacterial biopesticides
Quality control plays a key role in Bt production. Spore counts have been totally replaced
by bioassay and expression of potency in terms of International Units (IU). The international unit
is defined as a quantity of a biological toxin or its equivalent based on a bioassay that produces a
particular biological effect agreed upon internationally.
.

27
Suggested standards for Bacillus thuringiensis:
i) Immunological assay:
✓ i)ELISA/Dot Blot assay test for estimation of the  - endotoxin protein available.
ii) Routine test:
✓ Level of  - exotoxin to be identified by housefly bioassay method
✓ Potency of product by bioassay method
✓ Viable spore count
iii). Human pathogen like Salmonella, Shigella and Vibrio should be absent
iv). Other non-pathogenic microorganisms (not more than 104
/gm)
The product quality standards are as follows:
✓ Liquid formulation: 2000-4000 IU/l, 1 year stability period
✓ Powder formulation: 16000-32000 IU/l, 2 years stability period.
3. Quality control in mycoinsecticides
Entomogenous fungi are a versatile group of biological control agents, as they have a
wide host range, infective to different stages of the host and are capable of causing natural
epizootics under favourable environment conditions.
Suggested Indian standards for entomopathogenic fungi:
1) Colony forming units (CFU) count: 1x108
CFU /ml or gm
2) Contaminants: Salmonella, Shigella, Vibrio should be absent. Other contaminants should not
exceed 1x104
/ml or gm
3) Method of analysis:
✓ CFU count by serial dilution and plating on specific media
✓ Plating for contaminants on specific media
✓ Entomopathogenic capability on target insect by bioassay
4) The strain should be indigenous, naturally occurring not exotic or genetically modified
5) Maximum moisture content should not be more than 8% for dry formulation.

28
4. Quality control in entomophilic nematodes
Entomophilic nematodes (EPN) belonging to the genus Steinernema and Heterodera have
been used mostly in inundative biological control. They are most efficacious for insects residing
in soil or cryptic habitat, mushroom pests, berries, citrus, turf grass etc. In India, commercial
production of EPN has not taken shoot so far. Efficacy depends on many factors, but preservation
of high nematode viability and virulence during large-scale production and formulation are
essential components of a quality control strategy. The most important production factors
affecting nematode quality are the type of medium, amount and type of antifoam, bacterial phase,
delivery of oxygen, sheer stress, production and storage temperature, and contamination. The
quality of the nematodes that survive the rigors of the manufacturing process is analyzed by
determining their shelf life and virulence. Nematode shelf life is predicted from storage energy
reserves (eg. dry wt of total lipid content) of Infective juvenile (IJ) nematodes; virulence
potential is measured using insect bioassay.
Haemocytometer
Calculation
I) The number of polyhedra/ml is calculated by the formula
X x 400x10x1000XD
=
Y
Where,
X = Number of polyhedra counted totally
Y = Number of smaller (1/400) squares checked
10 = Depth factor
1000 = Conversion factor for mm to cm to ml
D = Dilution factor

29
II) Alternatively number of polyhedra / ml is calculated by
D x X
Number of NPV (POB) per ml/gm =
N x K
Where:
D = Dilution factor
X = Total number of polyhedra counted
N = Number of squares counted
K = Volume above one small square in cm3
= (2.5x10-7
cm3
)
Area of each small square is 1/400 mm2
= 0.0025 mm2
. Depth of chamber is 0.1 mm. Volume of
liquid above a single small square is 0.0025 mm2
x 0.1mm = 0.00025 mm3
. To covert to cm3
multiply by 1/1000 to get a volume of 2.5 x 10-7
cm3
above
1 small square. Hence, K=2.5x10-7
cm
Exercise
1. Enumerate the infective propagules count from any of the biopesticide product from the
pesticide shop dealer and compare with the Indian standards
2. A farmer brought Metarhizium anisopliae talc formulation from two pesticide shops A &
B with different trade names to control the rhinoceros beetle. Analyze the quality of the
product based on the haemocytometer count and write the inference.
Pesticide shop A Pesticide shop B
Spore count 300 185

30
Ex. No:08
VISIT TO BIO PESTICIDES PRODUCTION UNIT
Date:
===================================================
Aim: To visit bio pesticide industry and understand the production procedure of different
entomopathogens
Observations
Exercise:
1. Collect information on bio pesticide unit and products generated
2. Write in detail about the production methodology followed for each entomopathogen
production
3. Paste the photographs of the visit and bio pesticide products as well.

31
Annexure 1: Information on bio pesticide unit and products generated
Name of
the unit
Location/Address
Director/
Manager
details
Products sold
along with
concentration
Trade names
Cost/Unit
production
Cost /unit
selling
Target pests
1
2
3
4
5
6
7
8
9
10
11

32
REFERENCES
DS. Reddy. 2010. Applied Entomology. New Vishal Publications, West Patel Nagar, New Delhi.
227p.
G.S. Dhaliwal and Ramesh Arora. 2017. Integrated Pest management. Kalyani Publishers,
Ludhiana. 235-271p.
M. Johnson and V. Chandra sekhar. 2012. Practical Manual on Principles of Plant Pathology.
Acharya N.G.Ranga Agricultural University/ Professor Jayashankar Telangana state
Agricultural University, Rajendranagar, Hyderabad.79p.
Md. Arshad Anwer. 2018. Biopesicides and Bioagents: Novel tools for pest management. Apple
Academic Press, Mistwell Crescent, Oakville, Canada. 402p
S.Sridharan, P.A.Saravanan, S.Srinivasnaik, S.Sanghamitra, S.Sekar, R.Gowthami and Nikita
S.Awasthi. 2016. Practical manual on insect pathology. Tamilnadu Agricultural
University. Coimbatore. 62p.

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